May 1, 1981 - Divisions of Medical Oncology and Pediatric Oncology, and Laboratory of Clinical Microbiology, Sidney. Farber Cancer Institute; Division ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1982, p. 146-150 0066-4804/82/010146-05$02.00/0
Vol. 21, No. 1
Effect of Acyclovir and Interferon on Human Hematopoietic Progenitor Cells LEROY M. PARKER, JEFFREY M. LIPTON, NEIL BINDER, E. LINN CRAWFORD, MICHELE KUDISCH, AND MYRON J. LEVIN* Divisions of Medical Oncology and Pediatric Oncology, and Laboratory of Clinical Microbiology, Sidney Farber Cancer Institute; Division of Hematology and Oncology, Children's Hospital Medical Center; and Departments of Medicine and Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 Received 1 May 1981/Accepted 6 October 1981
Continuous in vitro exposure of human bone marrow cells to acyclovir (-200 ,uM) or human leukocyte interferon (-250 U/ml) caused 50% inhibition of granulocyte colony-forming cell differentiation. Colonies expressed in the presence of either agent were reduced both in size and number. Erythroid progenitors were more resistant than granulocyte progenitors to the antiproliferative effects of acyclovir. Progenitor cells of patients recovering from cytotoxic chemotherapy were no more sensitive to the effects of acyclovir or interferon than were cells obtained from patients before chemotherapy.
Acyclovir (ACV) [9-(2-hydroxyethoxymethyl)guanine] is being evaluated as topical or systemic therapy in local and disseminated herpesvirus infection. ACV inhibits herpes simplex virus types I and II and varicella-zoster virus at concentrations that have little effect on the division of mammalian fibroblasts (1, 4, 6, 19, 24) or lymphocytes (25). In mice, rabbits, and guinea pigs infected with herpes simplex virus types I and II ACV is both effective and nontoxic (7, 12, 20, 24). The favorable therapeutic effect of ACV is explained by its efficient phosphorylation to an active form by cells which contain virusspecified kinases (6, 8) and the high affinity of herpesvirus-specified DNA polymerase for this active ACV triphosphate (6). Although ACV is highly selective in comparison with other antimetabolites, we previously found that human fibroblasts and mitogen- and antigen-stimulated human lymphocytes can be inhibited by higher levels (100 to 200 ,uM) of ACV (14). McGuffin et al. (15) recently reported -20% inhibition of human granulocyte colony-forming unit (CFUC) differentiation at ACV concentrations of 100 puM. Because higher doses of ACV may be required to treat more resistant herpesviruses in patients myelosuppressed by chemotherapy, we have measured the effect of higher doses of ACV on human CFU-Cs. The myelosuppression caused by ACV was compared with that of human leukocyte interferon which is known to be myelosuppressive in man. To determine whether the kinetic state of the marrow altered the susceptibility of myeloid precursors we studied samples obtained before and after chemotherapy. We also determined possible effects of ACV on the immature erythroid progenitor, the 146
erythroid burst-forming unit (BFU-E), and its progeny, the erythroid colony-forming unit
(CFU-E). MATERIALS AND METHODS Patient samples. Bone marrow cells were obtained from seven patients (five with glioblastoma, one with melanoma, one with oat-cell carcinoma) before treatment with intensive chemotherapy and autologous bone marrow infusion. Marrow cells were also obtained from three patients with oat-cell carcinoma after combination chemotherapy, from one patient with acute lymphocytic leukemia in remission, and from two normal donors. Peripheral blood cells enriched for granulocyte progenitors during the period of leukocyte recovery after chemotherapy (23) were obtained from two patients with oat-cell carcinoma. Peripheral blood cells were also obtained from one patient with chronic myelogenous leukemia and acute lymphocytic leukemia in remission. Assay for peripheral blood and bone marrow CFUCs. Bone marrow and peripheral blood cells were processed as previously described and stored in 10% dimethyl sulfoxide in the vapor phase of liquid nitrogen (23, 26). Thawed bone marrow and peripheral blood colony-forming cells were assayed in 35-mm culture dishes (Falcon Plastics, Cockeysville, Md.) by the technique of Pike and Robinson (21) with the following modifications. Alpha medium without nucleosides or nucleotides (alpha minus; GIBCO Laboratories, Grand Island, N.Y.) was used in place of McCoys 5A medium. Mononuclear cells isolated from normal donors on a Ficoll-Hypaque gradient were irradiated (1,000 rad) with a cell irradiator containing a 137cs source (Gammacell; Atomic Energy Co. of Canada, Toronto) before combining them into agar-based feeder layers; the 1-ml upper layer contained 0.5 x 10i to 3.0 x 105 cells in 1.2% premium-grade methylcellulose (lot M0819728; Dow Chemicals, Midland, Mich.) in place of agar. Cultures were incubated for 14 days at
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37°C in a fully humidified 7.5% CO2 atmosphere. Colonies were counted at 7 and 14 days with an inverted microscope. Colonies containing 20 to 50 cells and those with more than 50 cells were scored separately. Assay for bone marrow CFU-Es and BFU-Es. Bone marrow cells were collected in preservative-free heparin, and mononuclear cells were separated on a FicollHypaque gradient. These were plated in plasma clots at a final concentration of 5 x 105 cells per ml by adding 0.1 ml of cell suspension in alpha-minus medium and 5% fetal calf serum (Flow Laboratories, Inc., Rockville, Md.) to 0.8 ml of the erthropoietin-dependent plasma clot system of McLeod and co-workers (16), as modified by Clarke and Housman (3) and Nathan and co-workers (18). Erthropoietin (Connaught step III; specific activity, approximately 5 IU/ ml of protein; Connaught Laboratories, Willowdale, Ontario) was used in a final concentration of 2 IU/ml of plasma clot. Cultures were incubated at 37°C in 100o humidity and 5% CO2. On day 7 and between days 11 and 14, clots were fixed and stained (red cell clusters composed of eight cells counted on day 7), and CFU-E and BFU-E colonies (composed of three or more clusters counted on days 11 to 14) were enumerated as previously described (18). Determination of the inhibitory effect. ACV was supplied by Burroughs Wellcome Co. (Research Triangle Park, N.C.). Human leukocyte interferon (HLI) was supplied by June Dunnick of the Antiviral Substance Program of the National Institute of Allergy and Infectious Diseases. In the granulocyte progenitor system, serial dilutions of ACV and HLI were added to the methylcellulose overlayers. Each concentration was tested in three replicate dishes; the average standard error of the mean for 115 triplicate assays was 13.5 ± 9%6. Inhibition was determined by averaging individual patient sample values from 3 to 10 separate experiments. In the erythroid progenitor system, serial dilutions of ACV were added to the plasma clot culture in three to six replicate wells. Results were expressed as the percentage of the untreated control. The surviving fraction was plotted as a function of drug dose. Residual ACV activity in culture supernatants was determined by radioimmunoassay (22) at Burroughs Wellcome Co. (P. De Miranda), and residual HLI activity was determined in a microtiter assay of antiviral activity with vesicular stomatitis virus and human embryonic lung fibroblasts (11). There was no loss of either type of antiviral activity from supermatants of cultures incubated for up to 14 days. Statistcal analysis. Mean values were calculated and compared by the Student's t test.
RESULTS Effect of ACV on CFU-Cs. Continuous exposure of human bone marrow cells to ACV caused a dose-dependent decrease in the number of CFU-C colonies observed at 7 (data not shown) and 14 days (Fig. 1B). CFU-C expression in the presence of 100 ,uM ACV was 73 + 18% of the control (P < 0.05); at 200 ,M ACV, growth was 50 ± 17% of the control (P < 0.01). An additional effect of ACV on CFU-C differentiation was observed when the number of large
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(>50 cells) and small (20 to 50 cells) colonies was compared. Control cultures contained 85% large colonies, whereas cultures exposed to 200 ,uM ACV had only 66% (P < 0.01). Bone marrow from three patients who had received chemotherapy contained 139 ± 45 CFU-Cs per 105 cells plated, whereas the bone marrow from five patients before chemotherapy contained 30 ± 14 CFU-Cs per 105 cells (P < 0.001). ACV inhibited CFU-C colony growth in these two patient populations to a similar degree. Marrow obtained before chemotherapy yielded 66 ± 20%o of the control number of CFUC colonies in the presence of 100 ,uM ACV, whereas post-chemotherapy marrow yielded 83 ± 8% (0.05 < P < 0.1) of the control number. Peripheral blood CFU-Cs obtained from patients 14 to 24 days after combination chemotherapy demonstrated ACV susceptibility very similar to CFU-Cs obtained from bone marrow (Fig. 1A). Effect of HLI on CFU-Cs. HLI produced dosedependent inhibition of bone marrow and peripheral blood CFU-Cs (Fig. 1C and D). In the presence of 100 U of HLI per ml, bone marrow CFU-C colony growth was 74% of the control; there was no difference between untreated (77 ± 17%) and recovering (68 ± 23%) bone marrow (P > 0.1). Similarly, peripheral blood and bone marrow CFU-Cs from recovering patients were equally sensitive to HLI. Effect of ACV on bone marrow erythroid progenitors (BFU-Es and CFU-Es). Figure 2 depicts the differentiation of CFU-Es and BFU-Es in the presence of ACV. CFU-E colony growth was unaffected by ACV at concentrations as high as 1,000 ,uM. BFU-E colony growth was also relatively resistant; colony numbers derived from BFU-Es were inhibited only 16% at 100 p.M (differs from control with P > 0.3), and colony numbers remained 80%o of normal at 500 ,uM (P > 0.1). There was no difference between patient and normal controls in these studies or in pilot studies (data not shown). DISCUSSION Optimal use of ACV and HLI to treat opportunistic infections with human herpesviruses, especially cytomegalovirus, may require prolonged maintenance of high serum levels of virus inhibitor (1, 2, 17). Patients at high risk for these infections frequently receive intensive chemotherapy for malignancy or are bone marrow transplant recipients. Recovering bone marrow in these patients is rapidly proliferating and therefore potentially very sensitive to antiviral agents which might inhibit cell division (5). The present studies were an attempt to reproduce this clinical situation. We exposed committed hematopoietic marrow progenitors (CFU-Cs, CFU-Es, and BFU-Es) to the stress of contin-
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Leukocyte Interferon (units/ml) FIG. 1. Effect of ACV (A and B) and HLI (C and D) on human bone marrow (A and B) and peripheral blood (A and D). Data from all patient samples at each dose level were combined and plotted as a percentage of the control (no drug added). Mean values at each drug concentration are indicated by a bar; brackets enclose 1 standard deviation of the mean. Bone marrow samples without drug added grew 85.6 ± 78.1 CFU-Cs per 105 nucleated cells plated (10 experiments). Peripheral blood grew 29.6 ± 14.5 CFU-Cs per 105 cells. (0) Samples obtained from patients before intensive chemotherapy. (0) Samples obtained from patients after chemotherapy. Peripheral blood CFU-Cs were studied only after chemotherapy when adequate numbers were present for assay.
ued in vitro proliferation in the presence of various concentrations of ACV. Bone marrow was obtained from patients both before and after treatment with chemotherapy. HLI was studied in the CFU-C assay for comparison. These agents were present throughout the process of colony development. ACV had a significant inhibitory effect on CFU-Cs at concentrations greater than 100 ,uM, but there was no evidence that bone marrow from patients after chemotherapy was more susceptible to ACV than pretreatment marrow. Peripheral blood and bone marrow CFU-Cs were equally sensitive. However, erythroid progenitors were less sensitive to ACV. It should also be noted that the more primitive progenitors, CFU-Cs and BFU-Es (9), were inhibited by ACV to a greater extent than the more mature CFU-Es. This suggests that earlier progenitors,
such as the common myeloid progenitor or the pluripotent stem cell, might be inhibited to an extent greater than these assays predict. It would appear from these in vitro measurements that myelosuppression should not be a complication of extended ACV therapy for herpes simplex virus and varicella-zoster virus, for which the 50%o infective doses are less than 1.0 and 5.0 p,M, respectively (1, 4). Nor is there any indication that preceding chemotherapy will alter this susceptibility. On the other hand, the levels of ACV required to inhibit cytomegalovirus (>100 ,M) (4) were inhibitory for committed granulocyte progenitor cells. HLI at 100 U/ml was as inhibitory to CFU-C development as ACV at a 100 FM concentration. This level of HLI circulating in the blood of humans is associated with reversible myelosuppression (2, 10, 17). A combination of ACV and HLI may result in a
ACV AND HLI EFFECTS ON CFU-Cs AND BFU-Es
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50 F
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Acyclovir (,iM) FIG. 2. Effect of ACV on human bone marrowderived CFU-Es and BFU-Es. The mean values of three to six determinations from two normal controls and one patient are plotted as percentages of the control (no drug added). The concentration of ACV is plotted on a logarithmic scale on the horizontal axis. Control bone marrow samples for CFU-Es were 236 14, 98 ± 23, and 57 ± 18 per 105 mononuclear cells; for BFU-Es they were 33 ± 5.2, 47 ± 12, and 54 ± 7 per 10' mononuclear cells. Mean values at each drug concentration are indicated by a bar; brackets enclose 1 standard deviation of the mean. (0) CFU-Es and (0) BFU-Es.
superior antiviral effect with a reduction in myelosuppressive and other toxicity (13). However, extrapolation of these in vitro studies to potential clinical effects should be made with caution. Inhibition of committed stem cell precursors followed continuous exposure to ACV or HLI, which differs from typical clinical dose schedules that produce wide variations in drug levels. Furthermore, a fall in polymorphonuclear leukocytes might not occur in vivo because of other regulatory influences on stem cell populations. It must also be emphasized that the in vitro culture assays measure cellular differentiation and give no direct information on the effect of ACV and HLI on progenitor self-renewal. ACKNOWLEDGMENTS This study was supported by Public Health Service Clinical Center grant 1-POI-CA-19589-06 from the National Cancer Institute, by Public Health Service grants HL-07146, AM15322, and CA-18662 from the National Institutes of Health, and by the Louis and Sidelle Bruckner Memorial Fund and the Amy Clare Potter Memorial Fund. J.M.L. is a Dyson Foundation investigator in pediatric oncology. We also thank David G. Nathan for his continued support.
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