Dec 30, 1988 - (zidovudine) was reported in clinical studies to prolong survival and reduce the frequency and severity of opportu- nistic infections in patients ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1989, 0066-4804/89/060920-04$02.00/0 Copyright C) 1989, American Society for Microbiology
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Vol. 33, No. 6
920-923
Human Immunodeficiency Virus Inhibition Is Prolonged by 3'Azido-3'-Deoxythymidine Alternating with 2',3'-Dideoxycytidine Compared with 3'-Azido-3'-Deoxythymidine Alone STEPHEN A. SPECTOR,* DANIEL RIPLEY, AND KAREN HSIA Division of Infectious Diseases, Department of Pediatrics, and Center for Molecular Genetics, University of California, San Diego, San Diego, California 92103 Received 30 December 1988/Accepted 29 March 1989
The inhibition of the lymphadenopathy-associated virus strain of human immunodeficiency virus (HIV) by alternating regimens of two dideoxynucleosides, 3'-azido-3'-deoxythymidine (AZT) (zidovudine) and 2',3'dideoxycytidine (ddC), was determined in CEM cells. Cultures infected with virus for 2 h were treated with clinically achievable concentrations of AZT, ddC, or a 3-day-alternating regimen of AZT and ddC. Media were completely changed every 3 days and replaced with antiviral agent, and virus production was assayed by p24 antigen and virus-specific DNA. Cells treated with no antiviral agent exhibited breakthrough infection by day 6 in culture, whereas cells treated with 0.1, 1.0, or 3.0 ,uM AZT had a prolonged time to viral breakthrough. For each regimen of AZT alternating with 0.05 or 0.1 ,uM ddC, there was consistently prolonged HIV inhibition compared with continuous treatment with AZT alone. The viral suppression achieved with the alternating combinations required AZT as well as ddC and was superior to 3 days of treatment with ddC alternating with 3 days of no antiretroviral treatment. Levels of unintegrated HIV DNA paralleled the detection of p24 antigen, with the most prolonged inhibition of virus-specific DNA occurring with AZT alternating with ddC (compared with all regimens except continuous treatment with ddC). These data suggest that alternating regimens of AZT and ddC not only might decrease toxicity associated with the two drugs but may prove to be more efficacious than AZT alone. pg of p24 antigen per ml) for 2 h, washed four times with phosphate-buffered saline, and grown at 37°C in 5% CO2 in 25-cm2 T flasks containing the desired concentration of antiviral agent. At each time point, cells were counted and percentage of viability was determined, and the cells were then suspended in fresh medium containing the appropriate antiviral agent at a density of approximately 2.0 x 105/ml. Supernatants were clarified and stored at -70°C for p24 antigen determination. Cells were suspended in 40% RPMI 1640-50% fetal bovine serum-10% dimethyl sulfoxide and frozen at -70°C. The Abbott human T-cell lymphotropic virus type III solid-phase enzyme immunoassay was used to detect HIV core p24 antigen (7). All tests were performed according to the specifications of the manufacturer. Complete viral breakthrough was considered to have occurred when greater than 105 pg of p24 antigen per ml was detected in culture supernatants, because once this level was reached, cultures treated with antiviral agents continued to produce the same quantity of virus as the untreated HIV cultures. DNA analysis. DNA was extracted by the method of Hirt (3), and 1 ptg of DNA was placed onto nitrocellulose filters by using a slot blot manifold (Schleicher & Schuell) as previously described (11). Filters were hybridized with a 32P-labeled HIV probe made from the full-length infectious clone pHXB-2D of HIV proviral DNA (kindly provided by Flossie Wong-Staal). The probe was labeled by random priming (1). Prehybridization and hybridization were performed in 50% formamide at 52°C as previously described (11). Filters were washed in 2x SSC (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate) with 0.1% sodium dodecyl sulfate at room temperature (-23°C) for 60 min and in 0.1x SSC at room temperature. Filters were dried and subjected to autoradiography at -70°C with Kodak XAR-5 film and an intensifying screen. For quantitation, autoradiographs were
The thymidine analog 3'-azido-3'-deoxythymidine (AZT) (zidovudine) was reported in clinical studies to prolong survival and reduce the frequency and severity of opportunistic infections in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) (2, 12, 13). AZT treatment, however, is associated with significant toxicity, primarily bone marrow suppression (8, 9). Another dideoxynucleoside, 2',3'-dideoxycytidine (ddC), is a potent inhibitor of human immunodeficiency virus (HIV) (5), but patients with AIDS and ARC treated with ddC develop a painful peripheral neuropathy which limits its use as a single agent taken daily (4a, 14). Because the toxicity profiles of AZT and ddC differ, administering the two drugs in an alternating regimen has been proposed to decrease drugrelated toxicity (4a, 14). The objective of these experiments was to examine the inhibition of HIV in vitro by alternating regimens of AZT and ddC, using clinically achievable concentrations of both drugs. MATERIALS AND METHODS Cells, virus strains, and antiviral agents. CEM cells obtained from the American Type Culture Collection were grown in RPMI 1640 with 10% fetal bovine serum-100 U of penicillin and 100 jig of streptomycin per ml. Viability determinations were performed by trypan blue exclusion. The lymphoadenopathy-associated virus (LAV-BRU) strain of HIV, originally obtained from Luc Montagnier (Institut Pasteur), was passaged in CEM cells. AZT was obtained courtesy of Burroughs Wellcome Co., and ddC was obtained courtesy of Hoffmann-La Roche Inc. Virus infection and antiviral agent assay. CEM cells (5.0 x 106) in 10-ml cultures were infected with 1 ml of HIV (2 x 104 *
Corresponding author. 920
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HIV INHIBITION BY AZT ALTERNATING WITH ddC
analyzed by densitometry with an LKB ultrascan densitometer. Values are expressed as absorbance area (millimeters2). RESULTS Suppression of HIV by AZT and by AZT alternating with ddC. CEM cells infected with HIV produced greater than 105 pg of p24 antigen per ml by day 6 in culture. Treatment of infected cells with clinically achievable AZT concentrations of 0.1, 1.0, or 3.0 ,uM prolonged the time to viral breakthrough, with greater than 105 pg of p24 antigen per ml reached at 9 days for cells treated with 3 puM AZT (Fig. 1). To determine whether HIV inhibition would be affected by treatment of infected cells with AZT alternating with ddC, CEM cells treated with 0.1, 1.0, or 3.0 puM AZT were treated every 3 days with 0.1 or 0.05 F.M ddC for another 3 days and vice versa. For each combination of AZT alternating with ddC, there was consistently prolonged HIV inhibition compared with continuous treatment with AZT alone (Fig. 1). As would be expected, the highest concentration of both antiretroviral agents (3 ,uM AZT alternating with 0.1 ,uM ddC) provided the longest period of viral inhibition. However, HIV breakthrough occurred at all dosage regimens evaluated. Inhibition of HIV by ddC alone. Because ddC is known to be a potent inhibitor of HIV replication, we next evaluated the inhibition of HIV by ddC alone. HIV inhibition, as determined by p24 antigen, was achieved most effectively by 0.1 ,uM ddC, with viral breakthrough occurring beyond 18 days postinfection. However, continual treatment of patients with ddC at doses which can achieve these concentrations in serum will not be possible because of dose-limiting toxicity. Therefore, the next set of experiments was designed to evaluate HIV inhibition by treatment with ddC alternating with a rest period with no antiretroviral agents. In these studies, when infected CEM cells were treated with alternating regimens of ddC for 3 days and no ddC for 3 days, breakthrough consistently occurred earlier than with continuous ddC treatment or with ddC alternating with AZT (Fig. 2). Inhibition of HIV DNA synthesis. Unintegrated viral DNA was extracted from infected cells originally frozen in 10% dimethyl sulfoxide and 50% fetal bovine serum at 3, 6, 9, and 12 days postinfection. Samples of DNA (1 p.g) were blotted onto a nitrocellulose filter and hybridized to the 32P-labeled HIV DNA probe. The autoradiographs were analyzed by densitometry to quantitate the relative amounts of viral DNA present at each point (Fig. 3). During the first 6 days, little HIV DNA was detected in infected cultures. By day 9, large amounts of viral DNA were detected in HIV-infected control cultures. An increase in HIV DNA was detected at 9 days in AZT-treated cells, in which HIV DNA dramatically increased by day 12. Cultures treated with AZT alternating with ddC demonstrated markedly lower amounts of HIV DNA at days 9 and 12 postinfection, with cultures in which 0.1 ,uM ddC was alternated with AZT having the least viral DNA. Alternating regimens of ddC for 3 days and no drug for 3 days resulted in a marked increase in viral DNA synthesis by day 12, indicating that AZT alternating with ddC contributes to prolonged inhibition of viral DNA synthesis compared with ddC alternating with 3 days of no antiviral treatment (Fig. 3 and 4).
DISCUSSION Treatment for persons infected with HIV will require drugs that inhibit viral replication which preferably can be
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21 18 15 12 9 Days Post-infection FIG. 1. Production of HIV in CEM cells with 0.1 (A), 1 (B), and 3 (C) puM AZT and regimens of AZT alternating every 3 days with 3 days of treatment with ddC. Cells were infected with HIV for 2 h, washed, and maintained in the presence or absence of antiviral agent. Medium with or without drug was completely replaced every 3 days for all cultures. Samples of supernatant medium were assayed for HIV p24 antigen.
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administered orally and which are well tolerated over prolonged periods of time. Both AZT and ddC inhibit HIV replication in vitro (5, 6) and have demonstrated an antiviral effect, as measured by p24 antigen decline, in patients with AIDS or ARC (2, 4, 4a, 14: S. A. Spector, C. Kennedy, J. A. McCutchan, S. A. Bozzette, R. C. Straube, J. D. Connor, and D. D. Richman, J. Infect. Dis., in press). Prolonged administration of either drug, however, is associated with significant toxicity, which limits their long-term usefulness when taken daily. The spectrum of clinical adverse experiences associated with AZT administration has been described in detail (8, 13), with the frequent side effects being nausea, headache, and insomnia. Granulocytopenia occurs monthly in 10 to 25% of
ANTIMICROB. AGENTS CHEMOTHER.
SPECTOR ET AL.
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