2-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies.

Earlier studies have shown guanine arabinoside (ara-G) is an effective agent against growth of T-cell lines and freshly isolated human T-leukemic cells. However, poor water solubility of ara-G limits clinical use. 2-Amino-6-methoxypurine arabinoside (506U) is a water-soluble prodrug converted to ara-G by adenosine deaminase. 506U is not a substrate for deoxycytidine kinase, adenosine kinase, or purine nucleoside phosphorylase and is phosphorylated by mitochondrial deoxyguanosine kinase at a rate 4% that of ara-G phosphorylation. Mitochondrial DNA polymerase was the least sensitive to ara-GTP inhibition of the five human DNA polymerases tested. [3H]506U was anabolized to ara-G 5'-phosphates in CEM cells but not to phosphorylated metabolites of 506U. 506U was selective for transformed T over B cells and also inhibited growth in two of three monocytic lines tested. 506U given i.v. to cynomolgus monkeys was rapidly converted to ara-G; the ara-G had a half-life of approximately 2 h. 506U had in vivo dose-dependent efficacy against human T-cell tumors in immunodeficient mice. A Phase 1 trial of 506U against refractory hematological malignancies is now in progress at two study sites.

Pharmacokinetics, oral bioavailability, and metabolism in mice and cynomolgus monkeys of (2'R,5'S-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, an agent active against human immunodeficiency virus and human hepatitis B virus.

(2'R,5'S-)-cis-5-Fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (524W91) is a nucleoside analog with potent anti-human immunodeficiency virus and anti-human hepatitis B virus activities in vitro. The pharmacokinetics and bioavailability of 524W91 after oral dosing were studied in mice dosed with 10, 100, and 600 mg of 524W91 per kg of body weight by the oral and intravenous routes. Cynomolgus monkeys were dosed with 10 and 80 mg of 524W91 per kg. In both species, the clearance of 524W91 was rapid, via the kidney, and was independent of dose. In monkeys, the total body clearance of 10 mg of 524W91 per kg was 0.7 +/- 0.1 liter/h/kg, and the volume of distribution at steady state was 0.8 +/- 0.02 liter/kg. The terminal elimination half-life was 1.0 +/- 0.2 h. The absolute bioavailability after oral dosing was 63% +/- 4% at 10 mg/kg. Concentrations of 524W91 in the cerebrospinal fluid were 4% +/- 0.7% of the corresponding levels in plasma. In mice, the total clearance of 10 mg of 524W91 per kg was 2.3 liters/kg/h, and the volume of distribution at steady state was 0.9 liter/kg. Absolute bioavailability in mice after oral dosing was 96% at a dose of 10 mg/kg. The metabolism of orally administered [6-3H]524W91 was studied in cynomolgus monkeys at a dose of 80 mg/kg and in mice at a dose of 120 mg/kg. Monkeys excreted 41% +/- 6% of the radioactive dose in the 0- to 72-h urine, 33% +/- 10% in the feces, and 10% +/- 7% in the cage wash. Unchanged 524W91 was 64% of the total radiolabeled drug recovered in the urine. The glucuronide was a minor urinary metabolite. 5-Fluorouracil was not detected (less than 0.02% of the dose). Mice dosed orally with 120 mg of [6-3H]524W91 per kg excreted 67% +/- 7% of the radiolable in the )- to 48-h urine. Small amounts of the 3' -sulfoxide and glucuronide metabolites were observed in the urine, but 5-fluorouracil was not detected. Good bioavailability after oral dosing and resistance to metabolism recommend 524W91 for further preclinical evaluation.

6-Dimethylamino-9-(beta-D-arabinofuranosyl)-9H-purine: pharmacokinetics and antiviral activity in simian varicella virus-infected monkeys.

6-Dimethylamino-9-(beta-D-arabinofuranosyl)-9H-purine (ara-DMAP) effectively prevented the development of rash and appreciably reduced viremia in simian varicella virus-infected monkeys. Doses of 100 and 50 mg/kg/day, administered orally, were highly effective. The lowest dose of 20 mg/kg/day was much less effective in preventing moderate viremia. However, the 20 mg/kg/day did prevent the development of rash in two of three monkeys. All three doses of ara-DMAP reduced liver infection as reflected by lower aspartate aminotransferase values in the sera of the African green monkeys. Orally administered ara-DMAP was rapidly absorbed. However, significant variation among individual monkeys in the AUC values, peak plasma levels, and plasma half-lives were observed.

Metabolism and pharmacokinetics of the anti-varicella-zoster virus agent 6-dimethylaminopurine arabinoside.

The metabolism of 6-dimethylaminopurine arabinoside (ara-DMAP), a potent inhibitor of varicella-zoster virus replication in vitro, was studied in rats and cynomolgus monkeys. Rats dosed intraperitoneally or orally with ara-DMAP excreted unchanged ara-DMAP and one major metabolite, 6-methylaminopurine arabinoside (ara-MAP), in the urine. They also excreted allantoin and small amounts (less than 4% of the dose each) of hypoxanthine arabinoside (ara-H) and adenine arabinoside (ara-A). The relative amount of each urinary metabolite excreted remained fairly constant for intraperitoneal ara-DMAP doses of 0.3 to 50 mg/kg of body weight. Rats pretreated with an inhibitor of microsomal N-demethylation, SKF-525-A, excreted more unchanged ara-DMAP and much less ara-MAP than did rats given ara-DMAP alone. Rats pretreated with the adenosine deaminase inhibitor deoxycoformycin excreted more ara-MAP and much less ara-H and allantoin. ara-MAP was shown to be a competitive alternative substrate inhibitor of adenosine deaminase (Ki = 16 microM). Rats given ara-DMAP intravenously rapidly converted it to ara-MAP and purine metabolism end products; however, ara-A generated from ara-DMAP had a half-life that was four times longer than that of ara-A given intravenously. In contrast to rats, cynomolgus monkeys dosed intravenously with ara-DMAP formed ara-H as the major plasma and urinary end metabolite. Rat liver microsomes demethylated ara-DMAP much more rapidly than human liver microsomes did. ara-DMAP is initially N-demethylated by microsomal enzymes to form ara-MAP. This metabolite is further metabolized by either adenosine deaminase, which removes methylamine to form ara-H, or by microsomal enzymes, which remove the second methyl group to form ara-A.

Inhibition of xanthine oxidase by 4-hydroxy-6-mercaptopyrazolo[3,4-d]pyrimidine.

Compound B103U, 4-hydroxy-6-mercaptopyrazolo[3,4-d]pyrimidine, was investigated as an inhibitor of human xanthine oxidase. Studies in vitro demonstrated that it was significantly more potent than oxypurinol, 4,6-dihydroxypyrazolo[3,4-d]pyrimidine. It formed an initial complex with electron-rich (reduced) human xanthine oxidase that was tighter than the corresponding complex formed by oxypurinol. The initial complexes with each inhibitor and reduced enzyme were internally rearranged into more stable complexes with first-order rate constants of 2.5 to 3 per min. However, the half-life of the isomerized (stable) complex with B103U was three to four times longer than the half-life of the analogous complex with oxypurinol. This stability was previously noted by Massey et al. (J. Biol Chem 254: 2837-2844, 1970) with B103U and bovine xanthine oxidase. The overall Ki values accounting for the initial and isomerized complexes were 5 nM for B103U and 100 nM for oxypurinol. B103U was also more potent as an inhibitor of bovine xanthine oxidase-catalyzed generation of superoxide radicals. Studies in mice revealed that the relative in vitro potency of B103U was not sustained in vivo. Compared to the inhibition of xanthine oxidase by oxypurinol, inhibition by B103U was neither more potent nor longer lasting. This shortcoming was not caused by weaker inhibition of mouse xanthine oxidase. Instead, it was the result of poor bioavailability. Plasma levels of available B103U rapidly decreased from samples of mouse and human blood because of reversible binding to serum proteins. B103U was also susceptible to oxidation. Two equivalents of H2O2 stoichiometrically oxidized the 6-thiol substituent to a sulfinic acid. This oxidized product was three orders of magnitude weaker as an inhibitor of xanthine oxidase than was B103U.

2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1110U), a potent inactivator of ribonucleotide reductases of herpes simplex and varicella-zoster viruses and a potentiator of acyclovir.

2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]thiocarbonohydrazone (A1110U) was found to be a potent inactivator of the ribonucleotide reductases (EC 1.17.4.1) encoded by herpes simplex virus types 1 and 2 and by varicella-zoster virus and to be a weaker inactivator of human ribonucleotide reductase. It also markedly potentiated the antiherpetic activity of acyclovir against these viruses in tissue culture. A1110U both decreased the dGTP pool that builds up when infected cells are treated with acyclovir and induced a large increase in the pool of acyclovir triphosphate. The resultant 100-fold increase in the ratio of the concentrations of acyclovir triphosphate to dGTP should facilitate the binding of the fraudulent nucleotide to its target enzyme, herpes virus-encoded DNA polymerase, and could account for the synergy between A1110U and acyclovir. A similar change in the acyclovir triphosphate-to-dGTP ratio was previously reported to be induced by another ribonucleotide reductase inhibitor, 2-acetylpyridine 4-(2-morpholinoethyl)thiosemicarbazone (A723U). However, A1110U is considerably more potent and may have better clinical potential. Synergistic toxic interactions between A1110U and acyclovir were not detected in uninfected cells.

Inhibition by ganciclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information.

The ability of LM cells, thymidine kinase-deficient LM cells (LMTK-), and LMTK- cells transformed to the LMTK+ phenotype by herpes simplex virus type 1 genetic information (LH7 cells) to anabolize the acyclovir congener ganciclovir was examined. About 50-fold more ganciclovir triphosphate was produced by LH7 cells than by either LM or LMTK- cells. Growth inhibition studies indicated that 180 and 120 microM ganciclovir were required to achieve 50% growth inhibition of LM and LMTK- cells, respectively; only 0.07 microM ganciclovir was necessary to achieve 50% inhibition of LH7 cells. DNA synthesis in the transformed cells was significantly reduced by ganciclovir treatment, whereas ganciclovir had little effect on DNA synthesis in the nontransformed cells. Alkaline sucrose gradient sedimentation analysis of transformed cellular DNA indicated that LH7 DNA synthesized in the presence of ganciclovir chased into mature DNA. Both LM and LH7 DNA synthesized in the presence of ganciclovir exhibited a concentration-dependent reduction in the rate of elongation into mature DNA. Finally, [14C]ganciclovir was incorporated internally into the growing chains of LH7 cells.

A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate.

We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate approximately equal to 1/10th the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed. The induction of normal nucleoside and deoxynucleoside triphosphates and certain cellular nucleoside kinases was comparable in mutant and wild-type virus infections. These results provide strong evidence for the importance of phosphorylation in the selectivity of this antiviral compound and raise the possibility that human cytomegalovirus encodes a nucleoside kinase. The mutant may identify the existence of a cytomegalovirus function whose properties could facilitate genetic analysis of this important pathogen.

Metabolic activation of 9([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human lymphoblastoid cell lines infected with Epstein-Barr virus.

9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) is more potent and has a more prolonged inhibitory effect against Epstein-Barr virus (EBV) in vitro than does acyclovir (ACV). To assess the mechanism of this difference, we first compared the extent of phosphorylation of the two drugs in superinfected Raji cells. BW B759U is phosphorylated to levels 100-fold higher than is ACV. In addition, lower levels of phosphorylation of BW B759U and ACV were observed in uninfected Raji cells. Studies on the kinetics of formation of BW B759U triphosphate in superinfected Raji cells indicated that drug-phosphorylating activity was detected as early as 3 h after superinfection; this activity was steadily maintained for the first 7 h, followed by a burst of activity between 7 and 10 h and a doubling of phosphorylation between 10 and 25 h. During the superinfection cycle, the pool sizes of deoxyribonucleoside and ribonucleoside triphosphates were increased and reached their maxima at 10 h after infection. The maximal amount of triphosphorylated drug in a virus producer cell, P3HR-1 (LS), was obtained at 21 h after drug treatment. During long-term drug treatment, approximately 44 and 77% reduction in EBV genome copies per cell was observed on days 3 and 7, respectively. In a separate experiment, after treatment of P3HR-1 (LS) cells with BW B759U for 36 h, 4.2 pmol of BW B759U triphosphate per 10(6) cells was achieved. After the cells were released into drug-free medium, drug triphosphate was rapidly decreased to 11% of the original level in 1 day. Thereafter, the decrease was slow but steady, down to 0.22 pmol/10(6) P3HR-1 cells by 5 days. We calculated that 0.22 pmol of BW B759U triphosphate per 10(6) cells represents a cellular concentration of 0.22 microM, which is theoretically enough to inhibit EBV replication. This is based upon a comparison with the 50% effective dose of BW B759U (0.05 microM) for inhibition of genome replication and a Ki of 0.08 microM for BW B759U triphosphate inhibition of EBV DNA polymerase.

Potentiation of antiherpetic activity of acyclovir by ribonucleotide reductase inhibition.

Compound A723U, a 2-acetylpyridine thiosemicarbazone, produced apparent inactivation of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase. Inactivation occurred after A723U formed a reversible complex with the enzyme and only while the enzyme was catalyzing the formation of deoxynucleotides. A723U inhibited HSV-1 replication at concentrations that were not toxic to the confluent host cells. Most importantly, A723U and acyclovir (ACV) were found to exhibit mutual potentiation of their antiviral activities. Subinhibitory concentrations of either compound greatly reduced the ED50 (median effective dose) of the other. Studies of the deoxynucleotide pool sizes and the levels of ACV triphosphate (ACV-P3) revealed that A723U not only significantly reduced the pool of dGTP but also increased the level of ACV-P3 in infected cells. The net result was an 80-fold increase in the ratio of ACV-P3 to dGTP. This should greatly facilitate the initial binding of ACV-P3 to HSV-1 DNA polymerase and probably accounts for the mechanism of potentiation.

Metabolic activation of the nucleoside analog 9-[( 2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human diploid fibroblasts infected with human cytomegalovirus.

9-[( 2-Hydroxy-1-(hydroxymethyl)ethoxy]-methyl)guanine (BW B759U) is a more potent inhibitor of human cytomegalovirus (HCMV) in vitro than is the related nucleoside analog acyclovir (ACV). BW B759U was selectively activated to the 5'-triphosphate (BW B759U-triphosphate) in cells infected with HCMV to levels at least 10-fold higher than those measured for ACV-triphosphate and up to as much as 100-fold higher than the levels found in uninfected cells. BW B759U-triphosphate accumulated in HCMV-infected cells with time; the rate of this increase was dependent upon the drug dose and virus multiplicity of infection. Enzyme activities that catalyzed the phosphorylation of thymidine and 2'-deoxycytidine increased 3- to 7-fold in extracts of cells early after HCMV infection but thereafter declined. No concomitant increase in the rate of BW B759U phosphorylation was detected under these assay conditions. Maximal rate of accumulation of both BW B759U-triphosphate and ACV-triphosphate after a short exposure to drug occurred in the late phase of the infective cycle, as the titer of extracellular virus reached a peak in untreated cultures, but after the decline of stimulated host deoxypyrimidine kinase activities. Once formed, the BW B759U-triphosphate pool decreased very slowly and thus it persisted for several days in both HCMV-infected and uninfected cells.

Inhibition of cellular alpha DNA polymerase and herpes simplex virus-induced DNA polymerases by the triphosphate of BW759U.

The triphosphate form of the acyclovir analog BW759U (9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine) inhibited the DNA polymerases (EC 2.7.7.7) from several strains of herpes simplex virus type 1. Two acyclovir triphosphate-resistant DNA polymerases were as sensitive to BW759U-triphosphate as were the DNA polymerases induced by wild-type viruses (Ki = 0.05 to 0.1 microM). The Ki value for cellular alpha DNA polymerase was 35- to 50-fold greater than those for the DNA polymerases induced by the various herpes simplex virus strains investigated. Incubation of Vero cells infected by the KOS strain of herpes simplex virus type 1 with the acyclovir analog resulted in the formation of substantial quantities of (9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine) triphosphate.

Effect of acyclovir on the deoxyribonucleoside triphosphate pool levels in Vero cells infected with herpes simplex virus type 1.

The effect of acyclovir on the deoxyribonucleoside triphosphate pools of Vero cells infected with herpes simplex virus type 1 was examined. Deoxyguanosine triphosphate and deoxyadenosine triphosphate pool levels in infected cells treated with acyclovir increased dramatically compared with pool levels in untreated infected cels. The increases were due, at least in part, to inhibition of viral DNA polymerase activity which resulted in reduced utilization of the deoxyribonucleoside triphosphates. Differences of as much as 26 times were detected in the sensitivity of herpes simplex virus type 1 to inhibition by acyclovir with different Vero cell cultures. These results were due to differences in acyclovir triphosphate levels, not to differences in deoxyguanosine triphosphate levels.

Pharmacokinetics of inhibition of adenosine deaminase by erythro-9-(2-hydroxy-3-nonyl)adenine in CBA mice.

The pharmacokinetics of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) inhibition of adenosine deaminase (ADA) was measured in vivo in CBA mice. The in vivo assay utilized injection of 10-100 nmoles [2-3H]adenosine and measurement of blood 3H2O 20 min later. A single oral dose of EHNA (50 mg/kg) totally inhibited ADA for 4 hr and caused a large increase in conversion of [2-3H]adenosine to [2-3H]ATP. EHNA (3 mg/kg) decreased deamination by 50% for 2-6 hr, depending on the dose of adenosine used. Mice dosed with EHNA (100 mg/kg) once daily for 7 days showed the same ADA recovery rate as mice dosed only once. High single oral doses of EHNA had no effect on blood ATP and GTP pools.