Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.

N-substituted pantothenamides are analogues of pantothenate, the precursor of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive target for drug discovery due to low sequence homology between bacterial and human PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is located within a highly aromatic pocket, suggesting that an aromatic substituent may enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved. The structure reveals that the pyridine ring adopts alternative conformations in the aromatic pocket, providing the structural basis for further improvement of pantothenamide-binding to KpPanK.

CYP2D6 metabolizes a range of centrally acting drugs, neurotoxins, and endogenous neurochemicals. Higher levels of brain, but not liver, CYP2D6 have been identified in alcoholics and smokers, suggesting exposure to ethanol and/or nicotine may induce brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression. METHODS: Monkeys were randomized into 4 groups of 10/group consisting of a control group, ethanol-only group, nicotine-only group, and a combined ethanol and nicotine group; treatments occurred for 64 days. RESULTS: Exposure to chronic ethanol and nicotine induced CYP2D across various brain regions and cell types, particularly when both drugs were given in combination. No changes in protein levels were observed in liver or in CYP2D mRNA levels in liver and brain. CONCLUSIONS: Ethanol and nicotine increase brain CYP2D levels, which may affect CNS drug response, neurodegeneration and personality among those exposed to alcohol and/or nicotine.

Endogenous brain compounds and xenobiotics, including the neurotoxins such as the amphetamine analogs 3,4-methylenedioxymethamphetamine (MDMA,Ecstasy), methamphetamine (METH, Speed) and methylenedioxyamphetamine (MDA, active metabolite of MDMA), may be bioactivated by prostaglandin H synthase (PHS) to free radicals that generate reactive oxygen species (ROS). In the absence of adequate antioxidant or repair mechanisms, ROS oxidize macromolecules such as DNA, protein and lipids, which can lead to toxicity. In vitro, we evaluated bioactivation using both purified ovine PHS-1 and cultured cells stably overexpressing either human PHS-1 or hPHS-2 isozymes. We found the neurotransmitter dopamine, its precursors and some metabolites, as well as METH and MDA, can be bioactivated by ovine and/or human PHS in an isozyme-dependent fashion that generates ROS, which oxidize DNA and protein and increase toxicity. This process is blocked by both the PHS inhibitor acetylsalicylic acid (ASA) and the ROS detoxifying enzyme catalase. Our data are the first to reveal isozyme-dependent bioactivation by PHS as a potential mechanism for enhanced susceptibility to both exogenous and endogenous neurotoxins, the latter of which may be particularly important in aging. METH-initiated ROS can also activate redox-sensitive transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in the induction of an array of protective mechanisms in both adult and fetal brain. Using Nrf2 knockout mice, we showed Nrf2 has a novel neuroprotective role in METH-initiated oxidative stress, neurotoxicity and functional deficits in both fetal development and adulthood, especially with multiple exposures allowing time for the induction of neuroprotective mechanisms. Our studies are the first to show that Nrf2 afforded protection against both motor coordination deficits and olfactory deficits caused by METH in utero and in adults, suggesting that deficiencies in Nrf2 activation constitute a risk factor for ROS-mediated neurotoxicity in the fetus and adult.

Cytochrome P450 3A isozymes (CYP3A) metabolize approximately 50% of therapeutic drugs. It has recently been discovered that human CYP3A mRNA levels can be induced by hypertonicity; a physiological state not previously linked to its regulation. The osmosensitive transcription factor, Nuclear Factor of Activated T-Cells 5 (NFAT5), regulates multiple genes that restore osmolyte homeostasis and promote cell protection during osmotic stress. In silico examinations and in vitro experiments using reporters, knockdown and binding assays in the human intestinal cell line C2bbe1 have revealed an active tonicity-responsive enhancer (TonE) within CYP3A7 intron (+5417/+5427 from CYP3A7 transcriptional start site) that is responsible for NFAT5 binding and NFAT5-dependent regulation of CYP3A isoforms. In addition, hypertonicity-mediated CYP3A induction is also observed in both hepatic and intestinal cell lines. Effects of tonicity changes on in vivo CYP3A expression and function were examined in a humanized CYP3A transgenic mouse with similar tissue expression in humans. More specifically, intervention with prolonged dehydration involving alternating between 24-hour cycles of water-deprivation and water ad lib for 1 week (cyclic water-deprivation; four 24-hour water-deprivation and three 24-hour water ad lib periods), increased expression of NFAT5 target genes Slc6a12 in the liver and kidney (2.5 ± 0.6-fold over water ad lib, n = 14, p = 0.04; and 3.1 ± 0.6-fold, n = 10, p = 0.02, respectively), Akr1b3 in the liver, and Slc5a3 in the kidney. Immunofluorescent microscopy revealed an increase of nuclear-distributed mouse NFAT5 in cyclic water-deprived animals, consistent with NFAT5 activation. Most importantly, CYP3A4 mRNA levels were noted to be elevated in the liver and kidney (11.8 ± 4.8-fold over water ad lib, n = 14, p = 0.04 and 2.2 ± 0.4-fold, n = 9, p = 0.02, respectively), with concurrent CYP3A protein and activity increase. Localized hypertonic environment in the gut was simulated by providing animals with a week-long high-salt diet. The effects of high-salt diet in the gut were similar to those of cyclic water-deprivation in the liver and kidney; where NFAT5 showed nuclear distribution and NFAT5 target gene expression (Slc6a12; 20.5 ± 6.7-fold over a week-long low-salt diet, n = 8, p = 0.02 and Slc6a6; 3.2 ± 0.7-fold, n = 10, p < 0.01, in the duodenum). Furthermore, an increase of CYP3A4 mRNA was observed (2.6 ± 0.5-fold over a week-long low-salt diet, n = 14, p = 0.03), with a corresponding rise in protein expression and activity levels. In summary, increased expression of in vitro and in vivo human CYP3A was achieved using a hypertonic stimulus; concurrent NFAT5 activation and NFAT5 target gene expression were observed. These results suggested a possible binding of activated NFAT5 to CYP3A TonE situated within the intronic region of CYP3A7. It could be further concluded that NFAT5 may be responsible for the hypertonic induction of human CYP3A.

N-substituted pantothenamides are analogues of pantothenate, the precursor of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive target for drug discovery due to low sequence homology between bacterial and human PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is located within a highly aromatic pocket, suggesting that an aromatic substituent may enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved. The structure reveals that the pyridine ring adopts alternative conformations in the aromatic pocket, providing the structural basis for further improvement of pantothenamide-binding to KpPanK.

Cocaethylene (CE) is a cocaine metabolite formed during alcohol and cocaine co-consumption. To our knowledge, no previous studies were conducted assessing CE as a biomarker indicating chronic excessive alcohol consumption in a suspected high-risk population. In this study, we hypothesized that hair CE can be an effective marker for alcohol consumption in a high-risk population. We recorded cocaine, benzoylecgonine, and CE levels in hair samples from individuals, establishing the predictive value of CE by comparing it to hair levels of the widely used hair fatty acid ethyl esters (FAEE), direct markers of chronic excessive alcohol consumption. CE had 14.04% sensitivity and 95.18% specificity in samples separating FAEE positive/negative results. The positive predictive value was 0.66, showing that the results for individuals with CE positive results were more than likely to be FAEE positive, but not conclusively. Thus, CE cannot be used as a definitive marker, indicating chronic excessive alcohol consumption.

Polybrominated diphenyl ethers (PBDEs) are synthetic chemicals that are added to a variety of consumer products as flame-retardants. The ubiquitous nature and endocrine disrupting properties of PBDEs are a public concern. A pilot study was performed to investigate whether in utero PBDE exposure, as measured in maternal hair, is associated with genitourinary tract malformations in male infants. In addition, we compared PBDE levels in maternal and infant hair and used segmental analysis to investigate how PBDEs varied along the shaft. Preliminary results suggest a trend toward an elevated PBDE body burden in mothers whose infants were born with genitourinary tract malformations; this was significant for some PBDEs. The sum of PBDEs (ΣPBDEs) in maternal hair did not correlate with infant hair levels; children had significantly greater levels. A significant increase in the ΣPBDEs was observed in distal hair suggesting hair PBDEs may be reflective of both internal and external exposure.

CYP2D6 metabolizes a range of centrally acting drugs, neurotoxins, and endogenous neurochemicals. Higher levels of brain, but not liver, CYP2D6 have been identified in alcoholics and smokers, suggesting exposure to ethanol and/or nicotine may induce brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression. METHODS: Monkeys were randomized into 4 groups of 10/group consisting of a control group, ethanol-only group, nicotine-only group, and a combined ethanol and nicotine group; treatments occurred for 64 days. RESULTS: Exposure to chronic ethanol and nicotine induced CYP2D across various brain regions and cell types, particularly when both drugs were given in combination. No changes in protein levels were observed in liver or in CYP2D mRNA levels in liver and brain. CONCLUSIONS: Ethanol and nicotine increase brain CYP2D levels, which may affect CNS drug response, neurodegeneration and personality among those exposed to alcohol and/or nicotine.

La variabilité interindividuelle dans la réponse aux médicaments constitue une problématique importante pouvant causer des effets indésirables ou l’échec d’un traitement. Ces variabilités peuvent être causées par une diminution de l’activité de l’enzyme responsable du métabolisme de certains médicaments, fréquemment les cytochromes P450, un système enzymatique majeur dans le métabolisme de ces derniers. Ces enzymes sont sujets à des mutations génétiques appelées polymorphismes, qui altèrent l’activité métabolique. Il est donc important d’évaluer le rôle de ces enzymes dans le métabolisme des médicaments afin d’identifier leur responsabilité dans la variabilité interindividuelle de la réponse au traitement. Parmi l’important système enzymatique que représentent les cytochromes P450, l’isoenzyme CYP2D6 est particulièrement étudiée, ses variations métaboliques revêtant une haute importance clinique. L’un des substrats du CYP2D6 est l’oxycodone, un analgésique narcotique largement prescrit en clinique. Une grande variabilité est observée dans la réponse analgésique à l’oxycodone, variabilité pouvant être causée par un polymorphisme génétique. Il est connu que des variations génétiques dans le CYP2D6 compromettent la réponse analgésique à la codéine en rendant moins importante la formation de son métabolite actif, la morphine. Par analogie, plusieurs études supportent l’hypothèse selon laquelle le métabolite oxymorphone, formée par l’isoenzyme CYP2D6, serait responsable de l’analgésie de l’oxycodone. Une déficience génétique de l’enzyme compromettrait la réponse analgésique au médicament. Les travaux effectués dans le cadre de ce mémoire ont démontré que l’inhibition du CYP2D6 chez des sujets volontaires réduit de moitié la production d’oxymorphone, confirmant l’importante implication de l’enzyme dans le métabolisme de l’oxycodone. Ces résultats démontrent une forte ressemblance avec le métabolisme de la codéine, suggérant que l’oxymorphone pourrait être responsable de l’analgésie. Cependant, les travaux effectués n’ont pu établir de relation entre la concentration plasmatique d’oxymorphone et le niveau d’analgésie ressenti par les sujets. La continuation des études sur le mécanisme d’action de l’oxycodone dans la réponse analgésique est essentielle afin d’établir la source des variabilités interindividuelles expérimentées par les patients et ainsi d’éviter des effets secondaires ou lacunes dans le traitement. / Intersubject variability in drug response is an important issue provoking side effects or treatment failure. Such variability may be caused by the decreased activity of the enzyme metabolising the drug, frequently cytochromes P450, a major enzyme system in drug metabolism. These enzymes are prone to genetic mutations called polymorphisms, which alter their metabolic activity. It is therefore important to assess the role of these enzymes to identify their responsibility in the intersubject variability of the drug. Among the important enzyme system that represents the cytochrome P450, CYP2D6 is particularly studied for its genetic polymorphisms, which are of clinical importance. One of CYP2D6 substrates is oxycodone, a narcotic analgesic widely prescribed in clinical practice. A large variability is observed in the analgesic response to oxycodone, which could be caused by genetic polymorphism. It is known that these variations affect the analgesic response to codeine, which form the active metabolite morphine by CYP2D6 to be effective. Several studies support the hypothesis that oxymorphone, a metabolite formed by CYP2D6, has the analgesia properties, in a similar mechanism to codeine. A genetic deficiency in the enzyme would compromise the analgesic response to the drug. Results obtained from our laboratory indicate that inhibition of CYP2D6 halved oxymorphone production, confirming the significant involvement of the enzyme in the metabolism of oxycodone. These results demonstrate a strong resemblance to codeine metabolism, suggesting that oxymorphone may be responsible for analgesia. We could not find a relationship between plasma concentration of oxymorphone and analgesia level experienced by subjects. Studies on oxycodone mecanism of action in the analgesic response should continue to establish the source of intersubject variability experienced by patients and thus avoid side effects or gaps in treatment.