The clinical use of xylazine may result in morbidity and mortality in small ruminants, and it was suspected that exposure to changes in environmental temperature may contribute to these effects. Xylazine hydrochloride was administered intravenously at a dose of 0.1 mg/kg to a group of six indigenous domestic goats with a mean body mass of 28.2 kg. Xylazine was administered at a room temperature of 14°C and relative humidity of 33%, at 24°C and a relative humidity of 55%, and at 34°C with a relative humidity of 65%. The following variables were evaluated: clinical behaviour, cardiopulmonary function, haematology, acid-base balance, plasma glucose and insulin, body temperature, and the pharmacokinetic characteristics of xylazine. Xylazine administration resulted in transient restlessness, followed by sedation, muscle relaxation, and salivation. The onset of these clinical signs was not influenced by environmental conditions. Administration of xylazine resulted in a transient increase in respiratory rate in the 24 and 34°C environments. In the 14°C environment, the respiratory rate decreased significantly (p<0.05) from baseline and continued to decrease for the full duration of the 60 minutes observation period. Heart rate decreased in all three environments, but this decrease was only significant in the 14°C environment for the duration of the observation period. Changes in haemoglobin concentration, haematocrit, red blood cell count and mean red blood cell volume were significantly (p<0.05) different 15 minutes after xylazine administration and continued to be so for the duration of the observation period. Total serum protein changed significantly (p<0.05) in the 24° and 34°C environments from 15 minutes after xylazine administration. The white cell count changed significantly (p<0.05) from 15 minutes after xylazine administration for the duration of the observation period in all three environments. Significant (p<0.05) changes occurred after xylazine administration in acid-base balance and arterial blood gas variables independent of environmental conditions. Arterial pH and the partial pressure of oxygen decreased significantly within 5 minutes of xylazine administration, and the partial pressure of carbon dioxide, total carbon dioxide and base excess increased significantly (p<0.05). Environmental conditions had no observable on plasma glucose and insulin concentration. Significant (p<0.05) changes occurred in all three environments. Environmental conditions had no influence on body temperature in the control (untreated) animals. Following the administration of xylazine, the body temperature of the goats in the 14 and 24°C environments was significantly (p<0.05) lower than that of the goats in the 34°C environment. The maximum decrease in oesophageal temperature of 1.57°C was observed 60 minutes after xy1azine administration to goats maintained in the 14°C environment. Environmental conditions had no influence on all of the pharrnacokinetic parameters of xylazine hydrochloride evaluated. It is concluded that apart from changes in body temperature, changes that occurred in clinical and pharmacokinetic variables after xylazine administration, were independent of the three environmental temperature and humidity conditions. / Thesis (DPhil (Surgery))--University of Pretoria, 1999. / Companion Animal Clinical Studies / unrestricted

Drug metabolism is a specialised subset of xenobiotic metabolism, pertaining to the breakdown and elimination of pharmaceutical drugs. The enzymes involved in these pathways are the cytochrome P450 family of isozymes. Metabolism is an important factor in determining the pharmacological effects of drugs. The main aim of this study was to develop a system whereby the major metabolites of drugs can be produced in vitro. An in vitro system was developed and optimised using commercially prepared microsomes from rat liver and coumarin (by monitoring its conversion to 7-hydroxycoumarin) as a model. The optimum running conditions for the incubations were 50 μM coumarin, 50 μg protein/ml microsomes, 1 mM NADP⁺, 5 mM G6P and 1U/ml G6PDH incubated for 30 minutes at 38℃. The HPLC method for the detection of coumarin and 7-hydroxycoumarin was also validated with respect to linearity, reproducibility, precision, accuracy and lower limits of detection and quantification. The system developed was then tested using microsomes prepared from fresh bovine liver on these ten drugs of interest in doping control in horse racing: diazepam, nordiazepam, oxazepam, promazine, acepromazine, chlorpromazine, morphine, codeine, etoricoxib and lumiracoxib. The bovine liver microsomes were prepared using differential centrifugation and had activity on a par with the commercial preparations. This in vitro system metabolised the drugs and produced both phase I and II metabolites, similar to those observed in humans and horses in vivo. For example, the major metabolites of the benzodiazepine drug, diazepam, nordiazepam, temazepam and oxazepam as well as the glucuronidated phase II products were all found after incubations with the bovine liver microsomes. The metabolism of the drugs was also investigated in silico using the computational procedure, MetaSite. MetaSite was able to successfully predict known metabolites for most of the drugs studied. Differences were observed from the in vitro incubations and this is most likely due to MetaSite using only human cytochrome P450s for analysis.

Indiana University-Purdue University Indianapolis (IUPUI) / Cytochrome P450 2D6 (CYP2D6) is an important drug metabolizing enzyme that is involved in the metabolism of 20-25% of commonly prescribed drugs. There is interindividual variability in CYP2D6 enzyme activity and this leads to compromised metabolism of many drugs. Genetic and environmental factors explain only a part of the interindividual variability; the other factors that contribute to this variability are largely unknown. Hence, it becomes important to study CYP2D6 to understand the endogenous and exogenous factors that control its activity. The specific objective of this research was to determine the contribution of genetic and epigenetic factors in the regulation of CYP2D6 expression and activity. The specific aims were: (1) to identify the common CYP2D6 polymorphisms in Vietnamese and Filipino women with breast cancer and evaluate its association with plasma concentrations of endoxifen (an active metabolite of the breast cancer therapeutic drug, tamoxifen); (2) to identify the CYP2D6 copy number variations (CNVs) in these women and evaluate their association with endoxifen concentration; and (3) to identify microRNAs (miRNAs) that regulate the expression of CYP2D6 directly or indirectly. The results of this study indicated that: (1) in Vietnamese and Filipino women, the reduced function allele CYP2D6*10 was frequent (~55%) and it was significantly associated with reduced endoxifen concentration; (2) in these women, only 39% carried two copies of the CYP2D6 gene, the rest had a genomic imbalance for CYP2D6, primarily involving the CYP2D6(*36)n-*10 allele. However, carrying multiple copies of CYP2D6*36 allele did not significantly affect CYP2D6 activity, suggesting that multiple copies of a gene does not always translate to additive effects; and (3) microRNAs were identified to target HNF4A, a transcriptional factor that regulates CYP2D6 expression. These miRNAs are likely to play an important role in the indirect regulation of CYP2D6. Taken together, these results emphasize on the role of polymorphisms, CNVs and possibly miRNAs in the regulation of CYP2D6. These clinically important biomarkers will help to improve the efficacy and reduce the side effects of many CYP2D6 substrate drugs and thus contribute to personalization of drug therapy.

Indiana University-Purdue University Indianapolis (IUPUI) / Aromatase is the cytochrome P450 enzyme that converts androgens to estrogens. Aromatase is the target of the aromatase inhibitor class of drugs widely used to treat estrogen-mediated conditions including breast cancer. Little is known about the role of this enzyme in drug metabolism or in drug interactions. Since this lack of knowledge has been an impediment to optimal therapy, it is important to understand these roles of aromatase. Therefore, a comprehensive series of studies was carried out to characterize its ability to metabolize drugs and its susceptibility to inhibition by xenobiotics. The overall objective of this work was to better understand the interactions of small molecules with aromatase and to use this new knowledge to predict aromatase-mediated drug interactions and anticipate novel molecular structures that interact with the enzyme. Aromatase was shown to be a drug metabolizing enzyme able to metabolize methadone both in vitro (Km of 314 μM) and in vivo (22% of methadone clearance). A number of novel aromatase inhibitors that employ diverse kinetic mechanisms were identified. These include a potent competitive inhibitor: norendoxifen (Ki of 35 nM), two non-competitive inhibitors: endoxifen (Ki of 4.0 μM) and N-desmethyl-tamoxifen (Ki of 15.9 μM), a mechanism-based inhibitor: methadone (KI of 40.6 ± 2.8 μM; kinact of 0.061 ± 0.001 min-1), and a stereoselective inhibitor: naringenin (IC50s of 2.8 μM for (R)-enatiomer and 1.4 μM for (S)-enatiomer). Through investigation of the structure-potency relationships so discovered, a series of new biochemical structures to be exploited as aromatase inhibitors were identified. These studies have identified new roles for aromatase as a catalyst for methadone metabolism and as a mediator of the effects of tamoxifen by demonstrating that a number of its metabolites can act as aromatase inhibitors. This work also provides a new mechanistic framework for the design of novel aromatase inhibitors that can be used in breast cancer. Overall, the data suggest ways to more consistently treat breast cancer with current medications, to better anticipate drug interactions, and therefore to improve the quality of life of patients in ways that minimize side effects, while optimizing therapeutic benefits, in each person treated.

The urinary excretion of D-glucaric acid (DGA) has been used as a nonspecific measure of the induction of hepatic enzymes associated with drug metabolism in man. A survey of 124 nonmedicated men (18-56 years of age), who kept a 5-day food and beverage intake record and collected their total urinary output for the last three days of the five, was conducted to assess the relationship between DGA excretion and various dietary factors. Eighteen nonmedicated, healthy women collected the same data, but started recording on the eighth day from the commencement of menstruation. DGA was determined by an enzymatic assay and recorded as micromoles D-glucaro-1,4-lactone/gm creatinine. There was a positive association between total DGA excreted and the use of marijuana, caffeine, and tobacco products, the heaviest users excreting significantly higher levels of urinary DGA than the moderate or low users of the same substance. Analysis of variance of mean DGA excretion also revealed significant differences between females (17.0±3.7) and males (14.3±5.2): male vegetarians (17.4±5.5) and nonvegetarians ( 13.9±5.1) ; and female vegetarians (19.8±4.6) and nonvegetarians (16.2±3.1). Alcohol consumption and family history of cancer incidences were not significantly related to DGA excretion in either sex. Multiple regression analyses revealed that consumption were the two vegetarianism and caffeine strongest predictors of DGA excretion, while alcohol and marijuana consumption affected DGA the least. These results indicate that dietary and environmental factors can exert a significant effect on DGA excretion, and these associations may identify dietary inducers of hepatic enzymes associated with xenobiotic biotransformations in humans. / Master of Science

The fate of halofuginone and its metabolites from poultry when they were refed to rats was investigated. During a 6 d accumulation period rats excreted .607 uCi or 53.8% of the administered dose in the feces. The urinary output accounted for .157 uCi or 13.7% of intake. The rats respired 1.5% of the administered 14 C. Level of radioactivity in the tissues after the accumulation period was not significantly different from that found in control animals. After the accumulation period the withdrawal pattern of halofuginone was investigated using two withdrawal times, 5 and 10 d. During the 5 d withdrawal period the rats excreted .105 uci in the feces and .03 uCi in the urine, or 9.5 and 2.7% of intake, respectively. By 24 h post-withdrawal, levels in the feces did not differ significantly from the controls. The amount of radioactivity in the urine, however, remained higher (P<.05) than in control animals. The greatest amounts of 14 C were found in the kidneys but tissue levels did not differ from levels in control animals. During the last 5 d of withdrawal fecal excretion accounted for 1.2% of intake with the urine contributing .7%. Levels of 14 C, significantly higher than the control, continued to be excreted in the urine. The kidneys contained modest amounts of radioactivity, although not significantly different from the control. By d 10 of the withdrawal period, 92.3% of the 14 C intake had been accounted for in the rat study. A sheep study was conducted using wether lambs consuming a silage composed of 60% excreta, 20% ground hay, 10% ground corn and 10% dry molasses. The feces contained detectable levels of the drug. Sheep were slaughtered after a 16 d accumulation period. None of the analyzed tissues except the eyes contained halofuginone. When the remaining sheep were started on a withdrawal period, the halofuginone disappeared from the feces between d 3 and 4. No traces of halofuginone were found in tissue samples from sheep slaughtered 5 and 10 d post-withdrawal. / M. S.

Thesis submitted in fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology 2014 / According to the World Health Organization, cancer is the leading cause of death in the developed world, while it is the second leading cause of death in the developing world. In particular, liver cancer is the fifth most commonly diagnosed cancer in men, however, it is the second most frequent cause of death, responsible for an estimated 700,000 deaths annually. General limited access to health services, including treatment and the overall management of cancer in developing countries often contribute to the increased mortality rates when compared to developed countries. For centuries, medicinal plants have been used to prevent, and to a certain extent, treat cancer as a readily available and affordable alternative. In many instances, the curative or preventative claims still remain anecdotal. However, increasing evidence suggest that polyphenolic components of plants possess antioxidant activities, which are credited with curative/beneficial properties of medicinal plants. The curative properties could either be related to the primary compounds present in the plant itself, or the bio-activation products of plant components affecting hepatic drug metabolising and antioxidant enzymes systems related to carcinogen metabolism and maintaining oxidative homeostasis, respectively. Similarly, chronic consumption of medicinal plants could also result in hepatotoxicity, either caused by the primary plant components or bio-activation products. Due to these observations it is paramount to understand the mechanisms involved in the metabolism of plant components to critically assess beneficial versus potential harmful properties associated with chronic consumption. The focus of the current study was aimed at elucidating the bio-activity of four multipurpose indigenous plants to Southern Africa, i.e. Adansonia digitata, Agathosma betulina, Siphonochilus aethiopicus and Myrothamnus flabellifolius. Traditionally, A. digitata has been used as an immunostimulant, anti-inflammatory and analgesic agent, while also as an antipyretic agent in the treatment of diarrhoea and dysentery. Similarly, traditional medicinal uses of A. betulina include treatment cholera, haematuria, calculus, kidney diseases, as well as infections of the bladder, urethra, and prostate among others. S. aethiopicus was traditionally employed to treat infections associated with pains and fevers, whereas M. flabellifolius served as treatment of conditions ranging from respiratory ailments, backache, kidney problems, haemorrhoids, chest pain, and asthma. In the first part of this study, the polyphenolic contents and antioxidant capacities of the four plants were characterised. The emphasis was placed on using different solvents, namely water, ethanol and acetone for the extraction of the plant material and different methodologies to assess the antioxidant contents and -capacities of the various extracts as both these factors can influence the outcome. When considering the antioxidant contents, total polyphenols, flavanols, and flavonols of the different solvent extracts prepared from the four plants were determined, whereas three different assays were used for the antioxidant capacities, i.e. oxygen radical absorbance capacity (ORAC), trolox equivalent antioxidant capacity (TEAC) and ferric-reducing antioxidant power (FRAP) assays. The A. digitata acetone extract had the highest (7.121 mg gallic acid equivalent (GAE)/milligram (mg) soluble solids), whereas the water extract of the same plant had the lowest total phenolic content (0.008 mg GAE/mg soluble solids). In general, the acetone extracts demonstrated the highest total polyphenol, flavanol, and flavonol contents, followed by the ethanol extracts, with the water extracts having the lowest contents. M. flabellifolius was the only distinct deviation from this rule, where the water extract demonstrated the highest total polyphenol content. Considering antioxidant capacities, the acetone extracts provided the highest antioxidant capacities for all plants when assessed using the TEAC (8.56-32.68 milimole (mmole) trolox equivalent (TE)/mg soluble solids) and FRAP (5.69-37.39 mmole ascorbic acid equivalent/mg soluble solids) antioxidant assays, with the exception of M. flabellifolius where the water extract demonstrated the highest activity (22.73 mmole ascorbic acid equivalent/mg soluble solids). Antioxidant capacity determinations with TEAC and FRAP assays followed similar patterns, which were different from capacities determined by the ORAC (0.46-533.54 mmoleTE/mg of soluble solids) assay. Corroborating the antioxidant content findings, the acetone extracts also demonstrated the highest antioxidant capacities (140.41-533.54 mmoleTE/mg of soluble solids), followed by ethanol (94.62-151.29 mmoleTE/mg of soluble solids) and water (0.46-134.02 mmoleTE/mg of soluble solids). Only M. flabellifolius (TEAC and FRAP) and S. aethiopicus (FRAP) deviated from this trend. Correlations between the polyphenolic contents and antioxidant capacities indicated that acetone and ethanol were more effective in extracting polyphenolic compounds than water, while also providing extracts with superior antioxidant activities. Furthermore, ORAC assay was the antioxidant capacity determining assay of choice for the aqueous plant extracts, whereas the TEAC and FRAP assays were more suitable when determining the antioxidant capacities of the acetone and ethanol plant extracts. These results confirm the notion that no single assay can comprehensively determine the antioxidant activities of plant extracts and that a battery of assays should be used, as the various antioxidant capacity determination techniques use different substrates with different targets for measurement. The second part of this study comprised an in vivo experimental animal model to assess the potential toxicity, antioxidant status and modulation of the hepatic phase 2 drug metabolising enzymes following chronic consumption of the various plant extracts in male Fisher rats. Rats consumed aqueous extracts of the various plants (2% and 5% (w/v)) as the sole source of drinking fluid for 90 days, and the serum chemical pathology parameters for monitoring liver and kidney function conducted. These included alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), total iron (Fe), and creatinine (CREA). Parameters for blood and hepatic redox status included total polyphenols, ORAC, reduced glutathione (GSH), oxidised glutathione (GSSG), their ratio (GSH:GSSG), conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS). Assessment of the phase 2 hepatic xenobiotic metabolising enzymes included glutathione S-transferase (GST)  and activity in the cytosolic fraction and, UDP-glucuronosyltransferase (UDP-GT) activity in liver microsomes. When considering the liver and kidney function none of the plant extracts induced any significant toxicity, while 2% A. digitata significantly increased serum Fe. When considering the redox status, the whole blood and liver samples yielded similar results, with significant decreases in oxidised glutathione (GSSG) in rats consuming the 2% M. flabellifolius (82.76 mole/L) and 5% A. digitata (90.42 mole/L) with a resultant significant increase in the glutathione redox status (GSH:GSSG ratio of 5.69 and 5.64, respectively) when compared to rats consuming water (4.77). The GSH:GSSG ratio was also significantly increased by consumption of 2% A. betulina (8.45) and 5% S. aethiopicus (5.99). The consumption of all plant extracts, except 5% A. betulina and M. flabellifolius, significantly increased lipid peroxidation in the plasma CDs assay. These results indicated an increased antioxidant capacity in the liver with/without an associated reduced cellular oxidative stress status, which could be interpreted as a reduced susceptibility to oxidative damage. When considering the phase 2 hepatic enzymes, none of the plant extracts caused any significant changes in GST, GST or UDP-GT activities. The third part investigated the chemoprotective properties against cancer promotion in the liver utilising diethylnitrosamine (DEN) as cancer initiator and maize culture material of Fusarium verticillioides, containing the fumonisin B mycotoxins, as promoters in male Fischer rats. The rats consumed 2% (w/v) aqueous extracts of A. digitata, A. betulina, and S. aethiopicus over 28 days after cancer initiation and liver sections subjected to glutathione-S-transferase placental form positive GSTP+ staining and pre-cancerous liver foci categorised according to size. In addition, blood and liver analyses were done as described in the chronic feeding study above. Consumption of the A. digitata and, to a certain extent, S. aethiopicus extracts, altered the oxidative stress status in the liver as indicated by the increased lipid peroxidation, as determined by significantly increased liver CDs and the decreased GSH:GSSG ratio in the blood. This can be related to a subchronic toxicity due to the high total polyphenol intake as mentioned above. These underlying sub chronic toxic effects of A. digitata and S. aethiopicus are likely to be responsible for the observed inhibitory effect on the proliferation of GSTP+ minifoci in the liver. Hepatic phase 2 metabolising enzyme activities were not significantly altered by A. digitata and S. aethiopicus consumption, while GST activity was significantly increased by A. betulina treatment. Based on the findings of the current study, aqueous extracts of A. digitata, A. betulina, and S. aethiopicus may serve as hepatoprotectors with a potential to modulate liver carcinogenesis, specifically cancer promotion. To our knowledge, no other studies have attempted to describe the possible chemoprevention mechanisms of these indigenous medicinal plants. Assessments of phase 1 hepatic enzymes and other antioxidant enzymes are suggested for future studies to further describe biochemical and molecular mechanisms associated with consumption of these extracts. Additionally, identifying main compounds present in the plant extracts could culminate in development of drugs and novel nutraceuticals. It is also recommended that increasing concentrations of the plant extracts and/or the ethanol extracts to be used in future studies to better describe dose-responses of the different plants in liver carcinogenesis.

When considering oral administration, drug release from its pharmaceutical form and its dissolution into gastrointestinal fluids generally precedes absorption and systemic availability. The solubility-dissolution behaviour of a drug is frequently the rate-limiting step to absorption of drugs from the gastrointestinal tract (BCS class II drugs). Poor aqueous solubility has always been a very challenging obstacle as it is, together with membrane permeability, an essential factor in the limitation of a drug’s bioavailability following oral administration. Since an increasing number of newly developed drug candidates in pre-clinical development phases present poor water-solubility characteristics, there is a great need for formulation approaches to overcome this factor.<p><p>Out of the many ways to increase a product’s solubility/dissolution rate characteristics with the aim of enhancing its oral bioavailability, drug formulation as nanoparticles has received much-increased interest over the last decade. The hypothesis behind dissolution rate enhancement, considering drug particle size reduction to nanometer range, lies primarily in a much-increased effective surface area (Noyes-Whitney) presented by the resulting drug nanoparticles. Out of the various technologies available for drug particle size reduction to nanometer range, milling using high pressure homogenization is regarded as one of the simplest and most effective techniques. High pressure homogenization is a solvent-free process and is relatively rapid (time-saving). Furthermore, and most importantly, the scaling up of this technique is already established; processing capacities ranging from 3 l/h (e.g. EmulsiFlex C3®: minimum sample volume - 10 ml) to 1000 l/h (e.g. EmulsiFlex C1000®: minimum sample volume - 2 l).<p><p>Four model drugs were studied in this work. Nifedipine (NIF), an extensively studied poorly water-soluble drug in the literature, was used as the main model on which most of the development was done. In parallel to the work carried out on NIF, three UCB S.A. molecules currently under development were also studied as poorly water-soluble drugs: these being ucb-35440-3, UCB-A and UCB-B (salt of UCB-A). These three UCB S.A. model drugs are, contrarily to NIF, predicted highly dosed drugs and are weak bases, and thus present pH-dependent solubility profiles, which allowed us to investigate model drugs with different profiles.<p><p>Firstly, investigations regarding appropriate formulation development (stabilizer (surfactant) selection) and appropriate high pressure homogenization operating conditions (pre-milling cycles, influence of the number of high pressure homogenizing cycles, influence of homogenizing pressure, influence of sample temperature) were made. It has been shown, through this development, for the four studied model drugs, that high pressure homogenization is an appropriate technique for reducing drug particle size to nanometer range (NIF &61566; 290 nm, ucb-35440-3 &61566; 180 nm, UCB-A &61566; 350 nm and UCB-B &61566; 250 nm). Investigations regarding water-removal from the nanosuspensions obtained and most importantly regarding the redispersion characteristics of the retrieved powders (i.e. nanoparticles) were then carried out. In that regard, it has been shown that the presence of carriers in the formulation is essential for limiting nanoparticles agglomeration during the water-removal operation.<p><p>Drug crystalline state characterizations before and following particle size reduction were then carried out on the three studied model drugs, mainly through DSC and PXRD studies. In fact, one of the advantages of this particle size reduction approach (using high pressure homogenization), versus other frequently studied solubility/dissolution rate enhancement technologies (e.g. such as solid dispersions), is that original crystalline state shall not be altered in such a way that the achieved increased solubility and dissolution rate characteristics do not rely on the presence of the amorphous form of the drug; this furthermore implying a greater time-stability of the developed formulations. Through the data obtained, it has been shown that original drug crystalline state seems to be unaltered following particle size reduction.<p><p>In vitro solubility and dissolution characteristics were then evaluated on the formulations developed in order to verify the posed hypothesis regarding effective surface area increase. It has been shown through these studies that drug solubility and most importantly drug dissolution rate can be significantly enhanced for nanoparticulate systems (verified for NIF, ucb-35440-3, UCB-A and UCB-B). For example, solubility was enhanced from 26 µg/ml vs. 19.5 µg/ml for NIF nanoparticles and the dissolution characteristics showed that 100% of the tested dose (equivalent to 10 mg NIF) was already dissolved following 10 min vs. less than 5% for un-milled NIF. Following these very interesting and promising results, and preliminary to the in vivo pharmacokinetic studies carried out, in vitro permeation studies (apical to basolateral transfer studies) across intestinal cell models (Caco-2 and HT29-5M21 cultures and co-cultures) were carried out. This evaluation was only carried out using NIF as a model drug and showed a 6-fold increase in the permeation rate for NIF nanoparticles. The influence of chitosan (permeability enhancer/bioadhesive polymer) in the NIF nanoparticle formulation with regard to in vitro NIF permeation rate was also evaluated.<p><p>In vivo pharmacokinetic studies in rats were conducted using NIF and ucb-35440-3 as model drugs. The very different profiles of these two model drugs allowed us to retrieve interesting information regarding the in vivo behaviour of the developed formulations. As expected from the in vitro (i.e. solubility/dissolution/permeation) studies and results obtained for NIF, an increased extent of exposure could be observed for NIF nanoparticles versus un-milled NIF; the difference being more pronounced when the formulations were orally administered into capsules (2.5-fold increase in extent of exposure and 6-fold increase in Cmax). For ucb-35440-3, a poorly water-soluble weak base with a reported significant food effect considering oral bioavailability, an increased extent of exposure for nanoparticles, versus the un-milled drug, could only be observed in fasted state (4-fold increase in extent of exposure and 2.7-fold increase in Cmax). These different, diet-relative observations allowed us to put forward some limitations and precautions (considering poorly water-soluble weak bases) relative to the possibility of drug reprecipitation following stomach’s exiting, particularly if dissolution in the stomach is quite fast (e.g. nanoparticulate systems).<p><p>In parallel to the in vivo pharmacokinetic evaluation of NIF nanoparticles, evaluation of the antihypertensive effect of the systems developed following oral administration, using spontaneously hypertensive rats, was also carried out and compared to un-millled NIF. The results obtained showed a significant drop in systolic blood pressure for NIF nanoparticles (32% reduction of initial SBP following 30 min vs. 1% for un-milled NIF) and nicely complemented the in vitro and in vivo results obtained for NIF nanoparticles.<p><p>Finally, a stability study of the optimized NIF nanoparticle formulation was carried out with respect to reported ICH conditions (25°C/60% RH; 30°C/65% RH; 40°C/75% RH). The results showed that the studied NIF nanoparticle formulation retains all its original characteristics (dissolution, crystalline state, redispersion characteristics); this being verified over time (12 months) and for each of the three storage conditions studied.<p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished

CAR (Constitutive Androstane Receptor, NR1I3) et PXR (Pregnane X Receptor, NR1I2) sont deux récepteurs nucléaires dédiés à la reconna issance et à l'élimination de molécules lipophiles potentiellement toxiques pour l'organisme. Ces facteurs de transcription peuvent être activés par des ligands d'origines et de structures diverses (médicaments, polluants environnementaux, produits de l'alimentation et de phytothérapies). L'activation de ces récepteurs entraîne l'expression des gènes majeurs de la fonction de détoxication entéro-hépatique (CYP450, transférases, transporteurs) permettant l'élimination de ces toxiques. Dans ce travail, nous avons dans un premier temps 1) montré que CAR contrôle l'expression de Spot14, une protéine pro-lipogénique, et 2) nous avons identifié une nouvelle isoforme de PXR (PXR-small) codant uniquement pour le domaine de liaison des ligands de PXR. Nous avons pu déterminer les origines de transcription par 5'-RACE PCR et montrer que PXR-small représente environ 10% de l'ensemble des transcrits de PXR dans le tissu hépatique sain par une approche de PCR qua ntitative. Nous avons pu détecter sa présence par western-blot sur des extraits de protéines nucléaires issus de tissus hépatiques et de lignées cellulaires hépatiques. Par des expériences de gel retard, nous avons observé que cette nouvelle isoforme tronquée, qui ne code que pour le LBD de PXR, ne peut pas se lier à l'ADN. Des expériences de gènes rapporteurs suggèrent que cette isoforme se comporte comme un dominant négatif de PXR. Enfin, la présence d'un ilot CpG situé juste en amont de PXR-small suggère que cette nouvelle isoforme pourrait être régulée épigénétiquement par méthylation, notamment dans les cellules tumorales. / CAR (Constitutive Androstane Receptor, NR1I3) and PXR (Pregnane X Receptor, NR1I2) are two nuclear receptors devoted to the recognition and elimination of lipohilic molecules potentially toxic to the body.These transcription factors can be activated by ligands of different origins and structures (drugs, environmental pollutants, food products and herbal medicine...). The activation of these receptors leads to the expression of major genes of the detoxification process (CYP450, transferases, transporters) leading to the elimination of these toxics. In this work, we 1) showed that Spot14, a pro-lipogenic protein, is a target gene of CAR, then 2) we identified a novel isoform of PXR (PXR-small), coding only the ligand binding domain of PXR. By using 5'-RACE PXR, we established the origins of transcription of PXR-small and by quantitative PCR we observed that PXR-small represents about 10% of all PXR transcripts in human liver. By using western blo t, we detect its presence on nuclear protein extracts from liver tissues and hepatic cell lines. In Electromobility shift essays experiments, we observed that PXR-small cannot bind to DNA, while reporter essay experiments suggest that this isoform acts as a dominant negative of PXR. Finally, the presence of a CpG island just upstream of PXR-small suggests that this novel isoform might be regulated epigenetically by methylation, more particularly in tumor cells.

La bio-transformation naturelle des médicaments peut produire des métabolites toxiques; l’identification de ces métabolites est essentielle dans la stratégie de choix de molécules thérapeutiques. En appliquant les technologies de fermentation en bioréacteur des cellules hétérologues (souches d’E. coli recombinantes exprimant une iso-enzyme de cytochrome P450 humain avec la réductase humain), la bioconversion du substrat (principe actif) en ses métabolites de dégradation, a été réalisée à grande échelle (& / Doctorat en Sciences / info:eu-repo/semantics/nonPublished