Selectivity--key to chemotherapy: presidential address.
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Acyclovir↗
Biomedical subjects
G B Elion
Publications and source records attributed to G B Elion.
Combination chemotherapy in vitro exploiting glutamine metabolism of human glioma and medulloblastoma.
The human glioma-derived cell line D-54 MG and the human medulloblastoma-derived cell line TE-671 have been shown to be sensitive in culture to the pharmacological interference with glutamine metabolism by acivicin, 6-diazo-5-oxo-L-norleucine, and methionine sulfoximine. Using as a guide the multiple contributions of glutamine to the biosynthesis of proteins, purines, and pyrimidines, we now have identified six additional antimetabolites active against these lines in vitro at clinically relevant concentrations. The 50% growth-inhibitory levels of the drugs against D-54 MG in 6-day continuous exposure experiments were: L-asparaginase, 0.057 IU/ml; 5-fluorouracil, 0.5 micrograms/ml; 6-mercaptopurine, 0.8 micrograms/ml; actinomycin D, 0.0007 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 2.3 micrograms/ml; and 5-azacytidine, 0.2 micrograms/ml (3-day exposure. The corresponding 50% growth-inhibitory values in TE-671 were: L-asparaginase, 0.54 IU/ml; 5-fluorouracil, 1.5 micrograms/ml; 6-mercaptopurine, 4.7 micrograms/ml; actinomycin D, 0.00044 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 4.5 micrograms/ml; and 5-azacytidine, 0.49 micrograms/ml. Dipyridamole up to 10 micrograms/ml was inactive against both lines. The isobologram method was used to evaluate the effectiveness of several two-drug combinations which were biochemically designed. The sums of the optimal fractional inhibitory concentrations for the pairs were: acivicin plus L-asparaginase, 0.14; acivicin plus methionine sulfoximine, 0.40; 6-diazo-5-oxo-L-norleucine plus methionine sulfoximine, 0.60; acivicin plus 6-mercaptopurine, 1.0, all in TE-671; and acivicin plus 5-fluorouracil, 0.79, in D-54 MG. Our findings suggest that an antimetabolite regimen exploiting glutamine sensitivity might improve the chemotherapy of some human gliomas and medulloblastomas.
Antimetabolites, Antineoplastic↗
Mercaptopurine "bioavailability".
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Biological Availability↗
Metabolic studies of high doses of allopurinol in humans.
In animals and in humans given high doses of allopurinol, the oxidation of allopurinol to oxipurinol is inhibited, resulting in a higher proportion of unchanged allopurinol and of allopurinol riboside in plasma and urine than is seen at low doses. The dose which produces this inhibition of allopurinol oxidation is higher in rodents than in man or in the dog. Urinary orotate and orotidine increased in proportion to the dose of allopurinol. These increased levels of orotate would be expected to compete more effectively with 5-fluorouracil for conversion to a nucleotide by orotate phosphoribosyltransferase. Since allopurinol and allopurinol riboside are active against leishmaniae in vitro, it may be possible to attain therapeutic levels of allopurinol and allopurinol riboside in vivo by using high doses of allopurinol.
Allopurinol↗
Identification of small DNA fragments synthesized in herpes simplex virus-infected cells in the presence of acyclovir.
The effect of acyclovir on DNA synthesized in cells infected with herpes simplex virus type 1 was examined. DNA that was synthesized in infected cells in the presence of acyclovir during a short pulse with [3H]thymidine remained near the top of an alkaline sucrose gradient after centrifugation. The sedimentation characteristics of labeled DNA were not changed after chasing in isotope-free medium. The slowly sedimenting DNA was identified as viral in origin by hybridization to purified herpes simplex virus nucleocapsid DNA. When cells were infected with acyclovir-resistant virus containing mutations in the polymerase gene, the viral DNA synthesized in the presence of acyclovir was chased into high-molecular-weight DNA. These findings are consistent with chain termination of herpes simplex virus DNA in virus-infected cells.
Acyclovir↗
Effects of acyclovir and its metabolites on hypoxanthine-guanine phosphoribosyltransferase.
Acyclovir [9-(2-hydroxyethoxymethyl)guanine], a clinically useful anti-herpesvirus agent, was a weak inhibitor (Ki = 190 microM) of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from human erythrocytes. Nevertheless, this acyclic nucleoside analog was a more effective inhibitor than were its natural counterparts, guanosine (Ki = 1400 microM) and deoxyguanosine (Ki = 570 microM). The two oxidized metabolites of acyclovir, 9-carboxymethoxymethylguanine (Ki = 720 microM) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine (Ki greater than 2000 microM), were less inhibitory than was the parent drug. None of the phosphorylated metabolites of acyclovir was as potent an inhibitor of HGPRTase as was GMP (Ki = 4 microM). However, the Ki value for acyclovir monophosphate was similar to that of dGMP (12 microM). The Ki values for acyclovir diphosphate (8.3 microM) and triphosphate (30 microM) were less than those for dGDP (110 microM) and dGTP (140 microM). The levels of these phosphate esters of acyclovir in cultured monkey kidney (Vero) and human embryo fibroblast (WI38) cells exposed to therapeutic levels of the drug were well below the observed Ki values. However, in herpesvirus-infected WI38 cells the levels of the phosphate esters of acyclovir were high enough potentially to inhibit the enzyme. Although inhibition of this enzyme by the phosphorylated metabolites of acyclovir may occur in these infected cells, concentrations of the drug very much higher than the EC50 concentration were required to achieve inhibitory levels. It is, therefore, unlikely that this inhibition contributes significantly to the antiviral activity.
Acyclovir↗
Ribonucleotide reductase induced by herpes simplex type 1 virus. Characterization of a distinct enzyme.
The ribonucleotide reductase induced by herpes simplex virus type 1 (HSV-1) was purified in high yield from serum-starved baby hamster kidney (BHK-21) cells infected with HSV-1 (strain H-29). The enzyme preparation was essentially free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could cause significant depletion of substrates. The HSV-1-induced enzyme was assayed in 0.2 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Na at the pH optimum of 8.1 and the optimal dithiothreitol concentration of 10 mM. Nucleoside diphosphates were the substrates of this enzyme. The HSV-1-induced ribonucleotide reductase was inhibited by anions. EDTA also inhibited the enzyme and this inhibition was not reversed by the addition of FeCl2. Hydroxyurea acted as a noncompetitive inhibitor versus CDP reduction (Kii = 1.3 mM Kis = 2.4 mM). The enzyme was inhibited by either free Mg2+ or free ATP. However, it was neither inhibited nor activated by the ATP X Mg complex. Reduction of either CDP or ADP was only weakly inhibited by dATP, dTTP, dGTP, and dCTP. No activation of this enzyme by these compounds was observed. The V'm values for reduction of CDP, UDP, and ADP were similar, while the GDP V'm was 2-fold greater. The K'm values were 80, 12, 1.2, and 0.65 microM for UDP, ADP, GDP, and CDP, respectively. The K'm values for CDP, GDP, and ADP were the lowest values observed for any ribonucleotide reductase. Each ribonucleoside diphosphate substrate competitively inhibited the reduction of each other substrate. The K'is values obtained for inhibition by a given ribonucleoside diphosphate were similar to the K'm value obtained for that compound as a substrate. Kinetic analysis of the combined rate of product formation when both CDP and ADP were simultaneously present as substrates produced patterns that were consistent with reduction at a common catalytic site. The 2'-deoxynucleoside diphosphate products were also competitive inhibitors versus the substrates. The K'is values versus CDP reduction were 310, 140, 9, and 5 microM for dUDP, dADP, dGDP, and dCDP, respectively. Similar K'is values were obtained when ADP was the substrate. All of these data are most consistent with the hypothesis that the HSV-1-induced ribonucleotide reductase catalyzes the reduction of all substrates at a common site. The apparent lack of significant allosteric modulation of HSV-1-induced ribonucleotide reductase, its kinetic behavior, and its low K'm for CDP, GDP, and ADP clearly differentiate this enzyme from other ribonucleotide reductases.
Adenosine Diphosphate↗
Inhibition by acyclovir of herpes simplex virus type 2 morphologically transformed cell growth in tissue culture and tumor-bearing animals.
Rat embryo fibroblasts (REF) morphologically transformed by herpes simplex virus type 2 (HSV-2) and tumor-derived cells were tested for ability to grow in the presence of 9-(2-hydroxyethoxymethyl) guanine (acyclovir). Results indicated that the effective dose of acyclovir (ACV) required to inhibit HSV-2-transformed and tumor-derived cell growth by 50% (ED50) compared to mock-treated control cells averaged 15 to 75 micrograms/ml. In contrast, the ED50 of acyclovir was more than HEp-2 cells. HSV-2-transformed and tumor-derived cells after both low (less than 30) and high (greater than 30) serial passages expressed detectable levels of the virus-coded thymidine kinase (TK) measured in cell extracts by serum neutralization assay. HSV-2-transformed or tumor-derived cells converted two- to ten-fold more acyclovir to phosphorylated forms than nontransformed REF cells. Preliminary data showed that the drug inhibited tumor development in newborn syngeneic rats inoculated with HSV-2-transformed cells. The inhibitory activity of acyclovir and presence of low levels of HSV-2 TK activity appeared to correlate.
Acyclovir↗
Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 3. Synthesis and activity of some nucleosides of 4-[(arylalkenyl)thio]pyrazolo[3,4-d]pyrimidines.
Ribonucleosides of 4-(alkylthio)-1H-pyrazolo[3,4-d]pyrimidines have been shown to be useful anticoccidial agents [Krenitsky, T. A.; Rideout, J. L.; Koszalka, G. W.; Inmon, R. B.; Chao, E. Y.; Elion, G. B.; Latter, V. S.; Williams, R. B. J. Med. Chem. 1982, 25, 32. Rideout, J. L.; Krenitsky, T. A.; Elion, G. B. U.S. Patent 4299 283, 1981]. In that study, the unsaturated 4-allylthio and 4-crotylthio derivatives (19 and 20) were shown to be more active in vivo against Eimeria tenella than their saturated congeners; therefore, some unsaturated (arylalkyl)thio derivatives were synthesized and investigated as anticoccidial agents. The novel compounds in this study (2 to 18) were prepared by the alkylation of 4-mercapto-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidine (1), which was prepared by an enzymatic method. The (E)-4-cinnamylthio derivative (2) and the 5'-monophosphate (18) were the most active compounds against E. tenella in vivo. None of the analogues with substituents in the aryl moiety (3 to 13) was more active than 2 in vivo. The geometry about the double bond was important, since the (Z)-4-cinnamylthio derivative (14) was inactive both in vitro and in vivo. The 4-(3-phenylpropynyl)thio and 4-(5-phenyl-2,4-pentadienyl)thio derivatives (15 and 16) were at least as active as 2 in vitro; however, they were less active than 2 in vivo. Compound 2 was effective in vivo against E. tenella, E. necatrix, E. maxima, and E. brunetti; these species of Eimeria were controlled when 2 was given in the diet at levels upt to 100 ppm. Infections in vivo due to E. acervulina were controlled by 2 only at about 800 ppm. The broad spectrum of anticoccidial activity shown by 2 represents a significant improvement over the activities reported for related compounds [Krenitsky, T. A.; Rideout, J. L.; Koszalka, G. W.; Inmon, R. B.; Chao, E. Y.; Elion, G. B.; Latter, V. S.; Williams, R. B. J. Med. Chem. 1982, 25, 32].
Animals↗
The biochemistry and mechanism of action of acyclovir.
Acyclovir, 9-(2-hydroxyethoxymethyl)guanine, is an acyclic nucleoside analogue which has a high activity and selectivity for herpes viruses, particularly herpes simplex viruses types 1 and 2 and varicella zoster virus. This selectivity is due to the initial activation of the drug by phosphorylation by a herpes virus-specified thymidine kinase. Normal cellular enzymes do not phosphorylate acyclovir to any significant degree. Acyclovir monophosphate is subsequently converted to a triphosphate which is a more potent inhibitor of herpes virus DNA polymerases than of cellular DNA polymerases. The relationship between the amount of acyclovir triphosphate formed and its inhibition constant (Ki) for the particular viral or cellular DNA polymerase is predictive of the inhibitory activity of acyclovir on DNA replication.
Acyclovir↗
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.
Acyclovir↗
Mechanism of action and selectivity of acyclovir.
Acyclovir, an acrylic purine nucleoside analog, is a highly potent inhibitor of herpes simplex virus (HSV), types 1 and 2, and varicella zoster virus, and has extremely low toxicity for the normal host cells. This selectivity is due to the ability of these viruses to code for a viral thymidine kinase capable of phosphorylating acyclovir to a monophosphate; this capability is essentially absent in uninfected cells. The acyclovir monophosphate (acyclo-GMP) is subsequently converted to acyclovir triphosphate (acyclo-GTP) by cellular enzymes. Acyclo-GTP persists in HSV-infected cells for many hours after acyclovir is removed from the medium. The amounts of acyclo-GTP formed in HSV-infected cells are 40 to 100 times greater than in uninfected Vero cells. Acyclo-GTP acts as a more potent inhibitor of the viral DNA polymerases than of the cellular polymerases. The DNA polymerases of HSV-1 and HSV-2 also use acyclo-GTP as a substrate and incorporate acyclo-GMP into the DNA primer-template to a much greater extent than do the cellular enzymes. The viral DNA polymerase binds strongly to the acyclo-GMP-terminated template, and in thereby inactivated.
Acyclovir↗
Species differences in the disposition of acyclovir.
The disposition of acyclovir was investigated in several species. The drug was well distributed into all the tissues, including the brain, in mice and rats. Binding of acyclovir to plasma proteins was 36 percent or lower. After intravenous bolus dosing (20 mg/kg) in dogs, the plasma acyclovir concentration-time profile, determined by radioimmunoassay, showed a biphasic decline with a half-life in the elimination phase of 2.3 +/- 0.1 hours. The volume of distribution (Vd beta) of 1.2 +/- 0.2 liters/kg indicated distribution of drug into the tissues. Marked species differences were observed in the gastrointestinal absorption and biotransformation of acyclovir. Oral dosing produced better absorption in dogs and mice than in rats and rhesus monkeys. More than 95 percent of the radioactivity in the urine derived from a 14C-acyclovir parenteral dose was recovered as the unchanged drug in mice, rats, and dogs. Minor urinary metabolites were characterized by high-performance liquid chromatography as 9-carboxymethoxymethylguanine (CMMG) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine. In other species--guinea pig, rabbit, and rhesus monkey--up to 40 percent of the radioactivity in the urine consisted of these metabolites.
Absorption↗
Effect of acyclovir combined with other antiherpetic agents on varicella zoster virus in vitro.
The sensitivity of varicella zoster virus (VZV) strain Ellen to acyclovir in combination with other antiherpetic agents in vitro has been examined by the plaque-reduction and infectious center assay methods. (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), trifluorothymidine (TFT), or 5-iodo-2'-deoxyuridine (IdUrd) produced, in general, additive antiviral activity when examined by either assay method. Acyclovir in combination with phosphonoacetate (PAA) resulted in additive effects as measured by the plaque-reduction assay. The combination of acyclovir with 9-beta-D-arabinofuranosyladenine (ara-A) gave generally additive or occasionally synergistic antiviral activity when tested in MRC-5 or WI-38 cells respectively, regardless of assay method.
Acyclovir↗
Activation and antiviral effect of acyclovir in cells infected with a varicella-like simian virus.
Acyclovir inhibited the replication of a varicella-like simian virus (DHV-1) in cell culture (Vero cells) with an ED50 of 38 +/- 2 microM. The activation of acyclovir in this cell culture system was compared with that in the cell system with human varicella zoster virus (VZV). Extracts of cells infected with DHV-1 catalyzed the phosphorylation of acyclovir. The phosphorylation was inhibited by dThd, suggesting the catalyst was a dThd kinase. Electrophoresis of cytosol fractions on polyacrylamide gels corroborated the existence of a virus-associated dThd kinase. This enzyme copurified with an acyclovir-phosphorylating activity. The enzyme catalyzed the phosphorylation of acyclovir at a greater relative rate than that with the VZV enzyme, but with a higher apparent Km value for acyclovir. The relative efficiencies for the two enzymes with acyclovir were similar. Anabolic studies with cells infected with DHV-1 and incubated with [14C]acyclovir indicated that triphosphate of acyclovir did accumulate. The results indicate that acyclovir is activated in cells infected with DHV-1 in a manner similar to that in cells infected with VZV.
Acyclovir↗
Effect of acyclovir on various murine in vivo and in vitro immunologic assay systems.
In two in vitro tests, lymphocyte-mediated cytotoxicity and neutrophil chemotaxis, acyclovir showed no inhibitory effects at concentrations as high as 600 microM. The compound inhibited rosette formation with nonimmune mouse lymphocytes in vitro by approximately 50 percent at 15.8 microM. The significance of this inhibition is unclear. In four in vivo tests in mice which measured humoral and cell-mediated immunity (complement-dependent cellular cytotoxicity, complement-independent cellular cytotoxicity, delayed hypersensitivity and graft versus host reaction) acyclovir showed no inhibitory effects at single doses up to 200 mg/kg given on day 2 after antigenic stimulation. Four daily doses of acyclovir at 50 mg/kg per day had no effect on the numbers of hemolytic IgM antibody-forming cells in the spleen when assayed on day 4. At the higher dosage of 100 mg/kg per day for four days, there was a slight reduction in the numbers of these cells. There was no significant decrease in hemagglutinin or hemolysin antibody titers after four daily doses of acyclovir up to 200 mg/kg.
Acyclovir↗
Selection and preliminary characterization of acyclovir-resistant mutants of varicella zoster virus.
A series of acyclovir-resistant mutants of varicella zoster virus (VZV) were selected in vitro by serial passage of VZV-infected human fibroblasts in increasing drug concentrations, or by continuous exposure of cultures infected at high multiplicity to 100 microM acyclovir. The in vitro susceptibility of these mutants to several antiherpetic agents was measured by the plaque-reduction assay. The capacity of extracts of cells infected with these mutants to phosphorylate acyclovir was examined and compared with that of their acyclovir-sensitive parent strains. Based on these studies, VZV could be shown to acquire resistance to acyclovir through diminished acyclovir phosphorylation. This was presumable due to loss of viral specific thymidine kinase (TK) function. Two acyclovir-resistant mutants remained TK competent but demonstrated phenotypic changes in sensitivity to antiviral agents known to act at the herpes simplex virus (HSV)-specific DNA polymerase level. These results suggest that the resistance of VZV to acyclovir results from qualitative or quantitative alterations in the virus-specified TK or DNA polymerase.
Acyclovir↗
A radioimmunoassay for herpes simplex virus (HSV) thymidine kinase.
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Cell Transformation, Viral↗