Papers by Bernard Monsarrat
Taxol metabolism. Isolation and identification of three major metabolites of taxol in rat bile
PubMed, Nov 1, 1990
The elimination of nonradioactive taxol in bile and urine was investigated in the rat after admin... more The elimination of nonradioactive taxol in bile and urine was investigated in the rat after administration via the caudal vein (10 mg/kg). As in humans, no metabolites of taxol were detected by HPLC in rat urine, and only 10% of the injected taxol was recovered in urine over a 24-hr period. In contrast, 11.5% and 29% of the injected taxol was recovered in rat bile as unchanged taxol and metabolites, respectively. Among the nine taxol metabolites detected by HPLC, the side chain at C13, which is required for pharmacological activity, had been removed in only one minor metabolite, baccatin III. The chemical structures of the two major hydroxylated metabolites were determined by mass spectrometry (fast atom bombardment and desorption chemical ionization) and 1H-NMR spectroscopy. One was a taxol derivative hydroxylated on the phenyl group at C3' of the side chain at C13, while the other corresponded to a taxol derivative hydroxylated in the m-position on the benzoate of the side chain at C2. Although these two major taxol metabolites were as active as taxol in preventing cold microtubule disassembly, they were, respectively, 9 and 39 times less cytotoxic as taxol on in vitro L1210 leukemia growth. These results show for the first time that there is a significant hepatic metabolism of taxol.
Biotransformation des taxoïdes par les cytochromes P450 humains :relation structure-activité
Bulletin Du Cancer, Feb 4, 1997
Le metabolisme des taxoides a ete etudie in vitro en utilisant des microsomes hepatiques humains.... more Le metabolisme des taxoides a ete etudie in vitro en utilisant des microsomes hepatiques humains. La formation du 6a-hydroxypaclitaxel est beaucoup plus importante que celle des autres metabolites hydroxyles sur la chaine laterale initialement caracterises chez le rat. Contrairement au paclitaxel, les etudes realisees in vitro avec des microsomes hepatiques humains et in vivo chez l’animal montrent que le metabolisme du docetaxel est independant de l’espece et que la premiere etape de metabolisation conduit a la formation du derive hydroxyle sur le tert-butyl de la chaine laterale en C13. La comparaison de la biotransformation des taxoides avec le contenu en differents CYP des microsomes humains et avec leurs activites enzymatiques respectives montre que 2 isoformes sont impliquees dans le metabolisme des taxoides. Le CYP2C est responsable de l'hydroxylation en position 6a et le CYP3A4 des hydroxylations intervenant sur la chaine laterale du paclitaxel et du docetaxel. Ces observations ont ete confirmees par des experiences d’inhibition faisant appel a des inhibiteurs chimiques specifiques et a des anticorps specifiques de cytochrome P450. L’effet d’agents antineoplasiques frequemment associes aux taxoides en chimiotherapie a ete teste in vitro sur le metabolisme du paclitaxel et du docetaxel. Utilises a dose therapeutique, la vincristine, la vinblastine, la doxorubicine et le cisplatine ont un effet inhibiteur modere ou nul, de meme que la cimetidine, la ranitidine et la diphenylhydramine utilisees pour reduire les effets secondaires des traitements par les taxoides. Chez des patients recevant une medication a base de barbituriques, l’hydroxylation de la chaine laterale du paclitaxel et du docetaxel est fortement stimulee a la suite de l’induction des isoformes 3A. Ces resultats demontrent que le metabolisme du paclitaxel et du docetaxel est realise par 2 isoformes differentes de CYP dans le foie humain et que des interactions peuvent modifier l’efficacite therapeutique des taxoides lors d’une chimiotherapie.
Experimental Dermatology, Aug 12, 2010
PURPOSE: C-reactive protein (CRP), an acute-phase protein, has been implicated in various inflamm... more PURPOSE: C-reactive protein (CRP), an acute-phase protein, has been implicated in various inflammatory and advanced malignant states. Increased serum CRP (s-CRP) levels have been shown to be associated with independent prognostic factors for survival in patients with advanced lung cancer. However, only few studies have focused on the role of CRP in pleural effusions. This study aimed to evaluate the diagnostic and prognostic value of pleural CRP (p-CRP) in lung cancer patients with malignant pleural effusion (MPE). Pleural effusion (PE) samples were collected from patients with MPE (68 lung cancers; 12 extrathoracic tumors), and from 68 patients with various benign conditions (31 with pneumonia; 37 with tuberculosis). Concentrations of p-and s-CRP were measured by enzyme-linked immunosorbent assay. The expression profile of CRP in pleural fluid and its association with survival were investigated. RESULTS: p-CRP levels correlated with s-CRP levels (r = 0.82, P < 0.0001). The area under the receiver operating characteristic curve, representing diagnostic accuracy in differentiating lung cancer with MPE from benign pleural effusion, was greater for p-CRP (0.86) than for s-CRP (0.77). High p-CRP expression significantly correlated with shorter overall survival (P = 0.006). In a multivariate Cox regression analysis, p-CRP was independent prognostic factor significantly associated with overall survival (P = 0.0001). The relative risk of overall survival for lung cancer patients with high p-CRP levels was 3.909 (95% confidence interval, 2.000-7.639). In conclusion, p-CRP is superior to s-CRP in determining pleural fluid etiology. CLINICAL IMPLICATIONS: Quantitative measurement of p-CRP might be a useful complementary diagnostic and prognostic test for lung cancer patients with MPE.
Biochimica et biophysica acta, Aug 1, 1983
Side-chain degradation of sterols by bacteria is known to proceed via oxidation of a terminal met... more Side-chain degradation of sterols by bacteria is known to proceed via oxidation of a terminal methyl group followed by a succession of /3-oxidative steps. By this pathway, the pregnane backbone is not produced. However, examination of cholesterol degradation products using a strain of Mycobucterium aunrm shows that progesterone and 1-dehydroprogesterone are present at low levels. These pregnane derivatives were identified by gas-liquid chromatography combined with mass spectrometry. This indicates that an alternative pathway for sterol side-chain degradation occurs in bacteria, which could be of great interest for the biological production of corticosteroid precursors.
Journal of Pharmacy and Pharmacology, Sep 1, 1988
Analysis of the degradation mechanisms of MHC class I-presented tumor antigenic peptides by high performance liquid chromatography/electrospray ionization mass spectrometry: application to the design of peptidase-resistant analogs
Rapid Communications in Mass Spectrometry, May 15, 1998
Peptide vaccines based on the use of MHC class I restricted epitopes are currently assayed for an... more Peptide vaccines based on the use of MHC class I restricted epitopes are currently assayed for anti-tumor and anti-viral immunotherapy. With the aim of designing minimally modified, peptidase-resistant analogs, we developed a rational approach based on a detailed understanding of the degradation mechanism of peptides in serum. Degradation of murine tumor antigen P198 and human tumor antigen MAGE-3.A1 was followed by on line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). This method provided high precision and sensitivity for rapid and direct analysis of degradation fragments in a complex mixture and, very importantly, precise identification of transient degradation fragments present at low concentrations. The design of structurally modified analogs, and the analysis of their degradation by on-line HPLC/ESI-MS, allowed us to to demonstrate the efficiency of local modifications in the protection of a given peptide bond towards a specific peptidase activity.
European Journal of Clinical Pharmacology, Apr 1, 1991
The pharmacokinetic parameters in the CSF of baclofen given to 4 patients as an intrathecal bolus... more The pharmacokinetic parameters in the CSF of baclofen given to 4 patients as an intrathecal bolus are reported. Considerable inter-individual variability in the parameters was observed. The elimination half-life ranged from 0.9 to 5 h and the clearance from 0.013 to 0.08 l'h -1. In order to optimize treatment, it is suggested that CSF baclofen levels be matched to changes in Hoffman's monosynaptic reflex (H reflex).
Paclitaxel metabolites in human plasma and urine: Identification of 6α-hydroxytaxol, 7-epitaxol and taxol hydrolysis products using liquid chromatography/atmospheric-pressure chemical ionization mass spectrometry
Rapid Communications in Mass Spectrometry, 1995
Reversed-phase high-performance liquid chromatography/mass spectrometry (LC/MS), with an atmosphe... more Reversed-phase high-performance liquid chromatography/mass spectrometry (LC/MS), with an atmospheric-pressure chemical ionization (APCI) interface, has been applied to the identification of metabolites and derivatives of paclitaxel (taxol) in plasma and urine of patients treated with this new anticancer drug. Protonated molecules with substantial fragmentation were obtained using this ionization technique. The three ion series observed are characteristic of the intact molecule, the taxane ring, and the side chain at C13. Their analysis gives information about chemical modifications of the taxane structure at different positions of the molecule. Urine and plasma extracts were evaluated using the capacity to perform MS analysis directly on the entire effluent from conventional LC columns. Excellent spectra were obtained with 50 pmol of separated compounds in full scan mode. This technique allowed highly sensitive identification of 6 alpha-hydroxytaxol, the major human biliary metabolite, and of 7-epitaxol in extracts of plasma and urine from patients. Taxol hydrolysis derivatives were observed for the first time in urine 24 hours after the end of the infusion period. Sensitivity could be increased further using single ion monitoring (SIM) mode, once a target derivative was identified. These results demonstrate that LC/MS with an APCI interface is useful for the characterization and pharmacokinetic analysis of taxoids in biological matrices.
HAL (Le Centre pour la Communication Scientifique Directe), 2005
Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA... more Rad51 protein plays an essential role in recombination repair of DNA double-strand breaks and DNA crosslinking adducts. It is part of complexes which can vary with the stage of the cell cycle and the nature of the DNA lesions. During a search for Rad51-associated proteins in CHO nuclear extracts of S-phase cells by mass spectrometry of proteins immunoprecipitated with Rad51 antibodies, we identified a centrosomal protein, c-tubulin. This association was confirmed by the reverse immunoprecipitation with c-tubulin antibodies. Both proteins copurified from HeLa cells nuclear extracts following a tandem affinity purification of double-tagged Rad51. Immunofluorescence analysis showed colocalization of both Rad51 and c-tubulin in discrete foci in mammalian cell nuclei. The number of colocalized foci and their overlapping area increased in the presence of DNA damage produced by genotoxic treatments either during S phase or in exponentially growing cells. These variations did not result from an overall stress because microtubule cytoskeleton poisons devoid of direct interactions with DNA, such as taxol or colcemid, did not lead to an increase of this association. The recruitment of Rad51 and c-tubulin in the same nuclear complex suggests a link between DNA recombination repair and the centrosome function during the cell cycle.
Identification of proNeuropeptide FFA peptides processed in neuronal and non-neuronal cells and in nervous tissue
European journal of biochemistry, Sep 30, 2003
Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in a... more Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in a neuronal (human neuroblastoma SH-SY5Y) cell line and in COS-7 cells after transient transfection of the human proNPFFA cDNA and were compared with those detected in the mouse spinal cord. After reverse-phase high performance liquid chromatography of soluble material, NPFF-related peptides were immunodetected with antisera raised against NPFF and identified by using on-line capillary liquid chromatography/nanospray ion trap tandem mass spectrometry. Neuronal and non-neuronal cells generated different peptides from the same precursor. In addition to NPFF, SQA-NPFF (Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPAF were identified in the human neuroblastoma while only NPFF was clearly identified in COS-7 cells. In mouse, in addition to previously detected NPFF and NPSF, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide), the homologous peptide of SQA-NPFF, were characterized. These data on intracellular processing of proNeuropeptide FFA are discussed in regard to the known enzymatic processing mechanisms.
Proteomics Clinical Applications, Aug 1, 2014
Tetralogy of Fallot (TOF) results in chronic progressive right ventricular (RV) pressure overload... more Tetralogy of Fallot (TOF) results in chronic progressive right ventricular (RV) pressure overload and shunt hypoxemia. We investigated the global changes in the proteome of RV among infant patients with and without TOF to gain an insight into early RV remodeling. One hundred and thirty-six differentially expressed proteins were identified using label-free LC-ESI-MS/MS analysis. Western blot results revealed that the expression of 6-phosphofructo-2-kinase/ fructose-2,6-biphosphatase 2 (PFKFB2) increased significantly in TOF patients; and levels of lysocardiolipin acyltransferase 1 (LCLAT1), lumican (LUM), and versican (VCAN) decreased significantly. QRT-PCR analysis showed that levels of PFKFB2 mRNA were markedly increased, but those of LCLAT1 and LUM were significantly decreased. VCAN mRNA showed no significant change in response to pathophysiology of TOF. The results of immunohistochemical staining were similar to those of Western blot analysis. Results of the proteomic analysis indicated that the level of glycolysis-related proteins had increased and levels of lipid-metabolism-related proteins had decreased. ECM proteins were found to be more down-regulated in TOF in the present study than in previous reports. Taken together, our findings may provide clues to both the metabolic inflexibility and ECM remodeling during the early RV remodeling, which occur in response to chronic hypoxia and long-term pressure overload in TOF patients.
Eupa Open Proteomics, Sep 1, 2014
Protein complexes are the main molecular machines that support all major cellular pathways and th... more Protein complexes are the main molecular machines that support all major cellular pathways and their in-depth characterization are essential to understand their functions. Determining the stoichiometry of the different subunits of a protein complex still remains challenging. Recently, many label-free quantitative proteomic approaches have been developed to study the composition of protein complexes. It is therefore of great interest to evaluate these different methods in a stoichiometry oriented objective. Here we compare the ability of four absolute quantitative label-free methods currently used in proteomic studies to determine the stoichiometry of a well-characterized protein complex, the 26S proteasome.
Analytical Chemistry, Aug 16, 2001
Mycolic acids, major and specific long-chain fatty (C70-C90) acid components of the mycobacterial... more Mycolic acids, major and specific long-chain fatty (C70-C90) acid components of the mycobacterial cell envelope, were analyzed for the first time using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry operating in a reflectron mode. The various types of purified mycolates from representative mycobacterial species were analyzed using 2,5-DHB as matrix, because less than 10 pmol of mycolates was sufficient to obtain well-resolved mass spectra composed exclusively of pseudomolecular [M + Na] + ions consistent with the structures deduced from the chemical analytical techniques applied to these molecules. Examination of the MALDI mass spectra demonstrated that the chain lengths of the various mycolates correlated with the growth rate of mycobacterial strains. Although slow growers, such as Mycobacterium tuberculosis and Mycobacterium ulcerans, produced a series of odd carbon numbers (C74-C82) of r-mycolic acids, rapid growers synthesized both odd and even carbon numbers. In addition, the main chain of oxygenated mycolic acids from slow growers were four to six carbon atoms longer than the corresponding r-mycolic acids, whereas rapid growers elaborated oxygenated homologues possessing the same chain lengths as their r-mycolic acids. Furthermore, a comparative analysis of the crude fatty acid mixtures from a wild-type strain of M. tuberculosis and its isogenic mutant effected in the synthesis of oxygenated mycolates by MALDI mass spectrometry revealed structural differences between the r-mycolates from the two strains. Thus, this technique appeared to be a rapid and highly sensitive technique for the analysis of mycolic acids, not only by providing accurate molecular masses and new structural information, but also by both reducing sample consumption and saving time.
Proceedings of the National Academy of Sciences of the United States of America, Jan 17, 2012
CDC25B phosphorylated by pEG3 localises to the centrosome and the spindle pole at mitosis
HAL (Le Centre pour la Communication Scientifique Directe), 2005
The phosphatase CDC25B is one of the key regulators that control entry into mitosis through the d... more The phosphatase CDC25B is one of the key regulators that control entry into mitosis through the dephosphorylation and subsequent activation of the cyclin-dependent kinases. Here we study the phosphorylation of CDC25B at mitosis by the kinase pEg3, a member of the KIN1/PAR-1/MARK family. Using mass spectrometry analysis we demonstrate that CDC25B is phosphorylated in vitro by pEg3 on serine 169, a residue that lies within the B domain. Moreover, using phosphoepitope-specific antibodies we show that serine 169 is phosphorylated in vivo, that this phosphorylated form of CDC25B accumulates during mitosis, and is localized to the centrosomes. This labelling is abrogated when pEg3 expression is repressed by RNA interference. Taken together, these results support a model in which pEg3 contributes to the control of progression through mitosis by phosphorylation of the CDC25 phosphatases.
Clinical pharmacology and metabolism of Taxol (paclitaxel): update 1993
PubMed, 1994
Paclitaxel may be one of the most important anticancer agents to be developed over the past 2 dec... more Paclitaxel may be one of the most important anticancer agents to be developed over the past 2 decades. With its unique mechanism of action as an inducer of tubulin assembly, paclitaxel has demonstrated impressive antitumor activity in patients with breast, lung (both non-small cell and small cell), head and neck, and advanced and platinum-refractory ovarian carcinomas. Unfortunately, there has been a relative lack of pharmacologic data available for paclitaxel, compared with other agents in similar phases of development. This scarcity of data is due, in part, to the aqueous insolubility of paclitaxel and to difficulties in developing sensitive analytic assays to measure the full range of drug concentrations achieved in small animals, both of which have limited preclinical pharmacologic studies. This report reviews the pharmacology of paclitaxel as ascertained during early clinical trials. Although most early studies used prolonged intravenous administration schedules of the agent as both monotherapy and in chemotherapy combinations, more recent studies have evaluated shorter administration schedules. In addition, available information pertaining to the pharmacodynamic and metabolic profiles of paclitaxel are discussed. Such information may be useful in designing rational treatment regimens of paclitaxel as a single agent and in chemotherapy combinations, potentially resulting in the optimal utilization of this important agent in cancer chemotherapeutics.
IL-33 is processed into mature bioactive forms by neutrophil serine proteases (172.26)
Journal of Immunology, May 1, 2012
IL-33 is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to m... more IL-33 is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to many inflammatory diseases. IL-33 signals through the receptor ST2 and drives production of cytokines in innate effector cells and T helper type 2 lymphocytes. IL-33 is constitutively expressed in the nuclei of endothelial cells and in epithelial cells of tissues exposed to the environment. It was initially believed that IL-33, like IL-1β and IL-18, requires processing by caspase-1 for biological activity. On the contrary, we reported that full length IL-33 is biologically active, and that processing by apoptotic caspases results in IL-33 inactivation, rather than activation. We also found that full length IL-33 is released upon cellular damage and we proposed that IL-33 may function, similarly to HMGB1, as an endogenous danger signal. Because IL-33 plays important roles in inflammatory diseases, we hypothesized that IL-33 could be cleaved by proteases released during inflammation. We analyzed the effects of inflammatory proteases from innate effector cells, on IL-33 processing and biological activity. We found that IL-33 can be cleaved and activated by several inflammatory proteases. Interestingly, all forms generated by these proteases are “super-active” IL-33 forms that contain an intact IL1-like cytokine domain. We propose that the inflammatory microenvironment exacerbates disease-associated functions of IL-33 through the generation of “super-active” IL-33 forms.
Differential processing of tumor antigens by standard proteasome and the immunoproteasome
Metabolism of Taxoid Drugs
Acs Symposium Series, Dec 7, 1994
Journal of Medicinal Chemistry, Jun 1, 1985
The chemical synthesis of 9-hydroxyolivacine and 7-hydroxyolivacine based on a biomimetic approac... more The chemical synthesis of 9-hydroxyolivacine and 7-hydroxyolivacine based on a biomimetic approach is described. These two hydroxylated derivatives have been found as main in vitro metabolites of olivacine after incubation with rat hepatic microsomes. The pretreatment of animals with benzo[a]pyrene caused a large increase in both microsomal hydroxylations, whereas the pretreatment with phenobarbital caused a weak increase, with a preservation of 9-hydroxylation/7-hydroxylation ratio >1 in both cases. The two hydroxyolivacines have been also found as principal in vivo metabolites of olivacine in rat bile as glucuronide and sulfate conjugates. The pretreatment of animals with benzo[a]pyrene reverses the 9-hydroxyolivacine/7-hydroxyolivacine ratio excretion in bile to a value that is <1. In both in vitro and in vivo experiments, the free metabolites were identified by HPLC and UV-visible, MS, and 'H NMR spectra. Hydroxylation at position 9 increases the in vitro cytotoxicity against leukemia L1210 cells (ID, = 0.06 pM compared to 2.03 pM for olivacine) and an opposite effect is observed for hydroxylation at position 7 (ID5o = 12.8 MM). On the other hand, hydroxylation a t position 9 has no effect on the in vivo antitumor activity against L1210. This might be related to the oxidative and conjugative metabolic pathways that play an important role in antitumor activity and deactivation of olivacine and its hydroxy metabolites.
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Papers by Bernard Monsarrat