41 |
Gone Fishing: Synthesis and Design of a Superparamagnetic Nanobait for Trapping Reactive Metabolites In Vivo.Tayyabi, Ehsen January 2018 (has links)
Adverse drug reactions are common causes of medical injuries. Drug-induced hepatotoxicity remains one of the leading causes of emergency room visits, FDA non-approval, and drug withdrawal from the market. We have investigated the ability of endogenous nucleophilic amino acid residues (K, H, and C) to selectively bind to reactive electrophilic drug metabolites, focusing on acetyl-para-aminophenol (APAP, i.e. Tylenol®), for which hepatotoxicity has recently re- emerged as a major health concern for Canadians. Three peptide sequences were synthesized bearing terminal nucleophilic residues, brominated phenylalanine residues, and c-terminal amides. These peptides were coupled to carboxy methyl dextran coated iron oxide nanoparticles (CMX- IONPs) with a hepatocyte targeting group. IONPs are known for their ability to act as T2-weighted MRI contrast agents, giving us the ability to track them in vivo. This study begins to establish a nanotechnology-based method for the in vivo trapping of NAPQI, the reactive metabolite of APAP, using a cysteine bearing IONP.
|
42 |
Cytochrome P450 isoform-specific <em>in vitro</em> methods to predict drug metabolism and interactionsTaavitsainen, P. (Päivi) 13 February 2001 (has links)
Abstract
Cytochromes P450 (P450, CYP) are a superfamily of enzymes that participate
especially in the oxidative metabolism of various xenobiotics and endogenous
compounds.
The major goal of this study was to characterise suitable methods for routine
preclinical in vitro testing of new chemical entities (NCE)
and to test the methods for the affinity screening of selected drugs.
In vitro methods used involve the utilisation of human
liver microsomes for studies with P450-selective reference inhibitors,
inhibitory antibodies and cDNA-expressed enzymes in cytochrome P450-catalysed
activities and for studying the reactions of selegiline and entacapone.
In this project, the CYP-catalysed oxidative in vitro
biotransformation of selegiline into its primary metabolites desmethylselegiline
and l-methamphetamine and the transformation of entacapone
into its in vitro metabolite
N-desethylentacapone were studied. The affinities of
selegiline, desmethylselegiline, l-methamphetamine,
entacapone, candesartan, eprosartan, irbesartan, losartan and valsartan to P450
enzymes were also elucidated, and the selectivity of tranylcypromine as a
CYP2A6-selective reference inhibitor was characterised.
The most important findings were that the methodology developed during this work
is suitable for preclinical in vitro testing of NCEs and
that the results obtained for the studied compounds are in line with the
available in vivo data.
By the in vitro testing methodology, it is possible to
target the in vivo interaction studies to the relevant
groups of compounds. The in vitro methods presented in this
thesis could also make the early phases of drug development more cost-effective.
Further, the number of animals used for in vivo testing in
preclinical metabolism and interaction studies can be markedly reduced by
effectively using this methodology.
|
43 |
Micro-RNA regulation of hepatic drug metabolism : age-related changes in micro-RNA expression and genetic variants in micro-RNA target sitesBurgess, Kimberly Sherrelle 31 August 2017 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Developmental changes in the liver significantly impact drug disposition. Due to the
emergence of microRNAs as important regulators of drug disposition, we hypothesize that
age-dependent change in microRNA expression and genetic variants in microRNA target
sites contribute to variability in drug disposition. In human liver tissues, expression of 533
microRNAs and over 14,000 genes were measured. In all, 114 microRNAs were
upregulated and 72 downregulated from fetal to pediatric, and 2 and 3, respectively, from
pediatric to adult. Among these microRNAs, 99 microRNA-mRNA interactions were
predicted or have previously been validated to target drug disposition genes and over
1,000 significant negative correlations were observed between miRNA-mRNA pairs. We
validated these interactions using various cell culture models. Genetic variants in the
promoter and coding regions of drug disposition genes have also been shown to alter
enzyme expression and/or activity. However, these variants do not account for all
variability in enzyme activity. Emerging evidence has shown that variants in the 3’UTR
may explain variable drug response by altering microRNA regulation. Five 3’UTR variants
were associated with significantly altered CYP2B6 activity in healthy human volunteers.
The rs70950385 (AG>CA) variant was associated with decreased CYP2B6 activity among
normal metabolizers. In vitro luciferase assays confirmed that the CA allele altered miR
1275 targeting of CYP2B6 mRNA. Due to the large number of 3’UTR variants predicted
to alter microRNA regulation, a high-throughput method, PASSPORT-seq, was developed
to test over 100 3’UTR variants simultaneously in different cell lines. Thirty-eight variants resulted in FDR-significant altered expression between wild-type and variant sequences.
Our data suggest a mechanism for the marked changes in hepatic gene expression
between the fetal and pediatric developmental periods, support a role for these age
dependent microRNAs in regulating drug disposition, and provide strong evidence that
3’UTR variants are also an important source of variability in drug disposition.
|
44 |
Canine hepatic slices as a model for studying drug toxicity and metabolismScott, Maya Millicent 16 August 2006 (has links)
Tissue slices can be made from organs, such as liver, kidney, brain, and heart, and from various species including humans, dogs, non-human primates, rats and mice. It has been demonstrated that human and rat liver slices are viable for up to 2 days, and liver slices have been extensively used as an in vitro method to study hepatic drug metabolism and toxicity in humans. The objective of this study was to determine the utility of canine hepatic slices as an in vitro model for studying drug metabolism and hepatotoxicity in dogs. Canine hepatic slices were incubated in media containing various drugs to determine the hepatotoxicity of the agents and the ability of the slices to metabolize the drugs. The toxicity of phenobarbital, primidone, lidocaine and carprofen to canine hepatic slices was assessed by determining changes in supernatant concentrations of potassium ions and adenosine triphosphate (ATP); histologic lesions were determined as necrosis, extent of vacuolation and severity of vacuolation. Xenobiotic drug metabolizing enzymatic activity was investigated by determining the metabolism of lidocaine to monoethylglycinexylidide (MEGX), and administration of phenobarbital plus primidone was used as a positive control for hepatotoxicity in dogs. The function of drug-metabolizing enzymes was demonstrated by the successful metabolism of lidocaine to MEGX. Carprofen, a drug which causes idiosyncratic hepatic disease in dogs, did not show any hepatotoxicity at concentrations of 10, 50 and 100 µg/ml using potassium ion levels, ATP concentrations and histology as indicators of hepatotoxicity. Slices incubated in media without drug showed no toxicity over 24 hours based on potassium ion and ATP supernatant concentrations while significant increases in histologic lesions were noted at 8, 12 and 24 hours. Canine hepatic slices were a useful model for examining drug metabolism and toxicity for up to 24 hours.
|
45 |
Heterologous expression systems for metabolite production during early drug researchWynant, Inneke S.A. 05 July 2010 (has links)
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 (g-g). Notre choix s’est porté sur le complexe hCYP3A4/HR fonctionnel produit par un hôte E. coli. Les cellules intactes ou les membranes cellulaires peuvent être exploitées comme biocatalyseur dans un système bioréacteur. Cependant, la faible solubilité des principes actifs dans des milieux de bioconversion aqueuse limitent le rendement. Un bioréacteur biphasique a été étudié. En solution, plusieurs combinaisons eau/solvants organiques conciliant la viabilité des cellules, la solubilité des principes actifs et produits de réaction et la catalyse des complexes enzymatiques ont conduit à l’établissement d’un mélange approprié. Cependant, ces combinaisons présentent toujours une inhibition importante du pouvoir catalytique des complexes enzymatiques. Pour minimiser un effet dénaturant possible des solvants sur le système enzymatique, ce dernier a été maintenu dans un environnement aqueux en immobilisant les cellules et/ou les membranes cellulaires dans une matrice hydrophile. L’alginate de calcium apparaît être une matrice d’immobilisation idéale pour les membranes assurant la fonctionnalité du complexe CYP/HR et permettant en outre un stockage à long terme des préparations. Par contre, l’immobilisation des cellules dans diverses matrices, si elle permet une viabilité et une conservation à long terme des souches recombinantes, ne permet aucune expression de l’activité enzymatique présente dans les cellules. La combinaison d’une localisation du complexe hCYP/HR fonctionnel dans la membrane interne et d’une perméabilité réduite des cellules d’E. coli (immobilisées) en est une explication possible mais non-démontrée. Entre-temps, cette technologie de bioréacteur homogène biphasique ou par immobilisation des membranes cellulaires a été utilisée plusieurs reprises pour produire des métabolites humains à partir de divers principes actifs. Ces métabolites ont été purifiés avec succès, démontrant que cette approche technologique est compétitive comparée aux procédures conventionnelles. Néanmoins, de nouvelles pistes de recherche seraient extrêmement intéressantes. La localisation des complexes enzymatiques recombinants en surface des cellules permettrait de concilier les propriétés hydrophobes des principes actifs et l’environnement hydrophile nécessaire aux enzymes. D’autre part une investigation de complexes enzymatiques résistant aux solvants pourrait remplacer avantageusement l’immobilisation.
|
46 |
Investigation of a Metabolic Pathway Leading to an Idiosyncratic Drug Reaction: Is the Sulfate of 12-Hydroxynevirapine Responsible for the Skin Rash in Brown Norway rats?Novalen, Maria 13 January 2011 (has links)
An animal model of nevirapine (NVP)-induced skin rash was used to test the hypothesis that sulfonation of 12-OH NVP, a metabolite of NVP proven essential for rash development, is the link between 12-OH NVP and the skin rash. Female Brown Norway (BN) rats were co-treated with NVP or 12-OH NVP and sulfation inhibitors dehydroepiandrosterone (DHEA) and salicylamide. Co-treatment with salicylamide markedly decreased formation of the sulfate conjugate but did not prevent development of the rash suggesting that the sulfate is not involved. However, it is not known whether the sulfate formation in the skin was affected. Co-treatments with DHEA decreased the sulfate formation and prevented the rash but also had other effects on NVP metabolism. This implies that the sulfate metabolite is responsible for the rash. Additional studies will be required to resolve these conflicting results.
|
47 |
Investigation of a Metabolic Pathway Leading to an Idiosyncratic Drug Reaction: Is the Sulfate of 12-Hydroxynevirapine Responsible for the Skin Rash in Brown Norway rats?Novalen, Maria 13 January 2011 (has links)
An animal model of nevirapine (NVP)-induced skin rash was used to test the hypothesis that sulfonation of 12-OH NVP, a metabolite of NVP proven essential for rash development, is the link between 12-OH NVP and the skin rash. Female Brown Norway (BN) rats were co-treated with NVP or 12-OH NVP and sulfation inhibitors dehydroepiandrosterone (DHEA) and salicylamide. Co-treatment with salicylamide markedly decreased formation of the sulfate conjugate but did not prevent development of the rash suggesting that the sulfate is not involved. However, it is not known whether the sulfate formation in the skin was affected. Co-treatments with DHEA decreased the sulfate formation and prevented the rash but also had other effects on NVP metabolism. This implies that the sulfate metabolite is responsible for the rash. Additional studies will be required to resolve these conflicting results.
|
48 |
Canine hepatic slices as a model for studying drug toxicity and metabolismScott, Maya Millicent 16 August 2006 (has links)
Tissue slices can be made from organs, such as liver, kidney, brain, and heart, and from various species including humans, dogs, non-human primates, rats and mice. It has been demonstrated that human and rat liver slices are viable for up to 2 days, and liver slices have been extensively used as an in vitro method to study hepatic drug metabolism and toxicity in humans. The objective of this study was to determine the utility of canine hepatic slices as an in vitro model for studying drug metabolism and hepatotoxicity in dogs. Canine hepatic slices were incubated in media containing various drugs to determine the hepatotoxicity of the agents and the ability of the slices to metabolize the drugs. The toxicity of phenobarbital, primidone, lidocaine and carprofen to canine hepatic slices was assessed by determining changes in supernatant concentrations of potassium ions and adenosine triphosphate (ATP); histologic lesions were determined as necrosis, extent of vacuolation and severity of vacuolation. Xenobiotic drug metabolizing enzymatic activity was investigated by determining the metabolism of lidocaine to monoethylglycinexylidide (MEGX), and administration of phenobarbital plus primidone was used as a positive control for hepatotoxicity in dogs. The function of drug-metabolizing enzymes was demonstrated by the successful metabolism of lidocaine to MEGX. Carprofen, a drug which causes idiosyncratic hepatic disease in dogs, did not show any hepatotoxicity at concentrations of 10, 50 and 100 µg/ml using potassium ion levels, ATP concentrations and histology as indicators of hepatotoxicity. Slices incubated in media without drug showed no toxicity over 24 hours based on potassium ion and ATP supernatant concentrations while significant increases in histologic lesions were noted at 8, 12 and 24 hours. Canine hepatic slices were a useful model for examining drug metabolism and toxicity for up to 24 hours.
|
49 |
The role of alpha-methyldopamine thioethers in the serotonergic neurotoxicity of MDA and MDMAJones, Douglas Campbell 28 August 2008 (has links)
Not available / text
|
50 |
An Investigation of CYP2B in Rat Brain: Regulation and Role in Drug and Toxin ResponseKhokhar, Jibran Y. 17 December 2012 (has links)
INTRODUCTION: Cytochrome P450 2B (CYP2B) is a drug-metabolizing enzyme subfamily found in both the brain and liver, which metabolizes clinical drugs, drugs of abuse (e.g. nicotine), toxicants and endogenous neurochemicals. Brain CYP2B’s role in the local metabolism of centrally acting substrates is important to investigate because of its ability to metabolize a variety of centrally active substrates. Additionally, CYP2B regulation by genetics, and exposure to xenobiotics, results in great inter-individual differences in the brain expression of this enzyme. METHODS: We investigated the time-course of rat brain CYP2B induction after chronic nicotine treatment. Using the rat model of brain CYP2B induction, combined with intracerebroventricular (ICV) inhibition of CYP2B, we assessed the effects of brain CYP2B in the response to the anaesthetic substrate, propofol. We also investigated the role of brain CYP2B-mediated activation of the pesticide chlorpyrifos on its neurotoxicity. RESULTS: Nicotine’s induction of rat brain CYP2B was long lasting, returning to basal levels by day 7, and was unaffected by nicotinic receptor blockade. Induction of CYP2B in rat brain, by chronic nicotine treatment, reduced the anaesthetic efficacy of propofol, through increased brain CYP2B-mediated metabolic inactivation. Inhibition of brain CYP2B, using mechanism based inhibitors of the enzyme, inhibited both basal and induced brain CYP2B activity, and prolonged propofol sleep time by reducing the local brain inactivation of the anaesthetic. Inhibition of rat brain, and not hepatic, CYP2B was able to effectively block local brain production of the toxic chlorpyrifos oxon, significantly attenuating the reductions in brain acetylcholinesterase activity and body temperature. Additionally, inhibition of brain CYP2B also significantly reduced the behavioural toxicity after chlorpyrifos exposure in a chlorpyrifos (CP) dose- and time-dependent manner. CONCLUSION: These studies indicate that rat brain CYP2B enzymes are active in vivo and play a meaningful role in the local metabolism of, and the response to, centrally acting substrates (i.e. propofol, chlorpyrifos). These data provide a first demonstration of the important role that brain CYP-mediated metabolism plays in the response to centrally acting substrates (i.e. clinical drugs, toxicants, endogenous neurochemicals), potentially contributing to the inter-individual variability seen in human responses to centrally active drugs and toxicants.
|
Page generated in 0.0864 seconds