A Neuro-hormonal Circuit for Paternal Behavior Controlled by a Hypothalamic Network Oscillation.

Parental behavior is pervasive throughout the animal kingdom and essential for species survival. However, the relative contribution of the father to offspring care differs markedly across animals, even between related species. The mechanisms that organize and control paternal behavior remain poorly understood. Using Sprague-Dawley rats and C57BL/6 mice, two species at opposite ends of the paternal spectrum, we identified that distinct electrical oscillation patterns in neuroendocrine dopamine neurons link to a chain of low dopamine release, high circulating prolactin, prolactin receptor-dependent activation of medial preoptic area galanin neurons, and paternal care behavior in male mice. In rats, the same parameters exhibit inverse profiles. Optogenetic manipulation of these rhythms in mice dramatically shifted serum prolactin and paternal behavior, whereas injecting prolactin into non-paternal rat sires triggered expression of parental care. These findings identify a frequency-tuned brain-endocrine-brain circuit that can act as a gain control system determining a species' parental strategy.

Validation of the Absorption-Enhancing Ability of Oligoarginines Grafted onto a Backbone of Hyaluronic Acid through Animal Studies from Rodents to Primates.

The purpose of our research is to develop functional additives that enhance mucosal absorption of biologics, such as peptide/protein and antibody drugs, to provide their non-to-poor invasive dosage forms self-managed by patients. Our previous in vivo and in vitro studies demonstrated that the intranasal absorption of biologics in mice was significantly improved when coadministered with oligoarginines anchored chemically to hyaluronic acid via a glycine spacer, presumably through syndecan-4-mediated macropinocytosis under activation by oligoarginines. The present mouse experiments first revealed that diglycine-L-tetraarginine-linked hyaluronic acid significantly enhanced the intranasal absorption of sulpiride, which is a poor-absorptive organic compound with a low molecular weight. However, similar enhancement was not observed for levofloxacin, which has a similarly low molecular weight but is a well-absorptive organic compound, probably because its absorption was mostly dominated by passive diffusion. The subsequent monkey experiments revealed that there was no species difference in the absorption-enhancing ability of diglycine-L-tetraarginine-linked hyaluronic acid for not only organic compounds but also biologics. This was presumably because the expression levels of endocytosis-associated membrane proteins on the nasal mucosa in monkeys were almost equivalent to those in mice, and poorly membrane-permeable/membrane-impermeable drugs were mainly absorbed via syndecan-4-mediated macropinocytosis, regardless of animal species. Drug concentrations in the brain assessed in mice and monkeys and those in the cerebral spinal fluids (CSFs) assessed in monkeys indicated that drugs would be delivered from the systemic circulation to the central nervous system by crossing the blood-brain and the blood-CSF barriers under coadministration with the hyaluronic acid derivative. In line with our original hypothesis, this new set of data supported that our oligoarginine-linked hyaluronic acid would locally perform on the mucosal surface and enhance the membrane permeation of drugs under its colocalization.

Pharmacological differences between human and mouse P2X4 receptor explored using old and new tools.

There is growing interest in the P2X4 receptor as a therapeutic target for several cardiovascular, inflammatory and neurological conditions. Key to exploring the physiological and pathophysiological roles of P2X4 is access to selective compounds to probe function in cells, tissues and animal models. There has been a recent growth in selective antagonists for P2X4, though agonist selectivity is less well studied. As there are some known pharmacological differences between P2X receptors from different species, it is important to understand these differences when designing a pharmacological strategy to probe P2X4 function in human tissue and mouse models. Here, we provide a systematic comparison of agonist and antagonist pharmacology in 1321N1 cells expressing either human or mouse P2X4 orthologues. We identify a rank order of agonist potency of ATP > 2-MeSATP > αßmeATP = BzATP > CTP = γ-[(propargyl)-imido]-ATP for human P2X4 and ATP > 2-MeSATP = CTP > ATPγS = γ-[(propargyl)-imido]-ATP = BzATP for mouse. Human P2X4 is not activated by ATPγS but can be activated by αßmeATP. We identify a rank order of antagonist potency of BAY-1797 = PSB-12062 = BX-430 > 5-BDBD > TNP-ATP = PPADS for human P2X4 and BAY-1797 > PSB-12062 = PPADS > TNP-ATP for mouse. Mouse P2X4 is not antagonised by 5-BDBD or BX-430. The study reveals key pharmacological differences between human and mouse P2X4, highlighting caution when selecting tools for comparative studies between human and mouse and ascribing cellular responses of some commonly used agonists to P2X4.

Indirubin mediates adverse intestinal reactions in guinea pigs by downregulating the expression of AchE through AhR.

Indirubin is the main component of the traditional Chinese medicine Indigo naturalis (IN), a potent agonist of aryl hydrocarbon receptors (AhRs). In China, IN is used to treat psoriasis and ulcerative colitis, and indirubin is used for the treatment of chronic myelogenous leukaemia. However, IN and indirubin have adverse reactions, such as abdominal pain, diarrhoea, and intussusception, and their specific mechanism is unclear.The purpose of our research was to determine the specific mechanism underlying the adverse effects of IN and indirubin. By tracking the modifications in guinea pigs after the intragastric administration of indirubin for 28 days.The results demonstrate that indirubin could accelerate bowel movements and decrease intestinal acetylcholinesterase (AchE) expression. Experiments with NCM460 cells revealed that indirubin significantly reduced the expression of AchE, and the AchE levels were increased after the silencing of AhR and re-exposure to indirubin.This study showed that the inhibition of AchE expression by indirubin plays a key role in the occurrence of adverse reactions to indirubin and that the underlying mechanism is related to AhR-mediated AchE downregulation.

Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms.

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 µM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.

Identification of the amino acid residues involved in the species-dependent differences in the pyridoxine transport function of SLC19A3.

Recent studies have shown that human solute carrier SLC19A3 (hSLC19A3) can transport pyridoxine (vitamin B6) in addition to thiamine (vitamin B1), its originally identified substrate, whereas rat and mouse orthologs of hSLC19A3 can transport thiamine but not pyridoxine. This finding implies that some amino acid residues required for pyridoxine transport, but not for thiamine transport, are specific to hSLC19A3. Here, we sought to identify these residues to help clarify the unique operational mechanism of SLC19A3 through analyses comparing hSLC19A3 and mouse Slc19a3 (mSlc19a3). For our analyses, hSLC19A3 mutants were prepared by replacing selected amino acid residues with their counterparts in mSlc19a3, and mSlc19a3 mutants were prepared by substituting selected residues with their hSLC19A3 counterparts. We assessed pyridoxine and thiamine transport by these mutants in transiently transfected human embryonic kidney 293 cells. Our analyses indicated that the hSLC19A3-specific amino acid residues of Gln86, Gly87, Ile91, Thr93, Trp94, Ser168, and Asn173 are critical for pyridoxine transport. These seven amino acid residues were found to be mostly conserved in the SLC19A3 orthologs that can transport pyridoxine but not in orthologs that are unable to transport pyridoxine. In addition, these residues were also found to be conserved in several SLC19A2 orthologs, including rat, mouse, and human orthologs, which were all found to effectively transport both pyridoxine and thiamine, exhibiting no species-dependent differences. Together, these findings provide a molecular basis for the unique functional characteristics of SLC19A3 and also of SLC19A2.

In vivo mouse models to study bile acid synthesis and signaling.

The synthesis of bile acids (BAs) is carried out by complex pathways characterized by sequential chemical reactions in the liver through various cytochromes P450 (CYP) and other enzymes. Maintaining the integrity of these pathways is crucial for normal physiological function in mammals, encompassing hepatic and neurological processes. Studying on the deficiencies in BA synthesis genes offers valuable insights into the significance of BAs in modulating farnesoid X receptor (FXR) signaling and metabolic homeostasis. By creating mouse knockout (KO) models, researchers can manipulate deficiencies in genes involved in BA synthesis, which can be used to study human diseases with BA dysregulation. These KO mouse models allow for a more profound understanding of the functions and regulations of genes responsible for BA synthesis. Furthermore, KO mouse models shed light on the distinct characteristics of individual BA and their roles in nuclear receptor signaling. Notably, alterations of BA synthesis genes in mouse models have distinct differences when compared to human diseases caused by the same BA synthesis gene deficiencies. This review summarizes several mouse KO models used to study BA synthesis and related human diseases, including mice deficient in Cyp7a1, Cyp27a1, Cyp7a1/Cyp27a1, Cyp8b1, Cyp7b1, Cyp2c70, Cyp2a12, and Cyp2c70/Cyp2a12, as well as germ-free mice.

Bisphenol A Analogues Inhibit Human and Rat 11ß-Hydroxysteroid Dehydrogenase 1 Depending on Its Lipophilicity.

Bisphenol A (BPA) analogues substituted on the benzene ring are widely used in a variety of industrial and consumer materials. However, their effects on the glucocorticoid-metabolizing enzyme 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) remain unclear. The inhibitory effects of 6 BPA analogues on the inhibition of human and rat 11ß-HSD1 were investigated. The potencies of inhibition on human 11ß-HSD1 were bisphenol H (IC50, 0.75 µM) > bisphenol G (IC50, 5.06 µM) > diallyl bisphenol A (IC50, 13.36 µM) > dimethyl bisphenol A (IC50, 30.18 µM) > bisphenol A dimethyl ether (IC50, 33.08 µM) > tetramethyl bisphenol A (>100 µM). The inhibitory strength of these chemicals on rat 11ß-HSD1 was much weaker than that on the human enzyme, ranging from 74.22 to 205.7 µM. All BPA analogues are mixed/competitive inhibitors of both human and rat enzymes. Molecular docking studies predict that bisphenol H and bisphenol G both bind to the active site of human 11ß-HSD1, forming a hydrogen bond with catalytic residue Ser170. The bivariate correlation of IC50 values with LogP (lipophilicity), molecular weight, heavy atoms, and molecular volume revealed a significant inverse regression and the correlation of IC50 values with ΔG (low binding energy) revealed a positive regression. In conclusion, the lipophilicity, molecular weight, heavy atoms, molecular volume, and binding affinity of a BPA analogue determine the inhibitory strength of human and rat 11ß-HSD isoforms.

Species-independent stimulation of osteogenic differentiation induced by osteoclasts.

The coupling of bone resorption and bone formation is well-recognized in the bone remodeling process, in which osteoblasts and osteoclasts are key players. However, the anabolic effect of human primary osteoclasts has rarely been reported as mouse and cell line derived osteoclasts were mostly used in previous reports. Therefore, a comprehensive comparison of mouse and human osteoclasts and their corresponding functions is needed to study cell-cell interactions between osteoclasts and osteoblasts. Osteoclasts from mouse and human origin were generated, characterized and compared, after which their anabolic effects on the osteogenic differentiation of mouse and human MSCs were assessed. Both murine RAW264.7 derived osteoclasts (mOCs) and primary human osteoclasts (hOCs) derived from buffy coats characteristically displayed multinuclearity, marked integrin ß3 expression and enhanced TRAP activity. Despite comparable cell size, mOCs showed higher osteoclast density (number of osteoclasts per cm2 culture dish) and osteoclast nuclearity (average number of nuclei per osteoclast), but lower TRAP activity compared to hOCs. Culturing primary rat and human bone marrow MSCs with the conditioned medium of mOCs or hOCs showed anabolic effects regarding the osteogenic differentiation of MSCs with superiority of hOCs over mOCs. We conclude that despite morphological and functional differences between mouse and human osteoclasts, their secretory factors evoke similar anabolic effects on MSC osteogenic differentiation.

Species-Specific Activation of Transient Receptor Potential Ankyrin 1 by Phthalic Acid Monoesters.

Phthalic acid (PA) diesters are widely used in consumer products, as plasticizers, and are ubiquitous environmental pollutants. There is a growing concern about their adjuvant effect on allergic diseases. Although its precise mechanism remains unknown, possible involvement of transient receptor potential ankyrin 1 (TRPA1) has been suggested. Hence, in this study, the activation of human and mouse TRPA1s by a series of PA di- and monoesters was investigated using a heterologous expression system in vitro. Consequently, it was found that monoesters activated human TRPA1, where EC50 values were in the order of mono-hexyl > mono-heptyl > mono-n-octyl > mono-2-ethylhexyl > mono-isononyl and mono-isodecyl esters. Significant species differences in TRPA1 activation by PA monoesters were also discovered; PA monoesters activated human TRPA1 but not mouse TRPA1 in a concentration-dependent manner up to 50 µM. These findings suggest that PA esters may exert TRPA1-dependent adverse effects on humans, which have never been demonstrated in experimental animals.

Elucidation of clearance mechanism of TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor: does a species difference in excretion routes exist between humans and animals?

1. TP0463518, a novel hypoxia-inducible factor prolyl hydroxylase inhibitor, is reportedly excreted predominantly through urinary excretion in an unchanged form in humans, with partial biliary excretion also possible. However, the clearance mechanisms remain unclear. The aim of this study was to investigate the clearance mechanisms in humans and to assess species differences in the excretion routes.2. TP0463518 was not metabolised in rat, dog, or human hepatocytes. TP0463518 is a substrate for human BCRP, OATP1B1, OATP1B3, and OAT3, suggesting that renal uptake by OAT3 is probably the predominant clearance route, with hepatic uptake by OATP1B1 and OATP1B3 contributing partially to clearance in humans.3. A species difference in excretion routes was observed. The unchanged urinary excretion rates in humans, male rats, female rats, dogs, and monkeys were 80.7%, 0.1%, 40.9%, 15.2%, and 72.6%, respectively. Urinary excretion was predominant in humans and monkeys, while only biliary excretion was observed in male rats. Uptake studies using hepatocytes showed that the hepatic uptake clearance in rats was 13.6-fold higher than that in humans. Therefore, not only reabsorption via renal tubules, but also hepatic uptake seems to be involved in the species differences in excretion routes between rats and humans.

In vitro differences in toddalolactone metabolism in various species and its effect on cytochrome P450 expression.

CONTEXT: Toddalolactone, the main component of Toddalia asiatica (L.) Lam. (Rutaceae), has anticancer, antihypertension, anti-inflammatory, and antifungal activities. OBJECTIVE: This study investigated the metabolic characteristics of toddalolactone. MATERIALS AND METHODS: Toddalolactone metabolic stabilities were investigated by incubating toddalolactone (20 µM) with liver microsomes from humans, rabbits, mice, rats, dogs, minipigs, and monkeys for 0, 30, 60, and 90 min. The CYP isoforms involved in toddalolactone metabolism were characterized based on chemical inhibition studies and screening assays. The effects of toddalolactone (0, 10, and 50 µM) on CYP1A1 and CYP3A5 protein expression were investigated by immunoblotting. After injecting toddalolactone (10 mg/kg), in vivo pharmacokinetic profiles using six Sprague-Dawley rats were investigated by taking 9-time points, including 0, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h. RESULTS: Monkeys showed the greatest metabolic capacity in CYP-mediated and UGT-mediated reaction systems with short half-lives (T1/2) of 245 and 66 min, respectively, while T1/2 of humans in two reaction systems were 673 and 83 min, respectively. CYP1A1 and CYP3A5 were the major CYP isoforms involved in toddalolactone biotransformation. Induction of CYP1A1 protein expression by 50 µM toddalolactone was approximately 50% greater than that of the control (0 µM). Peak plasma concentration (Cmax) for toddalolactone was 0.42 µg/mL, and Tmax occurred at 0.25 h post-dosing. The elimination t1/2 was 1.05 h, and the AUC0-t was 0.46 µg/mL/h. CONCLUSIONS: These findings demonstrated the significant species differences of toddalolactone metabolic profiles, which will promote appropriate species selection in further toddalolactone studies.

Species-specific differences in the inhibition of 11ß-hydroxysteroid dehydrogenase 2 by itraconazole and posaconazole.

11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) converts active 11ß-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11ß-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11ß-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11ß-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11ß-HSD2 was potently inhibited by all three compounds (IC50 values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC50 values compared to human), and mouse and zebrafish 11ß-HSD2 were very weakly inhibited (IC50 values above 7 µM). Sequence alignment and application of newly generated homology models for human and mouse 11ß-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11ß-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11ß-HSD2. The results provide an explanation for the observed species-specific 11ß-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11ß-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies.

Species Difference in Hydrolysis of an Ester-type Prodrug of Levodopa in Human and Animal Plasma: Different Contributions of Alpha-1 Acid Glycoprotein.

Previously, we found that ONO-2160, an ester-type prodrug of levodopa (3-hydroxy-l-tyrosine), was mainly hydrolyzed in human plasma by α1-acid glycoprotein (AGP) with a partial contribution of albumin. In this study, we investigated whether ONO-2160 was hydrolyzed in the plasma of preclinical species (dog, rabbit, rat, and mouse) and humans and whether AGP and albumin are involved in its hydrolysis. ONO-2160 was hydrolyzed to some extent in the plasma of all tested species with the order of magnitude mouse > human > rabbit > rat > dog. Except for dogs, ONO-2160 was partially hydrolyzed by animal AGP and albumin. This indicated that, similar to albumin, AGP possesses esterase-like activity in mice, rats, and rabbits, as well as humans. A comparison of the values of intrinsic clearance per milliliter of plasma demonstrated that AGP was the major contributor to the hydrolysis of ONO-2160 in rabbit plasma, whereas albumin was primarily responsible for the hydrolysis of ONO-2160 in mouse plasma. This was confirmed by experiments using AGP-knockout mouse plasma. This study reports the first evidence for the existence of species differences in the hydrolysis of ONO-2160 in plasma related to the different contributions of AGP and albumin.

A nodal point for brain-state transitions: the mesopontine tegmental anesthesia area (MPTA) in mice.

The mesopontine tegmental anesthesia area (MPTA) was identified in rats as a singular brainstem locus at which microinjection of minute quantities of GABAergic agents rapidly and reversibly induces loss-of-consciousness and a state of general anesthesia, while lesioning renders animals insensitive to anesthetics at normal systemic doses. Obtaining similar results in mice has been challenging, however, slowing research progress on how anesthetics trigger brain-state transitions. We have identified roadblocks that impeded translation from rat to mouse and tentatively located the MPTA equivalent in this second species. We describe here a series of modifications to the rat protocol that allowed us to document pro-anesthetic changes in mice following localized stereotactic delivery of minute quantities (20 nL) of the GABAA-receptor agonist muscimol into the brainstem mesopontine tegmentum. The optimal locus identified proved to be homologous to the MPTA in rats, and local neuronal populations in rats and mice were similar in size and shape. This outcome should facilitate application of the many innovative gene-based methodologies available primarily in mice to the study of how activity in brainstem MPTA neurons brings about anesthetic loss-of-consciousness and permits pain-free surgery.

Species differences in the CYP3A-catalyzed metabolism of TPN729, a novel PDE5 inhibitor.

TPN729 is a novel phosphodiesterase 5 (PDE5) inhibitor used to treat erectile dysfunction in men. Our previous study shows that the plasma exposure of metabolite M3 (N-dealkylation of TPN729) in humans is much higher than that of TPN729. In this study, we compared its metabolism and pharmacokinetics in different species and explored the contribution of its main metabolite M3 to pharmacological effect. We conducted a combinatory approach of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolite identification, and examined pharmacokinetic profiles in monkeys, dogs, and rats following TPN729 administration. A remarkable species difference was observed in the relative abundance of major metabolite M3: i.e., the plasma exposure of M3 was 7.6-fold higher than that of TPN729 in humans, and 3.5-, 1.2-, 1.1-fold in monkeys, dogs, and rats, respectively. We incubated liver S9 and liver microsomes with TPN729 and CYP3A inhibitors, and demonstrated that CYP3A was responsible for TPN729 metabolism and M3 formation in humans. The inhibitory activity of M3 on PDE5 was 0.78-fold that of TPN729 (The IC50 values of TPN729 and M3 for PDE5A were 6.17 ± 0.48 and 7.94 ± 0.07 nM, respectively.). The plasma protein binding rates of TPN729 and M3 in humans were 92.7% and 98.7%, respectively. It was astonishing that the catalyzing capability of CYP3A4 in M3 formation exhibited seven-fold disparity between different species. M3 was an active metabolite, and its pharmacological contribution was equal to that of TPN729 in humans. These findings provide new insights into the limitation and selection of animal model for predicting the clinical pharmacokinetics of drug candidates metabolized by CYP3A4.

Methyl-hydroxylation and subsequent oxidation to produce carboxylic acid is the major metabolic pathway of tolbutamide in chimeric TK-NOG mice transplanted with human hepatocytes.

Tolbutamide is an oral anti-hyperglycaemic agent used to treat non-insulin-dependent diabetes mellitus with species-dependent metabolic profiles. In this study, we investigated tolbutamide metabolism in chimeric TK-NOG mice transplanted with human hepatocytes (humanised-liver mice).Substantial 4-hydroxytolbutamide and 4-carboxytolbutamide production was observed in hepatocytes from humanised-liver mice (Hu-Liver cells) and humans, whereas 4-carboxytolbutamide production was not detected in mouse hepatocytes. In Hu-Liver cells, 4-hydroxytolbutamide formation was inhibited by sulfaphenazole (CYP2C9 inhibitor), whereas 4-carboxytolbutamide formation was inhibited by raloxifene/ethinyloestradiol (aldehyde oxidase inhibitor) and disulfiram (aldehyde dehydrogenase inhibitor).After a single oral dose of tolbutamide (10 mg/kg), the plasma levels of 4-carboxytolbutamide and p-tolylsulfonylurea were higher in humanised-liver mice than in TK-NOG mice. Urinary excretion was the predominant route (>99% of unchanged drug and metabolites detected in excreta) of elimination in both groups. 4-Carboxytolbutamide was the most abundant metabolite in humanised-liver mouse urine, as similarly reported for humans, whereas 4-hydroxytolbutamide was predominantly excreted in TK-NOG mouse urine.These results suggest that humanised-liver mice might represent a suitable animal model for studying the successive oxidative metabolism of tolbutamide by multiple drug-metabolising enzymes. Future work is warranted to study the general nature of primary alcohol metabolism using humanised-liver mice.

Evaluation of species differences in the metabolism of the selective NaV1.7 inhibitor DS-1971a, a mixed substrate of cytochrome P450 and aldehyde oxidase.

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

Identification of the metabolites of tofacitinib in liver microsomes by liquid chromatography combined with high resolution mass spectrometry.

Tofacitinib is an orally available Janus kinase inhibitor. The aim of this study was to investigate the metabolism of tofacitinib in mouse, rat, monkey, and human liver microsomes fortified with ß-nicotinamide adenine dinucleotide phosphate tetrasodium salt and uridine diphosphate glucuronic acid. The biotransformation was executed at a temperature of 37°C for 60 min, and the samples were analyzed by ultra-high performance liquid chromatography combined with high-resolution mass spectrometry (UHPLC-HRMS) operated in positive electrospray ionization mode. The structures of the metabolites were elucidated according to their retention times, accurate masses, and MS/MS spectra. Under the current conditions, a total of 13 metabolites, including 1 glucuronide conjugate, were detected and structurally proposed. Oxygenation of the pyrrolopyrimidine ring, oxygenation of piperidine ring, N-demethylation, oxygenation of piperidine ring side chain, and glucuronidation were the primary metabolic pathways of tofacitinib. Among the tested species, tofacitinib showed significant species difference. Compared with other species, rat showed similar metabolic profiles to those of humans. The present study provides some new information regarding the metabolism of tofacitinib in animals and humans, which would bring us considerable benefits for the subsequent studies focusing on the pharmacological effect and toxicity of this drug.

Differences between Human and Mouse IgM Fc Receptor (FcµR).

Both non-immune "natural" and antigen-induced "immune" IgM are important for protection against pathogens and for regulation of immune responses to self-antigens. Since the bona fide IgM Fc receptor (FcµR) was identified in humans by a functional cloning strategy in 2009, the roles of FcµR in these IgM effector functions have begun to be explored. In this short essay, we describe the differences between human and mouse FcµRs in terms of their identification processes, cellular distributions and ligand binding activities with emphasis on our recent findings from the mutational analysis of human FcµR. We have identified at least three sites of human FcµR, i.e., Asn66 in the CDR2, Lys79 to Arg83 in the DE loop and Asn109 in the CDR3, responsible for its constitutive IgM-ligand binding. Results of computational structural modeling analysis are consistent with these mutational data and a model of the ligand binding, Ig-like domain of human FcµR is proposed. Serendipitously, substitution of Glu41 and Met42 in the CDR1 of human FcµR with mouse equivalents Gln and Leu, either single or more prominently in combination, enhances both the receptor expression and IgM binding. These findings would help in the future development of preventive and therapeutic interventions targeting FcµR.