Данная работа предполагает разработку методики анализа остаточных органических растворителей (ООР) в субстанциях натрия 3,8-диэтоксикарбонил-4-оксо-4Н-пиразоло[5,1-c][1,2,4]триазинид моногидрата (AB-19) и натрия 7-метилтио-4-оксо-3-циано-4Н-[1,2,4]триазоло[5,1-c][1,2,4]триазинида тригидрата (MNR-857). Анализ литературных данных показал, что наиболее предпочтительным методом определения ООР является метод газожидкостной хроматографии с парофазным вводом пробы. Для определения целевых аналитов в исследуемых субстанциях рассмотрены схемы синтеза и строение соединений, и определено, что образцы могут содержать органические растворители II (гексан, пиридин, метанол) и III (этанол, этилацетат, 2-пропанол) классов опасности. Однако опытные данные показали наличие в образцах субстанций только этанола и пропан-2-ола. В связи с этим проводили количественное определение только указанных растворителей. На основе литературных данных были определены исходные условия проведения хроматографического анализа. На практике исходная методика была скорректирована: изменение массы навески, разведения образца, времени выдерживания виалы, установление диапазона концентраций применения методики и метода количественного определения. Разработанную методику анализа подвергали испытаниям на пригодность по таким показателям как специфичность, линейность, предел количественного определения, сходимость, промежуточная прецизионность и правильность. Согласно проведенным исследованиям была доказана достоверность и надежность получаемых с использованием данной методики результатов. Количественное определение ООР с помощью разработанной методики в образцах субстанций AB-19 (3 партии синтеза) и MNR-857 (5 партий синтеза) подтвердило их соответствие требованиям ОФС.1.1.0008.15 по показателю остаточные органические растворители (не более 0,5 % для растворителей III класса опасности). / This paper discusses the development of a method for assay of residual organic solvents in substances in substances such as sodium 3,8-diethoxycarbonyl-4-oxo-4H-pyrazolo[5,1-c][1,2,4]triazinide monohydrate (AB-19) and sodium 7-methylthio-4-oxo-3-cyano-4H-[1,2,4]triazolo[5,1-c][1,2,4]triazinide trihydrate (MNR-857). An analysis of the literature data showed that the most preferred method for determining residual organic solvents is headspace gas-liquid chromatography. To determine the target analytes in the studied substances, synthesis schemes and the structure of compounds are considered. Thanks to this, it was decided that the samples might contain organic solvents of hazard classes II and III. Class II includes hexane, pyridine, methanol, and сlass III includes ethanol, ethyl acetate, 2-propanol. However, experimental data showed the presence of only ethanol and propan-2-ol in the samples of substances. In this regard, an assay was carried out only for these two solvents. Based on literature data, the initial conditions for the chromatographic analysis were determined. The original method was adjusted by changing the test portion of sample, diluting the samples, the holdup time of the vial, as well as by establishing ranges of concentrations and the method of quantitative measurement. The developed analysis method was tested for suitability by such indicators as specificity, linearity, limit of quantification, repeatability, intermediate precision, and accuracy. According to the research, it was demonstrated that the analytical technique is acceptable for solving the task. The residual organic solvents were quantified using the developed procedure for three batches of synthesis of substance AB-19 and for five batches of synthesis of substance MNR-857. The percentage of residual organic solvents in all samples was less than 0.5%. Thus, the results obtained satisfy the requirements of the article OFS.1.1.0008.15 of the State Pharmacopoeia of the Russian Federation in terms of residual organic solvents.

Contexte : L’organisme en développement est très sensible à son environnement. Plusieurs études épidémiologiques ont suggéré la toxicité développementale d’une dizaine de polluants suite à des expositions prénatales. L’objectif général de cette thèse est d’évaluer les conséquences des expositions à des polluants chimiques (consommation de produits de la mer comme vecteur de contamination, solvants organiques et insecticides organophosphorés) pendant la grossesse sur le développement de l’enfant à 2 ans en se focalisant sur le développement neuro-comportemental, le développement respiratoire et immunitaire et le développement staturo-pondéral. Matériel et méthodes : Ce travail s’est appuyé sur la cohorte mère-enfant PELAGIE, mise en place en Bretagne entre 2002 et 2006. Près de 3500 femmes ont été incluses en début de grossesse et environ 1500 couples mère-enfant ont été suivis à 2 ans. Les expositions ont été évaluées en début de grossesse à partir de questionnaires, de matrices emplois-expositions ou de dosage de biomarqueurs d’exposition. Les indicateurs de santé ont été mesurés lors du suivi à 2 ans par questionnaire permettant de recueillir des éléments sur le comportement, les manifestations respiratoires à type de sifflement, les maladies allergiques et la croissance entre la naissance et 2 ans. Résultats : L’exposition professionnelle prénatale aux solvants organiques était associée à des déficits de l’attention/hyperactivité et des niveaux d’agressivité plus importants chez l’enfant à 2 ans. Cette exposition n’était pas associée avec les manifestations respiratoires et allergiques. Nous avons aussi montré que la consommation maternelle de coquillages et crustacés pendant la grossesse était associée à une augmentation du risque d’allergie alimentaire chez l’enfant. Enfin, l’exposition prénatale aux insecticides organophosphorés a été associée à une diminution de la vitesse de croissance en taille à 2 ans. Conclusion : Ce travail renforce l’hypothèse de l’implication de l’exposition prénatale aux polluants chimiques de l’environnement, y compris à faible dose, dans l’origine développementale de la santé et des maladies. / Background: The organism is very sensitive to environment during its developmental period. Number of epidemiological studies has suggested the developmental toxicity of about ten chemical pollutants after prenatal exposure. The general objective of the thesis is to explore the effect of prenatal exposure to certain chemical pollutants (organic solvents, organophosphates pesticides and maternal consumption of fish and shellfish (vectors of pollutants)) on child development at age 2. Methods: This thesis is based on the PELAGIE mother-child cohort that was set up in 2002, in Brittany (FRANCE). In total, 3421 women were included in this cohort at the beginning of pregnancy and 1500 mother- child pairs were followed when the child was 2 years old. Exposures were evaluated at the beginning of pregnancy from questionnaires, job-exposure matrices or measurement of biomarkers of exposure. Health indicators were measured at the 2 years follow-up. At follow-up, questionnaires allowed to obtain information on child behaviour, respiratory manifestations like wheezing, allergies (eczema and food allergy) and growth between birth and the age of two. Results: Prenatal occupational exposure to solvents was associated with higher level of hyperactivity and attention deficit at age 2. This exposure was not associated neither with respiratory nor with allergic manifestations. We also observed that maternal shellfish consumption during pregnancy was associated with a higher risk of food allergy in preschoolers. Finally, higher prenatal exposure to organophosphate pesticides was associated with a decrease height growth velocity at age 2. Conclusion: The present thesis based on the PELAGIE study supported for the hypothesis that prenatal exposure to environmental chemicals may be implicated in the developmental origin of health and diseases.

MOF-5 ist der Archetyp einer neuartigen Klasse hochporöser Materialien, den Metal Organic Frameworks, die unter anderem zur Anwendung als effektive Gasspeicher geeignet sind. Im Rahmen dieser Arbeit werden die seiner Bildung zugrunde liegenden Reaktionen untersucht. Aus den Erkenntnissen dieser Untersuchungen wurden zwei neue Syntheseverfahren entwickelt. Eines der Verfahren beruht auf der Phasenumwandlung von Zinkterephthalaten und ermöglichte die Laborsynthese von MOF-5 in 100 g-Mengen. Ein zweites Verfahren bedient sich des Strukturaufbaus mit Hilfe vorgefertigter anorganischer Cluster, wodurch erstmals die Synthese von MOF-5-Homologen anderer Metalle als Zink gelang.

La dihyrofolate réductase de type II R67 (DHFR R67) est une enzyme bactérienne encodée par un plasmide donc aisément transmissible. Elle catalyse la réaction de réduction du dihydrofolate (DHF) en tétrahydrofolate (THFA) essentiel pour la prolifération cellulaire. La DHFR R67 est une enzyme qui dépend du cofacteur NADPH. La DHFR R67 est différente, structurellement et génétiquement, de l’enzyme DHFR chromosomale présente chez tous les organismes et elle est résistante au triméthoprime (TMP) qui est largement utilisé dans les traitements antibactériens chez l’Homme. Aucun inhibiteur sélectif contre la DHFR R67 n’est actuellement répertorié. Le but de cette étude a été d’identifier des molécules qui pourront inhiber la DHFR R67 sélectivement, sans affecter la DHFR humaine (DHFRh). La vérification de la qualité des essais enzymatiques en conditions déterminées pour le criblage d’inhibiteurs sur plusieurs lectrices à plaques a identifié des appareils appropriés pour l’analyse. L’étude de l’activité enzymatique de la DHFR R67 et de la DHFRh en présence des solvants organiques et liquides ioniques (LIs), comme des co-solvants pour le criblage rationnel d’inhibiteurs, a montré que certains LIs peuvent servir de milieu alternatif pour les essais enzymatiques. Le criblage rationnel basé sur l’approche du design d’un inhibiteur à partir de petites molécules, a révélé des molécules primaires qui inhibent la DHFR R67 de façon faible, mais sélective. Le test des composés biologiquement actifs qui comprennent des petits fragments, a montré l’augmentation de l’affinité entre la DHFR R67 et les composés testés. Trois composés ont été déterminés comme des inhibiteurs sélectifs prometteurs pour la DHFR R67. / Type II R-plasmid encoded dihyrofolate reductase (DHFR), R67 DHFR is a bacterial enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THFA) which is essential for cell proliferation. R67 DHFR is an enzyme that depends on the cofactor NADPH as the hydride donor. R67 DHFR is distinct, structurally and genetically, from E. coli chromosomal DHFR (DHFR Ec) and it provides drug resistance to the widely-administered antibiotic trimethoprim (TMP). No selective inhibitor against R67 DHFR exists currently. The goal of this study was to discover molecules that can selectively inhibit R67 DHFR, without affecting human DHFR (hDHFR). Verification of the quality of enzyme assays under defined conditions for inhibitor screening on plate readers found several appropriate instruments for analysis. The study of the enzymatic activity of R67 DHFR and hDHFR in the presence of organic solvents and ionic liquids (ILs), as co-solvents for rational screening of inhibitors, showed that ILs can provide alternative media for enzymatic assays. Rational screening based on the approach of fragment-based drug design, revealed primary molecules that inhibited DHFR R67 weakly, but selectively. The testing of more complex compounds with known biological activities gave ligands with increased affinity for R67 DHFR. Three compounds were identified as promising selective inhibitors for R67 DHFR.

科学研究費補助金 研究種目:基盤研究(C)(2) 課題番号:10670348 研究代表者:柴田 英治 研究期間:1998-1999年度

La dihyrofolate réductase de type II R67 (DHFR R67) est une enzyme bactérienne encodée par un plasmide donc aisément transmissible. Elle catalyse la réaction de réduction du dihydrofolate (DHF) en tétrahydrofolate (THFA) essentiel pour la prolifération cellulaire. La DHFR R67 est une enzyme qui dépend du cofacteur NADPH. La DHFR R67 est différente, structurellement et génétiquement, de l’enzyme DHFR chromosomale présente chez tous les organismes et elle est résistante au triméthoprime (TMP) qui est largement utilisé dans les traitements antibactériens chez l’Homme. Aucun inhibiteur sélectif contre la DHFR R67 n’est actuellement répertorié. Le but de cette étude a été d’identifier des molécules qui pourront inhiber la DHFR R67 sélectivement, sans affecter la DHFR humaine (DHFRh). La vérification de la qualité des essais enzymatiques en conditions déterminées pour le criblage d’inhibiteurs sur plusieurs lectrices à plaques a identifié des appareils appropriés pour l’analyse. L’étude de l’activité enzymatique de la DHFR R67 et de la DHFRh en présence des solvants organiques et liquides ioniques (LIs), comme des co-solvants pour le criblage rationnel d’inhibiteurs, a montré que certains LIs peuvent servir de milieu alternatif pour les essais enzymatiques. Le criblage rationnel basé sur l’approche du design d’un inhibiteur à partir de petites molécules, a révélé des molécules primaires qui inhibent la DHFR R67 de façon faible, mais sélective. Le test des composés biologiquement actifs qui comprennent des petits fragments, a montré l’augmentation de l’affinité entre la DHFR R67 et les composés testés. Trois composés ont été déterminés comme des inhibiteurs sélectifs prometteurs pour la DHFR R67. / Type II R-plasmid encoded dihyrofolate reductase (DHFR), R67 DHFR is a bacterial enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THFA) which is essential for cell proliferation. R67 DHFR is an enzyme that depends on the cofactor NADPH as the hydride donor. R67 DHFR is distinct, structurally and genetically, from E. coli chromosomal DHFR (DHFR Ec) and it provides drug resistance to the widely-administered antibiotic trimethoprim (TMP). No selective inhibitor against R67 DHFR exists currently. The goal of this study was to discover molecules that can selectively inhibit R67 DHFR, without affecting human DHFR (hDHFR). Verification of the quality of enzyme assays under defined conditions for inhibitor screening on plate readers found several appropriate instruments for analysis. The study of the enzymatic activity of R67 DHFR and hDHFR in the presence of organic solvents and ionic liquids (ILs), as co-solvents for rational screening of inhibitors, showed that ILs can provide alternative media for enzymatic assays. Rational screening based on the approach of fragment-based drug design, revealed primary molecules that inhibited DHFR R67 weakly, but selectively. The testing of more complex compounds with known biological activities gave ligands with increased affinity for R67 DHFR. Three compounds were identified as promising selective inhibitors for R67 DHFR.

MOF-5 ist der Archetyp einer neuartigen Klasse hochporöser Materialien, den Metal Organic Frameworks, die unter anderem zur Anwendung als effektive Gasspeicher geeignet sind. Im Rahmen dieser Arbeit werden die seiner Bildung zugrunde liegenden Reaktionen untersucht. Aus den Erkenntnissen dieser Untersuchungen wurden zwei neue Syntheseverfahren entwickelt. Eines der Verfahren beruht auf der Phasenumwandlung von Zinkterephthalaten und ermöglichte die Laborsynthese von MOF-5 in 100 g-Mengen. Ein zweites Verfahren bedient sich des Strukturaufbaus mit Hilfe vorgefertigter anorganischer Cluster, wodurch erstmals die Synthese von MOF-5-Homologen anderer Metalle als Zink gelang.

BACKGROUND: Many women of reproductive age are employed in industries involving exposure to organic solvents. Animal toxicological studies and human case reports demonstrate that exposure to organic solvents can cause neuropsychological deficits in exposed offspring; however, there is limited data from prospective controlled human studies. OBJECTIVE: To compare neuropsychological functioning between children whose mothers were occupationally exposed to organic solvents during pregnancy with a non-exposed matched comparison group. METHODS: Participants were 48 women who had previously contacted the Motherisk Program in Toronto, Canada during pregnancy regarding occupational exposure to organic solvents and a matched comparison group of women with no known exposure to teratogens during pregnancy. Children (18 months to 8 years 11 months at time of study) were compared in areas of cognitive, language, motor, and behavioral functioning. RESULTS: Children whose mothers were exposed to organic solvents during pregnancy displayed a lower level of functioning when compared with their matched peers in areas of cognitive, language, motor, and behavioral domains. Although the scores on measures of behavioral functioning were not in the clinical range, the mothers of exposed children reported more challenging behavioral problems. In order to determine whether exposure predicted neuropsychological outcomes above and beyond maternal intellectual functioning, hierarchical regressions were run with maternal IQ and maternal education at Step 1and exposure status added at Step 2. In utero exposure to organic solvents predicted lower sores on global measures of Verbal IQ, receptive and expressive language scales above and beyond maternal intellectual functioning. Factors associated with higher levels of exposure (detecting odor, longer duration and total number of toxicity symptoms) was associated with poorer outcome on behavioral and motor functioning tests. CONCLUSION: Despite the fact that the exposed mothers experienced minimal symptoms of toxicity, detrimental effects were still evident in their offspring. Current safety standards for exposure were designed for adults and need to be reevaluated. Further studies addressing exposure to specific organic solvents, dose, and gestational timing of exposure are warranted.

BACKGROUND: Many women of reproductive age are employed in industries involving exposure to organic solvents. Animal toxicological studies and human case reports demonstrate that exposure to organic solvents can cause neuropsychological deficits in exposed offspring; however, there is limited data from prospective controlled human studies. OBJECTIVE: To compare neuropsychological functioning between children whose mothers were occupationally exposed to organic solvents during pregnancy with a non-exposed matched comparison group. METHODS: Participants were 48 women who had previously contacted the Motherisk Program in Toronto, Canada during pregnancy regarding occupational exposure to organic solvents and a matched comparison group of women with no known exposure to teratogens during pregnancy. Children (18 months to 8 years 11 months at time of study) were compared in areas of cognitive, language, motor, and behavioral functioning. RESULTS: Children whose mothers were exposed to organic solvents during pregnancy displayed a lower level of functioning when compared with their matched peers in areas of cognitive, language, motor, and behavioral domains. Although the scores on measures of behavioral functioning were not in the clinical range, the mothers of exposed children reported more challenging behavioral problems. In order to determine whether exposure predicted neuropsychological outcomes above and beyond maternal intellectual functioning, hierarchical regressions were run with maternal IQ and maternal education at Step 1and exposure status added at Step 2. In utero exposure to organic solvents predicted lower sores on global measures of Verbal IQ, receptive and expressive language scales above and beyond maternal intellectual functioning. Factors associated with higher levels of exposure (detecting odor, longer duration and total number of toxicity symptoms) was associated with poorer outcome on behavioral and motor functioning tests. CONCLUSION: Despite the fact that the exposed mothers experienced minimal symptoms of toxicity, detrimental effects were still evident in their offspring. Current safety standards for exposure were designed for adults and need to be reevaluated. Further studies addressing exposure to specific organic solvents, dose, and gestational timing of exposure are warranted.