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DISS. ETH No. 17952

Novel

99m

Tc-Labeled Bombesin Analogues

with Improved Pharmacokinetics for Targeting of Gastrin-Releasing-Peptide Receptor-Positive Tumors

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of DOCTOR OF SCIENCES

presented by CHRISTIAN SCHWEINSBERG Dipl. Biochemist, Eberhard Karls University of T¨ ubingen (Germany) born 24.10.1976 German Citizen

accepted on the recommendation of Prof. Dr. P.A. Schubiger, examiner Prof. Dr. R. Schibli, co-examiner Dr. E. Garc´ıa Garayoa, co-examiner

2008

F¨ ur meine Eltern

Acknowledgements

I would like to thank Professor P. August Schubiger and Professor Roger Schibli for giving me the great opportunity to do my doctoral work in the Center for Radiopharmceutical Sciences at the Paul Scherrer Institute.

Very special thanks go to my supervisor Dr. Elisa Garcia-Garayoa for her support and her guidance during my work in the laboratory as well as for her helpful advice and proofreading during the preparation of publications, presentations and writing this thesis.

My appreciation goes to Dr. Peter Bläuenstein for his huge scientific knowledge, his support and always helpful advice.

I would like to thank Professor Dirk Tourwé, Dr. Veronique Maes and Luc Brans for our fruitful collaboration and for providing us with the peptides I investigated in my thesis.

Special thanks go to Olga Gasser, Alain Blanc, Susan Cohrs and Dr. J¨ urgen Gr¨ unberg for their support and the nice atmosphere in the lab, to Dr. Alexander Hohn for introducing me into the world of SPECT/CT imaging and Christine De Pasquale for her professional advice in animal experiments.

I want to thank all my friends and colleagues at the Center for Radiopharmceutical Science for the convenient working atmosphere.

Finally, I want to thank my parents for their trust and support during the last years.

Contents Summary

V

Zusammenfassung

IX

Abbreviations

XIII

1 Introduction 1.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 SPECT/CT and PET/CT for diagnostic imaging of cancer . . . . . . . 1.3 Radionuclides for radiotherapy . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Technetium-99m and Rhenium-186/188 for radiopharmaceutical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.1 The organometallic fac-[M(OH2 )3 (CO)3 ]+ (M= 99m Tc, Re) . . . . 1.4.2 Bifunctional chelating agents . . . . . . . . . . . . . . . . . . . . . 1.5 Peptide-receptor mediated tumor targeting . . . . . . . . . . . . . . . . . 1.6 Aim of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 8 9 10 18 19

2 New [99m Tc]Bombesin Analogues with Improved Biodistribution for Targeting Gastrin Releasing-Peptide Receptor-Positive Tumors 2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 28 29 30 33 36 41

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1 1 3 5

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 Influence of the Molecular Charge on the Biodistribution of Analogues Labeled with the [99m Tc(CO)3 ]-Core 3.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Experimental Procedures . . . . . . . . . . . . . . . . . . . . . 3.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Supporting Information . . . . . . . . . . . . . . . . . . . . . . 3.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 42

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47 48 49 50 56 60 65 66 67

4 Novel Glycated [99m Tc(CO)3 ]-labeled Bombesin Analogues for Improved Targeting of Gastrin-Releasing Peptide Receptor-Positive Tumors 73 4.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 4.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4.4 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 4.6 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 4.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 5 ”Click to Chelate”: Synthesis and Biomolecules in a Single Step 5.1 Abstract . . . . . . . . . . . . . . 5.2 Results and Discussion . . . . . . 5.3 Acknowledgements . . . . . . . . 5.4 References . . . . . . . . . . . . .

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6 ’Click’-Modified 99m Tc-Tricarbonyl- labeled BBS Analogues for Imaging of Prostate Cancer 107 6.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 6.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

II

6.3 6.4 6.5 6.6 6.7

Materials and Methods Results and Discussion Conclusion . . . . . . . Acknowledgements . . References . . . . . . .

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110 115 123 123 124

7 Conclusion

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Publications and Presentations

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Curriculum Vitae

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IV

Summary Peptide receptors are known to be excellent targets for in vivo cancer diagnosis and therapy and the search for peptide-based radiopharmaceuticals has experienced an important rise in the past decades. In the last 10 years, the gastrin-releasing peptide receptor (GRPR), the subtype 2 of the bombesin (BBS) receptor superfamily, is gaining increasing attention for its overexpression in some of the most prevalent types of human cancer, such as prostate, breast and colon. GRPR features a high affinity for radiotracers based on the C-terminal amino acid sequence of the neuropeptide bombesin, BBS(7-14). So far, radiolabeled BBS analogues exhibit two major drawbacks for diagnostic applications, one of them in form of the rapid degradation, naturally exhibited by most neuropeptides. The modification of an enzymatic cleavage site led to new BBS analogues with enhanced biological half-lives. The replacement of leucine (Leu) by cyclohexylalanine (Cha) and of methionine (Met) by norleucine (Nle) resulted in an increase of the in vitro stability in human plasma (20-fold) and in GRPR-expressing tumor cells (6-fold), while the binding affinity remained unaffected (Kd ≤ 1 nM). The second challenge is the general high liver uptake and the high intestinal background of radiolabeled BBS analogues, due to a preferential hepatobiliary excretion, both most unfavorable for the detection of abdominal tumor lesions with radiologic imaging techniques. Therefore, the major focus of this work was the investigation of the influence of novel polar linkers on the pharmacokinetic profile of 99m Tc(I)-labeled BBS analogues, in order to enhance their potential use for the detection of GRPR-positive tumors using single photon emission tomography (SPECT). Technetium-99m (99m Tc) is a readily available γ emitter with optimal physical properties for diagnostic SPECT imaging (t1/2 = 6.02 h; Eγ = 140 keV). For radiolabeling of the novel peptides we used the tricarbonyl technique, based on the well established attachment of the organometallic precursor fac-[99m Tc(CO)3 (OH2 )3 ]+ to tridentate bifunctional chelating agents (BFCA). In a first approach different polar amino acids were introduced between the stabilized seV

quence BBS(7-14) and the retro-Nα -carboxymethyl histidine chelator, (Nα His)Ac, which is a powerful BFCA for radiolabeling of biomolecules with the 99m Tc(CO)3 -core. All peptides kept the increased stability in human plasma (t1/2 ≥ 16 h) and tumor cells (t1/2 = 30-40 min). The new polar moieties led to a shift of hydrophilicity from a LogD of 0.9 to values between 0.4 and -2.2. A LogD between +1 and -1 resulted in the highest binding affinities (Kd ≤ 0.5 nM) as well as the highest in vitro and in vivo uptake in GRPR-expressing tumor cells. A higher hydrophilicity (LogD ≤ -1.8) led to lower affinity and substantial decrease of internalization. The introduction of a positive charge (β 3 -homo-Lysine) caused unfavorable high kidney and liver uptake. The introduction of a single negative charge (β 3 -homo-glutamic acid) showed a significant increase in specific tumor uptake (2.1 ± 0.6 vs. 0.8 ± 0.4 %ID/g compared to the lipophilic reference) and significantly higher tumor-to-tissue ratios. Moreover, this analogue provided a much clearer image of the tumor xenografts in SPECT/CT studies. Considering the improved biodistribution profile with an increased tumor uptake and enhanced imaging, the introduction of a single negative charge proved to be most useful in the development of new BBS analogues. A second approach was the glycation of our peptides by the introduction of hydrophilic carbohydrates between the stabilized BBS(7-14)-fragment and the (Nα His)Ac-chelator. The new peptides showed LogD-values between -0.2 and -0.5. They exhibited high affinity for GRPR (Kd ≤ 0.5 nM) and kept the increased stability in tumor cells (t1/2 ≥ 35 min). In vivo all carbohydrated compounds exhibited higher tumor-to-background ratios compared to the non-glycated reference. The best results were obtained with a triazole coupled glucose, showing a 4-fold increased uptake and retention in the tumor (3.6 and 2.5 %ID/g at 1.5 h and 5 h p.i, respectively) and a significantly reduced accumulation in the liver (0.6 vs. 2.4 %ID/g, 1.5 h p.i., respectively). Additionally, tumor-to-kidney and tumor-to-blood ratios could be significantly improved by a factor of 1.5 and 2.7, respectively (1.5 h p.i., P ≤ 0.05). The imaging studies proved the reduction of abdominal background and tumor xenografts could clearly be visualized. Finally, we were interested in the potential of novel triazole-based chelators formed by the Cu(I)-catalyzed [3+2] cycloaddition of alkynes and azides (click-chemistry). The clickreaction proved to offer an intriguing new way for 99m Tc-labeling of biomolecules and the formation of the required efficient chelators in a single step. We compared the established (Nα His)Ac-chelator with two novel N2click- and N3click-chelators for labeling of our stabilized BBS analogues with 99m Tc. Furthermore, we investigated the influence of VI

glycation on the promising N3click-peptide. The N3click-BBS analogue exhibited a high affinity for GRPR (Kd ≤ 0.6 nM) and similar in vitro and in vivo properties compared to the (Nα His)Ac-peptide. Glycation of the N3click-compound by formation of a stable triazole-bond resulted in an increased accumulation in GRPR-expressing tissues, in a reduction of liver uptake and higher tumor-to-blood and tumor-to-liver ratios. Tumor uptake and retention were clearly enhanced (4.4 ± 0.6 and 2.6 ± 0.8 %ID/g; at 1.5 h and 5 h p.i., respectively). The biodistribution and imaging studies could underline the powerful positive impact of glycation on the in vivo properties of 99m Tc-labeled BBS. In summary, this work provides a valuable new inside into opportunities for improved GRPR targeting with polar 99m Tc(CO)3 -labeled BBS analogues.

VII

VIII

Zusammenfassung Peptidrezeptoren sind als hervorragende Angriffspunkte f¨ ur die in vivo Diagnose und die Therapie von Krebs bekannt, und die Suche nach auf Peptiden basierenden Radiopharmazeutika hat in den letzten Jahrzehnten bedeutend zugenommen. Aufgrund ¨ seiner Uberexpression in einigen der häufigsten humanen Krebsarten, unter anderem Prostata-, Brust- and Darmkrebs, hat der Gastrin freisetzende Peptidrezeptor (GRPR), der Subtyp 2 der Bombesin (BBS) Rezeptorfamilie, zunehmend an Aufmerksamkeit gewonnen. GRPR besitzt eine hohe Affinität zu Radiotracern, die auf der C-terminalen Aminosäuresequenz des Neuropeptids Bombesin, BBS(7-14), basieren. Bisher weisen radioaktiv markierte BBS-Analoga allerdings zwei bedeutende Nachteile f¨ ur radiologische Anwendungen auf. Einer davon ist der nat¨ urliche schnelle Abbau der meisten Neuropeptide. Die Modifizierung einer enzymatischen Schnittstelle f¨ uhrte zu BBS-Analoga mit einer erhöhten biologischen Halbwertszeit: Durch den Austausch von Leucin (Leu) durch Cyclohexylalanin (Cha) und von Methionin (Met) durch Norleucin (Nle) stieg die in vitro Stabilität in Humanplasma um das Zwanzigfache und in GRPR-exprimierenden Tumorzellen um das Sechsfache, ohne die Bindungsaffinität zu beeinträchtigen (Kd ≤ 1 nM). Eine weitere Herausforderung ist die im Generellen hohe Leberanreicherung von BBS-Analoga und der hohe radioaktive Hintergrund in den Eingeweiden aufgrund der starken hepatobiliaren Ausscheidung. Beides ist unvorteilhaft f¨ ur die Detektion von abdominalen Tumoren mittels radiologischer Bildgebungsverfahren. Der Schwerpunkt dieser Arbeit lag daher in der Untersuchung der Auswirkungen neuartiger polarer Linker auf das pharmakokinetische Profil 99m Tc-markierter BBS-Analoga. Das Ziel bestand in der Erhöhung des Potentials dieser Peptide f¨ ur die Detektion von GRPR-positiven Tumoren durch Einzelphotonen-Emissions-Computer-tomographie (SPECT). Technetium-99m (99m Tc) ist ein leicht verf¨ ugbarer γ-Strahler mit optimalen physikalischen Eigenschaften f¨ ur die SPECT-Diagnostik (t1/2 = 6.02 h; Eγ = 140 keV). Die radioaktive Markierung der neuen Peptide wurde gemäss der Tricarbonyl-Methode IX

durchgef¨ uhrt, die auf der Bindung des organometallischen Precursors fac-[99m Tc(OH2 )3 (CO)3 ]+ an dreizähnige bifunktionale Chelatoren (BFCA) basiert. Ein erster Ansatz war der Einbau verschiedener polarer Aminosäuren zwischen die stabilisierte BBS(7-14)-Sequenz und den Retro-Nα -carboxymethyl-Histidin-Chelator, (Nα His)Ac, einem potenten BFCA f¨ ur die Radiomarkierung von Biomolek¨ ulen mit dem 99m Tc (CO)3 -Kern. Alle neuen Peptide behielten eine erhöhte Stabilität in Humanplasma (t1/2 ≥ 16 h) und Tumorzellen (t1/2 = 30-40 min). Die zusätzlichen polaren Gruppen bewirkten eine erhöhte Hydrophilizität und die Verschiebung der Polaritäten von einem LogD-Wert von 0.9 zu Werten zwischen 0.4 und -2.2. LogD-Werte zwischen +1 und -1 waren mit der höchsten Bindungsaffinität (Kd ≤ 0.5 nM) und der höchsten in vitro und in vivo Aufnahme in GRPR-exprimierende Tumorzellen verbunden. Eine höhere Hydrophilizität (LogD ≤ -1.8) f¨ uhrte zu einer substanziellen Abnahme der Internalisierung. Der Einbau einer positiven Ladung (β 3 -homo-Lysin) bewirkte eine unvorteilhaft hohe Nieren- und Leberanreicherung. Die Einf¨ uhrung einer einzelnen negativen Ladung (β 3 homo-Glutamat) bewirkte einen signifikanten Anstieg der spezifischen Tumoraufnahme (2.1 ± 0.6 vs. 0.8 ± 0.4 %ID/g verglichen zum lipophilen Referenzpeptid) und signifikant höhere Tumor-zu-Gewebe-Quotienten. Desweiteren lieferte dieses Peptid klarere Bilder der Tumorxenografte in SPECT/CT-Studien. Im Hinblick auf das verbesserte Bioverteilungsprofil mit höherer Tumoranreicherung und deutlicherer Bildgebung erwies sich die Einf¨ uhrung einer einzeln negativer Ladung als positiv f¨ ur die Entwicklung neuer BBS-Analoga. Ein weiterer Ansatz war die Glykierung der Peptide durch den Einbau hydrophiler Kohlenhydrate zwischen die stabilisierte BBS(7-14)-sequenz und den (Nα His)Ac-chelator. Die neuen Peptide zeigten LogD-Werte zwischen -0.2 und -0.5. Sie wiesen eine hohe Affinität zu GRPR auf (LogD ≤ 0.5 nm) und bewahrten die erhöhte Zellstabilität (t1/2 ≥ 35 min). In vivo ergaben sich f¨ ur alle glykierten Peptide höhere Tumor-zuGewebe-Quotienten als f¨ ur die unglykierte Referenz. Die besten Ergebnisse wurden mittels triazol-gekoppelter Glukose erzielt, die eine Vervierfachung der Aufnahme und der Retention in den Tumoren (3.6 %ID/g, 1.5 h p.i.; 2.5 %ID/g, 5 h p.i.) sowie eine signifikante Reduktion der Leberanreicherung (0.6 vs. 2.4 %ID/g, 1.5 h p.i.) bewirkte. Zusätzlich wurden die Tumor-zu-Nieren- und Tumor-zu-Blut-Quotienten signifikant um den Faktor 1.5 bzw. 2.7 verbessert (1.5 h p.i., P ¡ 0.05). In Tomographiestudien konnten die Tumorxenografte und eine Verringerung des abdomnialen Hintergrundes deutlich visualisiert werden. X

Im Weiteren waren wir am Potential neuer Triazol-enthaltender Chelatoren interessiert, die durch die Cu(+I)-katalysierte Addition von Alkinen und Aziden (Click-Chemie)gebildet wurden. Die Click-Reaktion erwies sich als vielversprechender neuer Weg der Radio- markierung von Biomolek¨ ulen, da sie in einem Schritt eine einfache Synthese neuer effizienter Chelatoren und die Markierung mit 99m Tc ermöglichte. Wir verglichen das Potential des etablierten (Nα His)Ac-Chelators mit den neuen N2click- und N3clickChelatoren f¨ ur die 99m Tc-Markierung unserer BBS-Analoga und untersuchten im Anschluss die Glykierung des vielversprechenden N3click-Peptids. Das N3click-Peptid wies eine hohe Affinität zu GRPR (Kd ≤ 0.6 nM) auf und zeigte in vitro und in vivo vergleichbare Eigenschaften zum (Nα His)Ac-Peptid. Nach der Glykierung mittels triazolgekoppelter Glukose kam es zur Erhöhung der spezifischen Aufnahme in GRPR- exprimierenden Geweben, zu einer verringerten Leberanreicherung und erhöhten Tumorzu-Blut- und Tumor-zu-Leber- Quotienten. Aktivitätsaufnahme und -retention in den Tumoren waren signifikant erhöht (4.4 ± 0.6 %ID/g, 1.5 h p.i.; 2.6 ± 0.8 %ID/g,5 h p.i.). Die Bioverteilungs- und Tomographiestudien konnten deutlich den positiven Einfluss der Glykosilierung auf die in vivo Eigenschaften von 99m Tc-markiertem BBS unterstreichen. Zusammenfassend bietet diese Arbeit wertvolle neue Einblicke in die Möglichkeiten eines verbesserten GRPR-Targetings mit polaren 99m Tc(CO)3 -markierter BBS-Analoga.

XI

XII

Abbreviations ACE ACN Asp bb3 bb4 BBS BOC Bq BSA CyHAla DMEM EDTA FCS Fmoc Glu GRP GRPR h His HPLC kDa Lys M min (Nα His) NMB NLeu PBS PET SPECT TFA

Angiotensin converting enzyme (ACE) Acetonitrile Aspartic acid Bombesin receptor subtype 3 Bombesin receptor subtype 4 Bombesin Tertiary-butyloxy-carbonyl Bequerel Bovine serum albumin Cyclohexylalanin Dulbecco’s minimum essential medium Ethylenediaminetetraacetic acid Fetal calf serum N-(9-flurenyl)methoxycarbonyl Glutamic acid Gastrin releasing peptide Gastrin releasing peptide receptor Hour(s) Histidin High performance liquid chromatography Kilodalton Lysine Molar Minute(s) retro-Nα -carboxymethyl-histidine Neuromedin B Norleucin Phosphate buffered saline Positron emission tomography Single photon emission computed tomography Trifluoroacetic acid

XIII

XIV

Chapter 1 Introduction 1.1

General Considerations

The World Health Organization estimated in 2006 that about 13 % (7.6 million) of the total deaths worldwide were related to cancer, and both, proportion and the total number of cases, were projected to continue rising in the next decades (1). Intense efforts are being undertaken to find new effective strategies for the diagnosis and treatment of this disease. One of the major problems in the management of cancer patients is the late detection, whereas diagnosis of stage 1 cancer is associated with a more than 90 % 5-year survival rate and an even earlier detection is often curative (2, 3). Cancer is a class of diseases based on a genetic transformation of healthy cells, which can occur in almost any kind of tissue. The main properties of the malignant forms of cancer are the uncontrolled division of cells in combination with the ability to invade adjacent tissues (4). The dissemination of single cells into other parts of the body via blood and lymphatic vessels leads to the formation of secondary (metastatic) cancer and counts as the main reason for a lethal etiopathology (1). To date conventional strategies like chemotherapy, surgery and external radiation are limited, especially in the treatment of the numerous and often small metastases (5, 6). In the last decades, a remarkable change occurred in the understanding of the genetic, molecular and cellular basis of cancer pathogenesis (7). Today it is known to be rooted in the mutation of two groups of genes: the proto-oncogenes, responsible for promoting cell division, and the tumor suppressor genes, whose purpose is to block aberrant cell growth (4). The altered gene expression leads to a higher energy consumption, an uncontrolled

1

growth and an upregulation of the synthesis of various biomolecules, e.g. enzymes, nucleotides, phospholipids and transmembrane receptors (6, 8-10). Regarding to this there has been a shift in the management of cancer treatment from an organ-system approach to targeting specific molecular abnormalities. Today much of nuclear medicine is focused on the management of patients through the injection of target-specific radiolabeled substances (11). These radiopharmaceuticals are composed of carriermolecules linked to a diagnostic (β + - and γ-emitter) or therapeutic radionuclide (α-, β − -, Auger electron emitter) (Table 1.2) (12, 13). They allow a site-specific delivery of radioactivity to appropriate targets, which thereby can either be monitored or ambushed, depending on the attached radionuclide and its emitted radiation.

Table 1.1: Selected target-specific diagnostic and therapeutic radiopharmaceuticals Radiopharmaceutical Trade name

Primary use

ProstaScintR

Imaging of prostate cancer

Indium-111 pentetreotide

OctreoscanR

Imaging of neuroendocrine tumors

Indium-111 satumomab

OncoScintR

Imaging of metastatic disease associated

Indium-111 Cabromab pendetide

pendetide Tc-99m Apcitide

with colorectal and ovarian cancer AcuTectR

Syntheic peptide for imaging deep vein thrombosis

Tc-99m Arcitumomab

CEA-ScanR

Monoclonal antibody for imaging colorectal cancer

Tc-99m Depreotide

NeotectR

Y-90 Ibitumomab

ZevalinR

Treatment of Non-Hopkin’s Lymphoma

BexxarR

Treatment of Non-Hodgkin Lymphoma

Imaging of somatostatin receptor-positive tumors

Tiuxetan I-131 Tositumomab

2

Various radionuclides have been proposed for systemic tumor therapy (12, 14, 15) and encouraging clinical results have already been reported, e.g. for the 177 Lu-labeled peptide analogues DOTATATE and DOTATOC in the treatment of neuroendocrine tumors (16, 17) and 90 Y- or 131 I-labeled antibodies against non-Hodgkin lymphomas (18, 19). Together with therapeutic approaches, the evaluation of the response to any kind of treatment is of high interest for planning and monitoring of further therapy (6, 7). Much of the current radiologic research focuses on adapting the conventional anatomical imaging methods computed tomography (CT) and magnetic resolution imaging (MRI) to new physiological imaging techniques like single photon emission computed tomography (SPECT) or positron emission tomography (PET) (9, 10). Following their recent introduction into clinical use the hybrid technologies SPECT/CT and PET/CT are playing an increasingly important role in the diagnosis and staging of human diseases (20-25). These new methods are going to have a major impact on cancer detection, individualized treatment and drug development, as well as understanding of how cancer arises (Figure 1.1. The use of a variety of radiotracers is already standard for clinical oncology (13, 22) (a limited selection is depicted in Table 1.1). However, despite the fact that many diagnostic applications have significantly been ameliorated, there is still need for additional radiopharmaceuticals with a high and tumor specific accumulation (6). To advance both, the early stage diagnosis as well as the image-guided therapy, more appropriate molecules have to be established for the particular molecular characteristics of the numerous different types of cancer (6, 9).

1.2

SPECT/CT and PET/CT for diagnostic imaging of cancer

The advantage of diagnostic radiopharmaceuticals is the detailed non-invasive description of tissue morphology and the test of physiological function through the specific accumulation of radioactivity (26). In general, they are used in low concentrations (10−6 -10−7 M) and are not intended to have any pharmacological effects. Radionuclide imaging technologies are commonly devised into two modalities; single photon emission computed tomography (SPECT) and positron emission tomography (PET). Despite the fact that both techniques are accurate methods for the detection of cancer, 3

Figure 1.1: Improved anatomical-functional image fusion with combined SPECT/CT (a) imaging infection with 67 Ga-citrate with fever and aortic valve infection and (b) imaging bone disease with 99m Tc-methylene diphosphonate [21]

they do not provide the anatomical landmarks needed for precise tumor localization. This drawback has been overcome by the development of fused imaging modalities, combining PET and SPECT with morphologic imaging techniques like CT (20, 22, 24, 25, 27). PET radionuclides of first choice are depicted in Table 1.2. They emit a positive electron (β + ) which undergoes a reaction with a nearby negative electron. Both particles are annihilated and their masses are converted into a pair of γ photons moving in nearly opposite directions with energy of 511 keV. Besides mere qualitative imaging, the high sensitivity allows a quantitative evaluation of the distribution of radioactivity. The exact point of origin of the dual photons can be detected based on trajectories of both particles (coincidence detection) and the timing with which they arrive at the surface of the tomograph’s circular detector. The highest spatial resolution of PET is around 4 mm and is limited only by the positron range and the minimal non-collinearity of each pair of photons. Single-photon tomographs for SPECT applications are designed to detect single photons (γ rays) and to determine their point of origin based solely on their trajectory. A γϕ camera acquires multiple images from different angles and a tomographic reconstruction algorithm can be used to yield a three dimensional dataset (SPECT). 4

The need for a collimator for the accurate determination of the origin of the detected γ ray reduces the geometric efficiency (28). Thus, in general the sensitivity of SPECT is about 2-3 orders of magnitude below the one of PET and limited to lesions of the size between 5 and 10 mm. So far, SPECT technologies allow only a semi-quantitative evaluation of the detected radioactivity. However, recently published data describe promising advances in the use of SPECT/CT for the quantification of radionuclide kinetics, e.g. in the treatment of thyroid cancer (29). The most favorable radionuclides used for diagnostic purposes with SPECT are emitters of γ-radiation in a range of 100-200 keV (Table 1.2). Photons with a lower energy would readily be absorbed in the tissue, while a higher energy would result in higher radiation dose for the patients, less imaging resolution and lower signal-to-noise ratios. PET/CT has been used for several years in oncology and is accepted as an important tool for diagnosis, staging and response evaluation (22, 30, 31). It provided a novel type of information in radiology by producing three-dimensional images of functional processes. However, although SPECT/CT has been largely overshadowed by the huge clinical and commercial success of PET/CT, a major drawback of PET is its cost-intensiveness. The production of most PET tracers requires a cyclotron and a technical crew for on-site production, due to the generally very short half-lives of positron-emitting radionuclides (22). In contrast to this, SPECT/CT offers the advantage of a more convenient production of radiotracers. The longer half-lives and the easy availability of some of the most important SPECT nuclides by the use of generator systems are a major strength for their clinical use in nuclear medicine. Furthermore, recently a range of dual-modality SPECT/CT cameras has been developed, which enable hybrid imaging with enhanced detection of recurrent and metastatic disease and have been shown to be equally useful in selected oncological applications as PET/CT systems (32-35).

1.3

Radionuclides for radiotherapy

Therapeutic radiopharmaceuticals for treatment of cancer patients are designed for target-specific delivery of therapeutic doses of ionizing radiation to tumor sites, while producing only a minimal damage to healthy tissue (36, 37). The systemic administration of these drugs is especially interesting for treatment of secondary (metastatic) cancer, since the conventional therapeutic applications are not effective in the treatment of disseminated metastases (38). 5

Table 1.2: Radionuclides suitable for diagnosis or therapy [12]

Radiation

Nuclide

Physical

EM ax

Eγ

Eγ

Maximum range

half-life

of particle

[MeV]

(%)

of particle

[MeV] I-123

γ

In-111 Tc-99m C-11

β

+

F-18 Ga-68 N-13 O-15 Ag-111 Cu-67 I-131

β

−

Lu-177 Re-186 Re-188 Y-90

α

At-211 Bi-212 Ga-67 I-123

Auger

I-125 In-111

13.2 h 2.8 d 6.0 h 20 min 110 min 68 min 10 min 2 min 7.5 d 2.6 d 8.0 d 6.7 d 3.8 d 17.0 h 2.7 d 7.2 h 1.0 h 3.3 d 13.2 h 60.0 d 2.8 d

1.0 0.6 1.9 1.2 1.7 1.1 0.6 0.8 0.5 1.1 2.1 2.3 6.8 7.8 0.008 0.023 0.023 0.019

in tissue

0.159 0.173 0.247 0.140 0.511 0.511 0.511 0.511 0.511 0.342 0.184 0.364 0.208 0.137 0.155 0.720 0.093 0.159 0.027 0.173 0.247

83 88 94 85 6 48 81 11 9 15 7 39 83 74 90 94

0.56 mm 0.44 mm 2.28 mm 0.78 mm 2.10 mm 4.8 mm 1.8 mm 2 mm 1.5 mm 5 mm 11 mm 12 mm 65 µm 70 µm 2 µm 15 µm 15 µm 10 µm

Radionuclides used for therapeutic applications are α-, β − - or Auger-electron emitters (Table 1.2). The choice of the radionuclide depends on nuclear emission properties, the physical half-life, the decay characteristics, the in vivo pharmacokinetics of the carriermolecule, and the cost and availability (39). An important issue for the preparation of 6

site-specific compounds for targeting low-capacity systems, e.g. receptors or antigens overexpressed on tumor cells, is a high specific activity (GBq/µg) for a sufficient accumulation of radioactivity at the target sites (40). Each type of the emitted particles shows different effective range and linear energy-transfer (LET) properties, and the appropriate type of emission depends on the tumor size, the heterogeneity of radiotracer deposition in the tumor, the pharmacokinetics of the tracer and other factors (38). αparticles are high-energy helium nuclei with high LET but a relatively short penetration range (50-80 µm) (12). This makes them attractive for the treatment of tumors with small diameters and homogenously distributed target sites. Low-energy Auger electrons deposit energy with a high LET over subcellular dimensions (below 10 µm) (41). Thus, a DNA-associated decay is necessary and every single tumor cell has to internalize the radionuclides into the nucleus. A better understanding of Auger radiation dosimetry and biological efficacy remain a major task (41). β − -emitters emit high-energy electrons with a lower LET and their relatively high particle range (1.5 - 12 mm) is most favorable for heterogeneously distributed targets in the tumor tissue. A considerable crossfire effect does not only harm cells directly targeted but also untargeted neighboring cancer cells. Interacting with atoms, mainly in water molecules, the β − -particels lose their energy and form exited and ionized atoms and free radicals, which cause damage by inducing single strand breaks of DNA (42). The favorable LET in combination with the relatively easy availability turned β − emitters into the most widely utilized radionuclides in radiotherapy (12). Some βradiation emitting nuclides are available with a high specific activity from radionuclide generator systems (e.g. 90 Y and 188 Re). The availability of 188 Re by a 188 Re/188 W generator system and the chemical similarity of Re and Tc offer the possibility to use them for radiolabeling of the same carrier molecules and will be discussed below.

1.4

Technetium-99m and Rhenium-186/188 for radiopharmaceutical applications

The discovery of 99m Tc brought about a fundamental progress in diagnostic nuclear medicine. 99m Tc is so far the most commonly used radionuclide in nuclear imaging and more than 80 % of all radiopharmaceuticals contain this short-lived metastable radionuclide (43). The physical properties with a half-life of 6 h and a γ photon emission of 140 7

keV (89%) are advantageous for both effective imaging with SPECT and patient safety (43). Today, 99m Tc can readily be delivered from a 99 Mo/99m Tc-generator at low cost and with a high specific activity (26). 99 Mo is a fission product with a half-life of 2.8 days and decays to 99m Tc, which can be eluted with physiological sodium chloride solution (0.15 M) in oxidation state VII as sodium pertechnetate (Na[99m TcO4 ]). Interestingly, 99m Tc and the β − -emitters 186 Re and 188 Re are members of the same transition metal group (group VIIb). These radionuclides exhibit a similar coordination chemistry and physico-chemical and chemical properties as consequence of the lanthanide contraction (44). This turns them into a ’matched pair’ of radionuclides for diagnostic and therapeutic purposes (45). Both 186 Re and 188 Re have attractive physical properties for radiotherapeutic applications (Table 1.2). 186 Re has a half-life of 3.7 days, which is suitable for carrier-molecules requiring a longer time for tumor localization and clearance from non-target tissues. 188 Re has a relatively short half-life of 17 h, which allows high dose rates and repeated applications, turning it into a favorable nuclide for radiolabeling of radiotracers with a fast tissue and blood clearance (e.g. peptides and other small molecules) (44). While the production of 186 Re with high specific activity is rather complex, 188 Re can be obtained in a good yield from 188 W/188 Re-generator systems (44).

1.4.1

The organometallic fac-[M(OH2 )3 (CO)3 ]+ (M= 99m Tc, Re)

Technetium has 8 possible oxidation states (-I to + VII) and can easily be reduced in the presence of chelating agents (43), which facilitates the labeling of various types of biomolecules and the production of kits for versatile diagnostic applications. The metal core exhibits different affinities depending on the oxidation state. A core in state +VII is hard and has a high affinity for donor ligands like imido- or nitride-groups. The softer core of state +V shows affinity for both, donor and acceptor ligands through O, N, S or P atoms. In the oxidation state +I the metal core is soft and has a high affinity for acceptor ligands like CO, NO+ , Isonitrile or the ”soft” sp2 N of aromatic amines. The most common 99m Tc- and 186/188 Relabeled radiotracers in nuclear medicine have a metal center in oxidation state +V. Examples are the metal-oxo core [M+V =O]3+ ], the metal-nitrido core [M+V =N]2+ and the hydrazine nicotinic acid core (HYNIC) (M=N=NR) (M =

8

Figure 1.2: Preparation of the biomolecules (R) bearing the

99m Tc-tricarbonyl-core

(Nα His)Ac-chelator

using the Isolink Kit (A). Labeling of with the 99m Tc-tricarbonyl (B)

99m

Tc, 186/188 Re)(44). An alternative with a metal core in a different oxidation state is the more recently developed organometallic tricarbonyl core [M+I (CO)3]+ in state +I (Figure 1.2). In this complex the carbonyl part is very inert, while the water molecules can rather easily be exchanged by a variety of ligands. The obtained tricarbonyl-complexes exhibit unique features in terms of in vivo stability, kinetic inertness and small size. The latter is especially important to minimize the impact of the organometallic complex on the biological activity of small carrier molecules like peptides, amino acids or nucleosides. The [99m Tc(OH2 )3 (CO)3 ]+ -core can readily be produced by the reduction of 99m TcO4 − (which can directly be eluted from a 99 Mo/99m Tc-generator) with [H3 BCO2 ]2− in 10 min at 90°C (46). The reaction kinetics and radiolabeling conditions of 99m Tc and 186/188 Re differ, due to the lower redox-potential of Re compared to Tc (37). Thus, the often rather harsh conditions, which are necessary for the attachment of Re to biomolecules, turn especially small molecules, such as peptide derivatives, into favorable molecules for radiolabeling following the tricarbonyl technique, due to their in general high chemical stability (44). The fact that the radiometal-tricarbonyl-core can relatively easily be incorporated into biomolecules, which bear specific bifunctional chelating agents (BFCA), offered a novel convenient way for the on-site synthesis of 99m Tc/186/188 Re-labeled radiopharmaceuticals for clinical applications (Figure 1.2) (47).

1.4.2

Bifunctional chelating agents

The possible methods for labeling of biomolecules with the metal core (M) of Tc and Re involve bifunctional chelating agents (BFCA) which include the ”3+1” ligand sys9

tem, M(CO)3 -containing chelates, hydrazine nicotinamide acid (HYNIC), water-soluble phosphines, and other Tc/Re-carrying moieties (43). BFCAs consist of a conjugation group for a covalent attachment to a carrier molecule, a linker as pharmacokinetic modifier and a chelating unit for the coordination of the metallic radionuclide. The ideal BFCA should form stoichiometrically well-defined complexes, which feature kinetic and thermodynamic stability with respect to dissociation and stabilization of the current oxidation state of the radionuclide (45). A most favorable characteristic of the final radioconjugate is a high specific activity, which can be achieved by high labeling efficiencies and radiolabeling yields higher than 90%. In general, there are two ways for the attachment of radionuclides to carrier molecules. The formation of a complex of BFCA and radionuclide can be performed before (prelabeling approach) or after (postlabeling approach) the attachment of the BFCA to the carrier molecule. The latter is more suitable for clinical applications, because it usually involves less reaction steps and offers a combination of high effectiveness and a welldefined chemistry (48). Recently the development of the tridentate bifunctional chelator Nα -histidinyl acetate, (Nα His)Ac, for labeling with the organometallic precursor [M(CO)3 (OH2 )3 ]+ introduced a novel postlabeling approach for the incorporation of the 99m Tc/186/188 Re(+I)-tricarbonyl core into biomolecules with high in vitro and in vivo stability of the radiocomplex (47). This is of particular interest for the use of small biomolecules like radiopeptides for diagnostic and therapeutic targeting of cancer with the matched pair 99mTc/186/188Re.

1.5

Peptide-receptor mediated tumor targeting

In general, peptides are small molecules consisting of 2 to 50 amino acids. Peptide derivatives are easy to synthesize following standard solid phase synthesis strategies and resist the harsh chemical conditions required for chelation and radiolabeling of radiotracers. The small size is a major advantage for diagnostic imaging compared to macromolecules, such as proteins, antibodies or antibody fragments. A low molecular weight is supporting an easy penetration of blood vessel membranes and a rapid distribution in the body (and tumor) tissues, except for the brain, since the blood-brain-barrier is not permeable for more hydrophilic peptides (48). Thus, in combination with the high binding affinity for peptide receptors and a fast clearance from blood and receptor-negative tissues, high target-to-non-target ratios can be achieved (49). 10

Table 1.3: Advantages and Disadvantages of peptides as radiotracers [44] Advantages

Disadvantages

High affinity and specificity for their receptors

Small changes can affect binding affinity

Rapid clearance from blood and non-target tissues

Rapid degradation

Small size and low molecular weight Easy synthesis, modification and labeling Low immunogenicity and low general toxicity

High kidney uptake can cause renal toxicity

High penetration into tumor tissues Modifiable rate and route of excretion

The solubility, structure and function of peptides are characteristics of their amino acid sequence. Lipophilic peptides are usually cleared from the body by the hepatobiliary route, hydrophilic peptides mainly via the kidneys (50). A variety of functional groups (such as specific hydrophilic or lipophilic amino acids, carbohydrates or polyethylene glycol (PEG) chains) can be used to modify the route and rate of peptide excretion. Generally increased hydrophilicity and enhanced urinary excretion are more favorable compared to the slower hepatobiliary excretion followed by an accumulation of radioactivity in the intestines resulting in a high abdominal background (48). However, the peptide-receptor interaction is an extremely sensible process and the attachment of BFCA for radiolabeling as well as any other kind of modification bears the risk of a decrease or loss of binding affinity. Moreover, most bioactive peptides have only a short biological half-life and they are readily metabolized in plasma by endo- and exopeptidases, since their role as flexible messengers requires a rapid degradation in the blood to prevent a prolonged action (50). A higher plasma stability would be most favorable for a satisfying targeting. The resistance to endopeptidases can be achieved by a modification of amino acid side chains, the substitution of amino acids by nonnatural counterparts like β-amino acids or D-amino acids, by the introduction of spacers, or cyclization or methylation of amide nitrogens. The resistance to exopeptidases can be achieved by endcapping (e.g. acetylation of the N-terminus, amidation or reduction of the C-terminus, or N to C cyclization) (44). In summary, several modifications are feasible to turn peptide derivatives into powerful 11

Figure 1.3: Principle of in vivo peptide receptor targeting of cancer. The radiopeptide (P) is distributed in the whole body after i.v. injection and binds to tumor cells expressing the corresponding peptide receptors (P-R). After binding it internalizes and accumulates in the cancer cells (arrows). The remaining activity in the body is rapidly cleared through the kidneys. Whole body scans detect the specificly accumulated radioactivity in the tumor [52]

tools for radiopharmaceutical applications. However, the challenge for the development of new peptide-based radiotracers is to avoid a negative effect on their receptor binding properties and pharmacokinetics after necessary modifications. Neuropeptides in Oncology Neuropeptides are a group of neurohormones, neurotransmitters, and neuromodulators controlling physiological homeostasis and behavioral patterns (48, 51). They affect nonneural tissues and organs as well as neurons and are involved in a broad range of functions, e.g. the regulation of growth, food and water intake, water and salt metabolism, temperature control, reproduction, cardiovascular, gastrointestinal and respiratory control, behavior, memory, and affective states (51). Receptors for endogenous substances such as neuropeptides have a dual purpose. They recognize the ligand with high specificity and affinity and transduce the information from this messenger into cellular function. Interestingly the over-expression of various peptide receptors is involved in different pathological processes, such as inflammation or cancer. Their high expression in breast, prostate, ovary, pancreas and other tumors (Table 1.4)

12

turns them into potential targets for imaging and therapy with the appropriate radiolabeled peptide derivatives (52). About 20 years ago the development of radiolabeled somatostatin (sst) analogues led to a major breakthrough for the application of radiolabeled peptides for the early stage detection and treatment of human neoplasms (52). Neuroendocrine tumors over-expressing sst receptors could be successfully targeted in vivo by the intravenous injection of 111 Inlabeled sst analogues (e.g. 111 In-OctreoScan) (53), and this sensitive technique is still superior to all standard diagnostic tools available for the detection of this kind of cancer (54). Furthermore, clinical studies with sst analogues labeled with therapeutic radionuclides, such as 177 Lu- and 90 Y-DOTATOC and -DOTATATE, have shown an effective therapeutic response of sst receptor-positive tumors (16, 17, 55). However, the application of sst derivatives is naturally limited to tumors over-expressing sst receptors. Some individual tumors may not express them in a homogenous way and many types of cancer show in general no sst receptor expression. Moreover, numerous tumors overexpress other neuropeptide receptors, several not only one but even different types (Figure 1.4)(52). Therefore there is a strong need for the development of further, novel radiopeptides, whose application (maybe even in form of a mix of several peptides for multireceptor targeting), would offer the possibility to cover more tumor types and the complete mass of single tumors (55). Since a variety of peptide receptors is known to be over-expressed on human cancer cells, several neuropeptides are intensively studied to increase the number of radiolabeled tools for tumor targeting in nuclear oncology (Table 1.4) (48, 56). endfigure The bombesin receptor superfamily as targets for radiopharmaceutical applications Bombesin (BBS) was first isolated from the skin of the amphibians of the genus bombina by Espamer et al (57-60). It is a 14-amino acid neuropeptide with an N-terminal pyroglutamate residue and a C-terminal amidation (Figure 1.5). To date, two BBS-like peptides have been isolated from mammalian tissues: neuromedin B (NMB) consisting of 10 amino acids and gastrin-releasing peptide (GRP) containing 27 amino acids. Both prefer particular receptor subtypes but share a similar C-terminal sequence of eight amino acids, which is essential for receptor binding affinity and biological activity (51, 61).

13

Table 1.4: Neuropeptides and their targets in different tumors Peptide

Receptor

Bombesin

GRP

Biological effects

Tumor targets

Gut hormone release

Prostate, breast, pancreas, gastric, small cell lung cancer, colorectal cancer

Cholecystokinin

CCK2

Gallbladder contractions

Medullary thyroid

exocrine pancreatic

cancer, small cell lung cancer,

excretion

gastrointestinal stromal cancer, stromal ovarian cancer, astrocytomas

Neuropeptide Y

Y1 and Y2

(NPY)

Regulation of energy

Adrenal cortical tumors,

balance, memory and

renal cell carcinomas,

learning

ovarian sex cord-stromal tumors, breast cancer

Neurotensin

NTR1

Vasoconstriction, raise

Small cell lung cancer,

in vascular permeability

colon, exocrine pancreatic cancer, Ewing’s sarcoma, breast, prostate cancer

Somatostatin

sst2

(Octreotide,

Inhibition of hormone

Neuroendocrine tumors

and exocrine secretion

(gastroenteropancreatic

Octreotate)

tumors, pituitary tumors), lymphomas, carcinoids, paragangliomas, breast, brain, small cell lung cancer

Vasoactive

VPAC1

Vasodilation, water and

Adenocarcinomas of colon,

intestinal

electrolyte secretion

breast, endocrine tumors

peptide (VIP)

in gut

So far, four BBS receptor subtypes have been identified, all of them typical G-protein coupled seven-transmembrane-domain receptors (51). They trigger a signal transduction by the specific binding of the appropriate peptide to the extracellular receptor domain, which triggers an intracellular signal cascade by activation of the guanine nucleotide 14

Figure 1.4: Multiple receptor over-expression in the same breast cancer tissue sample. A = Hematoxylin-eosin-stained tissue section (Scale bar 1 mm). Autoradiograms show total binding of 125 I-[Tyr3 ]-octreotide (B) to somatostatin receptors (SS-R), of 125 I-[Tyr4 ]-bombesin (C) to the Gastrin-Releasing Peptide receptors (GRP-R), of 125 I-[Leu31 , Pro 34 ]-PYY (D)to Neuropeptide-Y receptors (NPY(Y1)-R) and of 125 I-VIP (E) to Vasoactive Intestinal Peptide receptors(VPAC1-R) [52].

binding proteins (G proteins) (48). Three of the BBS-receptor subtypes are expressed in mammalian tissues: The NMBreceptors (NMB-R or bbr-1) have a high binding affinity for NMB and a lower affinity for GRP. GRP-receptors (GRP-R or bbr-2) have a higher affinity for GRP than for NMB. The human ligand for the closely related orphan BBS-receptor 3 (bbr-3) has so far not been identified (29, 61, 62). Immunoreactivity and mRNA studies demonstrated the expression of NMB-R and GRP-R in the nervous system and peripheral tissues, especially the gastrointestinal tract (63, 64). Furthermore minimal expression of all three receptor subtypes in human pancreatic tissue has been reported (61). The receptor bbr-4 is only expressed in amphibians and therefore not relevant for clinical applications (65). 15

Figure 1.5: Degradation of the binding sequence BBS(7-14) of Bombesin by endopeptidases (arrows indicate known cleavage sites).

In most tissues, the effects of NMB and GRP overlap and demonstrate a broad spectrum of pharmacological and biological responses. Their physiological roles include stimulation of enzyme secretion from exocrine glands or stimulation of release of a series of gastrointestinal peptide hormones; stimulation of smooth muscle contraction in the gastrointestinal tract and urogenital system; potent CNS effects (e.g. regulation of circadian rhythm, thermoregulation, regulation of anxiety and fear response) as well as potent growth effects on normal and tumor tissue (66, 67). Considering pathological over-expression in cancer, the NMB-R is predominantly expressed on gastrointestinal carcinoids (29), while bbr-3 is particularly relevant in neuroendocrine lung tumors (68). The most interesting subtype is the GRP-R, which is over-expressed in a broad range of tumors, many of them among the most prevalent types of human cancer, e.g. prostate and breast carcinomas (52, 69). In general, the expression of BBS-receptor subtypes in healthy tissues is much lower compared to the high expression in many malignancies. The identical composition of the C-terminal binding sequence of GRP and BBS(7-14), turns BBS analogues into optimal ligands for targeting GRP-R. Therefore, and regarding to the analogy with the clinical usefulness of somatostatin receptor targeting in oncology, radiolabeled BBS analogues have been studied intensively for targeting cancer-related GRP-R over-expression (61). Various modifications have been tested to improve the potential of BBS analogues for optimal tumor targeting. Since the C-terminal binding sequence BBS(7-14) is essential to maintain receptor binding, the N-terminal region is usually used for radiolabeling and the introduction of pharmacokinetic modifiers. Especially the introduction of a linker (e.g. β-Alanine) between the binding sequence and a chelator proved to increase the specific accumulation in receptor-positive tissues (69).

16

On the other hand, BBS (like sst) has only a half-life in plasma of 2-3 min and is rapidly metabolized by endo- and exopeptidases (Figure 1.5) (56)and, therefore, a modification of the amino acid sequence and prolongation of the plasma half-life is of high interest (70). Finally, 99m Tc/186/188 Re-tricarbonyl-labeled BBS conjugates are often associated with a high hepatobiliary clearance (70). The resulting high abdominal background would be unfavorable for both imaging with 99mTc and therapy with 186/188 Re. Thus, the shift of the excretion route towards the renal/urinary pathway is a major important quest in the development of new 99m Tc/186/188 Re-labeled BBS analogues for targeting GRP-R-positive tumors.

17

1.6

Aim of the thesis

Radiolabeled analogues of the neuropeptide Bombesin (BBS) reveal a great potential for imaging and therapy of prostate, breast and other cancers over-expressing the gastrinreleasing peptide-receptor (GRP-R). This project is focused on the development of new BBS-based 99m Tc(I)-radiopharmaceuticals for diagnostic purposes by the application of single photon emission computed tomography (SPECT). The main goal of this work was to provide a better insight into the influence of different polar groups on the pharmacokinetic profiles of 99m Tc(I)-tricarbonyl-labeled BBS analogues. Thus, a spectrum of new peptides was synthesized bearing either charged groups or carbohydrates in a linker between the stabilized BBS binding sequence BBS(7-14) and the established (Nα His)Ac-chelator, used for labeling with the 99m Tc(I)tricarbonyl-core. The new peptides were tested in vitro for receptor binding, internalization and degradation and in vivo for biodistribution and imaging with SPECT/CT. A second aim was the evaluation of novel click modified BBS derivatives. Two novel ligand systems were attached to the stabilized BBS(7-14) sequence and tested in vitro and in vivo after labeling with the 99m Tc(I)tricarbonyl-core. Additionally, click- chemistry was applied for sugar conjugation of the two best peptides, with (Nα His)Ac-chelator and the most promising click-chelator, to increase their hydrophilic characters.

18

1.7

References

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22. Boss, D. S., Olmos, R. V., Sinaasappel, M., Beijnen, J. H., and Schellens, J. H. M. (2008) Application of PET/CT in the Development of Novel Anticancer Drugs. Oncologist 13, 25-38. 23. Blodgett, T. M., Meltzer, C. C., and Townsend, D. W. (2007) PET/CT: Form and Function. Radiology 242, 360-385. 24. Gabriel, M., Hausler, F., Bale, R., Moncayo, R., Decristoforo, C., Kovacs, P., and Virgolini, I. (2005) Image fusion analysis of 99m Tc-HYNIC-Tyr3 -octreotide SPECT and diagnostic CT using an immobilisation device with external markers in patients with endocrine tumours. European Journal of Nuclear Medicine and Molecular Imaging 32, 1440-1451. 25. Schillaci, O., and Simonetti, G. (2004) Fusion imaging in nuclear medicine - applications of dual-modality systems in oncology. Cancer Biother. Radiopharm. 19, 1-10. 26. Liu, S. (2008) Bifunctional coupling agents for radiolabeling of biomolecules and target-specific delivery of metallic radionuclides. Adv. Drug Deliv. Rev. In Press, Corrected Proof. 27. Pfannenberg, A. C., Eschmann, S. M., Horger, M., Lamberts, R., Vonthein, R., Claussen, C. D., and Bares, R. (2003) Benefit of anatomical-functional image fusion in the diagnostic work-up of neuroendocrine neoplasms. European Journal of Nuclear Medicine and Molecular Imaging 30, 835-843. 28. Moore, S. C., Kouris, K., and Cullum, I. (1992) Collimator design for single photon emission tomography. Eur. J. Nucl. Med. 19, 138-150. 29. Prideaux, A. R., Song, H., Hobbs, R. F., He, B., Frey, E. C., Ladenson, P. W., Wahl, R. L., and Sgouros, G. (2007) Three-Dimensional Radiobiologic Dosimetry: Application of Radiobiologic Modeling to Patient-Specific 3-Dimensional ImagingBased Internal Dosimetry. J. Nucl. Med. 48, 1008-1016. 30. Jerusalem, G., Withofs, N., Rorive, A., and Hustinx, R. (2007) Positron emission tomography in oncology: an update. Rev Prat 57, 1864-70.

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31. Rembielak, A., and Price, P. (2008) The role of PET in target localization for radiotherapy treatment planning. Onkologie 31, 57-62. 32. Patel, C. N., Chowdhury, F. U., and Scarsbrook, A. F. (2008) Clinical Utility of Hybrid SPECT-CT in Endocrine Neoplasia. Am. J. Roentgenol. 190, 815-824. 33. Schillaci, O., Danieli, R., Manni, C., and G., S. (2004) Is SPECT/CT with a hybrid camera useful to improve scintigraphic imaging interpretation? Nucl Med Commun. 25, 705-710. 34. Schillaci, O. (2005) Hybrid SPECT/CT: a new era for SPECT imaging? Eur. J. Nucl. Med. Mol. Imag. 32, 521-524. 35. Roach, P. J., Schembri, G. P., HoShon, I. A., Bailey, E. A., and Bailey, D. L. (2006) SPECT/CT imaging using a spiral CT scanner for anatomical localization: Impact on diagnostic accuracy and reporter confidence in clinical practice. Nucl Med Commun. 27, 977-987. 36. Hoefnagel, C. A. (1991) Anti-cancer radiopharmaceuticals. Anti-Cancer Drugs 2, 107-132. 37. Liu, G., and Hnatowich, D. J. (2007) Labeling Biomolecules with Radiorhenium - A Review of the Bifunctional Chelators. Anticancer Agents Med. Chem. 7, 367-377. 38. Volkert, W. A., and Hoffman, T. J. (1999) Therapeutic Radiopharmaceuticals. Chem. Rev. 99, 2269-2292. 39. Volkert, W. A., Goeckeler, W. F., Ehrhardt, G. J., and Ketring, A. R. (1991) Therapeutic Radionuclides: Production and Decay Property Considerations. J. Nucl. Med. 32, 174-185. 40. Sgouros, G. (1995) Radioimmunotherapy of Micrometastases: Sidestepping the Solid-Tumor Hurdle. J. Nucl. Med. 36, 1910-1912. 41. Buchegger, F., Perillo-Adamer, F., Dupertuis, Y. M., and Bischof-Delaloye, A. (2006) Auger radiation targeted into DNA: a therapy perspective Eur. J. Nucl. Med. 33, 1352-1363. 22

42. Lliakis, G. (1991) The role of DNA double strand brakes in ionizing radioationinduced killing of eukaryotic cells. BioEssays 13, 641-648. 43. Banerjee, S., Pillai, M. R., and Ramamoorthy, N. (2001) Evolution of Tc-99m in diagnostic radiopharmaceuticals. Semin Nucl Med. 31, 260-277. 44. Garcia-Garayoa, E., Schibli, R., and Schubiger, P. A. (2007) Peptides radiolabeled with Re-186/188 and Tc-99m as potential diagnostic and therapeutic agents. Nucl. Sci. Tech. 18, 88-100. 45. Liu, S., and Edwards, D. S. (1999) 99mTc-labeld small peptides as diagnostic radiopharmaceuticals. Chem. Rev. 99, 2235-2268. 46. Alberto, R., Ortner, K., Wheatley, N., Schibli, R., and Schubiger, A. P. (2001) Synthesis and properties of boranocarbonate: a convenient in situ CO source for the aqueous preparation of [99mTc(OH2)3(CO)3]+. J. Am. Chem. Soc. 123, 3135-3136. 47. Schibli, R., La Bella, R., Alberto, R., Garcia-Garayoa, E., Ortner, K., Abram, U., and Schubiger, P. A. (2000) Influence of the Denticity of Ligand Systems on the in Vitro and in Vivo Behavior of 99m Tc(I)-Tricarbonyl Complexes: A Hint for the Future Functionalization of Biomolecules. Bioconjugate Chem. 11, 345-351. 48. Dijkgraaf, I., Boerman, O. C., Oyen, W. J. G., Corstens, F. H. M., and Gotthardt, M. (2007) Development and Application of Peptide-Based Radiopharmaceuticals Anti. Canc. Agents Med. Chem. 7, 543-551. 49. Khan, I. U., and Beck-Sickinger, A. G. (2008) Targeted tumor diagnosis and therapy with peptide hormones as radiopharmaceuticals. Anticancer Agents Med. Chem. 8, 186-199. 50. Okarvi, S. M. (2004) Peptide-based radiopharmaceuticals: Future tools for diagnostic imaging of cancers and other diseases. Med. Res. Rev. 24, 357-397. 51. Strand, F. L. (1999) Neuropeptides - Regulators of Physiological Processes, MIT Press, Cambridge Massachusetts. 52. Reubi, J. C. (2003) Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy. Endocr Rev 24, 389-427. 23

53. Krenning, E. P., Kwekkeboom, D. J., Bakker, W. H., Breeman, W. A. P., Kooij, P. P. M., Oei, H. Y., Vanhagen, M., Postema, P. T. E., Dejong, M., Reubi, J. C., Visser, T. J., Reijs, A. E. M., Hofland, L. J., Koper, J. W., and Lamberts, S. W. J. (1993) Somatostatin Receptor Scintigraphy with [111 In-DTPA-D-Phe1 ]- and [123 I-Tyr3 ]-Octreotide - the Rotterdam Experience with More Than 1000 Patients. Eur. J. Nucl. Med. Mol. Imag. 20, 716-731. 54. Gibril, F., Reynolds, J. C., Doppman, J. L., Chen, C. C., Venzon, D. J., Termanini, B., Weber, H. C., Stewart, C. A., and Jensen, R. T. (1996) Somatostatin Receptor Scintigraphy: Its Sensitivity Compared with That of Other Imaging Methods in Detecting Primary and Metastatic Gastrinomas: A Prospective Study. Ann Intern Med 125, 26-34. 55. Krenning, E. P., Kwekkeboom, D. J., Valkema, R., Pauwels, S., Kvols, L. K., and De Jong, M. (2004) Peptide Receptor Radionuclide Therapy. Ann. NY Acad. Sci. 1014, 234-245. 56. Okarvi, S. M. (2008) Peptide-based radiopharmaceuticals and cytotoxic conjugates: Potential tools against cancer. Canc. Treat. Rev. 34, 13-26. 57. Nagalla, S. R., Barry, B. J., Falick, A. M., Gibson, B. W., Taylor, J. E., Dong, J. Z., and Spindel, E. R. (1996) There are three distinct forms of bombesin. Identification of [Leu13 ]bombesin, [Phe13 ]bombesin, and [Ser3 ,Arg10 ,Phe13 ]bombesin in the frog Bombina orientalis. J. Biol. Chem. 271, 7731-7737. 58. Erspamer, V., Erpamer, G. F., and Inselvini, M. (1970) Some pharmacological actions of alytesin and bombesin. J. Pharm. Pharmacol. 22, 875-876. 59. Falconieri-Erspamer, G., Severini, C., Erspamer, V., Melchiorri, P., Delle-Fave, G., and Nakajima, T. (1988) Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes. Regul. Pept. 21, 1-11. 60. Esparmer, V. (1988) Discovery, isolation, and characterization of bombesin-like peptides. Ann. NY Acad. Sci. 547, 3-9.

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61. Smith, C. J., Volkert, W. A., and Hoffman, T. J. (2005) Radiolabeled peptide conjugates for targeting of the bombesin receptor superfamily subtypes. Nucl. Med. Biol. 32, 733-740. 62. Weber, D., Berger, C., Heinrich, T., Eickelmann, P., Antel, J., and Kessler, H. (2002) Systematic optimization of a lead-structure identities for a selective short peptide agonist for the human orphan receptor BRS-3. J. Pept. Sci. 8, 461-475. 63. Moody, T. W., and Merali, Z. (2004) Bombesin-like peptides and associated receptors within the brain: distribution and behavioral implications. Peptides 25, 511-520. 64. Battey, J., and Wada, E. (1991) Two distinct receptor subtypes for mammalian bombesin-like peptides. Trends Neurosci. 14, 524-528. 65. Nagalla, S. R., Barry, B. J., Creswick, K. C., Eden, P., Taylor, J. T., and Spindel, E. R. (1995) Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4). Proc. Natl. Acad. Sci. USA 92, 6205-6209. 66. Matusiak, D., Glover, S., Nathaniel, R., Matkowskyj, K., Yang, J., and Benya, R. V. (2005) Neuromedin B and its receptor are mitogens in both normal and malignant epithelial cells lining the colon. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G718-728. 67. Jensen, R. T., Battey, J. F., Spindel, E. R., and Benya, R. V. (2008) International Union of Pharmacology. LXVIII. Mammalian Bombesin Receptors: Nomenclature, Distribution, Pharmacology, Signaling, and Functions in Normal and Disease States. Pharmacol Rev 60, 1-42. 68. Fathi, Z., Corjay, M. H., Shapira, H., Wada, E., Benya, R., Jensen, R., Viallet, J., Sausville, E. A., and Battey, J. F. (1993) BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J. Biol. Chem. 268, 5979-5984. 69. Smith, C. J., Sieckman, G. L., Owen, N. K., Hayes, D. L., Mazuru, D. G., Kannan, R., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical Investigations of

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Gastrin-releasing Peptide Receptor-specific [99m Tc(X)(CO)3 -Dpr-Ser-Ser-Ser-GlnTrp-Ala-Val-Gly-His-Leu-Met-(NH2 )] in PC-3, Tumor-bearing, Rodent Models: Syntheses, Radiolabeling, and in Vitro/in Vivo Studies where Dpr = 2,3-Diaminopropionic acid and X = H2 O or P(CH2 OH)3 . Cancer Res. 63, 4082-4088. 70. Garayoa, E. G., Ruegg, D., Blauenstein, P., Zwimpfer, M., Khan, I. U., Maes, V., Blanc, A., Beck-Sickinger, A. G., Tourwe, D. A., and Schubiger, P. A. (2007) Chemical and biological characterization of new Re(CO)3 /[99m Tc](CO)3 bombesin analogues. Nuc. Med. Biol. 34, 17-28.

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Chapter 2 New [99mTc]Bombesin Analogues with Improved Biodistribution for Targeting Gastrin Releasing-Peptide Receptor-Positive Tumors

Elisa Garc´ıa Garayoa1 , Christian Schweinsberg1 , Veronique Maes2 , Dominique R¨ uegg1 , Alain Blanc1 , Peter Bläuenstein1 , Dirk A. Tourwé2 , Annette G. Beck-Sickinger3 , P. August Schubiger1

1

Paul Scherrer Institute, Center for Radiopharmaceutical Science, CH-5232 Villigen PSI, Switzerland 2 Department of Organic Chemistry, Vrije Universiteit Brussel, 1050 Brussels, Belgium 3 Institute of Biochemistry, University of Leipzig, 04103 Leipzig, Germany

Q J NUCL MED MOL IMAGING 2007; 51:42-50

27

2.1

Abstract

Aim. Bombesin (BBS) receptors are potential targets for diagnosis and therapy of breast and prostate tumors. To overcome the rapid degradation of natural BBS some modifications were introduced at positions 13 and 14. Additionally, a spacer was inserted between the chelator and the binding sequence in order to further improve the in vivo uptake. The analogues were labeled with the [99m Tc(CO)3 ]-core and tested. Methods. Stability was analyzed in vitro in human plasma. Binding affinity and internalization were determined in vitro in prostate carcinoma PC-3 cells. Biodistribution studies and SPECT/CT imaging were performed in nude mice with PC-3 tumor xenografts. Results. The changes introduced in the BBS(7-14) sequence substantially increased plasma stability. Affinity for GRP receptors on PC-3 cells was comparable to that of the unmodified analogue with Kd < 1 nM. The presence of a spacer in the molecule induced an increment in the in vivo uptake in pancreas and PC-3 xenografts (GRP receptorpositive tissues). The increase in pancreas and tumor uptake was higher when both spacer and stabilization are present in the same molecule. Moreover, in vivo uptake was highly specific. The tumor was clearly visualized by SPECT/CT. Conclusions. The modifications in the BBS(7-14) sequence led to a higher plasma stability while binding affinity remained unaffected. Stabilization resulted in improved biodistribution with better tumor to non-tumor ratios. However, the insertion of a spacer had a greater influence on the biodistribution. Analogues with both spacer and stabilization are the most promising radiopharmaceuticals for targeting GRP receptor-positive tumors.

KEY WORDS: bombesin, spacer, technetium, tumor uptake, stabilization

28

2.2

Introduction

The search for peptide-based radiopharmaceuticals has experienced an important rise in the past decades. Neuropeptide receptors are over-expressed in a variety of human tumors and are, therefore, interesting targets for imaging and therapy with radiolabeled analogues (1-4). Since the first introduction of Octreoscan (an 111 In-labeled somatostatin analogue) in 1994, it has been successfully used to visualize a large range of tumors, mainly of neuro-endocrine origin (5-8). However, the use of sst analogues is restricted to somatostatin (sst) receptor-positive tumors. Thus, the search for peptide-based radiopharmaceuticals has been extended to other neuropeptides such as vasoactive intestinal peptide (VIP), neurotensin (NT), bombesin (BBS), cholecystokinin (CCK)/gastrin and others (9-12). Bombesin (BBS) is a 14 amino acid peptide (13), the amphibian counterpart of the human gastrin releasing-peptide (GRP). Their structures differ by only one of the 10 carboxy-terminal residues and the sequence 8-14 of BBS is the minimal fragment required for specific binding to the receptors. BBS/GRP mediate their actions through G protein-coupled receptors with at least four different subtypes: neuromedin B (NMB), GRP, BBS3 and bb4 receptors (14-17). BBS and GRP show a similar biological activity acting both in the central and enteric nervous systems (18, 19). Besides the important role in physiological processes, it has also been observed that GRP/BBS play a role in stimulating the growth of different types of cancers (20-22) as well as in promoting proliferation of various tumor cell lines such as neuroblastoma, or prostatic carcinoma (23, 24). This action involves the expression of these peptides and their receptors in tumor cells (21). Over-expression of the GRP receptor has been demonstrated in a large number of tumors, including breast, prostate, pancreatic and small cell lung cancers (21, 22, 25, 26), which are among the most frequent cancers. Radiolabeled BBS/GRP peptides are therefore potential candidates for targeting GRP receptor-positive tumors. In order to increase the plasma stability of natural BBS, we have synthesized new BBS analogues in which the amino acids Leu13 and/or Met14 have been replaced by the nonnatural amino acids cyclohexylalanine (Cha) and norleucine (Nle), respectively. Smith et al. (27) reported on the influence of aliphatic spacer groups on the pharmacokinetics and in vivo pancreatic uptake for different BBS analogues. Therefore, we also inserted spacers between the chelator and the binding sequence in some derivatives. 99m Tc is the most commonly used radioisotope for diagnostic applications in nuclear medicine because of its availability at low cost from a 99 Mo/99m Tc generator and its ideal physical

29

properties (t1/2 = 6 h; E = 140 keV, 89 %), that make it optimal for diagnostic imaging (28). 188 Re has favorable physical characteristics (t1/2 = 17 h; E max = 2.12 MeV; E = 155 keV, 15 %) for potential therapeutic applications. All the analogues include the chelator retroNα -carboxymethyl histidine, (Nα His)Ac, and were labeled with fac[99m Tc(H2 O)3 (CO)3 ]+ using the ”organometallic” labeling technique, which has shown to be suitable for small biomolecules (29, 30). We herein report on the in vitro and in vivo characterization of these new BBS analogues.

2.3

Materials and Methods

Synthesis and labeling Peptide solid-phase synthesis was carried out on a Merrifield resin with the Fmoc/tBustrategy, using a semi-automatic Labortec Peptide Synthesizer SP640B. The (Nα His)Ac (retro[Nα -carboxymethyl-histidine]) was coupled to the N-terminus for labeling to 99m Tc and the spacers βAla-βAla or Lys(sha)-βAla-βAla (sha = shikimic acid: C7 H10 O5 , (3R,4S,5R)-3,4,5-Trihydroxy-1-cyclohexencarboxylic acid) as described recently in detail (31). Radiolabeling was performed as recently described with few modifications (32, 33). Briefly, 1ml of a technetium generator eluate in saline (up to 5 GBq) was added to a mixture of 4.5 mg sodium-boranocarbonate, 2.9 mg borax, 9 mg potassium-sodium

Table 2.1: Sequence of the BBS analogues and binding affinity (Kd ) for GRP receptors on human prostate carcinoma PC-3 cells. (Cha = Cyclohexylalanine; sha = shikimic acid. Data are mean ± SD; (n) = number of experiments in triplicate.) Analogue

Sequence

Kd (pM)

BBS-II

(Nα His)Ac-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2

186 ± 91 (2)

BBS-IV

(Nα His)Ac-Gln-Trp-Ala-Val-Gly-His-Cha-Met-NH2

84 ± 39 (2)

BBS-IX

(Nα His)Ac-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH2

392 ± 233 (2)

BBS-XXX

(Nα His)Ac-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH

513 ± 283 (3)

BBS-37

(Nα His)Ac-βAla-βAla-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2

BBS-38

(

BBS-42

2

Nα His)Ac-βAla-βAla-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH

2

(Nα His)Ac-Lys(sha)-βAla-βAla-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH2

30

36 (1) 181 ± 120 (2) 22 ± 2 (2)

tartrate tetrahydrate and 7-8 mg sodiumcarbonate. The solution was heated for 20 min at 100°C. The tricarbonyl-solution was adjusted to pH 6.5 (±0.3) with a mixture of HCl and phosphate buffer 1 M/0.6 M (3:2). Then 500 µl were mixed with 15 to 30 µl of the peptide solution (1 mM) and heated for 1 h at 75°C. The final product was analyzed and purified with reversed phase high performance liquid chromatography (RP-HPLC, C18 column) equipped with a radioactivity detector (Radiomatic Model 525TR, Packard Canberra). Cell culture The human prostate adenocarcinoma cell line PC-3 was purchased from European Collection of Cell Culture (ECACC; Salisbury, England). Cells were maintained in DMEM GLUTAMAX-I supplemented with 1-10% FCS, 100 IU/ml penicillin G sodium, 100 µg/ml streptomycin sulphate, 0.25 µg/ml amphotericin B. Cell culture was incubated at 37°C in an atmosphere containing 5% CO2 . The cells were subcultured weekly. Animals Animal studies were conducted in compliance with the Swiss animal protection laws and with the ethical principles and guidelines for scientific animal trials established by the Swiss Academy of Medical Sciences and the Swiss Academy of Natural Sciences. Female CD-1 nu/nu mice (6 to 8 weeks old), purchased from Charles River (Sulzfeld, Germany), were used for the in vivo experiments. For the induction of tumor xenografts, PC-3 cells (5x106 cells/mouse) were injected subcutaneously and allowed to grow for at least 2 weeks. Metabolic stability in human plasma The degradation of the radiolabeled analogues was investigated in vitro in human plasma from healthy donors. The analogues (3 to 4 MBq/ml, 1.5-2 pmol/ml) were incubated for different time periods (from 0 to 24 h) at 37°C. After incubation, proteins were precipitated with acetonitrile/ethanol (1:1) and TFA (0.01%) and centrifuged (10 min, 20000g) at 4°C. The supernatant was filtered and analyzed by RP-HPLC. Different peaks corresponding to the intact peptide and the different degradation products were obtained in the radioactivity chromatograms. The percentage of intact peptide was determined for each incubation time. 31

Binding assays PC-3 cells at confluence were placed in 48-well plates (106 cells/well). Cells were incubated in triplicate with increasing concentrations of the labeled analogues (0.001-1 nM) for 1 h at 37°C. The concentration of total technetium was estimated according to Bauer and Pabst (34). After 2 washing steps with cold PBS, the cells were solubilized with 400 µl (2x) of NaOH 1 N. Radioactivity was measured in an Auto-γ-counter (Packard Canberra Cobra II). Nonspecific binding was determined in the presence of 1 µM unlabeled BBS(8-14). The protein content was determined with a BIO RAD microplate reader (570 nm) using the Pierce Micro BCA Protein Assay Reagent (Socochim SA, Lausanne, Switzerland). Internalization PC-3 cells at confluence were placed in 6-well plates (106 cells/well). Cells were incubated with the labeled analogues (4 kBq/well) in culture medium for 5, 15, 30, 60 and 120 min at 37°C to allow binding and internalization. Nonspecific internalization was evaluated in the presence of 1 µM unlabeled BBS(8-14). After the various incubation times, cells were washed 3 times with cold PBS. Surface-bound activity was removed by acid wash (50 mM glycine-HCl, 100 mM NaCl, pH 2.8, 600 µl 2x, 5 min at room temperature). Internalized activity was recovered by adding 600 µl (2x) 1 N NaOH per well. Surface bound and internalized activities were measured in the γ-counter. The results are either presented in relation to the total added or as % of the activity associated with the cells. All experiments were carried out 2-3 times in triplicate. Biodistribution studies The radioactive analogues (0.4 to 3.5 MBq/mouse) were injected intravenously into the tail vein. The animals were sacrificed by cervical dislocation at different post-injection times (from 30 min to 5 h) and dissected. Blood, tumors and other tissues were removed and weighed and the amount of radioactivity was determined with the γ-counter. Results were expressed as percentage of injected dose per gram of tissue (% I.D./g). In addition, a group of 3 mice per analogue received 100 µg of cold BBS(1-14) co-injected with the radiolabeled analogue and was sacrificed at 1.5 h p.i. to determine in vivo non-specific uptake. Single photon emission computed tomography/X-ray computed tomography (SPECT/CT) images were performed post-mortem 1.5 h after i.v. injection of 99m Tc32

BBS-42 (3.5 MBq). Images were obtained on an X-SPECTT M -system (Gamma Medica Inc.) equipped with a single head SPECT device and a CT-device. SPECT data were acquired and reconstructed with LumaGEM (version 5.407). CT data were acquired by X-Ray CT-system (Gamma MedicaT M ) and reconstructed with Cobra (version 4.5.1). Fusion of SPECT- and CT-data was performed with IDL Virtual MachineT M (version 6.0). Images were generated with AmiraT M (version 3.1.1).

2.4

Results

Radiolabeling The sequences of the new analogues are shown in Table 2.1. Yields higher than 90% were obtained after labeling. The main product obtained after labeling corresponded to the 99m Tc-labeled analogues. Pertechnetate and Tc-tricarbonyl represented a maximum of 5% and 1%, respectively, of the total activity and were not detectable after HPLC purification. Metabolic stability in human plasma The unmodified analogue was rapidly metabolized with a half-life of 30 min (35). The replacement of the amino acids Leu13 and/or Met14 by the non-natural amino acids cyclohexylalanine (Cha) and Norleucine (Nle) resulted in a much slower degradation in human plasma. Binding assays Binding of the 99m Tc-labeled BBS analogues to GRP receptor on PC-3 cells was saturable and highly specific. BBS-II, the unmodified sequence, showed a Kd of 186 pM, similar to that reported for BBS (36). Replacement of Met14 by Nle (BBS-IX and BBS-XXX) decreased the affinity (Kd of 392 and 513 pM, respectively). The additional insertion of a spacer, either βAla-βAla (BBS-37, BBS-38) or Lys(sha)-βAla-βAla (BBS-42) improved the receptor affinity. Thus, BBS-38 had a similar affinity to BBS-II whereas BBS-37 and BBS-42 showed even higher affinity: Kd of 181, 36 and 22 pM, respectively (Table 2.1).

33

Figure 2.1: Representative curve of the time-course degradation of the BBS analogues in human plasma at 37°C.

Internalization The distribution of the bound activity was similar for the different BBS analogues. The percentage of internalized activity increased strongly within the first 30 min reaching a maximum of 70-80% and then remained constant for at least 2 h. However, the internalized activity related to the total activity added differ from one analogue to the other and ranged from 6-8% of BBS-IX and BBS-XXX to 30% of BBS-37 (Figure 2.2). Biodistribution and tumor uptake The biodistribution of the different analogues was compared at 1.5 h p.i.. The biggest differences were found in the uptake in pancreas, liver and tumor (Figure 2.3). Activity in blood was very low for the analogues with double stabilization and/or spacer. Only stabilization had a slight increase in pancreas uptake for one of the peptides but no differences were seen in tumor uptake. The insertion of a spacer importantly increased both pancreas and tumor uptake. The combination of a hydrophilic spacer and stabilization

34

(BBS-42) had an even greater effect in the uptake of those tissues. For this compound liver uptake was also reduced as expected. The highest uptake in GRP receptor-positive tissues was obtained for the analogue BBS-42 (18.5% I.D./g and 3.2% I.D./g in pancreas and tumor, respectively). Colon uptake was relatively high for all the derivatives and no big differences were seen among the analogues. The tumor-to-background ratios were calculated (Table 2.2). We observed that stabilization contributed to increase tumor-to-blood ratios and tumor-to-muscle ratios when both positions 13 and 14 are modified. With the insertion of βAla-βAla (BBS-37), tumor-to-blood ratios increased by a factor of 10 compared to the unmodified analogue (BBS-II). The spacer in BBS-42 is more hydrophilic and resulted in 11-fold higher tumor-to-liver ratios vs. BBS-II (Table 2.2). Tumor-to-kidney ratios were also improved after insertion of spacer (2.50, 2.25 and 3.25 times higher for BBS-37, BBS-38 and BBS-42, respectively). A time-course biodistribution (up to 5 h) was performed with the analogues BBS-38 and BBS-42 with both stabilization and spacer (Figure 2.4). In these studies we found that the analogue BBS-38 was more rapidly cleared from GRP receptor-positive tissues than the analogue BBS-42.

Figure 2.2: Internalization of the BBS analogues in PC-3 cells after incubation at 37°C. Data represent the percentage of internalized activity related to the total activity added to the cells.

35

Figure 2.3: Comparative biodistribution of the different BBS analogues (0.4-3.5 MBq/mouse) in nude mice with PC-3 tumor xenografts at 1.5 h p.i.

2.5

Discussion

BBS analogues are interesting carrier molecules to selectively deliver radionuclides in BBS receptor-rich tumors. The rapid degradation of the fragment BBS(7-14) led us to the synthesis of new analogues with some changes in the natural sequence in order to increase the in vivo stability. It is known that modifications at the C-terminal domain do not affect importantly the binding affinity if the final molecule retains the lipophilic character (37, 38). The modifications of the new analogues consisted of the replacement of Leu13 and /or Met14 with Cha and Nle, respectively. Additionally, some analogues include a spacer, either βAla-βAla (BBS-37 and BBS-38) or Lys(sha)-βAla-βAla (BBS42). Finally, a retroNα -carboxymethyl histidine ((Nα His)Ac) was linked to the analogues as a chelating system since it forms stable complexes with 99m Tc-tricarbonyl (32, 35). In stability studies we found that the unmodified analogue (BBS-II) was rapidly metabolized with a half-life of 30 minutes (35). The replacement of Met14 by Nle (BBS-IX) led to an increase of stability with a half-life 12 times longer. However, the replacement of Leu13 by Cha (BBS-IV) was more effective and it resulted in a more than 30-fold

36

Table 2.2: Tumor to non-tumor ratios in nude mice with PC-3 tumor xenografts 1.5 h after i.v. injection of the 99m Tc-labeled BBS analogues (0.4-3.5 MBq/mouse). (Tu = tumor; Bl = blood; Mu = muscle; Ki = kidney; Li = liver; Pa = pancreas) Analogue

Tu/Bl

Tu/Mu

Tu/Ki

Tu/Li

Tu/Pa

BBS-II

1.1±0.03

6.1±0.5

0.4±0.2

0.5±0.1

0.4±0.2

BBS-IV

1.5±1.4

5.7±6.7

0.4±0.2

0.2±0.1

0.2±01

BBS-IX

1.2±1.4

5.8±6.9

0.4±0.4

0.2±0.1

0.6±0.7

BBS-XXX

4.0±1.6

10.5±9.1

0.6±0.2

0.2±0.1

0.2±0.1

BBS-37

11.1±10.2

18.8±11.8

1.0±0.6

2.7±1.9

0.2±0.1

BBS-38

4.3±3.4

14.5±8.1

0.9±0.4

0.4±0.4

0.1±0.04

BBS-42

9.5±5.4

20.1±13.3

1.3±0.7

5.6±3.3

0.2±0.04

increase of the stability with a plasma half-life of 16 h. A combination of both modifications (BBS-XXX) did not further improve the metabolic stability. The spacer βAla-βAla slightly decreased plasma stability (t1/2 = 10 h) whereas the spacer Lys(sha)-βAla-βAla did not have any influence (Figure 2.1). Higher plasma stability would translate into a higher amount of intact radiolabeled analogues at the tumor area, increasing the chances of targeting the receptors on the tumor cells surface. However, in order to also attain a higher amount of radioactivity in the tumor cells, the binding affinity to the receptors should be preserved. The affinity of the new analogues to GRP receptors on PC-3 cells was comparable to that reported for 125 I-BBS (36). The analogues including the non-natural aminoacid Cha in the molecule showed a decrease of affinity although the values of the Kd remained at the sub-nanomolar range. In turn, the presence of the spacer βAla-βAla had a positive effect on binding affinity. Thus, the analogues with this spacer had a better affinity than the corresponding analogues without spacer (Kd of 36 and 181 pM for BBS-37 and BBS-38, respectively, compared to 86 and 513 pM for BBS-II and BBS-XXX, respectively, Table 2.1). Moreover, an additional Lys(sha) in the spacer showed a greater influence on the binding affinity (Kd of 22 pM for BBS-42 compared to 181 pM for BBS-38, Table 2.1). In blockade studies we found that in vivo uptake in receptor-rich tissues (pancreas, colon and tumor) was specific especially for the analogues BBS-38 and BBS-42 with both stabilization and spacer in their molecules (Table 2.3). Finally, the tumor xenograft could be clearly visualized by SPECT/CT 37

imaging 1.5 h after i.v. injection of BBS-42 (Figure 2.5). After binding to the receptors the analogues underwent a rapid internalization reaching a maximum at 30 min. The percentages of internalization differ for every analogue (Figure 2.2) being highest for the analogue BBS-37 (around 28%) and lowest for the analogues BBS-IX and BBS-XXX (6 to 8%). With the exception of BBS-IV and BBS-42, internalization matched with the affinities of the different analogues for GRP receptors (Table 2.1). No differences, however, were found in the distribution of the cell-associated radioactivity, which in a 70 to 80% corresponded to internalized peptide within the first 15 min. This is interesting since radiolabeled peptides that internalize would be preferable, especially if they are bound to therapeutic radionuclides and are intended for tumor targeted radiotherapy. The rapid and high degree of endocytosis of the receptor-bound radiolabeled analogues would indicate their agonistic nature as described for agonist but not for antagonists in native GRP receptor containing cells (39, 40). Murine pancreas and intestine are GRP receptor-expressing tissues. In biodistribution studies, 1.5 h after the i.v. injection of the different analogues, the biggest differences were found in pancreas and tumor uptakes, two receptor-positive tissues, as well as in liver uptake (Figure 2.3). Only stabilization improved neither pancreas uptake nor tumor uptake although it did result in higher tumor-to-blood and tumor-to-muscle ratios (Table 2.2). The presence of a spacer, however, had a greater influence in pancreas uptake and tumor uptake (Figure

and 99m Tc-BBS-42 in nude mice bearing PC-3 tumor xenografts (3.5 MBq/ml). Bl = blood; Ki = kidney; Pa = pancreas; Sto = stomach; SI = small intestine; Co = colon; Li = liver; Mu = muscle; Tu = tumor.

Figure 2.4: Time-course biodistribution of

99m Tc-BBS-38

38

Table 2.3: Specificity of the in vivo uptake in GRP receptor-positive tissues in nude mice with PC-3 tumor xenografts at 1.5 h p.i. Animals received i.v. the 99m Tc-labeled BBS analogues (0.4-3.5 MBq/mouse) alone or together with BBS (0.1 mg/mouse). Analogue

Inhibition of

Inhibition of

Inhibition of

tumor uptake (%)

pancreas uptake (%)

colon uptake (%)

BBS-II

41.2

55.2

41.3

BBS-IV

11.3

50.0

52.0

BBS-IX

61.2

83.5

82.6

BBS-38

53.8

80.7

78.8

BBS-42

60.7

88.0

89.3

2.3), leading to better tumor-to-background ratios (Table 2.2). Higher receptor densities in pancreas compared to tumor would entail the higher uptake found in pancreas in the in vivo experiments, which has also been reported for other BBS analogues (11, 41-43). Colon uptake was rather similar for all the analogues (Figure 2.3). This may be explained by a saturation of the receptors. Schumacher et al. (44) reported saturation of intestinal receptors with a maximal uptake at doses equal or lower than 5 pmol/animal. In our in vivo experiments each mouse received around 2 pmol and, therefore, saturation of colonic receptors can not be excluded. The specificity of the in vivo uptake was determined in the blockade experiments in which the animals received the radiolabeled BBS analogues and the natural BBS (100 µg/mouse) in co-injection. Uptake in GRP receptor-positive tissues (pancreas, colon and tumor) was significantly reduced and it was more pronounced for the analogues BBS-38 and BBS-42, with both stabilization and spacer in their molecules (Table 3). Time-course biodistribution studies were carried out with the analogues BBS-38 and BBS-42 in order to test how the characteristics of the spacer influence the in vivo behavior (Figure 2.4). Clearance from blood and normal GRP receptor-negative tissues was fast for both analogues. However, within the first 1.5 h, BBS-38 was more rapidly cleared from GRP receptor-expressing tissues than BBS-42 (Figure 2.4). The spacer Lys(sha)-βAla-βAla- contributed to improve both the in vivo pancreas uptake and tumor uptake. Moreover, it led to lower liver uptake, which was indeed rapidly cleared. Despite of a higher kidney uptake for BBS-42, clearance from this organ was 39

also rather fast. Interestingly, BBS-42 showed better tumor-to-background ratios than other reported BBS analogues (11, 42, 43, 45). These results may be ascribed to the higher hydrophilicity of the spacer Lys(sha)-βAla-βAla-, which would counteract the high lipophilicity of the [99m Tc(CO)3 (Nα His)Ac] complex, and are in agreement with Smith et al. (27) who considered that the lipophilicity of the final molecule was decisive for the in vivo behavior. Finally, the tumor was easily detectable by means of SPECT/CT imaging (Figure 2.5). A high radioactivity accumulation was found in the abdominal area, similar to the biodistribution studies in which pancreas and intestinal uptake were higher than tumor uptake (Figure 2.4).

Figure 2.5: SPECT/CT imaging of a nude mouse with a PC-3 tumor xenograft. Images were taken 1.5 h after i.v. administration of

99m Tc-BBS-42

40

(3.5 MBq).

2.6

Conclusion

Technetium-tricarbonyl linked to the tridentate ligand (Nα His)Ac is a useful method for labeling BBS analogues. Modifications at the positions 13 and 14 contributed to increase plasma stability with little changes in the binding properties. The additional insertion of a spacer permitted to modulate the lipophilicity of the analogues improving the biodistribution and leading to better tumor to non-tumor ratios, especially in the case of the spacer Lys(sha)-βAla-βAla. The analogue BBS-42 holds a higher potential as radiopharmaceutical for targeting GRP receptor-positive tumors.

2.7

Acknowledgements

The authors thank Ms. Margaretha Lutz and Ms. Olga Gasser for technical assistance. This work was funded by an Oncosuisse grant (Nr. OCS 01311-02-2003) and by the Fund for Scientific Research-Flanders, Belgium, (grant Nr. G.0036.04).

41

2.8

References

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11. Nock B, Nikolopoulou A, Chiotellis E, Loudos G, Maintas D, Reubi J C, Maina T. 99m Tc Demobesin 1, a novel potent bombesin analogue for GRP receptor-targeted tumour imaging. Eur J Nucl Med Mol Imaging 2003; 30: 247-258. 12. von Guggenberg E, Behe M, Behr TM, Saurer M, Seppi T, Decristoforo C. 99m Tclabeling and in vitro and in vivo evaluation of HYNIC- and (Nα -His)acetic acidmodified [D-Glu1]-minigastrin. Bioconjugate Chem 2004; 15: 864-871. 13. Nagalla SR, Barry BJ, Falick AM, Gibson BW, Taylor JE, Dong JZ, Spindel ER. There are three distinct forms of bombesin. Identification of [Leu13 ]bombesin, [Phe13 ]bombesin, and [Ser3 ,Arg10 ,Phe13 ]bombesin in the frog Bombina orientalis. J Biol Chem 1996; 271: 7731-7737. 14. Spindel ER, Giladi E, Brehm P, Goodman RH, Segerson TP. Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor. Mol Endocrinol 1990; 4: 1956-1963. 15. Wada E, Way J, Shapira H, Kusano K, Lebacq-Verheyden AM, Coy D, Jensen R, Battey J. cDNA cloning, characterization and brain region-specific expression of a neuromedin-B preferring bombesin receptor. Neuron 1991; 6: 421-430. 16. Fathi Z, Corjay MH, Shapira H, Wada E, Benya R, Jensen R, Viallet J, Sausville EA, Battey JF. BRS-3: novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J Biol Chem 1993; 268: 5979-5984. 17. Nagalla SR, Barry BJ, Creswick KC, Eden P, Taylor JE, Spindel ER. Cloning of a receptor for amphibian [Phe13 ]bombesin distinct from the receptor for gastrinreleasing peptide: identification of a fourth bombesin receptor subtype (BB4). Proc Natl Acad Sci USA 1995; 92: 6205-6209. 18. Bunnett G. Gastrin-releasing peptide. In: Walsh JH, Dockray GJ editors. Gut peptides: biochemistry and physiology. New York: Raven Press, Ltd; 1994.p.423445. 19. Delle Fave G, Annibale B, De Magistris L, Severi C, Bruzzone R, Puoti M, Melchiori P, Torsoli A, Erspamer V. Bombesin effects on human GI functions. Peptides 1985; 6: 113-116.

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20. Alexander RW, Upp Jr JR, Poston GJ, Gupta V, Townsend Jr CM, Thompson JC. Effects of bombesin on growth of human small cell lung carcinoma in vivo. Cancer Res 1988; 48: 1439-1441. 21. Moody TW, Cuttitta F. Growth factor and peptide receptors in small cell lung cancer. Life Sci 1993; 52: 1161-1173. 22. Wang QJ, Knezetic JA, Schally AV, Pour PM, Adrian TE. Bombesin may stimulate proliferation of human pancreatic cancer cells through an autocrine pathway. Int J Cancer 1996; 236: 528-534. 23. Kim S, Hu W, Kelly DR, Hellmilch MR, Evers BM, Chung DH. Gastrin-releasing peptide is a growth factor for human neuroblastomas. Ann Surg 2002; 235: 621630. 24. Bologna M, Festuccia C, Muzi P, Biordi L, Ciomei M. Bombesin stimulates growth of human prostatic cancer cells in vitro. Cancer 1989; 63: 1714-1720. 25. Gugger M and Reubi JC. Gastrin-releasing peptide receptors in non-neoplastic and neoplastic human breast. Am J Pathol 1999; 155: 2067-2076. 26. Markwalder R, Reubi JC, Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res 1999; 59: 1152-1159. 27. Smith CJ, Gali H, Sieckman GL, Higginbotham C, Volkert WA, Hoffmann TJ. Tadiochemical Investigations of 99m Tc-N3 S-X-BBN[7-14]NH2 : An in vitro/in vivo structure activity relationship study where x= 0-, 3-, 5-, 8-, and 11-carbon tethering moieties. Bioconjug chem 2003; 14: 93-102. 28. Liu S, Edwards DS. 99m Tc-labeled small peptides as diagnostic radiopharmaceuticals. Chem Rev 1999; 99: 2235-2268. 29. Alberto R, Schibli R and Schubiger PA. Reactions with the technetium and rhenium carbonyl complexes (N(Et)4 )2 [MX3 (CO)3 ]. Synthesis and structure of [Tc(CNBut)3 (CO)3 ](NO3 ) and (NEt4 )[Tc2 ( -SCH2 CH2 OH)3 (CO)6 ]. Polyhedron 1996; 15: 1079-1089. 30. Egli A, Alberto R, Tannahill L, Schibli R, Abram U, Schaffland A, Waibel R, Tourwé D, Jeannin L, Iterbeke K, Schubiger PA. Organometallic 99m Tc-aquaion 44

labels peptide to an unprecedented high specific activity. J Nucl Med 1999; 40: 1913-1917. 31. Maes V, Garc´ıa-Garayoa E, Bläuenstein P, Tourwé D. Novel 99m Tc-labelled neurotensin analogs with optimized biodistribution properties. J Med Chem 2006; 49: 1833-1836. 32. Schibli R, La Bella R, Alberto R, Garcia Garayoa E, Ortner K, Abram U, Schubiger PA. Influence of the denticity of ligand systems on the in vitro and in vivo behavior of 99m Tc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules. Bioconj Chem 2000; 11: 345-351. 33. Garc´ıa-Garayoa E, Allemann-Tannahill L, Bläuenstein P, Willmann M, CarrelRémy N, Tourwé D, Iterbeke K, Conrath P, Schubiger PA. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors. Nucl Med Biol 2001; 28: 75-84. 34. Bauer R and Pabst H. Tc-generators - yield of Eur J Nucl Med 1982; 7: 35-36.

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35. La Bella R, Garcia-Garayoa E, Bähler M, Bläuenstein P, Schibli R, Conrath P, Tourwé D, Schubiger PA. A 99m Tc(I)-postlabeled high affinity bombesin analogue as a potential tumor imaging agent. Bioconjugate Chem 2002; 13: 599-604. 36. Reile H, Armatis PE, Schally AV. Characterization of high affinity receptors for bombesin/gastrin-releasing peptide on the human prostate cancer cell lines PC3 and DU-145: internalization of receptor bound [125I-Tyr4]bombesin by tumor cells. Prostate 1994; 25: 29-38. 37. Lin JT, Coy DH, Mantey SA and Jensen RT. Comparison of the peptide structural requirements for high affinity interaction with bombesin receptors. Eur J Pharmacol 1995; 294: 55-69. 38. Devin C, Bernad N, Cristau M, Artis-Noel A, Heitz A, Fehrentz J.-A, Martinez J. Synthesis and Biological Evaluation of C-Terminal Hydroxyamide Analogues of Bombesin. J. Peptide Sci. 1999; 5: 176-184.

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39. Mantey S, Frucht H, Coy DH, Jensen RT. Characterization of bombesin receptors using a novel, potent, radiolabeled antagonist that distinguishes bombesin receptor subtypes. Mol Pharmacol 1993; 45: 762-774. 40. Zhu WY, Goke B, Williams JA. Binding, internalization and processing of bombesin by rat pancreatic acini. Am J Physiol 1991; 261: G57-G64. 41. La Bella R, Garcia-Garayoa E, Langer M, Bläuenstein P, Beck-Sickinger AG, Schubiger PA. In vitro and in vivo evaluation of a 99m Tc(I)-labeled bombesin analogue for imaging of gastrin releasing peptide receptor-positive tumors. Nucl Med Biol 2002; 29: 553-560. 42. Smith CJ, Sieckman GL, Owen NK, Hayes DL, Mazuru DG, Kannan R, Volkert WA, Hoffman TJ. Radiochemical investigations of gastrin-releasing peptide receptor-specific [99m Tc(X)(CO)3 -Dpr-Ser-Ser-Ser-Gln-Trp-Ala-Val-Gly-His-Leu-Met(NH2 )] in PC-3 tumor-bearing, rodent models: syntheses, radiolabeling, and in vitro / in vivo studies where Dpr = 2,3-diaminopropionic acid and X = H2 O or P(CH2 OH)3 . Cancer Res 2003; 63: 4082-4088. 43. Nock B, Nikolopoulou A, Galanis A, Cordopatis P, Waser B, Reubi JC, Maina T. Potent bombesin-like peptides for GRP-receptor targeting of tumors with 99m Tc: a preclinical study. J Med Chem 2005; 48: 100-110. 44. Schuhmacher J, Zhang H, Doll J, Mäcke HR, Matys R, Hauser H, Henze M, Haberkorn U, Eisenhut M. GRP receptor-targeted PET of a rat pancreas carcinoma xenograft in nude mice with a 68 Ga-labeled bombesin(6-14) analog. J Nucl Med 2005; 46: 691-699. 45. Zhang X, Cai W, Cao F, Schreibmann E, Wu Y, Wu JC, Xing L, Chen X. 18 FLabeled bombesin analogs for targeting GRP receptor-expressing prostate cancer. J Nucl Med 2006; 47: 492-501.

46

Chapter 3 Influence of the Molecular Charge on the Biodistribution of Bombesin Analogues Labeled with the [99mTc(CO)3]-Core

Elisa Garc´ıa Garayoa1 , Christian Schweinsberg1 , Veronique Maes2 , Luc Blanc2 , Peter Bläuenstein1 , Dirk A. Tourwé2 , P. August Schubiger1,3 , R. Schibli1,3

1

Paul Scherrer Institute, Center for Radiopharmaceutical Science, CH-5232 Villigen PSI, Switzerland 2 Department of Organic Chemistry, Vrije Universiteit Brussel, 1050 Brussels, Belgium 3 Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland

Bioconjugate Chemistry (accepted)

47

3.1

Abstract

The overexpression of Bombesin (BBS) receptors on a variety of human cancers make them interesting targets for tumor imaging and therapy. Analogues of the neuropeptide BBS have been functionalized with the (Nα His)-chelator for labeling with the 99m Tctricarbonyl-core. The introduction of a βAla-βAla linker between the stabilized BBS binding sequence and the chelator led to increased tumor uptake but still rather unfavorable in vivo properties. Novel polar linkers, with different charge, have been introduced in the molecule and tested for their influence on the biodistribution. The new analogues showed a shift in hydrophilicity from a LogD = 0.9 to LogD values between 0.4 and -2.2. All compounds kept the increased stability in both human plasma (t1/2 > 16 h) and in tumor cells (t1/2 = 30-40 min). The compounds with LogD values between +1 and -1 showed the highest binding affinities with Kd values < 0.5 nM, as well as the highest cellular uptake. However, higher hydrophilicity (LogD < -1.8) led to lower affinity and substantial decrease of internalization. The introduction of a positive charge (β 3 hLys) resulted in unfavorable biodistribution, with increased kidney uptake. The introduction of an uncharged hydroxyl group (β 3 hSer) improved the biodistribution, resulting in significantly better tumor-to-tissue ratios. The compound with one single negative charge (β 3 hGlu) showed a significant increase in the tumor uptake (2.1 ± 0.6 vs. 0.80 ± 0.35 % ID/g in comparison to the βAla-βAla analogue), and also significantly higher tumorto-tissue ratios. The specificity of the in vivo uptake was confirmed by co-injection with natural BBS. Moreover, it provided a much clearer image of the tumor xenografts in the SPECT/CT studies. The introduction of a single negative charge may be useful in the development of new BBS analogues to obtain an improved biodistribution profile, with increased tumor uptake and better imaging.

KEY WORDS: gastrin-releasing-peptide receptor, bombesin, spacer, technetium-99m, tumor uptake

48

3.2

Introduction

A variety of human carcinomas show an overexpression of peptide receptors for which natural neuropeptides feature high affinities. Thus, peptide-based radiopharmaceuticals exhibit a great potential for imaging and therapy of cancer (1-5). Their small size allows a good penetration into tumor tissue and promotes a more rapid blood clearance compared to other macromolecular carriers such as antibodies (4). This can result in high specific uptake and optimal tumor-to-background ratios for the visualization of tumors and small metastases as well as for therapeutic applications, depending on the radionuclide used (4). Peptides are readily synthesized and can withstand harsh conditions for modification and labeling. Their chemical characteristics offer a broad range for functionalization and modification like the introduction of chelating systems and pharmacokinetic modifiers (4-6). There is evidence that for some cancer types not only one but several peptide receptors may be overexpressed in varying levels and regions in the tumor tissue (7). Therefore, it is of great interest to develop more than one peptide radiopharmaceutical to cover a broader range of possible targets as well as to obtain a more homogeneous distribution of the activity within the tumor mass. Bombesin (BBS) is a tetradecapeptide with high affinity for the mammalian gastrin releasing peptide receptor (GRP-R), a subtype of the BBS receptor superfamily, which is overexpressed in most human prostate and breast tumors as well as small-cell lung cancer and several other cancer types (8-10). A variety of BBS analogues has been designed, based either on the complete original peptide sequence or on the fragment (7-14). Several BBS analogues containing the appropriate chelating systems for radiolabeling with different radionuclides (e.g., 99m Tc or 186 Re/188 Re, 64 Cu, 111 In, 177 Lu and 18 F) show promising pharmacological properties (4-6, 11-14). Of the different radionuclides useful in nuclear medicine 99m Tc is still the one with the broadest clinical application due to its favorable dosimetry, high specific activity, low cost and ready availability from a 99 Mo/99m −Tcgenerator. Its physical properties (t1/2 = 6 h, Energy γ = 140 keV, 89 % abundance) are optimal for imaging using single photon emission computed tomography (SPECT) (15-17). In addition, a metal chelator applicable for 99m Tc is usually also suitable for the therapeutic radionuclides 186 Re and 188 Re due to their similar chemical properties. Thus, a successful candidate for targeting GRP-R with 99m Tc could be interesting for radiotherapeutic applications with Re isotopes (16). Our group successfully established a chelating system for both 99m Tc(I) and 186 Re/188 Re(I) in the form of a tridentate

49

chelator, the retro(Nα His)Ac-chelator, for labeling of biomolecules with the tricarbonyl technique (18, 19). Some modifications of the BBS amino acid sequence, such as the exchange of Met14 with norleucine (Nle) and Leu13 with cyclohexylalanine (Cha), resulted in an improved stability in human plasma and in prostate and breast cancer cells. These modifications did not affect the binding affinity and cell internalization, but led to increased tumor-tobackground ratios although tumor uptake did not significantly change (20, 21). Smith et al reported on the positive influence of aliphatic spacers on the uptake of 99m Tclabeled BSS analogues into GRP-R-expressing tissues (14). With the introduction of a βAla-βAla linker we showed an additional improvement of the in vivo tumor uptake of stabilized BBS analogues labeled with the 99m Tc-tricarbonyl core (20)(βAla is the exception to the general nomenclature of the β-amino acids according to Abele and Seebach (25, 26)). Likewise, other recent papers report on the positive effects of polar linker moieties on 64 Cu-labeled DOTA-, NOTA- and 99m Tc(III)-labeled BBS conjugates (22-24). The BBS analogues with the Tc(CO)3 -His complex are more lipophilic than the above mentioned peptides since the His-Imidazol residue (pK = 6) is not protonated at physiological pH values. An increase of the hydrophilicity of the molecule as well as the presence of charges may lead to new analogues with improved biodistribution profiles. New BBS derivatives were synthesized with different polar linkers inserted between the stabilized binding sequence and the retro(Nα His)Ac-chelator. The spacers contain different βamino acids (25, 26) either neutral or with a positive or negative charge in the side chain. In this study we investigated the influence of these polar linkers and charges on the in vitro and in vivo behavior of new 99m Tc(I)-labeled BBS analogues.

3.3

Experimental Procedures

Chemicals and Equipment Cell culture media DMEM GLUTAMAXT M -I, trypsin/EDTA, bovine serum albumin and soybean trypsin inhibitor were obtained from Invitrogen (Basel, Switzerland), fetal calf serum (FCS) and antibiotic/antimycotic solution were obtained from Bioconcept (Allschwil, Switzerland). Bacitracin, HEPES and Chymostatin were purchased from SIGMA (Buchs, Switzerland). All other chemicals were obtained from Fluka (Buchs, Switzerland) or Merck (Dietkon, Switzerland). For the determination of protein concen-

50

trations a Pierce BCA Protein Assay kit from Socochim (Lausanne, Switzerland) was used. Na[99m TcO4 ] was eluted from a Mallinckrodt 99 Mo/99m Tc generator (Tyco Healthcare, Petten, The Netherlands) using 0.9 % saline. Sodium boranocarbonate was a gift from the provider of the generator. The radioactivity of in vitro and in vivo experiments was measured with a NaI-γ-counter (Packard Canberra Cobra II, Meriden, USA) or a NaI ionization chamber. The Fmoc-β 3 homo-amino acids were purchased from Fluka. Peptide Synthesis and radiolabeling with

99m

Tc

Peptide solid-phase synthesis was carried out on a Rink Amide Resin with the Fmocstrategy as previously described (20). Purification of the peptides was done on a semipreparative HPLC system (Gilson) on a reverse phase C18 column (DiscoveryBIO SULPELCO Wide Pore, 25 cm x 2.21 cm, 5 µm) with a linear gradient of 3 % B to 100 % B (solvent A = H2 O + 0.1 % TFA as and as solvent B = CH3 CN + 0.1 % TFA) in 30 min, a flow rate of 1 mL/min and UV detection at 215 nm. Each of the new spacers introduced between the binding sequence and the chelator contained three β-amino acids in form of a βAlanine (βAla), β 3 -homoserine (βSer), β 3 -homolysine (βLys) or β 3 -homoglutamic acid (βhGlu) (Figure 3.1). The (Nα His)Ac moiety (retro[Nα -carboxymethyl-histidine]) was coupled to the N-terminus for labeling to [99m Tc(CO)3 ] (Figure 3.1)(25, 26) Radiolabeling of the analogues was performed as previously described (20). For analyses and purification of the labeled peptides from the unlabeled compounds a Macherey-Nagel CC Nucleosil 100-5 C18 RP column (5 µm, 250 x 4.6 mm) was used. Preparative HPLC for peptide purification was performed using a Merck-Hitachi L-7100-system with an equipped 2-channel scintillation detector interface (John Caunt scientific Ltd, Eynsham, England) and a Rainin Dynamax absorbance detector model UV-1. HPLC solvents consisted of H2 O + 0.1 % TFA (solvent A) and CH3 OH + 0.1 % TFA (solvent B). The separation was performed with a linear gradient 50 % A / 50 % B to 20 % A / 80 % B in 25 min (1.0 mL/min). For the analytical HPLC of the labeled peptides a Varian ProStar system was used with a Varian ProStar 230 Solvent Delivery-Pump, a Varian Photodiode Array Detector Model 330, a Varian ProStar Autosampler Model 410 and a radiomatic Flo-one Beta Detector (Packard Canberra). HPLC solvents consisted of CH3 CN + 0.1 % TFA (solvent A) and H2 O + 0.1 % TFA (solvent B). The labeling control was performed with a linear gradient of 30 % A / 70 % B to 80 % A / 20 % B in 25 min (1.0 mL/min).

51

Figure 3.1: Chemical structure of the new

99m Tc-labeled

BBN analogues.

Octanol/water partition coefficient study To compare the polarity of the different new analogues the octanol/water partition coefficients were determined at pH = 7.4 by measuring the distribution of the radiolabeled peptides in 1-octanol and PBS. Five µL containing 500 kBq of radiolabeled compound in PBS were added to a vial containing 1.2 ml 1-octanol and PBS (1:1). After vortexing for 1 min the vial was centrifuged for 5 min at 10000 rpm to ensure complete separation of layers. Then 40 µL of each layer were taken in a preweighed vial and measured in the γ-counter. Counts per unit weight of sample were calculated, and logD values were calculated using the formula:

in 1 g of octanol Log10 D = log10 ( counts ) counts in 1 g of P BS

52

Cell culture PC-3 human prostate cancer cell line was obtained from European Collection of Cell Cultures (Salisbury, United Kingdom). Cells were maintained in Dulbecco’s MEM with GLUTAMAXT M -I supplemented with 10 % FCS, 100 IU/mL penicillin G sodium, 100 µg/mL streptomycin sulphate, 0.25 µg/mL amphotericin B. Cells were incubated at 37°C in an atmosphere containing 5 % CO2 , and subcultured twice a week after detaching with trypsin-EDTA. Metabolic stability in human plasma and in PC-3 cells The degradation of the radiolabeled analogues was investigated in vitro in human plasma from healthy donors. The analogues (3 MBq/mL, 1.5-2 pmol/mL) were incubated for different time periods (from 0 to 24 h) at 37°C. After incubation, proteins were precipitated as follows: 250 µL of sample + 750 µL of acetonitrile/ethanol (1:1) and TFA (0.01%). The mixture was vortexed and centrifuged (10 min, 20000g) at 4°C. The supernatant was filtered and analyzed by RP-HPLC equipped with a radioactivity detector. The stability in cell culture was evaluated with 2 x 106 cells in PBS. The analogues (3 MBq/mL, 1.5-2 pmol/mL) were incubated from 0 to 5 h at 37°C. Afterwards proteins were precipitated, and the supernatant filtered and analyzed as above. All experiments were carried out 2-3 times. Different peaks corresponding to the intact peptide and the different degradation products were obtained in the radioactivity chromatograms. The percentage of intact peptide was determined for each incubation time. Receptor inhibition studies One day prior to the assay PC-3 cells at confluence were placed in 48-well plates. Cells were incubated in a special binding buffer (50 mM HEPES, 125 mM NaCl, 7.5 mM KCl, 5.5 mM MgCl*6H2 O, 1 mM EGTA, 5 g/L BSA, 2 mg/L Chymostatin, 100 mg/L Soybean Trypsin inhibitor, 50 mg/L Bacitracin, pH 7.4) with increasing concentrations of the BBS analogues (0-30000 nM) in presence of 4 kBq of 99m Tc-BBS(7-14), which is known to express a high binding affinity to the GRP receptor. After 1 h incubation at 37°C the cells were washed twice with cold PBS. Bound radioactivity was recovered by solubilizing the cells in 1 N NaOH (2 x 400 µL). The radioactivity was measured in the γ-counter. All experiments were carried out 2-3 times in triplicate.

53

Receptor saturation studies One day prior to the assay PC-3 cells at confluence were placed in 48-well plates and incubated for 1 h at 37°C in the binding buffer described above with increasing concentrations of the labeled BBS analogues (0.01-2 nM). The concentration of total technetium-labeled peptide was calculated according to Bauer et al. (17). Non-specific binding was determined by co-incubation with 1 µM natural BBS. After incubation the cells were washed twice with cold PBS, solubilized in 1 N NaOH (2 x 400 µL) and the radioactivity was measured in the γ-counter. Experiments were performed 2-3 times in triplicate. Internalization One day prior to the assay PC-3 cells at confluence were placed in 6 well plates. Cells were incubated with the labeled analogues (4 kBq/well) in binding buffer for 5, 15, 30, 60 and 120 min at 37°C to allow binding and internalization. Non-specific binding was determined in the presence of 1 µM natural BBS. After incubation cells were washed twice with cold PBS. Surface bound activity was removed by acid wash (50 mM glycine/HCL, 100 mM NaCl, pH 2.8, 2 x 600 µL for 5 min at room temperature). Afterwards the plates were neutralized with 600 µL PBS and cells were lysed with 1 N NaOH (2 x 600 µL). Surface bound and internalized activities were measured in the γ-counter. The protein concentration was determined and results were calculated as percentage of total added radioactivity per mg protein. All experiments were carried out 2-3 times in triplicate. Externalization One day prior to the assay PC-3 cells at confluence were placed in 6 well plates. Cells were incubated with the labeled analogues (20 kBq/well) in culture medium for 1 h at 37°C to allow binding and maximal internalization. After incubation the supernatant was discarded and each well washed twice with cold PBS. Afterwards the cells were incubated again at 37°C in 2 mL culture medium for 30, 60, 150 or 300 min. At each time point the supernatant was collected, the cells were washed twice with cold PBS and the radioactivity remaining in the cells was recovered by lysing the cells with 1 N NaOH (2 x 600 µL). Radioactivity in the supernatant (released activity) and in the lysate (internalized activity) was determined in the γ-counter. Results are expressed as percentage of the total activity associated with the cells. The protein concentration was determined and results were calculated as percentage of total added radioactivity per 54

mg protein. All experiments were carried out 2-3 times in triplicate. Biodistribution studies All animal experiments were conducted in compliance with the Swiss animal protection laws and with the ethical principles and guidelines for scientific animal trials established by the Swiss Academy of Medical Sciences and the Swiss Academy of Natural Sciences. Female CD-1 nu/nu mice 6-8 week-old (Charles River Laboratories, Sulzfeld, Germany) were subcutaneously injected with 8 x 106 cells in 150 µL culture medium without supplements. About 3 weeks after tumor implantation the mice (3-6 per group) were injected i.v. with 3.7 MBq of the radiolabeled peptides. At 0.5 h, 1.5 h or 5 h postinjection (p.i.), the animals were killed by cervical dislocation. The different organs (heart, lung, spleen, kidneys, pancreas, stomach, small intestine, large intestine, liver, muscle, bone) and blood were collected from the animals, weighed and the radioactivity was measured in a γ-counter. To determine specificity of the in vivo uptake one group of mice received a co-injection of 100 µg of unlabeled natural BBS and the radiolabeled analogue and sacrificed 1.5 h p.i. Results are presented as percentage of injected dose per gram tissue (%ID/g). SPECT/CT studies Single photon emission computed tomography / X-ray computed tomography (SPECT/ CT) images were performed post-mortem 1.5 h after i.v. injection of the 99m Tc-BBS analogues (20 MBq). Images were obtained on an X-SPECTT M -system (Gamma Medica Inc.) equipped with a single head SPECT device and a CT-device. SPECT data were acquired and reconstructed with LumaGEM (version 5.407). CT data were acquired by an X-Ray CT-system (Gamma MedicaT M ) and reconstructed with the software Cobra (version 4.5.1). Fusion of SPECT- and CT-data was performed with IDL Virtual MachineT M (version 6.0). Images were generated with AmiraT M (version 3.1.1). Statistical Analysis All data are presented as the mean ± SD. Differences in the in vivo uptake in the biodostribution studies were analyzed by one-way ANOVA followed by Dunnett’s comparison test. Differences between blocked and control groups were analyzed by unpaired t-test. P ≤ 0.05 was considered statistically significant. 55

Table 3.1: Characteristics of the new BBS analogues: retention time and polarity (LogD) of the 99m Tc-labeled analogues, and binding affinity for GRP-receptors in PC-3 cells of the labeled (Kd values) and unlabeled (IC50 values) compounds (n = 2-3; n.d. = non-determined). HPLC Analogue

γ-trace

LogD

Kd [nM]

IC50 [nM]

tR [min]

3.4

1 (Reference)

16.8

0.9 ± 0.2

0.19 ± 0.12

5.1 ± 1.7

2

16.6

0.4 ± 0.1

0.05 ± 0.03

6.8 ± 3.2

3

16.6

-0.5 ± 0.1

0.08 ± 0.01

13.3 ± 3.0

4

14.9

-0.5 ± 0.1

0.14 ± 0.06

23.6 ± 12.0

5

16.7

-2.0 ± 0.2

0.36 ± 0.07

16.3 ± 8.3

6

15.7

-2.2 ± 0.4

n.d.

634.0 ± 221.7

Results

Peptide Synthesis and Radiolabeling In the peptide syntheses yields of 50-58 % were obtained for the cold purified peptides (Table 3.4 in supporting information). The labeling yields with the 99m Tc-tricarbonyl core were higher than 90 %. Pertechnetate and tricarbonyl represented a maximum of 3 % and 5 %, respectively. For both in vitro and in vivo studies labeled and unlabeled peptide were separated by preparative RP-HPLC. After the separation the labeled analogue was analyzed with analytical RP-HPLC and corresponded to more than 98 % of the radioactivity. Octanol/Water-Coefficient The reference compound (1) with the linker βAla-βAla showed a rather lipophilic character with a LogD of +0.9. The introduction of a hydroxyl group (2) changed the polarity to +0.4. The insertion of additional positive or negative charges resulted in a clear increase of hydrophilicity up to -2.2 for compound 6 (bearing three negative charges) (Table 3.1).

56

Metabolic stability All new analogues, based on the fragment BBS(7-14) with the same changes at positions 13 and 14 (Cha and Nle for Leu and Met, respectively) showed increased stability compared to the unmodified sequence with a t1/2 of approximately 16 h in human plasma. Stability in PC-3 tumor cells was also higher for the new analogues (t1/2 = 30-40 min) especially for compound 4, which showed a clear increase in cell stability with a t1/2 of 80 min. Binding assays The binding affinity was tested for both the labeled and the unlabeled analogues. In inhibition experiments compounds 1 and 2 showed the highest affinity, with IC50 values of 5.1 and 6.8 nM, respectively. Compounds 3 to 5 had slightly lower affinities, whereas compound 6 showed a high decrease of affinity (IC50 = 634 nM) (Table 3.1). The 99m Tclabeled compounds 1 to 5 exhibited a saturable and highly specific binding. These analogues had high affinity for GRP receptors, with Kd values in subnanomolar range under 0.5 nM (Table 3.1). Under the set conditions the binding of the labeled compound 6 was very low in the range of unspecific binding and the Kd value was not determined. Internalization/Efflux For compounds 1 to 5, more than 80 % of the activity bound to PC-3 cells was internalized after 30 min. Internalization of compounds bearing a neutral spacer (2) or a single negative charge in the spacer (3) was comparable to the reference peptide 1 after 1 h (30 % of total dose/mg protein). Compound 4 (with a single positive charge) showed the highest internalization value (40 % of total dose/mg protein after 1 h), whereas compound 5 and especially compound 6 showed a lower internalization (15 % and 1 % of total dose/mg protein after 1 h, respectively). Results are depicted in Figure 3.2. Due to its low affinity and internalization compound 6 was not tested for externalization. In the externalization experiments the analogues 3, 4 and 5 were rapidly externalized with a release of approximately 70% in the first 2.5 h after maximal internalization. Release of compounds 1 and 2 was slower (45 % of the internalized activity was in the first 2.5 h). Percentages of 20-30 % of the analogues 3, 4 and 5 remained in the cells after 5 h, whereas the retention for the analogues 1 and 2 was slightly higher with 40 % of activity still associated with the cells at this time (Figure 3.2). 57

Figure 3.2: Time-course internalization (A) and externalization after maximal internalization (B) of the 99m Tc-labeled BBS analogues in PC-3 cells after incubation at 37°C. Data represent the percentage of internalized activity per mg protein related to the total activity added to the cells (n = 2-3).

Biodistribution and SPECT/CT studies The neutral or negatively charged compounds (analogues 1, 2, 3 and 5) were further tested in mice with PC-3 tumor xenografts (Tables 3.2 and 3.3). The compound 4 with a positive charge was excluded from these studies due to its unfavorable high retention in kidneys and liver found in previous biodistribution studies in normal mice (Table 3.5 in supporting information). Compared to compound 1 the analogues 2 (neutral) and 3 (one negative charge) showed higher uptake in the receptor-expressing tissues (pancreas, colon and tumor) at 0.5 h p.i. time (Tables 3.2 and 3.3). Interestingly, the tumor uptake was significantly higher for analogue 3 compared to the reference 1 (P ≤ 0.05) and the activity was longer retained (1 %ID/g at 5 h p.i.) than for the other analogues ( 0.5 %ID/g at 5 h p.i.). The analogues 2, 3 and 5 showed a highly significant decrease in liver accumulation compared to the reference peptide 1 (P ≤ 0.01) (Tables 3.2 and 3.3). In spite of the higher kidney uptake of all new peptides at 0.5 h p.i. the activity washout from this organ was very fast, with the exception of compound 5. Thus, the activity in the kidneys at later p.i. times was lower than 1 %ID/g and similar for compounds 1, 2 and 3 (Tables 3.2 and 3.3). 58

Figure 3.3: Tumor to non-tumor ratios of the

99m Tc-labeled

BBS analogues in mice bearing PC-3 tumor xenografts (3.7 MBq/mouse i.v.) (n = 3-13). (A) Tumor-to-blood. (B) Tumor-tokidney. (C) Tumor-to-liver. * (P ≤ 0.05), ** (P ≤ 0.01) vs. reference compound 1 (one-way ANOVA and Dunnett’s post hoc test)

The higher tumor uptake in combination with a lower liver uptake and a faster clearance from blood and normal tissues led to high tumor-to-non tumor ratios for the new analogues especially for compound 3 after 1.5 h (Tumor/Kidney = 2.3 ± 0.8, Tumor/Liver = 6.3 ± 0.6) and 5 h (Tumor/Kidney = 3.5 ± 0.5 , Tumor/Liver = 11.1 ± 1.3) (Figure 3.3). In the blocking experiments, an excess of natural BBS was co-injected with the labeled analogues at 1.5 h p.i. and the uptake in the receptor-positive tissues pancreas, colon and tumor was significantly reduced, thus indicating clearly a specific accumulation of radioactive tracer in these tissues (Tables 3.2 and 3.3). SPECT/CT images were acquired with the compounds 1, 3 and 5 at 1.5 h p.i. The visualization of the tumor xenograft was clearer with 3 than with 1 and 5, which corroborated the results obtained in the biodistribution studies (Figure 3.4). Compound 1 exhibited high uptake in the abdominal cavity due to the high hepatic, pancreatic and intestinal uptakes (Figure 3.4 A). The radioactivity in the abdominal area for compound 5 would be determined by the uptake in the kidneys, pancreas and bowel (Figure 3.4 C). Compound 3 showed lower renal and hepatic uptakes and the abdominal activity would correspond to the uptake in pancreas and intestinal tract (Figure 3.4 B).

59

3.5

Discussion

A variety of BBS analogues has been labeled with different radionuclides to investigate their potential for the detection and therapy of GRP-R-expressing neoplasms (4-6, 11, 13, 14, 22.24, 27). Our work is focused on the development and characterization of new BBS derivatives radiolabeled with 99m Tc using the tricarbonyl labeling technique (18, 19). The amino acid sequence of our peptides is based on the fragment 7-14 of BBS (Figure 3.1), which has been modified as previously published to increase the stability (replacement of Leu13 with Cha, and Met14 with Nle). The additional introduction of a βAla-βAla linker (1) led to improved in vivo tumor uptake (14, 20). However, the accumulation of activity in the liver increased and the tumor-to-non tumor ratios were still rather low. High uptake in the liver and in the kidneys has also been reported for other 99m Tc-labeled BBS analogues (11, 23) and may obscure the detection of lesions in the abdomen. Moreover, high renal uptake could also induce radiation nephrotoxicity. The modification of the polarity can change the pharmacokinetics of biomolecules and have a significant impact on the biodistribution as reported for several radiolabeled peptides (21, 22, 28-30). Therefore, new hydrophilic amino acid linkers were introduced into our BBS analogues to improve the in vivo distribution and to increase the tumorto-background ratios (Figure 3.1). To avoid the introduction of new enzymatic cleavage sites β 3 -amino acids were chosen for the syntheses of these linkers (26). All new peptides showed a higher hydrophilicity compared to our reference peptide 1 (Table 3.1).A single hydroxyl group (β 3 hSer, 2) resulted only in a slight change of polarity. The insertion of β 3 hGlu (3) and β 3 hLys (4) provided the molecule with a single negative and a single positive charge, respectively, and resulted in higher hydrophilicity. As expected, the increase in polarity was even more evident for the analogues 5 and 6, with two and three negative charges. The linkers did not influence the metabolic stability of the peptides. Thus, the new BBS analogues showed high plasma stability in vitro (t1/2 = 16 h) as well as in PC-3 cells (t1/2 = 30-40 min), similar to that of stabilized BBS analogues recently published (21). Besides a high stability, compounds 2 to 5 exhibited high binding affinities for GRP-R, in the range of 125 I-BBS and our previous BBS analogues (Table 3.1) (21), while the affinity of the unlabeled compound 6 was more than 100-fold lower than that of the reference 1. In vitro internalization studies in human prostate carcinoma PC-3 cells reflected an ago-

60

nistic behavior for the new conjugates, which is consistent with data previously reported for BBS constructs with a similar amino acid sequence (Figure 3.3) (11, 21, 23, 29). The internalization of the analogues 1, 2 (neutral) and 3 (one negative charge) was comparable, with a maximum of 30 % of the total dose/mg protein. Since these three BBS analogues showed big differences in the molecular polarity and no differences in both affinity and internalization, polarity does not seem to be crucial for the internalization. However, analogue 4 (one positive charge) showed a slightly higher internalization than 1 to 3, whereas the internalization of analogue 5 (two negative charges) was significantly decreased and that of compound 6 (three negative charges) was negligible. These results would indicate that a positive charge may favor internalization of the tricarbonyl labeled analogues, while more than one negative charge would have an adverse influence, which is consistent with the results Parry et al. reported for the insertion of one and two glutamic acids into the linker of a 64 Cu-labeled DOTA(BBS7-14). The Cu-labeled DOTA-complex already bears a single negative charge and the introduction of the additional charges also led to a loss of binding affinity and internalization (22). This would underline that not only the linker but also the chelating system may affect the properties of biomolecules, which has already been discussed by Decristoforo et al. for 99m Tc-labeled peptides (31). Furthermore, the influence of different polar groups might be specific for each particular labeling approach. The results of the in vitro studies would indicate that the overall molecular charge may have a strong influence on the binding affinity and internalization of radiolabeled BBS analogues. Biodistribution studies were performed in mice bearing PC-3 tumor xenografts to investigate the influence of the charge on the in vivo uptake into human tumor cells. For the analogues 2 (neutral), 3 (one negative charge) and 5 (two negative charges), the hydrophilic character translated into a highly significant reduction of activity in the liver, which was comparable to or even clearly lower than that of other recently published 99m Tc-labeled BBS analogues (11, 21, 23, 32). The increased polarity resulted in higher kidney uptake, especially for analogue 5. The biodistribution of 5 was less favorable since it showed not only high uptake and retention in the kidneys but also lower uptake in receptor-positive tissues, which confirmed the in vitro results. In contrast, compounds 2 and 3 exhibited a faster clearance from kidney which led to the highest tumor-to-kidney ratios reported for 99m Tc-labeled BBS agonists (11, 29). Analogue 3 showed higher tumor-to-liver ratios (Figure 3.3), thus indicating that a single negative charge would be more favorable. Compound 4 (one positive charge) was not included in these experiments because of its unsatisfying in vivo behavior in pre61

Table 3.2: Biodistribution of the

99m Tc-labeled

reference (3.7 MBq/mouse i.v.) in mice with PC-3 xenografts. The animals of the blocked group received a co-injection of 0.1 mg BBS(1-14). Data (mean ± SD) are expressed as percentage of injected dose per gram of tissue. Compound

1 (Reference)

Organs

0.5 h

1.5 h

5h

1.5 h blocked

(n = 3)

(n = 13)

(n = 3)

(n = 3)

Blood

0.58 ± 0.21

0.36 ± 0.66

0.23 ± 0.11

0.47 ± 0.07

Stomach

1.37 ± 0.62

0.73 ± 0.47

0.60 ± 0.33

1.23 ± 0.08

Kidney

1.82 ± 0.51

0.79 ± 0.35

0.39 ± 0.20

0.76 ± 0.19

Liver

5.26 ± 1.26

2.36 ± 0.87

1.83 ± 0.75

2.13 ± 0.36

Small intestines

5.00 ± 0.69

1.83 ± 0.57

0.32 ± 0.19

6.48 ± 2.88

Pancreas

19.00 ± 7.60

6.16 ± 1.80

0.69 ± 0.53

1.19 ± 0.07

Colon

2.65 ± 0.30

4.36 ± 3.60

2.55 ± 1.77

1.11 ± 0.17

Tumor

1.53 ± 0.26

0.80 ± 0.35

0.41 ± 0.27

0.37 ± 0.09

liminary biodistribution studies in normal mice (Table 3.5 in supporting information). This analogue showed high kidney uptake and slow washout. The influence of the charge on renal uptake has been reported for radiolabeled peptides (30, 33, 34). It is known that interaction between positively charged peptides and the negatively charged surface of the proximal tubular cells may increase renal accumulation (33, 34). The positive charge on the Lys side chain of analogue 4 may contribute to the higher and persistent accumulation of radioactivity in the kidneys. High renal uptake may cause radiation nephrotoxicity, the most important dose-limiting factor that reduces the therapeutic index of radiopeptides. Analogue 3 provided a better imaging of the tumor xenografts with a SPECT/CT system than analogues 1 and 5 (Figure 3.4) with an enhanced delineation of the xenografts and decreased radioactivity in the abdominal area. High abdominal accumulation of radioactivity may obscure the detection of tumors and metastases localized in this region, and it is a general observation reported not only for 99m Tc-labeled BBS analogues (11, 14, 29, 35) but also for 64 Cu-labeled DOTA-BBS(7-14) (22), 64 Cu-NOTA-8-Aoc-BBN(714)NH2 (24) or 68 Ga-labeled DOTA-BBS(6-14) (36). Therefore, in order to increase the potential of new radiolabeled BBS analogues in tumor targeting, it is very important to reduce the abdominal uptake. 62

Table 3.3: Biodistribution of the 99m Tc-labeled BBS analogues (3.7 MBq/mouse i.v.) in mice bearing PC-3 xenografts. The animals of the blocked group received a co-injection of 0.1 mg natural BBS. Data (mean ± SD) are expressed as percentage of injected dose per gram of tissue (n = 3-4; * (P ≤ 0.05), ** (P ≤ 0.01) vs. reference compound 1 (one-way ANOVA and Dunnett’s post hoc test); § (P ≤ 0.05), §§ (P ≤ 0.01) vs. unblocked group (unpaired t-test)). Compound

Organs

0.5 h

1.5 h

5h

1.5 h blocked

Blood

1.05 ± 0.27

0.11 ± 0.01

0.04 ± 0.00

0.14 ± 0.03

Kidney

3.51 ± 0.87

0.59 ± 0.10

0.24 ± 0.05

0.66 ± 0.30

1.56 ± 0.19 **

0.35 ± 0.23 **

0.46 ± 0.16 **

1.02 ± 0.36

Stomach

2.29 ± 1.18

0.62 ± 0.04

0.16 ± 0.06

0.39 ± 0.26

Small intestines

5.54 ± 0.72

2.90 ± 1.27

0.50 ± 0.09

3.19 ± 0.29

Colon

4.94 ± 0.24 *

1.74 ± 0.39

1.79 ± 0.74

0.25 ± 0.20

§§

Pancreas

28.37 ± 2.82

10.07 ± 1.82

2.54 ± 0.93

1.12 ± 0.73

§§

Tumor

2.85 ± 0.15

0.97 ± 0.04

0.53 ± 0.09

0.45 ± 0.06

§§

Blood

1.64 ± 0.23

0.25 ± 0.07

0.05 ± 0.00

0.41 ± 0.18

Kidney

7.05 ± 1.69 **

0.90 ± 0.10

0.28 ± 0.03

1.57 ± 1.31

Liver

1.90 ± 0.24 **

0.33 ± 0.12 **

0.09 ± 0.02 **

2.40 ± 0.67

Stomach

2.12 ± 0.14

1.17 ± 0.19

0.30 ± 0.05

0.38 ± 0.31

Small intestines

7.25 ± 0.67

3.49 ± 1.25

0.52 ± 0.08

1.30 ± 0.21

6.71 ± 1.49 **

3.07 ± 0.51

2.96 ± 1.78

0.18 ± 0.03

§§

22.98 ± 6.95

11.01 ± 3.72

3.46 ± 2.20

0.42 ± 0.10

§§

Tumor

3.19 ± 0.68 **

2.11 ± 0.60 **

0.98 ± 0.23 *

0.49 ± 0.29

§§

Blood

2.00 ± 0.39

0.41 ± 0.01

0.19 ± 0.04

0.60 ± 0.23

Kidney

7.37 ± 0.75 **

3.53 ± 0.38 **

2.17 ± 0.27 **

3.99 ± 0.96

Liver

1.16 ± 0.11 **

0.61 ± 0.06 **

0.47 ± 0.02 **

0.54 ± 0.22

Stomach

1.23 ± 1.06

0.69 ± 0.09

0.67 ± 0.28

0.81 ± 0.11

Small intestines

2.28 ± 0.48

0.83 ± 0.15

0.35 ± 0.08

1.03 ± 0.34

Colon

2.38 ± 0.38

1.30 ± 0.15

0.68 ± 0.13 *

0.30 ± 0.12

§§

Pancreas

9.05 ± 1.89

2.09 ± 1.08

2.33 ± 0.22

0.33 ± 0.10

§§

Tumor

2.29 ± 0.80

0.76 ± 0.33

0.48 ± 0.22

Liver

2

3

Colon Pancreas

4

63

0.55 ± 0.27

Figure 3.4: SPECT/CT image of nude mice bearing PC-3 tumor xenografts at 1.5 h p.i. of 20 MBq of the 99m Tc-labeled BBS analogue. (A) Three dimensional image (left) and coronal slice (right) of compound 1 (neutral). (B) Three dimensional image (left) and coronal slice (right) of compound 3 (one negative charge). (C) Three dimensional image (left) and coronal slice (right) of compound 5 (two negative charges). T = tumor; K = kidney; L = liver; I = intestinal activity, including pancreas.

In conclusion, we evaluated five new BBS analogues for their usefulness in imaging GRP-R-expressing neoplasms and could confirm the great impact of the polarity and charge on their in vivo behavior. Different linkers, consisting of polar β 3 homo-amino acids, were introduced between the binding sequence and the retro(Nα His)Ac-chelator. A positive charge in the linker resulted in higher uptake and retention in kidney and liver. A hydroxyl group and especially a single negative charge in form of a β-homo glutamic acid considerably ameliorated the biodistribution profile, with higher tumor uptake and retention, and significantly improved tumor-to-background ratios. However, additional negative charges led to a loss of binding affinity and internalization, and unfavorable biodistribution. The introduction of polar groups seems to be of great interest to improve the in vivo properties of 99m Tcor 186/188 Re-labeled BBS analogues and increase their potential for imaging and therapy of GRP-R-expressing tumors.

64

3.6

Acknowledgements

The authors thank Ms. Olga Gasser, Dr. Alexander Hohn and Mr. Alain Blanc for their technical assistance. This work was funded by an Oncosuisse grant (Nr. OCS 01311-02-2003) and by the Fund for Scientific Research-Flanders, Belgium, (grant Nr. G.0036.04).

65

3.7

Supporting Information

Supporting Information Available: Additional experimental data available via the Internet at http://pubs.acs.org. Table 3.4: Analytical data of the unlabeled BBS analogues.(1 Gradient: 3 % CH3 CN to 100 % CH3 CN in 20 min (flow 1 mL/min)) Molecular Peptide

Yield of

formula

peptide

MW calcd m/z

ES-MS [M+H+ ]

HPLC

[M+2H+ ]/2

UV-trace

synthesis

tR [min]

1

C61 H90 N18 O14

55 %

1298.7

1299.5

650.5

12.01

2

C65 H96 N18 O17

50 %

1399.7

1401.0

701.0

14.4

3

C67 H98 N18 O18

53 %

1441.7

1443.0

722.0

14.5

4

C68 H103 N19 O16

58 %

1440.8

1442.0

722.0

14.2

5

C70 H103 N19 O19

55 %

1513.8

1514.2

758.1

10.8

6

C73 H107 N19 O21

56 %

1585.8

1586.9

793.9

10.8

Table 3.5: Biodistribution of

99m Tc-labeled

compound 4 (3 MBq/mouse i.v.) in BALB/c mice. The animals of the blocked group received a co-injection of 0.1 mg natural BBS. Data (mean ± SD) are expressed as percentage of injected dose per gram of tissue (n = 3; * (P ≤ 0.01), ** (P ≤ 0.001) vs. unblocked group (unpaired t-test). BBS

4

Organs

Blood Muscle Kidney Liver Stomach Small intestines Colon Pancreas

0.5 h

1.5 h

5h

0.5 h blocked

3.64 ± 1.23

0.22 ± 0.02

0.10 ± 0.02

3.7 ± 0.62

0.86 ± 0.18

0.15 ± 0.10

0.06 ± 0.03

0.78 ± 0.07

12.88 ± 2.84

7.08 ± 0.51

5.53 ± 1.60

11.93 ± 1.82

10.66 ± 0.43

8.41 ± 0.29

4.82 ± 1.01

13.05 ± 1.63

3.21 ± 0.48

1.22 ± 0.11

0.69 ± 0.11

1.88 ± 0.26

6.54 ± 0.27

5.41 ± 1.46

1.29 ± 0.08

5.65 ± 1.28

4.82 ± 0.55

4.01 ± 0.96

4.67 ± 1.07

1.92 ± 0.28 *

24.21 ± 4.45

10.45 ± 8.63

5.44 ± 0.99

1.42 ± 0.12 **

66

3.8

References

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11. Nock, B. A., Nikolopoulou, A., Galanis, A., Cordopatis, P., Waser, B., Reubi, J. C., and Maina, T. (2005) Potent Bombesin-like Peptides for GRP-Receptor Targeting of Tumors with 99m Tc: A Preclinical Study. J. Med. Chem. 48, 100-110. 12. Zhang, H., Chen, J., Waldherr, C., Hinni, K., Waser, B., Reubi, J. C., and Maecke, H. R. (2004) Synthesis and Evaluation of Bombesin Derivatives on the Basis of PanBombesin Peptides Labeled with Indium-111, Lutetium-177, and Yttrium-90 for Targeting Bombesin Receptor-Expressing Tumors. Cancer Res. 64, 6707-6715. 13. Smith, C. J., Gali, H., Sieckman, G. L., Hayes, D. L., Owen, N. K., Mazuru, D. G., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical investigations of 177 Lu-DOTA-8-Aoc-BBN[7-14]NH2 : an in vitro/in vivo assessment of the targeting ability of this new radiopharmaceutical for PC-3 human prostate cancer cells. Nucl. Med. Biol. 30, 101-109. 14. Smith, C. J., Gali, H., Sieckman, G. L., Higginbotham, C., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical Investigations of 99m Tc-N3 S-X-BBN[714]NH2 : An in Vitro/in Vivo Structure-Activity Relationship Study Where X = 0-, 3-, 5-, 8-, and 11-Carbon Tethering Moieties. Bioconjugate Chem. 14, 93-102. 15. Bauer, R., and Pabst, H. W. (1982) Tc-Genrators - Yield of ’Inactive’ 99 Tc. Eur. J. Nucl. Med. 7, 35-36.

99m

Tc and Ratio to

16. Garcia-Garayoa, E., Schibli, R., and Schubiger, P. A. (2007) Peptides radiolabeled with Re-186/188 and Tc-99m as potential diagnostic and therapeutic agents. Nucl. Sci. Tech. 18, 88-100. 17. Liu, S., and Edwards, D. S. (1999) 99m Tc-Labeled Small Peptides as Diagnostic Radiopharmaceuticals. Chem. Rev. 99, 2235-2268. 18. Schibli, R., La Bella, R., Alberto, R., Garcia-Garayoa, E., Ortner, K., Abram, U., and Schubiger, P. A. (2000) Influence of the Denticity of Ligand Systems on the in Vitro and in Vivo Behavior of 99m Tc(I)-Tricarbonyl Complexes: A Hint for the Future Functionalization of Biomolecules. Bioconjugate Chem. 11, 345-351. 19. La Bella, R. L., Garcia-Garayoa, E., Bahler, M. B., Blauenstein, P., Schibli, R., Conrath, P., Tourwe, D., and Schubiger, P. A. (2002) A 99m Tc(I)-Postlabeled High

68

Affinity Bombesin Analogue as a Potential Tumor Imaging Agent. Bioconjugate Chem. 13, 599-604. 20. Garcia Garayoa, E., Ruegg, D., Blauenstein, P., Zwimpfer, M., Khan, I. U., Maes, V., Blanc, A., Beck-Sickinger, A. G., Tourwe, D. A., and Schubiger, P. A. (2007) Chemical and biological characterization of new Re(CO)3 /[99m Tc](CO)3 bombesin analogues. Nucl. Med. Biol. 34, 17-28. 21. Garcia Garayoa, E., Schweinsberg, C., Maes, V., Ruegg, D., Blanc, A., Blauenstein, P., Tourwe, D. A., Beck-Sickinger, A. G., and Schubiger, P. A. (2007) New [Tc-99m]bombesin analogues with improved biodistribution for targeting gastrin releasing-peptide receptor-positive tumors. Q. J. Nucl. Med. Mol. Imag. 51, 42-50. 22. Parry, J. J., Kelly, T. S., Andrews, R., and Rogers, B. E. (2007) In Vitro and in Vivo Evaluation of 64 Cu-Labeled DOTA-Linker-Bombesin(7-14) Analogues Containing Different Amino Acid Linker Moieties. Bioconjugate Chem. 18, 1110-1117. 23. Kunstler, J. U., Veerendra, B., Figueroa, S. D., Sieckman, G. L., Rold, T. L., Hoffman, T. J., Smith, C. J., and Pietzsch, H. J. (2007) Organometallic 99m Tc(III) ’4 + 1’ Bombesin(7-14) Conjugates: Synthesis, Radiolabeling, and in Vitro/in Vivo Studies. Bioconjugate Chem. 18, 1651-1661. 24. Prasanphanich, A. F., Nanda, P. K., Rold, T. L., Ma, L., Lewis, M. R., Garrison, J. C., Hoffman, T. J., Sieckman, G. L., Figueroa, S. D., and Smith, C. J. (2007) [64 CuNOTA-8-Aoc-BBN(7-14)NH2 ] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues. Proc. Natl. Acad. Sci. U S A 104, 12462-12467. 25. Abele, S., and Seebach, D. (2000) Preparation of achiral and of enantiopure geminally disubstituted beta-amino acids for beta-peptide synthesis. Eur. J. Org. Chem., 1-15. 26. Seebach, D., Beck, A. K., and Bierbaum, D. J. (2004) The World of beta- and gamma-Peptides Comprised of Homologated Proteinogenic Amino Acids and Other Components. Chem Biodivers. 1, 1111-1239.

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27. Smith, C. J., Volkert, W. A., and Hoffman, T. J. (2003) Gastrin releasing peptide (GRP) receptor targeted radiopharmaceuticals: A concise update. Nucl. Med. Biol. 30, 861-868. 28. Schottelius, M., Wester, H. J., Reubi, J. C., Senekowitsch-Schmidtke, R., and Schwaiger, M. (2002) Improvement of Pharmacokinetics of Radioiodinated Tyr3 Octreotide by Conjugation with Carbohydrates. Bioconjugate Chem. 13, 10211030. 29. Alves, S., Correia, J. D. G., Santos, I., Veerendra, B., Sieckman, G. L., Hoffman, T. J., Rold, T. L., Figueroa, S. D., Retzloff, L., McCrate, J., Prasanphanich, A., and Smith, C. J. (2006) Pyrazolyl conjugates of bombesin: a new tridentate ligand framework for the stabilization of fac-[M(CO)3 ]+ moiety. Nucl. Med. Biol. 33, 625-634. 30. Christensen, E. I., Rennke, H. G., and Carone, F. A. (1983) Renal tubular uptake of protein: effect of molecular charge. Am J Physiol Renal Physiol 244, F436-441. 31. Decristoforo, C., and Mather, S. J. (2002) The influence of chelator on the pharmacokinetics of Tc-99m-labelled peptides. Q. J. Nucl. Med. Mol. Imag. 46, 195-205. 32. Rogers, B. E., Bigott, H. M., McCarthy, D. W., Manna, D. D., Kim, J., Sharp, T. L., and Welch, M. J. (2003) MicroPET Imaging of a Gastrin-Releasing Peptide Receptor-Positive Tumor in a Mouse Model of Human Prostate Cancer Using a 64 Cu-Labeled Bombesin Analogue. Bioconjugate Chem. 14, 756-763. 33. Antunes, P., Ginj, M., Walter, M. A., Chen, J., Reubi, J. C., and Maecke, H. R. (2007) Influence of Different Spacers on the Biological Profile of a DOTASomatostatin Analogue. Bioconjugate Chem. 18, 84-92. 34. Akizawa, H., Arano, Y., Mifune, M., Iwado, A., Saito, Y., Mukai, T., Uehara, T., Ono, M., Fujioka, Y., Ogawa, K., Kiso, Y., and Saji, H. (2001) Effect of molecular charges on renal uptake of 111In-DTPA-conjugated peptides. Nucl Med Biol 28, 761-768.

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35. Reubi, J. C., Wenger, S., Schmuckli-Maurer, J., Schaer, J.-C., and Gugger, M. (2002) Bombesin Receptor Subtypes in Human Cancers: Detection with the Universal Radioligand 125 I-[D-TYR6 , β-ALA11 , PHE13 , NLE14 ] Bombesin(6-14). Clin. Cancer Res. 8, 1139-1146. 36. Schuhmacher, J., Zhang, H., Doll, J., Macke, H. R., Matys, R., Hauser, H., Henze, M., Haberkorn, U., and Eisenhut, M. (2005) GRP Receptor-Targeted PET of a Rat Pancreas Carcinoma Xenograft in Nude Mice with a 68 Ga-Labeled Bombesin(6-14) Analog. J Nucl Med 46, 691-699.

71

72

Chapter 4 Novel Glycated [99mTc(CO)3]-labeled Bombesin Analogues for Improved Targeting of Gastrin-Releasing Peptide Receptor-Positive Tumors

Christian Schweinsberg1 , Veronique Maes2 , Luc Brans2 , Peter Bläuenstein1 , Dirk A. Tourwé2 , P. August Schubiger1,3 , R. Schibli1,3 , Elisa Garc´ıa Garayoa1

1

Paul Scherrer Institute, Center for Radiopharmaceutical Science, CH-5232 Villigen PSI, Switzerland 2 Department of Organic Chemistry, Vrije Universiteit Brussel, 1050 Brussels, Belgium 3 Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland

Bioconjugate Chemistry (accepted)

73

4.1

Abstract

Radiolabeled bombesin (BBS) analogues are promising pharmaceuticals for imaging of cancer cells expressing gastrin-releasing peptide receptors (GRPR). However, most of the radiolabeled BBS derivatives show a high accumulation of activity in the liver and a strong hepatobiliary excretion, both unfavorable for imaging and therapy of abdominal lesions. For this reason we introduced hydrophilic carbohydrated linker moieties into our BBS analogues to reduce the abdominal accumulation and to improve the tumor-tobackground ratios. A stabilized BBS(7-14) sequence bearing the (Nα His)Ac-chelator was modified with amino acid linkers containing a lysine or propargylglycine residue. The -amino group of a lysine was either coupled to shikimic acid, or reacted with glucose to form the Amadori conjugate. Alternatively a glucose was attached to the peptide via ’click’ chemistry with the propargylglycine side chain. The peptides were synthesized on Rink-amide-resin using solid-phase peptide synthesis and labeled with 99m Tc using the tricarbonyl technique Binding and degradation were tested in vitro in GRPR-expressing PC-3 cells. Biodistribution and SPECT/CT imaging studies were performed in nude mice bearing PC-3 tumor xenografts. The new peptides showed a LogD between -0.2 and -0.5 and kept the high affinity for GRPR with Kd values ≤< 0.5 nM. In vitro they were rapidly internalized into the tumor cells and showed an increased cellular retention and stability (t1/2 ¿ 35 min). In vivo all new compounds exhibited higher tumor-tobackground ratios compared to the non-glycated reference. Thus, the best results were obtained with the triazole coupled glucose with a 4-fold increased uptake and retention in tumor tissue (3.6 and 2.5 %ID/g at 1.5h and 5h p.i, respectively) and a significantly reduced accumulation in the liver (0.6 vs. 2.4 %ID/g, 1.5h p.i., respectively). Apart from higher tumor-to-liver ratios (17-fold, 1.5h p.i.), both tumor-to-kidney and tumorto-blood ratios could be significantly improved by a factor of 1.5 and 2.7, respectively (1.5h, p.i., P ≤ 0.05). The imaging studies proved the reduction of abdominal background and tumor xenografts could clearly be visualized. In conclusion, the introduction of a carbohydrated linker substantially improved the biodistribution properties of BBS analogues labeled with the 99m Tc-tricarbonyl core. KEY WORDS: carbohydration, bombesin, gastrin-releasing-peptide receptor, linker, technetium-99m, tumor uptake

74

4.2

Introduction

Modified neuropeptides have gained an increasing attention for radiopharmaceutical applications like peptide receptor imaging (PRI) and peptide receptor radiotherapy (PRRT) (1-5). They bind with high affinity and selectivity to their corresponding receptors, which are over-expressed on a considerable number of cancers (1, 5, 6). Apart from somatostatin (sst) analogues like 111 In-DTPA-Octreotide (OctreoScan) or 90 Y- and 177 LuDOTA-TOC, which already are successful in the clinical treatment of neuroendocrine tumors and gastrinomas (7-10), additional receptor ligands are under investigation for targeting a broader range of tumor types. The tetradecapeptide bombesin (BBS) has recently been studied intensively due to its high affinity for the mammalian gastrin-releasing peptide receptor (GRPR) (11). GRPR shows a high expression in most human prostate and breast tumors as well as small-cell lung, ovarian, endometrial and other cancers (12-14). BBS derivatives have been labeled with various radionuclides like 99m Tc, 64 Cu, 68 Ga, 111 In, 177 Lu or 18 F and proved to be successful candidates for both PRI and PRRT (15-21). However, the modification of their pharmacokinetic profile is of high interest to reduce their frequent high hepatic accumulation and hepatobiliary excretion which is problematic for imaging and therapy of abdominal lesions (15, 16, 18, 20-23). Different modifiers offer the possibility to influence these unfavorable in vivo properties. Smith et al investigated the positive effects of aliphatic and amino acid linkers for BBS analogues (18, 24, 25). Moreover, glycation by the Amadori reaction has been reported to provide radiohalogenated sst analogues with excellent physicochemical characteristics (26-28). These compounds were more hydrophilic, but still exhibited a high receptor binding affinity, improved tumor uptake and tumor-to-non-tumor ratios, in addition to a reduced hepatobiliary transfer and liver accumulation (26, 28-31). Recent studies with 111 In- and 177 Lu-labeled DOTA-sst analogues confirmed the positive effects of glycation via Amadori rearrangement for radiolabeled metallopeptides (32) and, interestingly, also the modification of neurotensin (NT) with the glycomimetic 3,4,5,trihydroxyl-1-cyclohexene-1-carboxylic acid (shikimic acid) induced a reduction of both, hepatic uptake and renal retention (33). Our group could already show an improvement of 99m Tc-labeled BBS analogues by the introduction of a double β-Alanine sequence and recently published first promising results of a linker coupled to shikimic acid (34). Surprisingly, so far no further data of glycated BBS analogues have been published.

75

In this study we investigated for the first time the influence of glycation on the biodistribution profile of radiolabeled BBS derivatives. We inserted carbohydrated linker moieties between the stabilized binding sequence BBS(7-14) and the Nα Histidin-chelator by the application of established methods like the Amadori reaction, by the formation of a stable triazole bond via ’click’ chemistry and by coupling of shikimic acid to a lysine residue (33, 35, 36). The peptides were labeled with 99m Tc (t1/2 = 6 h, 140 keV γ photon energy, 89% abundance) featuring a favorable dosimetry, high specific activity, low cost and ready availability from a 99 Mo/99m Tc-generator, and radiochemical properties optimal for single photon emission tomography (SPECT) (37). Moreover, the chemical similarities of the “mateched pair” 99m Tc/188 Re offer the opportunity to use successful 99m Tc-labeled candidates for tumor targeting also for therapeutic applications labeled with 188 Re (t1/2 = 17 h; 2.12 MeV β- max-radiation) (37). The new compounds were tested in vitro for binding affinity, internalization, efflux and metabolic stability in the GRPR-expressing human prostate carcinoma cell line PC-3. In vivo biodistribution and SPECT/CT studies were performed in nude mice bearing PC-3 tumor xenografts.

4.3


Chemicals and Equipment All chemicals were obtained from Fluka (Buchs, Switzerland) or Merck (Dietkon, Switzerland). Cell culture media DMEM GLUTAMAXT M -I, trypsin/EDTA, bovine serum albumin and soybean trypsin inhibitor were obtained from Invitrogen (Basel, Switzerland), fetal calf serum (FCS) and antibiotic/antimycotic solution were obtained from Bioconcept (Allschwil, Switzerland). Bacitracin, HEPES and Chymostatin were purchased from SIGMA (Buchs, Switzerland). For the determination of protein concentrations a Pierce BCA Protein Assay kit from Socochim (Lausanne, Switzerland) was used. Na[99m TcO4 ] was eluted from a Mallinckrodt 99 Mo/99m Tc generator (Tyco Healthcare, Petten, The Netherlands) using 0.9 % saline. Sodium boranocarbonate was a gift from the provider of the generator. The radioactivity of in vitro and in vivo experiments was measured with a NaI-γ-counter (Packard Canberra Cobra II, Meriden, USA) or a NaI ionization chamber.

76

Table 4.1: Sequence of the BBS analogues with new carbohydrated groups in the linker. (Lys(sha) = Lysine-coupled shikimic acid; Lys(Amd) = Amadori-Product; Pra(glu) = triazole-coupled glucose)

Peptide

Chelator

Linker

Binding Sequence

1 (Reference)

(Nα His)Ac

βAla-βAla

-Gln-Trp-Ala-Val-Gly-Cha-Nle-Met-NH2

α

2

(N His)Ac

Lys(sha)-βAla-βAla


3

(Nα His)Ac

Lys(Amd)-βAla-βAla


4

(Nα His)Ac

Pra(glu)-βAla-βAla


Peptide Synthesis and radiolabeling with

99m

Tc

The peptide solid-phase synthesis was carried out on a Rink Amide Resin with the Fmoc-strategy as previously described (38). The introduction of different carbohydrates (Figure 4.1) in form of glucose or shikimic acid was performed as recently published (33, 35, 36). The purification of the peptides was done on a semipreparative HPLC system (Gilson) on a reverse phase C18 column (DiscoveryBIO SULPELCO Wide Pore, 25 cm x 2.21 cm, 5 µm) with a linear gradient of 3 % B to 100 % B (solvent A = H2 O + 0.1 % TFA as and as solvent B = CH3 CN + 0.1 % TFA) in 30 min, a flow rate of 1 mL/min and UV detection at 215 nm. Radiolabeling of the peptides with [99m Tc(OH2 )3 (CO)3 ]+ was performed as previously described (22). For analyses and purification of the labeled peptides from the unlabeled compounds a Macherey-Nagel CC Nucleosil 100-5 C18 RP column (5µm, 250 x 4.6 mm) was used. Preparative HPLC for peptide purification was performed using a Merck-Hitachi L-7100-system with an equipped 2-channel scintillation detector interface (John Caunt scientific Ltd, Eynsham, England) and a Rainin Dynamax absorbance detector model UV-1. HPLC solvents consisted of H2 O + 0.1 % TFA (solvent A) and CH3 OH + 0.1 % TFA (solvent B). The separation was performed with a linear gradient 50 % A / 50 % B to 20 % A / 80 % B in 25 min (1.0 mL/min). For the analytical HPLC of the labeled peptides a Varian ProStar system was used with a Varian ProStar 230 Solvent Delivery-Pump, a Varian Photodiode Array Detector Model 330, a Varian ProStar Autosampler Model 410 and a radiomatic Flo-one Beta Detector (Packard Canberra). HPLC solvents consisted of CH3 CN + 0.1 % TFA (solvent A) and H2 O + 0.1 % TFA (solvent B). The labeling

77

Figure 4.1: Chemical structure of the carbohydrated groups introduced into the linker sequence of the BBS analogues. 2 = Lysine-coupled shikimic acid; 3 = Amadori-product; 4 = triazolecoupled glucose

control was performed with a linear gradient of 30 % A / 70 % B to 80 % A / 20 % B in 25 min (1.0 mL/min). Octanol/water partition coefficient study The octanol/water partition coefficients were determined at pH = 7.4. Five µL containing 500 kBq of the radiolabeled compound in PBS were added to a vial containing 1.2 ml 1-octanol and PBS (1:1). After vortexing for 1 min the vial was centrifuged for 5 min at 10000 rpm to ensure complete separation of layers. Then 40 µL of each layer were taken in a preweighed vial and measured in the γ-counter. Counts per unit weight of sample were calculated, and logD values were calculated as described by Pillarsetty et al (39) using the formula in 1 g of octanol Log10 D = log10 ( counts ) counts in 1 g of P BS

Cell culture PC-3 human prostate cancer cell line was obtained from European Collection of Cell Cultures (Salisbury, United Kingdom). Cells were maintained in Dulbecco’s MEM with GLUTAMAXT M -I supplemented with 10 % FCS, 100 IU/mL penicillin G sodium, 100 78

µg/mL streptomycin sulphate, 0.25 µg/mL amphotericin B. Cells were incubated at 37°C in an atmosphere containing 5 % CO2, and subcultured twice a week after detaching with trypsin-EDTA. Metabolic stability in human plasma and in PC-3 cells The stability in cell culture was evaluated with 2 x 106 cells in PBS. The analogues (3 MBq/mL, 1.5-2 pmol/mL) were incubated for 0.5, 1, 2.5 and 5 h at 37°C. Afterwards, proteins were precipitated as follows: 250 µL of sample + 750 µL of CH3 CN /ethanol (1:1) and TFA (0.01 %). The mixture was vortexed and centrifuged (10 min, 20000g) at 4°C. The supernatant was filtered and analyzed by RP-HPLC equipped with a radioactivity detector. All experiments were carried out 2-3 times. Receptor inhibition studies One day prior to the assay PC-3 cells at confluence were placed in 48-well plates. Cells were incubated in a special binding buffer (50 mM HEPES, 125 mM NaCl, 7.5 mM KCl, 5.5 mM MgCl*6H2 O, 1 mM EGTA, 5g/L BSA, 2 mg/L Chymostatin, 100 mg/L Soybean Trypsin inhibitor, 50 mg/L Bacitracin, pH 7.4) with increasing concentrations of the BBS analogues (0-30000 nM) in presence of 4 kBq of 99m Tc-BBS(7-14). After 1 h incubation at 37°C the cells were washed twice with cold PBS. Bound radioactivity was recovered by solubilizing the cells in 1 N NaOH (2 x 400 µL). The radioactivity was measured in the γ-counter. All experiments were carried out 2-3 times in triplicate. Receptor saturation studies One day prior to the assay PC-3 cells at confluence were placed in 48-well plates and incubated for 1 h at 37°C in the binding buffer described above with increasing concentrations of the labeled BBS analogues 2 - 4 (0.01-2 nM). The concentration of total technetium-labeled peptide was calculated according to Bauer et al. (40). Non-specific binding was determined by co-incubation with 1 µM BBS(1-14). After incubation the cells were washed twice with cold PBS, solubilized in 1 N NaOH (2 x 400 µL) and the radioactivity was measured in the γ-counter. Experiments were performed 2-3 times in triplicate.

79

Internalization and Externalization One day prior to the assay PC-3 cells at confluence were placed in 6 well plates. For internalization studies cells were incubated with the labeled analogues (4 kBq/well) in binding buffer for 5, 15, 30, 60 and 120 min at 37°C to allow binding and internalization. Non-specific binding was determined in the presence of 1 µM BBS(1-14). After incubation cells were washed twice with cold PBS. Surface bound activity was removed by acid wash (50 mM glycine/HCL, 100 mM NaCl, pH 2.8, 2 x 600 µL for 5 min at room temperature). Afterwards the plates were neutralized with 600 µL PBS and cells were lysed with 1 N NaOH (2 x 600 µL). Surface bound and internalized activities were measured in the γ-counter. The protein concentration was determined and results were calculated as percentage of total added radioactivity per mg protein. For externalization studies PC-3 cells were incubated with the labeled analogues (20 kBq/well) in culture medium for 1 h at 37°C to allow binding and maximal internalization. After incubation the supernatant was discarded and each well washed twice with cold PBS. Afterwards the cells were incubated again at 37°C in 2 mL culture medium for 30, 60, 150 or 300 min. At each time point the supernatant was collected, the cells were washed twice with cold PBS and the radioactivity remaining in the cells was recovered by lysing the cells with 1 N NaOH (2 x 600 µL). Radioactivity in the supernatant (released activity) and in the lysate (internalized activity) was determined in the γ-counter. Results are expressed as percentage of the total activity associated with the cells. The protein concentration was determined and results were calculated as percentage of total added radioactivity per mg protein. All experiments were carried out 2-3 times in triplicate. Biodistribution studies All animal experiments were conducted in compliance with the Swiss animal protection laws and with the ethical principles and guidelines for scientific animal trials established by the Swiss Academy of Medical Sciences and the Swiss Academy of Natural Sciences. Female CD-1 nu/nu mice 6-8 week-old (Charles River Laboratories, Sulzfeld, Germany) were subcutaneously injected with 8 x 106 PC-3 cells in 150 µL culture medium without supplements. About 3 weeks after tumor implantation the mice (3-6 per group) were injected i.v. with 3.7 MBq radiolabeled peptides. At 0.5 h, 1.5 h or 5 h post-injection (p.i.) the animals were killed by cervical dislocation. The different organs (heart, lung, 80

spleen, kidneys, pancreas, stomach, small intestine, large intestine, liver, muscle, bone) and blood were collected, weighed and the radioactivity was measured in a γ-counter. Results are presented as percentage of injected dose per gram of tissue (%ID/g). To determine the specificity of the in vivo uptake one group of mice received a co-injection of 100 µg of unlabeled BBS(1-14) and the radiolabeled analogue and sacrificed at 1.5 h p.i.. SPECT/CT studies Single photon emission computed tomography / X-ray computed tomography (SPECT/ CT) images were performed post-mortem 1.5 h after i.v. injection of the 99m Tc-BBS analogues (20 MBq). Images were obtained on an X-SPECTT M -system (Gamma Medica Inc.) equipped with a single head SPECT device and a CT-device. SPECT data were acquired and reconstructed with LumaGEM (version 5.407). CT data were reconstructed with the software Cobra (version 4.5.1). Fusion of SPECT- and CT-data was performed with IDL Virtual MachineT M (version 6.0). Images were generated with AmiraT M (version 3.1.1). Statistical Analysis All data are presented as the mean ± SD. Differences in the in vivo uptake in the biodostribution studies were analyzed by one-way ANOVA followed by Dunnett’s comparison test. Differences between blocked and control groups were analyzed by unpaired t-test. P = 0.05 was considered statistically significant.

4.4

Results and Discussion

The modification of the biodistribution profiles of BBS analogues towards a reduction of abdominal accumulation is of high interest for both diagnostic and therapeutical applications. In this study we investigated the influence of different glycated linkers on the in vivo behavior of 99m Tc(CO)3 -labeled BBS derivatives. The amino acid sequence of the new peptides was based on the fragment 7-14 of BBS , which had been successfully modified as previously published to increase the metabolic stability in human plasma and PC-3 tumor cells (replacement of Leu13 with Cha, and Met14 with Nle) (34). The additional introduction of a βAla-βAla linker (compound 1) led to improved in vivo 81

Table 4.2: Characteristics of the new BBS analogues: retention time and polarity (LogD) of the 99m Tc-labeled peptides, and binding affinity for GRP-receptors in PC-3 cells of the labeled (Kd values) and unlabeled (IC50 values) compounds (n = 2-4). HPLC Compound

γ-trace

t1/2 in LogD

Kd [nM]

IC50 [nM]

tR [min]

PC-3 cells [min]

1 (Reference)

16.8

0.9 ± 0.2

0.19 ± 0.12

5.1 ± 1.7

30 ± 06

2

16.4

-0.2 ± 0.1

0.02 ± 0.01

6.5 ± 1.7

35 ± 11

3

16.7

-0.5 ± 0.1

0.18 ± 0.03

3.7 ± 1.7

37 ± 08

4

16.8

-0.5 ± 0.1

0.44 ± 0.38

3.2 ± 1.2

38 ± 05

tumor uptake (18, 38). However, the accumulation of activity in the liver was increased and the tumor-to-tissue ratios were still rather poor (tumor-to-blood = 4.3 ± 3.4; tumorto-kidney = 0.9 ± 0.3; tumor-to-liver = 0.4 ± 0.3; at 1.5 h p.i.). A significant impact of polar linker moieties on the biodistribution was reported for several radiolabeled peptides and especially carbohydrated compounds exhibited clearly improved pharmacokinetics (21, 26, 41-43). Therefore we set out to synthesize three glycated BBS analogues (2, 3 and 4) for comparison with the unglycated 99m Tc(CO)3 -labeled compound 1, which has recently been characterized by our group (22) (Table 4.1). Compound 2 was modified with a linker bearing a Lysine coupled to the glycomimetic shikimic acid at the -amino group, as recently published (33). Compound 3 was glycated by the ”Amadori rearrangement”, the Schiffbase reaction of an aldose with a free amino group, known to form extremely stable carbohydrated peptide derivatives (36, 44). Finally we investigated a clicked, carbohydrated BBS(7-14) analogue by the formation of a stable triazole bond between an alkyne in the linker and an azido glucose derivative (35) (Figure 4.1). Radiolabeling of these novel carbohydrated peptides with [99m Tc(OH2 )3 (CO)3 ]+ was performed at ligand concentrations of 10−5 M for 1 h at 75 °C. The HPLC traces of the 99m Tc(CO)3 -complexes showed generally a shift to longer retention times compared to the unlabeled peptides ( 14.5 min) and the precursor [99m Tc(OH2 )3 (CO)3 ]+ ( 9 min). Radiolabeling yields were higher than 95 % for all derivatives and the active complexes 2 - 4 were successfully separated from the unlabeled peptides to avoid a saturation of 82

the GRPR in subsequent in vitro/in vivo experiments. To compare the polarity of the [99m Tc(CO)3 ]-labeled new analogues 2 - 4 the octanol/water partition coefficients were determined. The reference compound 1 bearing only the βAlaβAla-linker showed a rather lipophilic character with a LogD of +0.9. The introduction of the shikimic acid changed the polarity of peptide 2 to -0.2, while the coupling to glucose resulted in LogD values of -0.5 for both analogues 3 and 4 (Table 4.2). In vitro Studies The in vitro experiments were performed with PC-3 cells, which express a high level of GRPR. In order to investigate the potential of our three novel carbohydrated peptides 2 - 4 for specific targeting of GRPR, studies on receptor binding (Table 4.2) and cell internalization/cellular efflux (Figure 4.2) were performed. Additionally, the metabolic stability in tumor cells was investigated. We already published an increased stability in PC-3 cells for compound 1 compared to the unmodified sequence (30 ± 6 min vs. 5 ± 2 min, respectively) (34). The t1/2 of the novel peptides 2 (35 ± 11 min), 3 (37 ± 8 min) and 4 (38 ± 5 min) was slightly higher than for the reference 1, but the differences were not statistically significant (Table 4.2).

Figure 4.2: Time-course internalization (A) and externalization after maximal internalization (B) of the 99m Tc-labeled BBS analogues in PC-3 cells after incubation at 37°C. Data represent the percentage of internalized activity per mg protein related to the total activity added to the cells (n = 2-3).

83

The affinity of the unlabeled analogues for GRPR was tested in inhibition experiments in competition with 4 kBq of 99m Tc-BBS(7-14), which is known to express a high affinity for this receptor (45). All peptides exhibited a similar high affinity with IC50 values between 3 and 7 nM (Table 4.2). Furthermore, the receptor binding of the new analogues proved to be saturable and highly specific after radiolabeling with the 99m Tc(CO)3 -core, with Kd values lower than 0.5 nM (Table 4.2). Interestingly, peptide 2 showed the highest affinity (Kd = 0.02 ± 0.01 nM), about ten times higher than the reference 1 (Kd = 0.19 ± 0.12 nM) and the new peptides 3 (Kd = 0.18 ± 0.03 nM) and 4 (Kd = 0.44 ± 0.38 nM, respectively). The internalization experiments showed a similar and rapid uptake into PC-3 cells reaching a plateau between 60 and 120 min (Figure refglycfig2). We found 29 - 43 % of the total radioactivity per mg protein in the cell lysate after stripping the surface bound activity off the cells with acidic buffer (pH 2.8). These results would reflect an agonistic behavior for the new compounds and are consistent with recently published BBS analogues with similar amino acid sequences (15, 20, 41, 42). The almost complete blockade (≤ 1 % of the added radioactivity per mg protein) by co-incubation with an excess of BBS(1-14) demonstrated the high specificity for all new glycated BBS radiotracers. Variations in the bound radioactivity among the new peptides were statistically not significant. Additionally, efflux studies revealed a similar intracellular retention of the internalized activity of 35 % up to 5 h for all new peptides 2 - 4 and the reference compound 1 (Figure refglycfig2). These results are in agreement with the similar metabolic stability of the new peptides we found in the degradation experiments with PC-3 cells (Table 4.2). Biodistribution Time-dependent biodistribution studies with the new radiotracers 2 - 4 have been performed in athymic mice, bearing PC-3 tumor xenografts (Table 4.4). The new peptides 2 and 4 exhibited higher blood levels at 0.5 h p.i. (2.1 ± 0.7 %ID/g and 1.8 ± 0.2 %ID/g, respectively) than the reference 1 (0.6 ± 0.2 %ID/g). However, the clearance from the blood pool was fast and efficient for all new derivatives, providing low levels at 1.5 h p.i. for all new compounds (≤ 0.4 %ID/g), similar to those published for the non-glycated ligand 1. (Table 4.3) All three new ligands 2 - 4 showed a relatively low liver uptake between 2.3 ± 0.3 %ID/g

84

(peptide 2) and 1.3 ± 0.3 %ID/g (peptide 3) after 0.5 h. Peptides 2 and 4 showed a rapid elimination of activity from this organ (0.6 ± 0.1 %ID/g and 0.6 ± 0.3 %ID/g at 1.5 h p.i., respectively). Peptide 3 showed a slightly slower washout with 1.0 ± 0.1 %ID/g (1.5 h p.i.) and 0.6 ± 0.2 %ID/g (5 h p.i.). However, the uptake and retention of the carbohydrated peptides was significantly lower than the data reported for the non-glycated analogue 1 (5.3 ± 1.3 %ID/g at 0.5 h p.i. and 2.1 ± 0.4 %ID/g at 5 h p.i.). Maximal tumor uptake was found at 0.5 h for all complexes. Compound 4 featured the highest accumulation at all p.i. times (5.6 ± 2.1 %, 3.6 ± 1.1 and 2.5 ± 1.3 %ID/g at 0.5 h, 1.5 h and 5 h p.i., respectively), which was significantly higher than that reported for peptide 1 (P ≤ 0.01) (Table 4.3). Interestingly, the retention of 4 in the tumor was significantly higher than that of the other new peptides 2 and 3 and the reference 1. Thus, in case of complex 4, 45 % of the tumor localized activity after 0.5 h still remained inside the tumor at 5 h p.i., compared to 16 - 29 % for the other analogues (Table 4.4). Besides the increased tumor uptake, we observed high uptake of the radiopeptides in pancreas and colon, which are known to express GRPR in mice. The highest values in both organs were obtained for peptide 4 at 0.5 h p.i. (pancreas = 63 %ID/g and colon 13.0 %ID/g). Moreover, the specificity of tracer accumulation in GRPR-positive tissues was determined by co-injection with 0.1 mg BBS(1-14) per mouse. These experiments showed a clear reduction of radioactivity in the tumor xenografts and in pancreas and colon (Table 4.4), what would confirm that the in vivo uptake was due to receptorspecific interaction. Despite the higher kidney uptake found for the new carbohydrated BBS analogues at early p.i. times (due to their increased hydrophilicity), it rapidly decreased and the activity in the kidneys at later p.i. times was relatively low for all peptides (≤ 1 %ID/g, at 1.5 and 5 h p.i.) (Table 4.4). This would indicate that the tracers were not trapped in the renal tissue and that renal excretion of the radiopeptides was very fast. The increased hydrophilicity of the new peptides resulted in a decrease in hepatic activity. However, a relatively high and unspecific accumulation of activity was observed in the small intestines, which would indicate that hepatobiliary excretion partially contributes to the overall tracer elimination (Table 4.4). Biodistribution experiments in normal mice have been reported for 99m Tc(III)-labeled BBS analogues, all modified with different lipophilic linkers, and, in the case of two compounds, an additional carboxylic group. All these complexes featured high uptake in hepatic tissue (7.2 - 9.4 %ID/g) and relatively high renal accumulation between 1.8 and 6.0 %ID/g at 1 h p.i. (20). 85

Table 4.3: Biodistribution of the non-glycated, 99m Tc-labeled reference (3.7 MBq/mouse i.v.) in mice with PC-3 xenografts. The animals of the blocked group received a co-injection of 0.1 mg BBS(1-14). Data (mean ± SD) are expressed as percentage of injected dose per gram of tissue. Compound

1 (Reference)

Organs

0.5 h

1.5 h

5h

1.5 h blocked

(n = 3)

(n = 13)

(n = 3)

(n = 3)

Blood

0.58 ± 0.21

0.36 ± 0.66

0.23 ± 0.11

0.47 ± 0.07

Stomach

1.37 ± 0.62

0.73 ± 0.47

0.60 ± 0.33

1.23 ± 0.08

Kidney

1.82 ± 0.51

0.79 ± 0.35

0.39 ± 0.20

0.76 ± 0.19

Liver

5.26 ± 1.26

2.36 ± 0.87

1.83 ± 0.75

2.13 ± 0.36

Small intestines

5.00 ± 0.69

1.83 ± 0.57

0.32 ± 0.19

6.48 ± 2.88

Pancreas

19.00 ± 7.60

6.16 ± 1.80

0.69 ± 0.53

1.19 ± 0.07

Colon

2.65 ± 0.30

4.36 ± 3.60

2.55 ± 1.77

1.11 ± 0.17

Tumor

1.53 ± 0.26

0.80 ± 0.35

0.41 ± 0.27

0.37 ± 0.09

Calculation of tumor-to-blood, tumor-to-liver and tumor-to-kidney ratios (Figure 4.3) allowed comparisons with other radiolabeled BBS analogues, recently described in the literature. The highest tumor-to-blood ratios corresponded to compound 4 and were significantly higher than those of the reference peptide 1 at 1.5 h p.i. (11.5 ± 4.8 vs. 4.3 ± 3.4, respectively, P ≤ 0.05) and at 5 h p.i. (33.4 ± 4.2 vs. 1.9 ± 0.5, P ≤ 0.01). The compounds 2 and 3 showed similar tumor-to-blood ratios compared to peptide 1 at 0.5 and slightly higher at 1.5 p.i and 5 h p.i. time, but the differences were not statistically significant, with the exception of compound 3 at 1.5 h (13.0 ± 7.1 vs. 4.3 ± 3.4, respectively, P ≤ 0.01). Interestingly, compound 4 featured significantly increased tumor-to-kidney ratios already at 1.5 h p.i. (1.4 ± 0.4 for 4 vs. 0.9 ± 0.3 for 1; P ≤ 0.05) but more remarkably at 5 h p.i. (2.6 ± 0.4 for 4 vs. 1.1 ± 0.3 for 1; P ≤ 0.01). Tumor-to-kidney ratios for peptides 2 and 3 were no significantly different compared to the reference 1 at any p.i. time (Figure 4.3). The most notable finding was the improvement of tumor-to-liver ratios for all new glycated radiotracers at all p.i. times compared to the non-glycated peptide 1 (Figure 4.3). This indicates a significant impact of carbohydration on tracer accumulation in the 86

Table 4.4: Biodistribution of the new

99m Tc-labeled

BBS analogues (3.7 MBq/mouse i.v.) in mice bearing PC-3 xenografts. The animals of the blocked group received a co-injection of 0.1 mg BBS(1-14). Data (mean ± SD) are expressed as percentage of injected dose per gram of tissue (n = 3; 1 n = 6; * (P = 0.05), ** (P = 0.01) vs. reference compound 1 (one-way ANOVA and Dunnett’s post hoc test);

Compound

2

3

§

(P = 0.05),

Organs

(P = 0.01) vs. unblocked group (unpaired t-test)).

0.5 h

1.5 h

5h

1.5 h blocked

Blood

2.12 ± 0.72

0.34 ± 0.10

0.11 ± 0.05

0.36 ± 0.08

Stomach

3.01 ± 0.23

1.50 ± 0.33

0.30 ± 0.10

0.90 ± 0.26

Kidney

7.20 ± 1.96

2.21 ± 0.38

0.99 ± 0.29

1.96 ± 0.62

2.28 ± 0.33 **

0.57 ± 0.14 *

0.22 ± 0.04 **

0.87 ± 0.37

Small intestines

5.95 ± 1.68

5.52 ± 1.00

0.47 ± 0.07

2.84 ± 1.13

Pancreas

22.38 ±8.10

16.05 ±7.41

1.96 ± 1.64

2.16 ± 0.58

§§

Colon

5.67 ± 2.59

4.57 ± 1.84

3.15 ± 1.31

0.48 ± 0.06

§§

Tumor

3.79 ± 1.09 *

2.83 ± 1.26 **

0.62 ± 0.23

1.27 ± 0.62

Blood

0.66 ± 0.21

0.16 ± 0.07

0.04 ± 0.01

0.15 ± 0.06

Stomach

1.53 ± 0.45

1.07 ± 0.10

0.58 ± 0.22

0.28 ± 0.09

Kidney

2.98 ± 0.84

1.89 ± 0.55

0.96 ± 0.15

1.52 ± 0.38

1.34 ± 0.33 **

0.95 ± 0.10

0.61 ± 0.18 **

2.07 ± 0.69

Small intestines

3.42 ± 0.15

2.88 ± 0.27

0.89 ± 0.08

1.88 ± 0.78

Pancreas

20.12 ± 6.60

18.56 ± 1.42

4.32 ± 1.58

1.52 ± 1.64

§§

Colon

5.05 ± 1.10

4.53 ± 1.12

3.36 ± 0.46

0.52 ± 0.23

§§

Tumor

2.37 ± 0.64

1.82 ± 0.43

0.71 ± 0.53

0.67 ± 0.20

§§

Blood

1.77 ± 0.16

0.34 ± 0.11

1

0.07 ± 0.01

0.49 ± 0.15

Stomach

4.18 ± 0.20

1.82 ± 0.33

1

0.54 ± 0.12

0.47 ± 0.11

7.48 ± 0.53

2.65 ± 0.80

1

0.95 ± 0.05

2.06 ± 0.71

1.74 ± 0.07 **

0.64 ± 0.33 **

0.22 ± 0.01 **

0.98 ± 0.80

8.51 ± 1.49

4.55 ± 1.24

1.77 ± 0.46

3.64 ± 1.15

Liver

Liver

Kidney 4

§§

Liver Small intestines

1 1

Pancreas

63.17 ± 8.23

26.96 ± 2.53

Colon

12.93 ± 1.88

7.31 ± 1.22

Tumor

5.64 ± 2.06 **

3.62 ± 1.09 **

87

1

1 1

3.70 ± 1.15

3.33 ± 0.28

§§

6.43 ± 0.79

0.74 ± 0.07

§§

2.49 ± 1.32 **

1.01 ± 0.09

§§

Figure 4.3: Tumor to tissue ratios of the

99m Tc-labeled

BBS analogues in mice bearing PC-3 tumor xenografts (3.7 MBq/mouse i.v.) (n = 3-13). (A) Tumor-to-blood. (B) Tumor-tokidney. (C) Tumor-to-liver. * (P = 0.05), ** (P = 0.01) vs. reference compound 1 (one-way ANOVA and Dunnett’s post hoc test)

liver, which is particularly important since high accumulation of radioactivity in the liver has been reported not only for 99m Tc-labeled BBS analogues but also for 64 Cu-DOTABBS(7-14), 64 Cu-NOTA-8-Aoc-BBN(7-14)NH2 or 68 Ga-DOTA-BBS(6-14) (19, 21, 23). The positive effect of carbohydration on liver accumulation has already been published for other peptides, e.g. radiolabeled sst analogues. Carbohydrated conjugates of Tyr3 octreotide (TOC) showed a significant decrease of tracer accumulation in the liver and the intestines, without effect on the binding affinity and similar or even increased uptake in sst receptor expressing tissues and tumor xenografts (26). There are relatively few biodistribution data in the literature to compare 99m Tc-labeled BBS analogues with an agonistic character. Maina et al reported on in vivo data for several 99m Tc(V)-labeled BBS(1-14) analogues either with a hydrophilic or with a lipophilic character. Their results indicated a strong impact of the polarity on the accumulation of the tracers in liver, kidneys and tumors (15), the hydrophilic peptides showing the best tumor-to-non-tumor ratios. Tumor-to-liver and tumor-to-blood ratios were comparable to those of our best compound (4). However the glycated 99m Tc(I)-labeled peptide 4 showed higher tumor-to-kidney values than the 99m Tc(V)-labeled compound, which could be of interest for therapeutical applications with 186/188 Re-labeled BBS.

88

SPECT/CT imaging studies High accumulation of activity in the liver and gastrointestinal tract may hide the localization of cancerous lesions in the abdomen. Therefore, the reduced abdominal uptake by introduction of carbohydrates in addition to the increased tumor uptake is very interesting to increase the potential of radiolabeled BBS, in general, and of 99m Tc(CO)3 -labeled peptides, in particular. The new glycated analogues were further evaluated in vivo in SPECT/CT imaging experiments. Representative SPECT/CT images of the novel glycated radiotracer 4 and the non-glycated ligand 1 are depicted in Figure 4.4. The new conjugate 4 clearly provided a better imaging of the tumor xenografts than the reference compound 1. The higher tumor uptake of conjugate 4 led to a clearer visualization of the xenografts compared to the non-glycated 1. Furthermore the lower liver and kidney uptakes resulted in decreased radioactive background in the abdominal area. Accumulation in small intestines due to hepatobiliary excretion as well as uptake in the GRPR-positive pancreatic and colon tissue were still high, in accordance with our ex vivo biodistribution data.

Figure 4.4: SPECT/CT image of nude mice bearing PC-3 tumor xenografts at 1.5 h p.i. of 20 MBq of the 99m Tc-labeled BBS analogues. (A) coronal (left) and sagittal (right) slizes of compound 1 (non-glycated). (B) coronal (left) and sagittal (right) slizes of compound 4 (glycated via click reaction). T = tumor; L = liver; I = intestinal activity.

89

4.5

Conclusion

We evaluated three novel carbohydrated BBS derivatives for their usefulness in imaging GRPR-expressing neoplasms and we could confirm the positive impact of glycation on the in vivo behavior of BBS analogues. In summary, the introduction of the polar carbohydrates had no effect on internalization, efflux or metabolic stability in vitro but led to a significant reduction of abdominal accumulation, higher tumor uptake and improved tumor-to-background ratios in vivo. The best results were obtained with a triazole coupled glucose, which ameliorated the biodistribution profile significantly. In this respect the relative easy accessibility of carbohydrates, especially of the glycation via click reaction, clearly offers an easy opportunity to improve the pharmacokinetic profiles of 99m Tcor 188 Re-labeled BBS analogues and increase their potential for imaging and therapy of GRPR-expressing tumors.

4.6

Acknowledgements

The authors thank Ms. Olga Gasser, Dr. Alexander Hohn and Mr. Alain Blanc for their technical assistance. This work was funded by an Oncosuisse grant (Nr. OCS 01311-02-2003) and by the Fund for Scientific Research-Flanders, Belgium, (grant Nr. G.0036.04).

90

4.7

References

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9. Otte, A., Mueller-Brand, J., Dellas, S., Nitzsche, E. U., Herrmann, R., and Maecke, H. R. (1998) Yttrium-90-labelled somatostatin-analogue for cancer treatment. Lancet 351, 417-418. 10. de Jong, M., Breeman, W. A. P., Valkema, R., Bernard, B. F., and Krenning, E. P. (2005) Combination radionuclide therapy using Lu-177- and Y-90-Labeled somatostatin analogs. J. Nucl. Med. 46, 13S-17S. 11. Reubi, J. C., Wenger, S., Schmuckli-Maurer, J., Schaer, J.-C., and Gugger, M. (2002) Bombesin Receptor Subtypes in Human Cancers: Detection with the Universal Radioligand 125 I-[D-TYR6 , β-ALA11 , PHE13 , NLE14 ] Bombesin(6-14). Clin. Cancer Res. 8, 1139-1146. 12. Smith, C. J., Volkert, W. A., and Hoffman, T. J. (2005) Radiolabeled peptide conjugates for targeting of the bombesin receptor superfamily subtypes. Nucl. Med. Biol. 32, 733-740. 13. Markwalder, R., and Reubi, J. C. (1999) Gastrin-releasing Peptide Receptors in the Human Prostate: Relation to Neoplastic Transformation. Canc. Res. 59, 1152-1159. 14. Zhou, J., Chen, J., Mokotoff, M., Zhong, R., Shultz, L. D., and Ball, E. D. (2003) Bombesin/Gastrin-Releasing Peptide Receptor: A Potential Target for AntibodyMediated Therapy of Small Cell Lung Cancer. Clin. Cancer Res. 9, 4953-4960. 15. Nock, B. A., Nikolopoulou, A., Galanis, A., Cordopatis, P., Waser, B., Reubi, J. C., and Maina, T. (2005) Potent Bombesin-like Peptides for GRP-Receptor Targeting of Tumors with 99m Tc: A Preclinical Study. J. Med. Chem. 48, 100-110. 16. Zhang, H., Chen, J., Waldherr, C., Hinni, K., Waser, B., Reubi, J. C., and Maecke, H. R. (2004) Synthesis and Evaluation of Bombesin Derivatives on the Basis of PanBombesin Peptides Labeled with Indium-111, Lutetium-177, and Yttrium-90 for Targeting Bombesin Receptor-Expressing Tumors. Cancer Res. 64, 6707-6715. 17. Zhang, X., Cai, W., Cao, F., Schreibmann, E., Wu, Y., Wu, J. C., Xing, L., and Chen, X. (2006) 18 F-Labeled Bombesin Analogs for Targeting GRP ReceptorExpressing Prostate Cancer. J Nucl Med 47, 492-501.

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18. Smith, C. J., Gali, H., Sieckman, G. L., Higginbotham, C., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical Investigations of 99m Tc-N3 S-X-BBN[714]NH2 : An in Vitro/in Vivo Structure-Activity Relationship Study Where X = 0-, 3-, 5-, 8-, and 11-Carbon Tethering Moieties. Bioconjugate Chem. 14, 93-102. 19. Schuhmacher, J., Zhang, H., Doll, J., Macke, H. R., Matys, R., Hauser, H., Henze, M., Haberkorn, U., and Eisenhut, M. (2005) GRP Receptor-Targeted PET of a Rat Pancreas Carcinoma Xenograft in Nude Mice with a 68 Ga-Labeled Bombesin(6-14) Analog. J. Nucl. Med. 46, 691-699. 20. Kunstler, J. U., Veerendra, B., Figueroa, S. D., Sieckman, G. L., Rold, T. L., Hoffman, T. J., Smith, C. J., and Pietzsch, H. J. (2007) Organometallic 99m Tc(III) ’4 + 1’ Bombesin(7-14) Conjugates: Synthesis, Radiolabeling, and in Vitro/in Vivo Studies. Bioconjugate Chem. 18, 1651-1661. 21. Parry, J. J., Kelly, T. S., Andrews, R., and Rogers, B. E. (2007) In Vitro and in Vivo Evaluation of 64 Cu-Labeled DOTA-Linker-Bombesin(7-14) Analogues Containing Different Amino Acid Linker Moieties. Bioconjugate Chem. 18, 1110-1117. 22. Garayoa, E. G., Ruegg, D., Blauenstein, P., Zwimpfer, M., Khan, I. U., Maes, V., Blanc, A., Beck-Sickinger, A. G., Tourwe, D. A., and Schubiger, P. A. (2007) Chemical and biological characterization of new Re(CO)3 /[99m Tc](CO)3 bombesin analogues. Nuc. Med. Biol. 34, 17-28. 23. Prasanphanich, A. F., Nanda, P. K., Rold, T. L., Ma, L., Lewis, M. R., Garrison, J. C., Hoffman, T. J., Sieckman, G. L., Figueroa, S. D., and Smith, C. J. (2007) [64 CuNOTA-8-Aoc-BBN(7-14)NH2 ] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues. Proc. Natl. Acad. Sci. U S A 104, 12462-12467. 24. Smith, C. J., Sieckman, G. L., Owen, N. K., Hayes, D. L., Mazuru, D. G., Kannan, R., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical Investigations of Gastrin-releasing Peptide Receptor-specific [99m Tc(X)(CO)3 -Dpr-Ser-Ser-Ser-GlnTrp-Ala-Val-Gly-His-Leu-Met-(NH2 )] in PC-3, Tumor-bearing, Rodent Models: Syntheses, Radiolabeling, and in Vitro/in Vivo Studies where Dpr = 2,3-Diaminopropionic acid and X = H2 O or P(CH2 OH)3 . Cancer Res. 63, 4082-4088.

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25. Smith, C. J., Gali, H., Sieckman, G. L., Hayes, D. L., Owen, N. K., Mazuru, D. G., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical investigations of 177 Lu-DOTA-8-Aoc-BBN[7-14]NH2 : an in vitro/in vivo assessment of the targeting ability of this new radiopharmaceutical for PC-3 human prostate cancer cells. Nucl. Med. Biol. 30, 101-109. 26. Schottelius, M., Wester, H. J., Reubi, J. C., Senekowitsch-Schmidtke, R., and Schwaiger, M. (2002) Improvement of Pharmacokinetics of Radioiodinated Tyr3 Octreotide by Conjugation with Carbohydrates. Bioconjugate Chem. 13, 10211030. 27. Wester, H. J., Schottelius, M., Scheidhauer, K., Meisetschlager, G., Herz, M., Rau, F. C., Reubi, J. C., and Schwaiger, M. (2003) PET imaging of somatostatin receptors: design, synthesis and preclinical evaluation of a novel 18 F-labelled, carbohydrated analogue of octreotide. Europ. J. Nucl. Med. Mol. Imaging 30, 117-122. 28. Wester, H. J., Schottelius, M. K. Scheidhauer, J.C. Reubi, I. Wolf, and Schwaiger, M. (2002) Comparison of radioiodinated TOC, TOCA and Mtr-TOCA: the effect of carbohydration on the pharmacokinetics. Eur. J. Nucl. Med. Mol. Imaging 29, 28-38. 29. Haubner, R., Wester, H. J., Weber, W. A., Mang, C., Ziegler, S. I., Goodman, S. L., Senekowitsch-Schmidtke, R., Kessler, H., and Schwaiger, M. (2001) Noninvasive imaging of alpha(v)beta(3) integrin expression using F-18-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Research 61, 1781-1785. 30. Schottelius, M., Rau, F., Reubi, J. C., Schwaiger, M., and Wester, H. A. (2005) Modulation of pharmacokinetics of radioiodinated sugar-conjugated somatostatin analogues by variation of peptide net charge and carbohydration chemistry. Bioconjugate Chem. 16, 429-437. 31. Wester, H. J., Schottelius, M., Poethko, T., Bruus-Jensen, K., and Schwaiger, M. (2004) Radiolabeled carbohydrated somatostatin analogs: A review of the current status. Canc. Biother. Radiopharmac. 19, 231-244.

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32. Antunes, P., Ginj, M., Walter, M. A., Chen, J., Reubi, J. C., and Maecke, H. R. (2007) Influence of Different Spacers on the Biological Profile of a DOTASomatostatin Analogue. Bioconjugate Chem. 18, 84-92. 33. Maes, V., Garcia-Garayoa, E., Blauenstein, P., and Tourwe, D. (2006) Novel 99m TcLabeled Neurotensin Analogues with Optimized Biodistribution Properties. J. Med. Chem. 49, 1833-1836. 34. Garayoa, E. G., Schweinsberg, C., Maes, V., Ruegg, D., Blanc, A., Blauenstein, P., Tourwe, D. A., Beck-Sickinger, A. G., and Schubiger, P. A. (2007) New [99m Tc]bombesin analogues with improved biodistribution for targeting gastrin releasing-peptide receptor-positive tumors. Q. J. Nucl. Med. Mol. Imaging 51, 42-50. 35. Maes, V., Brans, L., Schweinsberg, C., Garcia-Garayoa, E., Blauenstein, P., Schubiger, P. A., and Tourwe, D. (2007) Click-carbohydrated [99m Tc(CO)3 ](Nα His)AcBombesin(7-14) analogs. Biopolymers 88, 547-547. 36. Maes, V., and Tourwe, D. (2006) Maillard glycation of peptides containing the (Nα His)Ac chelator for 99mTc(CO)3 labeling. Int. J. Pept. Res. Therap. 12, 197-202. 37. Garcia-Garayoa, E., Schibli, R., and Schubiger, P. A. (2007) Peptides radiolabeled with Re-186/188 and Tc-99m as potential diagnostic and therapeutic agents. Nucl. Sci. Tech. 18, 88-100. 38. Garcia Garayoa, E., Ruegg, D., Blauenstein, P., Zwimpfer, M., Khan, I. U., Maes, V., Blanc, A., Beck-Sickinger, A. G., Tourwe, D. A., and Schubiger, P. A. (2007) Chemical and biological characterization of new Re(CO)3 /[99m Tc](CO)3 bombesin analogues. Nucl. Med. Biol. 34, 17-28. 39. Pillarsetty, N., Cai, S., Ageyeva, L., Finn, R. D., and Blasberg, R. G. (2006) Synthesis and Evaluation of [18 F] Labeled Pyrimidine Nucleosides for Positron Emission Tomography Imaging of Herpes Simplex Virus 1 Thymidine Kinase Gene Expression. J. Med. Chem. 49, 5377-5381. 40. Bauer, R., and Pabst, H. W. (1982) Tc-Genrators - Yield of 99mTc and Ratio to ’Inactive’ 99Tc. Eur. J. Nucl. Med. 7, 35-36.

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41. Garcia Garayoa, E., Schweinsberg, C., Maes, V., Ruegg, D., Blanc, A., Blauenstein, P., Tourwe, D. A., Beck-Sickinger, A. G., and Schubiger, P. A. (2007) New [Tc-99m]bombesin analogues with improved biodistribution for targeting gastrin releasing-peptide receptor-positive tumors. Q. J. Nucl. Med. Mol. Imag. 51, 42-50. 42. Alves, S., Correia, J. D. G., Santos, I., Veerendra, B., Sieckman, G. L., Hoffman, T. J., Rold, T. L., Figueroa, S. D., Retzloff, L., McCrate, J., Prasanphanich, A., and Smith, C. J. (2006) Pyrazolyl conjugates of bombesin: a new tridentate ligand framework for the stabilization of fac-[M(CO)3 ]+ moiety. Nucl. Med. Biol. 33, 625-634. 43. Christensen, E. I., Rennke, H. G., and Carone, F. A. (1983) Renal tubular uptake of protein: effect of molecular charge. Am J Physiol Renal Physiol 244, F436-441. 44. Albert, R., Marbach, P., Bauer, W., Briner, U., Fricker, G., Bruns, C., and Pless, J. (1993) Sdz-Co-611 - a Highly Potent Glycated Analog of Somatostatin with Improved Oral Activity. Life Sci. 53, 517-525. 45. La Bella, R. L., Garcia-Garayoa, E., Bahler, M. B., Blauenstein, P., Schibli, R., Conrath, P., Tourwe, D., and Schubiger, P. A. (2002) A 99m Tc(I)-Postlabeled High Affinity Bombesin Analogue as a Potential Tumor Imaging Agent. Bioconjugate Chem. 13, 599-604.

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Chapter 5 ”Click to Chelate”: Synthesis and Installation of Metal Chelates into Biomolecules in a Single Step

Thomas L. Mindt1 , Harriet Struthers2 , Luc Brans3 , Todor Anguelov2 , Christian Schweinsberg2 , Veronique Maes3 , Dirk Tourwé3 , and Roger Schibli1,2

1

2

Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland Center for Radiopharmaceutical Science, Paul Scherrer Institute, 5232 Villigen PSI, Switzerland 3 Faculty of Sciences, Vrije Universiteit Brussels, 1050 Brussels, Belgium

J AM CHEM SOC 2006; 128:15096-15097

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5.1

Abstract

Click chemistry has been employed for the assembly of novel and efficient triazole-based multidentate chelating systems while simultaneously attaching them to molecules of biological interest. The ”click-to-chelate” approach offers a powerful new tool for the modification of (bio)molecules with metal chelators for potential diagnostic and therapeutic applications.

Figure 5.1: Mechanism of the single step reaction for radiolabeling of biomolecules via click chemistry.

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5.2


The radiolabeling of biologically active molecules has become an indispensable tool for the assessment of novel drug candidates. To keep pace with the growing number of new targets, innovative and efficient methodologies are needed for the firm attachment of molecules of biomedical interest to readily available radionuclides with suitable decay characteristics. In recent years, new cores of technetium-99m (T1/2 = 6 h, 140 keV -radiation) have been explored for the radiolabeling of biomolecules.1 The organometallic precursor [Tc(OH2 )3 (CO)3 ]+ , is a prominent example.2 A variety of bifunctional tridentate ligand systems (e.g. based on amino acid scaffolds like cysteine, lysine and histidine) have been designed for the tricarbonyl precursor.3−6 However, the preparation of such chelators generally requires multiple-step syntheses and their incorporation into biomolecules often lacks efficiency and is complicated by cross-reactivity with other functional groups present. To circumvent the problems associated with current strategies for functionalizing (bio)molecules with metal chelators, we set out to investigate the use of Sharpless’ click chemistry in this context.7,8

Figure 5.2: a) Cu(OAc)2 , Na(ascorbate), water, 25 °C (15 h) or 100 °C (30 min). b) M = 99m Tc:

[99m Tc(OH2 )3 (CO)3 ]+ , PBS pH 7.4, 30 min 100 °C; M = Re: [ReBr3 (CO)3 ]2− , water or alcohols, 50-65 °C, 1-4 h.

99

Click chemistry (the Cu(I)-catalyzed [3+2] cycloaddition of alkynes and azides forming stable 1,2,3-triazole linkages) meets the requirements of an innovative functionalization strategy for biomolecules because it is efficient, selective and devoid of side reactions. The mild reaction conditions are well suited for the modification of a wide variety of (bio)molecules, into which the required azide and alkyne functionalities can be incorporated by standard synthetic transformations or biochemical methods.9 In addition, we recognized that 1,4-disubstituted 1,2,3-triazoles share structural and electronic features with 1,4-disubstituted imidazoles of Nα -derivatized histidines, which have been shown to be extraordinarily good chelators, particularly for organometallic cores of Mo, Tc and Re.10−12 Surprisingly few examples of 1,2,3-triazole chelators obtained via click chemistry are reported13,14 even though triazoles are known to be potent ligand systems for various transition metals.15

Figure 5.3: Derivatives of biomolecules functionalized via click chemistry with a tridentate 1,2,3-triazole containing metal chelator.

To probe the viability of a ”click-to-chelate” approach, various commercial and noncommercial azide and alkyne derivatives were reacted with either L-propargyl glycine (1) or L-azido alanine (2)16 to form 1,2,3-triazole-4-yl-alanines 5/6 (referred to as a ”regular” click ligand), or the isomeric products 7/8 (”inverse” click ligand, Figure 5.2). In addition, representatives of four different classes of biomolecules (tumor affine peptide 100

bombesin 9, thymidine 10, carbohydrate 1117 and phospholipid 12) were selected to demonstrate the versatility of the new approach (Figure 5.3). Unlike the multi-step syntheses required for the preparation of Nα -derivatized histidines,18 the click strategy avoids protective groups, which allowed the synthesis of optically pure histidine-like, 1,4-functionalized triazoles 5-8 and 10-12 in a single step and in quantitative yields.19 These features are particularly appealing for the functionalization of biomolecules such as carbohydrates with polydentate metal chelates, which is notoriously inefficient using common synthetic strategies as we and others have experienced.20,21 The azido-bombesin derivative was prepared and clicked on solid support22 to afford product 9.

Figure 5.4: Labeling capacities of ligands 5-8 and Nα -methyl histidine (Me-His) with [99m Tc(OH2 )3 (CO)3 ]+ .

Reaction of ligands 5-12 with [ReBr3 (CO)3 ]2− in aqueous or alcoholic media yielded the well defined neutral complexes [Re(CO)3 (5-12)] (Figure 5.2). 1 H- and 13 C-NMR analyses indicate that metal chelation occurs via N(3) (regular click) or N(2) (inverse click) of the 1,2,3-triazole heterocycle, the N -amine and the carboxylate.19 The characteristic features of the NMR spectra of isolated complexes are consistent with those reported for Tc/Re(CO)3 -complexes of Nα -functionalized histidine derivatives.12 Radioactive complexes [99m Tc(CO)3 (5-12)] were also obtained as single products.23,24

101

For ligands 5-8, varying the ligand concentration gave rise to step-sigmoid curves (Figure 5.4). EC50 values of investigated regular click ligands ranged from 2-3*10−7 M. These values were comparable to that of Nα -methyl histidine (EC50 = 10−7 M), demonstrating the potency of the regular triazole ligands 5/6. Values of inverse click ligands 7/8 were approximately two orders of magnitude higher (EC50 = 10−5 M), presumably a consequence of the lower electron density at N(2) compared to N(3) of the triazole heterocycle (DFT calculations).19 While the click products represent a class of extraordinarily good chelators, the individual substrates (e.g. alkyne 1, azide 2) do not form stable or defined complexes with [M(OH2 )3 (CO)3 ]+ . This observation spurred the idea of a one-pot procedure to avoid isolation of the functionalized (bio)molecule prior to labeling with [M(OH2 )3 (CO)3 ]+ . Thus, aqueous solutions of 3’-azido thymidine or 1-azido-1-deoxyβ-D-galactopyranose and alkyne 1, Cu(OAc)2 and Na(ascorbate) were heated to 100 99m °C for 30 min. [ Tc(OH2 )3 (CO)3 ]+ was added and the mixtures were heated for further 30 min (Figure 5.5). HPLC analysis confirmed the clean formation of complexes [99m Tc(CO)3 (10)] and [99m Tc(CO)3 (11)], identical to the products obtained with presynthesized and purified ligands 10 and 11.19

Figure 5.5: One-pot procedure to yield radiolabeled conjugates directly. The radiolabeled bombesin derivative [99m Tc(CO)3 (9)] was assessed in vitro and in vivo for its stability and receptor affinity.19 These preliminary experiments revealed a high in vivo stability of the conjugate and an almost identical pharmacological profile to [99m Tc(CO)3 Nα AcHis-bombesin],25 a previously tested stable bombesin analog with sequence homology but a histidine chelate (detailed results will be published elsewhere). 102

These results suggest that the new triazole ligands represent a valuable alternative to histidine-derived chelators. We have shown that click chemistry both simplifies the synthesis of efficient bifunctional ligands in which 1,4-disubstituted triazoles form an integral part of the metal chelating system and facilitates their incorporation into (bio)molecules. Labeling of the chelators and bioconjugates with fac-”Tc/Re(CO)3 ” is efficient and results in complexes that are stable in vitro and in vivo. The one-pot procedure represents a remarkable improvement for the synthesis of metal-labeled conjugates e.g. for diagnostic purposes. Extension of the ”click-to-chelate” approach to the preparation of structurally diverse ligand systems suitable for complexation of other metals is currently being investigated.

5.3

Acknowledgements

We thank Judith Stahel and Elisa Garcia-Garayoa for technical assistance and Jason Holland for DFT.

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5.4

References

1. Banerjee, S. R.; Maresca, K. P.; Francesconi, L.; Valliant, J.; Babich, J. W.; Zubieta, J. Nucl. Med. Biol. 2005, 32, 1-20. 2. Alberto, R.; Schibli, R.; Egli, A.; Schubiger, A. P.; Abram, U.; Kaden, T. A. J. Am. Chem. Soc. 1998, 120, 7987-7988. 3. Alberto, R., New organometallic technetium complexes for radiopharmaceutical imaging. In Contrast Agents III: Radiopharmaceuticals - from Diagnostics to Therapeutics, 2005; Vol. 252, pp 1-44. 4. van Staveren, D. R.; Benny, P. D.; Waibel, R.; Kurz, P.; Pak, J. K.; Alberto, R. Helv. Chim. Acta 2005, 88, 447-460. 5. Stephenson, K. A.; Zubieta, J.; Banerjee, S. R.; Levadala, M. K.; Taggart, L.; Ryan, L.; McFarlane, N.; Boreham, D. R.; Maresca, K. P.; Babich, J. W.; Valliant, J. F. Bioconjugate Chem. 2004, 15, 128-136. 6. Levadala, M. K.; Banerjee, S. R.; Maresca, K. P.; Babich, J. W.; Zubieta, J. Synthesis 2004, 1759-1766. 7. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem.-Int. Edit. 2002, 41, 2596-2599. 8. Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193. 9. Prescher, J. A.; Bertozzi, C. R. Nature Chemical Biology 2005, 1, 13-21. 10. van Staveren, D. R.; Metzler-Nolte, N. Chem. Commun. 2002, 1406-1407. 11. van Staveren, D. R.; Bill, E.; Bothe, E.; Buhl, M.; Weyhermuller, T.; MetzlerNolte, N. Chem.-Eur. J. 2002, 8, 1649-1662. 12. Pak, J. K.; Benny, P.; Spingler, B.; Ortner, K.; Alberto, R. Chem.-Eur. J. 2003, 9, 2053-2061. 13. Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853-2855. 104

14. Dai, Q.; Gao, W. Z.; Liu, D.; Kapes, L. M.; Zhang, X. M. J. Org. Chem. 2006, 71, 3928-3934. 15. Moore, D. S.; Robinson, S. D. Adv. in Inorg. Chem. 1988, 32, 171-239. 16. Link, A. J.; Vink, M. K. S.; Tirrell, D. A. J. Am. Chem. Soc. 2004, 126, 1059810602. 17. Kuijpers, B. H. M.; Groothuys, S.; Keereweer, A. R.; Quaedflieg, P.; Blaauw, R. H.; van Delft, F. L.; Rutjes, F. Org. Lett. 2004, 6, 3123-3126. 18. van Staveren, D. R.; Mundwiler, S.; Hoffmanns, U.; Pak, J. K.; Spingler, B.; Metzler-Nolte, N.; Alberto, R. Org. Biomol. Chem. 2004, 2, 2593-2603. 19. See Supporting Information for details. 20. Dumas, C.; Petrig, J.; Frei, L.; Spingler, B.; Schibli, R. Bioconjugate Chem. 2005, 16, 421-428. 21. Storr, T.; Obata, M.; Fisher, C. L.; Bayly, S. R.; Green, D. E.; Brudzinska, I.; Mikata, Y.; Patrick, B. O.; Adam, M. J.; Yano, S.; Orvig, C. Chem.-Eur. J. 2004, 11, 195-203. 22. Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057-3064. 23. The identity of the technetium species was verified by comparison of the g-HPLC trace (99m Tc) with the UV-HPLC trace (254 nm) of the corresponding Re-complexes. See Supporting Information for details. 24. We were able to show that [99m Tc(CO)3 (5-12)] can be quantitatively synthesized directly, by reacting 5-12 in presence of [99m TcO4 ]− in a vial containing the IsoLinkT M components necessary for formation of the [99m Tc(OH2 )3 (CO)3 ]+ . See Supporting Information for details. 25. Garcia-Garayoa, E.; Ruegg, D.; Bläuenstein, P.; Zwimpfer, M.; Maes, V.; Khan, I. U.; Blanc, A.; Beck-Sickinger, A.; Tourwe, D.; Schubiger, A. P. Nucl. Med. Biol. 2006, in press.

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Chapter 6 ’Click’-Modified 99mTc-Tricarbonyllabeled BBS Analogues for Imaging of Prostate Cancer

107

6.1

Abstract

Gastrin-releasing peptide receptors (GRP-R) are known to be over-expressed in numerous human cancers, e.g. prostate and breast, which turns them into interesting targets for in vivo imaging with the 99m Tc-labeled GRP analogue bombesin (BBS). The Cu(I)catalyzed [3+2] cycloaddition of alkynes and azides (’Click’-chemistry) offers a convenient way to functionalize peptides by the formation of chelating systems for radiolabeling or for the introduction of pharmacokinetic modifiers (1, 2). In this study we compared the established retro(Nα His)Ac-chelator with two novel triazole-chelating systems and investigated the carbohydration of the most promising of the novel peptides via ’Click’chemistry. The BBS analogues were synthesized manually on a Rink amide resin by solid phase peptide synthesis using Fmoc-strategy. Both triazole-chelators were attached using ”Click”-chemistry. Additionally, the compound with the most promising biodistribution was glycated via the click-reaction. All peptides were labeled with the [99m Tc(CO)3 ]core and tested in vitro with the BBS-receptor expressing human prostate carcinoma cell line PC-3 for receptor binding and cell internalization. In vivo biodistribution and SPECT/CT imaging studies experiments were carried out in nude mice bearing PC-3 tumor xenografts. The new peptides with the N3click-chelator exhibited high affinity for BBS receptors in unlabeled and labeled form with Kd values between 0.19 and 0.6 nM and were rapidly internalized within 1 h. Moreover, the new derivatives showed improved in vivo properties such as higher specific uptake, lower liver accumulation and higher tumor-to-blood and tumor-to-liver ratios in comparison to the non-glycated (Nα His)Acanalogue (2 to 8-fold at 1.5 h and 5 h p.i.). Tumor-to-kidney ratios were similar at 1.5 h p.i. for all compounds but improved up to 3-fold at 5 h p.i. Glycation by ’click’-chemistry resulted in a significantly higher tumor uptake for both the (Nα His)Ac- and the ’click’compound (3.4 ± 1.0 and 4.4 ± 0.6 %ID/g, respectively at 1.5 h p.i.). Additionally, the ’click’-glycated peptides showed a relatively long retention of activity in the tumors (2.4 ± 1.2 and 2.6 ± 0.8 %ID/g at 5 h p.i.). In the imaging studies the tumor xenografts could be clearly visualized. In conclusion, the application of ’click’-chemistry provided a new stable chelating system for BBS analogues with favorable attributes compared to the (Nα His)Ac-system. Furthermore, we could underline the significant improvement of the in vivo properties of 99m Tc-tricarbonyl-labeled BBS analogues by glycation.

108

6.2

Introduction

The modification of numerous biomolecules, such as peptides, amino acids or nucleosides, holds a huge potential to create new powerful tools for radiopharmaceutical applications (3-6). However, the alteration of the molecular structure, e.g. the introduction of pharmacokinetic modifiers or the attachment of chelators for the incorporation of radiometals, are often challenging and time consuming processes. Sharpless et al. introduced ’click’chemistry (the Cu(I) catalyzed [3+2] cycloaddition of alkynes and azides) as a novel approach in organic chemistry that involves several almost perfect chemical reactions (7, 8). Recently our group could apply the ’click’-reaction as an efficient, convenient way for the modification of biomolecules, amongst others peptide derivatives, for radiopharmaceutical applications (1, 2, 9, 10). Neuropeptides are known to bind with high affinity and selectivity to their corresponding receptors, which are over-expressed on a considerable number of cancers (11-13). Many radiopeptides have been modified by the incorporation of polar groups, such as charged amino acids or carbohydrates and especially the latter revealed a great impact on the in vivo characteristics, e.g. a clear reduction of liver uptake and hepatobiliary excretion in the case of radiolabeled sst analogues (14-19). Bombesin (BBS) is a tetradecapeptide with high affinity for the mammalian gastrin releasing peptide receptor (GRPR), a subtype of the BBS receptor superfamily, which is overexpressed in numerous human tumors, such as prostate, breast, small-cell lung cancer and others (19-21). Of the different radionuclides useful in nuclear medicine 99m Tc is still the one with the broadest clinical application due to its favorable dosimetry, high specific activity, low cost and ready availability from a 99 Mo/99mT c -generator. Its physical properties (t1/2 = 6 h, Energy γ = 140 keV, 89 % abundance) are optimal for imaging using single photon emission computed tomography (SPECT) (22-24). We reported the applicability of the ’click’-reaction for the modification of BBS derivatives, namely the formation of two new chelators as part of an efficient bifunctional chelating system for labeling with 99m Tc/186/188 Re(CO)3 as well as the introduction of a glucose moiety into the linker between the chelator and the BBS(7-14) binding sequence (1, 2, 9). Preliminary experiments with a 99m Tc(CO)3 -labeled N3click-chelator attached to the stabilized BBS(7-14)-fragment revealed a similar in vivo behavior compared to the reference 99m Tc(CO)3 (Nα His)Ac-βAla-βAla-BBS(7-14), with a high specific uptake in GRPR expressing tissues pancreas and colon (1). Furthermore, the glycation of the linker by

109

Figure 6.1: Chemical structure of the chelators. (A) reference chelator

99m Tc(CO)

(Nα His)Ac

3 -complexes

formed with the tridentade (B) N2click-chelator (c) N3click-chelator

formation of a stable triazole bond using ’click’-chemistry led to a significant improvement of the biodistribution of the 99m Tc(CO)3 -labeled BBS(7-14) analogue with higher tumor uptake, reduction of hepatic activity and clearly improved tumor-to-nontumor ratios (2). In this study we intensified the investigation of the ’click’-chelators (Figure 6.1) with regard to their impact on the in vitro characteristics and the biodistribution of the 99m Tc(CO)3 -labeled BBS analogues in tumor xenografted mice. Additionally, we investigated the influence of a ’click’-coupled glucose moiety on the in vitro and in vivo properties compared to the carbohydrated 99m Tc(CO)3 (Nα His)Ac-counterpart.

6.3


Chemicals and Equipment The chemicals were obtained from Merck (Dietkon, Switzerland) or Fluka (Buchs, Switzerland). The determination of protein concentrations was performed using a Pierce BCA Protein Assay kit from Socochim (Lausanne, Switzerland). Na[99m TcO4 ] was eluted from a Mallinckrodt 99 Mo/99m Tc generator (Tyco Healthcare, Petten, The Netherlands) using 0.9 % saline. Sodium boranocarbonate was a gift from the provider of the generator. The radioactivity of in vitro and in vivo experiments was measured with a NaI-γ-counter (Packard Canberra Cobra II, Meriden, USA) or a NaI ionization chamber. DMEM GLUTAMAXT M -I, trypsin/EDTA (0.25 % trypsin, 1 mM EDTA), bovine serum albumin and soybean trypsin inhibitor were obtained from Invitrogen (Basel, Switzerland), fetal calf serum (FCS) and antibiotic/antimycotic solution were from Bioconcept 110

(Allschwil, Switzerland). Bacitracin, HEPES and Chymostatin were purchased from SIGMA (Buchs, Switzerland). Peptide Synthesis and radiolabeling with

99m

Tc

Peptide solid-phase synthesis was carried out on a Rink Amide Resin with the Fmocstrategy as previously described (3). The attachment of the new chelators and the introduction of the glucose moiety (Figure 6.1) were performed as recently published (1, 4). The purification of the peptides was done on a semipreparative HPLC system (Gilson) on a reverse phase C18 column (DiscoveryBIO SULPELCO Wide Pore, 25 cm x 2.21 cm, 5 µm) with a linear gradient of 3 % B to 100 % B (solvent A = H2 O + 0.1 % TFA as and as solvent B = CH3 CN + 0.1 % TFA) in 30 min, a flow rate of 1 mL/min and UV detection at 215 nm. Radiolabeling of the peptides with [99m Tc(OH2 )3 (CO)3 ]+ was performed as previously described (22). For analyses and purification of the labeled peptides from the unlabeled compounds a Macherey-Nagel CC Nucleosil 100-5 C18 RP column (5µm, 250 x 4.6 mm) was used. Preparative HPLC for peptide purification was performed using a Merck-Hitachi L-7100-system with an equipped 2-channel scintillation detector interface (John Caunt scientific Ltd, Eynsham, England) and a Rainin Dynamax absorbance detector model UV-1. HPLC solvents consisted of H2 O + 0.1 % TFA (solvent A) and CH3 OH + 0.1 % TFA (solvent B). The separation was performed with a linear gradient 50 % A / 50 % B to 20 % A / 80 % B in 25 min (1.0 mL/min). For the analytical HPLC of the labeled peptides a Varian ProStar system was used with a Varian ProStar 230 Solvent Delivery-Pump, a Varian Photodiode Array Detector Model

Figure 6.2: Chemical structure of the glycated BBS analogues (A) (Nα His)Ac-chelator plus lysine-coupled glucose B) (Nα His)Ac-chelator plus triazole-coupled glucose (C) N3clickchelator plus triazole-coupled glucose

111

Table 6.1: Sequence of the ’Click’-modified BBS analogues. Peptide

Chelator

Linker

Binding Sequence

1 (Reference)

(Nα His)Ac

βAla-βAla


2

N2Click

βAla


3

N3Click

βAla


4 (Reference)

(Nα His)Ac

Lys(Gluc)βAla-βAla


α

5 (Reference)

(N His)Ac

Glu(pra)βAla-βAla


6

N3Click

Glu(pra)βAla-βAla


330, a Varian ProStar Autosampler Model 410 and a radiomatic Flo-one Beta Detector (Packard Canberra). HPLC solvents consisted of CH3 CN + 0.1 % TFA (solvent A) and H2 O + 0.1 % TFA (solvent B). The labeling control was performed with a linear gradient of 30 % A / 70 % B to 80 % A / 20 % B in 25 min (1.0 mL/min). Octanol/water partition coefficient study Octanol/water partition coefficients were determined at pH = 7.4. Five µL containing 500 kBq of the radiolabeled compound in PBS were added to a vial containing 1.2 ml 1-octanol and PBS (1:1). After vortexing for 1 min the vial was centrifuged for 5 min at 10000 rpm to ensure complete separation of layers. Then 40 µL of each layer were taken in a preweighed vial and measured in the γ-counter. Counts per unit weight of sample were calculated, and logD values were calculated as described by Pillarsetty et al (6) using the formula in 1 g of octanol Log10 D = log10 ( counts ) counts in 1 g of P BS

Cell culture The human prostate cancer cell line PC-3 was purchased from European Collection of Cell Cultures (Salisbury, United Kingdom) and maintained in Dulbecco’s MEM with GLUTAMAXT M -I supplemented with 10% FCS, 100 IU/mL penicillin G sodium, 100 µg/mL streptomycin sulphate, 0.25 µg/mL amphotericin B. Cells were subcultured twice 112

a week after detaching with trypsin-EDTA and incubated at 37°C in an atmosphere containing 5% CO2 . Receptor inhibition studies PC-3 cells at confluence were placed in 48-well plates one day prior to the assay. During the experiment the cells were incubated in a special binding buffer (50 mM HEPES, 125 mM NaCl, 7.5 mM KCl, 5.5 mM MgCl*6H2 O, 1 mM EGTA, 5g/L BSA, 2 mg/L Chymostatin, 100 mg/L Soybean Trypsin inhibitor, 50 mg/L Bacitracin, pH 7.4) with increasing concentrations of the BBS analogues (0-30000 nM) in presence of 4 kBq of 99m Tc-BBS(7-14). After 1 h incubation at 37°C the cells were washed twice with cold PBS. Bound radioactivity was recovered by solubilizing the cells in 1 N NaOH (2 x 400 µL). The radioactivity was measured in the γ-counter. All experiments were carried out 2-3 times in triplicate. Receptor saturation studies One day prior to the assay PC-3 cells at confluence were placed in 48-well plates and incubated for 1 h at 37°C in the binding buffer described above with increasing concentrations of the labeled BBS analogues (0.01-2 nM). After the HPLC separation from unlabeled peptide the concentration of total Tc-labeled peptide was calculated according to Bauer et Pabst (7). Non-specific binding was determined by co-incubation with 1µM BBS(1-14). After incubation the cells were washed twice with cold PBS, solubilized in 1 N NaOH (2 x 400 µL) and the radioactivity was measured in the γ-counter. Experiments were performed 2-3 times in triplicate. Internalization For internalization studies PC-3 cells were placed in 6 well plates and incubated with the labeled analogues (4 kBq/well) in binding buffer for 5, 15, 30, 60 and 120 min at 37°C to allow binding and internalization. Non-specific binding was determined in the presence of 1 µM BBS(1-14). After incubation cells were washed twice with cold PBS. Surface bound activity was removed by acid wash (50 mM glycine/HCL, 100 mM NaCl, pH 2.8, 2 x 600 µL for 5 min at room temperature). Afterwards the plates were neutralized with 600 µL PBS and cells were lysed with 1 N NaOH (2 x 600 µL). Surface bound and internalized activities were measured in the γ-counter. The protein concentration was 113

determined and results were calculated as percentage of total added radioactivity per mg protein.

Biodistribution studies The animal experiments were conducted in compliance with the Swiss animal protection laws and with the ethical principles and guidelines for scientific animal trials established by the Swiss Academy of Medical Sciences and the Swiss Academy of Natural Sciences. Female CD-1 nu/nu mice 6-8 week-old (Charles River Laboratories, Sulzfeld, Germany) were subcutaneously injected with 8 x 106 PC-3 cells in 150 µL culture medium without supplements. About 3 weeks after tumor implantation the mice (3-6 per group) were injected i.v. with 3.7 MBq radiolabeled peptides. At 0.5 h, 1.5 h or 5 h post-injection (p.i.) the animals were sacrificed by cervical dislocation. The different organs (heart, lung, spleen, kidneys, pancreas, stomach, small intestine, large intestine, liver, muscle, bone) and blood were collected, weighed and the radioactivity was measured in a γcounter. Results are presented as percentage of injected dose per gram of tissue (%ID/g). To determine the specificity of the in vivo uptake one group of mice received a co-injection of 100 µg of unlabeled BBS(1-14) and the radiolabeled analogue and sacrificed at 1.5 h p.i.. SPECT/CT studies Single photon emission computed tomography / X-ray computed tomography (SPECT/ CT) images were performed post-mortem 1.5 h after i.v. injection of the 99m Tc-BBS analogues (20 MBq). Images were obtained on an X-SPECTT M -system (Gamma Medica Inc.) equipped with a single head SPECT device and a CT-device. SPECT data were acquired and reconstructed with LumaGEM (version 5.407). CT data were reconstructed with the software Cobra (version 4.5.1). Fusion of SPECT- and CT-data was performed with IDL Virtual MachineT M (version 6.0). Images were generated with AmiraT M (version 3.1.1).

114

6.4


The development of novel simpler methods for the syntheses and functionalization of small modified biomolecules is of high interest for the production of new tracers for radiopharmaceutical applications. Recently, the ’click’-reaction (Figure 6.1) was introduced as a convenient and cheap way for the easy incorporation of 1,4-disubstituted triazoles into the stabilized BBS fragment (7-14) for labeling with Tc/Re(CO)3 (1). Preliminary in vitro and in vivo data revealed a similar profile to the reference compound 1, a homologous peptide with a histidine chelator. Compound 1 featured rather poor tumor-to-tissue ratios (tumor-to-blood = 4.3 ± 3.4, tumor-to-kidney = 0.9 ± 0.3 and tumor-to-liver = 0.4 ± 0.3, 1.5 h p.i.). Its high accumulation in the liver is especially unfavorable for both the detection and radiotherapeutic treatment of abdominal leasons. We synthesized new peptides with novel chelating systems coupled by ’click’-chemistry. The new analogues are based on the same stabilized sequence (replacement of Leu13 with Cha, and Met14 with Nle) (Table 6.1), which has been reported to exhibit an increased metabolic stability in human plasma and tumor cells (17). Likewise, βAla-groups were inserted between the chelator and the peptide binding sequence, due to their positive influence on the in vivo behavior (7, 28). The two new ’click’-chelators were incorporated into the BBS-fragment as recently published: the N2click-chelator by the attachment of L-propargyl glycine to an BBS-precursor with a N-terminal alkyne (compound 2), the N3-chelator by the reaction of L-azido alanine with an N-terminal azide (compound 3) (1). Finally, compound 6 was synthesized

Table 6.2: Binding affinity of the unlabeled (IC50 values) and labeled (Kd values) compounds for GRP-receptors in PC-3 cells (n = 2-3).

Compound

IC50 [nM]

Kd [nM]

1 (Reference)

3.9 ± 1.1

0.19 ± 0.12

2

4.2 ± 0.7

n.d.

3

1.9 ± 1.1

0.19 ± 0.06

4 (Reference)

3.7 ± 0.7

0.18 ± 0.06

5 (Reference)

4.2 ± 0.1

0.29 ± 0.16

6

5.2 ± 0.1

0.60 ± 0.27

115

Figure 6.3: Time-course internalization of

99m Tc-labeled

BBS analogues after incubation at 37°C. Data represent the percentage of internalized activity per mg protein related to the total activity added to the cells (n = 2-3)

by the formation of a stable triazole bond between an alkyne in the linker and an azido glucose derivative, in analogy to the synthesis recently published for peptide 5 (9). Radiolabeling of these novel carbohydrated peptides with [99m Tc(OH2 )3 (CO)3 ]+ was performed at ligand concentrations of 10-5 M for 1 h at 75 °C. Radiolabeling yields were higher than 95 % for the peptides 2, 3 and 6, which were similar to those of the reference compounds 1, 4 and 5. The radioactive complexes were successfully separated from the unlabeled peptides to avoid a saturation of the GRP-R in subsequent in vitro/in vivo experiments. The polarity of the 99m Tc(CO)3 -labeled carbohydrated compound was evaluated by determinig the octanol/water partition coefficient. Coupling of glucose to the N3chelator-bearing peptide 6 resulted in a LogD value of -0.6, comparable to the values for peptides 4 and 5 (-0.5) (2).

116

In vitro studies The in vitro experiments were performed with the human prostate carcinoma cell line PC-3, which expresses a high level of GRPR. The affinity of the unlabeled analogues was tested in inhibition experiments in competition with 4 kBq of 99m Tc-BBS(7-14), which has been reported to exhibit a high affinity for the GRPR (29). The two new chelating systems as well as the additional carbohydration showed no influence on the high binding affinity with IC50 values between 1.9 and 5.2 nM, similar to the tracers 1, 4 and 5 (Table 6.2). However, after radiolabeling with 99m Tc(CO)3 only the experiments with peptides with the N3click chelator led to saturable and specific receptor binding to the tumor cells. The Kd values of the N3click-radioconjugate 3 were identical to the reference 1 (0.19 ± 0.06 and 0.19 ± 0.12 nM, respectively), whereas for the glycated analogue 6 (0.60 ± 0.27 nM) was slightly higher than those of the glycated references 4 and 5 (0.18 ± 0.06 and 0.29 ± 0.16 nM, respectively) (Table 6.2). Binding affinity could not be determined for the new radiolabeled peptide 2, due to lack of specificly bound cell associated activity. Considering the stability of the complex in PBS, the high affinity found for the unlabeled compound 2 in the inhibition studies and the general low impact of the organometallic complex on the binding affinity of our BBS(7-14) derivatives, the loss of binding affinity due to radiolabeling may be ascribed to the reported lower complex stability of the N2-click chelating systems (1). It is possible that a rapid decomposition of the 99m Tc(CO)3 -N2click-complex occurred under the conditions of the in vitro tests (binding buffer and tumor cells). Therefore, compound 2 was excluded from further in vitro or in vivo investigations. The internalization experiments showed a similar and rapid uptake into the tumor cells for both N3click-compounds 3 and 6. Between 25 and 35 % of the applied dose per mg protein corresponded to internalized radioactivity after 60 min (Figure 6.3). The differences between the glycated and non-glycated peptides as well as between the new N3click-chelator and the reference peptides with an (Nα His)Ac-chelator were statistically not significant. The fast internalization indicates an agonistic behavior and is consistent with other structurally similar BBS derivatives, which have been published recently (25, 30-32).

117

Table 6.3: Biodistribution of the new

99m Tc-labeled

BBS analogues (3.7 MBq/mouse i.v.) in mice bearing PC-3 xenografts. The animals of the blocked group received a co-injection of 0.1 mg BBS(1-14). Data (mean ± SD) are expressed as percentage of injected dose per gram of tissue (n = 3-4) Compound

3

6

Organs

0.5 h

1.5 h

5h

1.5 h blocked

Blood

1.54 ± 0.30

0.18 ± 0.01

0.06 ± 0.01

0.19 ± 0.08

Stomach

2.11 ± 0.50

0.92 ± 0.26

0.36 ± 0.01

0.28 ± 0.10

Kidney

4.86 ± 1.10

1.00 ± 0.11

0.35 ± 0.07

0.84 ± 0.40

Liver

4.05 ± 0.81

1.15 ± 0.17

0.38 ± 0.06

1.59 ± 0.31

Small intestines

10.05 ± 3.90

7.29 ± 1.99

7.41 ± 3.04

0.87 ± 0.13

Pancreas

29.52 ± 6.82

12.46 ± 2.91

4.20 ± 0.28

0.80 ± 0.27

Colon

6.99 ± 1.92

2.67 ± 1.01

3.27 ± 1.25

0.40 ± 0.15

Tumor

3.76 ± 1.47

1.20 ± 0.75

0.80 ± 0.44

1.02 ± 0.44

Blood

1.40 ± 0.39

0.19 ± 0.06

0.08 ± 0.01

0.16 ± 0.03

Stomach

4.67 ± 2.06

1.46 ± 0.21

0.96 ± 0.33

0.04 ± 0.96

Kidney

6.36 ± 0.81

2.20 ± 0.60

0.89 ± 0.15

1.63 ± 0.20

Liver

0.96 ± 0.09

0.31 ± 0.05

0.23 ± 0.07

0.77 ± 0.08

Small intestines

7.21 ± 2.47

3.34 ± 0.61

0.66 ± 0.14

1.32 ± 0.32

Pancreas

51.99 ± 5.52

31.40 ± 2.81

17.65 ± 2.00

1.91 ± 0.25

Colon

9.21 ± 2.07

6.14 ± 0.78

4.65 ± 1.75

0.36 ± 0.14

Tumor

4.97 ± 1.14

4.41 ± 0.55

2.61 ± 0.81

0.68 ± 0.22

Biodistribution studies The N3click-compounds 3 and 6 were tested in biodistribution studies performed with mice bearing PC-3 tumor xenografts and compared with data obtained for the reference peptides 1, 4, and 5 (2, 28). For both new peptides 3 and 6 a higher radioactivity was found in the blood at 0.5 h p.i. (1.5 ± 0.3 %ID/g and 1.4 ± 0.4 %ID/g, respectively) compared the non-glycated reference 1 (0.6 ± 0.2 %ID/g). These values were similar to the ’click’-glycated reference compounds 5 (1.8 ± 0.2 %ID/g) but higher than for peptide 4 (0.7 ± 0.2). All peptides featured a rapid blood clearance and activity was (t1/2 0.4 %ID/g) after 1.5 h p.i.

118

The presence of the new ’click’-chelator in compound 3 showed no significant effect on the accumulation in the liver (4.0 ± 0.8 %ID/g; 0.5 h p.i.), which was in the range of that published for reference 1 (5.3 ± 1.3 %ID/g). The clearance from hepatic tissue was slightly faster until 1.5 h p.i. for peptide 3 (1.2 ± 0.2 %ID/g) than for 1 (2.1 ± 0.4 %ID/g). Interestingly, the uptake of the novel glycated compound 6 in hepatic tissue at 0.5 h p.i. (1.0 ± 0.1 %ID/g) was clearly reduced compared to its non-glycated counterpart peptide 3 (4.0 ± 0.8 %ID/g) as well as to the glycated reference 5 (1.7 ± 0.1 %ID/g). These results underline the effect of the polar carbohydrate towards the urinary/renal excretion of radiotracers as well as a low impact of the N3click-chelator alone on a decrease of liver accumulation. The relatively high and unspecific accumulation of activity in the small intestines observed for both peptides 3 and 6 at 0.5 h p.i. (10.1 ± 3.9 and 7.2 ± 2.5 %ID/g, respectively) is a hint for a partial hepatobiliary excretion (Table 6.3). However, the retention of the intestinal radioactivity was long for analogue 3 (7.3 ± 2.0 and 7.4 ± 3.0 %ID/g at 1.5 h and 5 h, respectively), while a faster depletion was observed for analogue 6 (3.3 ± 0.6 %ID/g at 1.5 h, 0.7 ± 0.1 %ID/g at 5 h p.i.). This clearly indicates a shift from the hepatobiliary excretion towards the urinary/renal system similar to the carbohydrated compounds 4 and 5 (3.4 ± 0.2 vs. 8.5 ± 1.5 %ID/g at 0.5 h, 2.9 ± 0.3 vs. 4.6 ± 1.2 %ID/g at 1.5 h, 0.9 ± 0.1 vs. 1.8 ± 0.5 %ID/g at 5 h p.i., respectively). In accordance with the rapid blood clearance of all radiopeptides 1 and 3 - 6, the highest tumor uptake was always at the earliest p.i. time (0.5 h). Peptide 3 exhibited a clearly higher tumor uptake at this time compared to peptide 1 but both showed a similar rapid washout from tumor tissue (3.8 ± 1.5 vs. 1.5 ± 0.3 %ID/g at 0.5 p.i., 1.2 ± 0.8 vs. 0.8 ± 0.4 at 1.5 h p.i. and 0.8 ± 0.4 vs. 0.4 ± 0.3 %ID/g at 5 h p.i., respectively) (Table 6.3). Peptide 6 featured similar high uptake and retention of activity in the tumor tissue compared to conjugate 5, with a high uptake at 0.5 h p.i. (5.0 ± 1.1 and 5.6 ± 2.1 %ID/g, respectively) and relatively high values at 5 h p.i. (2.6 ± 0.8 and 2.5 ± 1.3 %ID/g, respectively). Thus, interestingly, the retention of both ’click’-glycated radioconjugates 5 and 6 in the tumor was significantly higher than that of the peptide 4, which was glycated via a lysine residue. Therefore, the glycation via ’click’-chemistry seems to be more favorable for the in vivo properties than the attachment of glucose to the side chain of lysine. Besides the increased tumor uptake, we observed high uptake of the radiopeptides in pancreas and colon, which are known to express GRPR in mice. Accumulation of the 119

new tracer 3 at 0.5 h p.i. was again higher in these organs compared to peptide 1 (Table 6.3). However, the fast clearance of 3 was comparable to the depletion found for both tracers in the tumor tissue, and indicates a general fast in vivo washout from receptorexpressing tissues for non-glycated compounds (Table 6.3). Incorporation in pancreas and colon was similar for the ’click’-glycated radiotracers 5 and 6 with high values at the early p.i. time points (63.2 ± 8.2 vs. 52.0 ± 5.5 %ID/g at 0.5 h p.i. and 27.0 ± 2.5 vs. 31.4 ± 2.8 %ID/g at 1.5 h p.i., respectively), but a significantly higher retention in pancreatic tissue was found for 6 compared to 5 (17.7 ± 2.0 vs. 3.7 ± 1.2 %ID/g at 5 h p.i., respectively). The specificity of tracer accumulation in GRPR-positive tissues was determined by co-injection with 100 µg BBS(1-14) per mouse. The clear decrease of radioactivity in all receptor-expressing tissues including the tumors (Figure 6.5) confirmed that the in vivo uptake was due to receptor-specific interaction. Kidney uptake was higher for the new peptides 3 and 6 at 0.5 h p.i. (4.9 ± 1.1 and 6.4 ± 0.8 %ID/g, respetively) than for the nonglycated conjugate 1 (1.8 ± 0.5 %ID/g). This may be because of an increased renal excretion of the radiopeptides underlined by the reduced hepatic accumulation described above. The kidney values importantly decreased at later times (Table 6.3), indicating that the peptides were not absorbed into the renal tissue but rapidly excreted into the bladder. Tumor-to-tissue ratios were calculated for a further comparison of the biodistribution results with the reference compounds 1, 4 and 5 (Figure 6.4). The tumor-to-blood ratios are interesting in terms of the overall radioactive background caused by the circulation of a radiotracer in the blood pool. These ratios were similar for all radiolabeled BBS analogues 1 and 3 - 6 at 0.5 h p.i. (T/B ≈ 4). However, an improved tumor-to-blood ratio was found at 1.5 h p.i. for the glycated compounds, being highest for peptide 6 (12 for 4, 11 for 5 and 23 for 6). At a later time point (5 h p.i.) the ratios were improved for both new peptides 3 and 6 compared to the reference 1. Concerning the glycated analogues, the ratio of 6 was again similar to that of 5 andhigher than that of 4 (Figure 6.4). High tumor-to-kidney ratios are of particular interest for a potential use of the radiotracers for therapeutic applications with 186/188 Re. The tumor-to-kidney ratios found in this study showed a similar pattern as described for the tumor-to-blood ratios above. Again the values were almost identical for all analogues at 0.5 h p.i. (T/K ≈ 1). This ratio increased for peptide 6 up to 2.2 at 1.5 h p.i. and was higher than that of the reference peptide 5 (1.4 at 1.5 p.i.). High uptake of radiotracers in the liver is problematic for imaging and therapeutic treatment 120

Figure 6.4: Tumor to non-tumor ratios of the

99m -labeled

BBS analogues in mice bearing PC-3 tumor xenografts (3.7 MBq/mouse i.v.) (n = 3-13). (A) Tumor-to-blood. (B) Tumorto-kidney. (C) Tumor-to-liver

of abdominal lesions, such as liver metastases, which is a general drawback of radiolabeled BBS analogues and has been published not only for 99m Tc-labeled BBS derivatives but also for 64 Cu-DOTA-BBS(7-14), 64 Cu-NOTA-8-Aoc-BBN(7-14)NH2 or 68 Ga-DOTABBS(6-14) (19, 31, 33, 34). Interestingly, the tumor-to-liver ratios of compound 6 were significantly superior at 0.5 and 1.5 h p.i. compared to both the non-glycated counterpart 3 as well as the glycated peptides 4 and 5 (Figure 6.5). Similar results on the reduction of hepatic uptake after glycation were published for radiolabeled sst analogues. Sugar moieties attached to Tyr3 -octreotide (TOC) exhibited a significant decrease of tracer accumulation in the liver and intestines in combination with an increased uptake in sst receptor expressing tissues and tumor xenografts (15, 35). SPECT/CT studies Biodistribution and SPECT/CT studies performed with the carbohydrated 99m Tc(CO3 )labeled BBS analogue 4 and 5 revealed a significant impact of sugar moieties on the pharmacokinetic profiles (2). The reduction of activity found in the liver and gastrointestinal tract increased the potential of these peptides for diagnostic applications. In order to examine the capability of the novel compound 6 for diagnostic purposes, SPECT/CT imaging experiments were performed after the i.v. injection of 70 MBq of the 99m Tclabeled peptide. Coronal imaging slices of the animal showed a clear localization of the

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Figure 6.5: Coronal slices of a SPECT/CT of a nude mouse with PC-3 xenografts 1.5 h after injection of 70 MBq of the 99mTc-labeled click-glycated N3click-BBS (compound 6)

tumor xenografts inoculated into the shoulders. A relatively low activity was found in liver and intestines (Figure 6.5). The relatively low radioactivity detected in the abdomen was in accordance with the expression of GRPR in the murine pancreas and a limited hepatobiliary excretion. Overall the abdominal accumulation was significantly reduced compared to the 99m Tc(CO3 )-labeled analogue 1 (2). The results were in agreement with both the biodistribution experiments as well as data obtained with the ’click’glycated 99m Tc-labeled analogue 5 (2). Thus, the carbohydration via ’click’-chemistry again showed a significant impact on the biodistribution profile of 99m Tc(CO3 )-labeled BBS derivatives. The new ’click’-glycated and N3click-chelated BBS analogue 6 revealed a huge potential for imaging of GRPR-expressing tumors.

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6.5

Conclusion

In this work we evaluated the applicability of ’click’-chemistry for the functionalization of 99m Tc(CO)3 -labeled BBS analogues for targeting of GRPR-positive tumors. We studied the implication of two chelating systems attached by ’click’-chemistry to the N-terminal end of a modified BBS(7-14) peptide and further investigated the influence of an additional glucose moiety attached by the ’click’-reaction to the linker. The new N3click-chelator clearly offered a convenient way for radiolabeling of BBS derivatives with the organometallic 99m Tc-tricarbonyl-core and may even be slightly favorable compared to the established (Nα His)Ac-chelator with regard to a reduced liver accumulation. Furthermore, the data confirmed the positive impact of a triazole coupled glucose on the pharmacokinetic profile of BBS analogues labeled with the 99m Tc(CO)3 -core, namely on the uptake and in vivo retention in receptor-expressing organs and tumor xenografts. Especially the low hepatic uptake and the improved tumor-to-liver and tumor-to-kidney ratios turn the ’click’-glycated and N3click-chelated BBS analogue into a promising radiotracer for diagnostic tumor targeting. In summary, ’click’-chemistry provides a valuable tool for both modification and functionalization of 99m Tc-labeled BBS analogues for improved tumor targeting of GRPR-expressing tumors.

6.6

Acknowledgements

This work was funded by an Oncosuisse grant (Nr. OCS 01311-02-2003).

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6.7

References

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18. Markwalder, R., and Reubi, J. C. (1999) Gastrin-releasing Peptide Receptors in the Human Prostate: Relation to Neoplastic Transformation. Canc. Res. 59, 1152-1159. 19. Zhou, J., Chen, J., Mokotoff, M., Zhong, R., Shultz, L. D., and Ball, E. D. (2003) Bombesin/Gastrin-Releasing Peptide Receptor: A Potential Target for AntibodyMediated Therapy of Small Cell Lung Cancer. Clin. Cancer Res. 9, 4953-4960. 20. Bauer, R., and Pabst, H. W. (1982) Tc-Genrators - Yield of ’Inactive’ 99 Tc. Eur. J. Nucl. Med. 7, 35-36.

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27. La Bella, R. L., Garcia-Garayoa, E., Bahler, M. B., Blauenstein, P., Schibli, R., Conrath, P., Tourwe, D., and Schubiger, P. A. (2002) A 99mTc(I)-Postlabeled High Affinity Bombesin Analogue as a Potential Tumor Imaging Agent. Bioconjugate Chem. 13, 599-604. 28. Alves, S., Correia, J. D. G., Santos, I., Veerendra, B., Sieckman, G. L., Hoffman, T. J., Rold, T. L., Figueroa, S. D., Retzloff, L., McCrate, J., Prasanphanich, A., and Smith, C. J. (2006) Pyrazolyl conjugates of bombesin: a new tridentate ligand framework for the stabilization of fac-[M(CO)(3)](+) moiety. Nucl. Med. Biol. 33, 625-634. 29. Nock, B. A., Nikolopoulou, A., Galanis, A., Cordopatis, P., Waser, B., Reubi, J. C., and Maina, T. (2005) Potent Bombesin-like Peptides for GRP-Receptor Targeting of Tumors with 99mTc: A Preclinical Study. J. Med. Chem. 48, 100-110. 30. Kunstler, J. U., Veerendra, B., Figueroa, S. D., Sieckman, G. L., Rold, T. L., Hoffman, T. J., Smith, C. J., and Pietzsch, H. J. (2007) Organometallic 99mTc(III) ’4 + 1’ Bombesin(7-14) Conjugates: Synthesis, Radiolabeling, and in Vitro/in Vivo Studies. Bioconjugate Chem. 18, 1651-1661. 31. Schuhmacher, J., Zhang, H., Doll, J., Macke, H. R., Matys, R., Hauser, H., Henze, M., Haberkorn, U., and Eisenhut, M. (2005) GRP Receptor-Targeted PET of a Rat Pancreas Carcinoma Xenograft in Nude Mice with a 68Ga-Labeled Bombesin(6-14) Analog. J. Nucl. Med. 46, 691-699. 32. Prasanphanich, A. F., Nanda, P. K., Rold, T. L., Ma, L., Lewis, M. R., Garrison, J. C., Hoffman, T. J., Sieckman, G. L., Figueroa, S. D., and Smith, C. J. (2007) [64CuNOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues. Proc. Natl. Acad. Sci. U S A 104, 12462-12467. 33. Wester, H. J., Schottelius, M. K. Scheidhauer, J.C. Reubi, I. Wolf, and Schwaiger, M. (2002) Comparison of radioiodinated TOC, TOCA and Mtr-TOCA: the effect of carbohydration on the pharmacokinetics. Eur. J. Nucl. Med. Mol. Imaging 29, 28-38.

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Chapter 7 Conclusion The major focus of this work was to investigate the influence of polar linkers on the biodistribution profile of radiolabeled BBS analogues. The over-expression of different peptide receptors has been confirmed for a considerable number of human cancers. A prominent example is the GRP-receptor, which is expressed in high levels on prostate and breast tumors as well as colon, small-cell lung, ovarian cancer and other carcinoids. The fragment 7-14 of BBS is the minimal sequence required for high affinity binding to GRP receptors. Therefore, radiolabeled derivatives of BBS(7-14) hold a remarkable potential as powerful agents for tumor targeting, either peptide receptor imaging (PRI) or peptide receptor radiotherapy (PRRT). Targeting of GRPR-expressing tumors with BBS analogues has two major drawbacks. On the one hand, these peptides show a short biological half-life. On the other hand, their high hepatic accumulation and hepatobiliary excretion is problematic for imaging and therapy of abdominal lesions. The modification of the BBS(7-14) sequence at positions 13 and 14 led to more stable analogues with significantly slower degradation in human plasma and tumor cells. The changes in their binding properties were negligible but they showed a moderately higher tumor accumulation and tumor-to-non-tumor ratios in biodistribution studies performed in nude mice with human tumor xenografts. The introduction of polar pharmacokinetic modifiers is a promising approach to reduce the hepatic and intestinal accumulation and promote the urinary/renal excretion of the radiotracer. In a first study we evaluated five new polar 99m Tc(CO)3 -labeled BBS analogues and could confirm the great impact of polarity and charge on their in vitro and

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in vivo characteristics. Different linkers, consisting of polar β 3 homo-amino acids, were introduced between the binding sequence and the retro(Nα His)Ac-chelator. Despite the fact that the different modifiers exhibited rather similar in vitro properties, we found a remarkable variation among them in vivo. A positive charge in the linker resulted in higher uptake and retention in kidney and liver. A neutral linker with a hydroxyl group resulted in a moderate improvement of the biodistribution in form of decreased hepatic accumulation. However, a single negative charge, after insertion of a β 3 -homo-glutamic acid in the linker, considerably ameliorated the biodistribution profile with higher tumor uptake and retention and significantly improved tumor-to-background ratios, which could be confirmed by SPECT/CT imaging studies. Additional negative charges led to a reduction of binding affinity and internalization and unfavorable biodistribution with reduced tumor uptake and increased kidney uptake. The results indicate that the introduction of a single negative charge may be useful in the development of new BBS analogues, in order to obtain an improved biodistribution profile with enhanced imaging. Our second approach covered the influence of carbohydration on the pharmacokinetic profiles of 99m Tc(CO)3 -labeled BBS analogues. We tested three novel glycated BBS derivatives. The sugar moieties were attached to the linker either via the -amino group of lysine or the formation of a triazole-bond with propargylglycine by ’click’-chemistry. The new compounds were more hydrophilic, but still exhibited a high receptor binding affinity. The introduction of the polar carbohydrates had no effect on internalization, efflux or metabolic stability in vitro but led to a significant reduction of abdominal accumulation, higher tumor uptake and improved tumor-to-background ratios in vivo. The increased hydrophilicity of the new conjugates resulted in high kidney uptake at early p.i. times, which decreased fast indicating that the tracers did not remain trapped in renal tissues but were rapidly released. In general, coupling of glucose via a triazole-bond seems to be superior compared to coupling sugar via a lysine as well as the insertion of a single negative charge. Apart from much higher tumor-to-liver ratios, both tumorto-kidney and tumor-to-blood ratios of the triazole-coupled compound were significantly increased. Additional imaging studies proved the reduction of abdominal background and enhanced tumor delineation.

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The radiolabeling of biologically active molecules has become an indispensable tool for the assessment of novel drug candidates in nuclear medicine. To keep pace with the growing number of new targets, innovative and efficient methodologies are needed for the firm attachment of molecules of biomedical interest to readily available radionuclides with suitable decay characteristics. We could show that ’click’-chemistry offers an exciting new opportunity for the design of efficient probes for radiopharmaceutical applications by innovative chemistry. Labeling of the novel ’click’-chelators and -bioconjugates with Tc/Re(CO)3 was efficient and the one-pot procedure represents a remarkable improvement for the synthesis of metal-labeled conjugates for diagnostic and therapeutic purposes. We intensified our studies on the applicability of ’click’-chemistry by the functionalization of BBS(7-14) with two novel chelating systems for radiolabeling with 99m Tc(CO)3 . The new chelators were attached by ’click’-chemistry to the N-terminus of the peptide sequence. The N3click-chelator clearly offered a convenient way for radiolabeling of BBS derivatives with the organometallic 99m Tc(CO)3 -core, which may even be slightly favorable compared to the established (Nα His)Ac-chelator regarding a reduced liver accumulation. The N2click-ligand did not reveal sufficient complex-stability after radiolabeling for further in vitro and in vivo studies. Furthermore, our data confirmed the positive influence of a triazole-coupled sugar moiety on the uptake and in vivo retention in receptor-expressing organs and tumor xenografts. Especially the low hepatic uptake and the improved tumor-to-liver and tumor-to-kidney ratios turn the click-glycated and N3click-chelated BBS analogue into a promising radiotracer for diagnostic and therapeutic tumor targeting. In summary, the presence of polar linkers, especially in form of a triazole-coupled glucose moiety, significantly improved the biodistribution and imaging properties of 99m Tc(CO)3 labeled BBS derivatives. ’Click’-chemistry provided a convenient method for the formation of a new stable chelating system for 99m Tc(CO)3 -labeling as well as for the incorporation of triazole-coupled glucose. Both glycated N3click-BBS and glycated (Nα His)AcBBS analogues showed the highest potential for targeting of GRP-R-expressing tumors.

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Publications and Presentations Publications 1. T.L. Mindt, H. Struthers, L. Brans, T. Anguelov, C. Schweinsberg, V. Maes, D. Tourwé, R. Schibli ”Click to chelate”: Synthesis and Installation of Metal Chelates into biomolecules in a single step J Am. Chem. Soc. 2006, 128 15096-15097 2. E. Garc´ıa Garayoa, C. Schweinsberg, V. Maes, D. R¨ uegg, A. Blanc, P. Bläuenstein, D. Tourwé, A.G. Beck-Sickinger, P.A. Schubiger New [99m Tc]bombesin analogues with improved biodistribution for targeting gastrin releasingpeptide receptor-positive tumors Q J Nucl Med Mol Imaging 2007; 51:42-50 3. E. Garc´ıa Garayoa, C. Schweinsberg, V. Maes, L. Brans, P. Bläuenstein, D. Tourwé, R. Schibli, P.A. Schubiger Influence of the Molecular Charge on the Biodistribution of Bombesin Analogues Labeled with the [99m Tc(CO)3 ]-Core Bioconjugate Chem (accepted) 4. C. Schweinsberg, E. Garc´ıa Garayoa, V. Maes, P. Bläuenstein, D. Tourwé, P.A. Schubiger Novel Glycated [99m Tc(CO)3 ]-labeled Bombesin Analogues for Improved Targeting of Gastrin-Releasing Peptide Receptor-Positive Tumors Bioconjugate Chem (accepted)

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Oral Presentations 1. C. Schweinsberg, E. Garc´ıa Garayoa, V. Maes, L. Brans, A. Blanc, P. Bläuenstein, D. Tourwé, P.A. Schubiger Preclinical evaluation of stabilized Bombesin analogues with new polar spacers. 8th Annual Congress of the Swiss Society of Nuclear Medicine (SGNM/SSMN), June 7. - 9. 2007, Basel, Switzerland 2. C. Schweinsberg, E. Garc´ıa Garayoa, P. Bläuenstein, R. Schibli, P.A. Schubiger Preclinical Evaluation of New Bombesin Analogues for Targeting of GRP-Receptor Expressing Tumors. PhD Seminar Day 2007, ETH Zurich, Switzerland 3. C. Schweinsberg, L. Brans, V. Maes, T.L. Mindt, E. Garcia Garayoa, P. Bläuenstein, D. Tourwé, P.A. Schubiger, R. Schibli Application of ’Click’-Chemistry to Improve Tumor Targeting with 99m Tc-labeled Bombesin Analogues. 15. Joined Meeting of the Society of Radiochemistry and Radiopharmacy, 27.-29. September 2007, Swissholidaypark, Morschach, Switzerland 4. C. Schweinsberg, E. Garc´ıa Garayoa, L. Brans, V. Maes, T.L. Mindt, P. Bläuenstein, D. Tourwé, P.A. Schubiger, R Schibli Preclinical evaluation of stabilized Bombesin analogues with new polar spacers. 9th Annual Congress of the Swiss Society of Nuclear Medicine (SGNM/SSMN), May 27. - 29. 2008, St.Gallen, Switzerland

Poster Presentations 1. C. Schweinsberg, T. Ebenhan, W. Ehrlichmann, M. Kuntzsch, C. Solbach, H.J. Machulla In vitro evaluation of choline and fluoro-analouges of choline for imaging prostate cancer with PET. Anual Meeting of the Germand Pharmaceutical Society, 2004, Regensburg, Germany 2. C. Schweinsberg, E. Garc´ıa Garayoa, P. Bläuenstein, A. Blanc, P.A. Schubiger Bombesin (BBS) Analogues for Targeting of Gastrin Releasing-Peptide (GRP) Receptors Overexpressed in Human Tumor Tissues. 10th Swiss Receptor Workshop, 2006, Basel, Switzerland

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3. E. Garc´ıa Garayoa, C. Schweinsberg*, A. Blanc, P. Bläuenstein Technetium99m-tricarbonyl Bombesin: preclinical evaluation of new analogues with different spacers. 7th Annual Congress of the Swiss Society of Nuclear Medicine (SGNM/SSMN), 2007, Lausanne, Switzerland 4. C. Schweinsberg, L. Brans, V. Maes, E. Garc´ıa Garayoa, P. Bläuenstein, D. Tourwé, P.A. Schubiger, R. Schibli Functionalization and Modification of Bombesin Analogues using Click-Chemistry. 17th International Symposium on Radiopharmaceutical Sciences, 2007, Aachen, Germany 5. C. Schweinsberg, E. Garc´ıa Garayoa, V. Maes, L. Brans, A. Blanc, P. Bläuenstein, D Tourwé, P.A. Schubiger Influence of polar Spacers on the Biodistribution of radiolabeled Bombesin Analogues. 17th International Symposium on Radiopharmaceutical Sciences, 2007, Aachen, Germany

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Curriculum Vitae Name Born

Christian Schweinsberg October 24th 1976 in Bad Nauheim, Germany

1983-1987 1987-1996 1996-1997

Elementary school in Freigericht Altenmittlau, Germany Kopernikus High School Freigericht, Germany Civilian Service as Rescue Assistant at the Red Cross Rescue Service Center Gelnhausen, Germany Studies in law at the Johann-Wolfgang-Goethe University, Frankfurt, Germany Studies in Biochemistry at the Eberhard Karls University, T¨ ubingen, Germany Internships at the National Kappodestrian University, the National Center for Scientific Research “Demokritos” and the Hellenic Pasteur Institute, Athens, Greece Diploma Thesis at the Center for Radiopharmacy, University Hospital T¨ ubingen, T¨ ubingen, Germany Position as scientific researcher at the Center for Radiopharmacy, University Hospital T¨ ubingen, T¨ ubingen, Germany PhD. student at the Center for Radiopharmaceutical Science, PSI-ETH-UCZ, Villigen/Z¨ urich, Switzerland

1997-1998 1998-2003 2003

2003-2004 2004-2005 2005-2008

Formation 2004 2006 2006 2007

Course for shipping of radioactive materials (corresponding to IATA/ICAO) Expert in radioprotection of ionizing sources LTK Module 1 introductory course in laboratory animal science (corresponding to FELASA-category B) LTK Module 4 training course for anaesthesia of laboratory animals

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