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Stem Cells Translational Medicine Jan 2017Liver disease affects large numbers of patients, yet there are limited treatments available to replace absent or ineffective cellular function of this crucial organ....
Liver disease affects large numbers of patients, yet there are limited treatments available to replace absent or ineffective cellular function of this crucial organ. Donor scarcity and the necessity for immunosuppression limit one effective therapy, orthotopic liver transplantation. But in some conditions such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. After transplanting multicellular liver organoid units composed of a heterogeneous cellular population that includes adult stem and progenitor cells, both mouse and human tissue-engineered liver (TELi) form in vivo. TELi contains normal liver components such as hepatocytes with albumin expression, CK19-expressing bile ducts and vascular structures with α-smooth muscle actin expression, desmin-expressing stellate cells, and CD31-expressing endothelial cells. At 4 weeks, TELi contains proliferating albumin-expressing cells and identification of β2-microglobulin-expressing cells demonstrates that the majority of human TELi is composed of transplanted human cells. Human albumin is detected in the host mouse serum, indicating in vivo secretory function. Liquid chromatography/mass spectrometric analysis of mouse serum after debrisoquine administration is followed by a significant increase in the level of the human metabolite, 4-OH-debrisoquine, which supports the metabolic and xenobiotic capability of human TELi in vivo. Implanted TELi grew in a mouse model of inducible liver failure. Stem Cells Translational Medicine 2017;6:238-248.
Topics: Adult Stem Cells; Animals; Arginase; Cell Proliferation; Disease Models, Animal; Female; Hepatocytes; Humans; Liver; Mice, SCID; Organoids; Tissue Engineering
PubMed: 28170183
DOI: 10.5966/sctm.2016-0205 -
Bulletin of Experimental Biology and... Mar 2016Polymorphic variants of CYP1A2 and CYP2D6 genes of the cytochrome P450 system were studied in patients with schizophrenia with drug-induced motor disorders and...
Polymorphic variants of CYP1A2 and CYP2D6 genes of the cytochrome P450 system were studied in patients with schizophrenia with drug-induced motor disorders and hyperprolactinemia against the background of long-term neuroleptic therapy. We revealed an association of polymorphic variant C-163A CYP1A2*1F of CYP1A2 gene with tardive dyskinesia and association of polymorphic variant 1846G>A CY2D6*4 and genotype A/A of CYP2D6 gene (responsible for debrisoquin-4-hydroxylase synthesis) with limbotruncal tardive dyskinesia in patients with schizophrenia receiving neuroleptics for a long time.
Topics: Antipsychotic Agents; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2D6; Drug-Related Side Effects and Adverse Reactions; Female; Genetic Predisposition to Disease; Humans; Hyperprolactinemia; Male; Motor Disorders; Polymorphism, Single Nucleotide; Schizophrenia; Tardive Dyskinesia
PubMed: 27021090
DOI: 10.1007/s10517-016-3250-4 -
Pharmacogenomics 2016This study was aimed to functionally characterize four novel CYP2D6 alleles identified in Chinese Han population.
AIM
This study was aimed to functionally characterize four novel CYP2D6 alleles identified in Chinese Han population.
MATERIALS & METHODS
CYP2D6 proteins of wild-type and the four novel variants along with CYP2D6.2 and CYP2D6.10 were heterologously expressed in yeast cells and the kinetic parameters were determined.
RESULTS
Compared with CYP2D6.1 (frequency in Chinese 24.65%), CYP2D6.X (1.63%), CYP2D6.Y (1.50%), CYP2D6.Z (0.81%), CYP2D6.10 (52.53%) and CYP2D6.75 (0.13%) exhibited low activity at different degrees, whereas the kinetic parameters of CYP2D6.2 (11.06%) were much the same with CYP2D6.1. The novel allele CYP2D6.75 showed decreased enzyme activity.
CONCLUSION
This is the first study to conduct functional analysis of CYP2D6 four novel alleles in Chinese Han population, which might be helpful for optimizing pharmacotherapy and the design of personalized medicine.
Topics: Alleles; Asian People; Cytochrome P-450 CYP2D6; Debrisoquin; Genetic Variation; Humans; Recombinant Proteins; Saccharomyces cerevisiae
PubMed: 26652007
DOI: 10.2217/pgs.15.148 -
ACS Medicinal Chemistry Letters Jun 2023The COVID-19 pandemic has highlighted the need for new antiviral approaches because many of the currently approved drugs have proven ineffective against mitigating...
The COVID-19 pandemic has highlighted the need for new antiviral approaches because many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a promising antiviral target because it plays a role in priming the spike protein before viral entry occurs for the most virulent variants. Further, TMPRSS2 has no established physiological role, thereby increasing its attractiveness as a target for antiviral agents. Here, we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we identify new noncovalent TMPRSS2 inhibitors that block SARS-CoV-2 infectivity in a cellular model. One such inhibitor, debrisoquine, has high ligand efficiency, and an initial structure-activity relationship study demonstrates that debrisoquine is a tractable hit compound for TMPRSS2.
PubMed: 37284689
DOI: 10.1021/acsmedchemlett.3c00035 -
American Journal of Veterinary Research Sep 2016OBJECTIVE To characterize polymorphisms of the gene for cytochrome P450 isozyme 2D50 (CYP2D50) and the disposition of 2 CYP2D50 probe drugs, dextromethorphan and...
OBJECTIVE To characterize polymorphisms of the gene for cytochrome P450 isozyme 2D50 (CYP2D50) and the disposition of 2 CYP2D50 probe drugs, dextromethorphan and debrisoquine, in horses. ANIMALS 23 healthy horses (22 Thoroughbreds and 1 Standardbred). PROCEDURES Single-nucleotide polymorphisms (SNPs) in CYP2D50 were identified. Disposition of dextromethorphan (2 mg/kg) and debrisoquine (0.2 mg/kg) were determined after oral (dextromethorphan) or nasogastric (debrisoquine) administration to the horses. Metabolic ratios of plasma dextromethorphan and total dextrorphan (dextrorphan plus dextrorphan-O-β-glucuronide) and 4-hydroxydebrisoquine concentrations were calculated on the basis of the area under the plasma concentration-versus-time curve extrapolated to infinity for the parent drug divided by that for the corresponding metabolite. Pharmacokinetic data were used to categorize horses into the phenotypic drug-metabolism categories poor, extensive, and ultrarapid. Disposition patterns were compared among categories, and relationships between SNPs and metabolism categories were explored. RESULTS Gene sequencing identified 51 SNPs, including 27 nonsynonymous SNPs. Debrisoquine was minimally detected after oral administration. Disposition of dextromethorphan varied markedly among horses. Metabolic ratios for dextromethorphan ranged from 0.03 to 0.46 (mean, 0.12). On the basis of these data, 1 horse was characterized as a poor metabolizer, 18 were characterized as extensive metabolizers, and 3 were characterized as ultrarapid metabolizers. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that CYP2D50 is polymorphic and that the disposition of the probe drug varies markedly in horses. The polymorphisms may be related to rates of drug metabolism. Additional research involving more horses of various breeds is needed to fully explore the functional implication of polymorphisms in CYP2D50.
Topics: Animals; Cytochrome P-450 Enzyme System; Debrisoquin; Dextromethorphan; Female; Horses; Isoenzymes; Male; Polymorphism, Single Nucleotide
PubMed: 27580115
DOI: 10.2460/ajvr.77.9.1029 -
International Journal of Clinical... Jun 2018Propafenone is an antiarrhythmic drug applied to ventricular arrhythmias, initially recognized as a sodium channel blocker. This study aims to evaluate the... (Randomized Controlled Trial)
Randomized Controlled Trial
Propafenone is an antiarrhythmic drug applied to ventricular arrhythmias, initially recognized as a sodium channel blocker. This study aims to evaluate the bioequivalence of two propafenone formulations (300 mg tablet) in healthy subjects under non-fasting conditions. The study was conducted as an open, randomized, 2-period design with a 2-sequence (RT, TR) with a 1-week washout interval. The subjects were selected for the study after having their health status previously assessed by a clinical evaluation and laboratory tests (biochemical and hematological parameters, and urinalysis). Debrisoquine phenotype of healthy subjects was determined by analysis of urinary excretion of debrisoquine and its major metabolite, 4-hydroxydebrisoquine. A single propafenone tablet (300 mg) was given in each occasion. Plasma propafenone concentrations were analyzed by liquid chromatography coupled to tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization using multiple reactions monitoring (MRM). The geometric mean and 90% confidence intervals (CI) of propafenone/Ritmonorm (T/R) percent ratio were 100.44% (88.39 - 114.13%) for AUC, 99.84% (90.31 - 110.36%) for AUC, and 99.30% (90.08 - 109.47%) for C. Since the 90% CI for C, AUC, and AUC ratios were all inside the 80 - 125% interval proposed by the US Food and Drug Administration Agency, it was concluded that the propafenone formulation elaborated by Biolab Sanus Farmacêutica Ltda. is bioequivalent to Ritmonorm formulation for both the rate and the extent of absorption. The drug was well tolerated by the subjects, indicating that it is safe to perform propafenone bioequivalence studies in healthy subjects with intermediate/extensive metabolism. .
Topics: Adolescent; Adult; Chromatography, High Pressure Liquid; Humans; Middle Aged; Propafenone; Tandem Mass Spectrometry; Therapeutic Equivalency; Young Adult
PubMed: 29648531
DOI: 10.5414/CP203181 -
Therapeutic Advances in Urology Jul 2017The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride...
BACKGROUND
The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride on the seven major human cytochrome P450 enzymes (CYP) by using a standardized and validated seven-in-one cytochrome P450 cocktail inhibition assay.
METHODS
An cocktail of seven highly selective probe substrates was incubated with human liver microsomes and varying concentrations of the seven test compounds. The major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Enzyme kinetics were estimated by determining IC and values nonlinear regression. Obtained values were used for predictions of potential clinical impact of the inhibition using a static mechanistic prediction model.
RESULTS
In this study, 49 IC experiments were conducted. In six cases, IC values lower than the calculated threshold for drug-drug interactions (DDIs) in the gut wall were observed. In these cases, no increase in inhibition was determined after a 30 min preincubation. Considering a typical dosing regimen and applying the obtained values of 0.72 µM (darifenacin, 15 mg daily) and 7.2 µM [propiverine, 30 mg daily, immediate release (IR)] for the inhibition of CYP2D6 yielded a predicted 1.9-fold and 1.4-fold increase in the area under the curve (AUC) of debrisoquine (CYP2D6 substrate), respectively. Due to the inhibition of the particular intestinal CYP3A4, the obtained values of 14 µM of propiverine (30 mg daily, IR) resulted in a predicted doubling of the AUC for midazolam (CYP3A4 substrate).
CONCLUSIONS
/ extrapolation based on pharmacokinetic data and the conducted screening experiments yielded similar effects of darifenacin on CYP2D6 and propiverine on CYP3A4 as obtained in separately conducted DDI studies. As a novel finding, propiverine was identified to potentially inhibit CYP2D6 at clinically occurring concentrations.
PubMed: 28747995
DOI: 10.1177/1756287217708951 -
Genes and Environment : the Official... 2017Genetic and environmental risk factors play an important role for the susceptibility to sporadic Parkinson's disease (PD). It was hypothesized that a splice variant of...
BACKGROUND
Genetic and environmental risk factors play an important role for the susceptibility to sporadic Parkinson's disease (PD). It was hypothesized that a splice variant of the gene ( allele) is associated with PD because it alters the ability to metabolize toxins and in particular neurotoxins. codes for the drug metabolizing enzyme debrisoquine 4-hydroxylase. The CYP2D6*4 variant results in an undetectable enzyme activity and consequently in a reduction in metabolism of some toxins.
METHODS
Some of agricultural chemicals have neurotoxic potential and CYP2D6 is involved in their detoxification. Thus, we conducted a case control study to investigate the association of the CYP2D6*4 with PD in a Pakistani subpopulation that is known to be exposed to high levels of some agricultural pesticides, insecticides and herbicides.
RESULTS
We found a significantly higher allele and genotype frequency of the variant in 174 sporadic PD patients when compared to 200 controls. In addition, there was a trend to an earlier age of PD onset and a tremor dominant phenotype in variant carriers.
CONCLUSION
Our data provide further evidence that a poor metabolizer status may increase the risk to develop PD especially in populations that are exposed to environmental toxins.
PubMed: 28680508
DOI: 10.1186/s41021-017-0078-8 -
Drug Metabolism and Disposition: the... Sep 2014Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed...
Cilengitide is a stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αVβ3 and αVβ5 integrins. The candidate drug showed unexpected inhibition of cytochrome P450 (P450) 3A4 at high concentrations, that is, a 15-mM concentration caused attenuation of P450 3A4 activity (depending on the probe substrate): 15-19% direct inhibition, 10-23% time-dependent inhibition (30-minute preincubation), and 54-60% metabolism-dependent inhibition (30-minute preincubation). The inactivation efficiency determined with human liver microsomes was 0.003 ± 0.001 min(-1) mM(-1) and was 0.04 ± 0.01 min(-1) mM(-1) with baculovirus-based microsomes containing recombinant P450 3A4. Neither heme loss nor covalent binding to apoprotein could explain the observed reductions in residual activity. Slowly forming type II difference spectra were observed, with maximum spectral changes after 2 hours. Binding to both reduced and oxidized P450 3A4 was observed, with apparent Kd values of 0.66 μM and 6 μM. The significance of the guanidine group in inhibition was demonstrated using ligand binding spectral changes and inactivation assays with guanidine analogs (debrisoquine, N-acetylarginine-O-methyl ester) and the acetylated ornithine derivative of cilengitide. The observed inhibition could be explained by direct inhibition, plus by formation of stable complexes with both ferric and ferrous forms of heme iron and to some extent by the formation of reactive species capable to react to the protein or heme. Formation of the complex required time and NADPH and is attributed to the guanidino group. Thus, the NADPH-dependent inhibition is considered to be mainly due to the formation of a stable complex rather than the formation of reactive species.
Topics: Adult; Cytochrome P-450 CYP3A; Enzyme Inhibitors; Female; Guanidine; Heme; Humans; Male; Microsomes, Liver; NADP; Oxidation-Reduction; Snake Venoms
PubMed: 24985702
DOI: 10.1124/dmd.114.059295 -
Biochimica Et Biophysica Acta Jul 2015Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly...
Expression in yeast, new substrates, and construction of a first 3D model of human orphan cytochrome P450 2U1: Interpretation of substrate hydroxylation regioselectivity from docking studies.
BACKGROUND
Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an "orphan" enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin.
METHODS
We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography-mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions.
RESULTS
We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site.
MAJOR CONCLUSION
Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model.
GENERAL SIGNIFICANCE
These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.
Topics: Blotting, Western; Catalytic Domain; Chromatography, High Pressure Liquid; Computer Simulation; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Debrisoquin; Kinetics; Mass Spectrometry; Models, Molecular; Molecular Structure; Oxidation-Reduction; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Saccharomyces cerevisiae; Substrate Specificity
PubMed: 25857771
DOI: 10.1016/j.bbagen.2015.03.014