Phosphonomethoxy)propyl]adenine Research Articles

Impact of Antiretroviral Treatment on Gene Expression in Peripheral Blood Mononuclear Cells from SIVmac251–Infected Macaques

A survey of gene expression in peripheral blood mononuclear cells from cynomolgus macaques infected with SIVmac251 was conducted to ascertain the impact of viral infection and successful antiretroviral (ARV) intervention on gene transcription at peak seroconversion, viral set point, and after treatment with 9-R 2 phosphonomethoxypropyl adenine and beta -2'3' dideoxy-3'-thia-5 fluorocytidine. Robust multichip average-normalized data sets generated on Affymetrix GeneChips were analyzed using Significance Analysis of Microarrays (SAM), to determine differential gene expression. Unsupervised learning algorithms and gene-ontology tools were used to elucidate hierarchical relationships and to define the function of significantly enriched biological categories of differentially regulated genes. Gene networks associated with immune response and inflammation impacted by ARV treatment were derived by use of Pathway Architect software. Viral infection results in down-regulation of gene expression, which is greatest by the viral set point. Of the 3647 genes down-regulated at the viral set point, 1033 were up-regulated as the result of successful ARV treatment. There is significant overlap in the identity of these genes. Intervention with successful ARV treatment in macaques infected with SIVmac251 results in the partial reversal of the down-regulated gene expression characteristic of early viral infection.

Administration of Fludarabine-Loaded Autologous Red Blood Cells in Simian Immunodeficiency Virus-Infected Sooty Mangabeys Depletes pSTAT-1-Expressing Macrophages and Delays the Rebound of Viremia after Suspension of Antiretroviral Therapy

A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4+ T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4+ or CD8+ T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.

Ex vivo culture of human colorectal tissue for the evaluation of candidate microbicides

Establishment of an in vitro model to evaluate rectal safety and the efficacy of microbicide candidates. An investigation and characterization of human colorectal explant culture for screening candidate microbicides to prevent rectal transmission of HIV-1 infection. Human colorectal explants were cultured at the liquid-air interface on gelfoam rafts. Phenotypic characterization of HIV-1 target cells was performed by fluorescence-activated cell sorter analysis. HIV-1 infection was determined by the measurement of p24 antigen release, viral RNA, and proviral DNA accumulation. Colorectal explant CD4 T cells expressed higher CCR5 and CXCR4 levels compared with blood. Minor differences between the rectal and sigmoid colon were observed with a trend for slightly higher CCR5 and HLA-DR expression in cells from the sigmoid colon. Favourable culture conditions were established for colorectal tissue. Although tissue structure degenerated with time, CD4: CD8 cell ratios remained constant, and tissue supported productive HIV-1 infection. The ability of candidate microbicides to inhibit R5 HIV-1 infection was evaluated. Polyanion candidates, PRO2000 and dextrin sulphate, provided 99% protection at 1 microg/ml and 1 mg/ml, respectively, equivalent to 1/5000 and 1/40 of the vaginal formulations. The nucleotide reverse transcriptase inhibitor (NRTI) 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) provided protection at concentrations 1000-fold lower (10 microg/ml) than the proposed vaginal formulation (1%). Furthermore, non-NRTI UC-781 and TMC-120 provided greater than 99% inhibition at 3.3 or 0.33 microg/ml, respectively. No products demonstrated toxicity to rectal mucosa at inhibitory concentrations. Colorectal explant culture was shown to be a useful tool for the preclinical evaluation of potential microbicides. The data suggest that rectally applied microbicides might provide protection from HIV-1 transmission.

Transient early post-inoculation anti-retroviral treatment facilitates controlled infection with sparing of CD4+ T cells in gut-associated lymphoid tissues in SIVmac239-infected rhesus macaques, but not resistance to rechallenge.

Like human immunodeficiency virus infection of humans, infection of rhesus macaques with pathogenic simian immunodeficiency virus (SIV) strains typically results in persistent progressive infection, leading to clinically significant immunosuppression. In previous studies, we administered short term anti-retroviral treatment, shortly after intravenous inoculation with SIVsmE660, in an effort to allow immunologic sensitization under conditions not characterized by overwhelming cytopathic infection compromising the developing immune response. We showed that such treatment allowed control of off treatment viremia and was associated with resistance to rechallenge. Control of off treatment viremia was associated, at least in part, with CD8+ lymphocytes, based on in vivo CD8 depletion studies. In the present study, six rhesus macaques were infected intravenously with 100 MID50 of SIVmac239; four then received 30 days of treatment with tenofovir 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA); 20-30 mg/kg, subcutaneously) starting 24 hours post-inoculation. Tenofovir-treated animals showed low (<500 copy Eq/ml) or undetectable (<100 copy Eq/ml) plasma SIV RNA levels during treatment, with undetectable plasma viremia following discontinuation of treatment. Plasma SIV RNA remained <100 copy Eq/ml, even after depletion of CD8+ lymphocytes, 6 weeks after discontinuation of tenofovir treatment. In contrast to untreated infected control animals that showed substantial depletion of CD4+ T cells from gut-associated lymphoid tissues (GALT), tenofovir-treated animals showed sparing of GALT CD4+ T cells both during the treatment period and in the off treatment follow-up period. However, in contrast to earlier results with animals infected with SIVsmE660, in the present study, the animals did not develop readily measurable cellular anti-SIV immune responses, and did not resist homologous rechallenge with SIVmac239, administered 44 weeks after the initial infection. Differences in the animals and virus strains employed may in part account for the differences in results observed. Comparative analysis of virologic and immunologic parameters in this model system may provide important insights for understanding the basis of effective immunologic control of SIV infection.

Feline immunodeficiency virus is concentrated in milk early in lactation.

We studied mother-to-offspring transmission of feline immunodeficiency virus (FIV), focusing on milk-borne virus transmission in order to assess its similarities to perinatal HIV transmission. We also attempted to evaluate the influence of intragestational treatment with 9-[2-(phosphono-methoxy)-propyl]adenine (PMPA) on virus transmission to offspring. Eleven female cats (queens), chronically infected with FIV-B-2542 and bred to an FIV-negative male, produced a total of 25 viable and 18 nonviable term kittens. Overall, the vertical transmission rate by untreated queens was 22%, similar to that for HIV, which unfortunately precluded adequate assessment of PMPA efficacy. However, at delivery 9 of 10 queens (90%) had higher viral RNA loads in milk (4 x 10(4) to 4 x 10(8) viral copies/ml) than in plasma (5 x 10(3) to 2.5 x 10(6) viral copies/ml). Conversely, 10 of 11 queens (91%) had lower proviral loads in milk cells (0 to 10(2) proviral copies/microg DNA) than blood cells (10(2) to 10(4) proviral copies/microg DNA). Thus, FIV is concentrated in early milk despite relatively low proviral loads in milk cells, suggesting that virus may be actively secreted by the mammary gland for dissemination to offspring. FIV provides a model for the study of milk-borne lentivirus transmission and assessment of strategies to reduce postnatal HIV vertical transmission.

IL-4 increases Simian immunodeficiency virus replication despite enhanced SIV immune responses in infected rhesus macaques

It is widely believed that a Th1 type CD4 response is critical for enhancement of CD8 immunity and for controlling HIV-1 infection. Th2 type responses, such as what might be seen in a chronic parasitic infection, would sacrifice cellular immunity and thus benefit the virus at the expense of the host. However, there has been little direct examination of the hypothesis in a primate model system. Accordingly, the simian immunodeficiency virus (SIV) infected rhesus macaque model was used to investigate the impact of immunisation with SIV expressing DNA constructs and co-injection with IL-4 on the SIV specific immunological responses, lymphocyte cell counts, as well as the impact on viral load. IL-4 is a Th2 type cytokine, which enhances antibody production and inhibits a CD4 Th1 phenotype. Rhesus macaques were infected with 10 AID50 of SIVmac239 and treated with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) 9 weeks post-infection. During PMPA treatment, animals were immunised with plasmids that expressed the SIV proteins, env, rev, gag and pol. In addition, they were immunised with a construct that encoded the gene for IL-4. IL-4 co-immunisation increased the neutralizing antibody titres in this group. Importantly, the viral loads in animals vaccinated with IL-4 expressing plasmid increased during the immunisation regimens despite the higher neutralizing antibody titres. In addition, neutralizing antibodies did not correlate with viral set point prior to PMPA treatment, however, there was a correlation between viral loads and antibody titres following the treatment with PMPA. Antibody titres decreased following the suppression of viral load. Importantly, vaccination in the absence of IL-4 protected CD4 levels without increasing viral load. The data support the hypothesis that inappropriate immune bias toward a Th2 pathway would ultimately enhance disease progression.

Activation by 9-(R)-[2-(phosphonomethoxy)propyl]adenine of chemokine (RANTES, macrophage inflammatory protein 1alpha) and cytokine (tumor necrosis factor alpha, interleukin-10 [IL-10], IL-1beta) production.

Development of a novel group of antiviral agents, acyclic nucleoside phosphonates, has provided a new perspective for treating human immunodeficiency virus (HIV) infection. One of the compounds, 9-(R)-[2-(phosphonomethoxy)propyl]adenine (PMPA) (tenofovir), has been shown to confer complete protection against AIDS in a simian model of the infection. The aim of our study was to investigate whether the antiviral efficacy of PMPA, which depends mainly on inhibition of virus-induced DNA polymerase or of reverse transcriptase, could be contributed by immunomodulatory potential of this drug. We screened for its ability to activate production of cytokines and chemokines that are known to interfere with the replication and/or the entry of HIV in cells. Using the in vitro test system of mouse macrophages and lymphocytes, it has been found that PMPA stimulates macrophage secretion of interleukin-1beta (IL-1beta), IL-10, and tumor necrosis factor alpha. Production of the chemokines RANTES and macrophage inflammatory protein 1alpha was activated in both macrophages and lymphocytes, and also in human cell line U937. Other cytokines--i.e., IL-2, IL-12, IL-13, and gamma interferon-remained uninfluenced by PMPA. The cytokines were stimulated in a dose-dependent fashion, with rapid onset, and peak concentrations were achieved within 5 to 24 h. The findings contribute to a more complex understanding of mechanisms of antiviral effectiveness of PMPA and support the view that this drug could become a promising candidate for therapeutic exploitation in anti-HIV preventive medicine.

Prophylactic and therapeutic benefits of short-term 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) administration to newborn macaques following oral inoculation with simian immunodeficiency virus with reduced susceptibility to PMPA.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model of human pediatric AIDS to study pathogenesis and to develop intervention strategies aimed at preventing infection or delaying disease progression. In previous studies, we demonstrated that 9-¿2-(R)-(phosphonomethoxy)propylădenine (PMPA; tenofovir) was highly effective in protecting newborn macaques against infection with virulent wild-type (i.e., drug-susceptible) SIVmac251. In the present study, we determined how reduced drug susceptibility of the virus inoculum affects the chemoprophylactic success. SIVmac055 is a virulent isolate that has a fivefold-reduced in vitro susceptibility to PMPA, associated with a K65R mutation and additional amino acid changes (N69T, R82K, A158S, S211N) in reverse transcriptase (RT). Eight newborn macaques were inoculated orally with SIVmac055. The three untreated control animals became SIVmac055 infected; these animals had persistently high viremia and developed fatal immunodeficiency within 3 months. Five animals were treated once daily with PMPA (at 30 mg/kg of body weight) for 4 weeks, starting 24 h prior to oral SIVmac055 inoculation. Two of the five PMPA-treated animals had no evidence of infection. The other three PMPA-treated infant macaques became infected but had a delayed viremia, enhanced antiviral antibody responses, and a slower disease course (AIDS in 5 to 15 months). No reversion to wild-type susceptibility or loss of the K65R mutation was detected in virus isolates from any of the PMPA-treated or untreated SIVmac055-infected animals. Several additional amino acid changes developed in RT, but they were not exclusively associated with PMPA therapy. The results of this study suggest that prophylactic administration of PMPA to human newborns and to adults following exposure to human immunodeficiency virus will still be beneficial even in the presence of viral variants with reduced susceptibility to PMPA.

Administration of 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) to gravid and infant rhesus macaques (Macaca mulatta): safety and efficacy studies.

9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) significantly inhibits viral reverse transcription and has been reported to sustain low virus load in SIV-infected rhesus monkeys. Based on these findings, studies were conducted to assess the safety, efficacy, and placental transfer of PMPA when administered once daily subcutaneously to gravid rhesus monkeys during the second and third trimesters and their offspring (30 mg/kg/day). Fetuses (SIV-infected, N = 6; noninfected, N = 6) were monitored sonographically, and maternal/fetal blood samples were collected at select time points for hematologic, clinical chemical, virologic, immunologic, and pharmacologic assessments. Newborns were delivered by cesarean section at term and nursery reared for postnatal studies. Infants were administered PMPA once daily beginning on day 2 of life until 9 months postnatal age. Results of these studies have shown significant placental transport of PMPA, with peak fetal levels at 1 to 3 hours post-maternal administration; a significant and sustained reduction in viral load in SIV-infected fetuses and infants; and marked improvements in outcome (e.g., survival, growth, health) in SIV-infected offspring. However, decreased infant body weights and alterations of select serum biochemical parameters (e.g., decreased phosphorus levels, elevated alkaline phosphatase) have been shown to occur in approximately 67% of PMPA-treated infants, with severe growth restriction and bone-related toxicity in approximately 25% of animals studied. These data suggest that although PMPA holds great promise for HIV-infected patients, there is the potential for bone-related toxicity at chronic, high dosages, particularly in infants.

9-[2-(Phosphonomethoxy)propyl]adenine (PMPA) therapy prolongs survival of infant macaques inoculated with simian immunodeficiency virus with reduced susceptibility to PMPA.

Simian immunodeficiency virus (SIV) infection of newborn rhesus macaques is a useful animal model of human immunodeficiency virus infection for the study of the emergence and clinical implications of drug-resistant viral mutants. We previously demonstrated that SIV-infected infant macaques receiving prolonged treatment with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) developed viral mutants with fivefold reduced susceptibility to PMPA in vitro and that the development of these mutants was associated with the development of a K65R mutation and additional compensatory mutations in reverse transcriptase (RT). To study directly the virulence and clinical implications of these SIV mutants, two uncloned SIVmac isolates with similar fivefold reduced in vitro susceptibilities to PMPA but distinct RT genotypes, SIVmac055 (K65R, N69T, R82K A158S,S211N) and SIVmac385 (K65R, N69S, I118V), were each inoculated intravenously into six newborn rhesus macaques; 3 weeks later, three animals of each group were started on PMPA treatment. All six untreated animals developed persistently high levels of viremia and fatal immunodeficiency within 4 months. In contrast, the six PMPA-treated animals, despite having persistently high virus levels, survived significantly longer: 5 to 9 months for the three SIVmac055-infected infants and > or = 21 months for the three SIVmac385-infected infants. Virus from only one untreated animal demonstrated reversion to wild-type susceptibility and loss of the K65R mutation. In several other animals, additional RT mutations, including K64R and Y115F, were detected, but the biological role of these mutations is unclear since they did not affect the in vitro susceptibility of the virus to PMPA. In conclusion, this study demonstrates that although SIVmac mutants with the PMPA-selected K65R mutation in RT were highly virulent, PMPA treatment still offered strong therapeutic benefits. These results suggest that the potential emergence of HIV mutants with reduced susceptibility to PMPA in patients during prolonged PMPA therapy may not eliminate its therapeutic benefits.

Early short-term 9-[2-(R)-(phosphonomethoxy)propyl]adenine treatment favorably alters the subsequent disease course in simian immunodeficiency virus-infected newborn Rhesus macaques.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model of human pediatric AIDS to study disease pathogenesis and to develop intervention strategies aimed at delaying disease. In the present study, we demonstrate that very early events of infection greatly determine the ultimate disease course, as short-term antiviral drug administration during the initial viremia stage significantly delayed the onset of AIDS. Fourteen newborn macaques were inoculated orally with uncloned, highly virulent SIVmac251. The four untreated control animals showed persistently high virus levels and poor antiviral immune responses; they developed fatal immunodeficiency within 15 weeks. In contrast, SIV-infected newborn macaques which were started on 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) treatment at 5 days of age and continued for either 14 or 60 days showed reduced virus levels and enhanced antiviral immune responses. This short-term PMPA treatment did not induce detectable emergence of SIV mutants with reduced in vitro susceptibility to PMPA. Although viremia increased in most animals after PMPA treatment was withdrawn, all animals remained disease-free for at least 6 months. Our data suggest that short-term treatment with a potent antiviral drug regimen during the initial viremia will significantly prolong AIDS-free survival for HIV-infected infants and adults.

Two doses of PMPA protect newborn macaques against oral simian immunodeficiency virus infection.

Simple and affordable intervention strategies are needed to reduce the rate of HIV transmission from mother to infant in developing countries. Simian immunodeficiency virus (SIV) infection of newborn rhesus macaques is considered to be a useful model of human pediatric HIV infection. To investigate whether short-term 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) administration can protect newborn rhesus macaques against perinatal SIV infection. Eight newborn macaques were inoculated orally with highly virulent SIVmac within the first 3 days of life. Four of these animals were untreated controls. The other four animals were given one dose of PMPA (30 mg/kg subcutaneously) 4 h before oral SIV inoculation, and were then given a second and final dose of PMPA 24 h later. All four untreated control animals were persistently SIV-positive within 2 weeks after virus inoculation. In contrast, no virus could be detected in the four animals that received two doses of PMPA; these animals were seronegative and healthy at 10 months. Two doses of PMPA prevented SIV infection of newborn macaques. Our data suggest that short-term administration of PMPA to HIV-infected pregnant women at the onset of labor and to their newborns after delivery may reduce the rate of intrapartum HIV transmission.

Administration of 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) for prevention of perinatal simian immunodeficiency virus infection in rhesus macaques.

Simian immunodeficiency virus (SIV) infection of newborn macaques is a useful animal model to explore novel strategies to reduce perinatal human immunodeficiency virus (HIV) infection. The availability of two easily distinguishable virus isolates, SIVmac251 and the simian/human immunodeficiency virus chimera SHIV-SF33, allows tracing the source of infection following inoculation with both viruses by different routes. In the present study, we evaluated the efficacy of pre- and postinoculation treatment regimens with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) to protect newborn macaques against simultaneous oral SIVmac251 and intravenous SHIV-SF33 inoculation. Untreated newborns became persistently infected following virus inoculation. When three pregnant macaques were given a single subcutaneous dose of PMPA 2 hr before cesarean section, their newborns became SIV-infected following SIV and SHIV inoculation shortly after birth. In contrast, when four newborn macaques were inoculated simultaneously with SIV and SHIV, and started immediately on PMPA treatment for 2 weeks, only one animal became persistently SIV-infected; the remaining three PMPA-treated newborns, however, had some evidence of an initial transient virus infection but were seronegative and healthy at 8 months of age. Our data demonstrate that PMPA treatment can reduce perinatal SIV infection and suggest that similar strategies may also be effective against HIV.

Pharmacokinetics and bioavailability of the anti-human immunodeficiency virus nucleotide analog 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) in dogs.

The pharmacokinetics, bioavailability, and metabolism of the anti-human immunodeficiency virus nucleotide analog 9[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) were determined in beagle dogs following intravenous, intraperitoneal, and oral administration. Fasted male beagle dogs (n = 5) were pretreated with pentagastrin and received PMPA (10 mg/kg of body weight) by the intravenous and oral routes with a washout period of 1 week between doses. A further group of male dogs received PMPA as a single dose via the intravenous (1 mg/kg; n = 5) and the intraperitoneal (10 mg/kg; n = 3) routes, with 1-week washout period between doses. The concentrations of PMPA in plasma and urine were determined over 48 h postdosing by fluorescence derivatization and high-performance liquid chromatography (HPLC). The potential for metabolism or biliary excretion of PMPA was evaluated in a dog with a chronic indwelling bile cannula. Urine, feces, and bile were collected at intervals over 48 h following the intravenous administration of [14C]PMPA (10 mg/kg; 55 microCi/kg). The concentrations of PMPA in plasma after intravenous injection were best described by an open two-compartment model with a terminal half-life of approximately 10 h. PMPA was excreted unchanged in urine (70%); recovery in feces (0.42%) or bile (0.26%) was negligible. The plasma clearance of PMPA (0.28+/-0.05 liter/h/kg) was substantially greater than the glomerular filtration rate in this species, suggesting active tubular secretion of PMPA. No metabolites of [14C]PMPA were observed in urine, feces, or bile on the basis of HPLC with radioactive flow detection. The remainder of the dose was probably excreted unchanged in urine beyond 48 h postdosing. The mean+/-standard deviation observed bioavailabilities of PMPA following oral and intraperitoneal administration at 10 mg/kg were 17.1%+/-1.88% and 73.5%+/-10.5%, respectively.

Synthesis, in Vitro Biological Evaluation and Oral Bioavailability of 9-[2-(Phosphonomethoxy)Propyl]Adenine (PMPA) Prodrugs

Potentially orally bioavailable prodrugs of the antiretroviral agent 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) were evaluated. Alkyl methyl carbamates were synthesized by alkylation of PMPA with the corresponding alkyl chloromethyl carbonate and N-alkyl chloromethyl carbamate reagents. The prodrugs were evaluated for in vitro antiviral activity in addition to chemical and enzymic stability. The inhibition of human immunodeficiency virus type 1 (HIV-1) strain IIIB replication in MT-2 cells by the carbonate prodrugs was found to be 2.5-500-fold increased compared to PMPA. The alkyl methyl carbonates, except t-butyl methyl carbonate, had reasonable chemical stability at pH 2.2 and 7.4, but were rapidly converted to the corresponding monoester of PMPA in the presence of dog plasma. The alkyl methyl carbamate prodrugs such as N-t-butyl methyl carbamate were found to have high stability in vitro. Based on its chemical stability and good oral bioavailability, bis(POC)PMPA (isopropyl methyl carbonate) was chos...

Metabolism and pharmacokinetics of novel oral prodrugs of 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) in dogs.

A series of prodrugs designed to enhance the oral bioavailability of the antiretroviral agent 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA; 1) have been synthesized, including a bis-(acyloxymethyl) ester 2 and a series of bis-(alkoxycarbonyloxymethyl) esters 3-9. The in vitro biological stability and in vivo pharmacokinetics of these prodrugs were evaluated to support selection of a prodrug candidate for clinical evaluation. The in vitro biological stability of the prodrugs was examined in dog tissues (intestinal homogenate, plasma and liver homogenate). The apparent half-lives were determined based on the disappearance of prodrug using reverse-phase HPLC with UV detection. Oral bioavailability of PMPA from each prodrug was determined in fasted beagle dogs. Concentrations of PMPA in plasma were determined by HPLC following fluorescence derivatization. Data for prodrugs were compared to historical data for intravenous PMPA. All prodrug were rapidly hydrolyzed in dog plasma and tissues (t1/2 < 60 min). In fasted beagle dogs, bis-[(pivaloyloxy)methyl] PMPA (bis-POM PMPA) 2 had the highest oral bioavailability as PMPA (37.8 +/- 5.1%). The oral bioavailabilities of PMPA from bis-(alkoxycarbonyloxymethyl) esters ranged from 16.0% to 30.7% and PMPA was the major metabolite formed. There was a correlation between oral bioavailability and intestinal stability of bis-(alkoxycarbonyloxymethyl) ester prodrugs (r2 = 0.96). Lipophilicity (log P) was not a good predictor of oral bioavailability. The most labile prodrugs in dog intestinal homogenates, bis-(n-butyloxycarbonyloxymethyl) PMPA 5 and bis-(neo-pentyloxy-carbonyloxymethyl) PMPA 8 (t1/2 < 5 min) had the lowest oral bioavailabilities. Based on good oral bioavailability (30.1%), chemical and intestinal stability bis-(isopropyloxycarbonyloxymethyl) PMPA (bis-POC PMPA) 4 was selected as a candidate for clinical evaluation.

9-[2-(Phosphonomethoxy)propyl]adenine therapy of established simian immunodeficiency virus infection in infant rhesus macaques.

The long-term therapeutic and toxic effects of 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) were evaluated in simian immunodeficiency virus (SIV)-infected newborn rhesus macaques. Four untreated SIV-infected newborn macaques developed persistently high levels of viremia, and three of the four animals had rapidly fatal disease within 3 months. In contrast, long-term PMPA treatment of four newborn macaques starting 3 weeks after virus inoculation resulted in a rapid, pronounced, and persistent reduction of viremia in three of the four animals. Emergence of virus with fivefold-decreased susceptibility to PMPA occurred in all four PMPA-treated animals and was associated with the development of a lysine-to-arginine substitution at amino acid 65 (K65R mutation) and additional mutations in the reverse transcriptase; however, the clinical implications of this low-level drug resistance are nuclear. No toxic side effects have been seen, and all PMPA-treated animals have remained disease-free for more than 13 months. Our data suggest that PMPA holds much promise for the treatment of human immunodeficiency virus-infected human infants and adults.