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Biomedical subjects

J Balzarini

Publications and source records attributed to J Balzarini.

At least 181 records · Page 10Linked to original sources
PubMed1998

Selection and characterisation of murine leukaemia L1210 cells with high-level resistance to the cytostatic activity of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl) adenine (PMEA).

An L1210 cell line showing a 300-fold resistance to the cytostatic effect of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) (designated L1210/PMEA-1) was selected in cell culture upon exposure of wild-type L1210/0 cells to stepwise-increased drug concentrations. The mutant L1210/PMEA-1 cell line was characterized by an unusual specificity in that the cytostatic activity was severely impaired only for PMEA and the closely related 2,6-diaminopurine derivative PMEDAP, but not for its guanine counterpart PMEG or for 9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA). The L1210/PMEA-1 cell line showed poor resistance to the cytostatic activity of the lipophilic PMEA prodrug bis(POM)PMEA and virtually kept its PMEA resistance profile in the presence of indomethacin, excluding resistance of the cells of PMEA and PMEDAP by an increased efflux of the drugs. Intracellular purine nucleotide pool labelling studies with adenine, hypoxanthine and glycine revealed that PMEA/PMEDAP resistance did not originate from a defect in the enzymatic pathways of purine nucleotides. ATP, AMP and cAMP, but not adenosine, adenine, HPMPA and inhibitors of nucleoside transport carriers markedly interfered with PMEA uptake in L1210/0 cells. The L1210/PMEA-1 cells proved to have less than 10% of the PMEA uptake capacity of wild-type L1210/0 cells as measured by rapid sampling kinetics as well as long-term incubation experiments. After a 24-h incubation period, the intracellular levels of [2,8-3H]PMEA and its phosphorylated metabolites were approximately 10-fold lower in L1210/PMEA-1 cells than in L1210/0 cells. Our observations point to a compromised and highly specific PMEA/PMEDAP uptake as the molecular basis for the pronounced PMEA resistance of the mutant L1210/PMEA-1 cells.

Adenine↗
PubMed1998

Retention of marked sensitivity to (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-di hydroquin oxaline-2(1H)-thione (HBY 097) by an azidothymidine (AZT)-resistant human immunodeficiency virus type 1 (HIV-1) strain subcultured in the combined presence of quinoxaline HBY 097 and 2',3'-dideoxy-3'-thiacytidine (lamivudine).

An azidothymidine (AZT)-resistant virus strain (HIV-1/AZT) (containing the 67 Asp --> Asn, 70 Lys --> Arg, 215 Thr --> Phe and 219 Lys --> Gln mutations into its reverse transcriptase) was grown in the combined presence of 2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) and the nonnucleoside reverse transcriptase inhibitor (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dih ydroquinoxaine-2(1H)-thione (quinoxaline HBY 097). Replication of HIV-1/AZT was inhibited to a significantly greater extent by the combination of 3TC and quinoxaline HBY 097 than by either drug alone. Virus breakthrough was markedly delayed in the combined presence of 3TC and HBY 097 at drug concentrations as low as 0.05 microg/mL and 0.0025 microg/mL, respectively. The virus that was recovered after exposure to the compounds (3TC and HBY 097) individually had acquired, in the genetic AZT-resistance background of HIV-1/AZT, 103 Lys --> Glu and 106 Val --> Ala mutations. The 103 Lys --> Glu mutation had not been observed before. However, both virus mutants retained marked sensitivity to HBY 097. In all cases, the genotypic AZT-resistance mutations were maintained in the mutant virus RT genomes, and the viruses also remained phenotypically resistant to AZT. Given the exquisite potency of a concomitant combination of 3TC and HBY 097 in suppressing virus replication, this drug combination should be further pursued in clinical trials in HIV-1-infected individuals.

Anti-HIV Agents↗
PubMed1998

A novel mutation (F227L) arises in the reverse transcriptase of human immunodeficiency virus type 1 on dose-escalating treatment of HIV type 1-infected cell cultures with the nonnucleoside reverse transcriptase inhibitor thiocarboxanilide UC-781.

Treatment of wild-type human immunodeficiency virus [HIV-1(IIIB)]-infected cell cultures with the thiocarboxanilide UC-781 under low selective pressure (i.e., 0.01 microg/ml) resulted in the emergence of V106A RT mutant virus. On increasing drug concentrations (stepwise up to 30 microg/ml) the virus retained the V106A RT mutation but acquired the novel F227L mutation in the RT genome in addition to the L100I, K1O1I, and Y181C mutations. This multiple-mutant virus proved highly resistant to virtually all nonnucleoside RT inhibitors (NNRTIs) (e.g., nevirapine, delavirdine, and loviride), but retained full sensitivity to nucleoside analogs such as AZT, ddI, (-)FTC, and 3TC. The F227 amino acid is highly conserved in HIV-1 strains and forms part of the NNRTI-binding pocket. Our model suggests a hydrophobic interaction between F227 and the chloro atom of UC-781.

Anilides↗
PubMed1998

Efficacy of the acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against feline immunodeficiency virus.

The acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were evaluated for their efficacy and side effects in a double-blind placebo-controlled trial using naturally occurring feline immunodeficiency virus (FIV)-infected cats. This natural retrovirus animal model is considered highly relevant for the pathogenesis and chemotherapy of HIV in humans. Both PMEA and FPMPA proved effective in ameliorating the clinical symptoms of FIV-infected cats, as measured by several clinical parameters including the incidence and severity of stomatitis, Karnofsky's score, immunologic parameters such as relative and absolute CD4+ lymphocyte counts, and virologic parameters including proviral DNA levels in peripheral blood mononuclear cells (PBMC) of drug-treated animals. In contrast with PMEA, FPMPA showed no hematologic side effects at a dose that was 2.5-fold higher than PMEA.

Adenine↗
PubMed1998

In vitro and in vivo inhibitory activity of the differentiation-inducing agent 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against rat choriocarcinoma.

The acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) has previously been shown to be a strong inducer of differentiation in several tumor cell lines. We have now investigated the in vitro differentiation-inducing and the in vivo antitumor, properties of PMEA in a rat choriocarcinoma tumor cell model. PMEA at 2 to 50 microM induced choriocarcinoma RCHO cell differentiation in vitro in a concentration-dependent manner, as monitored by morphological changes, induction of alkaline phosphatase and production and secretion of progesterone. Likewise, a clear dose-response relationship was established for the in vivo antitumor activity of PMEA in choriocarcinoma-bearing rats. (R)-PMPA, a structural analogue of PMEA which is much less effective than PMEA in inducing differentiation in vitro did not demonstrate any in vivo antitumor activity. This observation points to the specificity of the differentiation-inducing potential of PMEA.

Adenine↗
PubMed1998

Metabolism of EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide), a potent inhibitor of inosinate dehydrogenase.

The cytostatic agent 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) causes a rapid and marked inhibition of inosinate (IMP) dehydrogenase activity in intact tumor cells. [3H]EICAR is metabolised in L1210 cells to its 5'-mono-, 5'-di- and 5'-triphosphate in a concentration-dependent manner. The metabolites accumulate proportionally with the initial extracellular EICAR concentrations (ranging from 0.25 to 200 microM). The nicotinamide adenine dinucleotide (NAD) analogue of EICAR, designated EAD, also accumulates within the cells and becomes the major metabolite after 48 hr incubation with 5 microM [3H]EICAR. EAD has a markedly longer intracellular half-life than EICAR 5'-mono-, 5'-di- and 5'-triphosphate. An additional EICAR metabolite elutes on an anion exchange Partisphere SAX HPLC chromatogram between EICAR 5'-di- and 5'-triphosphate. Its intracellular levels are approximately 10-fold lower than those of EAD and the nature of this metabolite has still to be identified. The differential role of EAD and EICAR 5'-monophosphate in the inhibition of IMP dehydrogenase is currently under investigation.

Animals↗
PubMed1998

Metabolism and anti-HIV activity of phosphoramidate derivatives of D4T-MP with variations in the amino acid moiety.

The metabolism of different phosphoramidate prodrugs of d4T-MP, in which the phosphate group is linked to a phenyl group and the alkyl ester of an amino acid was studied in crude CEM cell extracts. Significant (80-100%) conversion to the amino acyl d4T-MP metabolite was obtained with derivatives containing L-alanine or methyl-L-aspartic acid. A lower degree of conversion was seen with derivatives containing L-phenylalanine, L-methionine, methyl-L-glutamic acid or L-leucine. Derivatives containing D-alanine, beta-alanine, glycine, L-valine or L-lactate showed no conversion to the amino acyl d4T-MP metabolite. Overall, there was a close correlation between the anti-HIV activity of these prodrugs and their conversion rate to the amino acyl d4T-MP metabolite. Our data suggest that the enzymes involved in the formation of the amino acyl d4T-MP metabolite have a rather stringent specificity for L-alanine as the amino acid moiety. In addition, these enzymes were found to be markedly species-dependent, their activities being highest in mouse serum, followed by guinea pig serum, but only minimal in human serum. Mouse serum therefore appears to be the medium of choice to isolate and identify the enzymes that are involved in the metabolism of these phosphoramidate prodrugs.

Amino Acids↗
PubMed1998

Anticonvulsant phenytoinergic pharmacophores and anti-HIV activity--preliminary evidence for the dual requirement of the 4-aminophthalimide platform and the N-(1-adamantyl) substitution for antiviral properties.

This work is aimed at further exploring the concept that phenytoin-related compounds might present with an anti-HIV potential. We screened for anti-HIV activity, selected compounds whose structural design rests on pharmacophores successfully shown to convey phenytoinergic anticonvulsant activity. We determined the corresponding anticonvulsant protective doses in mice via the i.p. route of administration using the maximal electroshock seizure test (a test in which the anticonvulsant activity of phenytoin is well expressed). Firstly, 4-aminophthalimide pharmacophores were utilized with either N-(2,6-dimethyl)phenyl or N-(1-adamantyl) substitutions. While the former was found to be highly potent, the latter was devoid of significant activity. Secondly, the pharmacophores N-(2,6-dimethylphenyl)phthalimide and N-(1-adamantyl)phthalimide were compared for antiviral (antiHIV-1 and antiHIV-2) properties in CEM (human T-lymphocyte) cells infected with HIV-1 or HIV-2 strains. Various phthalimide C4-substitutions (H, NO2, NH2, Cl, CH3, OCH3, COOH) of these pharmacophores were studied. From this set of experiments, 4-amino-N-(1-adamantyl)phthalimide emerged with EC50 (effective concentration-50) values of 16 and 27 microM against HIV-1 and HIV-2, respectively. The CC50 (cytostatic concentration-50) of this compound was 30 microM. Thirdly, the N-(2,6-dimethylphenyl) and N-(1-adamantyl) substitutions of the 4-aminobenzamide pharmacophore (another known phenytoinergic anticonvulsant platform) were shown to be devoid of anti-HIV activities. A similar negative result was obtained for amantadine. Taken as a whole, the present data indicate that both the 4-aminophthalimide pharmacophore and N-(1-adamantyl) substitutions are required for anti-HIV properties. Molecular modeling studies further provide clues for this dual requirement.

Adamantane↗
PubMed1998

Spectroscopic and biological properties of palladium(II) complexes of ethyl 2-quinolylmethylphosphonate.

Spectroscopic (1H NMR, UV-visible) and biological (cytostatic, antiviral activity) studies of palladium(II) complexes of monoethyl 2-quinolymethylphosphonate (2-Hmqmp): dihalide adducts trans-Pd(2-Hmqmp)2X2, chelate Pd(2-mqmp)2.2H2O and ion-pair salt complexes [2-H2mqmp]+[Pd(2-Hmqmp)X3]- (X = Cl, Br), have been carried out in order to determine structural and biological properties of these biologically interesting complex compounds. The complexes were evaluated in vitro for their cytostatic activity against murine L1210 and human KB and T-lymphoblast Molt4/C8 and CEM/0 cell lines, and the results obtained were compared with those obtained for the complexes of diethyl 2-quinolylmethylphosphonate (2-dqmp). The L1210 cell was the most responsive line and complexes of diester 2-dqmp were more active than complexes of monoester 2-Hmqmp. A good relationship was observed between the cytostatic activity of the complexes and their lypophilicity or solubility. Some complexes exhibited significant cell growth inhibitory effects, but none of the them was more cytostatic than cisplatin. Both 2-dqmp and 2-Hmqmp complexes were also evaluated in vitro for their antiviral activity in different assay systems, comprising a broad spectrum of DNA and RNA viruses, but no specific antiviral effects were noted. In addition, the complexes did not show any specific anti-HIV activity.

Animals↗
PubMed1998

New antivirals - mechanism of action and resistance development.

In recent years, several novel treatment modalities emerged for a number of virus infections, including lamivudine for hepatitis B virus, abacavir, adefovir dipivoxyl and apropovir disprometil for human immunodeficiency virus, cidofovir for cytomegalovirus, and famciclovir (the oral prodrug of penciclovir) and cidofovir for other herpesviruses (i.e. herpes simplex virus and varicella-zoster virus). For all drugs, resistance eventually develops upon prolonged administration to the infected individuals, albeit at a varying extent. In addition, new mutations related to multidrug resistance have recently been identified.

Antiviral Agents↗
PubMed1998

Superior cytostatic activity of the ganciclovir elaidic acid ester due to the prolonged intracellular retention of ganciclovir anabolites in herpes simplex virus type 1 thymidine kinase gene-transfected tumor cells.

Ganciclovir (GCV) and its lipophilic elaidic acid ester prodrug E-GCV were evaluated for their antiherpetic, cytostatic and metabolic properties, E-GCV proved exquisitely inhibitory to the replication of herpes simplex virus type 1 (HSV-1) and HSV-2 in cell cultures (50% effective concentration (EC50): 0.002 microM). It was five- to 10-fold more effective than its parent drug GCV. E-GCV was at least 2000-fold more cytostatic to HSV-1 or HSV-2 thymidine kinase (tk) gene-transfected mammary carcinoma FM3A tk-/HSVtk+ tumor cells than to the corresponding nontransfected tumor cells. The cytostatic activity of E-GCV to the HSVtk gene-transfected tumor cells was far superior to that of GCV. Metabolic studies revealed that both GCV and E-GCV were converted to the mono-, di- and tri-phosphate derivatives of GCV to a markedly higher extent in FM3Atk-/HSV-1 tk+ cells than in wild-type FM3A/0 cells. Strikingly, mono-, di- and tri-phosphate metabolites of GCV were retained for a substantially longer time in E-GCV-treated cells (half-life approximately 50 h) than in GCV-treated cells (half-life approximately 20 h). The longer retention time of the GCV metabolites most likely explains why E-GCV is superior to GCV against herpes simplex virus replication and HSVtk gene-transfected tumor cell proliferation. Taking into account the marked stability of E-GCV in human plasma and its much higher lipophilicity than GCV, E-GCV should be considered as an effective lipophilic prodrug of GCV with a markedly enhanced cytostatic activity in HSVtk gene-transfected tumor cells compared with parental ganciclovir. Its usefulness in the combined gene/chemotherapy of HSVtk gene-transfected tumors should be further pursued.

Animals↗
PubMed1998

Novel carbocyclic nucleosides containing a cyclobutyl ring. Guanosine and adenosine analogues.

(1R,cis)-2-(3-Amino-2,2-dimethylcyclobutyl)ethanol (4) was used as a precursor in the synthesis of cyclobutyl nucleoside analogues containing guanine, 8-azaguanine, adenine or 8-azaadenine. All the compounds were evaluated as antiviral agents in a variety of assay systems. Some activity was noted for compound 13, 17, 19 and 20 against vaccinia virus and for compounds 11, 12, 13, 17, 19 and 20 against herpes simplex virus, at concentrations that were up to 10-fold below the cytotoxic concentrations for the host cells.

Acids, Carbocyclic↗
PubMed1998

Synthesis, antiviral and cytostatic activities of carbocyclic nucleosides incorporating a modified cyclopentane ring. Part 2: Adenosine and uridine analogues.

Six new carbocyclic nucleosides were prepared by mounting a purine (compounds 5-7), 8-azapurine (compounds 9 and 10) or pyrimidine (compound 13) base on the amino group of (1R,cis)-3-(aminomethyl)-1,2,2-trimethylcyclopentylmethanol (2). The antiviral activity of compounds 5-7, 10 and 13, and their cytostatic activity, were evaluated. At subtoxic concentrations, the compounds showed no or marginal antiviral activity. Compound 5 showed moderate inhibition on tumor cell proliferation.

Adenine↗
PubMed1998

A proline-to-histidine substitution at position 225 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) sensitizes HIV-1 RT to BHAP U-90152.

Two mutant virus strains in which the novel P225H mutation appeared in a V106A reverse transcriptase (RT)-mutated genetic background upon treatment of human immunodeficiency virus type 1 (HIV-1) with quinoxaline S-2720 were isolated. Surprisingly, the addition of the P225H mutation to the V106A RT mutant genetic background resensitized the V106A RT mutant virus to the non-nucleoside RT inhibitor (NNRTI) BHAP U-90152, but not to other NNRTIs. Construction of both recombinant viruses and recombinant RTs containing the V106A, P225H and V106A+P225H mutations revealed that P225H was indeed responsible for the marked potentiation of the antiviral activity of BHAP against the P225H single-mutant virus and the V106A+P225H double-mutant virus when compared to wild-type and V106A single-mutant viruses, respectively. An explanation for the markedly increased sensitivity of the P225H mutant HIV-1 RT to BHAP and not to the other NNRTIs was provided by the unique features of the X-ray structure of the RT-BHAP complex.

Amino Acid Substitution↗
PubMed1998

Intracellular metabolism of the N7-substituted acyclic nucleoside analog 2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine, a potent inhibitor of herpesvirus replication.

We investigated the intracellular metabolism of S2242 (2-amino-7-(1,3-dihydroxy-2-propoxymethyl)purine), the only known antivirally active acyclic nucleoside analogue with the side chain substituted at the N7 position of the purine ring. Uptake of S2242 by CEM cells increased linearly with increasing extracellular concentrations of the compound and was blocked by inhibitors of nucleoside transport. S2242 was phosphorylated in a time- and concentration-dependent manner to its monophosphates, diphosphates, and triphosphates. Intracellular half-life of the diphosphates and triphosphates in CEM cells was approximately 3-6 hr. A strong correlation was found between the cytostatic action of the compound and its phosphorylation in different cell lines. In accord with the findings that (1) the cytostatic potential of S2242 is reversed by deoxycytidine (dCyd) and (2) the growth of deoxycytidine kinase-deficient (dCK-) cells is refractory to the inhibitory effect of S2242, the amount of metabolites formed from S2242 in the dCK- cell line was approximately one hundredth of that in the wild-type cells. The observation that purified dCK phosphorylates S2242 to its monophosphate further corroborates these results. The activity of S2242 against herpes simplex virus, varicella-zoster virus, and human herpesvirus type 6 was reversed by 50-100-fold on the addition of exogenous dCyd. Compound S2242 was not preferentially phosphorylated in herpes simplex virus 1-, varicella-zoster virus-, or human herpesvirus type 6-infected cells (Vero, human embryonic lung, and HSB-2 cells, respectively), and exogenously added dCyd reduced substantially the formation of S2242 metabolites in these cells. In human cytomegalovirus (HCMV)-infected human embryonic lung cells, a 5-25-fold increase in S2242 metabolite formation was observed compared with the noninfected cells, suggesting that an HCMV-encoded or -induced enzyme causes the specific phosphorylation of S2242. Exogenously added dCyd had little effect on the activity of S2242 against HCMV and on the phosphorylation of the compound in HCMV-infected cells. S2242 was not specifically phosphorylated by the HCMV-encoded UL-97 kinase in cells infected with a vaccinia/UL-97 recombinant. S2242 was found to be a substrate (K(m) = 90 microM) for purified human deoxyguanosine kinase; the latter enzyme was stimulated 3-4-fold in HCMV-infected cells.

Alkaline Phosphatase↗