Stavudine Resistance: an Update on Susceptibility Following Prolonged Therapy

Antiviral Therapy 4: 21–28 Stavudine resistance: an update on susceptibility following prolonged therapy

Pin-Fang Lin1*, Charles J González2, Brigitte Griffith2,3, Gerald Friedland2, Vincent Calvez4, Francoise Ferchal5, Raymond F Schinazi6, David H Shepp7, Amar B Ashraf7, Mark A Wainberg8, Vincent Soriano9, John W Mellors10 and Richard J Colonno1

1Bristol-Myers Squibb, Wallingford, Conn., USA 2Yale University School of Medicine, New Haven, Conn., USA 3VA Connecticut Healthcare System, West Haven, Conn., USA 4CERVI, Pitié-Salpêtrière Hospital and 5St Louis Hospital, Paris, France 6VA Medical Center, Emory University, Decatur, Ga., USA 7North Shore University Hospital, New York University School of Medicine, Manhasset, NY, USA 8AIDS Center, McGill University, Montreal, Canada 9Instituto de Salud Carlos III, Madrid, Spain 10University of Pittsburgh VA Medical Center, Pittsburgh, Pa., USA

*Corresponding author: Tel: +1 203 677 6437; Fax: +1 203 677 6088; E-mail: [email protected]

The current report summarizes the available published tivity. Of the four remaining isolates, two appeared to and unpublished data from several investigators on resis- have a multi-resistant phenotype to several nucleoside tance in clinical isolates following prolonged stavudine analogues and two had no detectable RT amino acid therapy. Results suggest that stavudine resistance is both changes to account for the observed change in stavudine modest in degree and infrequent in appearance. sensitivity. To date, clinical HIV-1 isolates displaying Phenotypic evaluation of 61 patients on stavudine stavudine-specific resistance have yet to be reported. therapy showed only modest changes in drug sensitivity Furthermore, full or partial RT sequence analysis of 194 following up to 29 months of treatment. The post-treat- post-treatment isolates failed to identify any consistent ment isolates from 15 patients exhibited an increase in amino acid changes. The strain-specific V75T mutation

EC50 value >fourfold (level above variability of assay) reported to confer stavudine resistance to the HXB2 HIV- when compared with the corresponding pretreatment 1 strain in vitro, was found in only six isolates and did isolates. However, the vast majority (11) of these not correlate with stavudine resistance. This low inci- pretreatment isolates either had unexpectedly low EC50 dence of stavudine resistance is in striking contrast to levels and/or had post-treatment isolates that lacked any that observed with other nucleoside analogues and amino acid changes within their reverse transcriptase further supports the use of stavudine in first-line combi- (RT) gene to account for the observed change in sensi- nation therapy for HIV patients.

Currently available anti-human immunodeficiency phorylated in resting cells) may account for the incom- virus (HIV) drugs include six nucleoside reverse tran- plete suppression of sensitive viruses. Furthermore, the scriptase (RT) inhibitors (zidovudine, didanosine, high replication rate and rapid turnover of HIV-1 stavudine, lamivudine, zalcitabine and abacavir), three combined with the frequent incorporation of muta- non-nucleoside RT inhibitors (nevirapine, delavirdine tions leads to the appearance of drug-resistant variants and efavirenz) as well as four peptidic protease and treatment failures when suboptimal drug concen- inhibitors (saquinavir, indinavir, ritonavir and nelfi- trations are present [1–5]. navir). When used in combination, these drugs have For the nucleoside RT inhibitors, viral resistance been very effective in enabling patients to live longer occurs at different rates and to various degrees. and healthier lives, with the number of hospitalizations Zidovudine treatment results in an ordered accumula- and deaths down significantly in the past 2 years. tion of RT mutations at codons 41L, 67N, 70R, 215Y However, these drugs can only transiently restrain viral and 219Q and resulting virions that are 10- to 1000- replication when used alone. Insufficient drug potency, fold resistant to the drug [6,7]. The selected mutations non-compliance, restricted tissue penetration and appear within months and persist for prolonged drug-specific limitations within certain cell types (for periods after drug removal [6,8]. Patients treated with example most nucleoside analogues cannot be phos- lamivudine, nevirapine or delavirdine can show high

Table 1. Phenotypic and genotypic changes of HIV-1 isolates following stavudine monotherapy

µ Treatment (months) EC50 ( M) Fold Reverse transcriptase Number Patient Study Other d4T Pre-d4T Post-d4T increase (region sequenced); post-d4T mutations 1001-AHS 006 0 18 0.03 0.03 0 (1–580); M41L, T215Y, K223Q 2002-PMS 006 0 18 0.49 0.45 0 (1–580); K20R, M41L, T215Y, A360T, L422I 3 004-TGF 006 0.3 AZT 21 0.41 0.87 2 (1–580); S68G, K70N, K73R, V75T, D158S 4 005-MDM 006 1 AZT 18 0.06 0.47 8 (1–580); T69N, V106I, T165I, I178L, D192N, A360T 5 006-JEW 006 2 AZT 18 0.30 0.58 2 (1–580); I135T, T215Y, G335C, A360T, L422I, Q547R 6007-MSB 006 3 AZT 18 0.29 0.12 0 (1–580); A360T 7 008-MDS 006 4 AZT 18 0.06 0.73 12 (1–580); T178L, T215Y, K219E 8 010-SMF 006 9 AZT 18 0.16 0.55 3 (1–580); K67N, K70R, Q145E, K166R, K219Q, K249R, A360T, L503I 9011-JMM 006 9 AZT 18 0.07 0.01 0 (1–580); D324E, V466I 10 012-LRB 006 10 AZT 22 0.02 0.06 3 (1–580); M41L, T139I, E203K, H208Y, L210W, T215Y, T286A, V365I, L484I, L491S 11 013-JEE 006 16 AZT 18 0.59 0.65 1 (1–580); M41L, E44D, D67N, V118I, L210W, T215Y, L341F, A360T 12 014-ATR 006 0 22 0.21 0.48 2 (24–73); T69A 13 016-MLB 006 0 18 0.15 0.13 0 (45–72); K70R 14 018-VRR 006 0 22 0.23 0.15 0 (20–73); none 15 019-MXC 006 0 23 0.35 0.22 0 (32–95); none 16 020-BT 006 0 14 0.39 0.18 0 (38–81); none 17 023-JKE 006 0 22 0.38 0.32 0 (19–93); none 18 024-BJ 006 25 AZT 22 0.04 0.07 2 (28–93); none 19 025-OLR 006 4 AZT 11 0.06 0.06 0 (28–93); M41L 20 026-ES 006 5 AZT 11 0.04 0.03 0 (28–93); K64R 21 027-BGB 006 0 11 0.06 0.04 0 (28–93); none 22 028-CBD 019 0 12 0.22 0.36 2 Not sequenced 23 029-MCC 019 0 28 0.11 0.12 1 Not sequenced 24 030-CLI 019 0 28 0.19 0.40 2 Not sequenced 25 031-JMA 019 0 26 0.42 0.34 0 Not sequenced 26 032-DPB 006 14 AZT 11 0.02 0.04 2 (28–93); D67N, T69A/D, V75M 27 033-PET 019 25 AZT 19 0.02 0.02 0 (28–93); none 28 034-BJK 019 34 AZT 29 0.03 0.07 2 (28–93); V60I, T69A/D 29 035-WLM 019 32 AZT 26 0.01 0.03 3 (1–580); M41L, K122E, D177E, I178M,T215Y 30 036-JPD 019 7 AZT 6 0.01 0.01 0 (28–93); none 31 041 OS 0 7 0.02 0.20 10 (1–580); none 32 042 OS 0 8.5 0.05 0.07 <2 (1–580); none 33 043 OS 0 8 0.01 0.05 5 (1–580); none 34 044 OS 0 8 0.07 0.55 8 (1–580); none 35 045 OS 0 7 0.02 0.03 <2 (1–580); none 36 046 OS 0 9 0.05 0.40 8 (1–580); none 37 047 OS 0 6.5 0.07 0.09 1 (1–580); M41L 38 048 OS 0 9 0.03 0.28 9 (1–580); none 39 049 OS 0 10 0.07 0.43 6 (1–580); none 40 050 OS 0 10 0.03 0.03 0 (1–580); none 41 051 OS 0 11 0.20 0.30 <2 (1–580); none 42 052 OS 0 12 0.05 0.45 9 (1–580); none 43 053 OS 0 12 0.13 1.01 8 (1–580); none 44 054 OS 0 10 0.03 0.11 4 (1–580); none 45 055 OS 0 12 0.08 0.53 7 (1–580); none 46 056-No12 024 0 12 0.09 0.12 1 Not analysed 47 057-No30 024 0 12 0.02 0.15 8 Not analysed 48 058-No18 024 0 12 0.90 0.95 1 Not analysed 49 059-No27 024 0 12 0.02 0.04 2 Not analysed 50 060-SEB EA 12 AZT 12 0.02 0.30 15 (15–232) T139A, R211K 51 061-TSD EA 12 AZT 12 0.05 0.80 16 (15–232) V35L, T39A, K70G, V75A, N81I, W88S, K103R,

Continued opposite

Table 1. Continued

µ Treatment (months) EC50 ( M) Fold Reverse transcriptase Number Patient Study Other d4T Pre-d4T Post-d4T increase (region sequenced); post-d4T mutations K122E, F124S, Q151M/L, Q174P 52 062-DW 009 0 12 0.32 0.27 <1 Not analysed 53 063-VA 009 3 AZT 12 0.31 2.13 7 (14–250); none 54 064-VSG 009 2 AZT 12 0.22 0.35 2 Not analysed 55 065-GAP 009 9 AZT 12 0.11 0.33 3 Not analysed 56 066-JT 009 22 AZT 12 0.26 0.05 <1 Not analysed 57 067-JSC 009 3 AZT 12 0.06 0.07 1 Not analysed 58 068-MZ 009 3 AZT 12 0.08 0.06 <1 Not analysed 59 069-VVW 009 1 AZT 12 0.41 0.63 2 Not analysed 60 070-EP 009 0 12 0.21 0.34 2 Not analysed 61 071-NL 009 16 AZT 12 0.19 0.24 1 Not analysed Average 14.72 0.16±0.17 0.31±0.35 Abbreviations: d4T, stavudine; AZT, zidovudine; EA, stavudine available through expanded access. Source of patient data for 001–013 [31], 014–036 [32], 041–055 [11,34], 056–059 ([V Calvez & F Ferchal, unpublished results]; viruses were isolated by co-culturing the patient PBMCs with the uninfected cells. Drug sensitivity was determined in PBMCs using RT activity as an endpoint [50]), 060–061 ([DH Shepp & AB Ashraf, unpublished results, 44]; virus stocks were obtained by co-culturing patient sera with PBMCs. Determination of virus titre and EC50 were performed in PBMCs using p24 as an endpoint. RT sequences were genotyped by the cycle sequencing method using an automated DNA sequencer [51]), 062–071 ([CJ González, B Griffith & G

Friedland, unpublished results]; clinical virus stocks were prepared by a PBMC co-culture method and the stavudine EC50 values were determined according to the standardized ACTG drug susceptibility assay [52]). levels of resistance after only a few weeks of drug of resistant viral strains, insufficient drug concentra- therapy. The major RT resistance mutations are 184V tions and/or non-compliance [23]. To maximize the for lamivudine [9–13], 103N, 181C, 188C and 190A clinical benefits of HIV drugs, it is important to under- for nevirapine [14–16], and 103N, 181C and 236L for stand their drug resistance profiles and to select delavirdine [17]. In contrast, the levels of resistance properly the initial combination therapies that preserve development to both didanosine and zalcitabine are future treatment options. relatively low (three- to 10-fold) and appear in- Stavudine (2′,3′-didehydro-3′-deoxythymidine; d4T) frequently in treated patients. The predominant resis- is a potent inhibitor of HIV-1 RT in vitro [24]. In clin- tance marker for didanosine is 74V, whereas the ical use, stavudine has been well tolerated and has genetic markers for zalcitabine resistance are 65R, 69D excellent oral bioavailability with cerebrospinal fluid and 184V [7,9,11,18,19]. penetration. The only notable dose-limiting toxicity is Unlike RT inhibitors, HIV protease inhibitors select peripheral neuropathy; mild macrocytosis and hepatic for distinct but overlapping constellations of amino function disturbances are seen occasionally. These side- acid mutations that alter the inhibitor binding site. The effects are observed largely in patients with advanced major resistance substitutions include 48V and 90M HIV disease [25–28]. The drug has been shown in for saquinavir, 82A/F/T, 84V and 90M for indinavir several clinical trials to increase CD4 cell numbers and and ritonavir, and 30N and 84V for nelfinavir. In addi- decrease viral titres in HIV-infected individuals tion to these mutations, all of the well-characterized [25–29]. It is particularly effective when used in combi- protease inhibitors have been shown to select for addi- nation with another nucleoside analogue and a tional overlapping sets of amino acid substitutions protease inhibitor [30]. elsewhere in the enzyme. These sites include residues Among the approved antiretrovirals, stavudine has 10, 46, 63 and 71, among others. These overlapping been the most difficult to demonstrate specific pheno- mutations are likely to play a role in stabilizing active typic and genotypic resistance in the clinic, despite site mutations and in the observed cross-resistance prolonged treatment periods [8,31–35]. A stavudine among the protease inhibitors [4,5,7]. resistance mutation (V75T) in the HIV-1 RT has been Owing to the propensity for resistance development, reported, following in vitro passage of the HIV-1 the long-term success of antiretroviral therapy clearly HXB2 laboratory strain in the presence of stavudine, relies on a high level of viral suppression [20]. that conferred sevenfold resistance [36]. However, this Combination drug therapies consisting of two RT putative resistance marker appears to be strain- inhibitors and one protease inhibitor have become the specific, since the HIV-1 NL4-3 strain containing the standard of care and have recently been shown to same V75T RT mutation remains sensitive to stavudine reduce viraemia to undetectable levels [1,2,21,22]. [31]. In addition, repeated attempts to select stavudine- However, a significant number of patients fail combi- resistant variants using the HIV-1 RF or other strains nation therapy, presumably because of the emergence have been unsuccessful [P-F Lin et al., unpublished

Antiviral Therapy 4:1 23 P-F Lin et al.

Table 2. Summary data for the stavudine-resistant isolates

µ Treatment (months) EC50 ( M) Fold Reverse transcriptase Number Patient Study Other d4T Pre-d4T Post-d4T increase (region sequenced); post-d4T mutations 4 005-MDM 006 1 AZT 18 0.06 0.47 8 (1–580); T69N, V106I, T165I, I178L, D192N, A360T 7 008-MDS 006 4 AZT 18 0.06 0.73 12 (1–580); T178L, T215Y, K219E 31 041 OS 0 7 0.02 0.20 10 (1–580); none 33 043 OS 0 8 0.01 0.05 5 (1–580); none 34 044 OS 0 8 0.07 0.55 8 (1–580); none 36 046 OS 0 9 0.05 0.40 8 (1–580); none 38 048 OS 0 9 0.03 0.28 9 (1–580); none 39 049 OS 0 10 0.07 0.43 6 (1–580); none 42 052 OS 0 12 0.05 0.45 9 (1–580); none 43 053 OS 0 12 0.13 1.01 8 (1–580); none 45 055 OS 0 12 0.08 0.53 7 (1–580); none 47 057 024 0 12 0.02 0.15 8 Not analysed 50 060-SEB EA 12 AZT 12 0.02 0.30 15 (15–232); T139A, R211K 51 061-TSD EA 12 AZT 12 0.05 0.80 16 (15–232); V35L, T39A, K70G, V75A, N81I, W88S, K103R, K122E, F124S, Q151M/L, Q174P 53 063-VA 009 3 AZT 12 0.31 2.13 7 (14–250); none Abbreviations: d4T, stavudine; AZT, zidovudine; EA, stavudine available through expanded access. results; 34]. Most importantly, the V75T change acid changes resulting from prolonged stavudine expo- appears very infrequently in clinical isolates from sure. In particular, the significance of the 75T stavudine-treated patients, and no apparent change in substitution can be more fully evaluated. stavudine sensitivity has been noted in these isolates Results from six independent studies (Table 1) that [8,31,32,34]. include phenotypic and partial genotypic analysis of 61 To date, there have been no published reports of matched pairs of isolates showed that the average clinical isolates that display resistance specific to effective concentrations needed to cause a 50% reduc- stavudine. Each of the isolates reported to have tion in viral replication (EC50) of the pre- and reduced sensitivity to stavudine also displays resis- post-treatment viruses were 0.16±0.17 (0.01–0.90) tance to several other nucleoside inhibitors. These and 0.31±0.35 (0.01–2.13) µM, respectively. multi-resistance variants emerge through several Therefore, the average stavudine sensitivity only mechanisms. They can arise through specific path- changes moderately following treatment for a mean of ways that involve either selection of the 151M 14.7 months (range 6–29 months). Nonetheless, 15 mutation [37–39] or insertion of two serine or post-treatment isolates out of the 61 patients displayed serine–alanine residues at amino acid residue 69 a notable (>fourfold) decrease in drug sensitivity. A [40–43]. Multi-resistance to nucleoside analogues can >fourfold change was deemed significant based on the also be achieved through changes that appear to map three- to fourfold variability typically encountered in outside of the RT gene [31]. However, in each case these assays. A closer examination of the data from the multi-resistant isolates emerged in patients who these 15 isolates (summarized in Table 2) shows that have been on sequential or combination nucleoside many of these isolates have pretreatment sensitivities analogue therapy for a prolonged period. significantly lower than expected (0.16 µM), which Because of the difficulties encountered in isolating may account for their observed stavudine resistance stavudine-resistant isolates from treated patients, this phenotype. In support of this interpretation is the report attempts to compile all of the currently available subsequent finding that 10 of these 15 post-treatment published and unpublished data from several investiga- isolates identified in Table 2 have no evidence of amino tors who have looked for stavudine-resistant isolates. acid changes within the RT gene to account for the In most cases, clinical resistance was determined by observed change in sensitivity. evaluating drug susceptibilities and RT genotypes of Four isolates (008, 053, 061 and 063) possess post- µ pre- and post-treatment virus isolates (Table 1). treatment EC50 values (0.73–2.13 M) significantly Alternatively, some investigators only sequenced the higher than the average pretreatment isolate (0.16 µM) RT genes of post-therapy isolates owing to the absence and seven- to 16-fold higher than their paired pretreat- of baseline samples or insufficient virus recovery (Table ment or baseline isolates. Post-treatment isolate 008, µ 3). RT sequence analysis of post-stavudine patient with an EC50 of 0.73 M, was previously reported to isolates allows the identification of common amino be a multi-resistant isolate that also had reduced sensi-

Table 3. Genotypic analysis of RT from stavudine monotherapy viral isolates

Number of Treatment (months) Residues clinical isolates Study* AZT d4T sequenced Comments 2 006 0–5 18 1–580† No mutation at codon 75 [31] 4 d4T EA 2–31 6–42 15–232‡§ No consistent mutation observed; 1 had V75 V/L [DH Shepp & AB Ashraf, unpublished results, 44] 35 020 >4 24 30–240‡¶ No common mutation observed; No mutation at codon 75 [8,33,35] 32 NR 62475 No V75T mutation observed [45] 8 Random patients 0–86 1–18 22–221 V75M, E138S or Q207G arose in various isolates [46] 69 VA-01 0–98 1–45 29–220 Only one had V75T change ([RF Schinazi, unpublished results]; the mutation was detected by the line probe assay [53]) *EA, expanded access; NR, not reported. †The controls for RT mutation are the pretreatment samples. ‡Data for the pretreatment isolates and phenotype of these patient isolates were not available. §The control for RT mutation was the SF-2 strain of HIV-1. ¶The control for RT mutation was the IIIB strain of HIV-1. tivity to zidovudine (33-fold), lamividine (ninefold), stavudine-specific resistance. The impact of the didanosine (29-fold) and zalcitabine (fourfold). V75L/M/T substitutions on stavudine sensitivity in the Interestingly, experiments using a chimeric virus other three isolates could not be accurately evaluated containing the entire RT gene from patient isolate 008- because of the lack of baseline samples (Table 3) MDS yielded a recombinant NL4-3 virus with normal [44,46]. Together, these results suggest that RT muta- sensitivity to all of the nucleoside analogues except tions at residue 75 are rarely selected by stavudine zidovudine [31]. This result suggested that the multi- therapy and that the 75T mutation alone is not suffi- resistance observed with this isolate maps outside of cient to confer stavudine resistance to clinical isolates. the RT gene and is likely due to a change in growth In earlier studies, zidovudine-associated mutations phenotype [31]. Post-treatment isolate 053 displayed a were observed in post-stavudine treatment samples, stavudine sensitivity of 1.01 µM, an eightfold change with further studies suggesting that the appearance of from baseline, but had no identified amino acid zidovudine resistant mutations may be caused by changes within the RT gene. The resistance pattern of covert usage of zidovudine by those on stavudine this isolate to other nucleoside analogues, if any, was monotherapy [31]. This conclusion is further strength- not determined. Post-treatment isolate 061-TSD had ened by the fact that stavudine did not select any µ an EC50 of 0.8 M and several amino acid changes, zidovudine-related RT mutation in vitro [36,48]. The including the known multi-resistance marker at residue cumulative data reported here from six independent 151, and is likely to be cross-resistant to several of the laboratories (Table 2) revealed that only one out of 15 nucleoside analogues. Finally, post-treatment isolate isolates with >fourfold reduced sensitivity to stavudine µ 063-VA had an EC50 of 2.13 M, only sevenfold higher contained known zidovudine resistance mutations. than the pretreatment isolate. No mutations were While there is insufficient data to rule out the possi- detected within the region between amino acids bility that stavudine may induce zidovudine-resistant 14–250 of RT as a result of therapy. Therefore, of the mutations, it is clear that zidovudine-resistant HIV aforementioned four putative resistant isolates, two strains remain sensitive to stavudine [31,47]. This were devoid of RT mutations and two isolates had finding questions the viral benefit of selecting for unrelated mutations and probably multi-resistant zidovudine mutations with stavudine. Additional phenotypes. studies will clearly be needed to further resolve this Attempts to identify a stavudine resistance marker issue. solely by RT genotypic analysis of an additional 150 An evaluation of whether stavudine resistance post-stavudine treatment isolates has not been fruitful, emerges during combination therapy is underway. In since no consistent mutations were observed following some patients treated with zidovudine plus either prolonged stavudine treatment in six studies didanosine or zalcitabine, viruses became simultane- [31,35,44–46, RF Schinazi, unpublished results]. A ously resistant to zidovudine, didanosine, zalcitabine specific emphasis was put on determining whether the and stavudine, and contained a novel set of RT muta- 75T mutation observed in vitro appeared in patient tions at positions 62V, 75I, 77L, 116Y and 151M isolates following prolonged stavudine therapy. [37–39]. However, the 75I mutation did not correlate Isolates from six out of the total 194 patients analysed with stavudine resistance because the early treatment contained a mutation at residue 75 (Tables 1 and 3). virus containing the 77L, 116Y and 151M substitu- Two of these viral isolates (004 and 032) evaluated tions showed an equal reduction in stavudine phenotypically remained sensitive to stavudine (Table sensitivity [38]. As mentioned above, the Q151M 1) [31,32], whereas a third (061) had a multi-resistant mutation appears to be essential for the multi-drug 151 mutation marker and could not be assessed for resistance phenotype. One study involving the combi-

Antiviral Therapy 4:1 25 P-F Lin et al.

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Received 4 November 1998; accepted 17 December 1998