Original Article Antiviral Activity and Tolerability of Amdoxovir with Zidovudine in a Randomized Double-Blind Placebo- Controlled Study in HIV-1-Infected Individuals

Antiviral Therapy 2010 15:185–192 (doi: 10.3851/IMP1514)

Original article Antiviral activity and tolerability of amdoxovir with zidovudine in a randomized double-blind placebo- controlled study in HIV-1-infected individuals

Robert L Murphy1, Nancy M Kivel2, Carlos Zala3, Claudia Ochoa3, Phillip Tharnish2, Judy Mathew 2, Maria Luz Pascual2, Raymond F Schinazi4*

1Northwestern University, Feinberg School of Medicine, Chicago, IL, USA 2RFS Pharma, LLC, Tucker, GA, USA 3Hospital Privado Modelo, Florida-Buenos Aires B1602DBG, Argentina 4Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA, USA

*Corresponding author e-mail: [email protected]

Background: Amdoxovir acts synergistically with zido- day 10 were determined. Laboratory and clinical safety vudine in vitro and the combination prevents or delays monitoring were performed. the selection of thymidine analogue and K65R muta- Results: Twenty-four patients were enrolled. The mean

tions. In silico studies have shown that a reduced dose VL log10 change was 0.10 with placebo, -0.69 with zido- of zidovudine (200 mg) results in decreased zidovudine- vudine 200 mg, -0.55 with zidovudine 300 mg, -1.09 monophosphate levels, associated with toxicity, while with amdoxovir, -2.00 with amdoxovir plus zidovudine maintaining zidovudine-triphosphate levels, which are (200 mg) and -1.69 with amdoxovir plus zidovudine associated with antiviral effects. Here, we aimed to (300 mg). Amdoxovir plus zidovudine (200 mg) was assess the short-term tolerability and antiviral activity significantly more potent than amdoxovir monotherapy

of amdoxovir in combination with reduced and standard in AUCVL and mean VL decline (P=0.019 and P=0.021, doses of zidovudine. respectively), suggesting synergy. There was markedly Methods: The study was a double-blind, placebo- decreased VL variability with the combination compared controlled study in HIV-1-infected patients not receiving with amdoxovir alone. All adverse events were mild to antiretroviral therapy and with plasma HIV-1 RNA ≥5,000 moderate. copies/ml. Patients were randomized to 10 days of twice- Conclusion: The combination of amdoxovir plus zidovu- daily treatment with 200 mg zidovudine, 300 mg zidovudine appeared synergistic with reduced VL variability. This dine, 500 mg amdoxovir, 500 mg amdoxovir plus 200 mg combined therapy, including the use of a lower zidovudine zidovudine or 500 mg amdoxovir plus 300 mg zidovu- dosage, warrants further development for the therapy of

dine. The mean change in viral load (VL) log10 and area HIV infection.

under the virus depletion curve (AUCVL) from baseline to

Introduction

Nucleoside reverse transcriptase inhibitors (NRTIs) Amdoxovir (AMDX, DAPD, (–)-β-d-2,6- form the backbone of currently used first-line regi- diaminopurine dioxolane) is an NRTI currently in mens for the treatment of HIV-1 infections and are the advanced Phase II clinical trials that has been safely preferred drug class of treatment in combination with administered to nearly 200 patients. This prodrug of other classes of drugs [1]. Suboptimal adherence mainly DXG (β-d-[2R,4R]-1,3-dioxolane guanosine) is the due to poor tolerability will lead to treatment failure only guanosine nucleoside analogue in clinical devel- in all regimens because of the subsequent development opment for the treatment of HIV-1 infections. DXG of drug resistance. Such failure would lead to limited has shown good activity in vitro against HIV-1 strains future therapeutic options [2]. resistant to lamivudine or emtricitabine (M184V/I)

AVT-09-OA-1380_Murphy.indd 185 23/3/10 15:36:48 RL Murphy et al.

and strains containing zidovudine/stavudine-resistant Amdoxovir has been well-tolerated in six human thymidine analogue mutations (TAMs), including the Phase I/II studies [21–24]. In one study involving 18 69SS double insert [3–5]. In vitro, DXG demonstrated individuals on amdoxovir therapy (300 or 500 mg synergistic antiviral activity in combination with other twice daily), non-gradable lens opacities were observed NRTIs, including zidovudine and lamivudine [3]. Selec- in four patients between weeks 6.8 and 15, and mild tion studies showed a delayed emergence of HIV-1- posterior subcapsular cataract was identified at week resistant viruses to DXG, which was associated with 3.6 in one patient; all five patients were withdrawn K65R or L74V mutations in MT-2 cells [6]. from the study. As ophthalmologic examinations were Previous reports suggest that the K65R mutation, added only mid-study, it is not known whether these which confers cross-resistance to DXG and amdoxo- findings were present at baseline. Lens abnormalities vir, can revert zidovudine-resistant virus to zidovudine- were visually insignificant in all five patients and there sensitive virus [3,5–7]. As observed for the K65R were no renal or glucose toxicities [21]. There were variant, virus containing the L74V mutation remained no lens abnormalities noted in the other 13 patients in sensitive to zidovudine [6]. In vitro, the combination this study, who were administered amdoxovir for up of amdoxovir plus zidovudine resulted in the absence to 96 weeks (n=4) or received amdoxovir at a dose of of drug-resistant mutations through week 28 [8]. The 300 mg twice daily (n=9) for up to 48 weeks in the bidirectional phenotypic antagonism between the K65R AIDS Clinical Trials Group (ACTG) study A5118; like- and TAM pathways has been elucidated previously, dem- wise, no abnormalities were seen in patients receiving onstrating that K65R reduces the enzymatic excision of amdoxovir at a dose of 500 mg twice daily (n=49) for chain-terminating analogues, such as zidovudine, and up to 96 weeks (n=5) in ACTG study A5165 [23,24]. that TAMs counteract K65R-mediated resistance by A recent in silico study demonstrated that reduced dose reducing the selectivity of the virus to incorporate nat- zidovudine at 200 mg twice daily produces decreased ural substrate over the nucleoside analogue [7,9–11]. zidovudine-monophosphate levels without reducing Retrospective analyses of clinical databases of patient zidovudine-triphosphate levels, which are necessary for genotypes also demonstrate the negative association of the antiviral effect [25]. Therefore, a proof-of-concept K65R with specific TAMs [7,9,12,13]. study was conducted in HIV-infected patients to evalu- Zidovudine is an attractive drug for use in combi- ate the short-term tolerability and antiviral activity of nation with amdoxovir, as these drugs are activated amdoxovir (500 mg twice daily) in combination with by different phosphorylation pathways (thymidine reduced and standard doses of zidovudine. kinase and 5′-nucleotidase, respectively) and the combination does not affect the nucleoside triphosphate Methods (NTP) levels of each drug in vitro [14–16]. On the basis of the favourable mutational resistance mecha- Approval nisms, counter-selection of K65R and TAMs, and The protocol was approved by the local ethics com- synergistic antiviral activity, it is expected that coad- mittees (Facultad de Medicina-Universidad de Buenos ministration of zidovudine with amdoxovir could Aires, Comité Independiente de Etica en Investigación delay the emergence of NRTI resistance and prolong [CIEI-FM-UBA]) and the Administración Nacional treatment response [17–20]. de Medicamentos, Alimentos y Tecnología Médica The major toxicity of amdoxovir in extensive animal (ANMAT) of Argentina. toxicology studies was obstructive nephropathy at high doses, secondary to the precipitation of amdoxovir/DXG Study design in the renal tubules. This was probably due to the poor This was a Phase Ib/IIa randomized, double-blind, aqueous solubility of DXG, similar to that observed for proof-of-concept study to evaluate antiviral activity the related nucleoside analogues acyclovir and cidofovir. and short-term tolerability of amdoxovir in combina- In a 52-week study, five cynomolgus monkeys, receiving tion with two different doses of zidovudine. Patients amdoxovir at 800 or 1,200 mg/kg per day, developed were randomly assigned (1:1:1) to one of three treat- obstructive nephropathy and associated uraemia after ment groups and within each treatment group patients 26 weeks of treatment, which was reversible after early were randomly assigned (3:1) to amdoxovir or placebo. identification and discontinuation of medication. In Treatment A was 500 mg amdoxovir or placebo twice addition, islet cell atrophy and hyperglycaemia occurred. daily, treatment B was 500 mg amdoxovir or placebo Lens opacities were observed, which was thought to plus 300 mg zidovudine twice daily and treatment C be secondary to hyperglycaemia and not a direct effect was 500 mg amdoxovir or placebo plus 200 mg zido- of amdoxovir. No patients enrolled in clinical trials of vudine twice daily. Computer-generated randomization amdoxovir have developed any renal abnormalities codes were used. Zidovudine administration was open- attributable to the drug [21]. label; amdoxovir administration was blinded. Patients,

AVT-09-OA-1380_Murphy.indd 186 23/3/10 15:36:48 Antiviral activity of amdoxovir in combination with zidovudine

investigators and laboratory personnel were blinded to Descriptive statistics were calculated for antiviral amdoxovir treatment assignment. Twenty-four patients activity, and immunological and safety parameters. The

were required to obtain eight evaluable patients per area under the virus depletion curve (AUCVL) and the treatment group. Within a group, six patients were ran- change in viral load from baseline were analysed using domized to amdoxovir and two to placebo. The proto- analysis of covariance (ANCOVA), including amdox- col allowed for replacement of a patient who withdrew ovir/placebo and zidovudine dose (none, 200 mg or consent or discontinued the study prior to day 10 for 300 mg) as factors, the amdoxovir plus zidovudine

any reason. interaction arm, and baseline log10 HIV-1 RNA as a covariate. The difference between least squares means Patients and the 95% confidence interval on the difference was Patients were HIV-1 infected men and women ≥18 years calculated for pairwise comparisons. The pairwise of age who were either antiretroviral therapy naive or comparisons examined the effect of amdoxovir sepa- had not received any antiretroviral therapy in the previ- rately with each dose of zidovudine (none, 200 mg or ous 90 days. All patients included had a plasma HIV-1 300 mg) and evaluated the effect of the three doses RNA (viral load, VL) ≥5,000 copies/ml and a CD4+ of zidovudine (20 0 mg versus none, 300 mg versus T-cell count ≥200 cells/mm3. Females of child-bearing none and 300 mg versus 200 mg) in combination with potential were required to have a negative pregnancy amdoxovir. Unadjusted P-values for the pairwise com- test and agree to adequate contraception. parisons between least squares means were obtained Exclusionary criteria were any active AIDS-defining from the ANCOVA; the Bonferroni adjustment was illness [26], active acute hepatitis A and/or chronic applied to protect the type I error rate for the pairwise hepatitis B or C with detectable viraemia, abnormal comparison of treatments. clinical laboratory tests, any active clinically sig- Several antiviral activity endpoints were assessed:

nificant disease or findings that in the investigator’s AUCVL from day 1 to day 10; change in viral load from opinion would compromise the outcome of the study, baseline through day 12 and on day 20; the lowest concomitant use of antiviral drugs, prophylactic drugs viral load value through day 12 and day 20; the maxi- for opportunistic infections, nephrotoxic drugs, com- mum reduction in viral load from baseline through petitors of renal excretion or immunosuppressive day 12 and day 20; the proportion of patients with

drugs within 30 days of study entry, visual abnormali- a decrease of at least 1.0 and 1.5 in log10 viral load ties (for example, cataracts or macular degeneration) on each day; the proportion of patients with a plasma other than non-organic decreased visual acuity, diabe- HIV-1 RNA value <400 copies/ml on each day; and tes mellitus type 1 or 2, active alcohol or drug use and the proportion of patients with a plasma HIV-1 RNA pregnancy or lactation. value <50 copies/ml on each day. The patients received the assigned study medication Immunological changes were determined by changes twice daily from days 1 to 9, but only one dose on in CD4+ and CD8+ T-cell counts from baseline at day 10 day 10. The morning dose was administered at the study by treatment arm. The incidence of adverse events and site, whereas the evening dose was taken by patients at laboratory toxicities were summarized by treatment home 12 h later. Those patients assigned to the amdoxo- group. The data were analysed using SAS version 9.1 vir plus zidovudine combination arms took both drugs (SAS Institute Inc, Cary, NC, USA). The adverse events at the same time. The time of the evening dose taken were coded in MedDRA (MSSO, Chantilly, VA, USA). at home was reported in a diary from days 2 to 9. The patients returned for follow-up visits on days 11, 12, 15 Results and 20. Plasma HIV-1 RNA was measured at baseline and on Baseline characteristics days 1–10, day 12 and day 20 using the standard PCR All 24 patients who were dosed completed the study and assay Amplicor HIV Monitor Assay, version 1.5 (Roche complied with the protocol. The primary baseline char- Diagnostics, Indianapolis, IN, USA). Viral genotyping acteristics of the patients according to treatment groups at baseline and day 10 was carried out using Trugene are summarized in Table 1. At baseline, the mean age

(Bayer Health Care LLC, Tarrytown, NY, USA). was 34 years, log10 viral load was 4.5 copies/ml and CD4+ and CD8+ T-cell counts were 417 cells/mm3 and Statistical analysis 975 cells/mm3 respectively. Of the total number of All patients who completed 10 days of treatment were patients, 54% were treatment-naive and 46% had pre- evaluable for antiviral activity analyses. All patients who vious antiretroviral therapy. Prior resistance testing was received at least one dose of study medication and had not available for the treatment-experienced patients as at least one post-baseline safety evaluation were included routine resistance testing is not necessarily part of the in the safety evaluation. Missing data were not imputed. clinical management of HIV-1 infection in Argentina.

Antiviral Therapy 15.2 187

AVT-09-OA-1380_Murphy.indd 187 23/3/10 15:36:48 RL Murphy et al.

Table 1. Baseline patient characteristics according to treatment group

Placebo ZDV 200 mg ZDV 300 mg AMDX 500 mg AMDX 500 mg + AMDX 500 mg + Characteristic (n=2) (n=2) (n=2) (n=6) ZDV 200 mg (n=6) ZDV 300 mg (n=6)

Gender Male, n (%) 2 (100) 0 1 (50) 5 (83) 4 (67) 0 Female, n (%) 0 2 (100) 1 (50) 1 (17) 2 (33) 6 (100) Mean age ±s d , years 27.0 ±4.2 47.5 ±3.5 24.0 ±2.8 36.7 ±10.4 37.3 ±9.7 30.0 ±5.2 (min–max)a (24.0–30.0) (45.0–50.0) (22.0–26.0) (21.0–49.0) (23.0–52.0) (21.0–36.0)

Mean log 10 viral load, copies/ml 4.1 3.8 5.0 4.7 4.5 4.5 (min–max)a (3.9–4.2) (3.6–3.9) (4.9–5.1) (4.3–5.0) (3.7–6.0) (4.0–5.1)

Patients with viral load >5 log10, n 0 0 1 0 1 1 Mean CD4+ T-cell count, cells/mm3 588 366 336 378 472 388 (min–max)a (570–606) (270–461) (227–445) (234–614) (237–1071) (201–580) Any previous ART, n (%) 1 (50) 0 1 (50) 2 (33) 2 (33) 5 (83) ATC class None, % 50 100 50 67 67 17 PI, % 50 0 0 33 0 0 NRTI, % 50 0 50 33 67 83 NNRTI, % 0 0 50 17 33 33 ART combination None, % 50 100 50 67 67 17 NRTI, % 0 0 0 0 17 50 PI+NRTI, % 50 0 0 17 0 0 NRTI+NNRTI, % 0 0 50 0 17 33 PI+NRTI+NNRTI, % 0 0 0 17 0 0 Patients with NRTI mutations, n 0 0 0 0 0 0 Patients with NNRTI mutations, n 0 0 0 0 0 2 Patients with PI mutations, nb 2 2 2 6 6 6

amin–max, minimum and maximum values. bMutations probably represent polymorphisms and not primary protease inhibitor mutations. AMDX, amdoxovir; ART, antiretroviral therapy; ATC, anatomical, therapeutic and chemical; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; ZDV, zidovudine.

Figure 1. Area under the virus depletion curve by treatment The patients, by choice or per discretion of treating physicians, were off treatment and not monitored for resistance. Of significance, no patients at baseline had 4 NRTI mutations, but two patients in the amdoxovir 2 plus zidovudine 300 mg arm had NNRTI mutations. 0 Protease inhibitor (PI) mutations were observed in all

change in -2 study participants; however, the PI mutations probably VL

copies/ml -4 represent polymorphisms and not primary mutations. In 10 -6 96% of patients, no baseline resistance to PI or boosted -8 PI regimens was detected. One patient in the amdoxovir -10 plus zidovudine 200 mg group had possible resistance to atazanavir and tipranavir/ritonavir at baseline. No -12

HIV-1 RNA, log HIV-1 new mutations were detectable at day 10. -14 Mean (min–max) AUC -16 Antiviral activity

The virus depletion AUCVL of plasma HIV-1 RNA over Placebo the 10 day treatment period according to treatment ZDV 200 mgZDV 300 mg s d AMDX 500 mg group is shown in Figure 1. The mean ± for AUCVL AMDX 500 mgAMDX + 500 mg + ZDV 200 mgZDV 300 mg was 0.41 1.17 with placebo, -4.50 0.19 with zidovu- Treatment group ± ± dine 200 mg, -2.42 ±1.80 with zidovudine 300 mg, -6.69 ±4.57 with amdoxovir, -11.98 ±1.86 with amdoxovir The mean is represented by a black diamond; bars represent minimum and maximum values. AMDX, amdoxovir; AUC, area under the curve; HIV-1, HIV plus zidovudine 200 mg and -11.32 ±1.44 with amdox- type-1; VL, viral load; ZDV, zidovudine. ovir plus zidovudine 300 mg. The two amdoxovir plus

AVT-09-OA-1380_Murphy.indd 188 23/3/10 15:36:49 Antiviral activity of amdoxovir in combination with zidovudine

Figure 2. Mean change in plasma viral load levels by treatment and day

0.5

0 copies/ml

10 -0.5

-1

-1.5

Mean change in HIV-1 RNA, log Mean change in HIV-1 -2

Treatment duration: days 1–10 -2.5 123456789 10 11 12 13 14 15 16 17 18 19 20

Time, days

Placebo ZDV 200 mg ZDV 300 mg AMDX 500 mg AMDX 500 mg + ZDV 200 mg AMDX 500 mg + ZDV 300 mg

AMDX, amdoxovir; HIV-1, HIV type-1; ZDV, zidovudine.

zidovudine combination groups had the greatest mean drugs of the combination) and persisted 2 days post- AUCs, which were comparable. The ANCOVA results treatment. Viral load decline was significantly improved showed amdoxovir/placebo (P<0.0001) and zidovudine with amdoxovir plus zidovudine 200 mg and 300 mg (P=0.013) to be significant cofactors with no significant combinations, compared with zidovudine monotherapy amdoxovir–zidovudine interaction effect (P=0.597). at days 11 (P=0.036 and P=0.015, respectively) and 12 Three significant pairwise comparisons were noted: (P=0.027 and P=0.024, respectively). There was no sig- amdoxovir plus zidovudine 300 mg versus zidovudine nificant difference between amdoxovir plus zidovudine 300 mg (P=0.005), amdoxovir plus zidovudine 200 mg 200 mg and amdoxovir plus zidovudine 300 mg com- versus amdoxovir (P=0.019) and amdoxovir plus zido- bination treatments. Furthermore, there was markedly vudine 300 mg versus amdoxovir (P=0.045). decreased viral load variability with both amdoxovir

The mean change in viral load (log10 copies/ml) by plus zidovudine combinations compared with amdoxo- treatment group over time is shown in Figure 2. The vir alone. Viral load returned to pre-treatment levels fol-

log10 mean change in viral load from baseline at the lowing treatment cessation, but there was a continued end of treatment was as follows: 0.10 (placebo), -0.69 post-treatment antiviral effect with amdoxovir in com- (zidovudine 200 mg), -0.55 (zidovudine 300 mg), -1.1 bination with zidovudine 200 or 300 mg groups. (amdoxovir 500 mg), -2.0 (amdoxovir plus zidovudine The proportion of patients with at least 1.0, 1.5

200 mg) and -1.7 (amdoxovir plus zidovudine 300 mg). log10 copies/ml decline in viral load or who had HIV-1 Viral load reductions for amdoxovir plus zidovudine 200 RNA<400 copies/ml on the last day of treatment is mg were significantly greater than for amdoxovir mono- shown in Figure 3. As anticipated, none of the patients therapy at days 8–12 (P=0.003, 0.008, 0.021, 0.005 and in the placebo or zidovudine 200 mg or 300 mg mon-

0.016, respectively), suggesting synergy (as calculated otherapy groups exhibited a ≥1.0 log10 decrease or

by adding log10 viral load declines for the individual HIV-1 RNA<400 copies/ml at the end of treatment,

Antiviral Therapy 15.2 189

AVT-09-OA-1380_Murphy.indd 189 23/3/10 15:36:50 RL Murphy et al.

Figure 3. Proportion of individuals (n=6 per group) with amdoxovir plus zidovudine 200 mg versus amdoxovir

at least 1.0, 1.5 log10 copies/ml viral load decline or HIV-1 monotherapy (P=0.015; P=0.015). RNA<400 copies/ml at day 10 During the short treatment period, there were no clinically significant changes in the absolute CD4+ or CD8+ T-cell counts in all treatment groups by day 10. HIV-1 RNA Changes in the absolute CD4+ and CD8+ T-cell counts <400 copies/ml ranged from -315 to 161 cells/mm3 and -402 to 817 1.0 log copies/ml decline 3 ≥ 10 cells/mm , respectively.

≥1.5 log10 copies/ml decline 100100 100 Tolerability 100 The 10-day treatment with amdoxovir/placebo with or 90 83 without zidovudine was well-tolerated; there were no 80 early withdrawals, discontinuations or serious adverse 70 events reported in the study. The adverse events assessed 60 50 50 by the principal investigator as at least remotely related 50 40 33 33 to the study medication are presented in Table 2.

Patients, % 30 17 20 Discussion 10 0 This 10-day monotherapy and dual-therapy design AMDX AMDX 500 mg AMDX 500 mg 500 mg bid + ZDV + ZDV provided a direct and rapid assessment of the poten- 200 mg bid 300 mg bid tial activity and tolerability of amdoxovir in combi- Treatment group nation with zidovudine and the short duration of the study limited the potential for the development of drug resistance. A direct characterization of efficacy, AMDX, amdoxovir; bid, twice daily; HIV-1, HIV type-1; ZDV, zidovudine. safety and tolerability without the confounding effects of other antiretroviral agents is possible with short- term monotherapy trials involving antiretroviral-naive whereas all of the patients in the two amdoxovir plus patients, although this study evaluated treatment-naive

zidovudine groups had a >1.0 log10 decrease with 50% and treatment-experienced patients who had not been achieving HIV-1 RNA<400 copies/ml. All patients in treated for at least 90 days [27]. the amdoxovir plus zidovudine 200 mg group and The significant antiviral activity observed for the two five out of six patients in the amdoxovir plus zidovu- combination groups compared with the amdoxovir or

dine 300 mg group exhibited a 1.5 log10 drop in viral zidovudine monotherapy groups after 10 days of treat- load. One patient in the amdoxovir plus zidovudine ment suggests a synergistic interaction between the

200 mg group reached HIV-1 RNA<50 copies/ml at two NRTIs, as noted by the greater than additive log10 days 11–12. The greatest mean maximum reduction reduction in viral load. Among all the actively treated in viral load value from baseline through day 12 was patients, those randomized to amdoxovir plus zidovu- seen in the amdoxovir plus zidovudine 200 mg group dine 200 mg exhibited the greatest reduction in viral

(-2.07 ±0.16 log10 copies/ml) followed by the amdoxo- load; 50% of the patients achieved at least a 2 log10

vir plus zidovudine 300 mg group (-1.84 ±0.28 log10 drop in viral load at day 10. As anticipated from the copies/ml) and the amdoxovir monotherapy group in silico studies [25], the antiviral activity observed in

(-1.22 ±0.30 log10 copies/ml). This coincided with the amdoxovir plus zidovudine 300 mg group was not the lowest viral load value observed through day 12: significantly different from amdoxovir plus zidovudine amdoxovir plus zidovudine 200 mg group (2.50 ±0.72 200 mg, although the magnitude of change was numer-

log10 copies/ml) followed by the amdoxovir plus zido- ically higher in the latter group at day 10. Likewise, the

vudine 300 mg group (2.70 ± 0.37 log10 copies/ml) magnitude of the overall incidence rate of treatment- and the amdoxovir monotherapy group (3.48 ±0.67 related adverse events in the amdoxovir plus zidovu-

log10 copies/ml). The ANCOVA results indicated that dine 300 mg group was comparable, but numerically amdoxovir/placebo and zidovudine had significant higher than for the amdoxovir plus zidovudine 200 mg effects on the maximum reduction in viral load and group. The results support the hypothesis that lowering produced the lowest viral load values. Significant pair- the dose of zidovudine when administered in combina- wise comparisons for both outcomes were observed tion with amdoxovir does not affect antiviral activity for amdoxovir plus zidovudine 300 mg versus zido- and is synergistic. These data were in agreement with vudine 300 mg monotherapy (P=0.008; P=0.008) and the in silico finding that a lower dose of zidovudine

AVT-09-OA-1380_Murphy.indd 190 23/3/10 15:36:50 Antiviral activity of amdoxovir in combination with zidovudine

Table 2. Treatment-emergent and treatment-related adverse events according to treatment group

Placebo ZDV 200 mg ZDV 300 mg AMDX 500 mg AMDX 500 mg plus AMDX 500 mg plus Total Adverse event (n=2) (n=2) (n=2) (n=6) ZDV 200 mg (n=6) ZDV 300 mg (n=6) (n=24)

At least one adverse event, n (%) 2 (100) 2 (100) 0 4 (67) 5 (83) 6 (100) 19 (79) Headache, n (%) 1 (50) 1 (50) 0 1 (17) 3 (50) 3 (50) 9 (37) Nausea, n (%) 0 0 0 0 3 (50) 3 (50) 6 (25) Dyspepsia, n (%) 2 (100) 1 (50) 0 1 (17) 0 1 (17) 5 (21) Abdominal pain upper, n (%) 0 1 (50) 0 0 1 (17) 0 2 (8) Back pain, n (%) 1 (50) 0 0 0 1 (17) 0 2 (8) Constipation, n (%) 0 0 0 1 (17) 0 1 (17) 2 (8) Stomach discomfort, n (%) 0 0 0 0 0 2 (33) 2 (8) Abdominal pain, n (%) 0 0 0 0 0 1 (17) 1 (4) Anxiety, n (%) 0 0 0 0 1 (17) 0 1 (4) Chest pain, n (%) 0 0 0 0 1 (17) 0 1 (4) Diarrhoea, n (%) 0 0 0 0 0 1 (17) 1 (4) Fatigue, n (%) 0 0 0 0 1 (17) 0 1 (4) Mucosal inflammation, n (%) 0 0 0 0 1 (17) 0 1 (4) Muscle contracture, n (%) 0 1 (50) 0 0 0 0 1 (4) Rhinitis seasonal, n (%) 0 0 0 1 (17) 0 0 1 (4) Vomiting, n (%) 0 0 0 0 0 1 (17) 1 (4)

AMDX, amdoxovir; ZDV, zidovudine.

retains the antiviral effect of the standard dose while reduced dose of zidovudine for the treatment of HIV-1 decreasing its toxicity [25]. infection. It should be noted that in certain countries The mean viral load decline from baseline of -1.1 like Thailand, the National Guidelines already recom-

log10 at day 10 observed with amdoxovir monotherapy mend zidovudine at the reduced dose of 200 mg twice was consistent with that reported previously. In a daily in persons <60 kg in weight [29]. In persons fail- short-term study involving treatment-naive patients, ing current first-line therapies, there is an ongoing need

the median viral load reduction was -1.3 log10 at day for new antiretroviral agents of existing classes with 15 [22]. Clinically significant improvements in + CD4 improved efficacy, tolerability and a high genetic bar- T-cell count would not be expected following 10 days rier to resistance that could prevent or delay the emer- of treatment and was not observed in this study. The gence of resistance. On the basis of the clinical study active metabolite of amdoxovir (DXG) produces rea- presented herein and previously published studies [21], sonably high levels of DXG-triphosphate, which has a the combination of amdoxovir plus zidovudine has the long intracellular half-life of 16 h in activated periph- potential to provide benefit as an important nucleoside eral blood mononuclear cells [16]. Thus, in combination treatment option, particularly in delaying the emer- with zidovudine, it is reasonable to observe a gradual gence of NRTI resistance. This combination also has decline in antiviral effect following cessation of therapy. the potential to provide a bridging regimen in failing There were no serious adverse events and no treatment persons with the option to postpone the introduction discontinuations. Likewise, the pharmacokinetic data of new drug classes. Additional long-term studies in from this study did not show any drug–drug interac- patients failing therapy is warranted to further evaluate tions [28]. This apparently benign tolerability profile the long-term efficacy and safety of this combination. and the potent antiviral activity observed in this study makes amdoxovir plus zidovudine a promising NRTI Acknowledgements combination product. However, the generalization of the results is limited by the small sample size and the The authors acknowledge the assistance of the following short treatment duration. individuals: Jose Luis Ippolito MD in managing this study In conclusion, amdoxovir in combination with (ACLIRES-Argentina, Buenos Aires, Argentina); Eliza- reduced or approved dose of zidovudine showed prom- beth Belsey PhD for statistical analysis and programming ising antiviral activity, good short-term tolerability and (SigmaClinical, Mas de Cause, France); Marina Machera a low propensity for resistance selection in patients PhD for clinical site monitoring (ACLIRES-Argentina, treated for 10 days. These results are significant and Buenos Aires, Argentina) and all the patients who par- provide initial clinical data to support the viability ticipated in this study. The work presented herein was of a combination of amdoxovir plus the approved or supported by RFS Pharma, LLC. RFS is supported by

Antiviral Therapy 15.2 191

AVT-09-OA-1380_Murphy.indd 191 23/3/10 15:36:50 RL Murphy et al.

13. Valer L, Martin-Carbonero L, de Mendoza C, Corral A, the National Institutes of Health sponsored Center for Soriano V. Predictors of selection of K65R: tenofovir use AIDS (2P30-AI-050409) and the Department of Veter- and lack of thymidine analogue mutations. AIDS 2004; 18:2094–2096. ans Affairs. ClinicalTrials.gov number: NCT00432016 14. Stein DS, Moore KHP. Phosphorylation of nucleoside analog antiretrovirals: a review for clinicians. Disclosure statement Pharmacotherapy 2001; 21:11–34. 15. Feng JY, Parker WB, Krajewski ML, et al. Anabolism of amdoxovir: phosphorylation of dioxolane guanosine and RFS, as an inventor of the amdoxovir technology and its 5′-phosphates by mammalian phosphotransferases. founder of RFS Pharma, has a financial interest that Biochem Pharmacol 2004; 68:1879–1888. has been reviewed and approved by Emory University 16. Hernandez-Santiago BI, Obikhod A, Fromentin E, Hurwitz SJ, Schinazi RF. Cellular pharmacology of in compliance with its conflict of interest policies. RLM 9-(beta-D-1,3-dioxolan-4-yl)guanine and its lack of is a consultant to RFS Pharma. The remaining authors drug interactions with zidovudine in primary human lymphocytes. Antivir Chem Chemother 2007; 18:343–346. declare no competing interests. 17. Gilliam BL, Sajadi MM, Amoroso A, Davis CE, Cleghorn FR, Redfield RR. Tenofovir and abacavir combination therapy: lessons learned from an urban clinic References population. AIDS Patient Care STDS 2007; 21:240–246. 1. Panel on Antiretroviral Guidelines for Adults and 18. Lanier ER, Givens N, Stone C, et al. Effect of concurrent Adolescents. Guidelines for the use of antiretroviral agents zidovudine use on the resistance pathway selected by in HIV-1-infected adults and adolescents. Department of abacavir containing regimens. HIV Med 2004; 5:394–399. Health and Human Services (Updated 3 November 2008; Accessed 12 December 2008). Available from: http://www. 19. Gulick RM, Ribaudo HJ, Shikuma CM, et al. Triple- aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf. nucleoside regimens versus efavirenz containing regimens for the initial treatment of HIV-1 infection. N Engl J Med 2. Pereira CF, Paridaen JT. Anti-HIV drug development – an 2004; 350:1850–1861. overview. Curr Pharm Des 2004; 10:4005–4037. 20. Elion R, Cohen C, DeJesus E, et al. COL40263: resistance 3. Gu Z, Wainberg MA, Nguyen-Ba N, et al. Mechanism and efficacy of once-daily trizivir and tenofovir DF of action and in vitro activity of 1′,3′-dioxolanylpurine in antiretroviral naive subjects. 11th Conference on nucleoside analogues against sensitive and drug-resistant Retroviruses and Opportunistic Infections. 8–11 February human immunodeficiency virus type 1 variants. Antimicrob Agents Chemother 1999; 43:2376–2382. 2004, San Francisco, CA, USA. Abstract 53. 4. Gu Z, Wainberg MA, Nguyen-Ba P, L’Heureux L, 21. Won SY, Kessler HA. Amdoxovir. The use of antibiotics: a de Muys JM, Rando RF. Anti-HIV-1 activities of 1,3- clinical review of antibacterial, antifungal and antiviral drugs. dioxolane guanine and 2,6-diaminopurine dioxolane. Sixth edition. The Bath Press, Avon, UK; in press 2009. Nucleosides Nucleotides 1999; 18:891–892. 22. Thompson MA, Kessler HA, Eron Jr JJ, et al. Short-term 5. Mewshaw JP, Myrick FT, Wakefield DACS, et al. Dioxolane safety and pharmacodynamics of amdoxovir in HIV- guanosine, the active form of the prodrug diaminopurine infected patients. AIDS 2005; 19:1607–1615. dioxolane, is a potent inhibitor of drug-resistant HIV-1 23. Gripshover BM, Ribaudo H, Santana J, et al. (A5118 isolates from patients for whom standard nucleoside therapy Team). Amdoxovir versus placebo with enfuvirtide plus fails. J Acquir Immune Defic Syndr 2002; 29:11–20. optimized background therapy for HIV-1-infected subjects 6. Bazmi HZ, Hammond JL, Cavalcanti SCH, Chu CK, failing current therapy (ACTG A5118). Antivir Ther 2006; Schinazi RF, Mellors JW. In vitro selection of mutations in the 11:619–623. human immunodeficiency virus type 1 reverse transcriptase that decrease susceptibility to (-)-beta-D-dioxolane-guanosine 24. Margolis DM, Mukherjee AL, Fletcher CV, et al. W for the ACTG 5165 team. The use of -D-2,6-diaminopurine and suppress resistance to 3′-azido-3′-deoxythymidine. β Antimicrob Agents Chemother 2000; 44:1783–1788. dioxolane with or without mycophenolate mofetil in drug- resistant HIV infection. AIDS 2007; 21:2025–2032. 7. Parikh UM, Bacheler L, Koontz DL, Mellors JW. The K65R mutation in human immunodeficiency virus type 25. Hurwitz SJ, Asif G, Kivel NM, Schinazi RF. Development 1 reverse transcriptase exhibits bidirectional phenotypic of an optimized dose for coformulation of zidovudine with antagonism with thymidine analog mutations. J Virol drugs that select for the K65R mutation using a population 2006; 80:4971–4977. pharmacokinetic and enzyme kinetic simulation model. Antimicrob Agents Chemother 2008; 52:4241–4250. 8. Rapp KL, Ruckstuhl M, Schinazi RF. The combination of zidovudine and amdoxovir prevents the selection 26. Centers of Disease Control and Prevention. 1993 Revised of thymidine analogue mutations in primary human classification system for HIV infection and expanded lymphocytes. 16th International HIV Drug Resistance surveillance case definition for AIDS among adolescents and Workshop. 12–16 June 2007, Barbados. Abstract 117. adults. JAMA 1993; 269:729–730. 9. Parikh UM, Barnas DC, Faruki H, Mellors JW. Antagonism 27. Wainberg MA, Sawyer JP, Montaner JS, et al. Challenges between the HIV-1 reverse transcriptase mutation K65R for the clinical development of new nucleoside reverse and thymidine-analogue mutations at the genomic level. transcriptase inhibitors for HIV infection. Antivir Ther J Infect Dis 2006; 194:651–660. 2005; 10:13–28. 10. Parikh UM, Sluis-Cremer N, Mellors JW. Kinetic mechanism 28. Hurwitz SJ, Asif G, Fromentin E, Tharnish PM, by which thymidine analogue mutations antagonize K65R in Schinazi RF. Lack of pharmacokinetic interaction between HIV-1 reverse transcriptase. Antivir Ther 2005; 10:S95. amdoxovir and reduced and standard close zidovudine in 11. Parikh UM, Zalina S, Sluis-Cramer N, Mellors JW. HIV-1 infected individuals. Antimicrob Agents Chemother Molecular mechanisms of bidirectional antagonism between 2009; 54:1248–1255. K65R and thymidine analog mutations in HIV-1 reverse 29. Thai Ministry of Public Health. National Guidelines for transcriptase. AIDS 2007; 21:1405–1407. The Clinical Management of HIV Infection in Children 12. Segondy M, Montes B. Prevalence and conditions of selection and Adults. 7th ed. Nonthaburi, Thailand: Department of of the K65R mutation in the reverse transcriptase gene of Disease Control, Ministry of Public Health 2002. HIV-1. J Acquir Immune Defic Syndr 2005; 38:110–111.

Accepted for publication 30 September 2009

AVT-09-OA-1380_Murphy.indd 192 23/3/10 15:36:50