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I Lefebvre

Publications and source records attributed to I Lefebvre.

At least 19 recordsLinked to original sources
PubMed2006

Diastereoisomeric resolution of a pronucleotide using solid phase extraction and high performance liquid chromatography: application to a stereoselective decomposition kinetic in cell extracts.

A stereospecific HPLC methodology has been developed for the diastereoisomeric resolution of a mononucleotide prodrug in cell extracts. This method involves the use of solid phase extraction on a C18 cartridge. Diastereoisomers and internal standard resolutions were performed on a cellulose based chiral column (Chiralcel OD-H) used in the normal phase mode. The method was validated in terms of specificity, recovery, linearity (diasteroisomers mixture concentration: 3-60 micromol L(-1)), precision and accuracy and detection limit (1.67 and 1.33 micromol L(-1) for first and second eluted diastereoisomer). This method was applied to the determination of the apparent rate constants of disappearance and half-lives of each stereoisomers. This permits to conclude to the stereoselectivity of the enzymatic activity involved in the decomposition pathway of 2.

Cell Line, Tumor↗
PubMed2005

Column selection and method development for the separation of nucleoside phosphotriester diastereoisomers, new potential anti-viral drugs. Application to cellular extract analysis.

Analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases used in normal and reversed-phase modes were developed for the diastereoisomeric separation of mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine (AZT). The resolutions were performed with two silica-based celluloses using normal and reversed-phase methodologies: Tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H and Chiracel OD-RH) and Tris-methylbenzoate (Chiralcel OJ and OJ-R). Two amyloses phases, Tris-3,5-dimethylphenylcarbamate (Chiralpak AD) and Tris-(S)-1-phenylethylcarbamate (Chiralpak AS), were used in normal-phase mode. Additionally, we developed separation using two stationary phases with immobilized cyclodextrins in reversed-phase and polar-organic modes. The mobile phase and the chiral stationary phase were varied to achieve the best resolution. Different types and concentration of aliphatic alcohols, acetonitrile or water in the mobile phase were also tested for the different separation modes. An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. The different columns gave complementary results in term of resolution. Limits of detection and quantification were 0.12-0.20 and 0.40-0.67 microm, respectively. This analytical method was applied in a preliminary study for the pronucleotide 2 quantification in cellular extract.

Amylose↗
PubMed2004

SATE pronucleotide approaches: an overview.

This review depicts in vitro and in vivo results obtained with nucleotide prodrugs (pronucleotides) bearing S-acyl-2-thioethyl (SATE) groups as esterase-labile phosphate protections. New developments are illustrated by the design of mononucleoside mixed phosphoester derivatives leading to the selective intracellular delivery of the corresponding 5'-mononucleotide through two different enzyme-mediated activation steps.

Animals↗
PubMed2001

Rational design of a new series of pronucleotide.

A new pronucleotide series is described involving a two-step degradation process mediated by, respectively, carboxylesterase and phosphoramidase. Taking AZT as nucleosidyl moiety, it is shown that most of the compounds inhibit HIV replication in TK(-) cell line, which proves 5'-AZTMP delivery.

Anti-HIV Agents↗
PubMed2001

Kinetics study of the biotransformation of an oligonucleotide prodrug in cells extract by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. We found a good fit between the calculated kinetics curves and the experimental points. The oligonucleotide prodrug was fully metabolized to yield the dodecathymidine phosphorothioate likely through a carboxyesterase mediated mechanism.

Biotransformation↗
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 effects of high-intensity and low-intensity cycle ergometry in older adults with knee osteoarthritis.

BACKGROUND: People with osteoarthritis (OA) of the knee experience pain and deconditioning that lead to disability. This study challenged the clinical belief that repetitive lower extremity exercise is not indicated in persons with knee OA. The effects of high-intensity and low-intensity stationary cycling on functional status, gait, overall and acute pain, and aerobic capacity were examined. METHODS: Thirty-nine adults (71+/-6.9 years old) with complaints of knee pain and diagnosis of OA were randomized to either a high-intensity (70% heart rate reserve [HRR]) or low-intensity (40% HRR) exercise group for 10 weeks of stationary cycling. Participants cycled for 25 minutes, 3 times per week. Before and after the exercise intervention they completed the Arthritis Impact Measurement Scale 2 for overall pain assessment, underwent timed chair rise, 6-minute walk test, gait, and graded exercise treadmill tests. Acute pain was reported daily with a visual analog scale and the Western Ontario and McMaster Universities Osteoarthritis Index scale. RESULTS: Analysis of variance revealed that participants in both groups significantly improved in the timed chair rise, in the 6-minute walk test, in the range of walking speeds, in the amount of overall pain relief, and in aerobic capacity. No differences between groups were found. Daily pain reports suggested that cycling did not increase acute pain in either group. CONCLUSIONS: Cycling may be considered as an alternative exercise modality for patients with knee OA. Low-intensity cycling was as effective as high-intensity cycling in improving function and gait, decreasing pain, and increasing aerobic capacity.

Activities of Daily Living↗
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

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↗
PubMed1997

[Enoxaparin-induced cutaneous necrosis localized on insulin lipodystrophies].

INTRODUCTION: Low-molecular weight heparin-induced cutaneous necrosis is exceptional. Pathogenesis remains unclear. We report an exceptional case with elective localization of the necrotic areas in insulin lipodystrophic tissue. CASE REPORT: A 69-year old patient developed areas of skin necrosis after starting enoxaparin therapy. These areas were located far from the points of injection and focalized on skin areas where the patient had been injecting insulin daily for the last four years. These areas had an aspect of insulin lipodystrophy. Biopsy specimens showed leukocytoclastic vasculitis. There were no associated biological anomalies. One month later, prick-tests were made with different low-molecular weight heparins and calcium heparinate in a lipodystrophic area together with an enoxaparin control test in healthy skin. The only positive test was for enoxaparin in an insulin lipodystrophic area (hard erythema at 24 hours). Histology at 72 hours demonstrated leukocytoclastic vasculitis. DISCUSSION: Six cases of cutaneous necrosis induced by low-molecular weight heparin have been reported, including three cases with enoxaparin. Two pathophysiological mechanisms could be involved: (i) localized heparin-dependent platelet aggregation, or (ii) vasculitis induced by type III hypersensitivity reaction. In our case, the leukocytoclastic aspect of the vasculitis was compatible with an immune complex hypersensitivity reaction. The localization of the necrotic areas would be explained by enoxaparin-induced preferential deposit of immune complexes in the vascular turbulences present in lipodystrophic areas.

Anticoagulants↗
PubMed1997

[Paraneoplastic pemphigus with tracheobronchial involvement].

BACKGROUND: Paraneoplastic pemphigus is a bullous skin disease with characteristic polymorphous clinical presentation and precise histological and immunological features. We report a case of paraneoplastic pemphigus associated with chronic lymphoid leukemia involving the tracheobronchial epithelium. CASE REPORT: A patient with chronic lymphoid leukemia developed pluriorificial lesions. There were several conjunctival, buccal and genital erosions associated with erosive plaques on the trunk, Nikolski's sign and bullous lesions suggestive of paraneoplastic pemphigus. Histology examination of a bulla showed intraepidermal blistering and suprabasal acantholysis. Direct immunofluorescence evidenced intercellular IgG and C3 deposits. Search for anti-intercellular substance antibodies was positive with fluorescence on specific paraneoplastic pemphigus substrates. At immunotransfer, the serum recognized several bands corresponding to 250, 230, 210 and 190 kD antigens, confirming the diagnosis of paraneoplastic pemphigus. Several days later, the patient's general condition deteriorated with bronchorrhea. Bronchial endoscopy visualized ulceronecrotic plaques. Tracheal biopsy evidenced acantholytic cells and intraepithelial cleavage. General corticosteroid therapy was initiated and led to improvement of the skin lesions but the patient died rapidly from pneumonia. Autopsy confirmed the presence of epithelial cleavage and acantholysis involving the trachea and bronchi. DISCUSSION: This case illustrates the difficulty of diagnosing paraneoplastic pemphigus in the early stages. The pluriorificial lesions were suggestive of a Stevens-Johnson syndrome. Besides the genital, conjunctival and buccal mucosa, other mucosa can be involved. In our case, despite the absence of an immunological element, histology was highly suggestive of specific tracheobronchial localizations. The presence of such lesions, which should be searched for in all cases with bronchopulmonary manifestations, worsens the prognosis.

Acantholysis↗
PubMed1996

New insights regarding the potential of the pronucleotide approach in antiviral chemotherapy.

The rationale for a pronucleotide approach based on the use of phosphotriesters which incorporate enzyme-mediated bio-labile protection is discussed in detail. Among the studied bio-labile phosphate protecting groups, the S-acyl-2-thioethyl (SATE) groups appeared the most promising as exemplified in cell culture systems in the case of the pronucleotides of 3'-azido-3'-deoxythymidine, 2',3'-didehydro-3'-deoxythymidine, 2',3'-dideoxyadenosine and acyclovir In vivo implementations of such bis(SATE) pronucleotides have been planned for future animal studies.

Acyclovir↗
PubMed1995

Mononucleoside phosphotriester derivatives with S-acyl-2-thioethyl bioreversible phosphate-protecting groups: intracellular delivery of 3'-azido-2',3'-dideoxythymidine 5'-monophosphate.

The synthesis, in vitro anti-HIV-1 activity, and decomposition pathways of several mononucleoside phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a new kind of carboxylate esterase-labile transient phosphate-protecting group, namely, S-acyl-2-thioethyl, are reported. All the described compounds showed marked antiviral activity in thymidine kinase-deficient CEM cells in which AZT was virtually inactive. The results strongly support the hypothesis that such pronucleotides exert their biological effects via intracellular delivery of the 5'-mononucleotide of AZT. This point was corroborated by decomposition studies in cell extracts and culture medium.

Antiviral Agents↗
PubMed1994

New insights into the resistance of alpha-oligonucleotides to nucleases.

Several studies have shown that alpha-oligonucleotides (alpha-ONs) are more resistant to degradation by nucleases than are beta-oligonucleotides (beta-ONs), but a few exceptions have been reported. The present work indicates that the resistance of alpha-ONs to 3'-exonucleases contained in calf or human sera strongly depends on their 3'-terminal sequence. When the 2 last residues were ...A-G, ...C-A, or ...C-T, the degradation rates in tissue culture media with fetal calf serum were similar to those observed for beta-ONs, but stabilization factors up to 200 were observed when the 2 last residues were ...T-C, ...A-C, or ...C-C, and intermediate stabilization factors were found with ...G-A, ...T-A, or ...T-T terminal sequences. Other data confirm that alpha-ONs are significantly more resistant than beta-ONs to purified 5'-exo- and endonucleases as well as to 3'-exonucleases contained in snake venom or CEM cell lysates, but suggest that sequence specificity may also exist in these media. These findings, which are consistent with literature data and explain the behavior of the aforementioned exceptions, also emphasize that the stability observed in the presence of purified nucleases cannot be extrapolated to sera. Other results show that handling, heat inactivation, and storage conditions have little effect on the 3'-exonuclease activity of serum-containing media, but can completely modify the nuclease activities of cell extracts. This study, which was carried out by means of the accurate "on-line ISRP cleaning" HPLC technique, brings new insights to the general problem of oligonucleotide stability.

Animals↗
PubMed1993

Comparative evaluation of seven oligonucleotide analogues as potential antisense agents.

12-Mer analogues, representative of seven different classes of structurally modified oligonucleotides and complementary to the same target, have been compared for their binding affinity for both single-stranded DNA and RNA, resistance to hydrolysis by nucleases in culture medium (RPMI 1640 + 10% inactivated fetal calf serum), and inhibition of HIV-1 replication in de novo infected MT4 T lymphocytes. The viral target was the splice acceptor site of the premessenger coding for the regulatory protein tat. The oligo(2'-O-alkyl)ribonucleotides (beta-2'O-allyl-RNA and beta-2'OMe-RNA) were shown to form the most stable hybrids with complementary RNA strands whereas the alpha-anomeric oligodeoxynucleoside phosphorothioate analogue displayed the highest stability in the culture medium. All the modified oligonucleotides examined in the present study exhibited a sequence-nonspecific inhibitory effect on HIV-1 replication, the phosphorothioate analogues being the most active ones (ED50 < 1 microM).

Antiviral Agents↗
PubMed1993

5'-Hydrogenphosphonates of anti-HIV nucleoside analogues revisited: controversial mode of action.

The monomeric and symmetrical dimeric 5'-hydrogenphosphonate derivatives of AZT were prepared and evaluated for their inhibitory properties against HIV-1 in several cell lines. The synthesis of the compounds was achieved by reaction of AZT with in situ prepared phosphorus tris(imidazolide) or with phosphonic acid in the presence of pivaloyl chloride. The two title compounds showed in vitro anti-HIV activity similar to (but not better than) that of AZT in three cell lines which were not deficient in thymidine kinase. On the other hand they were inactive in CEM-TK- cells. Pharmacokinetic studies in several media corroborate the assumption that these compounds must not be considered as 'true antiviral agents', but that they act by releasing their nucleoside entity.

Cell Line↗
PubMed1993

Intracellular delivery of nucleoside monophosphates through a reductase-mediated activation process.

On the basis of three different models (namely: ddU, AZT and PMEA), mononucleotide phosphotriester derivatives were designed to be able to liberate the corresponding monophosphate (or phosphonate) inside the cell through a reductase-mediated activation process. It was demonstrated that the use of bis[S-(2-hydroxyethylsulfidyl)-2-thioethyl] esters of ddUMP (11), AZTMP (12) and PMEA (17) resulted in intracellular delivery of the parent monophosphate (or phosphonate). This point was corroborated by observation of an anti-HIV effect of, 11 in various cell lines, for 12 in CEM TK- cells and by the enhanced activity observed for 17. Furthermore, the reported decomposition data in cell extracts fully confirm the validity of this approach and show unambiguously the potential for intracellular reductase-mediated activation of the starting drug.

Adenine↗