The relationships among treatment regimens, plasma human immunodeficiency virus (HIV) RNA levels, and resistance mutations to saquinavir (codons 48 and 90) and zidovudine (codon 215) were examined in a cohort of 144 patients from the AIDS Clinical Trials Group 229 study. After 24-40 weeks of therapy, no patients who had received the two-drug combination (zidovudine plus saquinavir) had only codon 48 mutations, 45.8% had only codon 90 mutations, and 8.3% had both codon 48 and 90 mutations. Mutations developed by patients who had received the three-drug combination (zidovudine and zalcitabine plus saquinavir) were codon 48 alone in 1.4%, codon 90 alone in 33.3%, and both codons 48 and 90 in 4.2%. The difference between the groups showed a trend toward reduced mutations with three versus two drugs but did not reach significance (p = .11, two-sided χ2). Higher baseline HIV RNA levels correlated with the development of protease mutations. Mutations at codon 215 were present in 82% of all patients at baseline and in 87% after therap

Collier, Ann C.

Coombs, Robert W.

Efron, Bradley

Lawrence, Jody

Merigan, Thomas C.

Schapiro, Jonathan M.

Speck, Roberto

Winters, Mark A.

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249Resistance Mutations to Zidovudine and Saquinavir in Patients ReceivingZidovudine plus Saquinavir or Zidovudine and Zalcitabine plus Saquinavir inAIDS Clinical Trials Group 229Jonathan M. Schapiro, Jody Lawrence, Roberto Speck,Mark A. Winters, Bradley Efron, Robert W. Coombs,Ann C. Collier, and Thomas C. MeriganDivision of Infectious Diseases and Geographic Medicine, StanfordUniversity School of Medicine, Stanford, California; Department ofMedicine, University of Washington School of Medicine, Seattle,Washington; Swiss National Science Foundation, Geneva, SwitzerlandThe relationships among treatment regimens, plasma human immunodeficiency virus (HIV)RNA levels, and resistance mutations to saquinavir (codons 48 and 90) and zidovudine (codon215) were examined in a cohort of 144 patients from the AIDS Clinical Trials Group 229study. After 24–40 weeks of therapy, no patients who had received the two-drug combination(zidovudine plus saquinavir) had only codon 48 mutations, 45.8% had only codon 90 mu-tations, and 8.3% had both codon 48 and 90 mutations. Mutations developed by patientswho had received the three-drug combination (zidovudine and zalcitabine plus saquinavir)were codon 48 alone in 1.4%, codon 90 alone in 33.3%, and both codons 48 and 90 in 4.2%.The difference between the groups showed a trend toward reduced mutations with three versustwo drugs but did not reach significance ( , two-sided x2). Higher baseline HIV RNAP 5 .11levels correlated with the development of protease mutations. Mutations at codon 215 werepresent in 82% of all patients at baseline and in 87% after therapy.Combination therapy is currently the standard of care inhuman immunodeficiency virus (HIV)–infected patients [1, 2].Due to the highly effective nature of potent antiretroviral drugregimens, insight into the development of resistance mutationsand loss of plasma HIV RNA suppression may require large,expensive studies with prolonged periods of follow-up. Studyof earlier clinical trials performed with less potent combinationsof drugs and in drug-experienced patients, in whom drug re-sistance and failure developed more rapidly than with antire-troviral-naive patients, may provide insight into some of thecauses leading to combination therapy failure [3].The importance of suppressing plasma HIV RNA with com-bination therapy to obtain a more durable response hasemerged from recent studies [4, 5]. This suppression reflectsreduced viral replication, thus minimizing the opportunities forHIV to develop drug resistance mutations [5, 6]. Although thecorrelation between virus load reduction and duration of re-sponse and improved prognosis has been well documented inlarge studies [5, 7], fewer data are available about these featuresand protease drug resistance mutations.Combinations including a greater number of antiretroviralagents are now commonly prescribed due to their clinical suc-cess [8–10]. Although three- and four-drug combinations arebeing studied and used [10, 11], the degree to which the additionReceived 19 February 1998; revised 17 July 1998.Reprints or correspondence: Dr. Thomas C. Merigan, Jr., Center ForAIDS Research, S-156 Grant Bldg., Stanford University School of Medicine,Stanford, CA 94305-5107.The Journal of Infectious Diseases 1999;179:249–53q 1999 by the Infectious Diseases Society of America. All rights reserved.0022-1899/99/7901-0035$02.00of a third agent to a double drug combination actually reducesthe rate at which resistance mutations develop has not beenwell studied.We sought to gain insight into these questions by examiningthe relationships among treatment regimens, plasma HIV RNAvirus load levels, and the presence of resistance mutations tosaquinavir and zidovudine in a large cohort of patients fromthe AIDS Clinical Trials Group (ACTG) 229 study in whichzidovudine-experienced patients were randomized to receivezidovudine plus zalcitabine, zidovudine plus saquinavir, or zi-dovudine and zalcitabine plus saquinavir.MethodsPatients. ACTG 229 was a double-blind randomized studythat enrolled 302 patients with a baseline CD4 T cell count of50–300/mL and at least 6 months of prior zidovudine therapy toreceive one of three treatments, zidovudine plus zalcitabine, zi-dovudine plus saquinavir (hard-gel capsule), or zidovudine andzalcitabine plus saquinavir (hard-gel capsule). Patients had a me-dian of 27 months of prior zidovudine therapy. Of the 302 enrolled,285 completed the initial 24 weeks of study [3]. We examined allavailable stored samples from the zidovudine plus saquinavir andzidovudine and zalcitabine plus saquinavir arms of the study forresistance mutations to saquinavir and zidovudine. Patients fromthe non–saquinavir-containing zidovudine plus zalcitabine armwere not studied, as they were not expected to develop saquinavirresistance mutations.Mutations. Mutations to saquinavir at codons 48 (G to V)and 90 (L to M) in the HIV-1 protease gene were determined fromfrozen plasma from samples taken at baseline and after 24–40weeks of therapy. The presence of a wild type (WT) or mutant250 Schapiro et al. JID 1999;179 (January)Table 1. Mutations at codons 48 and 90 in patients receiving either zidovudine (ZDV) plus saquinavir (SAQ)or ZDV and zalcitabine (ddC) plus SAQ.Baseline >24 weeksCodon 48 Codon 90 Codons 48 and 90 Codon 48 Codon 90 Codons 48 and 90ZDV 1 SAQ 0% (0/60) 0% (0/60) 0% (0/60) 0% (0/72) 45.8% (33/72) 8.3% (6/72)ZDV 1 ddC 1 SAQ 0% (0/57) 0% (0/57) 0% (0/57) 1.4% (1/72) 33.3% (24/72) 4.2% (3/72)All patients 0% (0/117) 0% (0/117) 0% (0/117) 0.7% (1/144) 39.6% (57/144) 6.3% (9/144)Figure 1. Changes in plasma human immunodeficiency virus RNA levels in patients receiving therapy with either zidovudine plus saquinavir( ) or zidovudine and zalcitabine plus saquinavir ( ).ZDV 1 SAQ ZDV 1 ddC 1 SAQ(MU) genotype was determined using a selective polymerase chainreaction method as previously described [12]. In addition, the pres-ence of the codon 215 mutation to zidovudine was determined bya similar technique [13].Statistical analysis. Plasma HIV RNA levels and CD4 T cellcounts had been previously determined for each patient as part ofthe ACTG 229 clinical trial [3]. These data were obtained from theACTG 229 database and were correlated with the presence of WTor MU genotype for the resistance mutations studied.ResultsSamples from 144 of the 149 patients who remained on ther-apy were analyzed for mutations at codons 48 and 90 of theprotease gene after 24–40 weeks of treatment. Of these patients,135 were at week 24, 7 at week 32, and 2 at week 40. Seventy-two patients had received treatment with zidovudine plus sa-quinavir and an equal number had received zidovudine andzalcitabine plus saquinavir. In addition, for 60 patients in thezidovudine plus saquinavir and for 57 in the zidovudine andzalcitabine plus saquinavir groups, baseline samples taken be-fore therapy started were also analyzed. All baseline samplesin both treatment groups were WT for codons 48 and 90 (table1).Following therapy with the two-drug combination in 72 pa-tients, none had developed the codon 48 mutation alone, 45.8%(33) had developed the codon 90 mutation alone, and 8.3% (6)had developed both codons 48 and 90 mutations. Of the 72patients receiving the triple combination, 1.4% (1 patient) haddeveloped the codon 48 mutation alone, 33.3% (24) had de-veloped the codon 90 mutation alone, and 4.2% (3) had de-veloped both codon 48 and 90 mutations. The rate of mutationsdeveloping after treatment for both groups ( ) combinedn 5 144was 0.7% (1 patient), 39.6% (57), and 6.3% (9) for codon 48alone, 90 alone, and 48 and 90 together, respectively. The de-JID 1999;179 (January) Resistance Mutations to Antivirals in ACTG 251Figure 2. Changes in plasma human immunodeficiency virus (HIV) RNA levels in patients who developed codon 90 mutation (MU) to saquinavirin their HIV protease gene following 24–40 weeks of therapy and in those who retained wild type (WT) at codon 90.velopment of the codon 90 mutation showed a trend towardstatistical significance for the two-drug (33/72, 45.8%) versusthe three-drug (24/72, 33.3%) regimen ( , two-sided x2).P 5 .11The total number of mutations to saquinavir (codons 48 and90) also showed a trend toward statistical significance for thetwo-drug (39/72, 54.2%) than for the three-drug (28/72, 38.9%)regimen ( , two-sided x2).P 5 .10Samples from 112 patients in both groups were also analyzedfor mutations in the reverse transcriptase gene at codon 215,both before and after therapy. Mutations at codon 215 werepresent in 82% (92 patients) at baseline and in 87% (97) aftertherapy. There was no statistical difference in the proportionwith codon 215 mutations before or after therapy between thezidovudine plus saquinavir and the zidovudine and zalcitabineplus saquinavir groups.As was seen in the parent study, the changes in plasma HIVvirus load for the group receiving zidovudine plus saquinavirand that receiving zidovudine and zalcitabine plus saquinavirshowed a greater reduction for the three- versus two-drug com-bination (figure 1). There was no statistical difference in thebaseline plasma HIV RNA values between the treatmentgroups.Changes in plasma HIV RNA were plotted for patients whodeveloped the codon 90 mutation following therapy and forthose who did not (figure 2). The group in which the codon 90mutation developed had a higher mean baseline plasma HIVRNA level (5.13 log RNA copies/mL, SD 0.58) than the groupin which the mutation did not develop (4.79 log RNA copies/mL, SD 0.66) ( ). The maximal reduction in plasma HIVP ! .01RNA in the patients developing the mutation was seen at week4 and was 0.36 log RNA copies/mL. In the group not devel-oping the codon 90 mutation, the maximal reduction was atweek 8 and was 0.45 log RNA copies/mL. The plasma HIVRNA levels remained consistently lower in the group that didnot develop the 90 mutation through week 48, although therewas no statistical difference in the magnitude of the reductionfrom baseline between the 2 groupsThe effect of the baseline reverse transcriptase codon 215mutational status on the subsequent development of the pro-tease codon 90 mutation was examined. There was no sta-tistical difference in the rate at which mutations at codon 90developed in patients with or without the codon 215 mutationat baseline.DiscussionWe studied the rate of development of key resistance mu-tations to saquinavir and zidovudine in highly zidovudine-252 Schapiro et al. JID 1999;179 (January)experienced patients receiving either a two-drug combination(zidovudine plus saquinavir) or a triple-drug combination (zi-dovudine and zalcitabine plus saquinavir). Although the dif-ference did not reach statistical significance ( ), there wasP 5 .11a trend toward a higher rate of development of mutations tosaquinavir in patients receiving the two- versus the three-drugcombination.The number of mutations developing to saquinavir in thisstudy was relatively high, considering that the great majorityof patients were assayed after 24 weeks of therapy. In a reviewof early saquinavir-containing studies, Jacobsen et al. [14]found that 33% of patients on monotherapy had developedmutations by 4–6 months and 12.5%–63% after 7–12 months.Of interest, in a small study of overlapping patients from ACTG229, Jacobsen et al. [14] reported that 12% and 22% of patientsin the triple-drug combination group had mutations after 4–6and 7–12 months, respectively. The differences between ourfindings and those previously reported may be due to the smallnumber of patients previously studied (25 at 4–6 months and18 at 7–12 months) and the specimens assayed (peripheralblood mononuclear cells vs. plasma) in the previous study. Thehigh rate of saquinavir mutations in our study also may berelated to the very high rate of baseline codon 215 mutationsto zidovudine. Since most of the patients were already resistantto zidovudine at the start of therapy, little or no reduction invirus load was likely produced by the zidovudine componentof the regimen, allowing mutations to saquinavir to develop ata higher rate.The two-drug combination ( ) resulted in a total of 33n 5 72(46%) patients developing the codon 90 mutation comparedwith 24 (33%) of 72 receiving three drugs ( ). This trendn 5 72may not have reached statistical significance due to the highrate of codon 90 mutation in both groups. This may be due tothe high baseline virus load, to suboptimal exposure to saquin-avir achieved with the standard dose used in this study, and tothe high rate of baseline codon 215 mutations. Therefore, manypatients in the triple-drug combination arm were essentiallyreceiving active antiretrovirals in the form of zalcitabine andlow-exposure saquinavir. This, coupled with the high virus load,would produce partial selective pressure on a highly replicatingvirus, a situation believed to result in high mutational rates [12,15]. More potent antiretroviral regimens with greater drug ex-posure in the setting of less baseline drug resistance would bemore likely to ultimately show benefit for three- versus two-drug regimens in reducing the development of mutations [4, 7].The higher baseline and nadir virus load found in patientswho developed the codon 90 mutation compared with thosewho did not supports the concept that higher viral replicationincreases the potential for resistance mutations to develop [15].Recently, Kempf et al. [16] showed a strong correlation betweenthe nadir of HIV RNA obtained after initiation of antiretroviraltherapy and the duration of virus load suppression [16]. Lowernadirs resulted in a more durable response. Our results showingthat lower nadirs result in a lower mutational rate would sup-port their findings, as the lower mutational rate links the lowernadir to a more durable response.In summary, we believe the results of this comprehensivemutational analysis of patients receiving dual versus triple com-bination therapy help substantiate our understanding of themechanisms leading to drug resistance and support the currentguidelines of administering triple combinations to obtain max-imal viral suppression [1, 2].AcknowledgmentsWe thank the volunteers who participated in the study, the membersof the ACTG 229 study team and ACTG staff, and Roland Bassetfrom Statistical and Data Analysis Center for his valued assistance inobtaining the study data.References1. Carpenter CC, Fishchl MA, Hammar SM, et al. Antiretroviral therapy forHIV infection in 1997: updated recommendations of the InternationalAIDS Society–USA panel. JAMA 1997;277:1962–9.2. Department of Health and Human Services, Henry J. Kaiser Family Foun-dation Panel on Clinical Practices for the Treatment of HIV Infection.Rockville, MD: HIV/AIDS Treatment Information Service, 1997.3. Collier AC, Coombs RW, Schoenfeld DA, et al. Treatment of human im-munodeficiency virus infection with saquinavir, zidovudine and zalcita-bine: AIDS Clinical Trials Group. N Engl J Med 1996;334:1011–7.4. Gulick RM, Mellors JW, Havlir D, et al. Treatment with indinavir, zidovudineand lamivudine in adults with human immunodeficiency virus infectionand prior antiretroviral therapy. N Engl J Med 1997;337:734–9.5. Katzenstein DA, Hammer SM, Hughes MD, et al. Virologic and immuno-logic markers and clinical outcomes after nucleoside therapy in adultswith 200 to 500 CD4 cells per cubic millimeter. NIAID Sponsored AIDSClinical Trials Group Study 175, a virology substudy. N Engl J Med1996;335:1091–8.6. Richman DD. HIV therapeutics. Science 1996;272:1886–8.7. Hammer SM, Squires KE, Hughes MD, et al. A controlled trial of twonucleoside analogues plus indinavir in persons with human immuno-deficiency virus infection and CD4 cell counts of 200 per cubic millimeteror less. AIDS Clinical Trials Group 320 Study Team. N Engl J Med1997;337:725–33.8. Moore RD, Keruly JC, Chaisson RE. Effectiveness of combination anti-retroviral therapy in clinical practice [abstract I-176]. In: Program andabstracts of the 37th Interscience Conference on Antimicrobial Agentsand Chemotherapy (Toronto). Washington, DC: American Society forMicrobiology, 1997.9. Abrams DI, Neaton J, Wentworth D, et al. Patterns of antiretroviral druguse over the past 6 years in patients enrolled in community-based clinicaltrials: the CPCRA experience. In: 4th Conference on Retroviruses andOpportunistic Infections (Washington, DC). Alexandria, VA: InfectiousDiseases Society of America, 1997.10. Coleman RL, Brosgart CL, Mitchell TF, Dyner T, Gee L, Abrams DI. Anti-retroviral prescribing patterns following the introduction of protease in-hibitors. In: 4th Conference on Retroviruses and Opportunistic Infections(Washington, DC). Alexandria, VA: Infectious Diseases Society of Amer-ica, 1997.11. Talal A, Cao Y, Hurley A. Saquinavir in combination with AZT/3TC andritonavir: a convenient BID regimen. In: Program and abstracts of the37th Interscience Conference on Antimicrobial Agents and Chemo-therapy (Toronto). Washington, DC: American Society for Microbiology,1997.12. Schapiro JM, Winters MA, Stewart F, et al. The effect of high dose saquinavirJID 1999;179 (January) Resistance Mutations to Antivirals in ACTG 253on viral load and CD41 T-cell counts in HIV-infected patients. Ann InternMed 1996;124:1039–50.13. Kozal MJ, Shafer RW, Winters MA, Katzenstein DA, Merigan TC. A mu-tation in human immunodeficiency virus reverse transcriptase and declinein CD4 lymphocyte numbers in long-term zidovudine recipients. J InfectDis 1993;167:526–32.14. Jacobsen H, Hanggi M, Ott M, et al. In vivo resistance to a human im-munodeficiency virus type 1 proteinase inhibitor: mutations, kinetics andfrequencies. J Infect Dis 1996;173:379–87.15. Feinberg MB, Carpenter C, Fauci AS, et al. Report of the NIH Panel toDefine Principles of Therapy of HIV Infection and Guidelines for the Useof Antiretroviral Agents in HIV-Infected Adults and Adolescents. AnnIntern Med 1997;128:1057–100.16. Kempf D, Rode R, Xu Y, et al. The durability of response to proteaseinhibitors therapy is predicted by viral load [abstract 62]. In: Programmeand abstracts of the International Workshop on HIV Drug Resistance,Treatment Strategies and Eradication (St. Petersburg). Antiviral therapy.St. Petersberg, FL: International Medical Press, 1997:41.

Resistance Mutations to Zidovudine and Saquinavir in Patients Receiving Zidovudine plus Saquinavir or Zidovudine and Zalcitabine plus Saquinavir in AIDS Clinical Trials Group 229

Resistance Mutations to Zidovudine and Saquinavir in Patients Receiving Zidovudine plus Saquinavir or Zidovudine and Zalcitabine plus Saquinavir in AIDS Clinical Trials Group 229

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