Lipophilic, acid-stable, adenosine deaminase-activated anti-HIV prodrugs for central nervous system delivery. 3. 6-Amino prodrugs of 2'-beta-fluoro-2',3'-dideoxyinosine

A series of 6-substituted amino analogs of 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) purines (F-ddN) has been synthesized and characterized with the objective of finding compounds which might be superior to existing drugs for the treatment of HIV in the central nervous system. These compounds are intended to be more lipophilic than the currently approved anti-HIV drugs for better blood-brain barrier penetration. Subsequent adenosine deaminase (ADA)-catalyzed hydrolysis of these prodrugs in the brain is expected to produce the anti-HIV agent, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine (F-ddI). The new compounds, synthesized from the corresponding 6-chloro analog, include F-ddN which contain methylamino, ethylamino, dimethylamino, hydroxylamino, methoxyamino, benzyloxyamino, hydrazino, and nitro substituents in the 6-position. The 6-nitro analog was isolated as an unexpected product during the preparation of the 6-chloro derivative. Among the analogs with anti-HIV activity, the ethylamino and dimethylamino compounds are ca. 100 times more lipophilic than ddI or F-ddI. As expected, 2'-fluoro substitution protects the compounds from acid-catalyzed glycosylic cleavage. Only the hydroxylamino and nitro analogs underwent any nonenzymatic hydrolysis at pH 1.0 or 7.4. This reaction, however, results in hydrolysis of the group in the 6-position rather than glycosylic bond cleavage. ADA catalyzes the hydrolysis of the 6-substituents at rates which vary from slightly slower (NO2, 1.7x) to much slower (NHEt, 5000x) than F-ddA. The 6-dimethylamino analog is the only compound which possesses anti-HIV activity (ED50 18 microM) without ADA hydrolysis. With the exception of the two inactive alkoxyamino compounds, the other prodrugs exhibited cellular protection in the HIV-1/PHA-PBM system with IC50 potencies of 7-40 microM.     

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.     

Effects of 2',3'-dideoxyinosine on Toxoplasma gondii cysts in mice

The activity against Toxoplasma gondii of 2',3' dideoxyinosine (ddI), an anti-human immunodeficiency virus drug, was examined in an in vitro and in vivo study. Cell cultures infected with a strain known to cause chronic infections were used to show the dose-dependent effect of this drug compared with spiramycin and sulfadiazine. When a dose of 4 microg/ml was used, no infected THP-1 cells or parasites were found after 60 h of incubation. An electron-microscopic study confirmed that after 12 h at 1 microg/ml, the few parasites observed were severely altered. The treatment of chronically infected mice 3 months postinfection showed that a 30-day treatment with 2 mg of ddI/ml induced a significant reduction in the number of T. gondii cysts in the cerebral tissue. These cysts were not viable, as confirmed by immunofluorescence and reinfection experiments. These experiments suggest a possible role for ddI in the treatment of toxoplasmosis, and this possibility deserves further investigation.     

Alterations of Toxoplasma gondii induced by 2',3'-dideoxyinosine in vitro

The time-course of action of the antiviral agent 2',3'-dideoxyinosine (ddI) against Toxoplasma gondii tachyzoites in vitro and its effects at the ultrastructural level were investigated. The very short latency of effect and high efficacy of ddI were evidenced by the fact that the drugs' effects on parasite growth occurred 2 hr after addition to the culture medium, and that an IL90 value of 0.5 microg/ml was reached after 72 hr. Although without apparent effect on uninfected cells, ddI clearly acted on the intracellular parasites, which tended to disappear. Remaining tachyzoites were almost exclusively extracellularly located and often exhibited a clustering of mitochondria-like bodies and subsequent deep alterations of their plasma membranes. These results confirm previous findings and emphasize the potential usefulness of ddI in the management of cerebral toxoplasmosis, a major health problem in acquired immune deficiency syndrome patients.     

Intracellular activation and cytotoxicity of three different combinations of 3'-azido-3'-deoxythymidine and 2',3'-dideoxyinosine

We measured the intracellular activation and cytotoxicity of 3'-azido-3'-deoxythymidine (zidovudine, ZDV) and 2',3'-dideoxyinosine (ddI) when combined at three different clinically relevant combinations of 1:1, 1:10, and 10:1 (ZDV:ddI). The activation of ddI to ddA-TP was increased in all three combinations with ZDV, compared to ddI alone. A maximum twofold increase in ddA-TP was observed, which could not be further increased by raising the concentration of ZDV in the combination. On the other hand, the concentration of ZDV in the combination could be reduced to one-tenth while retaining increased activation of ddI. We also examined the cytotoxicity of these combinations in CEM cells, phytohemagglutinin (PHA)-stimulated and resting human peripheral blood mononuclear cells (PBMCs). CEM cells were the least sensitive overall to the drugs. ZDV showed greater cytotoxicity in stimulated PBMCs than resting PBMCs, whereas the reverse was true for ddI. This could be explained by the different activation pathways of these two drugs. The 1:1 and 10:1 ZDV:ddI combinations showed reduced toxicity compared to the separate drugs. These results indicate that ZDV and ddI need not necessarily be combined together at a ratio of ZDV:ddI of 1:1, but that some alteration in the dosages of ZDV or ddI in patients could be possible without loss of the benefits of combined ZDV:ddI therapy.     

2',3'-O-(2,4,6- trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP)--a nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels

1. P2X receptor activation by alpha,beta-meATP evoked inward currents in acutely dissociated rat mesenteric artery smooth muscle cells and contractions of whole artery rings. 2. The selective P2X1 and P2X3 receptor antagonist TNP-ATP inhibited P2X receptor mediated inward currents in response to 3 microM alpha,beta-meATP (an approximately EC90 concentration) with an IC50 of approximately 2 nM. This provides further evidence that the P2X receptor underlying membrane depolarisation associated with P2X receptor activation can be accounted for by the expression of P2X1 receptors. 3. TNP-ATP inhibited alpha,beta-meATP induced contractions with an IC50 of approximately 30 microM and had non-specific effects on smooth muscle contraction. 4. The reduced potency of TNP-ATP in whole tissue experiments probably reflects the breakdown of TNP-ATP by nucleotidases. Thus, TNP-ATP is of limited use in whole tissue experiments as a P2X receptor antagonist.     

Enzymatic properties of the unnatural beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine

The beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine have been stereospecifically synthesized. In an attempt to explain the previously reported antiviral activities of these compounds, their enzymatic properties were studied with respect to adenosine kinase, deoxycytidine kinase, adenosine deaminase, and purine nucleoside phosphorylase. Adenosine deaminase was strictly enantioselective and favored beta-D-ddA and beta-D-d4A, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent substrate properties for the D- or L-enantiomers of beta-ddA or beta-d4A. Human deoxycytidine kinase showed a remarkable inversion of the expected enantioselectivity, with beta-L-ddA and beta-L-d4A having better substrate efficiencies than their corresponding beta-D-enantiomers. Our results demonstrate the potential of beta-L-adenosine analogues as antiviral agents and suggest that deoxycytidine kinase has a strategic importance in their cellular activation.     

Anti-hepatitis B virus activity and metabolism of 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine

2',3'-Dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine [L(-)Fd4C] was found to be at least 10 times more potent than beta-L-2',3'-dideoxy-3'-thiacytidine [L(-)SddC; also called 3TC, or lamivudine]against hepatitis B virus (HBV) in culture. Its cytotoxicity against HepG2 growth in culture was also greater than that of L(-)SddC (3TC). There was no activity of this compound against mitochondrial DNA synthesis in cells at concentrations upto 10 microM. The dynamics of recovery of virus from the medium of cells pretreated with equal drug concentrations were slower with L(-)Fd4C than with L(-)SddC (3TC). L(-)Fd4C could be metabolized to mono-, di-, and triphosphate forms. The degree of L(-)Fd4C phosphorylation to the 5'-triphosphate metabolite was higher than the degree of L(-)SddC (3TC) phosphorylation when equal extracellular concentrations of the two drugs were used. The apparent K(m) of L(-)Fd4C phosphorylated metabolites formed intracellularly was higher than that for L(-)SddC (3TC). This may be due in part to a difference in the behavior of L(-)Fd4C and L(-)SddC (3TC) towards cytosolic deoxycytidine kinase. Furthermore, L(-)Fd4C 5'-triphosphate was retained longer within cells than L(-)SddC (3TC) 5-triphosphate. L(-)Fd4C 5'-triphosphate inhibited HBV DNA polymerase in competition with dCTP with a Ki of 0.069 +/- 0.015 microM. Given the antiviral potency and unique pharmacodynamic properties of L(-)Fd4C, this compound should be considered for development as an expanded-spectrum anti-HBV drug.     

Pharmacokinetics and tissue distribution of (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine, a prodrug of 3'-azido-2',3'-dideoxythymidine (AZT) in mice

The in-vivo biodistribution and pharmacokinetics in mice of 3'-azido-2',3'-dideoxythymidine (1, AZT), 2-bromomyristic acid (2) and their common prodrug, (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine (3) are reported. The objectives of the work were to enhance the anti-human immunodeficiency virus and anti-fungal effects of 1 and 2 by improving their delivery to the brain and liver. The pharmacokinetics of AZT (beta t1/2 (elimination, or beta-phase, half-life) = 112.5 min; AUC (area under the plot of concentration against time) = 29.1 +/- 2.9 micromol g(-1) min; CL (blood clearance) = 10.5 +/- 1.1 mL min(-1) kg(-1)) and its ester prodrug (3, beta t1/2 = 428.5 min; AUC = 17.3 +/- 4.7 micromol g(-1) min; CL = 17.6 +/- 4.8 mL min(-1) kg(-1) were compared after intravenous injection of equimolar doses (0.3 mmol kg(-1)) via the tail vein of Balb/c mice (25-30 g). The prodrug was rapidly converted to AZT in-vivo, but plasma levels of AZT (peak concentration 0.17 micromol g(-1)) and AUC (12.3 micromol min g(-1)) were lower than observed after AZT administration (peak concentration 0.36 micromol g(-1); AUC 29.1 micromol min g(-1). The prodrug also accumulated rapidly in the liver immediately after injection, resulting in higher concentrations of AZT than observed after administration of AZT itself (respective peak concentrations 1.11 and 0.81 micromol g(-1); respective AUCs 42.5 and 12.7 micromol min g(-1)). Compared with doses of AZT itself, 3 also led to significantly higher brain concentration of AZT (25.7 compared with 9.8 nmol g(-1)) and AUCs (2.8 compared with 1.4 micromol min g(-1)). At the doses used in this study the antifungal agent 2-bromomyristic acid was measurable in plasma and brain within only 2 min of injection. Hepatic concentrations of 2-bromomyristic acid were higher for at least 2 h after dosing with 3 than after dosing with the acid itself. In summary, comparative biodistribution studies of AZT and its prodrug showed that the prodrug led to higher concentrations of AZT in the brain and liver. Although the prodrug did not result in measurably different concentrations of 2-bromomyristic acid in the blood and brain, it did lead to levels in the liver which were higher than those achieved by dosing with the acid itself.     

Characterization of adenosine receptors evoking excitation of mesenteric afferents in the rat

We examined the effects of adenosine receptor agonists and antagonists on the discharge of mesenteric afferent nerves supplying the jejunum in pentobarbitone sodium-anaesthetized rats. Adenosine (0.03-10 mg kg(-1), i.v.), NECA (0.3-300 microg kg(-1), i.v.) and the A1 receptor agonist, GR79236 (0.3-1000 microg kg(-1), i.v.), each induced dose-dependent increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. The A1 receptor antagonist, DPCPX (3 mg kg(-1), i.v.), antagonized all the effects of GR79236 but only the haemodynamic effects of adenosine and NECA. The A2A receptor antagonist, ZM241385 (3 mg kg(-1), i.v.), antagonized the hypotensive effect of NECA but none of the effects of GR79236. The A2A receptor agonist, CGS21680 (0.3-300 microg kg(-1), i.v.), and the A3 receptor agonist, IB-MECA (0.3-300 microg kg(-1), i.v.), each induced only a dose-dependent hypotension. Subsequent administration of adenosine (3 mg kg(-1), i.v.) induced increases in afferent nerve activity and intrajejunal pressure and bradycardia. ZM241385 (3 mg kg(-1), i.v.) antagonized the hypotensive effect of CGS21680 but not the effects of adenosine. Bethanechol (300 microg kg(-1), i.v.) evoked increases in afferent nerve activity and intrajejunal pressure, hypotension and bradycardia. However, adenosine (3 mg kg(-1), i.v.) evoked greater increases in afferent nerve activity than bethanechol despite inducing smaller increases in intrajejunal pressure. In summary, A1 and A2B and/or A2B-like receptors evoke adenosine-induced increases in mesenteric afferent nerve activity and intrajejunal pressure in the anaesthetized rat. Furthermore, elevations in intrajejunal pressure do not wholly account for adenosine-evoked excitation of mesenteric afferent nerves.     

Vasorelaxant effect of olprinone, an inhibitor of phosphodiesterase 3, on mesenteric small artery and vein of rabbits

An augmented synthesis of tetrahydroisoquinolines, such as salsolinol (SAL) or an increased N-methylation of these compounds has been addressed by various investigators as putative pathophysiologic mechanisms in Parkinson's disease (PD). Aim of this study was (1) to investigate putative relations between plasma levels of dopamine and R- and S-enantiomers of SAL and (2) whether these metabolic precursors of the neurotoxic N-methylated-SAL (NMSAL) are elevated in untreated "de-novo" Parkinsonian patients compared to age- and sex-matched healthy controls. Plasma levels of R- and S-SAL and dopamine did not significantly (R-SAL: p=0.61, S-SAL: p=0.51, dopamine: p=0.84) differ in both groups. Parkinsonian patients' R-SAL plasma levels were inversely related to intensity (p=0.03, r =-0.42) and duration of PD (p=0.03, r=-0.43) in contrast to S-SAL and dopamine. Dopamine levels were not associated to R-SAL (p=0.88, r2=0.0008) and S-SAL (p=0.088, r2=0.12) neither in Parkinsonian patients nor in controls. We conclude, that an upregulation of N-methylation of tetrahydroisoquinolines takes place in PD by enzymes such as neutral N-methyltransferase specific for R-SAL. The activity of this enzyme has been found elevated in parkinsonian lymphocytes. This increased N-methylation by the N-methyltransferase specific for R-SAL leads to the known augmented levels of neurotoxic R-NMSAL in Parkinsonian patients compared to controls in the cenral nervous system especially in the beginning of PD.     

Absorption, pharmacokinetics and metabolism of 14C-sumatriptan following intranasal administration to the rat

1. Studies have been carried out to investigate the absorption of sumatriptan after intranasal administration to rats. The pharmacokinetics, metabolism and excretion of 14C-sumatriptan were compared following intranasal and intravenous dosing to male and female albino rats using an aqueous buffered formulation at pH 5.5. 2. Following intravenous administration sumatriptan was eliminated from plasma with a half-life of about 1.1 h. After intranasal administration there was rapid absorption of part of the dose and two peak plasma concentrations were observed, initially at 0.5 and then at 1.5-2 h. The elimination half-life after the second peak was estimated as being about 4 h. 3. Radioactivity was largely excreted in urine (up to 89% of dose in 168 h) after both intravenous and intranasal administration, with a faster rate of excretion after intravenous dosage (73% males, 64% females within 6 h) than after intranasal dosage (37% males, 40% females within 6 h). 4. 14C-sumatriptan was the major component in urine and in extracts of faeces after both intravenous and intranasal administration. The major metabolite excreted in urine and faeces was GR49336, the indole acetic acid analogue. 5. The results of this in vivo rat study suggest that absorption of the dose via the nasal mucosa is incomplete after intranasal administration and that there is a secondary absorption phase probably reflecting oral absorption of part of the dose. The bioavailability is estimated as about 30%, for the period 0-6 h.     

cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant

Programmed cell death of antigen specific T cells (apoptosis) may be an important process in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Fas antigen was recognized as an apoptosis-related antigen. To investigate Fas antigen in multiple sclerosis (MS), we measured the expression rate of Fas antigen and activation markers on T cells in peripheral blood (PB) of 19 MS patients, 18 controls and in cerebrospinal fluid (CSF) of nine MS patients by flow cytometry. The positive rate of Fas antigen in MS patients was higher than that of healthy controls in PB. In patients with MS, the expression rates of Fas antigen and activation markers were higher in CSF than in PB. The above findings suggest that there is acceleration or impairment of apoptosis on activated T cells in MS.     

Differential in vitro hepatic and intestinal metabolism of ifosfamide in the rat

Influence of chitosan molecular weight on drug loading and drug release of drug-loaded chitosan microspheres was studied. Chitosans of 70,000 (LC), 750,000 (MC), and 2,000,000 (HC) molecular weight were employed alone or as mixtures (HC/LC 1:1-1:2 w/w). Ketoprofen (ket) was chosen as the model drug to be encapsulated. Microspheres characterized by different theoretical polymer/drug ratios were prepared (2:1, 1:1, 1:2 w/w). Satisfactory ket contents were obtained for all batches of chitosan microspheres with the theoretical polymer/drug ratio 1:2 w/w; microspheres made of HC/LC (1:2 w/w) were characterized by good drug content and encapsulation efficiency independent by polymer/drug ratio. Prepared chitosan microparticulate delivery systems can modulate ket release within 48 hr. Microspheres consisting of HC/LC (1:2 w/w) were the most suitable formulation in controlling drug release.     

Synthesis and biological properties of the four optical isomers of 5-o-carboranyl-2',3'-didehydro-2',3'-dideoxyuridine

The four isomers of the 5-o-carboranyl-2',3'-didehydro-2',3'-dideoxyuridine (d4CU) were synthesized and their antiviral activity and cytotoxicity in normal and cancer human cells determined. Coupling of silylated 5-o-carboranyluracil with the protected D/L 2,3-dideoxy-2-phenylselenenylribosylacetates provided after oxidative elimination and deprotection, the desired compounds. The presence of the electron deficient 5-o-carboranyl moiety on uracil influenced the yield of the various isomers. In general, the compounds demonstrated weak anti-human immunodeficiency virus activity in primary human lymphocytes. No marked difference in the biological profile was noted for the various optical isomers, suggesting that the high lipophilicity of these nucleosides imparted by the carboranyl moiety overrides stereochemical considerations in the 2',3'-didehydro-2',3'-dideoxyaglycon moiety.     

Molecular mechanics studies on the conformations of 2',3'-dideoxy-2',3'-didehydroguanine nucleoside, D4G

Conformational energy calculations have been presented on guanine nucleoside in which the furanose ring is replaced by 2',3'-dideoxy-2',3'-didehydrofuran using molecular mechanics and conformational analysis. Conformational energies have been evaluated using the MM2 and AMBER94 force field parameters at two different dielectric constants. The results are presented in terms of isoenergy contours in the conformational space of the glycosidic (chi) and C4'-C5' (gamma) bonds torsions. In general, the chi-gamma interrelationships differ from the corresponding plots for unmodified nucleosides and nucleotides, reported previously. Consistency of the calculated preferred conformations with the x-ray data is sensitive to the force field employed.