Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Cyclosporine forms the cornerstone of therapy to prevent rejection after organ transplantation. However, the clinical use of the drug is compromised by a narrow therapeutic window and a wide inter- and intra-individual variation in metabolism. Cyclosporine is metabolised by the CYP3A4 isoenzymes in both the liver and intestine, while it has been reported that the metabolism of the drug can be inhibited by certain furocoumarin derivatives in grapefruit juice. Methoxsalen (8-methoxypsoralen) is a furocoumarin and a potent inhibitor of the cytochrome P450 system in both the liver and intestine. The study was conducted to investigate the possibility whether methoxsalen may inhibit the metabolism of cyclosporine and thereby increase the bioavailability of the drug. The interaction is of clinical relevance since both drugs are used in the treatment of psoriases. The study, conducted in 12 healthy male volunteers, was a three-way comparative bioavailability study with a wash out period of one week between treatments. The patients received 40 mg methoxsalen, 200 mg cyclosporine or a combination of the two on three separate occasions. Blood samples of 10 ml were collected by venupuncture at the following times: 0, 0.5, 1, 1.5, 2, 2.5, 3.4, 5,6, 8, 12 and 24 hours after drug administration. Methoxsalen was analysed by a high pressure liquid chromatograph method (HPLC) with UV detection (LOQ = 10 ng/ml), while cyclosporine was analysed using a fluorescence polarisation immunoassay (FPIA) technique. There was a statistical significant difference in AUCo-00 and Cmax ' for cyclosporine when methoxsalen was added to the drug regimen. When the methoxsalen levels were compared with those in the presence of cyclosporine, the levels were lower, although the difference was not statistical significant. We conclude that methoxsalen increase the levels of cyclosporine by inhibiting the P450 system enzymes in the liver and intestine. However, the absorption of methoxsalen is highly variable in the same individual which needs to be considered before this interaction can be regarded as being of any clinical relevance. / Thesis (M.Sc.(Pharmacology))--North-West University, Potchefstroom Campus, 2004.

The impact of the intestine in determining the oral bioavailability of drugs has been extensively studied. Its large surface area, metabolic content and positioning at the first site of exposure for orally ingested xenobiotics means its contribution can be significant for certain drugs. However, prediction of the exact metabolic component of the intestine is limited, in part due to limitations in validation of in vitro tools as well as in vitro-in vivo extrapolation scaling factors. Microsomes are a well established in vitro tool for extrapolating hepatic metabolism, however standardised methodologies for preparation in the intestine are limited, in light of complexities in preparation (e.g. presence of multiple non-metabolic cells, proteases and mucus). Therefore, the aims of this study were to establish an optimised method of intestinal microsome preparation via elution in the proximal rat intestine, and to determine microsomal scaling factors by correcting for protein losses during preparation. In addition, to assess species in another preclinical species (dog) and human as well as assessing and regional differences in scaling factors and metabolism. Following optimisation of a reproducible intestinal microsome preparation method in the rat, the importance of heparin in limiting mucosal contamination was established. These microsomes were characterised for total cytochrome P450 (CYP) content, and CYP and uridine 5′-diphosphate glucuronosyltransferase (UGT) activities using maker probes of testosterone and 4-nitrophenol. Loss corrected microsomal scaling factors between two pools of n=9 rats was 9.6±3.5 (recovery 33%). A broad range of compounds (n=25) in terms of metabolic activity and physicochemical properties were screened in rat intestinal microsomes. The prediction accuracy relative to in house generated or literature in vivo estimates of the fraction escaping intestinal metabolism (FG) through in vitro-in vivo extrapolation of observed metabolism and the derived scaling factors and either Caco-2 permeability of physicochemical permeability estimates utilising the Qgut model. In the dog, regional differences in intestinal scaling factors and metabolic activities were explored, as well as relationships between the proximal intestine and liver in matched donors. Positive correlations in both hepatic activity and microsomal scalars were observed. Robust scaling factors were established using the 3 microsomal markers. A total of 24 compounds were screened for hepatic and intestinal metabolism in order to make in vivo estimates of FG, the fraction escaping hepatic metabolism (FH) and oral bioavailability (F). Estimates based on Caco-2 and physicochemical based scaling, as well as utilising a commercial PBPK software platform (ADAM model, Simcyp® v12) were broadly similar with generally reduced prediction accuracy in proximal physicochemical based Qgut scaling, and improved predictions using Caco-2 Qgut or PBPK approaches. Worse predictions were observed for compounds with high protein binding, transporter substrates and/or CYP3A inhibitors. Regional metabolism demonstrated peak metabolism in the proximal intestine, before declining distally. Human intestinal microsomes were prepared for jejunum and ileum tissue. Although samples were limited, regional differences in metabolic activities and scaling factors were also assessed, using correction markers and activity in 23 compounds. In all, 20 compounds overlapped between all three species. Comparison in Fa.FG between rat and human CYP3A substrates showed a modest relationship, however relationships between species and human were generally poor given the observed differing metabolic contributions of testosterone and 4-NP metabolite formation between species limited the observed relationships between species. However, within species, good estimates of oral bioavailability were observed. This is the largest know interspecies comparison of intestinal metabolism and scaling factors with microsomes prepared within the same lab.

Tout médicament administré par la voie orale doit être absorbé sans être métabolisé par l’intestin et le foie pour atteindre la circulation systémique. Malgré son impact majeur sur l’effet de premier passage de plusieurs médicaments, le métabolisme intestinal est souvent négligé comparativement au métabolisme hépatique. L’objectif de ces travaux de maîtrise est donc d’utiliser, caractériser et développer différents outils in vitro et in vivo pour mieux comprendre et prédire l’impact du métabolisme intestinal sur l’effet de premier passage des médicaments comparé au métabolisme hépatique. Pour se faire, différents substrats d’enzymes du métabolisme ont été incubés dans des microsomes intestinaux et hépatiques et des différences entre la vitesse de métabolisme et les métabolites produits ont été démontrés. Afin de mieux comprendre l’impact de ces différences in vivo, des études mécanistiques chez des animaux canulés et traités avec des inhibiteurs enzymatiques ont été conduites avec le substrat métoprolol. Ces études ont démontré l’impact du métabolisme intestinal sur le premier passage du métoprolol. De plus, elles ont révélé l’effet sur la vidange gastrique du 1-aminobenzotriazole, un inhibiteur des cytochromes p450, évitant ainsi une mauvaise utilisation de cet outil dans le futur. Ces travaux de maîtrise ont permis d’améliorer les connaissances des différents outils in vitro et in vivo pour étudier le métabolisme intestinal tout en permettant de mieux comprendre les différences entre le rôle de l’intestin et du foie sur l’effet de premier passage. / To reach the systemic circulation, orally administered drugs have to be absorbed and not metabolized by the intestine and the liver. Even though it has a major impact on the first pass effect of many xenobiotics, the intestinal metabolism is often neglect compare to the hepatic metabolism. The objective of this work is to use, characterize and develop multiple in vitro and in vivo tools to better understand and predict the impact of intestinal metabolism on the first pass effect of xenobiotics compared to the liver. To do so, multiple substrates of metabolic enzymes were incubated in intestinal and hepatic microsomes and differences between the rate of metabolism and the production of metabolites were demonstrated. To better understand the impact of these differences in vivo, mechanistic studies were undergone in rats cannulated or treated with enzymatic inhibitors with the substrate metoprolol. These studies demonstrated the impact of intestinal metabolism on the first pass of metoprolol. Moreover, they exposed the effect on gastric emptying of 1-aminobenzotriazole, a cytochrome p450 inhibitor, avoiding its wrong utilisation in future studies. This work helped increase the knowledge about the different in vitro and in vivo tools to study intestinal metabolism and to better understand the differences between the role of the intestine and the liver on the first pass effect.

Les derniers apports nutritionnels conseillés recommandent une consommation plus importante en acides gras polyinsaturés de la série n-3 que celle actuellement constatée dans l’alimentation française. Dans ce contexte, il convenait d’appréhender les facteurs susceptibles de moduler l’absorption et le devenir de leur chef de file, l’ALA, en faisant appel à deux approches, l’une in vivo (rat), l’autre in vitro (Caco-2). L’étude relative au devenir métabolique de l’ALA, selon sa forme physique et chimique de présentation, a été réalisée avec des lipides « modèles » (TAG structurés) ou naturels (huile de lin) riches en ALA, et selon différents systèmes lipidiques (huile en phase continue ou en émulsion de type huile dans eau et de composition en phospholipides variables). Les résultats obtenus in vivo et in vitro à l’égard de l’huile de lin (émulsionnée ou non) montrent que l’émulsification accélère non seulement le passage intestinal de l’ALA mais améliore également sa concentration lymphatique. L’étude cellulaire a par ailleurs démontré que la présence de lysophospholipides dans les micelles mixtes permet d’améliorer la sécrétion de l’ALA dans les lipoprotéines. D’autre part, le devenir métabolique de l’ALA dépend de sa régiolocalisation sur le triglycéride alimentaire. En effet, les résultats de l’étude faisant appel aux TAG structurés montrent que la position interne n’est que partiellement conservée dans la lymphe, suggérant qu’une hydrolyse des 2-MAG serait opérée par une MG lipase. En conséquence, l’ensemble des résultats obtenus lors de cette étude montre que l’absorption et le transport de l’ALA seraient uniquement modulés selon la forme physique de l’acide gras alors que son devenir et son utilisation métabolique dépendraient de sa régiolocalisation sur le TAG alimentaire. Ces deux facteurs réunis permettraient dés lors de prévenir l’ALA d’une β-oxydation précoce, en vue de favoriser son élongation en dérivés supérieurs dans les tissus cibles. / The last recommended nutrient intakes advise a higher consumption of n-3 polyunsaturated fatty acids series than currently found in French diet. In this context, it was appropriate to apprehend the factors that could modulate the absorption and fate of their leader, ALA, using two approaches, one in vivo (rat), the other one in vitro (Caco-2). The study on the metabolic fate of ALA, according to its physical and chemical submission form, was conducted with "models" lipids (structured TAG) or natural (flaxseed oil) rich in ALA, and according to different lipid systems (oil in continuous phase or in emulsion type oil in water and with variable phospholipid composition). Results obtained in vivo and in vitro for flaxseed oil (emulsion or not) show that emulsification enhances the recovery of ALA at the intestinal level but also improves its lymphatic concentration. The cell study also demonstrated that the presence of lysophospholipids in mixed micelles can improve the secretion of ALA in lipoproteins. On the other hand, the metabolic fate of ALA depends on its location on the glycerol backbone of the dietary triglyceride. The results of the study using structured TAG show that the internal position is partially preserved in lymph, suggesting that a hydrolysis of 2 - MAG by MG lipase could occur. Accordingly, all of the results obtained in this study shows that the absorption and transport of ALA would only be modulated according to the physical form of the fatty acid while its fate and its metabolic use would depend on its location on the dietary TAG. These two factors combined would then allow preventing the early β-oxidation of ALA in order to promote its elongation in higher derivatives in the target tissues.