Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography-tandem mass spectrometry.

An ion-paring HPLC-MS-MS method with positive ion mode electrospray ionization has been developed to simultaneously quantify Ziagen, carbovir monophosphate, carbovir diphosphate and carbovir triphosphate. N',N'-Dimethylhexylamine was used as the ion-pairing agent. The presence of this ion-pairing agent allowed the retention and separation of the four compounds on a reversed-phase HPLC column as well as the detection of the nucleotides with positive ion mode electrospray ionization. The limits of detection were found to be better than 25 nM for all the analytes. Calibration curves of the analytes showed excellent linearity over the range of 25 nM to 5 microM. The relative standard deviations and accuracies for replicate analyses of quality control samples were less than 15%. The method has been successfully applied to the analysis of these compounds in human liver cells treated with Ziagen.

Acyclovir is phosphorylated by the human cytomegalovirus UL97 protein.

Acyclovir (ACV) has shown efficacy in the prophylactic suppression of human cytomegalovirus (HCMV) reactivation in immunocompromised renal transplant patients without the toxicity associated with ganciclovir (GCV). The HCMV UL97 gene product, a protein kinase, is responsible for the phosphorylation of GCV in HCMV-infected cells. This report provides evidence for the phosphorylation of ACV by UL97. Anabolism studies with the HCMV wild-type strain AD169 and with recombinant mutants derived from marker transfer experiments performed by using mutant UL97 DNA from both clinical isolates and a laboratory-derived strain resistant to GCV showed that mutations in the UL97 gene cripple the ability of recombinant virus-infected cells to anabolize both GCV and ACV. These mutant UL97 recombinant viruses were less susceptible to both GCV and ACV than was the wild-type strain. A recombinant herpes simplex virus type 1 strain, in which the thymidine kinase gene is deleted and the UL13 gene is replaced with the HCMV UL97 gene, was able to induce the phosphorylation of ACV in infected cells. Finally, purified UL97 phosphorylated both GCV and ACV to their monophosphates. Our results indicate that UL97 promotes the selective activity of ACV against HCMV.

N-Phenylamidines as selective inhibitors of human neuronal nitric oxide synthase: structure-activity studies and demonstration of in vivo activity.

Selective inhibition of the neuronal isoform of nitric oxide synthase (NOS) compared to the endothelial and inducible isoforms may be required for treatment of neurological disorders caused by excessive production of nitric oxide. Recently, we described N-(3-(aminomethyl)benzyl)acetamidine (13) as a slow, tight-binding inhibitor, highly selective for human inducible nitric oxide synthase (iNOS). Removal of a single methylene bridge between the amidine nitrogen and phenyl ring to give N-(3-(aminomethyl)phenyl)acetamidine (14) dramatically altered the selectivity to give a neuronal selective nitric oxide synthase (nNOS) inhibitor. Part of this large shift in selectivity was due to 14 being a rapidly reversible inhibitor of iNOS in contrast to the essentially irreversible inhibition of iNOS observed with 13. Structure-activity studies revealed that a basic amine functionality tethered to an aromatic ring and a sterically compact amidine are key pharmacophores for this class of NOS inhibitors. Maximal nNOS inhibition potency was achieved with N-(3-(aminomethyl)phenyl)-2-furanylamidine (77) (Ki-nNOS = 0.006 microM; Ki-eNOS = 0.35 microM; Ki-iNOS = 0.16 microM). Finally, alpha-fluoro-N-(3-(aminomethyl)phenyl)acetamidine (74) (Ki-nNOS = 0. 011 microM; Ki-eNOS = 1.1 microM; Ki-iNOS = 0.48 microM) had excellent brain penetration and inhibited nNOS in a rat brain slice assay as well as in the rat brain (cerebellum) in vivo. Thus, N-phenylamidines should be useful in validating the role of nNOS in neurological disorders.

Partial substitution of the functions of the herpes simplex virus 1 U(L)13 gene by the human cytomegalovirus U(L)97 gene.

The predicted amino acid sequence of the human cytomegalovirus U(L)97 protein bears partial homology to the herpes simplex virus 1 U(L)13 protein, especially in regions that are homologous to conserved domains characteristic of protein kinases. Earlier studies showed that U(L)13 mediated the posttranslational processing of several herpes simplex virus 1 proteins including ICP22. Whereas no kinase activity has been specifically attributed to U(L)13, it has been shown that U(L)97 can phosphorylate ganciclovir. To examine whether U(L)97 can substitute for U(L)13, we constructed a herpes simplex virus 1 recombinant virus, R4970, in which U(L)13 was replaced by U(L)97 and in addition, the thymidine kinase gene was deleted. Characterization of this recombinant virus showed the following: (1) The recombinant virus grew as well as the wild-type virus in BHKTK+ cells, which restricted the growth of the U(L)13 virus. (2) U(L)97 could partially mediate the posttranslational modification of HSV-1 ICP22. This modification correlated with the restoration of the amounts of ICP0 and U(S)11 proteins, which were down regulated in the U(L)13- virus-infected cells. (3) The recombinant virus was sensitive to ganciclovir in Vero- and KHOS-infected cells but not in the 143 thymidine kinase minus cells derived from KHOS cells. Vero cells infected with this recombinant virus phosphorylated ganciclovir. We conclude that U(L)97 partially compensates for U(L)13 functions.

Purification and characterization of a rat liver enzyme that hydrolyzes valaciclovir, the L-valyl ester prodrug of acyclovir.

Valaciclovir is an oral prodrug of the antiherpetic agent acyclovir. An enzyme that hydrolyzes valaciclovir to acyclovir, valaciclovir hydrolase (VACVase), was purified from rat liver and characterized. VACVase was a basic (pI 9.4) protein associated with mitochondria. It was monomeric and had a molecular mass of 29 kDa. Amino acid sequences of six VACVase peptides, including its NH2 terminus (13 amino acids) and accounting for approximately 20% of its complete sequence, were not found in the SwissProt protein data base. VACVase hydrolyzed other amino acid esters of acyclovir in addition to valaciclovir (kcat/Km = 58 mM-1 s-1), with a preference for the L-alanyl (kcat/Km = 226 mM-1 s-1) and L-methionyl (kcat/Km = 200 mM-1 s-1) esters. It did not hydrolyze other types of esters or numerous di- and tripeptides and aminoacyl-beta-naphthylamides. Hydrolysis of valaciclovir by VACVase was not inhibited by amastatin, antipain, aprotinin, bestatin, chymostatin, E-64, EDTA, ebelactone A, ebelactone B, elastatinal, leupeptin, pepstatin, or phosphoramidon. It was neither inhibited nor activated by Ca2+, Co2+, Mg2+, Mn2+, or Zn2+. Therefore, this enzyme is not a typical esterase or peptidase and, to our knowledge, it has not been described previously. Its physiological function is not known; however, it may play a significant role in the biotransformation of valaciclovir to acyclovir.

Metabolic disposition of the acyclovir prodrug valaciclovir in the rat.

The prodrug valaciclovir demonstrated good oral absorption, rapid distribution and elimination, and extensive biotransformation to acyclovir in male CD rats. The mean urinary excretion of radioactivity following oral and intravenous administration of [8-14C]valaciclovir (25 mg/kg) was 65% and 95% of the dose, respectively. Acyclovir was the predominant radiolabeled urinary metabolite accounting for 57% and 65% of the dose, respectively, with valaciclovir accounting for 2% and 23% of the dose, respectively. Radioactivity from an oral dose of [8-14C]valaciclovir (10 mg/kg) was distributed to all 14 tissues examined 20 min postdose. The stomach, small intestine, kidney, liver, lymph nodes, and skin received the highest exposure to radioactivity, and the brain received the lowest exposure. Radioactivity in most tissues cleared by 24 hr postdose, and that in urine and feces accounted for essentially all of the administered dose by 48 hr postdose. Acyclovir derived from valaciclovir (10 and 25 mg/kg) exhibited dose-independent pharmacokinetics. The Cmax and AUC for acyclovir achieved with orally administered valaciclovir were 8- and 4-fold higher, respectively, than those estimated for an equivalent dose of acyclovir. The half-life of acyclovir derived from valaciclovir was approximately 1 hr, whereas that of valaciclovir was approximately 7 min. Valaciclovir was more efficiently metabolized when administered orally, indicating first-pass intestinal and/or hepatic metabolism. Rapid hydrolysis of valaciclovir in rat liver and intestinal homogenates further suggested the significance of presystemic metabolism. These studies indicate that valaciclovir is an efficient acyclovir prodrug particularly suited for oral administration.

Metabolic fate and pharmacokinetics of the acyclovir prodrug valaciclovir in cynomolgus monkeys.

Valaciclovir, the L-valyl ester of acyclovir (ZOVIRAX), demonstrated good oral absorption and nearly complete conversion to acyclovir in cynomolgus monkeys, indicating its suitability as an orally administered prodrug. The major urinary metabolites of [8-14C]valaciclovir, administered orally (10 and 25 mg/kg) or intravenously (10 mg/kg) to male monkeys, were acyclovir (46%-59% of urinary radioactivity), 8-hydroxyacyclovir (25%-30%), and 9-(carboxymethoxymethyl)guanine (CMMG) (11%-12%). Following oral and intravenous dosing, intact prodrug accounted for only 0.5% and 6% of urinary radioactivity, respectively. Dose-independent kinetics were observed for acyclovir derived from orally administered [8-14C]valaciclovir at the 10 and 25 mg/kg dose levels, with both AUC (24 and 60 microM.hr, respectively) and Cmax (8 and 23 microM, respectively) increasing nearly in proportion to the dose. Acyclovir was present in plasma at all sampling times (5 min to 7 hr postdose) after both oral doses, whereas the prodrug was not detected following either oral dose. The elimination of acyclovir after oral administration was monophasic, with an apparent half-life of 1.3-1.5 hr. Similar to acyclovir, both 8-hydroxyacyclovir and CMMG demonstrated dose-independent kinetics with apparent elimination half-lives of 1-1.6 hr. Intravenously administered [8-14C]valaciclovir (10 mg/kg) was rapidly converted to acyclovir, with the elimination half-life of acyclovir (0.9 hr) being 1.5-fold that of the prodrug (0.6 hr). The oral bioavailability of acyclovir derived from valaciclovir in cynomolgus monkey was 67 +/- 13%, representing a significant improvement over the limited bioavailability after acyclovir administration to primates.

Selective anabolism of 6-methoxypurine arabinoside in varicella-zoster virus-infected cells.

6-Methoxypurine arabinoside (ara-M) is a highly selective inhibitor of varicella-zoster virus (VZV). It belongs to a class of purine arabinosides whose anti-VZV activity in vitro correlates with substrate utilization by the VZV-encoded thymidine kinase (TK) (D. R. Averett, G. W. Koszalka, J. A. Fyfe, G. B. Roberts, D. J. M. Purifoy, and T. A. Krenitsky, Antimicrob Agents Chemother. 35:851-857, 1991). In this study, the mechanism of action of ara-M was explored. VZV-infected human fibroblasts selectively accumulated ara-M and its phosphorylated metabolites, whereas in uninfected fibroblasts or in those infected with a TK-deficient strain of VZV, there was virtually no cellular uptake of ara-M. The major intracellular metabolite of ara-M in VZV-infected cells was identified as the triphosphate of adenine arabinoside (ara-ATP). Appreciable levels of ara-ADP, ara-AMP, and ara-MMP were also detected. However, di- or triphosphorylated forms of ara-M were not detected. Moreover, in VZV-infected cells, the concentrations of ara-ATP which accumulated in the presence of ara-M were up to eightfold higher than those generated with ara-A itself. In contrast, in uninfected cells, the levels of ara-ATP which accumulated in the presence of ara-M were barely detectable. Clearly, Ara-M activation was dependent on the activity of the virus-encoded TK, while ara-A anabolism resulted primarily from the activity of host cell enzymes. Therefore, ara-M selectively generates the DNA polymerase inhibitor ara-ATP in the VZV-infected cell.

Anabolic pathway of 6-methoxypurine arabinoside in cells infected with varicella-zoster virus.

6-Methoxypurine arabinoside (ara-M) exhibits potent activity against varicella-zoster virus (VZV) as a result of ara-M's anabolism to the triphosphate of adenine arabinoside (ara-ATP) in VZV-infected cells. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) enhanced the formation of ara-ATP by inhibiting ara-M demethoxylation. In contrast, deoxycoformycin and coformycin, inhibitors of both adenosine deaminase and AMP deaminase, blocked the formation of ara-ATP and reversed the anti-VZV activity of ara-M. These results indicate that after the initial phosphorylation of ara-M by the VZV-coded thymidine kinase, the monophosphate is demethoxylated by AMP deaminase to form ara-IMP, which is converted to ara-ATP by the sequential actions of the cellular adenylosuccinate synthetase, adenylosuccinate lyase, and nucleotide kinases.

Metabolic disposition and pharmacokinetics of the antiviral agent 6-methoxypurine arabinoside in rats and monkeys.

The metabolism and pharmacokinetics of 6-methoxypurine arabinoside (ara-M), a potent and selective inhibitor of varicella-zoster virus, were investigated in rats and monkeys. In Long Evans rats, orally administered [8-14C]ara-M (10 mg/kg) was well absorbed but extensively metabolized to hypoxanthine arabinoside (ara-H), hypoxanthine, xanthine, uric acid, and allantoin. Only 4% of an oral dose was recovered in the urine as unchanged drug, compared with 40% of an intravenous dose, indicating significant presystemic metabolism. Pretreatment of rats with 1-aminobenzotriazole, an inhibitor of cytochrome P-450, did not alter this metabolism. Pretreatment with deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride, inhibitors of adenosine deaminase, resulted in a marked decrease in ara-M metabolism, indicating that adenosine deaminase plays a major role in the biotransformation of ara-M. In cynomolgus monkeys, [8-14C]ara-M (10 mg/kg) administered intravenously or orally was extensively metabolized to ara-H. Several minor urinary metabolites were detected in both rats and monkeys. However, adenine arabinoside was not found in urine or plasma from either rats or monkeys after administration of ara-M, except for a very low level detected in the urine of rats pretreated with deoxycoformycin. The elimination half-lives of intravenously administered ara-M in rats and monkeys were 29 and 45 min, respectively. The corresponding half-lives of the primary metabolite, ara-H, were 44 min and 2.3 h. Plasma profiles of orally administered ara-M in both rats and monkeys demonstrated the poor oral bioavailability of this arabinoside. The results of these studies indicate that ara-M is not well suited for oral administration because of extensive presystemic metabolism.

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.

Activity of 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine in the treatment of cytomegalovirus pneumonia.

Ten marrow transplant recipients with biopsy-proven cytomegalovirus pneumonia were treated with the acyclic nucleoside analog 9-[2-hydroxy-1-(hydroxymethyl) ethoxymethyl]guanine (BW B759U). Viruria and viremia ceased after 4 days of treatment in all patients with cultures initially positive from these sites. Cytomegalovirus was eliminated from respiratory secretions after a median of 8 days. Despite this antiviral effect, only one patient survived the pneumonia. Quantitative cultures of lung tissue before and after treatment confirmed that therapy with BW B759U was associated with substantial antiviral activity, with a mean decrease in viral titers of more than 99.99% after treatment. Neutropenia developed in three patients when mean peak and trough plasma levels exceeded 50 and 10 mu mol/L, respectively, but no other toxicity was seen. BW B759U is the first antiviral agent showing consistent activity against cytomegalovirus in vivo, and it should be evaluated in the earlier management of cytomegalovirus infections after marrow transplantation and in serious cytomegalovirus infections in other immunocompromised patients.