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Antimicrob Agents Chemother. 2006 February; 50(2): 625–631.
PMCID: PMC1366874

In Vitro Antiretroviral Activity and In Vitro Toxicity Profile of SPD754, a New Deoxycytidine Nucleoside Reverse Transcriptase Inhibitor for Treatment of Human Immunodeficiency Virus Infection

Abstract

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 μM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.

Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of highly active antiretroviral therapy (HAART) recommended for the treatment of human immunodeficiency virus (HIV) infection (7, 20), and the use of NRTI-containing combination therapy has significantly decreased the morbidity and mortality associated with HIV disease in treated patients (18, 19). NRTIs share a common mechanism of action. All undergo intracellular activation to the NRTI triphosphate (NRTI-TP) form, after which they compete with endogenous deoxynucleotide triphosphates for binding to the viral reverse transcriptase (RT) enzyme, and incorporation of the monophosphate (MP) into the nascent DNA. Since NRTIs lack a substituent capable of supporting further DNA elongation, the incorporation of the NRTI-MP results in the termination of chain elongation and inhibition of reverse transcription.

Many patients whose viral replication is effectively controlled by combination antiretroviral therapy ultimately experience virologic failure because of the development of antiretroviral resistance (for reviews, see references 23 and 24). A 6-year survey of viral genotypes in France found that almost 80% of clinical HIV samples collected until 2002 had mutations conferring resistance to NRTIs (29). Primary infection with resistant strains is also being increasingly recognized as a clinical problem in some countries (10, 14, 26, 31). NRTI resistance results from mutational changes within the RT gene. The resulting resistance mechanisms fall into two main categories (for reviews, see references 4 and 8). One group of RT mutations acts to increase the rate of RT-catalyzed phosphorolysis, i.e., the RT-catalyzed excision of the incorporated NRTI-MP from the chain-terminated DNA. These mutations include M41L, D67N, K70R, L210W, T215Y, and K219Q and are sometimes referred to collectively as thymidine analogue mutations (TAMs). The accumulation of these mutations confers high-level resistance to zidovudine and affects viral sensitivity to other NRTIs, including stavudine, tenofovir, and abacavir (4). The other mechanism by which mutations in RT can cause resistance to NRTIs is by altering the discrimination between deoxynucleoside triphosphate substrates and NRTI-TP inhibitors by the substrate binding site of RT. Examples of such mutations include the M184V mutation, which is found frequently in patients experiencing virologic failure during treatment with lamivudine-containing HAART (6, 15). This mutation causes high-level resistance to lamivudine and (in combination with other mutations) reduces sensitivity to didanosine, zalcitabine, and abacavir (32). Other mutations associated with resistance to multiple NRTIs include the Q151M and K65R mutations (4). Complex interactions occur between mutational patterns during combination therapy, complicating the evaluation of the clinical importance of individual resistance mutations. Cross-resistance among NRTIs limits the available options for salvage therapy, especially in patients who have experienced multiple episodes of virologic failure.

A second major obstacle to successful HAART is the poor long-term tolerability of many antiretrovirals. In particular, NRTIs have been associated with mitochondrial DNA (mtDNA) depletion and mitochondrial dysfunction within human cells (1, 13, 21). This depletion occurs because these agents are substrates for not only viral RT but also for human DNA polymerases, especially DNA polymerase γ (the enzyme responsible for the replication of mitochondrial DNA). Thus, there is a need for new NRTIs with improved long-term tolerability, as well as activity against HIV strains resistant to existing NRTIs.

SPD754 (also known as AVX754 and formerly known as BCH-10618) is a new deoxycytidine analogue NRTI in clinical development for the treatment of HIV infection (Fig. (Fig.1).1). SPD754 is the (−) enantiomer of 2′-deoxy-3′-oxa-4′-thiocytidine (dOTC or BCH-10652) (5, 30). SPD754-TP has been shown to inhibit wild-type HIV type 1 (HIV-1) RT in vitro with a Ki of 0.08 μM (compared with 0.16 μM for lamivudine-TP) (5), and SPD754 showed good activity against HIV-1 isolates in vitro (5, 30). SPD754-TP is highly selective for HIV-1 RT, having Ki values for HIV-1 RT that are 150- to 3,750-fold lower than those for human DNA polymerases (5). Preliminary reports suggest that SPD754 has a low potential for cellular toxicity (5; Z. Gu, N. Nguyen-Ba, C. Ren, J.-M. de Muys, D. Taylor, P. McKenna, M. A. Wainberg, and R. C. Bethell, Abstr. First Int. AIDS Soc. Conf. HIV Pathog. Treatment, abstr. 244, 2001). In a phase II study, SPD754 showed promising activity in antiretroviral-naïve, HIV-infected patients. After 10 days' monotherapy with SPD754 (1,200 mg/day), the mean reduction in HIV-1 RNA levels was −1.65 log10 copies/ml (P. Cahn, J. Lange, I. Cassetti, J. Sawyer, C. Zala, M. Rolon, R. Bologna, and L. Shiveley, Abstr. Second Int. AIDS Soc. Conf. HIV Pathog. Treatment, abstr. LB15, 2003).

FIG. 1.
Structural formula of SPD754.

In the present studies, the in vitro activity of SPD754 against NRTI-resistant HIV-1 clinical isolates and non-clade B isolates of HIV-1 subgroup M has been characterized. In addition, we report its activity in combination with other antiretroviral agents and its effects on host cell viability and mitochondrial DNA content.

MATERIALS AND METHODS

Materials. (i) Viruses.

HIV-1IIIB used in the antiviral combination studies was obtained from M. A. Wainberg, McGill University AIDS Centre, (Jewish General Hospital, Montreal, Québec, Canada). Clinical HIV-1 isolates used in the Antivirogram studies were obtained from the repository of genotyped HIV-1 clinical HIV isolates held by Virco (Mechelen, Belgium). The study of NRTI-resistant clinical isolates was performed with 50 clinical HIV-1 isolates that were selected at random from groups of viruses with predefined genotypes. Of these, five contained no known mutations in the RT gene conferring resistance to NRTIs (i.e., no mutations at codons 41, 44, 62, 65, 67, 69, 70, 74, 75, 77, 115, 116, 118, 151, 184, 210, 215, and 219), and 45 were NRTI resistant. Zidovudine-resistant isolates (n = 5) all had the M41L mutation and either T215Y or T215F and a median of three additional nucleoside analogue mutations (NAMs; range, 0 to 5; at codon 44, 67, 74, 75,118, 210, or 219). All lamivudine-resistant isolates (n = 5) contained the M184V mutation. Isolates resistant to both of these agents (n = 8) carried both M184V and a minimum of two TAMs (mutation M41L, D67N, K70R, L210W, T215Y/F, or K219E/H/N/Q/R). Abacavir-resistant isolates (n = 5) carried L74V, Y115F, and M184V mutations and a median of one other NAM (range, 0 to 4; at codon 44, 67, 70, 118, 210, or 215), while stavudine-resistant isolates (n = 5) all carried the V75T or V75M mutation, M41L, and T215F or T215Y, and a median of four other NAMs (range, 4 to 6; at codon 67, 69, 70, 118, 184, 210, or 219). Multidrug-resistant isolates (n = 17) that contained the Q151M substitution (with or without the M184V substitution) carried a median of four of the other mutations associated with Q151M (range, 2 to 4; at codons A62V, V75I, F77L, and F116Y). Multidrug-resistant isolates carrying a T69 insertion also carried the T215Y mutation and a median of two other NAMs (range, 1 to 3; at codon 41, 44, 62, 75, 184, or 210).

Recombinant group M HIV-1 isolates (n = 23) with pol genes corresponding to various clades other than clade B were selected randomly from the same repository. The isolates represented the following clades: CRF02_AG (n = 4 samples), B (n = 4), C (n = 4), D (n = 2), CRF05_DF (n = 2), CRF01_AE (n = 4) (9), F (n = 1), and H (n = 2). Two of these samples (Virco identification numbers 5 and 27) contained mutations at M184V and were resistant to lamivudine. The sensitivities of three of these isolates (Virco identification numbers 19, 22, and 25) were approximately fivefold lower than their sensitivity to zidovudine.

All recombinant viruses were prepared from these clinical isolates according to previously published procedures (12). In brief, RT and protease gene sequences from patient HIV RNA were isolated, amplified, and transfected into Molt-4 (MT-4) cells together with a standard laboratory isogenic (HXB2) HIV-1 DNA construct from which RT and protease gene sequences were deleted. After homologous recombination, new viruses whose diversity reflected the original clinical population were produced at high level from these cells. In common with the NRTI-resistant clinical isolates, the chimeric non-clade B viruses contained only the pol gene from the original clinical isolates. However, this gene is expected to be the only determinant of virus susceptibility to SPD754.

(ii) Cells.

MT-2 cells were obtained from the AIDS Research and Reference Reagent Program of the National Institutes of Health (Rockville, MD) and maintained as suspension cultures in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml) (all from Gibco, Burlington, Canada). MT-4 cells were cultured as previously described (30). Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors, isolated by centrifugation using Lymphocyte-Mammal medium (Cedarlane, Hornby, Canada) according to the manufacturer's instructions, and cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 U/ml interleukin-2 (Boehringer Mannheim and Roche, Laval, Québec, Canada). PBMCs were incubated at 37°C in an atmosphere of 5% CO2 in medium supplemented with phytohemagglutinin (5 μg/ml) (Roche, Laval, Québec, Canada) for 3 days prior to their use in antiviral assays.

Normal human bone marrow mononuclear cell samples were obtained from Poietic Technologies (Gaithersburg, MD). On the day of the assay, cells were thawed, washed with Iscove's modified Dulbecco's medium (IMDM) containing 10% FBS and 20 U/ml DNase I and centrifuged at room temperature for 15 min at 1,000 rpm. The pellet was then washed with IMDM plus 10% FBS and centrifuged again as described above. Cell pellets were resuspended, and the cells were left for at least 1 h at 37°C and in 5% carbon dioxide. The mononuclear cells were then counted with 3% acetic acid containing methylene blue, and the concentration was adjusted to 1.0 × 105 cells/ml.

(iii) Compounds and other reagents.

SPD754 was synthesized at Shire Biochem (Laval, Québec, Canada) as described previously (16), as were abacavir, tenofovir, and saquinavir. Zidovudine, stavudine, zalcitabine, and didanosine were purchased from Sigma (St. Louis, MO). Lamivudine was obtained from Glaxo-Wellcome (Greenford, United Kingdom).

Methods. (i) Antiviral assays.

The antiviral activity of the compounds was tested in the Antivirogram assay as previously described (12). Briefly, the phenotypic sensitivity of the chimeric viruses to SPD754 and a panel of approved NRTIs was analyzed using serial dilutions in an automated reporter gene-based MT-4 cell 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and quantified as the 50% inhibitory concentration (IC50) (22). The changes in sensitivity in NRTI-resistant recombinant isolates were calculated relative to the sensitivity of wild-type isolates. The antiviral activity of SPD754 against clinical HIV-1 isolates from clades other than clade B was also tested using the Antivirogram assay, and the changes were calculated compared to four wild-type, clade B clinical isolates used as controls.

The antiviral activity of SPD754 was assessed in combination with the NRTIs zidovudine, lamivudine, stavudine, didanosine, and abacavir, the nucleotide reverse transcriptase inhibitor tenofovir, the non-nucleoside reverse transcriptase inhibitor nevirapine, and the protease inhibitor saquinavir (11, 30). The combinations were performed using a checkerboard cross pattern of drug concentrations. Cells were incubated in the presence of virus for a period of 2 to 3 h at a multiplicity of infection of 0.005 for T-cell assays and at a multiplicity of infection of 0.5 for monocytic cell assays, and the infected cells were then incubated in the presence of the drug. The antiviral effects were determined by monitoring RT activity in the culture supernatant. The experiments were conducted in quadruplicate or quintuplicate. The drug interactions were analyzed by the methods of Chou and Talalay (2) and Pritchard et al. (25). The combination index (CI) for SPD754 and each coadministered antiretroviral was calculated using CalcuSyn software (Biosoft, Cambridge, United Kingdom) using the mutually exclusive method. A CI value of 1 indicates an additive effect, a value of >1.25 indicates antagonism, and a value of <0.8 indicates synergism. The cytotoxicity of SPD754 in combination with these agents was determined in parallel in the MT-2 cells using an MTT cell proliferation kit (Boehringer Mannheim, Laval, Québec, Canada).

(ii) Mitochondrial toxicity.

HepG2 cells at exponential growth were cultured in minimal essential medium (MEM) with nonessential amino acids supplemented with 10% heat-inactivated fetal bovine serum and 1% sodium pyruvate. Molt-4 cells at exponential growth (0.2 × 106 to 1.6 × 106 cells/ml) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The medium was changed every 3 or 4 days, and cells were subcultured once a week at a dilution of 1:3 to 1:5 for HepG2 cells and at a dilution of 1:10 for Molt-4 cells. All cultures were routinely checked for Mycoplasma infection and grown at 37°C in a humidified 5% CO2 atmosphere. All drugs tested were first dissolved at 100 mM in dimethyl sulfoxide (DMSO) before further dilution to the appropriate concentration in the culture medium. The analysis of mitochondrial DNA was performed as previously reported (5), with few modifications, and is only briefly described below.

The cells were seeded into 8.6-cm2 multidish culture plates at a cell density of 6,500 cells/cm2 in 3 ml of culture medium. In one experiment, HepG2 cells were consecutively treated for 28 days in duplicate with serial concentrations of SPD754 (1, 10, 50, and 100 μM). In a second experiment, HepG2 and Molt-4 cells were treated for the same duration with 10, 50, 100, and 200 μM SPD754. The NRTI zalcitabine, a specific inhibitor of mtDNA synthesis (17, 21), was used for comparison. The medium containing the freshly diluted compound was changed every 3 or 4 days, and cells were subcultured once a week. Cell viability was determined by visual inspection using an inverted microscope. After 28 days of drug exposure, cells were collected. Total cellular DNA was prepared using the Qiamp blood kit (QIAGEN, Chatsworth, CA), following the supplier's instructions. The cellular DNA was digested using SacI, separated with 0.8% agarose (0.5 μg cellular DNA/lane) in 1× modified TAE (40 mM Tris-acetate, pH 8.0, 0.1 mM EDTA), and then denatured. DNA was then transferred onto a nylon membrane using a standard upward capillary method (27). After transfer, the membrane was soaked in 20× SSC buffer (1.5 M sodium chloride, 0.15 M sodium citrate, pH 7.0) for 2 min, air dried for 10 min, and then baked at 80°C for 90 min. The membrane was hydrated for 2 min in 5× SSC buffer and then prehybridized with denatured salmon sperm DNA (100 μg/ml) at 42°C for 2 h in a buffer containing 5× SSC, 5× Denhardt's solution, 0.5% sodium dodecyl sulfate, and 50% deionized formamide. The DNA was then hybridized to an [α-32P]dCTP-labeled denatured 6-kb human mtDNA and a 1.5-kb human 28S rRNA gene probe (27) overnight at 42°C. The mtDNA and 28S rRNA gene probes were prepared from plasmid pA and plasmid N6, respectively, and radiolabeled using a random priming kit (Ready-to-Go; Amersham Pharmacia, Oakville, Ontario, Canada) following the supplier's instructions. The membrane was then washed and exposed to X-ray film for 1 to 24 h. The resulting autoradiograms were scanned with a CS9000U dual-wavelength flying spot densitometer, using Alpha Imager 2000 software (Packard Instrument Company, Meridien, CT). The mtDNA content on blots was determined as a ratio between the radioactive signals of the human mtDNA and 28S rRNA genes, which was independent of the amount of DNA applied to each lane.

(iii) Human myelotoxicity assay.

SPD754 was freshly dissolved first in 100% DMSO without filtration at a concentration of 518 mM and then diluted 3.5 times in IMDM to obtain a final concentration of 0.2% DMSO for the highest concentration of compound (1,000 μM). Serial dilutions were in 10-fold concentration increments. Sterile tubes were prepared with 4 ml of semisolid medium (MethoCult H4434) containing erythropoietin. To each tube was added 400 μl of normal human bone marrow mononuclear cells at a concentration of 1 × 105 cells/ml. Aliquots (30 μl) of SPD754 at various concentrations were added, and the tubes were vortexed vigorously and allowed to stand for 5 min to allow air bubbles to rise to the surface. Aliquots of the mixture (1.1 ml) were dispensed into each of two 35-mm sterile petri dishes. These dishes were placed inside a 100-ml petri dish, together with a third 35-mm lidless petri dish containing water, and incubated for 14 to 18 days at 37°C in an environment containing humidified carbon dioxide. Each experiment contained triplicate samples at each SPD754 dose, and each was run at least in triplicate using three different bone marrow samples. Doxorubicin was used as the positive control at a single concentration of 10 μM, at which 100% inhibition of colony formation was observed. IMDM medium was used as the negative control.

At the end of the incubation period, human CFU of granulocyte/macrophage colonies containing 30 cells or more were scored using an inverted microscope. The mean IC50 (together with the standard error of the mean) was calculated by nonlinear regression using a one-site competition equation (Prism Software, San Diego, CA).

RESULTS

Antiviral activity of SPD754 against NRTI-resistant clinical isolates of HIV-1.

The activity of SPD754 and other antiretrovirals was tested in 50 clinical HIV-1 isolates (45 NRTI-resistant isolates and 5 wild-type isolates) using the Antivirogram recombinant virus assay (12). Susceptibility to SPD754 was reduced 1.2- to 2.2-fold against HIV-1 isolates resistant to zidovudine, lamivudine, or both of these agents (Table (Table1)1) compared with that of wild-type isolates. Susceptibility to SPD754 was reduced by 1.3- to 2.8-fold against abacavir- or stavudine-resistant isolates. Multiresistant isolates with the Q151M mutation (with or without M184V) showed a 14- to 16-fold reduction in susceptibility to SPD754, while insertions at RT amino acid position 69 reduced SPD754 susceptibility by 5.2-fold. These changes in multiply resistant strains were similar to those observed for marketed NRTIs, with the exception of zidovudine and lamivudine (Table (Table1).1). Zidovudine showed a greater reduction in activity against strains containing the Q151M mutation and T69 insertions (all >38-fold). Lamivudine showed a similar reduction to SPD754 against strains with the Q151M mutation (14-fold) but a greater reduction against those with the M184V mutation or T69 insertions (>26-fold).

TABLE 1.
In vitro antiviral activity of SPD754 against HIV-1 clinical isolates resistant to nucleoside reverse transcriptase inhibitors in MT-4 cells

Combination antiviral activity.

In tests conducted in MT-2, MT-4, or PBMCs, SPD754 had additive activity in combination with zidovudine, lamivudine, stavudine, didanosine, abacavir, tenofovir, and nevirapine or over a range of concentration ratios, as indicated by the mean CI values in the range from 0.80 to 1.25 (Table (Table2).2). While the CI values in combination with saquinavir were >1.25 (indicating antagonism) at IC50, they were lower (indicating additivity) when using the more-sensitive definitions of IC75 (0.92 to 0.96) and IC95 (0.87 to 1.19). No evidence of synergistic toxicity was observed with any of the drug combinations tested (data not shown).

TABLE 2.
Effect of in vitro antiviral activity of SPD754 in combination with other antiretrovirals in MT-2, MT-4, and PBMCs as assessed by the combination index

Activity against non-clade B HIV-1.

The activity of SPD754 and other antiretrovirals was tested against HIV-1 clinical isolates representing various clades other than clade B using the Antivirogram assay. The IC50 values for SPD754 were between 10 and 50 μM for each of the individual HIV-1 samples tested. Table Table33 summarizes the mean IC50 value for each of the different clades of viruses tested. The mean IC50 value for SPD754 against clade B viruses was 27 μM ± 18 μM. This activity was identical to the results of previous tests of SPD754 against wild-type HIV-1 clinical isolates in the same assay (Table (Table1).1). The average IC50 values for SPD754 against non-B clades were 14 to 47 μM. Thus, these results indicate that SPD754 had equipotent activity against different clades of HIV-1 samples. In addition, the lamivudine-resistant and zidovudine-resistant viruses did not show significant decreased sensitivity to SPD754, which is consistent with the results reported above and in previous studies (32).

TABLE 3.
In vitro antiviral activity of SPD754 and marketed nucleoside reverse transcriptase inhibitors against clinical HIV-1 isolates of clades A to H

Mitochondrial toxicity.

No cellular toxicity on visual inspection was apparent after continuous 7- to 28-day exposure of HepG2 and Molt-4 cells to SPD754 (up to 200 μM). Quantitative analysis showed no significant effect of SPD754 concentrations of up to100 μM on cellular mtDNA content in HepG2 cells (data not shown). A second experiment showed no effect of SPD754 concentrations up to 200 μM in HepG2 or Molt-4 cells (Fig. (Fig.2).2). In comparison, zalcitabine produced marked and concentration-dependent reductions in cell viability and mitochondrial DNA content in these cell lines (Fig. (Fig.22).

FIG. 2.
Effect of 28 days of exposure to SPD754 (10 to 200 μM) and zalcitabine (0.8 to 5.4 μM) on mitochondrial DNA content in human HepG2 and Molt-4 cells in vitro, expressed as a percentage of mtDNA synthesis compared to that of controls. The ...

In study 2, no synergistic effects on cell proliferation were observed in this assay system when SPD754 was combined with any of the marketed antiretroviral agents tested at the following concentrations: SPD754 (20 μM) plus zidovudine (0.04 μM), abacavir (0.4 μM) or saquinavir (0.04 μM), and SPD754 (80 μM) plus lamivudine (8.0 μM), stavudine (80 μM), didanosine (80 μM), or nevirapine (8.0 μM).

Myelotoxicity.

During 16 to 19 days' exposure in human bone marrow cells, SPD754 showed a IC50 of 366.1 μM (±137.1 μM).

DISCUSSION

In the present studies, the deoxycytidine analogue SPD754 was shown to retain a high proportion of its antiviral activity against HIV-1 isolates resistant to a range of marketed NRTIs. These data support previous findings by de Muys et al. (5) and Taylor et al. (30) and suggest that SPD754 may have particular usefulness in the treatment of patients failing therapy and/or with resistance to one or more NRTIs.

The observation that HIV-1 isolates resistant to lamivudine showed little reduction in susceptibility to SPD754 is of particular interest. The M184V mutation is very common among patients failing initial therapy for a number of reasons. Firstly, the widespread use of lamivudine in first-line regimens means that there is a high level of exposure to the drug in the treated population. In addition, lamivudine is very well tolerated, suggesting that adherence to the lamivudine component of therapy may be higher than that to other less well tolerated medications, especially when it is not administered in the form of a fixed-dose combination. This may increase the frequency with which lamivudine may be taken in the absence of all the other components of a HAART regimen. Finally, the level of resistance to lamivudine conferred by the M184V mutation is very high, and the genetic barrier to the emergence of this mutation is low {6, 15; S. Bloor, S. D. Kemp, K. Hertogs, T. Alcorn, and B. A. Larder, Abstr. 4th Int. Workshop HIV Drug Resistance Treatment Strategies, abstr. 169 [Antivir. Ther. 5(Suppl. 3):132], 2000}.

The results of the present study therefore suggest that SPD754 may be used as a replacement deoxycytidine analogue for lamivudine in patients harboring the M184V mutation. However, clinical studies will be necessary to establish this effectiveness and to determine whether SPD754 maintains the M184V mutation in vivo when used as a replacement deoxycytidine analogue for lamivudine in patients who have developed resistance to lamivudine. According to the present data, SPD754 is less potent in vitro than lamivudine. However, in a randomized, double-blind, phase II clinical study, SPD754 daily doses of 400 to 1,600 mg produced statistically significant viral load reductions versus placebo of 1.18 to 1.65 log10 after 10 days in antiretroviral-naïve HIV-infected patients (P < 0.0001 versus placebo) (Cahn et al., Abstr. Second Int. AIDS Soc. Conf. HIV Pathog. Treatment). These data are similar to those reported with a 300-mg daily dose of lamivudine (J. Delehanty, C. Wakeford, L. Hulett, J. Quinn, B. McCreedy, M. Almond, D. Miralles, and F. Rousseau, Abstr. 6th Conf. Retrovir. Opportunistic Infect., abstr. 16, 1999). Therefore, available data suggest that in vitro differences in potency between SPD754 and lamivudine are not clinically significant. Moreover, the high selectivity shown by SPD754 for HIV-1 RT over human DNA polymerases (5) suggest that clinical doses of SPD754 are unlikely to be associated with significant mitochondrial damage (discussed further below).

Reductions in susceptibility to SPD754 against HIV-1 isolates containing T69 insertions and Q151M mutations were similar to or slightly less than those to marketed NRTIs. The Q151M mutation was recently found in 50% of a small sample of isolates containing the K65R mutation (C. Amiel, A. Kara, V. Schneider, G. Pialoux, W. Rozenbaum, and J. C. Nicolas, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 627, 2004). This suggests that an association may exist between these mutations, although none of isolates tested in the present study contained the K65R mutation. In previous in vitro studies in which HIV-1LAI was passaged in MT-2 cells in the presence of SPD754, the K65R mutation emerged after 20 passages. This mutation conferred a 3.6-fold reduction in sensitivity to SPD75, compared to the sensitivity of wild-type HIV-1, and the introduction of this mutation into wild-type HIV-1 background by site-directed mutagenesis resulted in a virus that was 4.6-fold less sensitive to SPD754 than the wild-type virus from which it was derived {Z. Gu, N. Nguyen-Ba, C. Ren, J. M. de Muys, B. Allard, M. A. Wainberg, P. McKenna, D. L. Taylor, and R. C. Bethell, Abstr. 5th Int. Workshop HIV Drug Resistance Treatment Strategies, abstr. 11 [Antivir. Ther. 6(Suppl. 1):8, 2001]}. The increased prevalence of the K65R mutation observed in recent years (L. Bacheler, H. Vermieren, P. McKenna, M. Van Houtte, and P. Lecoq, Abstr. 43rd Interscience Conf. Antimicrob. Agents Chemotherapy, abstr. H-917, 2003) warrants further investigation of its selection by SPD754 and its effect on viral susceptibility to this compound.

The K65R mutation appears to antagonize the selection of NAMs, such as 41L, 67N, 210W, and 215Y/F (U. Parikh, D. Koontz, N. Sluis-Cremer, J. Hammond, L. Bacheler, R. Schinazi, and J. Mellors, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 54, 2004). Although the clinical significance of these findings is presently unclear, they illustrate the complexity of determining the effects of NRTI combinations on resistance selection. It should be noted that the present studies were conducted using a small number of specific isolates. Further studies will be performed to better characterize the activity of SPD754 against larger numbers of isolates with defined mutational patterns.

HIV-1 isolates show considerable genetic diversity owing to the low fidelity of RT, the presence of HIV genomic RNA as a dimer (which allows recombination during reverse transcription), and the high rate of turnover of HIV-1 in vivo. HIV-1 strains are classified according to their genetic background as group M (major), group O (outlier), and group N (non-M/non-O). Group M strains are globally prevalent and have been subclassified into at least nine genetic clades, designated A to K, according to the nucleotide sequences of env, gag, and/or pol genes (28). Available evidence suggests that clade variation does not have a substantial effect on baseline antiretroviral sensitivity, although it may influence the rate of emergence of resistance to individual agents (28). Nevertheless, in order to evaluate thoroughly the therapeutic potential of a new agent, it is important to determine its relative activity against isolates of HIV-1 of different clades. In order to ensure that a proper comparison could be made between the viruses from different clades, we again chose to use the Antivirogram chimeric virus technology. The data reported here indicate that SPD754 has equipotent activity against clinical isolates of group M HIV-1 subtypes B, C, D, F, and H and circulating recombinant forms CRF01_AE, CRF02_AG, and CRF05_DF.

SPD754 demonstrated additive antiviral activity in combination with zidovudine, lamivudine, stavudine, didanosine, abacavir, tenofovir, nevirapine, and saquinavir. Although the CI value for saquinavir initially suggested a slight antagonistic effect, analyses of higher inhibition endpoints from the same experiments were consistent with additive rather than antagonistic antiviral activity. Recent in vitro data show that lamivudine markedly reduces intracellular SPD754-TP concentrations in a dose-dependent manner, owing to competition for intracellular phosphorylation. The IC50 value of SPD754 against a HIV-1 isolate with the M184V mutation in PBMCs was increased two- to fourfold by the addition of lamivudine. The human plasma pharmacokinetics of SPD754 and lamivudine and the intracellular pharmacokinetics of lamivudine-TP were unaffected by coadministration. However, intracellular levels of SPD754-TP were reduced approximately sixfold by coadministration of lamivudine (R. Bethell, J. Adams, J. de Muys, J. Lippens, A. Richard, B. Hamelin, C. Ren, P. Collins, C. Struthers-Semple, T. Holdich, and J. Sawyer, Abstr. 11th Conf. Retrovir. Opportunistic Infect., abstr. 138, 2004). This intracellular interaction is likely to result in a contraindication for the coadministration of SPD754 with lamivudine and probably all deoxycytidine analogues.

The present cytotoxicity studies demonstrate that SPD754 has a low potential to cause cellular damage or mitochondrial depletion. The recognition that many serious adverse effects of long-term NRTI treatment result from the inhibition of mitochondrial DNA synthesis has prompted research to better characterize the mitochondrial toxicity profiles of existing NRTIs (1, 13, 33). The clinical manifestations of mitochondrial depletion depend upon the tissue affected, as differences exist between cell types in NRTI metabolism and mitochondrial function. Mitochondrial toxicity is most likely to explain the occurrence of lipodystrophy, hepatic steatosis, lactic acidosis, mitochondrial myopathy, and peripheral myopathy during NRTI therapy, while further research is required to establish its potential role in other adverse effects (13, 33). Early data suggested that cellular mtDNA content was a more sensitive measure of mitochondrial toxicity than analyses of cell viability or mitochondrial morphology (17). No significant effects on either cell viability or mtDNA content were observed (either quantitatively or qualitatively relative to rRNA gene content) during continuous 28-day exposure to SPD754 concentrations of up to 200 μM, which is approximately 20 times the maximum plasma drug concentration expected in humans at clinical doses {R. J. Francis, L. Lanclos, L. Shiveley, and J. Sawyer, Abstr. Second Int. AIDS Soc. Conf. HIV Pathog. Treatment, abstr. 528 [Antivir. Ther. 8(Suppl. 1):S325], 2003}. In contrast, studies performed with zalcitabine, which is known to have a high potential for mitochondrial toxicity both in vitro and in clinical use (3, 17, 21) gave results that are in accord with the previous studies of the mitochondrial toxicity of this agent. The absence of observed effects of SPD754 on mtDNA content is consistent with the high level of selectivity of SPD754-TP for the HIV reverse transcriptase relative to human DNA polymerase γ (5). SPD754 also showed a low potential for myelotoxicity against human bone marrow cells, indicated by a cytotoxic concentration of drug which reduced the viable cell number by 50% 30-fold less than that of zidovudine and twofold less than that of lamivudine. The importance of these tests was emphasized by the evidence of myelotoxicity in an early clinical study of elvucitabine {L. M. Dunkle, J. C Gathe, D. E. Pedevillano, H. G. Robison, W. G. Rice, and J. C. Pottage, Jr., Abstr. 12th Int. HIV Drug Resistance Workshop, abstr. 2 [Antiviral Ther. 8:S5], 2003}.

In conclusion, SPD754 is a new NRTI that retains activity against NRTI-resistant HIV-1 strains and has a low propensity to cause mitochondrial DNA depletion or cytotoxicity. SPD754 is expected to offer a valuable addition to the therapeutic options available for people infected with HIV-1, and further clinical data concerning its effectiveness and tolerability are awaited.

Acknowledgments

This study was supported by Shire Biochem, Inc., Laval, Québec, Canada.

We acknowledge the assistance of Lee Baker (Prism Ideas, Nantwich, United Kingdom) in the development of this paper.

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