Dog Liver Research Articles

Discovery of Pyridone-Substituted Triazolopyrimidine Dual A2A/A1 AR Antagonists for the Treatment of Ischemic Stroke.

Ischemic stroke is a complex systemic disease characterized by high morbidity, disability, and mortality. The activation of the presynaptic adenosine A2A and A1 receptors modifies a variety of brain insults from excitotoxicity to stroke. Therefore, the discovery of dual A2A/A1 adenosine receptor (AR)-targeting therapeutic compounds could be a strategy for the treatment of ischemic stroke. Inspired by two clinical phase III drugs, ASP-5854 (dual A2A/A1 AR antagonist) and preladenant (selective A2A AR antagonist), and using the hybrid medicinal strategy, we characterized novel pyridone-substituted triazolopyrimidine scaffolds as dual A2A/A1 AR antagonists. Among them, compound 1a exerted excellent A2A/A1 AR binding affinity (Ki = 5.58/24.2 nM), an antagonistic effect (IC50 = 5.72/25.9 nM), and good metabolic stability in human liver microsomes, rat liver microsomes, and dog liver microsomes. Importantly, compound 1a demonstrated a dose–effect relationship in the oxygen-glucose deprivation/reperfusion (OGD/R)-treated HT22 cell model. These findings support the development of dual A2A/A1 AR antagonists as a potential treatment for ischemic stroke.

Preclinical safety profile of a liver-localized mitochondrial uncoupler: OPC-163493.

Mitochondrial uncouplers (mUncouplers) are known to exhibit a variety of toxic effects in animals. Here we report a safety profile of an mUncoupler, OPC-163493, recently synthesized at Otsuka Pharmaceutical Co, Ltd, and its development as a therapeutic agent for treating diabetes. To understand the acute and subchronic toxicity of OPC-163493, single and repeated oral dose studies in rats, dogs, and monkeys were performed. In the rat studies, rigor mortis and increased body temperatures were observed in the high dose group. Focal necrosis, fatty change, and granular eosinophilic cytoplasm of the hepatocytes were also observed in the high dose group. In the dog studies, gastrointestinal manifestations were observed with decreased body weight and decreased food consumption in the high dose group. Necrotizing arteritis was observed in multiple organs as well as meningitis with hemorrhage in the brain. In the monkey studies, vomiting, decreased food consumption, and decreased locomotor activity were observed in the high dose group. Degeneration of the proximal convoluted tubules and the straight tubular epithelium, regeneration of the proximal tubular epithelium, and degeneration of the collecting tubular epithelium were observed. The target organs of OPC-163493 were liver, blood vessels, and kidney in rats, dogs, and monkeys, respectively. In rats, dogs, and monkeys, safety ratios were 100:1, 13:1, and 20:1, respectively, in terms of total exposure (AUC24h). These safety ratios showed clear separation between exposure to OPC-163493 in animals at NOAEL and the exposure at the effective dose in ZDF rats. This information should contribute to the drug development of new and effective mUncoupler candidates.

Increases in serum carbonylated protein levels of dogs with hypercortisolism.

Protein carbonylation is an irreversible and degenerative modification that can be used to evaluate oxidative stress caused by glucocorticoids. In this study, we focused on protein carbonylation in dogs with hypercortisolism (HC). Sera samples were collected from 14 dogs diagnosed with HC and treated with trilostane, 12 dogs with inflammatory diseases (disease control group), and eight clinically healthy dogs. When the carbonylated protein levels were detected by the immunoblot analysis, one band of approximately 40 kDa was predominantly increased in the dogs with HC. The band was identified as haptoglobin using the liquid chromatography tandem mass spectrometry method. Furthermore, haptoglobin immune reactivity was higher in the dogs with HC. Although the average protein carbonylation level of the HC group was not significantly different from that of the other groups, the carbonylation level was significantly higher for the poorly controlled HC cases than for the well-controlled HC group. Additionally, the primary culture of canine hepatocytes was used to clarify the direct effect of glucocorticoids on protein carbonylation in dog livers. Both the carbonylated protein and haptoglobin clearly increased after 72 h. These findings suggest that haptoglobin and its carbonylated form are increased with canine HC, and that the protein carbonylation ratio and/or haptoglobin level could be related to disease management. These factors could be useful as biomarkers for an oxidative stress reaction, at least in the liver, and for treatment monitoring of HC.

Comparison of Tissue Abundance of Non-Cytochrome P450 Drug-Metabolizing Enzymes by Quantitative Proteomics between Humans and Laboratory Animal Species.

The use of animal pharmacokinetic models as surrogates for humans relies on the assumption that the drug disposition mechanisms are similar between preclinical species and humans. However, significant cross-species differences exist in the tissue distribution and protein abundance of drug-metabolizing enzymes (DMEs) and transporters. We quantified non-cytochrome P450 (non-CYP) DMEs across commonly used preclinical species (cynomolgus and rhesus monkeys, beagle dog, Sprague Dawley and Wistar Han rats, and CD1 mouse) and compared these data with previously obtained human data. Aldehyde oxidase was abundant in humans and monkeys while poorly expressed in rodents, and not expressed in dogs. Carboxylesterase (CES) 1 abundance was highest in the liver while CES2 was primarily expressed in the intestine in all species with notable species differences. For example, hepatic CES1 was 3× higher in humans than in monkeys, but hepatic CES2 was 3-5× higher in monkeys than in humans. Hepatic UDP-glucuronosyltransferase (UGT) 1A2 abundance was ∼4× higher in dogs compared with rats, whereas UGT1A3 abundance was 3-5× higher in dog livers than its ortholog in human and monkey livers. UGT1A6 abundance was 5-6× higher in human livers compared with monkey and dog livers. Hepatic sulfotransferase 1B1 abundance was 5-7× higher in rats compared with the rest of the species. These quantitative non-CYP proteomics data can be used to explain unique toxicological profiles across species and can be integrated into physiologically based pharmacokinetic models for the mechanistic explanation of pharmacokinetics and tissue distribution of xenobiotics in animal species. SIGNIFICANCE STATEMENT: We characterized the quantitative differences in non-cytochrome P450 (non-CYP) drug-metabolizing enzymes across commonly used preclinical species (cynomolgus and rhesus monkeys, beagle dogs, Sprague Dawley and Wistar Han rats, and CD1 mice) and compared these data with previously obtained human data. Unique differences in non-CYP enzymes across species were observed, which can be used to explain significant pharmacokinetic and toxicokinetic differences between experimental animals and humans.

Differences and Similarities in the Metabolism of Erlotinib across various Species: An Analysis by Liquid Chromatography - Tandem Mass Spectrometry

Erlotinib is an inhibitor of the epidermal growth factor receptor (EGFR), primarily used to treat non-small cell lung cancer (NSCLC) or pancreatic cancer. The main objective of the present study was to identify differences and similarities in the metabolism of erlotinib across various species and to identify new phase I metabolites. Metabolic characteristics of erlotinib were investigated in liver microsomes of human, mice, rat, dog, hamster, and S9-fraction of mice by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 19 phase I metabolites were detected in human liver microsomes; whereas, 12 metabolites in each of mice-, rat- liver microsomes and S9-fraction of mice; 10 in dog liver microsomes and 7 in hamster liver microsomes were detected. Out of these 19 metabolites, 8 metabolites were newly found including 1- novel metabolite (M23) which was identified with its putative structure in human liver microsomes. All phase I metabolites reported in healthy human volunteers were identified in human liver microsomes. Similar metabolic behavior had shown by liver microsomes of mice, rat, and S9-fraction of mice. Metabolites M6, M13, M14, M16, M22, and M25 were found in all tested species. These differences and similarities in the metabolism of erlotinib confirmed the role of CYP 450 enzymes and their distinct activity across various species.

Determination of six thyroid hormones in dog brain and liver using acidic extraction, mixed-mode cleanup, and liquid chromatography–tandem mass spectrometry

Thyroid hormones (THs) play a critical role in the regulation of biological processes, such as growth, metabolism, and development, in various animal species. Prohormone L-thyroxine (T4) is secreted from the thyroid gland and carried to peripheral tissues. T4 is then biotransformed to several metabolites which play different roles, mainly by iodothyronine deiodinases. Determination of deiodinated TH metabolites in key organs such as liver and brain would help to understand tissue-specific TH metabolism and homeostasis. In this study, we thus developed a highly sensitive method for the determination of six THs [T4, 3,5,3′-triodo-L-thyronine (T3), 3,3′,5′-triiodo-L-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2), 3,3′-diiodo-l-thyronine (3,3′-T2), and 3-iodo-l-thyronine (3-T1)] in the brain and liver by using stored dog samples. The analytical method consisted of ultrasonic-assisted extraction in acetone acidified with formic acid, cleanup with a EVOLUTE® EXPRESS CX cartridge (reversed-phase combined with strong cation-exchange cartridge), and quantification with liquid chromatography-tandem mass spectrometry. Acceptable accuracy (internal standard-corrected recovery: 80%–120%) and intra- and inter-day precision (coefficient of variation: <6% and <15%, respectively) (n = 3/ batch, three days) were obtained for both brain and liver samples. In addition, low method detection limits were achieved for both brain (0.013–0.12 ng g–1) and liver (0.030–0.78 ng g–1), which resulted in the quantitation of not only T4, T3, and rT3, but also 3,3′-T2 in both dog brain and liver samples. The developed method was successfully applied to the analysis of THs in the brain and liver of dogs (Canis lupus familiaris) which were exposed to polychlorinated biphenyls (PCBs). As a result, concentration ratios of rT3/T4 and 3,3′-T2/T3 in the PCB-exposed dogs were significantly higher than those in the control groups, suggesting the enhanced inner (tyrosyl)-ring deiodination (5-deiodination) by PCB exposure. The analytical method developed in the present study enables comprehensive evaluation of alterations in peripheral TH metabolism which are caused by exposure to environmental pollutants.

Diagnostic accuracy of ultrasonography to detect hepatic and splenic lymphomatous infiltration in dogs and cats.

To determine the diagnostic accuracy of ultrasonography in the detection of lymphomatous infiltration of the liver and spleen in a population of dogs and cats with lymphoma. To determine if specific ultrasonographic features of the liver and spleen in dogs are associated with lymphomatous infiltration or a specific immunophenotype of multi-centric lymphoma. A blinded retrospective evaluation of ultrasonographic images of the liver and/or spleen in dogs and cats with cytologically or histologically confirmed lymphoma was performed by two board-certified veterinary radiologists. A total of 161 animals met the inclusion criteria, comprising 132 dogs and 29 cats. Ultrasonography had a sensitivity, specificity, accuracy, positive predictive value and negative predictive value of 16.7%, 91.0%, 55.9%, 62.5% and 55.0% for the detection of lymphomatous infiltration of the liver, and 73.1%, 93.9%, 82.6%, 93.4% and 74.7% for the spleen. In dogs, an ultrasonographically normal liver was associated with not having lymphomatous infiltration, leopard-spotted splenic parenchyma and splenomegaly were independently associated with lymphomatous infiltration and leopard-spotted splenic parenchyma was also associated with the B cell immunophenotype of multi-centric lymphoma. Ultrasonography of the spleen and liver is specific but not sensitive in the detection of lymphomatous infiltration. A leopard-spotted splenic parenchyma in dogs is highly specific for lymphomatous infiltration and in this population predicted a specific immunophenotype of multi-centric lymphoma.

Pharmacokinetics of S-epacadostat, an indoleamine 2,3-dioxygenase 1 inhibitor, in dog plasma and identification of its metabolites in vivo and in vitro.

S-epacadostat (S-EPA) is an efficient and selective small-molecule inhibitor of indoleamine 2,3-dioxygenase 1. It is an EPA analog with a sulfur atom instead of a nitrogen atom at the furazan C3 position. This study documents the pharmacokinetics of S-EPA in dogs and its metabolic pathway. After an oral administration of 15 mg/kg of S-EPA in dogs, the time to peak concentration was 0.80 h, the mean elimination half-life was 7.3 h, and the absolute bioavailability was 55.8%. Furthermore, we identified S-EPA metabolites in dog plasma and dog liver microsomes by UPLC-Q Exactive Orbitrap HRMS. In dog plasma, we found five metabolites, which came from glucuronidation (M1 and M2), deoxygenation (the amidine M4), glucuronidation of M4 (M3), and desulfonamidation and oxidation of M4 (the carboxylic acid M5). In dog liver microsomes, we identified three major metabolites, namely, the glucuronide conjugate (M6), a mono-oxidation product (M7), and a desulfonamidation and oxidation product (M8). Gut microbiota may cause the differences between in vivo and in vitro oxidation metabolisms. Contrary to EPA, S-EPA did not undergo dealkylation, suggesting that substituting the nitrogen with sulfur affects the metabolism of the adjacent alkyl side chain.

Prevention and correction of postdecompression liver dysfunction in obstructive jaundice in experimental animals

Obstructive jaundice is a complex pathology that significantly complicates surgical interventions and is accompanied by a number of negative consequences. In connection with the danger of obstructive jaundice, it is very important to timely identify the severity of this condition and take appropriate measures to treat the patient. To date, methods for predicting the risk of postoperative complications in obstructive jaundice are being actively developed, and methods for treating this pathology are being developed. In this study, we studied the biochemical parameters of the functional state of the liver of dogs with simulated obstructive jaundice with different treatment regimens. 3 experimental groups were formed. Control dogs received no treatment. The dogs of the second group underwent decompression of the biliary tract without control of the rate of bile outflow. In animals of the third group, decompression of the biliary tract was carried out and the rate of bile outflow was controlled. As therapy, dogs of this experimental group received Cytoflavin (0.28 ml / kg body weight) and sulodexide (1 ml / kg body weight) under conditions of moderate hypothermia. All animals underwent laparotomy with puncture of the gallbladder and subsequent introduction of a catheter through the cystic duct into the common bile duct. Blood from the portal vein and lymph from the main lymphatic were taken for the analysis of the following biochemical parameters: direct bilirubin, C-reactive protein, alanine aminotransferase, gammaglutamyl transferase and glucose. The concentration of the listed indicators of the functional state of the liver was assessed at different times after the simulation of obstructive jaundice. The results of the study showed that in dogs of all groups, one day after modeling obstructive jaundice, biochemical parameters significantly increase both in the blood of the portal vein and in the lymph of the thoracic lymphatic duct, which is associated with a violation of the functional state of the liver. In animals in which the rate of bile outflow was not controlled, biochemical parameters remained at a high level, which reflected the development of liver failure and reperfusion syndrome. After administration of cytoflavin and sulodexide in moderate hypothermia, dogs showed a decrease in the concentration of biochemical parameters to the control level, which characterizes the normalization of the functional state of the liver. In general, the proposed treatment regimen has shown high efficiency and can be used to correct post-decompression liver dysfunction in dogs with obstructive jaundice. The results of the study are valuable for the development of methods for treating patients with obstructive jaundice and preventing the development of postoperative complications. This article can be informative for specialists in the field of surgery and anesthesiology who are involved in the correction of postoperative complications in patients with obstructive jaundice.

Comparison between computed tomographic and ultrasonographic findings of the liver and spleen in dogs with confirmed hepatic or splenic lymphoma involvement.

Computed tomography (CT) and ultrasonography (US) may be used to assist staging in dogs with lymphoma. The imaging features of splenic and hepatic infiltration have never been directly compared in the same population of dogs. The aim of this retrospective study was to describe and compare the CT and US findings of the liver and spleen in dogs with confirmed hepatic and/or splenic lymphoma and compare imaging and cytological diagnoses. CT and US studies of 18 dogs with multicentric lymphoma involving the liver and/or the spleen were retrospectively evaluated. US detected abnormalities more frequently than CT in the spleen with lymphoma, whereas CT detected abnormalities more frequently in the liver with lymphoma. The two diagnostic imaging modalities often disagreed in the findings, including the imaging classification of the organ as normal or abnormal. US and CT often disagree in the imaging findings present in hepatic and splenic lymphoma. The results of this study suggest that cytologic evaluation of both the liver and spleen is advisable regardless of their US or CT appearance for detection of lymphoma.

Metabolic Analysis of Triptolide Microspheres in Human, Dog, Rabbit and Rat Liver Microsomes with UPLC-MS/MS Method

Objectives: Riptolide (TPL) has been shown to have a good clinical effect on rheumatoid arthritis (RA). We designed TPL microspheres (TPL-MS) and investigated its metabolic behavior in human, dog, rabbit and rat liver microsomes (HLM, DLM, RLM and SDRLM) with UPLC- MS/MS method. Methods: First, a UPLC-MS/MS method was established to measure the concentration of TPL in samples. The sample was separated on a C18 column (2.1×100 mm, 1.8μm) and eluted with a gradient elution. The precursor ions/product ions were m/z 378.1/361.0 for TPL and 260.0/116.2 for the internal standard. Then T1/2, Vmax and CLint were calculated from the above data. Finally, the metabolites of TPL-MS were identified by high-resolution UPLC-MS/MS. The sample was separated on a C18 column (2.1×100 mm, 2.2 μm) and eluted with isocratic elution. Mass spectrometric detection was carried out on a thermo Q-exactive mass spectrometer with HESI. The scanning range of precursor ions was from m/z 50 to m/z 750. Results: Through several indicators including standard curve, precision, accuracy, stability, matrix effect and recovery rate, the enzymatic kinetics parameters including T1/2, Vmax and CLint were evaluated. Several metabolites of TPL-MS were identified. Discussion: UPLC-MS/MS method is an accurate and sensitive method for the determination of TPL in liver microsome samples with good precision, accuracy and stability. The variation of parameters indicated that the microspheres can delay the elimination of TPL in liver microsomes. Conclusion: The metabolism of TPL-MS varied among species, but no new metabolites appeared.

Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5&apos;-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 μL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 μL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 μL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 μL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 μL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 μL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 μL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Hepatic lead and copper concentrations in dogs with chronic hepatitis and their relationship with hematology, serum biochemistry, and histopathology.

BackgroundAlthough the influence of copper ([Cu]) on chronic hepatitis (CH) has been widely studied in dogs, little information is available about the accumulation of other metals.Hypothesis/ObjectivesWe assessed the concentration of lead ([Pb]) in the livers of dogs with CH with or without abnormal hepatic [Cu] to establish if any association existed between [Pb] and either hematologic or biochemical variables, fibrosis, necrosis and inflammation of the liver on histology.AnimalsThirty‐four dogs with CH that had hepatic [Cu] and [Pb] determined.MethodsRetrospective review of medical records of dogs with CH and hepatic [Cu] and [Pb]. Chronic hepatitis was defined using current American College of Veterinary Internal Medicine consensus statement guidelines. Hepatic [Cu] and [Pb] were determined using square wave anodic stripping voltammetry. Dogs were divided into 2 groups based on [Cu]: <400 ppm (LoCu) and ≥400 ppm (HiCu).ResultsThe median [Cu] and [Pb] were 357 ppm (range, 100‐7743 ppm) and 58.7 (range, 6.89‐224.4 ppm), respectively. Nineteen dogs had LoCu and 15 dogs had HiCu. Median [Pb] was significantly higher in HiCu compared to LoCu dogs (P < .001). Hepatic [Pb] and [Cu] were significantly correlated (rho = 0.7; P < .001). Dogs with microcytosis had higher [Pb] than did dogs with normal red cell volume (P = .02). Hepatic [Pb] was not correlated with either necroinflammatory or fibrosis scores.Conclusions and Clinical ImportanceAlthough additional studies are needed to better understand the clinical role of hepatic [Pb], dogs with abnormal hepatic [Cu] may also have higher hepatic [Pb]. In addition, in dogs with high hepatic [Pb], microcytosis may be present.

NON-HUMAN TRANSPLANTATION OF LIVING LIVER GRAFT IN DIFFERENT POSSIBLE SITES

Orthotopic liver transplantation became a standard operation. The recipients’ number wasincreasing because of the wide indications. In many centers, many of the waiting patients dieddue to shortage of liver grafts. This study implanted living dog liver graft, in three different sites.The results showed that implantation as orthotopic non- auxiliary was better than the other sites.

Quantitative mass spectrometry imaging of drugs and metabolites: a multiplatform comparison

Mass spectrometry imaging (MSI) provides insight into the molecular distribution of a broad range of compounds and, therefore, is frequently applied in the pharmaceutical industry. Pharmacokinetic and toxicological studies deploy MSI to localize potential drugs and their metabolites in biological tissues but currently require other analytical tools to quantify these pharmaceutical compounds in the same tissues. Quantitative mass spectrometry imaging (Q-MSI) is a field with challenges due to the high biological variability in samples combined with the limited sample cleanup and separation strategies available prior to MSI. In consequence, more selectivity in MSI instruments is required. This can be provided by multiple reaction monitoring (MRM) which uses specific precursor ion-product ion transitions. This targeted approach is in particular suitable for pharmaceutical compounds because their molecular identity is known prior to analysis. In this work, we compared different analytical platforms to assess the performance of MRM detection compared to other MS instruments/MS modes used in a Q-MSI workflow for two drug candidates (A and B). Limit of detection (LOD), linearity, and precision and accuracy of high and low quality control (QC) samples were compared between MS instruments/modes. MRM mode on a triple quadrupole mass spectrometer (QqQ) provided the best overall performance with the following results for compounds A and B: LOD 35.5 and 2.5 μg/g tissue, R2 0.97 and 0.98 linearity, relative standard deviation QC <13.6%, and 97–112% accuracy. Other MS modes resulted in LOD 6.7–569.4 and 2.6–119.1 μg/g tissue, R2 0.86–0.98 and 0.86–0.98 linearity, relative standard deviation QC < 19.4 and < 37.5%, and 70–356% and 64–398% accuracy for drug candidates A and B, respectively. In addition, we propose an optimized 3D printed mimetic tissue model to increase the overall analytical throughput of our approach for large animal studies. The MRM imaging platform was applied as proof-of-principle for quantitative detection of drug candidates A and B in four dog livers and compared to LC-MS. The Q-MSI concentrations differed <3.5 times with the concentrations observed by LC-MS. Our presented MRM-based Q-MSI approach provides a more selective and high-throughput analytical platform due to MRM specificity combined with an optimized 3D printed mimetic tissue model.Graphical abstract

Tertiary Oxidation of Deoxycholate Is Predictive of CYP3A Activity in Dogs.

Deoxycholic acid (DCA, 3α, 12α-dihydroxy-5β-cholan-24-oic acid) is the major circulating secondary bile acid, which is synthesized by gut flora in the lower gut and selectively oxidized by CYP3A into tertiary metabolites, including 1β,3α,12α-trihydroxy-5β-cholan-24-oic acid (DCA-1β-ol) and 3α,5β,12α-trihydroxy-5β-cholan-24-oic acid (DCA-5β-ol) in humans. Since DCA has the similar exogenous nature and disposition mechanisms as xenobiotics, this work aimed to investigate whether the tertiary oxidations of DCA are predictive of in vivo CYP3A activities in beagle dogs. In vitro metabolism of midazolam (MDZ) and DCA in recombinant canine CYP1A1, 1A2, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 enzymes clarified that CYP3A12 was primarily responsible for either the oxidation elimination of MDZ or the regioselective oxidation metabolism of DCA into DCA-1β-ol and DCA-5β-ol in dog liver microsomes. Six male dogs completed the CYP3A intervention studies including phases of baseline, inhibition (ketoconazole treatments), recovery, and induction (rifampicin treatments). The oral MDZ clearance after a single dose was determined on the last day of the baseline, inhibition, and induction phases, and subjected to correlation analysis with the tertiary oxidation ratios of DCA detected in serum and urine samples. The results confirmed that the predosing serum ratios of DCA oxidation, DCA-5β-ol/DCA, and DCA-1β-ol/DCA were significantly and positively correlated both intraindividually and interindividually with oral MDZ clearance. It was therefore concluded that the tertiary oxidation of DCA is predictive of CYP3A activity in beagle dogs. Clinical transitional studies following the preclinical evidence are promising to provide novel biomarkers of the enterohepatic CYP3A activities. SIGNIFICANCE STATEMENT: Drug development, clinical pharmacology, and therapeutics are under insistent demands of endogenous CYP3A biomarkers that avoid unnecessary drug exposure and invasive sampling. This work has provided the first proof-of-concept preclinical evidence that the CYP3A catalyzed tertiary oxidation of deoxycholate, the major circulating secondary bile acid synthesized in the lower gut by bacteria, may be developed as novel in vivo biomarkers of the enterohepatic CYP3A activities.

High‐resolution mass spectrometry‐based approach for the identification and profiling of the metabolites of taletrectinib formed in liver microsomes

Taletrectinib is a potent, orally active, and selective ROS1/NTRK kinase inhibitor. The aim of this study was to study the metabolism of taletrectinib in rat, dog, and human liver microsomes. The biotransformation of taletrectinib was carried out using rat, dog, and human liver microsomes supplemented with nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH) and uridine diphosphate glucuronic acid (UDPGA). The microsomal incubations were conducted at 37°C for 60min. The formed metabolites were identified by ultrahigh performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS) using electrospray ionization in the positive ion mode. They were identified by accurate masses and MS/MS spectra and based on their fragmentation pathways. With UHPLC-HRMS, a total of 10 metabolites including one glucuronide conjugate (M7) were structurally identified. M9 and M10 were unambiguously identified as taletrectinib alcohol and taletrectinib ketone, respectively, using reference standards. The phase I metabolic pathways of taletrectinib involved N-dealkylation, O-dealkylation, oxidative deamination, and oxygenation; the phase II metabolic pathways referred to glucuronidation. The current study investigated the in vitro metabolic fate of taletrectinib in animals and human species, which would bring us considerable benefits for the subsequent studies focusing on the pharmacological effect and toxicity of this drug.

Detoxication versus Bioactivation Pathways of Lapatinib In Vitro: UGT1A1 Catalyzes the Hepatic Glucuronidation of Debenzylated Lapatinib.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17β-estradiol 3-glucuronidation (R 2 = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase-mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

Toxocara canis Differentially Affects Hepatic MicroRNA Expression in Beagle Dogs at Different Stages of Infection.

Toxocara canis is a neglected zoonotic parasite, which threatens the health of dogs and humans worldwide. The molecular mechanisms that underlie the progression of T. canis infection remain mostly unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been identified in T. canis; however, the regulation and role of miRNAs in the host during infection remain incompletely understood. In this study, we determined hepatic miRNA expression at different stages of T. canis infection in beagle dogs. Individual dogs were infected by 300 embryonated T. canis eggs, and their livers were collected at 12 hpi (hours post-infection), 24 hpi, and 36 dpi (days post-infection). The expression profiles of liver miRNAs were determined using RNA-sequencing. Compared to the control groups, 9, 16, and 34 differentially expressed miRNAs (DEmiRNAs) were detected in the livers of infected dogs at the three infection stages, respectively. Among those DEmiRNAs, the novel-294 and cfa-miR-885 were predicted to regulate inflammation-related genes at the initial stage of infection (12 hpi). The cfa-miR-1839 was predicted to regulate the target gene TRIM71, which may influence the development of T. canis larvae at 24 hpi. Moreover, cfa-miR-370 and cfa-miR-133c were associated with immune response at the final stage of infection (36 dpi). Some immunity-related Gene Ontology terms were enriched particularly at 24 hpi. Likewise, Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that many significantly enriched pathways were involved in inflammation and immune responses. The expression level of nine DEmiRNAs was validated using quantitative real-time PCR (qRT-PCR). These results show that miRNAs play critical roles in the pathogenesis of T. canis during the hepatic phase of parasite development. Our data provide fundamental information for further investigation of the roles of miRNAs in the innate/adaptive immune response of dogs infected by T. canis.