Dog Liver Research Articles

Toxicological screening of zinc oxide nanoparticles in mongrel dogs after seven days of repeated subcutaneous injections

Zinc oxide nanoparticles (ZnO NPs) have recently been applied in various veterinary and medical fields, however, the toxicological evaluations of these NPs in dogs are lacking. Therefore, the current study is designed to assess the impact of exposure to daily subcutaneous (SC) injections of ZnO NPs at different concentrations on various organs of mongrel dogs. Nine dogs were randomly divided into three groups (n = 3 for each) as follows: group (1) served as the control group, whereas groups (2&3) received SC injections of 50 and 100 ppm ZnO NPs (8 and 16 μg/kg bwt), respectively, once/day for 7 days. Our results revealed that ZnO NPs disrupted the oxidant/antioxidant balance in the lungs, liver, and kidneys of dogs in a dose-dependent manner. ZnO NPs induced dose-dependent radiological, ultrasonographical, and histopathological alterations in various organs especially lungs, spleen, liver, and kidneys along with disturbance in both liver and kidney biomarkers levels. Most organs of both ZnO NPs receiving groups displayed strong caspase-3 protein expression. Additionally, it upregulates the transcriptase levels of TNF-α and VEGF, as well as downregulates the antiapoptotic gene IL-10 in lung, kidney, and liver tissue homogenates. It was concluded that the daily SC injections of dogs with ZnO NPs at concentrations of 50 and 100 ppm caused extensive oxidative stress damage in various organs which provoked serious pathological processes such as apoptosis and inflammation.

Investigation of Functional Cytochrome P450 4A Enzymes in Liver and Kidney of Pigs, Cats, Tree Shrews, and Dogs in Comparison with the Metabolic Capacity of Human P450 4A11.

Pigs are sometimes used in preclinical drug metabolism studies, with growing interest, and thus their drug-metabolizing enzymes, including the cytochromes P450 (P450 or CYP; EC 1.14.14.1), need to be examined. In the present study, novel CYP4A cDNAs were isolated and characterized, namely, pig CYP4A23 and CYP4A90; cat CYP4A37 and CYP4A106; and tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g. For comparison, the following known CYP4A cDNAs were also analyzed: pig CYP4A21 and dog CYP4A37, CYP4A38, and CYP4A39. These CYP4A cDNAs all contained open reading frames of 504-513 amino acids and had high amino acid sequence identity (74%-80%) with human CYP4As. Phylogenetic analysis of amino acid sequences revealed that these CYP4As were clustered in each species. All CYP4A genes contained 12 coding exons and formed a gene cluster in the corresponding genomic regions. A range of tissue types were analyzed, and these CYP4A mRNAs were preferentially expressed in liver and/or kidney, except for pig CYP4A90, which showed preferential expression in lung and duodenum. CYP4A enzymes, heterologously expressed in Escherichia coli, preferentially catalyzed lauric acid 12-hydroxylation and arachidonic acid 20-hydroxylation, just as human CYP4A11 does, with the same regioselectivity (i.e., at the ω-position of fatty acids). These results imply that dog, cat, pig, and tree shrew CYP4As have functional characteristics similar to those of human CYP4A11, with minor differences in lauric acid 12-hydroxylation. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450, CYP) 4As are important P450s in human biological processes because of their fatty acid-metabolizing ability. Pig CYP4A21, CYP4A23, and CYP4A90; cat CYP4A37 and CYP4A106; tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g; and dog CYP4A37, CYP4A38, and CYP4A39 cDNAs were isolated and analyzed. These CYP4A cDNAs shared relatively high sequence identities with human CYP4A11 and CYP4A22. Pig, cat, tree shrew, and dog CYP4As in the liver and kidneys are likely to catalyze the ω-hydroxylation of fatty acids.

Glucuronidation of tizoxanide, an active metabolite of nitazoxanide, in liver and small intestine: Species differences in humans, monkeys, dogs, rats, and mice and responsible UDP-glucuronosyltransferase isoforms in humans

Tizoxanide (TZX) is an active metabolite of nitazoxanide (NTZ) originally developed as an antiparasitic agent, and is predominantly metabolized into TZX glucuronide. In the present study, TZX glucuronidation by the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice, and recombinant human UDP-glucuronosyltransferase (UGT) were examined. The kinetics of TZX glucuronidation by the liver and intestinal microsomes followed the Michaelis–Menten or biphasic model, with species-specific variations in the intrinsic clearance (CLint). Rats and mice exhibited the highest CLint values for liver microsomes, while mice and rats were the highest for intestinal microsomes. Among human UGTs, UGT1A1 and UGT1A8 demonstrated significant glucuronidation activity. Estradiol and emodin inhibited TZX glucuronidation activities in the human liver and intestinal microsomes in a dose-dependent manner, with emodin showing stronger inhibition in the intestinal microsomes. These results suggest that the roles of UGT enzymes in TZX glucuronidation in the liver and small intestine differ extensively across species and that UGT1A1 and/or UGT1A8 mainly contribute to the metabolism and elimination of TZX in humans. This study presents the relevant and novel-appreciative report on TZX metabolism catalyzed by UGT enzymes, which may aid in the assessment of the antiparasitic, antibacterial, and antiviral activities of NTZ for the treatment of various infections.

Application of Electro-Activated Dissociation Fragmentation Technique to Identifying Glucuronidation and Oxidative Metabolism Sites of Vepdegestrant by Liquid Chromatography-High Resolution Mass Spectrometry.

Drug metabolite identification is an integrated part of drug metabolism and pharmacokinetics studies in drug discovery and development. Definitive identification of metabolic modification sides of test compounds such as screening metabolic soft spots and supporting metabolite synthesis are often required. Currently, liquid chromatography-high resolution mass spectrometry is the dominant analytical platform for metabolite identification. However, the interpretation of product ion spectra generated by commonly used collision-induced disassociation (CID) and higher-energy collisional dissociation (HCD) often fails to identify locations of metabolic modifications, especially glucuronidation. Recently, a ZenoTOF 7600 mass spectrometer equipped with electron-activated dissociation (EAD-HRMS) was introduced. The primary objective of this study was to apply EAD-HRMS to identify metabolism sites of vepdegestrant (ARV-471), a model compound that consists of multiple functional groups. ARV-471 was incubated in dog liver microsomes and 12 phase I metabolites and glucuronides were detected. EAD generated unique product ions via orthogonal fragmentation, which allowed for accurately determining the metabolism sites of ARV-471, including phenol glucuronidation, piperazine N-dealkylation, glutarimide hydrolysis, piperidine oxidation, and piperidine lactam formation. In contrast, CID and HCD spectral interpretation failed to identify modification sites of three O-glucuronides and three phase I metabolites. The results demonstrated that EAD has significant advantages over CID and HCD in definitive structural elucidation of glucuronides and phase I metabolites although the utility of EAD-HRMS in identifying various types of drug metabolites remains to be further evaluated. SIGNIFICANCE STATEMENT: Definitive identification of metabolic modification sites by liquid chromatography-high resolution mass spectrometry is highly needed in drug metabolism research, such as screening metabolic soft spots and supporting metabolite synthesis. However, commonly used collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation techniques often fail to provide critical information for definitive structural elucidation. In this study, the electron-activated dissociation (EAD) was applied to identifying glucuronidation and oxidative metabolism sites of vepdegestrant, which generated significantly better results than CID and HCD.

Metabolic profiling of lumateperone in vitro and in vivo by UPLC-Q Exactive Orbitrap HRMS, and its pharmacokinetic study in rat plasma by LC-MS/MS

Lumateperone is a novel agent approved by FDA for treatment of schizophrenia in adults. To elucidate the species differences in the of biotransformation of lumateperone and its pharmacokinetic (PK) characteristics in rats, the metabolite identification of lumateperone was carried out in rat, dog and human liver microsomes, and rat plasma after oral administration using UPLC-Q Exactive Orbitrap high-resolution mass spectrometry HRMS. Furtherly, the PK characteristics of lumateperone and its N-demethylated metabolite (M3) in rat plasma were investigated using a validated LC-MS/MS method following intravenous and oral administration. Fourteen phase I metabolites were found in liver microsomes and ten of them were observed in rat plasma. N-demethylation, carbonylation, dehydrogenation, and piperazine ring cleavage were main metabolic pathway of lumateperone. No unique metabolites were formed in human liver microsomes. After rapid absorption in rats, lumateperone was quickly metabolized and eliminated with bioavailability of less than 5%. The exposure level of M3 was about 1.5-fold higher than that of lumateperone in rat plasma. Lumatperone underwent extensive metabolism and was absorbed rapidly in rats. Metabolite M3 had equivalent or slightly higher exposure levels than lumateperone. This study provides essential PK information to facilitate further pharmacodynamic researches of lumateperone.

Identification of UDP-glucuronosyltransferase (UGT) isoforms involved in the metabolism of Chlorophenols (CPs)

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs’ mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.

Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer

AbstractGene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e−4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.

Radiolabel Uncovers Nonintuitive Metabolites of BIIB104: Novel Release of [14C]Cyanide from 2-Cyanothiophene and Subsequent Formation of [14C]Thiocyanate.

BIIB104 (formerly PF-04958242), N-((3S,4S)-4-(4-(5-cyanothiophen-2-yl)phenoxy)tetrahydrofuran-3-yl)propane-2-sulfonamide, is an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiator investigated for the treatment of cognitive impairment associated with schizophrenia. Preliminary in vitro metabolism studies with non-radiolabeled BIIB104 in rat, dog, and human liver microsomes (RLM, DLM, and HLM) showed O-dealkylation in all three species, tetrahydrofuran hydroxylation dominating in DLM and HLM, and thiophene hydroxylation prevalent in RLM. However, a subsequent rat mass balance study with [nitrile-14C]BIIB104 showed incomplete recovery of administered radioactivity (∼80%) from urine and feces over 7 days following an oral dose, and an exceptionally long plasma total radioactivity half-life. Radiochromatographic metabolite profiling and identification, including chemical derivation, revealed that [14C]cyanide was a major metabolite of [nitrile-14C]BIIB104 in RLM, but a minor and trace metabolite in DLM and HLM, respectively. Correspondingly in bile duct-cannulated rats, [14C]thiocyanate accounted for ∼53% of total radioactivity excreted over 48 hours postdose and it, as an endogenous substance, explained the exceptionally long plasma radioactivity half-life. The release of [14C]cyanide from the 2-cyanothiophene moiety is postulated to follow an epoxidation-initiated thiophene-opening based on the detection of non-radiolabeled counterpart metabolites in RLM. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate. Additionally, the potential cyanide metabolite of nitrile-containing drug molecules may be detected in liver microsomes with liquid chromatography-mass spectrometry following a chemical derivatization. SIGNIFICANCE STATEMENT: Using [nitrile-14C]BIIB104, non-intuitive metabolites of BIIB104 were discovered involving a novel cyanide release from the 2-cyanothiophene motif via a postulated epoxidation-initiated thiophene-opening. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate.

Integration of metabolic flux with hepatic glucagon signaling and gene expression profiles in the conscious dog.

Glucagon rapidly and profoundly stimulates hepatic glucose production (HGP), but for reasons that are unclear, this effect normally wanes after a few hours, despite sustained plasma glucagon levels. This study characterized the time course of glucagon-mediated molecular events and their relevance to metabolic flux in the livers of conscious dogs. Glucagon was either infused into the hepato-portal vein at a sixfold basal rate in the presence of somatostatin and basal insulin, or it was maintained at a basal level in control studies. In one control group, glucose remained at basal, whereas in the other, glucose was infused to match the hyperglycemia that occurred in the hyperglucagonemic group. Elevated glucagon caused a rapid (30 min) and largely sustained increase in hepatic cAMP over 4 h, a continued elevation in glucose-6-phosphate (G6P), and activation and deactivation of glycogen phosphorylase and synthase activities, respectively. Net hepatic glycogenolysis increased rapidly, peaking at 15 min due to activation of the cAMP/PKA pathway, then slowly returned to baseline over the next 3 h in line with allosteric inhibition by glucose and G6P. Glucagon's stimulatory effect on HGP was sustained relative to the hyperglycemic control group due to continued PKA activation. Hepatic gluconeogenic flux did not increase due to the lack of glucagon's effect on substrate supply to the liver. Global gene expression profiling highlighted glucagon-regulated activation of genes involved in cellular respiration, metabolic processes, and signaling, as well as downregulation of genes involved in extracellular matrix assembly and development.NEW & NOTEWORTHY Glucagon rapidly stimulates hepatic glucose production, but these effects are transient. This study links the molecular and metabolic flux changes that occur in the liver over time in response to a rise in glucagon, demonstrating the strength of the dog as a translational model to couple findings in small animals and humans. In addition, this study clarifies why the rapid effects of glucagon on liver glycogen metabolism are not sustained.

A phase I study of ATR inhibitor gartisertib (M4344) as a single agent and in combination with carboplatin in patients with advanced solid tumours

BackgroundGartisertib is an oral inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), a key kinase of the DNA damage response. We aimed to determine the safety and tolerability of gartisertib ± carboplatin in patients with advanced solid tumours.MethodsThis phase I open-label, multicenter, first-in-human study comprised four gartisertib cohorts: A (dose escalation [DE]; Q2W); A2 (DE; QD/BID); B1 (DE+carboplatin); and C (biomarker-selected patients).ResultsOverall, 97 patients were enroled into cohorts A (n = 42), A2 (n = 26), B1 (n = 16) and C (n = 13). The maximum tolerated dose and recommended phase II dose (RP2D) were not declared for cohorts A or B1. In cohort A2, the RP2D for gartisertib was determined as 250 mg QD. Gartisertib was generally well-tolerated; however, unexpected increased blood bilirubin in all study cohorts precluded further DE. Investigations showed that gartisertib and its metabolite M26 inhibit UGT1A1-mediated bilirubin glucuronidation in human but not dog or rat liver microsomes. Prolonged partial response (n = 1 [cohort B1]) and stable disease >6 months (n = 3) did not appear to be associated with biomarker status. Exposure generally increased dose-dependently without accumulation.ConclusionGartisertib was generally well-tolerated at lower doses; however, unexpected liver toxicity prevented further DE, potentially limiting antitumour activity. Gartisertib development was subsequently discontinued.ClinicalTrials.govNCT02278250.

Metabolite characterisation of the peptide–drug conjugate LN005 in liver S9s by UHPLC-Orbitrap-HRMS

LN005 is a peptide–drug conjugate (PDC) targeting glucose-regulated protein 78 (GRP78) to treat several types of cancer, such as breast, colon, and prostate cancer. As a new drug modality, understanding its metabolism and elimination pathways will help us to have a whole picture of it. Currently, there are no metabolic studies on LN005; therefore, this study aimed to investigate the metabolism of LN005, clarify its metabolic profile in the liver S9s of different species, and identify the major metabolic pathways and differences between species. The incubation samples were measured by ultra-high performance liquid chromatography combined with orbitrap tandem mass spectrometry (UHPLC-Orbitrap-HRMS). The results showed that LN005 was metabolised by liver S9s, and four metabolites were identified. The main metabolic pathway of LN005 in liver S9s was oxidative deamination to ketone or hydrolysis. Similar metabolic profiles were observed in mouse, rat, dog, monkey, and human liver S9s, indicating no differences between these four animal species and humans. This study provides information for the structural modification and optimisation of LN005 and affords a reference for subsequent animal experiments and human metabolism of other PDCs.

Experimental infection and co-infection with Chinese strains of Ehrlichia canis and Babesia vogeli in intact and splenectomized dogs: Insights on clinical, hematologic and treatment responses

Animal infection models are crucial for studying various aspects of Ehrlichia canis infections. To understand the pathogenesis of the first Chinese isolate of E. canis and simulate the natural progression of canine ehrlichiosis, we developed a model with 18 Beagle dogs that consisted of E. canis initial infection (days 0–17), treatment with doxycycline or rifampicin (days 18–32), recovery (days 33–66), E. canis reinfection (days 67–91), and Babesia vogeli superinfection (days 92–116). We measured body weight and rectal temperature every other day, drew blood every 4 days for routine hematology and biochemistry tests, and for quantification of E. canis and B. vogeli by quantitative PCRs. In this study, the first isolate of E. canis from China was used to experimentally infect dogs, and the infected dogs exhibited clinical signs of acute severe ehrlichiosis, including high fever, loss of appetite, dehydration, and body weight loss, confirming the similar pathogenicity of E. canis in China as compared to isolates from other regions. Infection with E. canis and B. vogeli led to reduced body weight and fever in dogs. Doxycycline treatment led to absence of E. canis DNA in infected dogs, while rifampicin treatment lowered the blood E. canis copy number up to 1.5 folds. E. canis-free infected dogs after doxycycline treatment were successfully re-infected with E. canis, indicating dogs with antibodies are still at risk of re-infection. Super-infection with B. vogeli resulted in higher fever, more severe anemia, and a reduced number of platelets. Splenectomized dogs showed significantly higher E. canis numbers during recovery and re-infection than intact dogs. The histological changes were observed in brain, lung, kidney, liver and spleen of the infected dogs. The findings in this study provide insights into clinical and hematologic responses, as well as effective treatment options, for dogs infected with the first Chinese isolate of E. canis, and may contribute to our understanding of the diagnosis and prevention of tick-borne diseases in dogs, including canine monocytic ehrlichiosis.

The in vitro metabolism of GMDTC in liver microsomes of human, monkey, dog, rat and mouse: Metabolic stability assessment, metabolite identification and interspecies comparison

Sodium (S)− 2-(dithiocarboxylato((2 S,3 R,4 R,5 R)− 2,3,4,5,6-pentahydroxyhexyl)amino)− 4(methylthio)butanoate (GMDTC) is a compound that removes cadmium from kidney cells. This study aims to investigate the metabolic stability and metabolite identification of GMDTC in various liver microsomes, including those from human, monkey, dog, rat and mouse. The results show that the T1/2 values of GMDTC in human, monkey, dog, rat and mouse liver microsomes were 16.54, 18.14, 16.58, 15.16 and 16.00 min, respectively. While the hepatic extraction ratios (ERh) of GMDTC measured after 60 min incubation in these liver microsomes were 0.82, 0.70, 0.80, 0.75 and 0.79, respectively, indicating that GMDTC exhibits rapid hepatic metabolism and high hepatic clearance with no significant interspecies differences. Subsequent metabolite identification by high-resolution mass spectrometry revealed the presence of three metabolites, designated M1∼M3. The major metabolite products of GMDTC were found to be M1 and M2. The relative abundances of the hydrolysis products (M1 and M2) in human, monkey, dog, rat and mouse liver microsomes were found to be 97.18%, 97.99%, 95.94%, 96.31% and 93.43%, respectively, indicating that hydrolysis is the primary metabolic pathway of GMDTC in liver microsomes in vitro, and with no significant interspecies differences.

Plasma Neurofilament Light Chains as Blood-Based Biomarkers for Early Diagnosis of Canine Cognitive Dysfunction Syndrome.

The number of elderly dogs is increasing significantly worldwide, and many elderly dogs develop canine cognitive dysfunction syndrome (CCDS). CCDS is the canine analog of Alzheimer's disease (AD) in humans. It is very important to develop techniques for detecting CDDS in dogs. Thus, we used the detection of neurofilament light chains (NfL) in plasma as a blood-based biomarker for the early diagnosis of canine Alzheimer's disease using immunomagnetic reduction (IMR) technology by immobilizing NfL antibodies on magnetic nanoparticles. According to the 50-point CCDS rating scale, we divided 36 dogs into 15 with CCDS and 21 without the disease. The results of our IMR assay showed that the plasma NfL levels of dogs with CCDS were significantly increased compared to normal dogs (p < 0.01). By plasma biochemical analysis, we further confirmed that the liver and renal dysfunction biomarkers of dogs with CCDS were significantly elevated compared to normal dogs (p < 0.01-0.05). On the basis of our preliminary study, we propose that IMR technology could be an ideal biosensor for detecting plasma NfL for the early diagnosis of CCDS.

Laparoscopic ultrasonography of the liver is feasible and safe in dogs.

To describe the feasibility and technique for performing laparoscopic ultrasound (LUS) of the liver in dogs. 12 client-owned dogs presenting for elective laparoscopic surgery from January 1, 2022, to October 31, 2022. Laparoscopic exploration and LUS of the liver were performed in all dogs. Dogs were positioned in reverse Trendelenburg and laterally rotated to facilitate access to all liver lobes. Time to perform laparoscopic exploration and LUS, ability to visualize and access each liver lobe entirely, and any complications were recorded. Each dog underwent an elective laparoscopic procedure. The surgeon completed a National Aeronautics and Space Administration Task Load Index (NASA-TLX) questionnaire after surgery. Mean body weight was 25.9 kg (SD, ± 4.1 kg; range, 5.7 to 62 kg). All liver lobes were scanned to the level of the hilus in 10/12 dogs. In 2 dogs, the caudate lobe could not be completely imaged. Median time to perform LUS was 9 minutes (IQR, 5 to 16.5 minutes), and median NASA-TLX score was 9/100 (IQR, 6.3 to 20). There was a significantly strong negative correlation between time to perform LUS (r = -0.77; P = .0037) and NASA-TLX score (r = -0.84; P = .0006) with trial number. Minor complications occurred in 2 dogs during laparoscopic exploration. No complications occurred during LUS. LUS was feasible and safe in all dogs. The right lateral and caudate lobes were occasionally challenging to access. Technical demand and time to perform LUS improved with experience, suggesting a learning curve. Evaluation of LUS in dogs with clinical disease is warranted.

Preparation of glucuronides using liver microsomes and their characterization by 1D/2D NMR spectroscopy and mass spectrometry: Application to fentanyl metabolites.

A simple, low-cost method for preparing glucuronic acid-conjugated metabolites was developed using fentanyl, a potent synthetic opioid, as a model drug. Five glucuronic acid-conjugated metabolites of fentanyl were measured in the culture medium of fresh human hepatocytes incubated with fentanyl. These glucuronides were also formed by incubation of their corresponding substrates (e.g., 4'-hydroxy-fentanyl and β-hydroxy-fentanyl) with uridine 5'-diphosphoglucuronic acid and human liver microsomes (HLM). Experiments using liver microsomes of several animals revealed that significant species differences exist in the glucuronide formation patterns; fentanyl glucuronide was only formed in HLM, and 4'-hydroxy-fentanyl glucuronide was formed much more in rat liver microsomes (RLM) than HLM and dog liver microsomes. Furthermore, surprisingly, HLM and RLM showed opposite substrate selectivity for the enantiomers of β-hydroxy-fentanyl. Submilligram amounts of three of these metabolites, namely, 4'-hydroxy-fentanyl glucuronide and two glucuronides of β-hydroxy-fentanyl, were prepared by using HLM or RLM. The products were readily purified with a reversed-phase/anion-exchange mixed-mode solid-phase extraction cartridge, and then, their chemical structures were confirmed by 1D/2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry data. In addition, the products were quantitated by quantitative NMR, and the yields were 3.6-69%.

Cannabidiol and cannabidiolic acid: Preliminary in vitro evaluation of metabolism and drug-drug interactions involving canine cytochrome P-450, UDP-glucuronosyltransferase, and P-glycoprotein.

Phytocannabinoid-rich hemp extracts containing cannabidiol (CBD) and cannabidiolic acid (CBDA) are increasingly being used to treat various disorders in dogs. The objectives of this study were to obtain preliminary information regarding the in vitro metabolism of these compounds and their capacity to inhibit canine cytochrome P450 (CYP)-mediated drug metabolism and canine P-glycoprotein-mediated transport. Pure CBD and CBDA, and hemp extracts enriched for CBD and for CBDA were evaluated. Substrate depletion assays using pooled dog liver microsomes showed CYP cofactor-dependent depletion of CBD (but not CBDA) and UDP-glucuronosytransferase cofactor-dependent depletion of CBDA (but not CBD) indicating major roles for CYP and UDP-glucuronosytransferase in the metabolism of these phytocannabinoids, respectively. Further studies using recombinant canine CYPs demonstrated substantial CBD depletion by the major hepatic P450 enzymes CYP1A2 and CYP2C21. These results were confirmed by showing increased CBD depletion by liver microsomes from dogs treated with a known CYP1A2 inducer (β-naphthoflavone) and with a known CYP2C21 inducer (phenobarbital). Cannabinoid-drug inhibition experiments showed inhibition (IC50 = 4.6-8.1 μM) of tramadol metabolism via CYP2B11-mediated N-demethylation (CBD and CBDA) and CYP2D15-mediated O-demethylation (CBDA only) by dog liver microsomes. CBD and CBDA did not inhibit CYP3A12-mediated midazolam 1'-hydroxylation (IC50 > 10 μM). CBD and CBDA were not substrates or competitive inhibitors of canine P-glycoprotein. Results for cannabinoid-enriched hemp extracts were identical to those for pure cannabinoids. These in vitro studies indicate the potential for cannabinoid-drug interactions involving certain CYPs (but not P-glycoprotein). Confirmatory in vivo studies are warranted.

Detection and identification of imperatorin metabolites in rat, dog, monkey, and human liver microsomes by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry and Compound Discoverer software.

Imperatorin, a furanocoumarin that widely exists in many umbelliferous herbs, has been demonstrated to have a variety of pharmacological effects, including anti-inflammatory, antiosteoporosis, and antitumor activities. The purpose of this study was to investigate the metabolism of imperatorin using liver microsomes. The metabolites were generated by individually incubating imperatorin with rat, dog, monkey, and human liver microsomes. To trap the reactive metabolites during microsomal metabolism, glutathione (GSH) was included in the incubation. A LC technique coupled with benchtop orbitrap MS with full mass/data-dependent tandem mass spectrometry acquisition mode was used to detect and identify the generated metabolites. The possible structures of the metabolites were characterized according to their accurate masses and fragment ions. Under the current conditions, a total of 10 metabolites, including four GSH adducts, were identified. The results indicated that imperatorin underwent extensive metabolic reactions including hydroxylation, oxidation, glucuronidation, and GSH conjugation. This study provides essential data on the metabolism of imperatorin, which will be helpful for us to understand the safety and efficacy of this bioactive compound.

Preclinical pharmacokinetics and metabolism study of SCO-267, a GPR40 full agonist, in beagle dogs using ultra-high performance liquid chromatography coupled to tandem mass spectrometry.

SCO-267 is a GPR40 full agonist that has been developed for the treatment of diabetes. To support its preclinical and clinical development, in this study, we developed an ultra-high performance liquid chromatography tandem mass spectrometric method for the determination of SCO-267 in dog plasma using cabozantinib as internal standard (IS). The chromatographic separation was obtained on a Waters acquity BEH C18 column (50×2.1 mm, I.D., 1.7 μm) and the detection was performed using Thermo TSQ triple quadrupole mass spectrometer with multiple reactions monitoring (MRM) positive mode at m/z 615.3>230.1 for SCO-267 and m/z 502.5>323.3 for IS. The method was validated over the concentration range of 1-2000 ng/mL with the lower limit of quantification of 1 ng/mL. The selectivity, linearity, precision and accuracy over this range were acceptable. The extraction recovery was more than 88.73% and no matrix effect was observed. SCO-267 was demonstrated stable during the storage and processing period. The new method was successfully applied to the pharmacokinetic study in beagle dogs following a single oral and intravenous administration. The oral bioavailability was 64.34%. In addition, the metabolites from dog liver microsomal incubation and plasma collected after an oral administration were identified by a UHPLC-HRMS method. The biotransformation pathways of SCO-267 involved oxygenation, O-demethylation, N-dealkylation and acyl glucuronidation.

Congenital Portosystemic Shunts in Dogs and Cats: Treatment, Complications and Prognosis.

Congenital portosystemic shunts (CPSS) are a common vascular anomaly of the liver in dogs and cats. Clinical signs of CPSS are non-specific and may wax and wane, while laboratory findings can raise the clinical suspicion for CPSS, but they are also not specific. Definitive diagnosis will be established by evaluation of liver function tests and diagnostic imaging. The aim of this article is to review the management, both medical and surgical, complications, and prognosis of CPSS in dogs and cats. Attenuation of the CPSS is the treatment of choice and may be performed by open surgical intervention using ameroid ring constrictors, thin film banding, and partial or complete suture ligation or by percutaneous transvenous coil embolization. There is no strong evidence to recommend one surgical technique over another. Medical treatment strategies include administration of non-absorbable disaccharides (i.e., lactulose), antibiotics, and dietary changes, and are indicated for pre-surgical stabilization or when surgical intervention is not feasible. After CPSS attenuation, short- and long-term post-surgical complications may be seen, such as post-operative seizures and recurrence of clinical signs, respectively. Prognosis after surgical attenuation of CPSS is generally favorable for dogs and fair for cats.