Safety, Tolerance, and Efficacy of Atevirdine in Asymptomatic Human

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1996, p. 2664–2668 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 40, No. 11

Safety, Tolerance, and Efficacy of Atevirdine in Asymptomatic Human Immunodeficiency Virus-Infected Individuals ANNE MIEKE M. BEEN-TIKTAK,1,2 IAN WILLIAMS,3 HENK M. VREHEN,1 JOHN RICHENS,3 DIANA ALDAM,3 ANTON M. VAN LOON,2,4 CLIVE LOVEDAY,5 CHARLES A. B. BOUCHER,2 PENELOPE WARD,6 IAN V. D. WELLER,3 AND JAN C. C. BORLEFFS1* Section of Infectious Diseases and Tissue Damage, Department of Internal Medicine,1 and Eijkman-Winkler Institute of Medical and Clinical Microbiology,2 University Hospital Utrecht, Utrecht, and Laboratory of Virology, National Institute of Public Health and Environmental Protection, Bilthoven,4 The Netherlands; Departments of Sexually Transmitted Diseases and Virology, Division of Pathology and Infectious Diseases, University College London Medical School,3 and Department of Retrovirology, Royal Free Hospital School of Medicine,5 London, United Kingdom; and Pharmacia & Upjohn Inc. Europe, Puurs, Belgium6 Received 25 March 1996/Returned for modification 15 May 1996/Accepted 27 August 1996

Atevirdine is a nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1). In this study we investigated the effect of atevirdine in asymptomatic antiretroviral naive HIV-infected patients with CD41 cell counts of between 200 and 750 cells per mm3. Patients were randomized to receive 600 mg of atevirdine (n 5 15) or a placebo (n 5 15) three times a day for 12 weeks. There was no statistically significant effect of atevirdine on viral loads (HIV p24 antigen and HIV-1 RNA levels by PCR) or CD41 cell counts. The data do not support the use of atevirdine as a monotherapy in the treatment of HIV-infected patients. consent. The study was conducted in agreement with the declaration of Helsinki and its revisions (21). Male individuals aged over 18 years were eligible for the study if they had (i) documented HIV-1 infection, (ii) a CD41 cell count greater than 200 cells per mm3 but lower than 750 cells per mm3 or a CD41 cell count lower than 200 cells per mm3 if they had declined AZT therapy but wished to participate in the study, and (iii) normal liver function. Exclusion criteria were as follows: evidence of AIDS according to the 1987 case definition of the Centers for Disease Control (4), hypersensitivity to piperazine-type drugs, any previous antiretroviral treatment, the abuse of hard drugs, and concomitant medications other than those for prophylaxis of Pneumocystis carinii pneumonia (cotrimoxazole, aerolized pentamidine, and dapsone) and therapy for oral candidiasis (clotrimazole and nystatin suspension) or herpes simplex infection (up to 1,000 mg of acyclovir per day). At a screening visit up to 6 weeks prior to enrollment, the patients’ medical histories were taken and physical examinations were performed. In addition, complete hematological screening, blood chemistry, urinalysis (including screening for the abuse of hard drugs), and electrocardiograms were performed. Baseline examinations of viral loads consisted of four separate quantitative HIV-1 RNA determinations, according to the method described by Semple et al. (17). The dynamic range of this immunocapture assay is 20 to 100,000 copies per ml. Since 1991 the assay has been used to analyze the dynamics of HIV and responses to antiretroviral therapy in several studies. It has been shown in longitudinal evaluations of drug efficacies that the immunocapture assay exhibits patterns of changing viral loads similar to those of commercial assays, such as the Roche RT PCR, the Chiron bDNA, and the Organon Teknika NASBA assays (10). CD41 cell counting was performed twice before entry and once at baseline by flow cytometry (FACStar; Becton Dickinson, Mountain View, Calif.). The safety parameters were repeated every week following institution of drug therapy. CD41 cell counts, HIV p24 antigen levels (immune complex dissociated), and viral loads were measured

Atevirdine is a bisheteroarylpiperazine (BHAP) that inhibits the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The compound has a 50% inhibitory concentration (IC50) of approximately 1 mM (11, 15). BHAPs are nonnucleoside RT inhibitors (NNRTIs), a structurally diverse group of antiretroviral agents that have a similar mechanism of RT inhibition by binding to a common region of RT near the nucleotide binding site (6, 12, 18). NNRTIs have exhibited synergy with zidovudine (AZT) in inhibiting the replication of AZT-resistant viral strains. By contrast, this combination had a mostly additive effect when tested against AZT-susceptible isolates (3, 15). The development of HIV-1 variants with reduced levels of sensitivity to BHAPs has been reported (13, 14, 19). Interestingly, however, virus strains resistant to BHAPs appear to be more susceptible to other NNRTIs than the corresponding wild-type strain (7). Furthermore, the concomitant use of two classes of RT inhibitors acting at different enzymatic sites, i.e., NRTIs and NNRTIs, may prevent or delay the development of viral resistance (20). A preceding escalating-single-dose study of atevirdine in HIV-infected individuals showed that the compound was safe and well tolerated (1). In this report, the results of a phase Ib clinical trial with atevirdine are presented. This is the first study performed with asymptomatic HIV-infected individuals comparing the results of atevirdine monotherapy and a placebo. Study design. The study was a multicenter, double-blind, placebo-controlled trial. Patients were randomized to receive either atevirdine or a matching placebo for 12 weeks. The dosage regimen, 600 mg three times a day, was based on the pharmacokinetic data available from the AIDS Clinical Trials Group 199 study (11). The protocol and the informed consent forms were approved by the investigational review board of each participating hospital. All patients gave written informed * Corresponding author. Mailing address: University Hospital Utrecht, Department of Internal Medicine, Section of Infectious Diseases and Tissue Damage, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Phone: 31 30 2506228/2509111. Fax: 31 30 2513828. 2664

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TABLE 1. Baseline characteristicsa of the patients in the two treatment groups Baseline level of patients in indicated treatment group

Parameter

Placebo

Atevirdine

CD4 cell count (cells/mm ) Mean SE

355 36.23

437 42.36

HIV-1 RNA (log copies/ml) Mean SE

2.19 0.71

2.46 0.90

HIV p24 antigen (pg/ml) Mean SE

363.90 157.60

249.43 138.60

1

3

a The differences between the two study groups were not statistically significant for any of the three parameters.

at monthly intervals during the treatment period and followup. In order to measure possible changes in viral sensitivity to atevirdine during the study, peripheral blood mononuclear cells from the 20 Dutch patients were collected at baseline and at weeks 11 and 23 and were stored frozen in liquid nitrogen. Virus stocks were obtained by cocultivation of the stored samples at the end of the study. These procedures were performed according to the AIDS Clinical Trials Group-Department of Defense consensus protocol (5, 9). Reduced sensitivity to atevirdine was defined as a 10-fold increase in the IC50 or an absolute IC50 of $10 mM (11). Trough serum atevirdine concentrations at days 1 and 21 were determined by a high-performance liquid chromatography procedure, as described earlier (8). Atevirdine was provided by The Upjohn Co. as 200 mg of nonmicronized powder in hand-filled hard gelatin capsules. Placebo capsules of identical appearance contained 100 mg of lactulose. The statistical analyses were performed according to the on-treatment principle by using SAS software in a mainframe environment. All tests were two sided. Quantitatively measured variables were analyzed by the Student t test. For the analysis of CD41 cell and viral load responses, repeated-measures analysis of variance was used on each variable. For the CD41 cell count variable, a square root transformation was performed before repeated-measures analysis of variance analysis. A baseline value was defined as the mean of the screening and the day 0 assessments. Efficacy indicators were summarized in terms of absolute values and changes from baseline values. Results and discussion. The 30 study participants were selected from the outpatient clinics of the University Hospital in Utrecht (n 5 20) and the University College London Medical School (n 5 10). We randomly selected 15 patients to receive atevirdine and 15 to receive the placebo. Demographic characteristics, risk factors for HIV infection, and body weights were similar in the two study groups. Baseline CD41 cell counts, HIV-1 RNA levels, and HIV p24 antigen levels are shown in Table 1. The mean CD41 cell count and HIV-1 RNA level were higher in the atevirdine group, but the mean p24 level was lower. None of these differences were statistically significant. In both the atevirdine- and the placebo-treated groups a modest transient rise in CD41 cell count was seen during the 12-week treatment phase of the study (Fig. 1). Figure 2 shows the results of the p24 assay. In both groups a decline in levels

FIG. 1. Mean changes (and standard errors [SE]) in CD41 cell counts from the baseline during 12 weeks of treatment with atevirdine or the placebo and the subsequent 11 weeks of follow-up.

of p24 antigen in sera was seen 3 weeks after the study start (Fig. 2). In the atevirdine-treated patients, the decline was more sustained. However, in patients receiving the placebo, the mean p24 antigen level was already 77 pg/ml above the baseline level at week 11. This difference in response to treatment was not statistically significant. The results of the quantitative HIV-1 RNA PCR with the 26 patients participating in the study from whom serum samples were available for testing are shown in Fig. 3. For four patients, samples were not available either because of early withdrawal from the study or because of inadequate collection of specimens. Of the patients taking atevirdine (Fig. 3, left side), one showed a 2-log drop in the number of HIV-1 RNA copies per milliliter. Unfortunately, no follow-up data are available because this patient developed an acute hepatitis and was withdrawn from the study. One other patient showed a 1-log drop in response to treatment, but RNA levels returned to the baseline level at week 7. A third subject had a 0.5-log drop that was sustained until the end of the dosing phase of the study. In

FIG. 2. Mean changes (and standard errors [SE]) in HIV p24 antigenemia from the baseline during 12 weeks of treatment with atevirdine or the placebo and the subsequent 11 weeks of follow-up.

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FIG. 3. Individual changes in HIV-1 RNA PCR from the baseline during 12 weeks of treatment with atevirdine (left side) or the placebo (right side) and the subsequent 11 weeks of follow-up.

two other patients receiving atevirdine, a nearly 0.5-log drop was found at week 2; for these subjects, however, no follow-up data are available since they were withdrawn from the study because of rashes. In the other patients some fluctuation in HIV-1 RNA levels was seen, but no substantial decrease occurred. In the subjects receiving the placebo (Fig. 3, right side), two patients showed extreme fluctuations in their HIV-1 RNA levels. These samples were rerun and showed similar results, representing true biological variability. Overall, there appeared to be no important difference in HIV-1 RNA levels of treated patients compared with those of the controls. In atevirdine-treated patients the mean trough level in sera was 10 mM (range, 6 to 18 mM). Pre- and poststudy peripheral blood mononuclear cells were available from 7 of the 10 Dutch patients that received atevirdine. In one of these seven patients no HIV-1 stock with a sufficiently high titer could be obtained from the stored cell samples. Consequently, six atevirdine-treated subjects and six randomly chosen placebo-treated subjects were tested for drug

susceptibility. The patterns of sensitivity to atevirdine of individuals during the course of the study are shown in Fig. 4. In the atevirdine arm of the study, the mean IC50 of atevirdinenaive virus strains was 0.8 mM at baseline (Fig. 4, left side). In one patient a 10-fold increase in the IC50 was detected at week 11. After discontinuation of the study drug at week 12, sensitivity to atevirdine improved within the period of the follow-up. In the other patients no significant change in sensitivity to atevirdine was measured during the treatment phase of the study. One patient developed a reduced sensitivity to atevirdine after the treatment phase of the study (week 23). In the other patients sensitivity to atevirdine remained unchanged during the period of the follow-up. The right part of Fig. 4 shows the patterns of IC50s of six placebo-treated patients. Three of them had a relatively high baseline IC50 (1.2, 1.5, and 1.7 mM) when compared with the mean baseline IC50 (0.8 mM) of patients enrolled in the atevirdine arm of the study. However, the sensitivities of all virus strains in the placebo-treated group remained unchanged throughout the study.

FIG. 4. Individual changes in IC50 from the baseline during 12 weeks of treatment with atevirdine (left side) or the placebo (right side) and the subsequent 11 weeks of follow-up.

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Eight patients were withdrawn from the study because of adverse events; i.e., six were withdrawn from the atevirdine arm, and two were withdrawn from the placebo arm. Among the atevirdine-treated patients, one developed an acute hepatitis after 3 weeks of dosing. Concomitant medication consisted of fluconazole and terbinafine, either of which may also have been a cause of liver function disturbances. The patient recovered after discontinuation of all medication. The other five patients had a maculopapular rash occasionally accompanied by fever which occurred during the second week of dosing. All rashes cleared up rapidly after therapy was stopped. In the placebo arm of the study one patient developed presyncopal episodes which ceased after capsule withdrawal. Another patient died from hemorrhage secondary to thrombocytopenia. This patient had a normal prestudy platelet count. During the study his platelet count slowly declined. At week 16 he had a liver biopsy for diagnostic purposes and, in spite of platelet cover, died from a hemorrhage as a complication of that procedure. In all other participants of the study no significant changes in hematology and biochemistry were found. Our study shows that atevirdine when given as monotherapy in asymptomatic HIV-infected patients has a rather poor antiretroviral effect. In two patients who received atevirdine therapy there may have been an effect on HIV-1 RNA levels, but similar fluctuations were seen in the placebo-treated group. Overall there appeared to be no important difference in HIV-1 RNA levels between treated patients and controls. The results were disappointing in light of promising preclinical research. The participants were asymptomatic, with relatively low viral burdens and high CD41 cell counts. These conditions may have hampered the chances of detecting an effect on surrogate markers within 12 weeks of dosing. Trough levels of atevirdine in sera were greater than the IC50 of the drug. However, if one assumes that the drug concentration required for an in vivo antiviral effect should have been much higher than the IC50 of the compound, the lack of efficacy may be explained by insufficient serum atevirdine concentrations. The slight increase in CD41 cell counts also found in the placebo-treated group appears irrational. However, this phenomenon has only statistical relevance and may be due to a regression to the mean. This occurs with variables, such as CD41 cells, that fluctuate within an individual (22). The absence of an anti-HIV effect of atevirdine is also reflected by the low level of resistance to HIV-1 to the drug which occurred during the study. For other NNRTIs the rapid emergence of drug-resistant HIV-1 strains has been reported (13, 14, 16, 19). However, in patients receiving these NNRTIs, the occurrence of resistance was always preceded by a significant antiviral effect. The low level of viral resistance may well be due to insufficient selective pressure of the drug (16). In one patient the presence of resistance to atevirdine was documented only after the treatment phase of the study, i.e., after discontinuation of the selective pressure of the drug. It can be speculated that part of the virus population had become resistant to atevirdine during treatment. However, the size of this atevirdine-resistant population was relatively small in comparison with the atevirdine-susceptible part of the population. Therefore, resistance might not have been detected at week 11. The resistant part of the virus population may have consisted of a more fit virus strain with a replication rate higher than that of the original strain, leading to the emergence of resistance at week 23 following the cessation of therapy. Unfortunately, additional peripheral blood mononuclear cell samples from this patient for genotyping and phenotyping experiments are not available. With respect to the safety of atevirdine, the study suggests that hepatotoxicity and skin rash may occur. However,

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in all subjects, the signs and symptoms of adverse events disappeared quickly after discontinuation of therapy. The results of our study do not support the use of atevirdine as monotherapy in the treatment of HIV-infected patients. One other study with atevirdine showed promising results on neurological function in patients with AIDS-related dementia (2). Therefore, only further research will establish whether it has a place in antiretroviral therapy. We are indebted to the study participants and to Henriette Beumer, Fiona Suggett, Evert Vijge, and Folko-Jan Wijnholds for technical assistance. REFERENCES 1. Been-Tiktak, A. M. M., H. M. Vrehen, M. M. E. Schneider, M. van der Feltz, T. Branger, P. Ward, S. R. Cox, J. D. Harry, and J. C. C. Borleffs. 1995. Safety, tolerance, and pharmacokinetics of atevirdine mesylate (U-87201E) in asymptomatic human immunodeficiency virus-infected patients. Antimicrob. Agents Chemother. 39:602–607. 2. Brew, J. B., N. Dunbar, J. Druett, J. Freund, and P. Ward. 1992. Pilot study of the efficacy of atevirdine in AIDS dementia complex. AIDS 8:S532. 3. Campbell, T. B., R. K. Young, J. J. Eron, R. T. D’Aquila, W. G. Tarpley, and D. R. Kuritzkes. 1993. Inhibition of human immunodeficiency virus type 1 replication in vitro by the bisheteroarylpiperazine atevirdine (U-87201E) in combination with zidovudine or didanosine. J. Infect. Dis. 168:318–326. 4. Centers for Disease Control. 1987. Revision of the CDC surveillance case definition of acquired immunodeficiency syndrome. Morbid. Mortal. Weekly Rep. 36(1S):1–15. 5. Chou, J., and T. C. Chou. 1987. Dose-effect analysis with microcomputers quantification of ED50, LD50, synergism, antagonism, low-dose risk, receptor ligand binding and enzyme kinetics, p. 19–32. A computer software for IBM-PC and manual. Elsevier-Biosoft, Cambridge, United Kingdom. 6. Dueweke, T. J., F. J. Ke´zdy, G. A. Waszak, M. R. Deibel, Jr., and W. G. Tarpley. 1992. The binding of a novel bisheteroarylpiperazine mediates inhibition of human immunodeficiency virus type 1 reverse transcriptase. J. Biol. Chem. 267:27–30. 7. Dueweke, T. J., T. Pushkarskaya, S. M. Poppe, S. M. Swaney, J. Q. Zhao, I. S. Chen, M. Stevenson, and W. G. Tarpley. 1993. A mutation in reverse transcriptase of bis(heteroaryl)piperazine-resistant human immunodeficiency virus type 1 that confers increased sensitivity to other nonnucleoside inhibitors. Proc. Natl. Acad. Sci. USA 90:4713–4717. 8. Howard, G. M., and F. J. Schwende. 1993. The development of HPLC-based analytical methods for atevirdine, a novel reverse transcriptase inhibitor for the treatment of AIDS, and associated problems with chromatographic performance, abstr. MP-E12. In Abstracts of the Fourth International Symposium on Pharmaceutical and Biomedical Analysis, 1993. 9. Japour, A. J., D. L. Mayers, V. A. Johnson, D. R. Kuritzkes, L. A. Beckett, J.-M. Arduino, J. Lane, R. J. Black, P. S. Reichelderfer, R. T. D’Aquila, C. S. Crumpacker, the RV-43 Study Group, and the AIDS Clinical Trials Group Virology Committee Resistance Working Group. 1993. Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. Antimicrob. Agents Chemother. 37:1095–1101. 10. Loveday, C. 1995. An approach to quantification of serum HIV-1 RNA load by PCR, p. 69–71. In M. R. Haeney (ed.), Proceedings of the National Scientific Meeting of the Association of Clinical Pathologists—1994. 11. Reichman, R. C., G. D. Morse, L. M. Demeter, L. Resnick, Y. Bassiakos, M. Fischl, M. Para, W. Powderly, J. Leedom, C. Greisberger, and the AIDS Clinical Trials Group 199 Study Team. 1995. Phase I study of atevirdine, a nonnucleoside reverse transcriptase inhibitor, in combination with zidovudine for human immunodeficiency virus type 1 infection. J. Infect. Dis. 171: 297–304. 12. Richman, D., C. K. Shih, I. Lowy, J. Rose, P. Prodanovich, S. Goff, and J. Griffin. 1991. Human immunodeficiency virus type 1 mutants resistant to nonnucleoside inhibitors of reverse transcriptase arise in tissue culture. Proc. Natl. Acad. Sci. USA 88:11241–11245. 13. Richman, D. D. 1993. Resistance of clinical isolates of human immunodeficiency virus to antiretroviral agents. Antimicrob. Agents Chemother. 37: 1207–1213. 14. Richman, D. D., D. Havlir, J. Corbeil, D. Looney, C. Ignacio, S. A. Spector, J. Sullivan, S. Cheeseman, K. Barringer, D. Pauletti, C.-K. Shih, M. Myers, and J. Griffin. 1994. Nevirapine resistance mutations of human immunodeficiency virus type 1 selected during therapy. J. Virol. 68:1660–1666. 15. Romero, D. L., M. Busso, C. K. Tan, F. Reusser, J. R. Palmer, S. M. Poppe, P. A. Aristoff, K. M. Downey, A. G. So, L. Resnick, et al. 1991. Nonnucleoside reverse transcriptase inhibitors that potently and specifically block human immunodeficiency virus type 1 replication. Proc. Natl. Acad. Sci. USA 88: 8806–8810.

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