Enhanced oral bioavailability of 2'- beta-fluoro-2',3'-dideoxyadenosine (F-ddA) through local inhibition of intestinal adenosine deaminase.

PURPOSE: Intestinal enzyme inhibition may be an effective tool to increase the oral bioavailability of compounds that undergo first-pass intestinal metabolism. However, systemic enzyme inhibition may be undesirable and therefore should be minimized. 2-Beta-fluoro-2',3'-dideoxyadenosine (F-ddA) is an adenosine deaminase (ADA) activated prodrug of 2-beta-fluoro-2',3'-dideoxyinosine (F-ddI) with enhanced delivery to the central nervous system (CNS) that has been tested clinically for the treatment of AIDS. Unfortunately, intestinally localized ADA constitutes a formidable enzymatic barrier to the oral absorption of F-ddA. This study explores various factors involved in inhibitor selection and dosage regimen design to achieve local ADA inhibition with minimal systemic inhibition. METHODS: In situ intestinal perfusions with mesenteric vein cannulation were performed in the rat ileum to determine the lumenal disappearance and venous blood appearance of F-ddA and F-ddI. Coperfusions with the ADA inhibitor erythro9-(2-hydroxy-3-nonyl)adenine [(+)-EHNA] over a range of concentrations were used to monitor inhibitor effects on F-ddA absorption and metabolism. RESULTS: High concentrations of EHNA in coperfusions with F-ddA completely inhibited intestinal ADA, increasing the permeability coefficient of F-ddA by nearly threefold but producing high systemic inhibition of ADA. Mathematical models were utilized to show that in full-length intestinal perfusions an optimal log mean lumenal EHNA perfusate concentration of 0.5 microg/ml could achieve an intestinal bioavailability of 80% with <20% systemic inhibition. CONCLUSIONS: Optimizing local enzyme inhibition may require careful selection of a suitable inhibitor, the dose of the inhibitor, and the inhibitor vs. drug absorption profiles.

Role of brain tissue localized purine metabolizing enzymes in the central nervous system delivery of anti-HIV agents 2'-beta-fluoro-2',3'-dideoxyinosine and 2'-beta-fluoro-2',3'-dideoxyadenosine in rats.

PURPOSE: This study examines the central nervous system (CNS) delivery of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) and 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), acid stable analogues of dideoxyadenosine (ddA) and dideoxyinosine (ddI) having reduced susceptibility to purine salvage pathway enzymes important in the metabolism of ddA and ddI, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), respectively. Their CNS delivery compared to that for ddI provides insight into the role of brain tissue ADA and PNP in these processes. METHODS: Brain and cerebrospinal fluid (CSF) concentration-time profiles were obtained for F-ddI during and after intravenous infusions of F-ddI, and for both F-ddA and F-ddI after F-ddA infusions in normal rats or rats pre-treated with the ADA inhibitor 2'-deoxycoformycin (DCF). Rate constants for CNS entry, efflux and metabolism were estimated by computer fits using plasma concentration-time profiles as the driving force functions. RESULTS: The CNS delivery of F-ddI did not differ significantly from that for ddI. F-ddA, which is more lipophilic than F-ddI, provided higher brain (approximately 8x) and CSF (approximately 11x) concentrations of total dideoxynucleoside (F-ddA and F-ddI) compared to F-ddI. Deamination by brain tissue ADA to form F-ddI reduced CNS levels of intact F-ddA but provided higher brain parenchyma (5x) and CSF/plasma (3x) ratios of F-ddI relative to F-ddI controls. Thus, F-ddA functions in part as a CNS-activated prodrug of F-ddI. DCF pre-treatment inhibited brain tissue ADA, abolishing the prodrug effect, and enhancing F-ddA concentrations in both brain parenchyma (5x) and CSF (6x). CONCLUSIONS: PNP metabolism does not appear to play a role in the low CNS delivery of ddI. On the other hand, deamination of F-ddA by brain tissue ADA is an important process, such that F-ddA functions in part as a CNS-activated prodrug of F-ddI. Enhanced CNS uptake of intact F-ddA can be achieved with ADA inhibition.

Role of altered metabolism in dideoxynucleoside pharmacokinetics. Studies of 2'-beta-fluoro-2',3'-dideoxyinosine and 2'-beta-fluoro-2',3'-dideoxyadenosine in rats.

Enzymes of the purine salvage pathway play an important role in altering the in vivo pharmacokinetics of 2',3'-dideoxypurine nucleosides. This study examines the pharmacokinetics of enzyme-resistant 2'-beta-fluoro analogues of 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxyadenosine (ddA). 2'-beta-Fluoro-2',3'-dideoxyinosine (F-ddI) is an acid-stable analogue of ddI that is highly resistant to purine nucleoside phosphorylase, the principal enzyme in ddI metabolism. 2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable and purine nucleoside phosphorylase-resistant analogue of ddA, is converted in vivo to F-ddI by adenosine deaminase (ADA) but is a much poorer substrate for this enzyme than is ddA. Both F-ddA and F-ddI have been shown to have activity against human immunodeficiency virus in vitro, and F-ddA has been selected by the National Cancer Institute for clinical trials as a new human immunodeficiency virus reverse transcriptase inhibitor. The pharmacokinetics of F-ddI and ddI were compared at equivalent doses in chronically catheterized rats. Because ddI and F-ddI are isosteres having nearly identical lipophilicity, this comparison is likely to reflect primarily metabolic differences. The clearance of F-ddI was substantially reduced, in comparison with that of ddI (27.3 ml/min/kg vs. 90.9 ml/min/kg), resulting in higher systemic concentrations at steady state and prolonged retention of F-ddI after termination of infusions, consistent with a significant metabolic component in the clearance of ddI. Concentrations of F-ddA and F-ddI during and after infusions of F-ddA were determined in both untreated and 2'-deoxycoformycin-pretreated rats. In untreated rats, F-ddA was rapidly eliminated from plasma, with a total clearance of 68.5 ml/kg/min. Metabolic clearance of F-ddA to F-ddI accounted for 58% of this value (bioconversion t1/2 = 9.8 +/- 1.9 min). Pretreatment with 2'-deoxycoformycin, an ADA inhibitor, reduced the clearance of F-ddA to 23.8 ml/min/kg, leading to 2.9 +/- 0.4-fold higher steady-state plasma concentrations of F-ddA, in agreement with a 2.5-fold enhancement predicted by a compartmental model assuming complete ADA inhibition.

Acute cardiotoxicity of the Anti-HIV dideoxynucleoside, F-ddA, in the rat.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable, purine dideoxynucleoside with in vitro anti-HIV activity, has been selected by the NCI as a clinical trial candidate. A recent report that high, single doses of F-ddA produce cardiotoxicity in rats prompted the present investigation whose objective was to quantitate this effect and establish a relationship between this toxicity and F-ddA plasma concentrations. Microscopic examination of cardiac tissues for degenerative lesions established the effects of F-ddA and ddA on three iv schedules [daily x 1(2.5-250 mg/kg); daily x 5(125, 250 mg/kg), and BID x 1 (250 mg/kg)] as well as one oral schedule [BID x 1 (500 mg/kg) using 8- to 12-week old female Sprague-Dawley rats. For both F-ddA and ddA, the group mean severity of the cardiac lesions was dose-dependent and proportional to the measured plasma concentrations of the undeaminated parent drugs. F-ddI and ddI, were essentially nontoxic in this study (iv, 250 mg/kg, daily x 1 and daily x 5), since plasma concentrations exceeding 2 mM produced only minimal cardiac lesions. The cardiomyopathy of F-ddA was minimal to mild for all iv doses except 250 mg/kg (daily x 1) and usually was greater than that of ddA at any given dose. This is a consequence of the fact that F-ddA is deaminated 20 times more slowly than ddA, resulting in higher plasma concentrations of F-ddA relative to ddA at any given time for any given dose. Neither F-ddA nor ddA was more cardiotoxic on a repeated iv schedule (daily x 5) than when administered only once, suggesting that rat cardiotoxicity is related Cmax rather than total exposure. In this most sensitive species, the formation of cardiac lesions above the background level is associated with i.v. F-ddA administration when the F-ddA plasma concentration approaches 300 microM, 30-50 times the anticipated therapeutic level in humans.

Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol.

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of dideoxypurine nucleoside analogs in plasma and cerebrospinal fluid of rhesus monkeys.

The pharmacokinetics of 2',3'-dideoxyadenosine (ddA), didanosine, 2',3'-dideoxyguanosine (ddG), and 6-halogenated prodrugs of ddG, 6-chloro-ddG and 6-iodo-ddG, in plasma and cerebrospinal fluid (CSF) were studied in a non-human primate model. ddA was rapidly and completely deaminated to didanosine, such that didanosine concentration profiles in plasma and CSF were identical following administration of ddA and didanosine. The mean clearance of didanosine was 0.50 liters/h/kg, the terminal half-life was 1.8 h, and the CSF-to-plasma ratio was 4.8%. The disposition of ddG was similar, with a clearance of 0.70 liters/h/kg and a half-life of 1.7 h. The adenosine deaminase-mediated conversion of the 6-halogenated-ddG prodrugs to ddG was rapid but incomplete (48% for 6-chloro-ddG and 29% for 6-iodo-ddG). The CSF-to-plasma ratios of ddG with equimolar doses of ddG, 6-chloro-ddG, and 6-iodo-ddG were 8.5, 24, and 17%, respectively, but the actual ddG exposures in CSF (area under the CSF concentration-time curve) were comparable for ddG (12.1 microM.h) and the 6-halogenated-ddG prodrugs (18.8 microM.h for 6-chloro-ddG, 9.3 microM.h for 6-iodo-ddG).6-Chloro-ddG was not detectable in plasma or CSF, and the CSF-to-plasma ratio of 6-iodo-ddG was 9.4%, so the higher CSF-to-plasma ratios of ddG with the administration of the 6-halogenated-ddG prodrugs does not appear to be the result of enhanced penetration of the prodrug and subsequent dehalogenation to ddG. The penetration of ddG into CSF exceeds that of didanosine and is enhanced by administration of the 6-halogenated prodrugs, although the mechanism of this enhanced penetration is unclear.

Enhancement by 2'-deoxycoformycin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of 2',3'-dideoxyadenosine and 2'-beta-fluoro-2',3'-dideoxyadenosine.

The anti-human immunodeficiency virus agents 2',3'-dideoxyadenosine (ddAdo) and 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo) are rapidly converted, both in vitro and in vivo, to the corresponding inosine analogs by the widely distributed enzyme adenosine deaminase (EC 3.5.4.4). We have determined the effects of the potent adenosine deaminase inhibitor 2'-deoxycoformycin (2'-dCF) on ddAdo and 2'-beta-F-ddAdo metabolism in MOLT-4 cells and on ddAdo antiviral activity in the ATH8 test system. At levels as low as 5 nM in the incubation medium, 2'-dCF effectively blocks the extracellular deamination of both agents, thus permitting their rapid cellular uptake as the unchanged parent compounds, rather than as the less lipid-soluble 2',3'-dideoxyinosine or 2'-beta-fluoro-2',3'-dideoxyinosine. The result is a significant increase in intracellular levels of the pharmacologically active forms 2',3'-dideoxyadenosine-5'-triphosphate and 2'-beta-fluoro-2',3'-dideoxyadenosine-5'-triphosphate. The effect becomes maximal over the range of 50-250 nM 2'-dCF and declines to control levels when extracellular 2'-dCF levels exceed 1 microM. This decrease in ddAdo and 2'-beta-F-ddAdo phosphorylation with higher levels of the inhibitor appears to result from intracellular penetration of 2'-dCF and consequent inhibition of intracellular deamination, a critical step in the activation of both agents through the 5'-nucleotidase pathway. In anti-human immunodeficiency virus assays, a 2.2-fold increase in ddAdo antiviral potency was seen at 2'-dCF levels of 20 and 50 nM.

Pharmacokinetics of 2',3'-dideoxyadenosine in dogs.

The pharmacokinetics of 2',3'-dideoxyadenosine (ddAdo) and 2'-3'-dideoxyinosine (ddIno) were determined after intravenous bolus administration and long-term intravenous infusion of ddAdo in dogs. ddAdo was rapidly deaminated to ddIno and ddAdo plasma concentrations were only a fraction of ddIno concentrations. The total body clearance of ddAdo exceeded the literature value for the cardiac output of the dog, indicating an extremely rapid metabolism, and the existence of extrahepatic metabolism. Urinary excretion of unchanged ddAdo was a minor route of elimination (approximately 1%). The pharmacokinetics of ddIno was determined assuming complete conversions of ddAdo to ddIno. ddIno elimination was dose-dependent with total body clearance ranging from 4 to 55 ml/min/kg in individual animals. The plasma half-life was approximately 30 min after most routes of administration, but increased to approximately 60 min in two animals receiving a large intravenous dose of 500 mg/kg. ddIno penetrated into the cerebrospinal fluid to a limited extent, reaching concentrations of 3-11% of those in plasma. Urinary excretion of unchanged ddIno accounted for approximately 20% of the administered dose of ddAdo, while uric acid and hypoxanthine were minor urinary metabolites. Concentrations exceeding the in vitro minimal viral inhibitory concentration (2.4 micrograms/mL) could be safely maintained in plasma for a 10-day period. Infusions which gave cerebrospinal fluid concentrations of 12 to 17 micrograms/mL resulted in dose limiting myelosuppression and intestinal toxicity, after less than 10 days of infusion. Orally administered ddAdo was absorbed as ddIno, with bioavailabilities ranging from 28 to 93% in experiments where no emesis occurred. These studies indicate the rapid in vivo conversion of ddAdo to ddIno, and support the selection of ddIno over ddAdo for further drug development.

Disposition and metabolism in mice of the potential antitumor and anti-human immunodeficiency virus-1 agent, 2-chloro-2',3'-dideoxyadenosine.

A high-performance liquid chromatographic (HPLC) procedure was developed to examine the preclinical pharmacology and pharmacokinetics of 2-chloro-2',3'-dideoxyadenosine (ClddAd). The HPLC assay for ClddAd in human plasma was linear from 0.25 to 500 micrograms ClddAd/ml. Coefficients of variation for the measurement of ClddAd in human plasma were 9.7%, 4.1%, and 2.7% at 2.5, 25, and 250 micrograms/ml, respectively. Binding of ClddAd to human and mouse plasma proteins was determined by filtration to be 26.9% and 34.4%, respectively. ClddAd concentrations decreased by less than 5% when ClddAd was stored for 126 h at 37 degrees C in 0.9% NaCl or 0.1 M NaH2PO4 (pH 7.4) or when ClddAd was stored for 24 h at 37 degrees C in citrate-buffered human blood or plasma. Estimates of the lethal dose for 50% (LD50) and 10% (LD10) of male CD2F1 mice that received a single i.v. dose of ClddAd were 27 and 24 mg/kg, respectively. Elimination of a 24-mg/kg i.v. bolus dose of ClddAd from mouse plasma was biphasic, with half-lives of 0.73 and 14.7 min. The apparent volume of distribution of ClddAd was 215 ml/kg and the total body clearance was 20 ml min-1 kg-1. No ClddAd metabolites were detected in mouse plasma after in vivo exposure or in human whole blood or plasma after in vitro incubation. ClddAd was detected in the urine of mice within 2 min after exposure, and the total urinary excretion of unchanged ClddAd for 24 h after exposure to 24 mg/kg was 3.4% of the delivered dose. At least two possible ClddAd metabolites were detected in mouse urine; they did not co-elute with 2-chloro-2',3'-dideoxyinosine,2-chloradenine, or 2-chlorohypoxanthine.

Overview of the preclinical development of an antiretroviral drug, 2',3'-dideoxyinosine.

AIDS has remained a significant and worsening medical problem since its first description as a new clinical entity in 1981. In the past 6 years, substantial progress has been made in the chemotherapy for this disease; such progress is likely to exert a major effect on the epidemic of human immunodeficiency virus infection in the coming decade. In this article, we overview the preclinical development of an antiretroviral drug, 2',3'-dideoxyinosine (didanosine; ddI), which has recently been shown in early phase I studies to have activity against human immunodeficiency virus in patients with AIDS or AIDS-related complex. Although we will not know the full clinical potential of ddI until we have the results of ongoing controlled clinical trials, this drug appears to possess desirable features for clinical use.

2'-Fluoro-2',3'-dideoxyarabinosyladenine: a metabolically stable analogue of the antiretroviral agent 2',3'-dideoxyadenosine.

In this report, we have compared the uptake, metabolism, and relevant enzymology of a novel anti-acquired immunodeficiency syndrome drug, 2'-fluoro-2',3'-dideoxyarabinosyladenine (2'-F-dd-ara-A) with the corresponding properties of its parent compound 2',3'-dideoxyadenosine (2',3'-ddAdo) in three human T cell lines, MOLT-4, ATH8, and CEM. In previous communications, we have reported that the primary route of metabolism of 2',3'-ddAdo in human T lymphoblasts is catabolic, i.e., deamination to 2',3'-dideoxyinosine (2',3'-ddlno). At this point, the metabolic pathway diverges, to result in either cleavage and inactivation of 2',3'-ddlno by purine nucleoside phosphorylase or in 5'-phosphorylation by a phosphotransferase, a reaction that generates 2',3'-inosine monophosphate and ultimately the putative active metabolite 2',3'-dideoxy-ATP. Studies with kinase-deficient mutant CEM lines indicate, however, that 2'-F-dd-ara-A favors a more direct anabolic route toward formation of 2'-fluoro-dideoxynucleotides, catalyzed initially by 2'-deoxycytidine kinase. In MOLT-4 cells, amounts of 2'-fluoro-dideoxyarabinosyladenine di- and triphosphate formed were approximately 20-fold and 5-fold greater than the respective accumulation of 2',3'-dideoxy-ADP and 2',3'-dideoxy-ATP over the same time of exposure. This metabolic profile was supported by enzymological studies, which revealed that 2'-F-dd-ara-A is deaminated 10 times less rapidly than ddAdo and that the resulting deaminated product is resistant to hydrolysis by purine nucleoside phosphorylase. Under similar conditions, ddAdo was rapidly degraded through cleavage of its deamination product ddlno. Like ddAdo, 2'-F-dd-ara-A was found to be transported by passive diffusion and does not enter cells via the purine nucleoside transport carrier system. However, the rate of entry of 2'-F-dd-ara-A was about half that of ddAdo (9.7 pmol/10(6) cells/min for 2'-F-dd-ara-A versus 18.4 pmol/10(6) cells/min for ddAdo). This investigation, therefore, demonstrates that, under the conditions studied, 2'-F-dd-ara-A and its deamination product 2'-fluoro-2',3'-dideoxyarabinosylhypoxanthine have metabolic properties that differ significantly from those of their parent compounds ddAdo and ddlno. These properties, combined with the previously reported resistance of the fluorinated nucleosides to acid degradation, make these compounds interesting candidates for further study as orally administered agents for the inhibition of human immunodeficiency virus replication in patients with acquired immunodeficiency syndrome.