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Gut Mar 2021Shortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in...
OBJECTIVE
Shortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo.
DESIGN
3D bioprinted hepatorganoids (3DP-HOs) were constructed using HepaRG cells and bioink, according to specific 3D printing procedures. Liver functions of 3DP-HOs were detected after 7 days of differentiation in vitro, which were later transplanted into Fah-deficient mice. The in vivo liver functions of 3DP-HOs were evaluated by survival time and liver damage of mice, human liver function markers and human-specific debrisoquine metabolite production.
RESULTS
3DP-HOs broadly acquired liver functions, such as ALBUMIN secretion, drug metabolism and glycogen storage after 7 days of differentiation. After transplantation into abdominal cavity of mouse model of liver injury, 3DP-HOs further matured and displayed increased synthesis of liver-specific proteins. Particularly, the mice acquired human-specific drug metabolism activities. Functional vascular systems were also formed in transplanted 3DP-HOs, further enhancing the material transport and liver functions of 3DP-HOs. Most importantly, transplantation of 3DP-HOs significantly improved the survival of mice.
CONCLUSIONS
Our results demonstrated a comprehensive proof of principle, which indicated that 3DP-HO model of liver tissues possessed in vivo hepatic functions and alleviated liver failure after transplantation, suggesting that 3D bioprinting could be used to generate human liver tissues as the alternative transplantation donors for treatment of liver diseases.
Topics: Animals; Bioprinting; Cell Differentiation; Cell Proliferation; Cell Survival; Disease Models, Animal; Graft Survival; Liver; Liver Failure; Liver Function Tests; Liver Transplantation; Mice; Printing, Three-Dimensional; Survival Rate
PubMed: 32434830
DOI: 10.1136/gutjnl-2019-319960 -
Pharmacogenomics Nov 2021Genetic variation in the gene encoding CYP2D6 is used to guide drug prescribing in clinical practice. However, genetic variants in CYP2D6 show substrate-specific effects...
Genetic variation in the gene encoding CYP2D6 is used to guide drug prescribing in clinical practice. However, genetic variants in CYP2D6 show substrate-specific effects that are currently not accounted for. With a systematic literature, we retrieved 22 original studies describing experiments focusing on alleles (*1, *2, *10 and *17) and substrates. Allele activity (clearance of the allele of interest divided by the clearance of the wildtype) was extracted. The results support the hypothesis of the existence of substrate specificity of the *17-allele (higher debrisoquine clearance), a subtle effect of the *10-allele (lower dextromethorphan clearance) but no substrate-specific effect of the *2-allele. Although our results support substrate specificity, for most substrates data are too sparse and require further studies.
Topics: Alleles; Cytochrome P-450 CYP2D6; Debrisoquin; Genetic Variation; Humans; Isoenzymes; Substrate Specificity
PubMed: 34569808
DOI: 10.2217/pgs-2021-0093 -
Current Drug Metabolism 2016Non-human primates are valuable animal models in drug discovery and biomedical research. Human CYP2D6 accounts for 1.3-4.3% of total hepatic CYP content in the liver,... (Review)
Review
BACKGROUND
Non-human primates are valuable animal models in drug discovery and biomedical research. Human CYP2D6 accounts for 1.3-4.3% of total hepatic CYP content in the liver, but is involved in the metabolism of more than 150 drugs. With the advancement of genomic sequencing and annotation, a panel of CYP2D genes have been cloned from non-human primates. This review highlights the similarities and differences of these CYP2D genes non-human primates.
METHODS
We conducted a structured PubMed search using a focused review question and proper inclusion/exclusion criteria. The quality of retrieved papers was assessed and briefed using standard tools and expert knowledge.
RESULTS
Most studies on CYP expression in non-human primates have been carried out in the cynomolgus and Rhesus monkeys. Deduced amino acid sequences of primate CYP2D cDNAs share high sequence identity (93-96%) with human CYP2D6. The chimpanzee genome has CYP2D6 and 2D7 but bonobos only contain CYP2D6. The CYP2D6 gene is located on chromosome 22 in the chimpanzee genome (human CYP2D6 maps to chromosome 22q13.1), and on chromosome 10 in the genome of the Rhesus monkey. Cynomolgus monkey CYP2D17 and Japanese monkey 2D29 metabolize bufuralol and dextromethorphan. CYP2D17 metabolizes bufuralol and dextromethorphan, whereas CYP2D29 metabolizes bufuralol and debrisoquine. In addition, quinidine inhibits both cynomolgus monkey CYP2D17 and Japanese monkey 2D29.
CONCLUSION
The CYP2D members from non-human primates show differential genomic contexts, catalytic activities toward substrates and inhibitory profiles. Further studies are warranted to elucidate the structural and functional features of CYP2D members in non-human primates and thus offer a solid base for the application of these animals in drug discovery.
Topics: Animals; Cytochrome P450 Family 2; Drug Discovery; Isoenzymes; Liver; Models, Animal; Primates; Protein Conformation; Species Specificity; Structure-Activity Relationship; Substrate Specificity; Xenobiotics
PubMed: 26892731
DOI: 10.2174/1389200217666160219114241 -
The Annals of Pharmacotherapy Feb 2022Assess the impact of interferons and interleukin (IL)-2 and IL-6 inhibitors on cytochrome P450 (CYP) drug metabolism in human subjects. (Review)
Review
OBJECTIVE
Assess the impact of interferons and interleukin (IL)-2 and IL-6 inhibitors on cytochrome P450 (CYP) drug metabolism in human subjects.
DATA SOURCES
PubMed search from 1980 to March 31, 2021, limited to human subjects and English language via search strategy: (biological drug names) [AND] (cytochrome [OR] CYP metabolism).
STUDY SELECTION AND DATA EXTRACTION
Narrative review of human studies assessing biological drugs in select classes that affect CYP drug metabolism.
DATA SYNTHESIS
Exogenous interferons suppress CYP1A2 (theophylline, caffeine, antipyrone) clearance by 20% to 49% in patients; have minimal impact on CYP3A4 (midazolam and dapsone), CYP2C9 (tolbutamide), or CYP2C19 (mephenytoin) metabolism; and increase CYP2D6 (debrisoquine, dextromethorphan) metabolism. Biological IL-2 inhibitors (basiliximab, daclizumab) have no effect on metabolism via CYP1A2 (caffeine), CYP2C9 (s-warfarin), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan), and CYP3A4 (midazolam, tacrolimus) but may enhance CYP3A4 (cyclosporin) metabolism over time. IL-6 inhibitors (sirukumab, tocilizumab, sarilumab) significantly enhance metabolism via CYP2C9 (s-warfarin), CYP2C19 (omeprazole), and CYP3A4 (simvastatin, midazolam) and reduce metabolism via CYP1A2 (caffeine).
RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE
Patients using interferons, IL-2, or IL-6 blocking drugs at steady state with CYP substrates could have altered drug metabolism and experience adverse events. With interferons and biological anti-inflammatory drugs, some isoenzymes will be inhibited, whereas others will be enhanced, and the magnitude of the effect can sometimes be significant. In clinical practice, clinicians may consider these metabolic changes as an additive effect to a patient's entire disease and medication profile when determining risk/benefit of treatment.
CONCLUSIONS
Interferon therapy or inflammatory suppression via IL-2 or IL-6 can alter steady-state concentrations of CYP-metabolized small-molecule drugs.
Topics: Biological Products; Cytochrome P-450 Enzyme System; Drug Interactions; Humans; Interferons; Interleukin-2; Interleukin-6; Pharmaceutical Preparations
PubMed: 34078115
DOI: 10.1177/10600280211022281 -
Biofabrication Jun 2022In recent decades, 3Dcultures of primary human hepatocytes (PHHs) have been increasingly developed to establish models capable of faithfully mimicking main liver...
In recent decades, 3Dcultures of primary human hepatocytes (PHHs) have been increasingly developed to establish models capable of faithfully mimicking main liver functions. The use of 3D bioprinting, capable of recreating structures composed of cells embedded in matrix with controlled microarchitectures, is an emergent key feature for tissue engineering. In this work, we used an extrusion-based system to print PHH in a methacrylated gelatin (GelMa) matrix. PHH bioprinted in GelMa rapidly organized into polarized hollow spheroids and were viable for at least 28 d of culture. These PHH were highly differentiated with maintenance of liver differentiation genes over time, as demonstrated by transcriptomic analysis and functional approaches. The cells were polarized with localization of apico/canalicular regions, and displayed activities of phase I and II biotransformation enzymes that could be regulated by inducers. Furthermore, the implantation of the bioprinted structures in mice demonstrated their capability to vascularize, and their ability to maintain human hepatic specific functions for at least 28 d was illustrated by albumin secretion and debrisoquine metabolism. This model could hold great promise for human liver tissue generation and its use in future biotechnological developments.
Topics: Animals; Bioprinting; Gelatin; Hepatocytes; Humans; Hydrogels; Mice; Printing, Three-Dimensional; Tissue Engineering; Tissue Scaffolds
PubMed: 35696992
DOI: 10.1088/1758-5090/ac7825 -
British Journal of Pharmacology Mar 2021The metabolic activity of cytochrome P450 (CYP) 2D6 is highly variable and CYP2D6 genotypes insufficiently explain the extensive and intermediate metabolic phenotypes,...
BACKGROUND AND PURPOSE
The metabolic activity of cytochrome P450 (CYP) 2D6 is highly variable and CYP2D6 genotypes insufficiently explain the extensive and intermediate metabolic phenotypes, limiting the prediction of drug response plus adverse drug reactions. Since CYP2D6 prototypic substrates are positively charged, the aim of this study was to evaluate the organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) as potential contributors to the variability of CYP2D6 hydroxylation of debrisoquine, dextromethorphan, diphenhydramine, perhexiline and sparteine.
EXPERIMENTAL APPROACH
OCT1/SLC22A1-, OCT2/SLC22A2-, OCT3/SLC22A3-, MATE1/SLC47A1-, and MATE2K/SLC47A2-overexpressing cell lines were used to investigate the transport of the selected drugs. Individuals from a study cohort, well defined with respect to CYP2D6 genotype and sparteine pharmacokinetics, were genotyped for the common OCT1 variants rs12208357 (OCT1-R61C), rs34130495 (OCT1-G401S), rs202220802 (OCT1-Met420del), rs34059508 (OCT1-G465R), OCT2 variant rs316019 (OCT2-A270S) and MATE1 variant rs2289669. Sparteine pharmacokinetics was stratified according to CYP2D6 and OCT1, OCT2 or MATE1 genotype.
KEY RESULTS
OCTs and MATE1 transport sparteine and debrisoquine with high affinity in vitro, but OCT- and MATE1-dependent transport of dextromethorphan, diphenhydramine and perhexiline was not detected. Sparteine and debrisoquine transport depends on OCT1 genotype; however, sparteine pharmacokinetics is independent from OCT1 genotype.
CONCLUSIONS AND IMPLICATIONS
Some drugs that are substrates of CYP2D6 are also substrates of OCTs and MATE1, suggesting overlapping specificities. Variability in sparteine hydroxylation in extensive and intermediate metabolizers cannot be explained by OCT1 genetic variants indicating presence of other factors. Dose-dependent toxicities of dextromethorphan, diphenhydramine and perhexiline appear to be independent from OCTs and MATEs.
Topics: Cations; Cytochrome P-450 CYP2D6; Humans; Organic Cation Transport Proteins; Organic Cation Transporter 2; Pharmaceutical Preparations; Phenotype
PubMed: 33434947
DOI: 10.1111/bph.15370 -
Advances in Experimental Medicine and... 2015Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous... (Review)
Review
Dopamine (DA) is a putative neurotransmitter in the carotid body engaged in the generation of the hypoxic ventilatory response (HVR). However, the action of endogenous DA is unsettled. This study seeks to determine the ventilatory effects of increased availability of endogenous DA caused by inhibition of DA enzymatic breakdown. The peripheral inhibitor of MAO - debrisoquine, or COMT - entacapone, or both combined were injected to conscious rats. Ventilation and its responses to acute 8 % O(2) in N(2) were investigated in a whole body plethysmograph. We found that inhibition of MAO augmented the hyperventilatory response to hypoxia. Inhibition of COMT failed to influence the hypoxic response. However, simultaneous inhibition of both enzymes, the case in which endogenous availability of DA should increase the most, reversed the hypoxic augmentation of ventilation induced by MAO-inhibition. The inference is that when MAO alone is blocked, COMT takes over DA degradation in a compensatory way, which lowers the availability of DA, resulting in a higher intensity of the HVR. We conclude that MAO is the enzyme predominantly engaged in the chemoventilatory effects of DA. Furthermore, the findings imply that endogenous DA is inhibitory, rather than stimulatory, for hypoxic ventilation.
Topics: Adaptation, Physiological; Animals; Blood Pressure; Carotid Body; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Catechols; Debrisoquin; Dopamine; Drug Synergism; Hyperventilation; Hypoxia; Male; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Nitriles; Plethysmography, Whole Body; Rats; Rats, Wistar; Respiration
PubMed: 25310955
DOI: 10.1007/5584_2014_72 -
Biochimica Et Biophysica Acta. General... Jan 2017Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data...
BACKGROUND
Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. It is considered as an "orphan" protein as few data are available on its physiological function(s) and spectral characteristics. Its only known substrates reported so far are unsaturated fatty acids such as arachidonic acid (AA), and, more recently, N-arachidonoylserotonin (AS) and some xenobiotics related to debrisoquine (Deb) and terfenadine.
METHODS
We have expressed CYP2U1 in E. coli and performed UV-vis and EPR spectroscopy experiments with purified CYP2U1 alone and in the presence of substrates and imidazole and pyridine derivatives. Docking experiments using a 3D homology model of CYP2U1 were done to explain the observed spectroscopic data and the different regioselectivities of the oxidations of AA and AS.
RESULTS
The UV-vis and EPR spectra of native recombinant human CYP2U1 revealed a predominant low-spin hexacoordinate Fe state. Imidazole (Im) derivatives, such as miconazole, acted as Fe ligands, contrary to ketoconazole, whereas the previously described substrates AS and Deb led to "reverse type I" difference UV-vis spectra. These data, as well as the different regioselectivities of AA and AS oxidations, were supported by docking experiments performed on our previously reported CYP2U1 3D model.
MAJOR CONCLUSION AND GENERAL SIGNIFICANCE
Our study describes for the first time the mode of interaction of several Fe-heme ligands and substrates with the active site of CYP2U1 on the basis of spectroscopic and molecular docking data. The good agreement between these data validates the used CYP2U1 3D model which should help the design of new substrates or inhibitors of this orphan CYP.
Topics: Arachidonic Acid; Arachidonic Acids; Biocatalysis; Cytochrome P450 Family 2; Debrisoquin; Electron Spin Resonance Spectroscopy; Escherichia coli; Humans; Imidazoles; Lauric Acids; Ligands; Models, Molecular; Molecular Docking Simulation; Oxidation-Reduction; Protein Binding; Pyridines; Recombinant Proteins; Serotonin; Spectrophotometry, Ultraviolet; Substrate Specificity
PubMed: 27456766
DOI: 10.1016/j.bbagen.2016.07.018 -
Medicina (Kaunas, Lithuania) Nov 2023: Tachycardia is a common cardiovascular disease. Drugs blocking β1-adrenergic receptors (ADRB1) are used in the therapy of arrhythmogenic heart diseases....
: Tachycardia is a common cardiovascular disease. Drugs blocking β1-adrenergic receptors (ADRB1) are used in the therapy of arrhythmogenic heart diseases. Disease-related polymorphisms can be observed within the gene. The two most important are Ser49Gly and Arg389Gly, and they influence the treatment efficacy. The family of the cytochrome P450 system consists of the isoenzyme CYP2D6 (Debrisoquine 4-hydroxylase), which is involved in phase I metabolism of almost 25% of clinically important drugs, including antiarrhythmic drugs. A study was conducted to detect the and gene polymorphisms. : The material for the test was whole blood from 30 patients with ventricular and supraventricular tachycardia and 20 controls. The samples were obtained from the Department of Pediatric Cardiology. The first to be made was the extraction of DNA using a GeneMATRIX Quick Blood DNA Purification Kit from EURx. The selected and gene polymorphisms were detected by high-resolution melting polymerase chain reaction (HRM-PCR) analysis. : Based on the analysis of melt profile data for each PCR product, the identification of polymorphisms was carried out. Heterozygotes and homozygotes were found in the examined alleles. : The frequency of the Arg389Gly polymorphism differs statistically significantly between the control group and patients with supraventricular and ventricular arrhythmias, as well as between these two groups of patients. Moreover, the Arg389Gly polymorphism was statistically more prevalent in the group of girls with SVT arrhythmia compared to girls with VT. A few carriers of homozygous and heterozygous systems of the S49G polymorphism were detected among patients with arrhythmias, as well as control group. The percentage of individuals carrying the allele as either homozygous or heterozygous was observed in the study and control groups. The high prevalence of the allele carriers in both groups prompts the optimization of beta-1 blocker therapy.
Topics: Child; Female; Humans; Adrenergic beta-Antagonists; Arrhythmias, Cardiac; Cytochrome P-450 CYP2D6; DNA; Polymorphism, Genetic; Receptors, Adrenergic, beta-1
PubMed: 38138160
DOI: 10.3390/medicina59122057 -
Drug Metabolism and Disposition: the... Jul 2022Cytochrome P450 2D6 (CYP2D6), is responsible for the metabolism and elimination of approximately 25% of clinically used drugs, including antidepressants and...
Cytochrome P450 2D6 (CYP2D6), is responsible for the metabolism and elimination of approximately 25% of clinically used drugs, including antidepressants and antipsychotics, and its activity varies considerably on a population basis primary due to genetic variation. CYP2D6 phenotype can be assessed following administration of an exogenous probe compound, such as dextromethorphan or debrisoquine, but use of a biomarker that does not require administration of an exogenous compound ( drug) has considerable appeal for assessing CYP2D6 activity in vulnerable populations, such as children. The goal of this study was to isolate, purify and identify an "endogenous" urinary biomarker (M1; m/z 444.3102) of CYP2D6 activity reported previously. Several chromatographic separation techniques (reverse phase HPLC, cation exchange and analytical reverse phase UPLC) were used to isolate and purify 96 μg of M1 from 40 L of urine. Subsequently, 1D and 2D NMR, and functional group modification reactions were used to elucidate its structure. Analysis of mass spectrometry and NMR data revealed M1 to have similar spectroscopic features to the nitrogen-containing steroidal alkaloid, solanidine. 2D NMR characterization by HMBC, COSY, TOCSY, and HSQC-TOCSY proved to be invaluable in the structural elucidation of M1; derivatization of M1 revealed the presence of two carboxylic acid moieties. M1 was determined to be a steroidal alkaloid with a solanidine backbone that had undergone C-C bond scission to yield 3,4-seco-solanidine-3,4-dioic acid (SSDA). SSDA may have value as a dietary biomarker of CYP2D6 activity in populations where potato consumption is common. Endogenous biomarkers of processes involved in drug disposition and response may allow improved individualization of drug treatment, especially in vulnerable populations, such as children. Given that several CYP2D6 substrates are commonly used in pediatrics and the ubiquitous nature of potato consumption in western diets, SSDA has considerable appeal as non-invasive biomarker of CYP2D6 activity to guide treatment with CYP2D6 substrates in children and adults.
PubMed: 35878926
DOI: 10.1124/dmd.122.000957