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Biomedical subjects

J L Imbach

Publications and source records attributed to J L Imbach.

At least 37 records · Page 2Linked to original sources
PubMed2000

Synthesis and antiviral evaluation of some beta-L-2', 3'-dideoxy-5-chloropyrimidine nucleosides and pronucleotides.

The synthesis and in vitro anti human immunodeficiency virus (HIV) and anti-hepatitis B virus (HBV) activities of some unnatural beta-L-nucleoside enantiomers related to the anti-HIV compound 2', 3'-dideoxy-3'-fluoro-5-chlorouridine (beta-D-3'Fdd5ClU) are reported. In contrast to beta-D-3'Fdd5ClU, beta-L-3'Fdd5ClU and the other L-congeners were devoid of significant anti-HIV effects, but beta-L-2',3'-dideoxy-5-chlorocytidine (beta-L-dd5ClC) and beta-L-2', 3'-dideoxy-3'-fluoro-cytidine (beta-L-3'FddC) showed a distinct anti-HBV activity. Three mononucleoside phosphotriester derivatives with S-pivaloyl-2-thioethyl (t-BuSATE) groups as biolabile phosphate protective groups were also synthesized. The bis(t-BuSATE) derivative of beta-D-3'Fdd5ClU retained anti-HIV activity in thymidine kinase deficient (TK(-)) CEM cells.

Antiviral Agents↗
PubMed2000

Effects of beta-L-3'-azido-3'-deoxythymidine 5'-triphosphate on host and viral DNA polymerases.

We have previously reported that several beta-L-thymidine analogues including beta-L-3'-azido-3'-deoxythymidine (beta-L-AZT), beta-L-3'-fluoro-2',3'-dideoxythymidine (beta-L-FLT) and beta-L-2', 3'-didehydro-2',3'-dideoxythymidine (beta-L-D4T) did not inhibit HIV replication in human peripheral blood mononuclear (PBM) cells whereas their corresponding beta-D-counterparts are known as potent and selective anti-HIV agents [Faraj et al., 1997. Nucleosides and Nucleotides 16, 1287-1290]. In order to gain insight on the lack of antiviral activities of these beta-L-derivatives, in vitro enzymatic steady state studies were conducted in the present study with beta-L-AZT. beta-L-AZT 5'-triphosphate (L-AZTTP) was chemically synthesized and found to moderately inhibit wild-type HIV reverse transcriptase (HIV-1 RT) with a K(i) value of 2 microM; while lacking any inhibitory effect towards human DNA polymerase alpha, beta or gamma. However, the inhibitory effect of L-AZTTP towards HIV-1 RT was very modest (266-fold less potent) when compared to its isomer beta-D-AZT 5'-triphosphate (D-AZTTP) which exhibits a K(i) value of 0.0075 microM and this finding was further confirmed by DNA chain termination assay. These data suggest that the absence of antiviral activity of the parent beta-L-AZT may in part be explained by the poor inhibition of the targeted viral enzyme by L-AZTTP, the active metabolite. Finally, L-AZTTP was found to lack affinity for the mutant RT at position 184 (M184V) demonstrating that this mutation confers resistance not only to beta-L-2',3'-dideoxycytidine analogs as previously reported by our group [Faraj et al., 1994. Antimicrob. Agents Chemother. 38, 2300-2305] but as well as to beta-L-2',3'-dideoxythymidine analogs.

Anti-HIV Agents↗
PubMed2000

Intracellular metabolism of beta-L-2',3'-dideoxyadenosine: relevance to its limited antiviral activity.

The intracellular metabolism of the beta-L- enantiomer of 2', 3'-dideoxyadenosine (beta-L-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 microM [2',3',8-(3)H]-beta-L-ddA resulted in an increased intracellular concentration of beta-L-ddA with time, demonstrating that these cells were able to transport beta-L-ddA. However, it did not result in the phosphorylation of beta-L-ddA to its pharmacologically active 5'-triphosphate (beta-L-ddATP). Five other intracellular metabolites were detected and identified as beta-L-2', 3'-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 +/- 0.95, 8.15 +/- 2.64, and 15.60 +/- 1.74 pmol/10(6) cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a beta-glucuronic derivative of beta-L-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with beta-L-ddA in the presence of increasing concentrations of 5'-methylthioadenosine resulted in decreased phosphorolysis of beta-L-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of beta-L-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of beta-L-ddA by methylthioadenosine phosphorylase (EC 2.4.2.28).

Adenosine↗
PubMed2000

Pharmacokinetics of bis(t-butyl-SATE)-AZTMP, a bispivaloylthioethyl prodrug for intracellular delivery of zidovudine monophosphate, in mice.

The pharmacokinetics of a bispivaloylthioethyl prodrug of zidovudine monophosphate (AZTMP), bis(t-butyl-SATE)-AZTMP, and intracellular conversion of the prodrug to AZTMP were characterized following intravenous (i.v.) and oral (p.o.) administration of the prodrug to mice. Concentrations of bis(t-butyl-SATE)-AZTMP, AZTMP and zidovudine (AZT) in blood, red blood cells, plasma, brain and lymph nodes were determined by HPLC. Following i.v. administration of bis(t-butyl-SATE)-AZTMP, concentrations of the prodrug declined rapidly with low levels of the prodrug detected until 4 h. Both bis(t-butyl-SATE)-AZTMP and AZTMP were detected in brain 3 min after dosing. AZTMP was found in both plasma and peripheral red blood cells, peaking at approximately 30 min and remaining detectable until 2 h. No AZTMP was detected in lymph nodes. Compared to the pharmacokinetics of AZT following its i.v. administration, i.v. administration of bis(t-butyl-SATE)-AZTMP produced lower peak concentrations of AZT in plasma, peripheral red blood cells, brain and lymph nodes. However, terminal half-lives of AZT were significantly prolonged following administration of the prodrug. Following p.o. administration of bis(t-butyl-SATE)-AZTMP, neither the prodrug nor AZTMP were detectable in whole blood. The conversion of AZT from bis(t-butyl-SATE)-AZTMP in plasma and peripheral red blood cells following p.o administration was 12.1% of that following i.v. administration of the prodrug. Bis(t-butyl-SATE)-AZTMP demonstrated promising potential for intracellular delivery of AZTMP. The prodrug also prolonged the retention of AZT in mice, and particularly increased delivery of AZT to the lymphatic and central nervous systems.

Administration, Oral↗
PubMed1999

Synthesis and in vitro anti-HIV activity in human monocyte-derived macrophages of 2-oxothiazolidine-4(R)-carboxylic acid derivatives.

Oxidative stress and glutathione (GSH) deficit may play an important role in HIV infection pathogenesis, and oral administration of GSH-replenishing drugs such as N-acetylcysteine (NAC) and 2-oxothiazolidine-4(R)-carboxylic acid (OTC) may be associated with an increased survival rate of HIV-infected patients. Nevertheless, beneficial effects of these molecules are restricted in vivo by the high concentrations that are necessary to obtain biological effects, rapid extracellular metabolization, and low availability and plasma concentrations. We synthesized OTC derivatives that are more lipophilic than OTC and theoretically able to overcome these limitations and to generate, in addition to cysteine, other substrates of the gamma-glutamyl cycle. Their antiviral effects were investigated in human HIV-1/Ba-L-infected monocyte-derived macrophages. In our experimental conditions, OTC exhibited anti-HIV-1 effects and little cytotoxicity at high doses. None of the nine tested derivatives showed higher cytotoxicity than OTC, nor anti-HIV-1/Ba-L activity.

Anti-HIV Agents↗
PubMed1999

Lipophilic pro-oligonucleotides are rapidly and efficiently internalized in HeLa cells.

Model t-Bu-SATE pro-dodecathymidines labeled with fluorescein and exhibiting various lipophilicities were evaluated for their uptake by cells in culture. Pro-oligonucleotides with appropriate lipophilicity were found to permeate across the HeLa cell membrane much more extensively than the control phosphorothioate oligo or than the hydrophilic pro-oligos. Fluorescence patterns of internalization were consistent with a diffusion mechanism resulting in the appearance of a uniform cytoplasmic distribution and nuclear accumulation, as confirmed by confocal microscopy.

Animals↗
PubMed1999

Molecular basis for the enantioselectivity of HIV-1 reverse transcriptase: role of the 3'-hydroxyl group of the L-(beta)-ribose in chiral discrimination between D- and L-enantiomers of deoxy- and dideoxy-nucleoside triphosphate analogs.

In order to identify the basis for the relaxed enantio-selectivity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and to evaluate possible cross-resistance patterns between L-nucleoside-, D-nucleoside- and non-nucleoside RT inhibitors, to be utilised in anti-HIV-1 combination therapy, we applied an in vitro approach based on the utilisation of six recom-binant HIV-1 RT mutants containing single amino acid substitutions known to confer Nevirapine resistance in treated patients. The mutants were compared on different RNA/DNA and DNA/DNA substrates to the wild type (wt) enzyme for their sensitivity towards inhibition by the D- and L-enantiomers of 2'-deoxy- and 2',3'-dideoxynucleoside triphosphate analogs. The results showed that the 3'-hydroxyl group of the L-(beta)-2'-deoxyribose moiety caused an unfavourable steric hindrance with critic residues in the HIV-1 RT active site and this steric barrier was increased by the Y181I mutation. Elimination of the 3'-hydroxyl group removed this hindrance and significantly improved binding to the HIV-1 RT wt and to the mutants. These results demonstrate the critical role of both the tyrosine 181 of RT and the 3'-position of the sugar ring, in chiral discrimination between D- and L-nucleoside triphosphates. Moreover, they provide an important rationale for the combination of D- and L-(beta)-dideoxynucleoside analogs with non-nucleoside RT inhibitors in anti-HIV chemotherapy, since non-nucleosideinhibitors resistance mutations did not confer cross-resistance to dideoxynucleoside analogs.

Anti-HIV Agents↗
PubMed1999

L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity?

We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 microM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase alpha, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 microM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy.

Adenosine Monophosphate↗
PubMed1999

Prooligonucleotide metabolism in a crude cell extract followed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) we studied the decomposition pathway of a prooligonucleotide during incubation in a crude cell extract. This study, involving no sample processing, except drop dailysis to remove salts, led us to identify more than thirty metabolites from several metabolic pathways.

Animals↗
PubMed1999

The S-acyl-2-thioethyl pronucleotide approach applied to acyclovir: part I. Synthesis and in vitro anti-hepatitis B virus activity of bis(S-acyl-2-thioethyl)phosphotriester derivatives of acyclovir.

The synthesis and in vitro anti-hepatitis B virus (HBV) activity of two mononucleoside phosphotriester derivatives of acyclovir incorporating S-acyl-2-thioethyl (SATE) groups are reported. In contrast to the parent nucleoside, the described phosphotriesters emerged as potent and selective inhibitors of HBV replication in HepG2.2.15 cells. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the delivery to acyclovir 5'-monophosphate inside the infected cells. Moreover, the in vitro anti-HBV activities of one of these bis(SATE)phosphotriesters and of (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) were compared alone and in combination. Analysis of the combination data indicates that 3TC and the studied SATE pronucleotide of acyclovir exhibited strong synergistic interactions. The present study provides an example where the use of a pronucleotide approach extends the antiviral spectrum of a nucleoside analogue. Given the potency of SATE pronucleotides of acyclovir against HBV in HepG2.2.15 cells, further studies including animal experiments seem warranted to evaluate the potential of these compounds as anti-HBV agents.

Acyclovir↗
PubMed1999

The SATE pronucleotide approach applied to acyclovir: part II. Effects of bis(SATE)phosphotriester derivatives of acyclovir on duck hepatitis B virus replication in vitro and in vivo.

The in vitro and in vivo antiviral activities of two mononucleoside phosphotriester derivatives of acyclovir (ACV) incorporating S-acyl-2-thioethyl (SATE) groups are reported using the duck model of hepatitis B (DHBV). In primary duck hepatocyte cultures, the described phosphotriesters significantly inhibited the replication of DHBV at submicromolar concentrations. They were found to be more potent than the parent nucleoside. This result was in agreement with our data concerning the anti-HBV activity of these pronucleotides in HepG2.2.15 cells (previous paper). In vivo, the studied SATE pronucleotide was also found to be more efficient than ACV in infected ducklings upon short-term oral therapy, while intraperitoneal treatment showed high anti-DHBV activity with both ACV and its SATE pronucleotide in this animal model. These findings demonstrate the potential of SATE pronucleotides of ACV as anti-HBV agents.

Acyclovir↗
PubMed1999

Low enantioselectivities of human deoxycytidine kinase and human deoxyguanosine kinase with respect to 2'-deoxyadenosine, 2'-deoxyguanosine and their analogs.

The antiviral activity of L-nucleoside analogs depends in part on the enantioselectivity of nucleoside kinases which catalyse their monophosphorylation. The substrate properties of human recombinant deoxycytidine kinase (dCK) and human recombinant deoxyguanosine kinase (dGK) with respect to L-adenosine and L-guanosine analogs, in the presence of saturating amounts of ATP and relatively high concentrations of substrates, demonstrated a marked lack of enantioselectivity of both these enzymes. Human dCK catalysed the phosphorylation of D- and L-enantiomers of beta-dA, beta-araA, and beta-dG with enantioselectivities favoring the unnatural enantiomer for the adenosine derivatives and the natural enantiomer for 2'-deoxyguanosine. No other tested L-adenosine or L-guanosine analog was a substrate of dCK. Similarly, D- and L-enantiomers of beta-dA, beta-araA, and beta-dG were substrates of human dGK but with different enantioselectivities compared to dCK, especially concerning beta-dA. The present results indicate that human dCK and dGK have similar properties including substrate properties, relaxed enantioselectivities, and possibly catalytic cycles.

Deoxyadenosines↗
PubMed1999

Synthesis and anti-retroviral activity of O,O'-bis(3'-azido-2',3'-dideoxythymidin-5'-yl) phosphoramidate derivatives.

A simple and efficient protocol for the preparation of various symmetrical dinucleoside phosphoramidates derived from AZT, is presented. It consists of the phosphonylation of AZT with phosphonic acid in the presence of DCC to produce the symmetrical H-phosphonate diester, followed by its oxidative conversion to various phosphoramidate analogues. The synthesized compounds were evaluated for their anti-HIV activity in different cell cultures.

Amides↗
PubMed1999

Pharmacokinetics of beta-L-2',3'-dideoxy-5-fluorocytidine in rhesus monkeys.

beta-L-2',3'-Dideoxy-5-fluorocytidine (beta-L-FddC), a novel cytidine analog with an unnatural beta-L sugar configuration, has been demonstrated by our group and others to exhibit highly selective in vitro activity against human immunodeficiency virus types 1 and 2 and hepatitis B virus. This encouraging in vitro antiviral activity prompted us to assess its pharmacokinetics in rhesus monkeys. Three monkeys were administered an intravenous dose of [3H] beta-L-FddC at 5 mg/kg of body weight. Following a 3-month washout period, an equivalent oral dose was administered. Plasma and urine samples were collected at various times for up to 24 h after dosing, and drug levels were quantitated by high-pressure liquid chromatography. Pharmacokinetic parameters were obtained on the basis of a two-compartment open model with a first-order elimination from the central compartment. After intravenous administration, the mean peak concentration in plasma (Cmax) was 29.8 +/- 10.5 microM. Total clearance, steady-state volume of distribution, terminal-phase plasma half-life (t1/2 beta), and mean residence time were 0.7 +/- 0.1 liters/h/kg, 1.3 +/- 0.1 liters/kg, 1.8 +/- 0.2 h, and 1.9 +/- 0.2 h, respectively. Approximately 47% +/- 16% of the intravenously administered radioactivity was recovered in the urine as the unchanged drug with no apparent metabolites. beta-L-FddC exhibited a Cmax of 3.2 microM after oral administration, with a time to peak drug concentration of approximately 1.5 h and a t1/2 of 2.2 h. One monkey in the oral administration arm of the study had a significant delay in the absorption of the aqueous administered dose. The absolute bioavailability of orally administered beta-L-FddC ranged from 56 to 66%.

Animals↗
PubMed1998

Rational design of a new series of mixed anti-HIV pronucleotides.

MonoSATE aryl phosphotriesters of AZT are able to deliver intracellularly the corresponding 5'-mononucleotide. This process requires activation by an esterase followed by a phosphodiesterase. This finding opens the way to the design of new pronucleotide series.

Anti-HIV Agents↗
PubMed1998

A comparison of the enantioselectivities of human deoxycytidine kinase and human cytidine deaminase.

The stereoselectivities of recombinant human deoxycytidine kinase (EC 2.7.1.74) (dCK) and of recombinant human cytidine deaminase (EC 3.5.4.5) (CDA) were investigated with respect to a series of cytidine analogs, most of them having the unnatural L-stereochemistry. The enantioselectivity of dCK was always low and generally favored the L-enantiomers in the case of beta-2',3'-dideoxycytidine (beta-ddC), 5-fluoro-beta-2',3'-dideoxycytidine (beta-FddC) and beta-cytidine (beta-riboC). Concerning beta-2'-deoxycytidine, dCK showed a preference for the D-enantiomer. All other examined beta-L-cytidine analogs, [1-beta-L-lyxofuranosyl cytosine (beta-L-lyxoC), l-beta-L-xylofuranosyl cytosine (beta-L-xyloC), and 5-fluoro-1-beta-L-xylofuranosyl cytosine (beta-L-Fxylo C)], were substrates of dCK regardless of the nature of the pentose. None of the studied alpha-L-anomers (alpha-L-riboC, alpha-L-araC, alpha-L-lyxoC, or alpha-L-xyloC) was a substrate of dCK. Contrasting with the relaxed enantioselectivity of dCK, CDA had a strict requirement for D-cytidine analogs since none of the already listed beta-L- or alpha-L analogs was a substrate or an inhibitor of the enzyme. The conjunction of the preceding stereochemical properties of dCK and CDA confers to L-cytidine analogs important potentialities in antiviral and anticancer therapies.

Antiviral Agents↗
PubMed1998

Anti-HIV pronucleotides: decomposition pathways and correlation with biological activities.

The purpose of the present study was to compare the decomposition pathways in CEM cell extracts of various phenyl phosphoramidate derivatives of AZT. In addition, the structures of their metabolites were identified. Correlations with their anti-HIV activities in a thymidine kinase deficient (TK-) CEM cell line have been established with a rationale of designing phosphoramidate pronucleotides capable of delivering intracellularly their respective 5'-nucleoside monophosphate derivatives.

Anti-HIV Agents↗