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Despite substantial progress in the development of antiretroviral regimens that durably suppress Human Immunodeficiency Virus (HIV) infection, new agents that maintain high efficacy while further optimizing the safety of lifelong, chronic therapy are needed. Tenofovir alafenamide (TAF; formerly known as GS-7340) is a novel prodrug of the antiviral acyclic nucleoside phosphonate tenofovir (TFV) with improved properties relative to tenofovir disoproxil fumarate (TDF). Although potent and generally well tolerated, TDF therapy has been associated with changes in markers of renal function, decreases in bone mineral density and a rare occurrence of serious renal adverse events, including Fanconi's Syndrome. The renal and bone toxicity observed with TDF is associated with high circulating plasma levels of TFV. TAF was discovered to be a more efficient prodrug able to further refine HIV therapy and better address life-long therapy in an older and increasingly comorbid HIV infected population. By enhancing stability in biological matrices while being rapidly activated in cells, TAF produces higher levels of intracellular TFV diphosphate, the pharmacologically active metabolite, in HIV-target cells at substantially reduced oral doses of TFV equivalents. All TFV released in the body is eventually eliminated renally; therefore, lowering the TFV equivalents administered reduces off-target kidney exposure. Effective therapy is thus achieved at approximately 90% lower systemic exposure to TFV, translating to statistically and clinically significant improvement in safety parameters associated with bone mineral density and markers of renal function., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The occasion of this 25th anniversary issue encouraged us to reminisce about the important history of the discovery of the dideoxynucleoside analogues for the treatment of HIV/AIDS and to chronicle our thoughts about a particular exciting and rewarding period of our scientific careers. Following the identification of the anti-HIV activity of zidovudine (AZT), we participated in the urgent quest to discover optimal treatments of HIV infection and AIDS. A number of previously synthesized nucleoside analogues were comparatively evaluated, and stavudine (D4T) emerged as a promising candidate for development. Following clinical evaluation, D4T became a mainstay of the initial antiretroviral combination therapy, prolonging and saving numerous lives. It has only recently been supplanted by better-tolerated treatments. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010., (Copyright 2009 Elsevier B.V. All rights reserved.)

In the event of a bioterrorism attack using smallpox virus, there currently is no approved drug for the treatment of infections with this virus. We have reported previously that (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC) (also known as cidofovir [CDV]) has good activity against poxvirus infections; however, a major limitation is the requirement for intravenous administration. Two related acyclic nucleoside phosphonates (ANPs), adefovir (PMEA) and tenofovir (PMPA), are active against human immunodeficiency virus or hepatitis B virus but do not have activity against the orthopoxviruses. Therefore, we have evaluated a number of analogs and potential oral prodrugs of these three compounds for their ability to inhibit the replication of vaccinia virus or cowpox virus in tissue culture cells. The most-active compounds within the CDV series were (S)-HPMPA and (butyl L-alaninyl) cyclic HPMPC, with 50% effective concentrations (EC(50)s) from 4 to 8 microM, compared with 33 to 43 microM for CDV. Although PMEA itself was not active, adefovir dipivoxil [bis[(pivaloyl)oxymethyl] PMEA] and bis(butyl L-alaninyl) PMEA were active against both viruses, and bis(butyl L-alaninyl) PME-N6-(cyclopropyl)DAP and (isopropyl L-alaninyl)phenyl PME-N6-(cyclopropyl)DAP were the most active compounds tested, with EC(50)s of 0.1 to 2.6 microM. In the PMPA series, none of the analogs tested had significantly better activity than PMPA itself. These data indicate that a number of these ANP derivatives have activity against vaccinia virus and cowpox virus in vitro and should be evaluated for their efficacies in animal models.

Clinical studies with tenofovir disoproxil fumarate, an oral prodrug of the nucleotide analog tenofovir, recently approved for the treatment of HIV, have demonstrated antiviral activity and good tolerability in HIV-infected patients. In order to better understand the cytotoxicity profile of tenofovir relative to the other nucleoside reverse transcriptase inhibitors (NRTIs), the in vitro effects of these agents were evaluated in various human cell types. Tenofovir inhibited the proliferation of liver-derived HepG2 cells and normal skeletal muscle cells with CC(50) values of 398 and 870 microM, respectively. In comparison, ZDV, ddC, ddI, d4T, and abacavir all showed lower CC(50) values in these two cell types. Evaluation of hematopoietic toxicity revealed that tenofovir was less cytotoxic towards erythroid progenitor cells (CC(50)>200 microM) than ZDV, d4T, and ddC (CC(50)=0.06-5 microM). Despite some degree of donor-to-donor variability, the inhibitory activity of the tested NRTIs against myeloid cell lineage, in the order of decreasing severity, was consistently ddC>ZDV>d4T>tenofovir>3TC. Finally, tenofovir showed substantially weaker effects on proliferation and viability of renal proximal tubule epithelial cells than cidofovir, a related nucleotide analog with the potential to induce renal tubular dysfunction. In conclusion, tenofovir exhibited weak cytotoxic effects in all cell types tested with less in vitro cytotoxicity than the majority of NRTIs currently used for the treatment of HIV disease.

Drug-associated dysfunction of mitochondria is believed to play a role in the etiology of the various adverse symptoms that occur in human immunodeficiency virus (HIV)-infected patients treated with the nucleoside reverse transcriptase inhibitors (NRTIs). Tenofovir, a nucleotide analog recently approved for use in the treatment of HIV infection, was evaluated in vitro for its potential to cause mitochondrial toxicity and was compared to currently used NRTIs. Treatment with tenofovir (3 to 300 microM) for up to 3 weeks produced no significant changes in mitochondrial DNA (mtDNA) levels in human hepatoblastoma (HepG2) cells, skeletal muscle cells (SkMCs), or renal proximal tubule epithelial cells. The potencies of inhibition of mtDNA synthesis by the NRTIs tested were zalcitabine (ddC) > didanosine (ddI) > stavudine > zidovudine (ZDV) > lamivudine = abacavir = tenofovir, with comparable relative effects in the three cell types. Unlike ddC and ddI, tenofovir did not affect cellular expression of COX II and COX IV, two components of the mitochondrial cytochrome c oxidase complex. Lactate production was elevated by less than 20% in HepG2 cells or SkMCs following treatment with 300 microM tenofovir. In contrast, lactate synthesis increased by >200% in the presence of 300 microM ZDV. Thus, treatment of various human cell types with tenofovir at concentrations that greatly exceed those required for it both to have in vitro anti-HIV type 1 activity in peripheral blood mononuclear cells (50% effective concentration, 0.2 microM) and to achieve therapeutically relevant levels in plasma (maximum concentrations in plasma, 0.8 to 1.3 microM) is not associated with mitochondrial toxicity.

In animals and humans, intravenous administration of the antiviral nucleotide analogue cidofovir results in a dose-limiting nephrotoxicity characterized by damage to the proximal tubular epithelial cells. Probenecid, a competitive inhibitor of organic anion transport in the proximal tubular epithelial cells, was evaluated for its effect on the chronic toxicity and pharmacokinetics of cidofovir. Cynomolgus monkeys (5/sex/group) received cidofovir for 52 consecutive weeks as a once weekly intravenous bolus injection at 0 (saline), 0.1, 0.5, or 2.5 mg/kg/dose alone or at 2.5 mg/kg/dose in combination with probenecid (30 mg/kg/dose via oral gavage 1 h prior to cidofovir administration). Cidofovir-associated histopathological changes were seen only in the kidneys, testes, and epididymides. Nephrotoxicity (mild to moderate cortical tubular epithelial cell karyomegaly, tubular dilation, basement membrane thickening) was present only in monkeys receiving 2.5 mg/kg/dose cidofovir without probenecid. The incidence and severity of testicular (hypo- and aspermatogenesis) and epididymal (severe oligo- and aspermia) changes were increased in monkeys administered cidofovir at 2.5 mg/kg/dose, either alone or in combination with oral probenecid. Renal drug clearance was decreased between Weeks 1 and 52 in the 2.5 mg/kg/dose groups and resulted in an increased systemic exposure to cidofovir (as measured by AUC) that was significantly greater in monkeys administered cidofovir alone (312% increase in males, 98% in females) than in those coadministered probenecid (32% increase in males, 3% in females). These results demonstrate that oral probenecid coadministration protects against the morphological evidence of nephrotoxicity and the accompanying decrease in renal clearance in monkeys receiving chronic intravenous cidofovir treatment.

After intravenous administration of [14C]cidofovir to African green monkeys (43 mg/kg, 29.5 microCi/kg), the drug distributed rapidly into extracellular fluid. Concentrations of radioactivity in plasma were described by a three-compartment model with alpha, beta, and gamma half-lives of 0.67, 3.02, and 36.0 hr, respectively (N = 3). These phases are believed to represent renal elimination, efflux of free cidofovir from cells, and efflux from cells of cidofovir produced from dephosphorylation of metabolites, respectively. Less than 5% of the dose was phosphorylated, based on the proportion of total AUC in the gamma-phase. The clearance of cidofovir (211 +/- 16.6 ml/hr/kg) was dependent on dose and exceeded the theoretical glomerular filtration rate. Concentrations of cidofovir in kidney declined with a half-life of 23 hr and were > 1,000-fold higher than plasma levels by 120 hr. Clearance of cidofovir after multiple intravenous doses of 4.9 mg/kg/day (18.5 microCi/kg/day) decreased significantly by day 10, consistent with the observed nephrotoxicity. Oral and subcutaneous bioavailabilities of cidofovir were 21.8 +/- 9.44 and 98.5 +/- 15.8%, respectively. After intravenous administration of [14C]cidofovir to cynomolgus monkeys (10 mg/kg, 100 microCi/kg) alone or 1 hr after oral probenecid (30 mg/kg), mean +/- SD (N = 3) urinary recoveries of total radioactive dose were 91.4 +/- 11.3% and 94.4 +/- 29.8%, respectively, at 7 days postdose. The mean +/- SD half-lives of the terminal elimination phases were 33.3 +/- 10.6 and 24.4 +/- 5.0 hr, respectively. Cidofovir accounted for 98% of the radioactivity recovered in urine; the remainder was attributed to cidofovir phosphocholine. The prolonged elimination phase observed in monkeys is consistent with the long intracellular half-life of phosphorylated cidofovir in vitro and supports infrequent dosing of the drug for antiviral therapy.

Purpose: This study was designed to evaluate the intraocular distribution and metabolism of the antiviral nucleotide analogs cidofovir and cyclic 1-[(S)-3-hydroxy-2-(phosphonomethoxy) propyl]cytosine (HPMPC) in New Zealand white rabbits following intravitreal administration., Methods: Male rabbits received either 14C-cidofovir or 14C-cyclic HPMPC by intravitreal injection into both eyes (50 micrograms/eye, 11 microCi/eye). Two animals per group were sacrificed at 24, 48, 72 or 240 h post-dose. Ocular tissues, kidney and liver were oxidized to determine total radioactivity and metabolites were determined by HPLC., Results: At 24 h post-dose, total radioactivity was 9.96 and 5.18 micrograms-equiv/g for cidofovir and cyclic HPMPC, respectively, in vitreous and 20.9 and 3.54 micrograms-equiv/g, respectively, in retina. Although the initial vitreal clearance was 2-fold faster for the cyclic analog, the estimated terminal elimination half-lives in vitreous (42 hr) and in retina (66-77 hr) were similar for both drugs. By 240 h post-dose, radioactivity in all ocular tissues was approximately ten-fold higher for cidofovir. Radioactivity in vitreous at 240 h after intravitreal dosing with either drug contained cidofovir, cyclic HPMPC and cidofovir-phosphocholine., Conclusions: The long retinal half-life observed presumably reflects formation of phosphorylated cidofovir within retinal cells. Cidofovir achieved a ten-fold higher level of phosphorylated drug in retina than cyclic HPMPC: Therefore, intravitreal cidofovir may be expected to suppress progression of retinitis for a longer period than an equivalent intravitreal dose of cyclic HPMPC: The intravitreal half-life of cidofovir was 20-fold longer than that of ganciclovir in the same animal model.

Cidofovir [1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine] susceptibility among 29 paired pre- and post-cidofovir exposure isolates from 22 patients enrolled in phase I/II clinical trial, GS-92-101, was determined. This trial was designed to evaluate the safety and antiviral effects of cidofovir in patients with AIDS and asymptomatic shedding of human cytomegalovirus (HCMV) in semen with no evidence of end-organ HCMV disease and no prior anti-HCMV therapy. These patients received a median cumulative dose of 30 mg/kg (range, 3-67) administered over a median of 8 weeks (range, 2-38). The IC50 values of preexposure isolates ranged from <0.5 to 1.85 and for postexposure isolates from <0.5 to 2 uM. Thus, no significant changes in cidofovir susceptibilities have been noted for HCMV during the various regimens of cidofovir administrated in this clinical trial.

The pharmacokinetics of cidofovir (HPMPC; (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine) were examined at five dose levels in three phase I/II studies in a total of 42 human immunodeficiency virus-infected patients (with or without asymptomatic cytomegalovirus infection). Levels of cidofovir in serum following intravenous infusion were dose proportional over the dose range of 1.0 to 10.0 mg/kg of body weight and declined biexponentially with an overall mean +/- standard deviation terminal half-life of 2.6 +/- 1.2 h (n = 25). Approximately 90% of the intravenous dose was recovered unchanged in the urine in 24 h. The overall mean +/- standard deviation total clearance of the drug from serum (148 +/- 25 ml/h/kg; n = 25) approximated renal clearance (129 +/- 42 ml/h/kg; n = 25), which was significantly higher (P < 0.001) than the baseline creatinine clearance in the same patients (83 +/- 21 ml/h/kg; n = 12). These data indicate that active tubular secretion played a significant role in the clearance of cidofovir. The steady-state volume of distribution of cidofovir was approximately 500 ml/kg, suggesting that the drug was distributed in total body water. Repeated dosing with cidofovir at 3.0 and 10.0 mg/kg/week did not alter the pharmacokinetics of the drug. Concomitant administration of intravenous cidofovir and oral probenecid to hydrated patients had no significant effect on the pharmacokinetics of cidofovir at a 3.0-mg/kg dose. At higher cidofovir doses, probenecid appeared to block tubular secretion of cidofovir and reduce its renal clearance to a level approaching glomerular filtration.

1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosi ne (cyclic [cHPMPC]) was evaluated as a novel antiviral agent in comparison with (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). Evaluation for in vitro activity against herpes simplex virus type 2 in MA-104 and MRC-5 cells showed that both cHPMPC and HPMPC have comparable activities and cytotoxicities. cHPMPC was found to be stable on incubation in human plasma and human liver homogenates. Intracellular metabolism studies revealed that cHPMPC was converted inside of the cells to HPMPC and then to the monophosphate, the diphosphate, and the monophosphate choline metabolites. In a mouse herpes simplex virus type 2 encephalitis model, both cHPMPC and HPMPC exhibited similar potencies in vivo. Nephrotoxicity, which is the dose-limiting toxicity of HPMPC, was assessed in a 14-day repeated-dose toxicity study in rats; cHPMPC has an improved safety margin of > or = 13-fold over that of HPMPC.

A series of phosphonate prodrugs were evaluated in an attempt to increase the oral bioavailability of the anti-HIV agent 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; 1). The majority of the bis(alkyl ester) and bis(alkyl amide) prodrugs were prepared by alcohol or amine displacement of dichlorophosphonate 2. Basic hydrolysis of the bis(esters) or bis(amides) provided the corresponding monoesters or monoamides. Synthesis of bis[(acyloxy)alkyl] phosphonates 10a-c was accomplished by alkylation of PMEA with the appropriate chloromethyl ether in the presence of N,N'-dicyclohexylmorpholinecarboxamidine. The systemic levels of PMEA following oral administration of a PMEA prodrug to rats were determined by measuring the concentration of PMEA in the urine for 48 h after administration of the prodrug. The oral bioavailability of PMEA employing this method was determined to be 7.8%. Oral dosing with bis(alkyl) phosphonates 3a,b resulted in apparent absorption of the prodrugs (> or = 40%), although neither of the esters were completely cleaved to liberate the parent phosphonate PMEA. The mono(alkyl esters) 7a-e and 8a,b exhibited poor oral bioavailability (< or = 5%). Phosphonamides 5, 6, and 9 were unstable under acidic conditions and provided levels of PMEA comparable to the parent compound after oral administration. Bis[(acyloxy)alkyl] phosphonates 10a-c demonstrated significantly improved oral bioavailabilities of 17.6%, 14.6%, and 15.4%, respectively. When evaluated in vitro against HSV-2, (acyloxy)alkyl phosphonates 10a-c were greater than 200-fold more active than PMEA.

A series of 5'-derivatives and modified pyrimidine analogues of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine, 1) were synthesized to determine their potential as oral prodrugs of d4T. Utilizing a screen developed for the rapid evaluation of a variety of prodrugs in mice, it was determined that 5'-acetate 2 provided comparable plasma levels of d4T after oral administration of the prodrug to that when d4T was administered alone. The relative oral bioavailability of methoxy acetate 3 and cyclohexyl carbonate 5 was 79 and 41%, respectively. Dihydropyridine ester 6 did not provide detectable levels of d4T up to 1 h after oral administration of 6. Thiopyrimidines 8 and 9, as well as aminopyrimidine 10 also failed to provide measurable levels of d4T after oral administration. 5'-Derivatives 3, 5, and 6 showed similar activity to that of d4T against HIV and MuLV, as did 5'-benzoyl-4-thio derivative 8. However, the corresponding 4-thio 5'-alcohol 9 was inactive.

Improved methods of human immunodeficiency virus isolation have sparked an increased interest in determination of the number and proportion of infected cells in plasma samples. The limiting dilution assay is a popular method of determining the proportion of infected cells. We propose a new estimator of the proportion of infected cells based on an underlying Markov chain model for the progression of infected cells to further flasks in the series. This method should improve both design and analyses of these assays.

A series of 2'-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and evaluated in vitro for anti-human immunodeficiency virus (HIV) activity in the XTT assay and for anti-herpes activity in the plaque reduction assay. It has been observed that the anti-HIV activity of these derivatives depends on the size and the nature of the substituent as well as the chirality at the 2'-position of PMEG. In addition, these compounds generally demonstrated greater activity against HIV than herpes viruses. The most interesting analogues which emerged from these studies are (R)-2'-(azidomethyl)-PMEG [(R)-5] and (R)-2'-vinyl-PMEG [(R)-11]. The former showed anti-HIV activity with an IC50 of 5 microM and a cytotoxicity (CC50) greater than 1.4 mM in CEM cells. The latter has an IC50 of 13 microM for anti-HIV activity and a CC50 of greater than 1.6 mM. Furthermore, we have demonstrated that replacement of the guanine base of these 2'-substituted PMEG analogues with cytosine drastically reduces anti-HIV and anti-herpes activity.

The disposition and metabolic fate of 3'-deoxy-2',3'-didehydrothymidine (D4T) were evaluated both in isolated hepatocytes and in nonhuman primates. Rapid formation of thymine and beta-aminoisobutyric acid (BAIBA) occurred following incubation of hepatocytes with 10 microM [5(-3)H]D4T. Substantial levels of tritiated water were also detected. Exposure of cells to D4T in the presence of either 1 mM thymine or 10 microM benzyloxybenzyluracil, an inhibitor of dihydropyrimidine dehydrogenase, decreased intracellular BAIBA levels by approximately 89 and 63%, respectively. Concurrently, [3H]thymine levels increased two- to fivefold. These results are consistent with D4T being cleaved to thymine, which is then degraded to BAIBA. A similar metabolic disposition was observed in monkeys following administration of 25 mg of [5(-3)H]D4T per kg of body weight. BAIBA, thymine, and tritiated water were identified in plasma and urine. Approximately 50% of the administered dose was recovered in urine within 24 h, with the majority of the radioactivity representing unchanged drug. After administration intravenously or orally of 25 mg of [4(-14)C]D4T per kg of body weight to monkeys, a novel metabolite, designated X, in addition to unchanged D4T, thymine, and BAIBA, was also detected. The sum of the three metabolites and unchanged drug accounted for virtually all of the radioactivity in plasma and urine. Thymine and X exhibited kinetic profiles similar to that of D4T, with plasma elimination half-life of 2 to 3 h, whereas BAIBA levels remained constant for extended periods and declined slowly; this metabolite could be detected 24 h after intravenous drug administration. Mean oral bioavailability of D4T was high at approximately 70%. As observed in the [5(-3)H]D4T study performed in monkeys, approximately half of the administered [4(-14)C]D4T was recovered unchanged. The remainder was not recovered in urine or feces collected up to 30 days after drug administration. These data suggest that D4T metabolites are further metabolized by salvage pathways and/or converted to biological macromolecules.

The in vitro activity of didanosine (2',3'-dideoxyinosine, ddI) against the human immunodeficiency virus (HIV) is generally less potent than that of zidovudine (3'-azidothymidine, AZT) and zalcitabine (2',3'-dideoxycytidine, ddC), often by an order of magnitude or more. However, in monocyte/macrophage cell cultures, the potency of didanosine is similar to, or greater than, that of zidovudine but still less than that of ddC. The cytotoxicity of didanosine, including cytotoxicity to bone marrow progenitor cells, is low, and endpoints are often not reached. (The concentration inhibiting 50% of the cell growth [IC50] is > 100 microM.) Thus, in spite of lower potency, didanosine has a high antiviral selective index in vitro. By contrast, zidovudine and ddC are more cytotoxic, with IC50 values < 5 microM. Clinically derived HIV isolates with as high as 30-fold decreases in susceptibility to didanosine in vitro have been described. However, in studies designed to assess the frequency of resistance development after prolonged didanosine therapy, more moderate changes (2- to 5-fold) are seen in general. By contrast, isolates with a 100-fold decrease in sensitivity to zidovudine are frequently found in strains from patients who have received prolonged zidovudine therapy.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA; 1) was acylated with chloromethyl pivalate to afford bis(pivaloyloxymethyl) PMEA (2). The ester prodrug demonstrated enhanced in vitro potency against HSV-2 greater than 150-fold higher than the parent compound. The antiviral activity of 2 was 50-fold better than PMEA against HSV-1, and equipotent against HIV and HCMV. The toxicity of 2 was studied in both resting and growing cells.

A number of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and tested in vitro for anti-herpes and anti-human immunodeficiency virus (HIV) activity. Among these analogues, (R)-2'-methyl-PMEG [(R)-3] and 2',2'-dimethyl-PMEG (7) demonstrated potent anti-HIV activity in the XTT assay with EC50 values of 1.0 and 2.6 microM, respectively. The corresponding (S)-2'-methyl-PMEG [(S)-3] was found to be less potent against HIV. In addition, the (R) and (S) enantiomers of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG, 8) were prepared for comparison of biological activity, and shown to be active and equipotent against herpesviruses, but inactive against HIV.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is an antiviral phosphonate nucleotide analogue that displays activity against a range of herpesviruses. Anion exchange high performance liquid chromatography analysis of the 60% methanol extract from [14C]HPMPC-treated cells reveals the formation of three major metabolites. Two of these were identified as phosphorylated forms of HPMPC, HPMPC phosphate, and HPMPC diphosphate, by liberation of HPMPC upon acid digestion and coelution with synthetic standards on high performance liquid chromatography. The third metabolite, which is resistant to alkaline phosphatase cleavage but sensitive to phosphodiesterase, is proposed to be an HPMPC phosphate adduct. In herpes simplex virus-1-infected cells the same three metabolites are detected, at concentrations comparable to those in uninfected cells. When HPMPC is removed from the medium, the concentrations of the metabolites in cells decrease slowly, with half-lives of approximately 6, 17, and 48 hr for HPMPC phosphate, HPMPC diphosphate, and the HPMPC phosphate adduct, respectively. HPMPC diphosphate inhibits herpes simplex virus-1 and -2 DNA polymerases with a lower Ki than that for DNA polymerase alpha, and enzyme inhibition is competitive in each case. The formation and the persistence of HPMPC phosphates in cells and the selective inhibition of viral DNA polymerases by HPMPC diphosphate can explain why cells pretreated with HPMPC remain refractory to viral infection even long after HPMPC is removed from the medium.

2',3'-Didehydro-2',3'-dideoxythymidine (D4T) is a potent inhibitor of human immunodeficiency virus (HIV), with low hematological toxicity. In the present study, the cellular pharmacology of D4T was investigated in human bone marrow cells (BMC), in an attempt to understand the mechanism of the observed low bone marrow toxicity. After exposure of human BMC to 10 microM [3H]D4T for 24 hr, D4T-5'-triphosphate (D4T-TP) was the predominant metabolite, reaching a concentration of 0.3 pmol/10(6) cells. The D4T-5'-monophosphate levels were slightly lower, whereas the D4T-5'-diphosphate levels were about 6-fold lower than those of D4T-TP at 24 hr. Nucleic acids of human BMC exposed to 10 microM [3H]D4T for 24 hr were purified and analyzed by cesium sulfate density gradient centrifugation. No radioactivity was detected in the RNA region, whereas a limited amount was associated with the DNA region. The amount of label incorporated into DNA correlated with the extracellular D4T concentration and the length of incubation time. Enzymatic hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated incorporation of both D4T and thymidine (dThd) into DNA. Degradation of D4T to thymine and subsequent formation of labeled dThd was also detected in human BMC. Pulse (24 hr)-chase (48 hr) experiments with 10 microM [3H]D4T demonstrated that the amount of radiolabel from D4T in DNA decreased over time during the chase. Under similar conditions, [3H]3'-azido-3'-deoxythymidine (AZT) incorporated into DNA of human BMC did not decrease during the chase. Although D4T-TP standard was demonstrated to be unstable at 37 degrees and neutral pH, D4T was much more stable in solution when incorporated into newly synthesized DNA isolated from human BMC, suggesting that enzymatic excision may be the mechanism for D4T removal from DNA. In summary, although higher concentrations of D4T-TP, compared with AZT-5'-triphosphate, are observed in human BMC, after exposure of cells to similar extracellular concentrations of parent drug, steady state levels of D4T incorporated into DNA are 10-50-fold lower, compared with AZT. Competition with dTTP formed by D4T metabolism and excision of D4T from DNA may be responsible, in part, for these effects. This study further demonstrates that incorporation of 2',3'-dideoxynucleosides into nuclear DNA of human BMC may be related to the ability of these anti-HIV agents to induce hematological side effects.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (S-HPMPC) was able to prevent simian varicella infection in African green monkeys inoculated intratracheally with virus. A dose of 50 mg/S-HPMPC/kg administered intravenously was shown to prevent the development of rash, reduce viremia and protect the monkeys from death. The 50 mg/kg dose was effective when treatments initiated on day 2 post-infection (p.i.) was given as ten daily doses of 5 mg/kg, as 10 mg/kg administered on five days on an alternate-day schedule, as two 25 mg/kg doses given on day 2 and on day 7 p.i., or as a single injection of 50 mg/kg on day 2. The single 50 mg/kg dose was also effective when treatment was delayed until four days p.i., but was ineffective when treatment was delayed until six days p.i. The 50 mg/kg dose was not effective when given orally by gavage. No evidence of toxicity was noted in daily clinical examinations, or in frequent hematology and clinical chemistry tests performed during the clinical evaluation of the infection.

Novel phosphonate isosteres of acyclovir (ACV) and ganciclovir (DHPG) monophosphates were found to be potent and selective antiherpesvirus agents. In the series of phosphonate analogues of ACV monophosphate, only the guanine analogue 20 exhibited activity against herpesviruses, similar to the structure-activity relationship observed for base modification of ACV analogues. The phosphonate isostere of ACV monophosphate (20) was more effective than ACV in the HSV-1 infected mouse model. The 3'-carba analogues of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]purines/pyrimidines (adenine, HPMPA; guanine, HPMPG; cytosine, HPMPC) are devoid of antiherpesvirus activity. This result confirms that the beta-oxygen atom of the phosphonomethyl ether functionality in HPMP-purines/pyrimidines plays a critical role for activity against herpesviruses.

Three analogs of thymidine, D4T [2',3'-didehydro-2',3'-dideoxythymidine; 1-(2,3-dideoxy-beta-D-glyceropent-2-enofuranosyl)thymine], FddT (3'-fluoro-3'-deoxythymidine), and AZT (3'-azido-3'-deoxythymidine), were compared in biological tests designed to assess their potential utility as anti-human immunodeficiency virus (HIV) agents. The in vitro potencies of these compounds against HIV infection in CEM cells were measured, with FddT and AZT being more potent than D4T. The cytotoxicities of D4T, FddT, and AZT for CEM cells were comparable. The triphosphates of these three derivatives inhibited purified HIV reverse transcriptase, and their affinities for this polymerase were found to be 1 or 2 orders of magnitude greater than that for the normal substrate, dTTP. D4T was less toxic than FddT or AZT for cultured human and mouse bone marrow cells (granulocyte-macrophage CFU). The three compounds had similar toxicities for human progenitor erythrocyte burst-forming units. In a 30-day mouse toxicity study, AZT and FddT produced a similar spectrum of hematopoietic toxicities. These toxic effects occurred at much lower doses of FddT than of AZT. At the higher doses of FddT, a significant incidence of lethality occurred. By contrast, D4T was considerably less toxic than both AZT and FddT in this study. The dose-limiting toxicity of D4T in mice was hepatotoxicity. The very different phosphorylation patterns of D4T, its lower toxicity, and its comparable potency relative to FddT and AZT suggest that the potential of D4T as an anti-HIV agent should be further explored.

A series of 9-(phosphonoalkyl)purines, which are analogues of 9-[2-(phosphonomethoxy)ethyl]purines (guanine, PMEG, 1; adenine, PMEA, 2), were synthesized. The analogues were tested for activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), Rauscher murine leukemia virus (R-MuLV), and human immunodeficiency virus type 1 (HIV-1). With variations in the length of the alkyl chain, the optimal activity was achieved with two carbons between the purine base and the phosphonomethoxy functionality. Despite the structural similarity and the close pKa2 value of 8 to that of PMEG, no phosphorylation of 8 was observed by the bovine brain guanylate kinase. Since all isosteric analogues of PMEG (7-9) were not inhibitory against HSV-1 and HSV-2, the presence of the 3'-oxygen atom in the PME purines proved critical for anti-HSV activity. Introduction of the 1'-methyl group on the PMEG side chain significantly reduced its anti-HSV activity. Analogue 11, which is a mimic of the phosphate by incorporation of the alpha,alpha-difluoro carbon, was ineffective against HSV-1 and HSV-2. These results suggest that the structural requirements of PME purines for anti-HSV activity appear to be very strict.

Our studies have shown that the acyclic nucleotide analogues PMEA and HPMPC are able to penetrate into cells and are then activated to mono- and diphosphate derivatives. The latter correspond to triphosphate analogues and presumably serve an important role in the biological activity exerted by these antiviral agents. In support of this idea, the inhibitory effect of PMEApp on HIV reverse transcriptase has been demonstrated with both RNA and DNA template-primer systems. Further studies will be undertaken to determine the effect of HPMPCpp on viral DNA polymerases. Whereas the metabolism of PMEA in CEM cells gives rise to only PMEAp and PMEApp, additional metabolites were obtained in MRC-5 cells; the identity of these metabolites remains to be determined. In the case of HPMPC, a third metabolite was obtained in addition to HPMPCp and HPMPCpp, which has been tentatively assigned as a phosphate-choline adduct by analogy with activation of cytosine-based nucleoside derivatives. The metabolism of HPMPC was unchanged between uninfected and infected cells, indicating that viral enzymes are not necessary for the activation of HPMPC. The long intracellular half-lives of the HPMPC metabolites may have implications for the antiviral efficacy of this compound. The persistence of activated metabolites suggests that infrequent dosing may be possible due to a prolonged antiviral effect. Our results on the effectiveness of infrequent dosing schedules with HPMPC in the treatment of HSV 2 infections in mice support this hypothesis. It is also possible that HPMPCp-choline may serve as a reservoir for HPMPC and therefore for the presumed active metabolite HPMPCpp.

The acyclic nucleotide analogue (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl] cytosine (2, HPMPC) was prepared on a multigram scale in 18% overall yield starting from (R)-2,3-O-isopropylideneglycerol. The key step in the nine-step synthetic route is coupling of cytosine with the side-chain derivative 8 which bears a protected phosphonylmethyl ether group. In vitro data showed that HPMPC has good activity against herpes simplex virus types 1 and 2, although it was 10-fold less potent than acyclovir [AVC, 9-[(2-hydroxyethoxy)methyl]guanine]. By comparison, HPMPC exhibited greater activity than ACV against a thymidine kinase deficient strain of HSV 1 and was more potent than ganciclovir [DHPG, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine] against human cytomegalovirus. In vivo, HPMPC showed exceptional potency against HSV 1 systemic infection in mice, having an ED50 of 0.1 mg/kg per day (ip) compared with 50 mg/kg per day for ACV. HPMPC was also more efficacious than ACV in the topical treatment of HSV 1 induced cutaneous lesions in guinea pigs.

Tryptophan dioxygenase, derived from Streptomyces parvulus, was purified to near homogeneity and shown to have a native Mr of 88,000. Kinetic parameters of the enzyme were determined and evidence suggesting that it is a hemoprotein was obtained. Tryptophan dioxygenase has a high specificity toward L-tryptophan with an apparent Km of 0.3 mM. L-3-Hydroxykynurenine was a competitive inhibitor with respect to L-tryptophan with a Ki of 0.16 mM. In vitro, the enzyme displayed little activity in the absence of a reducing agent; ascorbate, at 50 mM, was the preferred reductant providing almost a 50-fold increase in enzyme activity. The regulation of tryptophan dioxygenase synthesis and activity is described. The expression of the enzyme is correlated with the biosynthesis of actinomycin D in S. parvulus. These results support the hypothesis that tryptophan dioxygenase functions as the first enzyme in the sequence converting L-tryptophan to the chromophore of this antibiotic.

Two forms of kynurenine formamidase (EC 3.5.1.9; aryl-formylamine amidohydrolase) are present in extracts of Streptomyces parvulus. The higher molecular weight enzyme (Mr = 42 000), kynurenine formamidase I, appears to be constitutive and is present at relatively constant but low levels in antibiotic producing and nonproducing cultures, whereas the synthesis of the lower molecular weight form (Mr = 25 000), kynurenine formamidase II, is initiated just prior to the onset of actinomycin formation. It is postulated (i) that kynurenine formamidase II catalyzes the second step in the pathway from tryptophan----actinocin, and (ii) that it is regulated specifically for the specialized function of actinomycin biosynthesis. The role of kynurenine formamidase I is unknown. Formamidase I and II activities were purified from extracts of S. parvulus and kinetic parameters of the two enzymes were determined. Although some of the properties of the two enzymes are quite similar (substrate specificities, Km values), some striking differences were noted (pH and temperature optima, molecular size, chromatographic properties, sensitivity to certain ions and chemicals). Mutant studies suggest that expression of the gene(s) coding for formamidase II activity play an essential role in regulating the formation of actinocin and, hence, antibiotic synthesis. Kynurenine formamidase activity was also found in a representative number of Streptomyces species and related organisms suggesting that the enzyme may function in the degradative metabolism of tryptophan by certain actinomycetes in nature.

Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.

The nucleoside analogue 1-(2,3-dideoxy-beta-D-glycero-pent-2-enofuranosyl)thymine (d4T, 1) was prepared by ring opening of the 3',5'-anhydro compound 5. This method has been refined such that it can be used to prepare d4T on a large scale. The triphosphate of d4T was also synthesized from 1 in order to examine the mode of action. The in vitro inhibitory activity of d4T was found to be comparable to that of AZT in HIV-infected CEM cells. The triphosphate of d4T (8) and that of AZT inhibited the HIV reverse transcriptase with poly(rA):oligo(dT) as the template:primer with Ki values of 0.032 and 0.007 microM, respectively. The in vitro toxicity of d4T against normal human hematopoietic progenitor cells (CFU-GM) was measured in comparison to AZT. While d4T reduces colony-forming units by 50% at a concentration of 100 microM, it takes only 1 microM AZT to have a similar toxic effect. With erythrocyte burst forming units (BFU-E) the in vitro toxicities for d4T and AZT have comparable ID50 values of 10 and 6.7 microM, respectively.

A new method is described for purification of the dehydropeptidase I enzyme, renal dipeptidase (EC 3.4.13.11). The six steps used are homogenization of the tissue, extraction with Triton X-100, sedimentation of insoluble material, and ion-exchange, size-exclusion, and affinity chromatographies. The use of Triton X-100 to solubilize the enzyme is a major advantage over the previously described procedure using n-butanol and results in both improvements in yield and interexperimental consistency. Also, the affinity column method we have employed results in higher recovery of active enzyme at the final step and a product which is apparently homogeneous. The method has general utility for this class of enzymes.

2',3'-Dideoxy-2',3'-didehydrothymidine (D4T) is a thymidine nucleoside analog which has potent anti-human immunodeficiency virus activity in vitro. We have studied its metabolism in cells to assist in determining its mechanism of action. D4T is metabolized in cells to the mono-, di-, and triphosphate nucleotides. Our data suggest that the initial conversion to the monophosphate is catalyzed by thymidine kinase. This enzyme has an affinity for D4T 600-fold lower than for thymidine and catalyzes the rate-limiting step in production of the triphosphate. Nevertheless, intracellular concentrations of the triphosphate approximately equal to the reported Ki for human immunodeficiency virus reverse transcriptase are attained with extracellular concentrations of free drug as low as 0.05 microM. The pattern of phosphorylation is different from that of 3'-azido-3'-deoxythymidine (AZT), which has an affinity for thymidine kinase equivalent to that of thymidine and is easily phosphorylated. The rate-limiting step in formation of AZT triphosphate is conversion of mono- to diphosphate, and thus the monophosphate accumulates. On removal of D4T or AZT from the media, both triphosphates have an intracellular half-life of about 200 min, and this rate ultimately controls the rate of elimination of the drugs from cells. The differences in metabolism of D4T and AZT observed in vitro may be responsible for the differences in toxicity seen in vitro and in vivo and support the exploration of the clinical utility of D4T as an anti-human immunodeficiency virus agent.

A simple method is described for labeling carbapenems with [14C]dimethyl sulfate. Reverse-phase high-performance liquid chromatography of the reaction was used to purify the product. Carbapenems with 2-substituents containing pyrid-3-yl and -4-yl moieties could be labeled by this method, but those containing a pyrid-2-yl group could not. Nonreversible binding of these labeled carbapenems to human serum albumin was investigated. Pyridinium-3-yl compounds displayed low binding rates (0.028 to 0.044%/h), whereas three of four pyridinium-4-yl compounds bound much faster (0.38 to 0.62%/h). It is postulated that these differences are related to the ability of the compound to stabilize a deprotonated form transiently.

Further studies on the expression of the two aspartokinase activities in Bacillus bovis are presented. Aspartokinase I (previously shown to be inhibited and repressed by lysine) was found to be repressed by diaminopimelate in the wild-type strain. However, in a mutant unable to convert diaminopimelate to lysine, starvation for lysine resulted in an increase in aspartokinase I activity. Thus, lysine itself or an immediate metabolite was the true effector of repression. Aspartokinase II (previously shown to be inhibited by lysine plus threonine) was repressed by threonine. Studies with the parent strain and auxotrophs inidicated that only threonine or an immediate metabolite of threonine was involved in this repression. Methionine and isoleucine were not effectors of any of the detected aspartokinase activities. Apart from inhibition and repression controls, a third as yet undefined regulatory mechanism operated to decrease the levels of both aspartokinases as growth declined, even in mutants in which repression control was absent. In thiosine-resistant, lysine-excreting mutants with elevated levels of aspartokinase, the increase in activity could always be attributed to one enzyme or the other, never both. The existence of separate structural genes for each aspartokinase is therefore suggested.