Catherine U. Lambe

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.

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.

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.

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.

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.

6-Dimethylamino-9-(β-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.

The Biosynthesis of Deoxyguanosine Triphosphate in Herpes Simplex Type-1 Infected Vero Cells Treated with Acyclovir and Hydroxyurea

Herpes simplex virus type 1 (HSV-1) infection of cells causes induction of virally specified thymidine kinase, DNA polymerase, ribonucleotide reductase and deoxyribonuclease1-3. Also accompanying infection is a rise in deoxyribonucleoside triphosphates (dNTPs), particularly dTTP2,4. Acyclovir (ACV) is an acyclic deoxyguanosine analog which, in its triphosphate form, inhibits viral DNA polymerase5,6. ACV treatment dramatically increased the pool sizes of dATP and dGTP in infected Vero cells compared to infected untreated cells4. The source of these elevated purine deoxyribonucleotide pools could be from either enhanced denovo synthesis, salvage of deoxyribonucleosides derived from host cell DNA, or possibly a combination of the two pathways.

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.

ACYCLOVIR'S ANTIHERPETIC ACTIVITY IS POTENTIATED BY INHIBITION OF RIBONUCLEOTIDE REDUCTASE: 205

Compound A723U, a 2-acetylpyridine thiosemicarbazone, was found to inactivate herpes simplex virus (HSV) ribonucleotide reductase. Inactivation occurred only while the enzyme was catalyzing the reduction of substrate and at a maximum rate of 17 per hr. A723U inhibited virus replication (ED50 = 3-5 μM) at concentrations that were not toxic to the confluent host cells. It also exhibited mutually potentiated anti-HSV activity with acyclovir (ACV). Sub-inhibitory concentrations of either compound significantly decreased the ED50 of the other compound. As expected, A723U caused the dGTP pool to decrease. However, it also caused the level of ACV-triphosphate to markedly increase. The consequence is a facilitation of the binding of ACV-triphosphate to its target enzyme, HSV DNA polymerase.