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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 -
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 -
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 -
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 -
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 -
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 -
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 -
Stem Cell Research & Therapy Mar 2015Chimeric mice with humanized livers were recently established by transplanting human hepatocytes. This mouse model that is repopulated with functional human hepatocytes...
INTRODUCTION
Chimeric mice with humanized livers were recently established by transplanting human hepatocytes. This mouse model that is repopulated with functional human hepatocytes could be a useful tool for investigating human hepatic cell biology and drug metabolism and for other preclinical applications. Successfully transplanting human hepatocytes into mice requires that recipient mice with liver failure do not reject these human cells and provide a suitable microenvironment (supportive niche) to promote human donor cell expansion and differentiation. To overcome the limitations of current mouse models, we used Alb-TRECK/SCID mice for in vivo human immature hepatocyte differentiation and humanized liver generation.
METHODS
1.5 μg/kg diphtheria toxin was administrated into 8-week-old Alb-TRECK/SCID mice, and the degree of liver damage was assessed by serum aspartate aminotransferase activity levels. Forty-eight hours later, mice livers were sampled for histological analyses, and the human donor cells were then transplanted into mice livers on the same day. Chimeric rate and survival rate after cell transplantation was evaluated. Expressions of human hepatic-related genes were detected. A human albumin enzyme-linked immunosorbent assay was performed after 50 days of transplantation. On day 60 after transplantation, drug metabolism was examined in mice.
RESULTS
Both human primary fetal liver cells and hepatic stem cells were successfully repopulated in the livers of Alb-TRECK/SCID mice that developed lethal fulminant hepatic failure after administering diphtheria toxin; the repopulation rate in some mice was nearly 100%. Compared with human primary fetal liver cells, human hepatic stem cell transplantation rescued Alb-TRECK/SCID mice with lethal fulminant hepatic failure, and human hepatic stem cell-derived humanized livers secreted more human albumin into mouse sera and also functioned as a "human liver" that could metabolize the drugs ketoprofen and debrisoquine.
CONCLUSION
Our model of a humanized liver in Alb-TRECK/SCID mice may provide for functional applications such as drug metabolism, drug to drug interactions, and promote other in vivo and in vitro studies.
Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chimera; Debrisoquin; Diphtheria Toxin; Disease Models, Animal; Hepatocytes; Humans; Inactivation, Metabolic; Ketoprofen; Liver; Liver Failure, Acute; Liver Regeneration; Mice; Mice, Knockout; Mice, SCID; Stem Cell Transplantation; Stem Cells; Transplantation, Heterologous
PubMed: 25889844
DOI: 10.1186/s13287-015-0038-9