Glucosamine modulates propranolol ...

European Journal of Pharmaceutical Sciences 105 (2017) 137–143

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Glucosamine modulates propranolol pharmacokinetics via intestinal permeability in rats

MARK

Hanadi A. Al Shakera, Nidal A. Qinnaa,b,⁎, Mujtaba Badrb, Mahmoud M.H. Al Omaric, Nasir Idkaideka, Khalid Z. Matalkab,1, Adnan A. Badwanc a b c

Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan University of Petra Pharmaceutical Center (UPPC), Amman, Jordan Research and Innovation Centre, The Jordanian Pharmaceutical Manufacturing Company (PLC), Naor, Jordan

A R T I C L E I N F O

A B S T R A C T

Keywords: Propranolol Glucosamine Single-pass intestinal perfusion Pharmacokinetics Bioavailability Rat intestinal effective permeability Hepatocytes disposition

Propranolol (PROP) undergoes extensive first-pass metabolism by the liver resulting in a relatively low bioavailability (13–23%); thus, multiple oral doses are required to achieve therapeutic effect. Since some studies have reported that glucosamine (GlcN) can increase the bioavailability of some drugs, therefore, it is aimed to study whether GlcN can change the pharmacokinetic parameters of PROP, thus modulating its bioavailability. When PROP was orally co-administered with GlcN (200 mg/kg) to rats, PROP area under curve (AUC) and maximum concentration (Cmax) were significantly decreased by 43% (p < 0.01) and 33% (p < 0.05), respectively. In line with the in vivo results, in silico simulations confirmed that GlcN decreased rat intestinal effective permeability (Peff) and increased PROP clearance by 50%. However, in situ single pass intestinal perfusion (SPIP) experiments showed that GlcN significantly increased PROP serum levels (p < 0.05). Furthermore, GlcN decreased PROP disposition/distribution into cultured hepatocytes in concentration dependent manner. Such change in the interaction pattern between GlcN and PROP might be attributed to the environment of the physiological buffer used in the in vitro experiments (pH 7.2) versus the oral administration and thus, enhanced PROP permeability. Nevertheless, such enhancement was not detected when everted gut sacks were incubated with both drugs at the same pH in vitro. In conclusion, GlcN decreased PROP serum levels in rats in a dose-dependent manner. Such interaction might be attributed to decreased intestinal permeability and enhanced clearance of PROP in the presence of GlcN. Further investigations are still warranted to explain the in vitro inhibitory action of GlcN on PROP hepatocytes disposition and the involvement of GlcN in the intestinal and hepatic metabolizing enzymes of PROP at different experimental conditions.

1. Introduction Beta-blockers have been widely used for the treatment of many cardiovascular diseases (Mansoor and Kaul, 2009; Reiter, 2004). In humans, PROP is the first non-selective β-adrenergic blocker used for the treatment of essential hypertension, arrhythmias, congestive heart failure, and myocardial infraction (Chafin et al., 1999; Stephenson et al., 1980; Wang et al., 2013). Moreover, it has been proved that PROP possesses anti-inflammatory, antioxidant properties, lipid peroxidation inhibitory effect as well as anti-cancer activities (Nkontchou et al., 2012). PROP is also effective clinically in the treatment of some

neurologic diseases, such as headache and migraine (Katzung et al., 2004; Shields and Goadsby, 2005). PROP is a highly lipophilic drug that is almost completely absorbed from the gastrointestinal tract following oral administration (Salman et al., 2010). According to the Biopharmaceutics Classification System (BCS), PROP is classified as class 1 drug with rapid dissolution, highsolubility and high-permeability (Custodio et al., 2008). However, PROP in man is highly metabolized by the liver as it undergoes extensive first-pass metabolism resulting in a relatively low oral bioavailability of 13–23% (Cid et al., 1986; Ismail et al., 2004; Ludden, 1991; Sastry et al., 1993) with a half-life ranging from 3 to

Abbreviations: PROP, propranolol; GlcN, glucosamine; CIME, cimetidine; RIFA, Rifampin; SPIP, single-pass intestinal perfusion; ERIS, everted rat intestinal sac; SLS, sodium lauryl sulphate; HPV, hepatic portal vein; IVC, inferior vena cava; NCA, non-compartmental analysis; Cmax, maximum concentration; Tmax, time of higher concentration; AUC, area under the curve; AUMC, area under the first moment curve; MRT, mean residence time; Kel, the fraction of drug eliminated per time; t0.5, elimination half-life; Peff, rat intestinal effective permeability; Vd, volume of distribution; h, hour; min, minute; BCS, biopharmaceutics classification system ⁎ Corresponding author at: Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, University of Petra, P.O. Box 961343, Amman, Jordan. E-mail address: [email protected] (N.A. Qinna). 1 Current address: OncoTherapeutica, Inc., North Grafton, MA 01536, USA. http://dx.doi.org/10.1016/j.ejps.2017.05.012 Received 18 September 2016; Received in revised form 7 April 2017; Accepted 5 May 2017 Available online 11 May 2017 0928-0987/ © 2017 Published by Elsevier B.V.


H.A. Al Shaker et al.

(Carlsbad, CA, USA), whereas collegenase II and L-glutamine were purchased from Gibco BRL (Gaithersburg, MD, USA). PROP-HCl was a kind gift from The Arab Pharmaceutical Manufacturing (APM) (Salt, Jordan), whereas GlcN-HCl was obtained from Biocon (Bangalore, India; batch No: DA-B10-04-000650/02493). Sildenafil citrate and Tris base were kindly obtained from the Jordanian Pharmaceuticals Manufacturing company (JPM) (Amman, Jordan). RIFA and Florane® (isoflurane) were a kind gift from Hikma Pharmaceuticals (Amman, Jordan), whereas CIME was kindly donated by Jordan Sweden Medical and Sterilization Company (JOSWE) (Naur, Jordan).

6 h (Castleden and George, 1979; Ismail et al., 2004; Leahey et al., 1980) and a large volume of distribution (Vd) of 4 L/kg (Ismail et al., 2004). Thus, it shows marked variations in its bioavailability among patients. As a result, the connection between the amount of dose and the bioavailability of PROP reached after oral administration is still ambiguous (Kiriyama et al., 2008). Few studies have been conducted on rats to increase PROP bioavailability. For instance, Ryu et al. used rectal route of PROP and found the latter route yielded higher bioavailability of PROP than oral administration which may reflect partial bypassing hepatic first pass metabolism (Ryu et al., 1999; van Hoogdalem et al., 1991). Another study in dogs has shown that co-administering PROP with a lipid vehicle increased its bioavailability mainly by increasing PROP intestinal absorption (Aungst and Hussain, 1992). Therefore, PROP bioavailability can be increased in rats by either partial bypassing hepatic first pass metabolism and/or increasing PROP intestinal absorption. Glucosamine, 2-amino-2-deoxy-D-glucose, (GlcN) is a monosaccharide compound generated by hydrolysis of chitosan or chitin and is considered as an important amino sugar building block for mucoproteins, mucolipids and mucopolysaccharides (Al-Hamidi et al., 2010; Kirkham and Samarasinghe, 2009; Xing et al., 2006). GlcN is produced naturally in the cells as GlcN-6-phosphate via hexosamine biosynthetic pathway and is involved in the formation of glycolipids, glycosaminoglycans, and proteoglycans. This sugar is further used for O-linked glycosylation of several proteins causing changes in the biological activity (Anderson et al., 2005; Roseman, 2001; Uldry et al., 2002). Furthermore, several studies have reported the involvement of GlcN in modulating drug pharmacokinetics and pharmacodynamics. For example, GlcN combination with other non-opioid analgesics was found to have synergistic, sub-additive, or additive effect as well as pain relieving effect depending on the ratios of GlcN and certain nonsteroidal anti-inflammatory drugs (NSAIDs) (Tallarida et al., 2003). Yet, most of the previously reported investigations were based on pharmacodynamics actions. It has been previously reported that GlcN, to some extent, is capable of competing with the paracetamol molecules on the catalytic pocket of the human CYP2E1 protein and concluded that GlcN increased paracetamol bioavailability by decreasing its metabolism (Qinna et al., 2015). The same study confirmed that GlcN also reduced hepatocyte injury after the administration of high doses of paracetamol (Qinna et al., 2015). Therefore, the aim of this study was to evaluate the effect of GlcN on PROP absorption in rats by investigating whether there is any possible pharmacokinetic interaction between PROP and GlcN in vivo, in situ and in vitro. PROP-GlcN pharmacokinetic interaction was compared with the other known interactors; rifampicin (RIFA), a hepatic enzyme inducer that has been demonstrated to accelerates the metabolism of several drugs (Brunton et al., 2006), and cimetidine (CIME), a hepatic enzyme inhibitor responsible for PROP metabolism (Reimann et al., 1981).

2.2. Animals The protocols for the animal study were approved by the Ethics Committee of the Research Council at the Faculty of Pharmacy and Medical Sciences, University of Petra (Amman, Jordan). Adult male and non-pregnant female Sprague Dawley rats were supplied and housed at the Animal House of University of Petra. Rats with average weight of (220 ± 20 g) were used. Rats were kept in air-conditioned environment under controlled temperatures (22–24C), humidity (55–65%), and photoperiod cycles (12 h light/12 h dark). Rats were kept fasting overnight (for 18 to 22 h) without free access to water, unless otherwise stated. All experiments were achieved in accordance with the Institutional Guidelines on Animal Use of University of Petra, which adopts the guidelines of the Federation of European Laboratory Animal Science Association (FELASA). 2.3. In vivo GlcN, CIME and RIFA effect on PROP serum levels in rats

2. Materials and methods

Reference solutions of PROP, CIME, RIFA and GlcN were prepared by dissolving an accurately weighed amount of each in distilled water to obtain 4, 1, 2 and 40 mg/ml, respectively. All solutions were freshly prepared on the day of experiment and were administered to fasting rats by stainless steel oral gavage needles (Harvard Apparatus, Kent, UK). For all experiments, rats were marked on tail for identification, weighed, and randomized into four groups consisting 7 rats/group. Before administration, blood samples were pooled from lateral tail vein of all grouped rats. Later, the rats received 1 ml of either water alone, GlcN 40 mg/ml, CIME 1 mg/ml or RIFA 2 mg/ml. After 30 min, all groups received 4 mg/ml of PROP solution. Rats in GlcN group were maintained on drinking water containing 25 g/l GlcN for three days prior to GlcN administration. Rats in RIFA experiment received a daily single dose of RIFA solution 2 mg/ml for two weeks before the day of experiment (Herman et al., 1983). For all groups, blood samples collected at different time intervals (0.25, 0.5, 1, 2, 3, 6, 8 and 10 h). Blood was left to clot, centrifuged for 10 min at 14000 rpm. Then, serum was separated, transferred directly into eppendorf tubes, and kept in freezer at −20 °C until HPLC analysis. Six independent experiments were performed and analyzed for each group (n = 6).

2.1. Materials

2.4. HPLC analysis

Potassium chloride and ethylene diamine tetra acetic acid were purchased from Acros organics (BVBA Geel, Belgium). Nanopur™ deionized water, methanol advanced gradient grade, and acetonitrile were obtained from Fisher Scientific Ltd. (Loughborough, UK). Potassium dihydrophosphate, penicillin, streptomycin, fetal bovine serum, sodium lauryl sulphate, and magnesium sulphate were all purchased from Sigma-Aldrich (St. Louis, Missouri), sodium bicarbonate from Merck (Darmstadt, Germany), phosphoric acid from Kyowa Medex Co. (Tokyo, Japan), and triethylamine from Tedia Company, Inc. (USA) were also used. All chemicals were of analytical grade, whereas solvents were of HPLC grade. For hepatocyte isolation and incubation, Hank's balanced salt solution (HBSS), without and with Ca2 + and Mg2 +, and Williams's medium E were obtained from Invitrogen

HPLC system used from Thermo Separation Products (Waltham, MA, USA) was set at a wavelength of 214 nm and coupled to a BDS hypersil C18 column (150 × 4.6 mm2 and particle size 5 μm) from Thermo Electron Corporation (San Jose, CA, USA) with a flow rate of 1 ml/min. The mobile phase was prepared as a mixture of acetonitrile, methanol and triethylamine phosphate solution (15: 32.5: 52.5, v/v). Triethylamine phosphate solution was prepared by the addition of 900 μl triethylamine to 1 l of water then pH was adjusted to 2.75 using phosphoric acid. A volume of 100 μl of serum or Krebs buffer samples (pH 7.2) were transferred to tubes followed by the addition of 150 μl of the internal standard sildenafil (5.0 μg/ml) dissolved in acetonitrile. Samples were vortexed for 1 min and centrifuged at 14000 rpm for 15 min. The supernatant was injected to HPLC using 25 μl injection 138



(3 cm in length) were prepared from the intestine of adult male Sprague Dawley rats (n = 4 rats) they were randomly divided into three groups (n = 6 in each group) and incubated in Krebs buffer in a shaking water bath (60 cycles/min) for 10 min at 37 °C. The sacs were then transferred to soaking solutions; PROP only, PROP with GlcN, and PROP with SLS, and were incubated again. At the appropriate time points (20, 40 and 60 min) of incubation, sacs were removed and samples were drawn from inside the sacs, placed in Eppendorf tubes and kept frozen until analysis (Chan et al., 2006; Qinna et al., 2015).

volume. The method was validated and accuracy, precision, linearity, stability and recovery were within the accepted range according to EMEA guidelines of bioanalytical method validation (EMEA, 2012). Briefly, the calculated lower limit of detection and lower limit of quantification were 15 and 50 ng/ml, respectively. Linearity was studied over a concentration range of 50.0–3000 ng/ml. Calibration curves of PROP were analyzed and correlation coefficients were calculated. R2 values were > 0.99 for all six calibrations and for linearity curves of PROP in serum and in Krebs buffer. The method was linear over this concentration range (Al Shaker et al., 2017).

2.8. Rat hepatocytes isolation and incubation 2.5. Anesthesia and surgical protocol All perfusion buffers (pH 7.2) were freshly prepared using sterile technique and were warmed for 30 min in an Elmasonic S water bath (Elma, Germany) at 42 °C with an optimal temperature. Perfusion buffer I was prepared by adding 0.9 and 0.5 mM of MgCl2 and EDTA, respectively, to HBSS, without Ca2 + and Mg2 +. Perfusion buffer II was prepared by adding 0.5 mM Tris base to HBSS (with Ca2 + and Mg2 +) followed by dissolving 1000 U of collagenase II in 300 ml of perfusion buffer II. This buffer was kept warm in water-bath and used within 30 min after preparation. William's complete Medium was prepared by the addition of 2 mM of L-glutamine, 100 IU/ml penicillin, 100 mg/ml of streptomycin and 5% fetal bovine serum to William's Medium E (Shen et al., 2012). Healthy untreated adult male Sprague Dawley rat was anaesthetised (Section 2.5) and the abdominal contents were displaced to its left side in order to expose the liver. Hepatic portal vein (HPV) was revealed and two loose ligatures were passed, one around the HPV, while the other around the inferior vena cava (IVC). An 18-gauge angiocath by Becton Dickinson (Mountain View, CA, USA) was inserted into the HPV, whereas the perfusate tubing was connected to the needle and infusion was initiated at a flow rate of 10 ml/min with pre-warmed 37 °C perfusion buffer I. Once the liver was blanched to a light-brown color and all lobes began to swell, a cut at IVC was made to allow buffer efflux. Thereafter, the chest of the rat was cut to place a second cannula of 18-gauge connected to a soft tube into the vena cava above the liver in order to enable a recirculating system. All loose ligatures were tied securely (Sahin and Rowland, 1998). Perfusion solution was switched to perfusion buffer II plus collagenase II, flow rate was then increased to 25 ml/min and the liver became pale in color. The recirculating perfusion mode with collagenase solution lasted for 15 min. Once the liver looked mushy, it was dissected. Finally, the liver was minced, placed in pre-chilled sterile beaker with 20 ml collagenase and transferred to laminar flow hood. Hepatocyte cell isolation procedure was the same as that described elsewhere (Shen et al., 2012). The isolated hepatocytes were transferred into a 98 well plate and incubated for 20 h in a CO2 incubator at 37 °C. Rat hepatocytes were later pre-treated with GlcN (4, 40 and 200 mM), CIME (5 μM) or RIFA (50 μM), which were dissolved in the incubation medium to obtain a final concentrations of GlcN (2.87, 28.6 and 143.4 mg/ml), CIME (0.005 mg/ml) and RIFA (0.164 mg/ml). After 30 min, 0.0236 mg/ml PROP was added. Samples were then collected after 15, 30, 60 and 120 min of incubation and placed in Eppendorf tubes and prepared for PROP analysis.

A perfusion system consisting of an anesthesia system provided from SomnoSuite small Animal Anesthesia System, Kent Scientific Corporation (Torrington, USA) that is linked to a Dual flow oxygen concentrator for oxygen bar (Hebei, China), an Elmasonic S water bath (Elma, Germany) and a peristaltic perfusion pump (BT100-2 J, Hebei, China) were used. The rat was placed on a surgical board of Plas Labs (Lansing, MI, USA) above a heating pad maintained at 37 °C, and was anaesthetized by isoflurane (5% for induction and 2.5% for maintenance). Anesthesia was maintained while experiment and monitored by toe pinch. Following sterilization, a midline longitudinal incision was made 1 cm below sternum down to 1 cm above tail in the pelvic region, followed by two mid-transverse incisions to left and right of the midline to expose rat viscera. 2.6. In situ single-pass intestinal perfusion (SPIP) All drugs used in SPIP experiment (PROP, GlcN, CIME, and RIFA) were dissolved in normal saline solution at final concentration of 1, 10, 1, and 2 mg/ml, respectively. As for the procedure of SPIP, nonpregnant fasting female rat was anaesthetised and its intestine was exposed surgically as described in (Section 2.5). Female rat was used in such experiment in order to obtain large amount of blood and since male rat did not give much amount for the entire one hour procedure. A semi-circular incision was made and an inlet silicon tube (0.3 cm diameter) was inserted into the duodenum 4 cm away from the pylorus. A second incision was made in the ileocecal end and an outflow silicon tube was fitted. Both tube ends were tied securely using a surgical suture from Atramat® (Mexico City, Mexico) and linked with suitable tube fittings then inserted in the peristaltic pump at a flow rate preset to 3.6 ml/min. Initially, intestinal segments were rinsed with pre-warmed (37 °C) saline washing solution for 20 min until the outlet solution was clear then perfusion solution was switched. A pre-sample drug was drawn from perfusion solution as well as zero-time blood sample was drawn from lateral tail vein of the rat before perfusion solution was switched. Perfusion solution was perfused for 60 min and blood samples were quantitatively pooled from tail at different time intervals namely; at 10, 20, 30 and 60 min. All blood samples were left to clot, centrifuged for 10 min at 14000 rpm, then serum was separated, transferred directly into eppendorf tubes, and kept frozen at −20 °C until analysis. Care was taken throughout the experiment to avoid disturbance in the circulatory system, and the exposed intestinal segments were kept moist with body tempered saline (37 °C).

2.9. Pharmacokinetics and data analysis 2.7. Everted rat intestinal sac (ERIS) Pharmacokinetic parameters for PROP concentration in serum were calculated by non-compartmental analysis (NCA) using Kinetica™ software version 5 provided from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Pharmacokinetic parameters used were maximum serum concentration (Cmax) that is observed at the time (Tmax) following drug administration. The area under the curve (AUC) was calculated using the trapezoidal rule, whereas the area under the first moment curve (AUMC) was calculated by multiplying drug concentration in the serum by time. The mean residence time (MRT) was calculated by dividing

Three PROP solutions were prepared by dissolving 10 mg PROP in 100 ml Krebs buffer (pH 7.2) to obtain a final concentration of 0.1 mg/ ml, whereas two bathing solutions were prepared by dissolving 100 mg GlcN, and 1 mg sodium lauryl sulphate in two PROP solutions prepared to give a final concentration of 1 and 0.01 mg/ml, respectively. SLS is known to be an anionic surfactant that is used as an emulsifier (Lee and Maibach, 1995). Krebs buffer preparation and ERIS technique was conducted as described elsewhere (Qinna et al., 2015). Once the sacs 139



AUMC over AUC. Kel, represents the fraction of drug eliminated per time and is determined by the slopes of the terminal segments of logarithmically transformed serum levels against their corresponding times, where slope equals (Kel). Elimination half-life(t1/2) was then calculated using the following equation t1/2 = 0.693/Kel.

(a) PROP alone

Serum PROP (ng/ml)

700

2.10. Data analysis Rat intestinal effective permeability (Peff) values were estimated by Nelder–Mead algorithm of the Parameter Estimation module using SimCYP program V13, Rat model. SimCYP program was used under academic license from SimCYP Ltd., Sheffield, U.K. (Lic. # CLCLID – AKDI – LEEE – FECI). The Nelder–Mead method, which is also called downhill simplex, is a commonly used non-linear optimization algorithm. This was done by searching for the best parameter values that produce serum concentration that match the actual serum concentration at the same time. The objective function is the weighted sum of squared differences of observed and model predicted values. Polar surface area was used first to predict an initial estimate of Peff. All other physicochemical factors used in calculations were obtained from the literature and were kept constant during the minimization processes. In vitro dissolution rate, in vivo clearance and volume of distribution were input from actual dissolution and serum profiles. Statistical comparisons were obtained using one-way ANOVA followed by Tukey's to compare between more than two groups using SPSS statistical software, (IBM, USP); version 22. Each data point represents the mean ± SEM. p-Value < 0.05 was considered statistically significant (p < 0.05).

PROP with 100 mg/kg GlcN

600

PROP with 200 mg/kg GlcN

500 400 300 200 100 0 0

2

4

6

8

10

Time (h)

(b)

Serum PROP (ng/ml)

1400

3. Results 3.1. GlcN administration lowered PROP AUC and Cmax in vivo levels in rats

PROP alone

1200

PROP with CIME

1000

PROP with RIFA

800 600 400 200 0

Different sets of experiments were carried out to study the effect of GlcN on PROP serum levels in rats. The experiments were performed to compare the effect of different doses of GlcN (100 and 200 mg/kg) on the pharmacokinetic parameters of a single dose of 20 mg/kg PROP. The results showed that GlcN at 200 mg/kg significantly decreased PROP AUC and Cmax by 43% (p < 0.01) and 33% (p < 0.05), respectively (Table 1, Fig.1a). In comparison to a hepatic enzyme inducer, RIFA (9 mg/kg) did not change PROP AUC and Cmax (p > 0.05), whereas CIME (5 mg/kg), a hepatic enzyme inhibitor, significantly increased PROP Cmax by 86% (p < 0.01) in addition to nearly 20% change in AUC, although not statistically significant (Table 1, Fig. 1b). Conversely, neither GlcN nor CIME affected Tmax of PROP. Based on the above data, Peff values for PROP were reduced by 50% when administered with 200 mg/kg GlcN but not with 100 mg/kg. In addition, the PROP clearance increased without changing the Vd parameter (Table 2). In addition, the observed PROP serum concentra-

0

2

4

6

8

10

Time (h) Fig. 1. In vivo serum concentration versus time curves of PROP in rats after a single oral dose of PROP (20 mg/kg), PROP with GlcN (100 or 200 mg/kg) (a), CIME (5 mg/kg) or RIFA (9 mg/kg) (b). Each data point represents the mean ± SEM (n = 6).

tions were correlated with SimCYP predicted values and the results showed high coefficient of determinations (Fig. 2a and b). These values were consistent with low objective function values (Table 2). 3.2. GlcN increased serum PROP following in situ SPIP in rats Since GlcN lowered PROP AUC and Cmax following oral administration, it was decided to study if GlcN modulates PROP intestinal

Table 1 GlcN, CIME and RIFA effect on PROP pharmacokinetic parameters after an oral dose of PROP (20 mg/kg), PROP with GlcN (100 or 200 mg/kg), CIME (5 mg/kg) or RIFA (9 mg/kg) in rats. Each data point represents the mean ± SEM (n = 6). Treatment

AUC (ng/ml ∗ h)

Cmax (ng/ml)

AUMC (ng/ ml ∗ h2)

MRT (h)

T1/2 (h)

Kel (1/h)

Percent change in AUC of PROP

Percent change in Cmax of PROP

PROP (20 mg/kg) PROP with GlcN (100 mg/kg) PROP with GlcN (200 mg/kg) PROP with CIME (5 mg/ kg) PROP with RIFA (9 mg/ kg)

1540 ± 99 1427 ± 170

593 ± 45 615 ± 63

4172 ± 387 4096 ± 776

2.7 ± 0.2 2.7 ± 0.3

1.8 ± 0.2 2.4 ± 0.4

0.40 ± 0.04 0.34 ± 0.07

– −7%

– + 4%

881 ± 123⁎⁎

394 ± 37⁎

2375 ± 499

2.5 ± 0.3

1.9 ± 0.3

0.41 ± 0.07

−43%

− 33%

1854 ± 151

1105 ± 158⁎⁎

4586 ± 303

2.5 ± 0.1

+20%

+ 86%

−14%

+ 12%

⁎ ⁎⁎

1324 ± 33

664 ± 14

4594 ± 410

3.5 ± 0.3

p < 0.05. p < 0.01 in comparison to PROP alone.

140

2.4 ± 0.1 ⁎

2.8 ± 0.2

0.29 ± 0.02 ⁎

0.25 ± 0.02

⁎



isolated intestinal tissue without the contribution or interference of the liver. PROP intestinal flux in ERIS experiments was found time dependent and increased over time. In the presence of GlcN, the flux of PROP was not significantly affected (p > 0.05) in comparison to PROP alone (Fig. 4).

Table 2 Peff, clearance, and volume of distribution results in rats of PROP (20 mg/kg), PROP with GlcN (100 or 200 mg/kg) by the Nelder–Mead algorithm using the SimCYP program. Treatment

Peff (× 10−4 cm/s)

Clearance (CL/F) (l/h)

Volume (Vd/F) (l/ kg)

Objective function

PROP (20 mg/ kg) PROP with GlcN (100 mg/ kg) PROP with GlcN (200 mg/ kg)

25.28

23.88

0.49

1.06

23.99

25.11

0.43

0.00

12.65

35.75

0.55

1.37

3.3. GlcN decreased PROP disposition/distribution into hepatocytes Since SPIP technique measures serum PROP following intestinal perfusion, then it is likely that PROP disposition into liver (before its metabolism) could be factor that GlcN modulates. Therefore, PROP disposition/distribution into hepatocytes was evaluated with and without GlcN. Upon treating hepatocytes with PROP, it was estimated that nearly 25% of PROP concentration diffuses into cells and the remaining percent of PROP (~ 6000 ng/ml) was left in the incubation medium. In addition, PROP distribution into hepatocytes did not change within 30 to 120 min. This might indicate that distribution reached saturation earlier than 30 min increasing concentrations of GlcN in the incubation medium, however, significantly decreased PROP disposition into hepatocytes. This was clearly observed when 200 mM of GlcN was incubated with PROP, as the PROP disposition into hepatocytes was reduced by (25–40%) during 30–120 min of incubation with the maximum decrease at 60 min post incubation (p < 0.001) (Fig. 5). In comparison to liver enzymes inhibitor, CIME (5 μM), but not RIFA (a liver enzyme inducer), significantly decreased PROP disposition over the same period (p < 0.01) (Fig. 5).

(a) PROP predicted value

800

Serum PROP (ng/ml)

PROP with 100 mg/kg GlcN predicted values PROP with 200 mg/kg GlcN predicted value

600

PROP observed values PROP with 100 mg/kg GlcN observed values

400 PROP with 200 mg/kg GlcN observed value

200

4. Discussion A drug such as PROP is considered highly permeable and highly metabolized since the extent of its absorption and metabolism is > 90% of the administered dose based on a mass balance determination or in comparison to an intravenous reference dose (Benet et al., 2008; Custodio et al., 2008). Our permeability results are consistent with the in vivo findings and have indicated that oral administration of GlcN decreased PROP serum levels in a dose-dependent manner as evident by a significant reduction in the calculated Cmax and AUC values. Since PROP was used as a powder dissolved in solution and not as a tablet, hence, dissolution parameter would be excluded and the intestinal effective permeability is the main factor responsible for PROP bioavailability in the current research. Therefore, Peff values were optimized by SimCYP program to predict the actual average serum PROP profile in order to estimate Peff, Vd and clearance. This optimization takes into account all ADME processes since we are dealing with oral dosing. Indeed, clearance and volume parameters were optimized too. The predicted PROP concentrations were in a good fit with the observed serum levels showing also a low objective function. This type of topdown calculation is stronger than bottom-up one; because it fits unknown parameters to actual data rather than predicts data using known parameters. In other words, top-down calculations are a selfvalidating method. GlcN at 100 mg/kg did not change Peff or clearance of PROP, whereas GlcN at 200 mg/kg decreased Peff, and increased clearance, indicating a dose dependent effect. Although PROP along with other lipophilic amine drugs are substrates for transporter proteins, however, it has been reported that little consequence on changing their bioavailability is expected due to their permeability properties (Wu and Benet, 2005). Thus, we can explain that the decreased PROP serum concentration, as evident by the decreased AUC and Cmax, after the administration of GlcN (200 mg/kg) is mainly attributed to decreased intestinal permeability of PROP. This explanation was emphasized by calculating Peff and clearance of PROP using SimCYP program where GlcN decreased Peff nearly to the half as well as increased its clearance. According to the pH partition theory, absorption of oral drugs takes place mainly by a passive diffusion of the un-ionized form of the drug molecule through the lipophilic intestinal membrane (Palm et al., 1999;

0 0.0

2.0

4.0

6.0

8.0

10.0

Time (h)

(b) 700

Observation

600 R² = 0.8958

500

R² = 0.9657

400

300 R² = 0.9704 200 100 0 0

100

200

300

400

500

600

Individual Predictions Fig. 2. Measured (observed) and predicted serum mean concentration profiles of PROP, PROP with GlcN (100 mg/kg or 200 mg/kg) versus SimCYP-predicted values(a), along with the trend lines of individual predictions when fitted with observations (b).

absorption. Thus, an in situ SPIP technique was used and showed that serum PROP concentrations increased in a time-dependent manner (Fig. 3), when GlcN was infused with PROP, however, serum PROP concentrations in systemic blood significantly increased over time (p < 0.05) more than when PROP was infused alone. In order to evaluate if hepatic enzymes have a role in such technique and modulate PROP serum levels, CIME, a liver enzyme inhibitor, and RIFA, a liver enzyme inducer, were each infused with PROP. CIME slightly increased serum PROP concentrations over time whereas RIFA, decreased serum PROP overtime (Fig.3). The ERIS technique usually measures intestinal flux of drugs from 141



1200 PROP only PROP with GlcN

Serum PROP (ng/ml)

1000

*

PROP with CIME

PROP with RIFA

800 600

*

400 200 0 0

20

40

60

Time (min) Fig. 3. Time dependence curves of serum PROP concentration in rats using SPIP. PROP (1 mg/ml), PROP with GlcN (10 mg/ml), CIME (1 mg/ml), or RIFA (2 mg/ml) were infused. The data are presented as mean ± SEM (n = 6). Significant effect was seen between PROP and PROP with GlcN (*p < 0.05).

Intestinal Flux PROP (ng/ml)

60000 50000

PROP alone

120

PROP with GlcN

110

PROP with SLS

100

40000

90

30000

80

20000

70

*

*

*

60

10000

**

50 0

0 0

20

40

60

30

80

Time (min) Fig. 4. Flux of PROP in the everted rat intestinal sac (3 cm in length) versus time. Intestinal sacs were bathed either with PROP (0.1 mg/ml), PROP with GlcN (1 mg/ml) or sodium lauryl sulphate (0.01 mg/ml) for 60 min. Samples were taken from each sac at each time point. The data are presented as mean ± SEM (n = 6). Insignificant effect was seen between PROP and PROP with GlcN (*p > 0.05).

60 Time (h)

90

120

Hep

Hep+GlcN 4 mM

Hep+GlcN 40 mM

Hep+GlcN 200 mM

Hep+CIME

Hep RIFA

Fig. 5. Percent reduction in PROP disposition into primary isolated hepatocyte over time. Cells were incubated with PROP (20 μM), PROP with GlcN (4, 40 and 200 mM), CIME (5 μM), or RIFA (50 μM) for 120 min (b). The data are presented as mean ± SEM (**, p < 0.01; ***, p < 0.001). PROP concentration in the medium is equivalent to 7924 ± 202 ng/ml.

Ungell et al., 1998). Therefore, PROP given on an empty stomach would be absorbed mainly after the passage into the duodenum where pH is > 7 in an un-ionized form (Hurst et al., 2007; Liedholm and Melander, 1990; Palm et al., 1999). Moreover, GlcN has been shown to be absorbed throughout the gut, where the highest absorption occurs in the small intestine, mainly the duodenum (Ibrahim et al., 2012). Since the absorption sites of PROP and GlcN are different (colon and duodenum, respectively), then it may be suggested that passing both GlcN and PROP in acidic media before reaching their absorption sites resulted in lowering Peff of PROP. On the other hand, in SPIP where the pH of the infusion buffer of both PROP and GlcN was controlled to be 7.2, GlcN increased PROP permeability and absorption. In addition, since SPIP technique is affected by hepatic first-pass metabolism (Li et al., 2011), and PROP, a cationic lipophilic drug, we studied GlcN effect on PROP disposition into rat hepatocytes. GlcN was found to decrease PROP disposition/distribution into hepatocytes by 25–40% over time in a concentration-dependent manner. Recently, Zheng et al. have shown that PROP does not actually exhibit an active transport, as it was thought earlier (Dudley et al., 2000; Kubo et al., 2013), but rather passive diffusion (Zheng et al., 2015). Also, this

process is found to be pH-dependent. These results may suggest that GlcN interferes with PROP disposition/distribution into hepatocytes since GlcN binds to CYP2E1 protein and interferes with metabolism (Qinna et al., 2015). In comparison to CIME, which is also a hepatic enzyme inhibitor, both CIME and GlcN showed similar behavior of PROP disposition into hepatocytes. 5. Conclusion GlcN decreased PROP serum levels in rats in a dose-dependent manner as evident by a significant reduction in the calculated Cmax and AUC values. Such interaction might be attributed to decreased rat intestinal effective permeability and enhanced clearance of PROP in the presence of GlcN. The involvement of first pass metabolism seems to also control PROP due to GlcN interaction, however, further investigations are still warranted to explain the in vitro inhibitory action of GlcN on PROP disposition into hepatocytes and the involvement of GlcN in the intestinal and hepatic metabolizing enzymes of PROP at different 142



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