Synthesis and Antiviral Activity of Novel Phosphorylated Derivatives of Didanosine Against Newcastle Disease Virus in Chicken.

A series of novel phosphorylated derivatives of didanosine were designed and docking studies were performed with a fusion protein of the Newcastle disease virus (NDV), to develop antiviral compounds against NDV. Based on the docking scores and binding affinities, three derivatives were selected. These compounds were synthesized and characterized by IR, (1) H, (13) C, (31) P, and CHN analysis and mass spectra. They were assessed for their in vitro antiviral activity in DF-1 cells; DDI-10 showed better antiviral activity as evidenced by significant reduction in plaque formation and cytopathic effects. DDI-10 was further evaluated in NDV-infected chicken; the survival rates and antioxidant enzyme levels in brain, liver, and lung tissues were estimated. Superoxide dismutase and catalase were significantly raised, and lipid peroxidation and HA titer levels were decreased upon treatment with 1.5 mg/kg body weight of DDI-10 than with 3 mg/kg body weight of DDI. Further histopathological alterations in NDV-infected tissues were restored in chicken treated with DDI-10. Thus, based on the results from in silico, in vitro, and in vivo assays, the novel phosphorylated DDI-10 might be considered as potent antiviral compound for NDV infection in chicken.

Amino acid esters substituted phosphorylated emtricitabine and didanosine derivatives as antiviral and anticancer agents.

Owing to the promising antiviral activity of amino acid ester-substituted phosphorylated nucleosides in the present study, a series of phosphorylated derivatives of emtricitabine and didanosine substituted with bioactive amino acid esters at P-atom were synthesized. Initially, molecular docking studies were screened to predict their molecular interactions with hemagglutinin-neuraminidase protein of Newcastle disease virus and E2 protein of human papillomavirus. The title compounds were screened for their antiviral ability against Newcastle disease virus (NDV) by their in ovo study in embryonated chicken eggs. Compounds 5g and 9c exposed well mode of interactions with HN protein and also exhibited potential growth of NDV inhibition. The remaining compounds exhibited better growth of NDV inhibition than their parent molecules, i.e., emtricitabine (FTC) and didanosine (ddI). In addition, the in vitro anticancer activity of all the title compounds were screenedagainst HeLa cell lines at 10 and 100 µg/mL concentrations. The compounds 5g and 9c showed an effective anticancer activity than that of the remaining title compounds with IC50 values of 40 and 60 µg/mL, respectively. The present in silico and in ovo antiviral and in vitro anticancer results of the title compounds are suggesting that the amino acid ester-substituted phosphorylated FTC and ddI derivatives, especially 5g and 9c, can be used as NDV inhibitors and anticancer agents for the control and management of viral diseases with cancerous condition.

Novel oleic acid derivatives enhance buccal permeation of didanosine.

The aim of this study was to explore the potential of novel oleic acid (OA) derivatives as buccal permeation enhancers for the delivery of didanosine (ddI). The OA derivatives, i.e. ester derivative (OA1E), the dicarboxylic acid derivative (OA1A) and the bicephalous dianionic surfactant (OA1ANa) were synthesized and their effects were compared to the parent OA. OA, OA1E, OA1A and OA1ANa at 1% w/w all showed potential for enhancing the buccal permeability of ddI with enhancement ratio (ER) of 1.29, 1.33, 1.01 and 1.72, respectively. OA1ANa at 1% w/w demonstrated the highest flux (80.30 ± 10.37 µg cm(-2 )h), permeability coefficient (4.01 ± 0.57 × 10(-3) cm h(-1)) and ER (1.72). The highest flux for ddI (144.00 ± 53.54 µg cm(-2 )h) was reported with OA1ANa 2% w/w, which displayed an ER of 3.09 more than that with ddI alone. At equivalent concentrations, OA1ANa (ER = 3.09) had a significantly higher permeation-enhancing effect than its parent OA (ER = 1.54). Histomorphological studies confirmed that OA1ANa at all concentrations (0.5, 2.0 and 6.0% w/w) had no adverse effects on the mucosae. Morphological changes such as vacuoles formation and increased intercellular spaces were attributed to the buccal permeation-enhancing effect of OA1ANa. This study demonstrated the potential of novel OA derivatives as buccal permeation enhancers. OA1ANa at 2% w/w was also identified as the optimal novel OA derivative to widen the pool of fatty acid derivatives as chemical permeation enhancers for buccal drug delivery.

A new nanomedicine based on didanosine glycerolipidic prodrug enhances the long term accumulation of drug in a HIV sanctuary.

New nanomedicines could improve drug accumulation in HIV sanctuaries and ameliorate their antiretroviral efficiency. In this view, we propose herein a combined strategy based on a biomimetic prodrug of ddI and its formulation in well-characterized lipid nanoobjects. The glycerolipidic prodrug of ddI (ProddINP) has been synthesized and its bulk structure was characterized. An appropriate formulation of this prodrug has been designed using a rational approach combining different physicochemical techniques. The high incorporation ratio of the prodrug into dipalmitoylphosphatidylcholine (DPPC) bilayers was determined by DSC. Then two liposome preparation methods were compared, with respect to size, incorporation yield and molecular/supramolecular organization of vesicles. The best liposomal formulation of ProddINP has been checked to keep intact the anti-HIV activity of ddI. This formulation was finally compared to ddI after oral route in rat. The animal experiments evidenced the increase of ddI blood half life (3-fold) and its enhanced accumulation as prodrug form at 24h in numerous organs and especially intestine after administration of ProddINP in comparison with free drug. Finally, the tested liposomal formulation of ProddINP seems to be a promising approach to eradicate HIV infection from intestinal sanctuaries where the virus can concentrate.

Bifunctional peptidomimetic prodrugs of didanosine for improved intestinal permeability and enhanced acidic stability: synthesis, transepithelial transport, chemical stability and pharmacokinetics.

Five peptidomimetic prodrugs of didanosine (DDI) were synthesized and designed to improve bioavailability of DDI following oral administration via targeting intestinal oligopeptide transporter (PepT1) and enhancing chemical stability. The permeability of prodrugs was screened in Caco-2 cells grown on permeable supports. 5'-O-L-valyl ester prodrug of DDI (compound 4a) demonstrated the highest membrane permeability and was selected as the optimal target prodrug for further studies. The uptake of glycylsarcosine (Gly-Sar, a typical substrate of PepT1) by Caco-2 cells could be inhibited by compound 4a in a concentration-dependent manner. The Caco-2 cells were treated with 0.2 nM leptin for enhanced PepT1 expression. The uptake of compound 4a was markedly increased in the leptin-treated Caco-2 cells compared with the control Caco-2 cells, both of which were obviously inhibited by 20 mM Gly-Sar. The K(m) and V(max) values of kinetic study of compound 4a transported by PepT1 in Caco-2 cells were 0.91 mM and 11.94 nmol/mg of protein/10 min, respectively. The chemical stability studies were performed in simulated gastric fluid (SGF), phosphate buffers under various pH conditions, rat tissue homogenates and plasma at 37 °C. The concentrations of DDI could not be detected in the two minutes in SGF. But compound 4a could significantly increase DDI acidic stability, and its t(½) was extended to as long as 36 min in SGF. Compound 4a was stable in pH 6.0 phosphate buffer but could be quickly transformed into DDI in plasma and tissue homogenates. The oral absolute bioavailability of DDI was 47.2% and 7.9% after compound 4a and DDI were orally administered to rats at a dose of 15 mg/kg, respectively. The coadministration with antiacid agent could also suggest that compound 4a was more stable under harsh acidic conditions compared with DDI. Compound 4a bioavailability in rats was reduced to 33.9% when orally co-administered with Gly-Sar (100 mg/kg). The In Vivo bioactivation mechanism of compound 4a was investigated by comparing the levels of DDI and compound 4a in the jugular and portal veins in rats. The plasma concentration of intact compound 4a was very low in portal veins and could hardly be detected in the jugular vein. In conclusion, compound 4a could significantly improve the oral bioavailability of DDI in rats through PepT1-mediated absorption and enhanced acidic stability, followed by rapid and mostly intracellular bioactivation, the majority in the intestinal cells but the minority in the liver. Additionally, the prodrug strategy targeted to intestinal PepT1 could offer a promising strategy to improve oral bioavailability of poorly absorbed didanosine.

Reacciones cutáneas adversas a fármacos en los pacientes con infección por el VIH en la era TARGA / Cutaneous Drug Reactions in HIV-Infected Patients in the HAART Era

La introducción del tratamiento antirretroviral de gran actividad (TARGA) en 1996 supuso un cambio radical en la historia natural de la infección por el virus de la inmunodeficiencia humana (VIH) al lograr reducir drásticamente la mortalidad en estos pacientes. No obstante, estos tratamientos no están exentos de limitaciones que incluyen efectos adversos, fracaso del tratamiento, interacciones farmacocinéticas, aparición de resistencias y respuestas inmunes anómalas. En este artículo se revisa la situación actual de las reacciones cutáneas adversas a fármacos en los pacientes con infección por el VIH (AU)

The introduction of highly active antiretroviral treatment (HAART) in 1996 radically changed the clinical course of human immunodeficiency virus (HIV) infection as it led to a dramatic reduction in mortality in these patients. However, these treatments have their limitations, including adverse effects, therapeutic failure, pharmacokinetic interactions, the development of resistance, and abnormal immune responses. In this article we review the current situation of cutaneous drug reactions in HIV-infected patients (AU)

WR1065 mitigates AZT-ddI-induced mutagenesis and inhibits viral replication.

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)-didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 muM AZT-ddI (equimolar) for 3 days with or without 150 muM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 muM AZT in the absence or presence of 5-1,000 muM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation.

Self-assembled drug delivery systems: Part 3. In vitro/in vivo studies of the self-assembled nanoparticulates of cholesteryl acyl didanosine.

Self-assembled drug delivery systems (SADDS) are defined as the self-assemblies of amphiphilic prodrugs, integrating prodrugs, molecular self-assembly and nanotechnology for drug targeting and controlled release. Cholesteryl-succinyl didanosine (CSD) and cholesteryl-adipoyl didanosine (CAD) nanoparticulate systems in water were previously prepared and optimized. In this paper, the in vitro and in vivo behavior of them was investigated. Precipitation occurred when they were mixed with acid solutions due to rapid production of hypoxanthine and subsequent disruption of supramolecular structures. They showed pH-dependent degradation and kept relatively stable in the neutral pH range. CSD is more stable than CAD due to the shorter spacer and poloxamer protection. CSD showed different degradation rates in various plasma with the descending order of rat, mouse, rabbit, dog and human. The half-life (t(1/2)) of CSD is 9 days in rat plasma, and 5.9 days in rat liver homogenates. CAD has a faster degradation than CSD though the t(1/2) in rat liver homogenates is long to 23 h. CSD nanoparticulates showed no significant anti-HIV effect in MT4 cell model because of very slow degradation. CSD nanoparticulates showed the distribution t(1/2) of 7.6 min after bolus intravenous (i.v.) administration to rats, and the site-specific distribution in liver, lung and spleen with the high t(1/2) of 10 days in liver. The factors affecting achievement of successful SADDS are discussed.

Morphological transformation of self-assembled nanostructures prepared from cholesteryl acyl didanosine and the optimal formulation of nanoparticulate systems: effects of solvents, acyl chain length and poloxamer 188.

Self-assembled nanostructures were obtained through injecting cholesteryl-succinyl didanosine (CSD) solutions in tetrahydrofuran (THF) into water. The incorporation of THF leads to CSD flexible bilayers. Spherical vesicles of CSD initially formed based on hydrophobic interaction and transformed to short nanotubes with THF leaving. A large proportion of THF led to formation of long flexible nanoribbons, also transforming to short nanotubes after removing THF because of rigid bilayers recovering. Cholesteryl-adipoyl didanosine (CAD) only formed spherical vesicles due to its long and flexible tails. A triblock copolymer, poloxamer 188 (P188) could insert into CSD monolayers at the air/water interface. P188 stabilized CSD nanoparticulate systems by incorporation and adsorption. The optimal formulation of nanoparticulate systems was identified and the appropriate ratio of CSD/P188 and CSD concentration in injected solutions were the keys. A highly concentrated system had a narrow size distribution, allowing heat sterilization at 100 degrees C, and resisting aggregation under high gravity accelerations in spite of the low zeta potential of -18.5 mV. A gel-liquid crystalline phase transition at 47 degrees C occurred. The system may become promising self-assembled drug delivery systems (SADDS) for anti-HIV therapy.

Synthesis and in vitro anti-HIV activities of didanosine prodrugs.

A series of prodrugs of didanosine were synthesized in an effort to enhance the anti-HIV activity. The 5'-OH function of didanosine was esterified with different aryl piperazine acetic acid derivatives and evaluated for anti-HIV-1 activity in MT-4 cell line using the MTT assay method. Among the synthesized compounds, (tetrahydro-5-(1,6-dihydro-6-oxopurin-9-yl)furan-2-yl)methyl 2-(4-(4-chlorophenyl)piperazin-1-yl)acetate (4b) was found to be the most potent compound with EC50 of 0.64 microM and was not toxic to the MT-4 cells up to 1000 microM with a selectivity index of > 1562. Compound 4b was found to be seven times more potent than the parent drug didanosine (EC50 of 4.8 microM) in vitro. In vitro hydrolysis of the various esters in human plasma indicated that these agents were relatively stable toward plasma esterases with t1/2 ranging from 20-60 min.

Determination of carrier-mediated transport of 2',3'-dideoxypurine nucleosides in the rat ileum using a bidirectional perfusion technique.

PURPOSE: Previous attempts to ascertain the role of uptake and efflux transporters in the oral absorption of anti-HIV dideoxypurine nucleosides have been inconclusive. A novel in situ intestinal perfusion technique with complete mesenteric arterial/venous cannulation was used to examine the asymmetry of ileal dideoxynucleoside permeability under near in vivo conditions. METHODS: Intestinal perfusions were performed in the rat ileum, with cannulation of the artery and vein immediately entering and leaving the segment. Urea and mannitol were used as passive permeability markers, and the directional transport of 2',3'-dideoxyinosine (ddI), 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), and 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) were examined. RESULTS: Urea and mannitol exhibited symmetric permeability (PLtoB = PBtoL), whereas F-ddI and ddI showed significantly higher permeability in blood-to-lumen transport (PBtoL > PLtoB). PBtoL for F-ddA exceeded PLtoB, but the difference did not reach significance at p < 0.05. PBtoL for ddI was demonstrated to be saturable with increasing ddI concentrations, but PLtoB was independent of ddI concentration. PBtoL for ddI was shown to be dependent on sodium concentration and inhibited by probenecid. CONCLUSIONS: Symmetric transport was demonstrated for urea and mannitol as expected for these passive permeability markers. F-ddI and ddI were shown to be preferentially transported from blood to lumen. The basolateral to luminal transport of ddI is saturable, inhibited by probenecid, and sodium ion dependent. These results are consistent with carrier-mediated uptake on the basolateral membrane.

Synthesis of new homo and heterodimers of 2',3'-dideoxyinosine (ddI) using ester linkages.

A series of new homo and heterodimers of ddI has been synthesized. A glutarate diester spacer was used to covalently couple ddI onto ddI, AZT or d4T.

Improved method for the simultaneous determination of d4T, 3TC and ddl intracellular phosphorylated anabolites in human peripheral-blood mononuclear cells using high-performance liquid chromatography/tandem mass spectrometry.

There is still a need for direct determination (i.e. without dephosphorylation) of nucleoside reverse transcriptase inhibitor (NRTI) triphosphorylated nucleotides in the peripheral-blood mononuclear cells (PBMCs) of HIV-positive patients. The objective of this paper was first to improve our previously described direct liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for stavudine triphosphate (d4T-TP). Preparation of PBMCs was modified to reduce degradation of d4T-TP during cell preparation and to simplify this step for routine use in clinical units. The performance of several HPLC columns was compared in order to improve the stability of peak shape over time. The SMT C(18) column was replaced by a Supelcogel ODP-50, thereby reducing two-fold the concentration of the first standard. Various internal standards were compared to optimize peak shape and remove an interfering peak in LC. 2-Chloroadenosine 5prime prime or minute-triphosphate was chosen as the most appropriate internal standard. Substitution of the narrowbore column by a microbore column (150 x 0.32 mm) is also presented and discussed. Secondly, this improved method was successfully applied to the simultaneous determination of d4T-TP, dideoxyadenosine triphosphate (ddA-TP) and lamivudine triphosphate (3TC-TP) in PBMCs, which is useful in view of the common use of NRTI combinations. The method was subsequently applied to clinical samples from HIV-positive patients receiving antiretroviral therapy containing d4T, ddl and/or 3TC. This method can be used simply and routinely on approximately 200 samples per week, using commercially available instruments and with a simple cell lysis as sample treatment.

Investigation of the mechanism of enhancement of central nervous system delivery of 2'-beta-fluoro-2',3'-dideoxyinosine via a blood-brain barrier adenosine deaminase-activated prodrug.

Enhanced central nervous system (CNS) delivery of certain poorly penetrating 2',3'-dideoxynucleosides has been achieved by designing prodrugs that are substrates for enzymes, such as adenosine deaminase (ADA), that are present at high activities in brain tissue. In this study, the potential role of adenosine deaminase localized within the endothelial cells of the blood-brain barrier (BBB) in providing enhanced intracellular and CNS delivery of an ADA-activated prodrug is assessed in vitro using cell culture models of the BBB. The kinetics of uptake and bioconversion of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA), a model ADA-activated prodrug of 2'-beta-fluoro-2',3'-dideoxyinosine, were determined in primary cultured bovine brain microvascular endothelial cells. Model-based simulations of CNS availability derived from in vitro estimates of parameters for simple passive diffusion and ADA-catalyzed deamination suggest that ADA that is localized within the BBB plays an important role in the conversion of F-ddA to 2'-beta-fluoro-2',3'-dideoxyinosine during its passage across the BBB. Consistent with in vivo observations, these simulations demonstrate that elevated levels of certain enzymes, such as ADA, in the brain microvascular endothelial cells of the BBB may be exploited in the design of brain-targeted prodrugs or drug-carrier conjugates, which brain tissue selectively converts.

Fluorometric determination of 2'-beta-fluoro-2',3'-dideoxyadenosine 5'-triphosphate, the active metabolite of a new anti-human immunodeficiency virus drug, in human lymphocytes.

A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells.

Synthesis and anti-HIV evaluation of 2',3'-dideoxy imidazo- and nu-triazolo[4,5-d]pyridazine nucleosides.

The syntheses of the 2'-deoxy and 2',3'-dideoxynucleosides of 2,8-diaza-3-deazainosine and the 2',3'-dideoxynucleoside of 2-aza-3-deazainosine were achieved and the pathways leading to these novel nucleosides are described. The preparation of the 2',3'-dideoxynucleoside (1) of 2-aza-3-deazainosine involved deoxygenation of the 2'-deoxy-3'-imidazolide intermediate with n-Bu3SnH and AlBN. The latter nucleoside was synthesized from the known 2'-deoxy derivative of 2-aza-3-deazainosine. The three-step synthesis of 1 from the 2'-deoxy analogue was accomplished in 40% overall yield. Rather than synthesize the corresponding 2',3'-dideoxynucleoside (2) of 2,8-diaza-3-deazainosine in the same manner, i.e. deoxygenation of the 2'-deoxynucleoside, a more cost-effective route was chosen. This pathway involved reductive cleavage of the 5'-protected, 2',3'-thiocarbonate derivative to furnish a mixture of the 2'- and 3'-deoxy isomers. This mixture was not separated, but was deoxygenated by the aforementioned imidazolide method. Using this methodology, 2 was prepared in 23% overall yield from 2,8-diaza-3-deazainosine. Nucleosides 1 and 2 were evaluated for antiretroviral activity and were found to be inactive.

Single dose and multiple dose pharmacokinetics of 2'-fluoro-2',3'-dideoxyadenosine and 2'-fluoro-2',3'-dideoxyinosine, anti-HIV agents, in rats.

A single and multiple dose pharmacokinetic (PK) study was conducted in rats following oral administration of 2'-fluoro-2',3'-dideoxyadenosine (FddA) and 2'-fluoro-2',3'-dideoxyinosine (FddI) at three dose levels. Six rats/gender were assigned to one of the three FddA or FddI dose levels: 40, 250, and 1000 mg/kg/day. Three rats/gender were assigned to the PK study on day 1, while the remaining 3 rats/gender were assigned to the PK study on day 14. The rats received the appropriate doses of either FddA or FddI orally by gavage once a day for 14 days. Serial blood samples up to 24 h and cumulative urine samples (0-24 h) were collected on both days 1 and 14. Plasma and urine samples were analyzed for the concentrations of intact FddA and/or FddI using a validated assay. The data were subjected to non-compartmental PK analyses. Over the dose range of 40-1000 mg/kg. both FddA and FddI exhibited dose dependent pharmacokinetics in rats. Following FddA administration, there was a rapid and extensive in vivo conversion of FddA to FddI; FddI was the major circulating moiety as reflected by Cmax and AUC values (generally 2-3-fold greater than those of FddA at each dose level) as well as the amount excreted (%UR) in the urine. In contrast, following FddI administration, Cmax, AUC, and %UR values were 2-5-fold lower as compared to the FddI generated from FddA administration at each dose level, which also suggested that FddI was not absorbed as extensively as FddA. Based on the findings of this study, FddA is an excellent prodrug of FddI.

Absorption and intestinal metabolism of purine dideoxynucleosides and an adenosine deaminase-activated prodrug of 2',3'-dideoxyinosine in the mesenteric vein cannulated rat ileum.

This study investigates the mechanisms of absorption and the role of intestinally localized purine salvage pathway enzymes on the ileal availabilities of 2',3'-dideoxyinosine (ddI), a substrate for purine nucleoside phosphorylase (PNP); 2'-fluoro-2',3'-dideoxyinosine (F-ddI), a non-PNP substrate; and 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), an adenosine deaminase (ADA) activated prodrug of ddI. The potential for increasing the intestinal availability of 6-Cl-ddP through the use of ADA inhibitors, namely, 2'-deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), is also explored. Drug permeability coefficients across the intestinal epithelium were determined in in situ perfusions in the mesenteric vein cannulated rat ileum based on both drug appearance in blood (Pblood) and disappearance from the lumen (Plumen) and their paracellular and transcellular components were estimated by comparison to the permeabilities of two paracellular markers, mannitol and urea. Values of Pblood for ddI were determined to be (1.1 +/- 0.3) x 10(-6) cm/s, in close agreement with the value of (1.0 +/- 0.3) x 10(-6) cm/s obtained for F-ddI, a PNP resistant analogue of ddI having virtually the same molecular size and lipophilicity as ddI. This indicates that PNP may not play an important role in the low intestinal absorption of ddI. The Pblood for 6-Cl-ddP, (19 +/- 2) x 10(-6) cm/s, was 4.5-fold lower than Plumen, (84 +/- 12) x 10(-6) cm/s, which means that 77 +/- 6% of 6-Cl-ddP was metabolized during its intestinal transport, thus qualitatively accounting for the low oral bioavailability (7%) of 6-Cl-ddP observed in vivo in rats. Extensive intracellular metabolism of 6-Cl-ddP by ADA was confirmed by the high concentrations of ddI found both in the intestinal lumen and blood during 6-Cl-ddP perfusions and by a rate of ddI appearance in blood which was approximately 10-fold higher than ddI controls. Co-perfusion of the potent, hydrophilic ADA inhibitor DCF (Ki = 0. 001-0.05 nM) with 6-Cl-ddP led to only partial inhibition of intestinal ADA, while complete inhibition was obtained using the less potent but more lipophilic inhibitor EHNA (Ki = 1-20 nM). Hence, EHNA may be used to improve intestinal absorption of 6-Cl-ddP in vivo.

Comparative metabolism of the antiviral dimer 3'-azido-3'-deoxythymidine-P-2',3'-dideoxyinosine and the monomers zidovudine and didanosine by rat, monkey, and human hepatocytes.

AZT-P-ddI is an antiviral heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (AZT) and one molecule of 2',3'-dideoxyinosine (ddI) linked through their 5' positions by a phosphate bond. The metabolic fate of the dimer was studied with isolated rat, monkey, and human hepatocytes and was compared with that of its component monomers AZT and ddI. Upon incubation of double-labeled [14C]AZT-P-[3H]ddI in freshly isolated rat hepatocytes in suspension at a final concentration of 10 microM, the dimer was taken up intact by cells and then rapidly cleaved to AZT, AZT monophosphate, ddI, and ddI monophosphate. AZT and ddI so formed were then subject to their respective catabolisms. High-performance liquid chromatography analyses of the extracellular medium and cell extracts revealed the presence of unchanged dimer, AZT, 3'-azido-3'-deoxy-5'-beta-D-glucopyranosylthymidine (GAZT), 3'-amino-3'-deoxythymidine (AMT), ddI, and a previously unrecognized derivative of the dideoxyribose moiety of ddI, designated ddI-M. Trace extracellular but substantial intracellular levels of the glucuronide derivative of AMT (3'-amino-3'-deoxy-5'-beta-D-glucopyranosylthymidine [GAMT]) were also detected. Moreover, the extent of the formation of AMT, GAZT, and ddI-M from the dimer was markedly lower than that with AZT and ddI alone by the hepatocytes. With hepatocytes in primary culture obtained from rat, monkey, and human, large interspecies variations in the metabolism of AZT-P-ddI were observed. While GAZT and ddI-M, metabolites of AZT and ddI, respectively, as well as AZT 5'-monophosphate (MP) and ddI-MP were detected in the extracellular media of all species, AMT and GAMT were produced only by rat and monkey hepatocytes. No such metabolites were formed by human hepatocytes. The metabolic fate of the dimer by human hepatocytes was consistent with in vivo data recently obtained from human immunodeficiency virus-infected patients.

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.