Search

(-)-beta-D-2-Aminopurine dioxolane (APD), (-)-beta-D-2-amino-6-chloropurine dioxolane (ACPD) and dioxolane guanine (DXG) are nucleoside analogues possessing potent activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) in vitro. APD and ACPD are metabolized in vivo to yield DXG. Reversed-phase HPLC analytical methodologies were developed for the simultaneous determination of APD and DXG, and for ACPD and DXG in monkey serum, urine and cerebrospinal fluid (CSF). 2-Fluoro-2',3'-dideoxyinosine (FDDI) served as the internal standard. The extraction recoveries of the nucleoside analogues from serum samples were similar, averaging approximately 90%. The limit of quantitation of the analytical method for serum samples was 0.1 microg/ml for DXG, and 0.25 microg/ml for APD and ACPD. The intra- and inter-day relative standard deviations for each compound at low, medium and high nucleoside concentrations were less than 9.0%. The accuracy of the assay methods was greater than 90% for prodrugs and parent compound. Similar results were observed with urine and CSF samples. Thus, these methods provide sensitive, accurate and reproducible determination of the prodrugs and parent nucleoside in biological samples.

As a part of our efforts to design prodrugs for antiviral nucleosides, 9-(beta-D-arabinofuranosyl)-6-azidopurine (6-AAP) was synthesized as a prodrug for ara-A that utilizes the azide reduction biotransformation pathway. 6-AAP was synthesized from ara-A via its 6-chloro analogue 4. The bioconversion of the prodrug was investigated in vitro and in vivo, and the pharmacokinetic parameters were determined. For in vitro studies, 6-AAP was incubated in mouse serum and liver and brain homogenates. The half-lives of 6-AAP in serum and liver and brain homogenates were 3.73, 4.90, and 7.29 h, respectively. 6-AAP was metabolized primarily in the liver homogenate microsomal fraction by the reduction of the azido moiety to the amine, yielding ara-A. However, 6-AAP was found to be stable to adenosine deaminase in a separate in vitro study. The in vivo metabolism and disposition of ara-A and 6-AAP were conducted in mice. When 6-AAP was administered by either oral or intravenous route,the half-life of ara-A was 7-14 times higher than for ara-A administered intravenously. Ara-A could not be found in the brain after the intravenous administration of ara-A. However, after 6-AAP administration (by either oral or intravenous route), significant levels of ara-A were found in the brain. The results of this study demonstrate that 6-AAP is converted to ara-A, potentially increasing the half-life and the brain delivery of ara-A. Further studies to utilize the azide reduction approach on other clinically useful agents containing an amino group are in progress in our laboratories.

A series of the anti-HIV nucleoside conjugates of either (1-O-alkyl) and thioether (1-S-alkyl) lipids linked by a pyrophosphate diester bond has been synthesized as micelle-forming prodrugs of the nucleosides to improve their therapeutic efficiency. These include AZT 5'-diphosphate-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (1), 3'-azido-2',3'-dideoxyuridine 5'-diphosphate-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (2) 2',3'-dideoxycytidine 5'-diphosphate-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (3), and AZT 5'-diphosphate-rac-1-O-tetradecyl-2-O-palmitoylglycerol (4). The conjugates form micelles by sonication (mean diameters ranging 6.8-55.5 nm). Conjugate 1 protected 80% of HIV-infected CEM cells as low as 0.58 microM and lost the protection at 180 microM due to prevailing cytotoxicity, while the conjugate started to show the cytotoxicity at 100 microM. Pharmacokinetics studies showed a significant increase of half-life values (t1/2) of AZT and AZddU2 (respective t1/2 = 5.69 and 6.5 h) after administration of conjugates 1 and 2, while those after administration of AZT and AZddU were 0.28 and 0.89 h, respectively. The fractions of the prodrugs 1 and 2 converted to the parent compounds AZT and AZddU were 36% and 55%, respectively. The results indicate that AZT and AZddU thioether lipid conjugates 1 and 2 warrant further investigation.

Recently, it was shown that human immunodeficiency virus (HIV)-infected cells preferentially locate in lymphoid tissue early in the course of infection. Therefore, it is important to characterize the disposition of the anti-HIV agents, 3'-azido-3'-deoxythymidine (AZT) and 3'-azido-2', 3'-dideoxyuridine (AZdU), in the lymphatic system. The disposition of AZT and AZdU in serum and neck, axillary, and mesenteric lymph nodes was studied in mice after intravenous, oral, and intraperitoneal administrations of 50 mg/kg doses. Samples were collected at 0.08, 0.5, 1, 2, 3, 4, and 6 hr after dosing and nucleoside concentrations were determined by HPLC. Pharmacokinetic parameters were estimated using noncompartmental analysis. Maximum concentration, half-life, and area under the serum concentration vs. time curve (AUC) obtained from the serum concentration data were similar for both compounds after intravenous and intraperitoneal administrations; however, a difference in oral bioavailability for AZT and AZdU (49% and 76%, respectively) was noted. Patterns of regional distribution in lymph nodes were similar for both drugs; however, the accumulation of AZdU in the various lymph nodes, according to AUC values, was 3-76% greater than that for AZT. The relative exposure re = AUClymph/AUCserum) of both nucleosides exhibited a dependence on route of administration. Intravenous and oral administrations resulted in a greater distribution of nucleoside into axillary lymph nodes, compared with neck and mesenteric lymph nodes. Following intraperitoneal administration, however, distribution was similar in all three regions. AZT and AZdU distribute into the lymphatic system; however, AZdU accumulation was greater than that of AZT.(ABSTRACT TRUNCATED AT 250 WORDS)