1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89.

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity.

Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase.

The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of > 400 microM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo. Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regressions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.

In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase.

A human colorectal carcinoma cell line, WiDr, was genetically engineered to express the nonmammalian enzyme, cytosine deaminase (CD). Expression of CD in WiDr cells (WiDr/CD) did not alter the growth rate of these cells when grown in vitro or as solid tumor xenografts in nude mice. However, expression of CD did increase the sensitivity of these cells to the nontoxic prodrug, 5-fluorocytosine (FCyt), decreasing the 50% inhibitory concentration for FCyt from 26,000 microM in parental WiDr cells to 27 microM in WiDr/CD cells. The increase in sensitivity to FCyt in WiDr/CD cells was the result of the CD-mediated conversion of FCyt to 5-fluorouracil (FUra) and subsequent FUra anabolites. The half-life of the prodrug, FCyt, was determined to be approximately 40 min in nude mice. A single i.p. injection of 500 mg FCyt/kg body weight resulted in a transient FCyt plasma level of approximately 4000 microM while osmotic minipumps or constant tail vein infusions of FCyt achieved continual FCyt plasma levels of 5 microM and 50 microM, respectively, with no overt signs of toxicity. Significant antitumor effects were observed in nude mice bearing tumors derived from WiDr/CD cells when these animals were given 500 mg FCyt/kg i.p. for 10 consecutive days. These antitumor effects were demonstrated by decreases in tumor growth rate, tumor size, tumor weight, and thymidine incorporation into tumor DNA. This antitumor effect was significant but less profound if FCyt was administered by constant tail vein infusion. WiDr and WiDr/CD cells were very sensitive to FUra in vitro (50% inhibitory concentration approximately 5 microM). However, no significant antitumor effects were observed in nude mice bearing tumors derived from either WiDr or WiDr/CD cells when these animals were treated with various doses of FUra. Taken collectively, these data indicate that nontoxic plasma levels of FCyt can be attained which can produce profound antitumor effects on tumors engineered to express CD and that these antitumor effects are significantly better than those that can be achieved using FUra. These positive data support the continued development of a gene therapy approach to colorectal carcinoma involving the selective expression of CD in colorectal tumors with subsequent administration of FCyt.

Synthesis and biological evaluation of dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine and 2', 3'-dideoxycytidine.

The 5'----5' dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) were prepared and evaluated for their inhibitory properties against different viruses, including human immunodeficiency virus (HIV). The synthesis of the compounds was achieved by reaction of AZT or N4-(4-monomethoxytrityl)-2',3'-dideoxycytidine with in situ prepared methylphosphonic bis (triazolide), followed in the latter case by an acidic treatment. The two title compounds showed in vitro anti-HIV activity, that was 200- to 450-fold less pronounced that that shown by the corresponding monomeric nucleosides AZT and DDC. The decreased antiviral activity may be ascribed to nuclease resistance of the methylphosphonate linkage.

Tissue distribution and metabolic disposition of zidovudine in rats.

The tissue distribution and metabolic fate of [5'-3H]zidovudine was studied in rats after a single dose of 10 mg/kg by gavage. The drug was absorbed rapidly and distributed into all tissues. Peak blood and tissue levels were observed 0.25 hr post-dose. The level of peak radioactivity in the stomach, intestine, liver, spleen, adrenals, and kidney was higher than in plasma, while in the heart, lung, thymus, lymph nodes, muscle, bone, and skin it was similar to that in plasma. Only in the testes and the brain the radioactivity was lower than in plasma. Blood and plasma radioactivity levels were nearly equivalent. A biphasic disappearance of radioactive material was observed in blood and plasma, as well as in most tissues, with a rapid decline in the early phase (0.25-4 hr) and a slower decline thereafter. The 0-24-hr urinary and fecal recoveries (mean +/- SD) of radioactive material were 78 +/- 14% and 20 +/- 9% of dose, respectively, indicating virtually complete recovery of the radioactive dose. Reversed-phase HPLC analysis indicated that approximately 88% of urinary radioactivity corresponded to unchanged zidovudine, with the remaining radioactivity accounted for by five metabolites. One of these urinary metabolites was identified as 3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine and another as 3'-amino-3'-deoxythymidine (AMT). The majority of fecal radioactivity (greater than 70%) corresponded to AMT. There is a component of biliary excretion in the disposition of zidovudine. At least 7% of a parenteral dose of zidovudine was secreted in the bile, primarily as 3'-azido-3'-deoxy-5'-beta-D-glucuronylazidothymidine, which may be a source of fecal AMT.

Isolation and characterization of an ether glucuronide of zidovudine, a major metabolite in monkeys and humans.

A major metabolite of zidovudine (3'-azido-3'-deoxythymidine, AZT), which previously had not been observed in a variety of experimental animals, was identified in samples of plasma and urine from cynomolgus monkeys and a patient treated with AZT. The urinary recoveries of metabolite from the monkeys and the patient were, respectively, 1.5- and 6.9-fold higher than the recoveries of unchanged drug. The metabolite was purified in gram quantities from the urines of the monkeys and the patient and was identified enzymatically, using beta-glucuronidase and a specific inhibitor of the enzyme, as a glucuronide conjugate of AZT. The metabolite was formed in vitro by incubating AZT with preparations of human liver in the presence of UDP-glucuronic acid. In addition, the metabolite was prepared synthetically and physical characterizations--including microanalysis and UV, IR, NMR and mass spectra--of compound from all three sources were identical and confirmed the metabolite to be the 5'-O-beta-D-glucuronide of AZT.

Alteration of zidovudine pharmacokinetics by probenecid in patients with AIDS or AIDS-related complex.

The anti-human immunodeficiency virus drug zidovudine is metabolized extensively in human beings to the 5'-glucuronide (GAZT) and is cleared rapidly, resulting in a short half-life and the need for frequent dosing. This study explores whether probenecid, which is also metabolized by glucuronidation, reduces zidovudine clearance when zidovudine is administered orally to patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC). The mean zidovudine plasma levels were significantly higher after concurrent administration of probenecid than in its absence, resulting in a twofold increase in the mean AUC, a corresponding decline in the apparent total clearance, and a prolongation in the mean half-life. Similar alterations were observed in GAZT disposition. There was a marked reduction in the urinary excretion ratio of GAZT to zidovudine and a decline in the renal clearance of GAZT after probenecid coadministration. Probenecid inhibits zidovudine glucuronidation and renal excretion of GAZT.

Simultaneous quantification of zidovudine and its glucuronide in serum by high-performance liquid chromatography.

A sensitive high-performance liquid chromatography (HPLC) assay has been developed to simultaneously determine levels of the anti human immunodeficiency virus agent, zidovudine (AZT), and its major metabolite (the 5'-O-glucuronide) in serum. Samples were first mixed with an internal standard (a stereoisomer of AZT), then prepared for analysis using solid-phase extraction columns and chromatographed using a reversed-phase analytical column. Isocratic elution with a mobile phase of 15% acetonitrile, buffered to pH 2.70 with ammonium phosphate, gave good resolution of the three analytes and endogenous serum components. The HPLC analysis time required per sample was 34 min and analyte recoveries were reproducibly high (greater than 93%). Replicate analyses of prepared standards gave satisfactory precision and accuracy, with coefficients of variation less than 15% and deviations from expected concentrations less than 10%.

Pharmacokinetics and bioavailability of zidovudine in humans.

The basic pharmacokinetic and bioavailability information on zidovudine was obtained during the initial phase I study. Following intravenous doses of 1.0 mg/kg every eight hours to 7.5 mg/kg every four hours, zidovudine plasma levels decay in a biexponential manner, indicating two-compartment pharmacokinetics. The mean half-life was 1.1 hours over this dose range and the total body clearance was approximately 1,900 ml/minute/70 kg, up to doses of 5 mg/kg. At 7.5 mg/kg, total body clearance decreased by 35 percent. The 5'-O-glucuronide was identified as a major metabolite of zidovudine in plasma and urine. This inactive metabolite is rapidly formed and cleared from plasma, with a half-life of one hour. No other metabolites have been found in humans. Renal clearance of zidovudine was estimated at 350 ml/minute/70 kg. Zidovudine penetrated the blood brain barrier as indicated by a cerebrospinal fluid:plasma ratio averaging 0.5, determined two to four hours after dosing. Following oral administration of zidovudine at doses from 2.0 mg/kg every eight hours to 10 mg/kg every four hours, peak plasma levels increased proportionately with dose; the average bioavailability was 65 percent. Since 90 percent of the drug was recovered in the urine as zidovudine or the 5'-O-glucuronide, the incomplete bioavailability is assumed to be the result of first-pass metabolism rather than incomplete absorption. Pharmacokinetic questions related to optimal use of the drug are currently being addressed.

Disposition in the dog and the rat of 2, 6-diamino-9-(2-hydroxyethoxymethyl)purine (A134U), a potential prodrug of acyclovir.

2,6-Diamino-9-(2-hydroxyethoxymethyl)purine (A134U), the 6-deoxy-6-amino analog of the antiviral agent acyclovir (ACV), was administered orally to dogs and rats. Plasma concentration-time profiles and urinary excretion of A134U and its deamination product, ACV, were determined. Mean peak plasma ACV concentrations achieved in the dog were 57, 156 and 285 microM after A134U doses of 20, 50 and 120 mg/kg, respectively, and increased in near proportion to the dose. The urinary recovery of ACV accounted for 60-92% of the two lower doses, but only 40-58% of the highest dose. In the rat, peak plasma ACV concentrations were 3.1 and 10.7 microM, respectively, after 20- and 50-mg/kg doses of A134U. After 5- and 20-mg/kg oral doses of [2-14C]A134U, the urinary recovery of ACV (20-27%) accounted for 59 to 76% of the absorbed dose. The remainder was excreted largely as unchanged A134U, with negligible (0.4-1.3%) biotransformation to inactive metabolites. Except for small decreases in absorption and increases in deamination, no change in the metabolism of A134U was observed after its repeated oral administration to rats. Oral dosing of dogs and rats with A134U resulted in peak plasma concentrations and total urinary recoveries of ACV greater than those observed after equivalent oral doses of ACV, suggesting that A134U might be an effective prodrug of ACV for use in the oral therapy of herpes simplex virus infections.

A single-tube radioimmunoassay for the antiviral agent 2,6-diamino-9-(2-hydroxyethoxymethyl)purine (A134U).

A direct radioimmunoassay for the detection of A134U in biological fluids and extracts has been developed. The entire assay, including scintillation counting, is performed using 12 X 55-mm centrifuge tubes and results in a sensitive, reliable, and relatively inexpensive procedure. The log-logit transformation is linear over a range of 1 to 30 pmol per sample. Intra-assay precision was found to be excellent with a coefficient of variation ranging from 4.1 to 14.5% for the standard curve with plasma and a coefficient of variation ranging from 4.0 to 17.8% for the standard curve with urine. Interassay precision and accuracy were also found to be good. With the antisera chosen for use, no cross-reactivity was found with acyclovir or its two known metabolites, while some cross-reactivity was seen with the corresponding two derivatives of A134U. Only very minor cross-reactivities were seen with a small number of other compounds out of a large number tested.

Metabolic fate of radioactive acyclovir in humans.

The metabolic fate and the kinetics of elimination of [8-14C]acyclovir in plasma and blood was investigated in five cancer patients. Doses of 0.5 and 2.5 mg/kg were administered by one-hour intravenous infusion. Radioactivity was distributed nearly equally in blood and plasma. The plasma and blood concentration-time data were defined by a two-compartment open pharmacokinetic model. The overall mean acyclovir plasma half-life and total body clearance +/- SD were 2.1 +/- 0.5 hours and 297 +/- 53 ml/min/1.73 m2. Binding of acyclovir to plasma proteins was 15.4 +/- 4.4 percent. The radioactive dose was excreted predominantly in the urine (71 to 99 percent) with less than 2 percent excretion in the feces and only trace amounts of radioactivity in the expired air. Reverse-phase high-performance liquid chromatography indicated that 9-carboxymethoxymethylguanine was the only significant urinary metabolite of acyclovir accounting for 8.5 to 14.1 percent of the dose. A minor metabolite (less than 0.2 percent of dose) had the retention time of 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine. Unchanged urinary acyclovir ranged from 62 to 91 percent of the dose. There was no indication of acyclovir cleavage to guanine. The renal clearances of acyclovir were three times higher than the corresponding creatinine clearances.

Metabolic disposition of acyclovir in the guinea pig, rabbit, and monkey.

Two guinea pigs and two rabbits were each inoculated subcutaneously with 14C-labeled acyclovir (25 mg/kg). Both species excreted the entire amount within 72 hours. The rabbits excreted all of the radioactivity in the urine while the guinea pigs excreted an average of 14.2 percent in the feces. The rabbits excreted an average of 71.0 percent of the dose as unchanged drug; 25.1 percent was excreted as 9-carboxymethoxymethylguanine (CMMG) and 3.5 percent as 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine (8-hydroxyacyclovir). An average of 60.7 percent of the dose was recovered from the guinea pigs as acyclovir; 32.3 percent was excreted as CMMG and 3.1 percent as 8-hydroxyacyclovir. The two rabbits showed elimination-phase half-lives (t 1/2 beta) for plasma acyclovir of 0.8 and 2.2 hours. Mean t 1/2 beta for acyclovir in two rhesus, four patas, and four african green monkeys, each receiving acyclovir (10 mg/kg) as a bolus intravenous injection, were 1.2, 1.7, and 1.8 hours respectively. The average 48 hour urinary excretion of acyclovir, 8-hydroxyacyclovir, and CMMG in the rhesus monkey was estimated to be 21.3 percent, 15.3 percent, and 7.1 percent, respectively, of the total administered amount. The patas and african green species excreted the dose mostly as acyclovir and CMMG.

Disposition of intravenous radioactive acyclovir.

The kinetic and metabolic disposition of (8-14C)acyclovir (ACV) was investigated in five subjects with advanced malignancy. The drug was administered by 1-hr intravenous infusion at doses of 0.5 and 2.5 mg/kg. Plasma and blood radioactivity-time, and plasma concentration-time data were defined by a two-compartment open kinetic model. There was nearly equivalent distribution of radioactivity in blood and plasma. The overall mean plasma half-life and total body clearance +/- SD of ACV were 2.1 +/- 0.5 hr and 297 +/- 53 ml/min/1.73 m2. Binding of ACV to plasma proteins was 15.4 +/- 4.4%. Most of the radioactive dose excreted was recovered in the urine (71% to 99%) with less than 2% excretion in the feces and only trace amounts in the expired Co2. Analyses by reverse-phase high-performance liquid chromatography indicated that 9-(carboxymethoxymethyl)guanine was the only significant urinary metabolite of ACV, accounting for 8.5% to 14.1% of the dose. A minor metabolite (less than 0.2% of dose) had the retention time of 8-hydroxy-9-[(2-hydroxyethoxy)methyl]guanine. Unchanged urinary ACV ranged from 62% to 91% of the dose. There was no indication of ACV cleavage to guanine. Renal clearance of ACV was approximately three times the corresponding creatinine clearances.