Pharmacology Research & Perspectives

来自 : 发布时间：2025-06-11

Pharmacology Research PerspectivesPublisher: Wiley (John Wiley Sons)Additional detailsCited half-lifedata not availableImmediacy indexdata not availableEigenfactordata not availableArticle influencedata not availableWebsiteWebsite descriptionElectronic ISSN2052-1707Publications in this journalSourceModerate, chronic ethanol feeding exacerbates carbon tetrachloride–induced hepatic fibrosis via hepatocyte-specific hypoxia-inducible factor 1αArticleOct 2014Sanjoy RoychowdhuryDian J ChiangMegan R McMullenLaura NagyThe hypoxia-sensing transcriptional factor HIF1α is implicated in a variety of hepato-pathological conditions; however, the contribution of hepatocyte-derived HIF1α during progression of alcoholic liver injury is still controversial. HIF1α induces a variety of genes including those involved in apoptosis via p53 activation. Increased hepatocyte apoptosis is critical for progression of liver inflammation, stellate cell activation, and fibrosis. Using hepatocyte-specific HIF1α-deficient mice (ΔHepHIF1α−/−), here we investigated the contribution of HIF1α to ethanol-induced hepatocyte apoptosis and its role in amplification of fibrosis after carbon tetrachloride (CCl4) exposure. Moderate ethanol feeding (11% of kcal) induced accumulation of hypoxia-sensitive pimonidazole adducts and HIF1α expression in the liver within 4 days of ethanol feeding. Chronic CCl4 treatment increased M30-positive cells, a marker of hepatocyte apoptosis in pair-fed control mice. Concomitant ethanol feeding (11% of kcal) amplified CCl4-induced hepatocyte apoptosis in livers of wild-type mice, associated with elevated p53K386 acetylation, PUMA expression, and Ly6c+ cell infiltration. Subsequent to increased apoptosis, ethanol-enhanced induction of profibrotic markers, including stellate cell activation, collagen 1 expression, and extracellular matrix deposition following CCl4 exposure. Ethanol-induced exacerbation of hepatocyte apoptosis, p53K386 acetylation, and PUMA expression following CCl4 exposure was attenuated in livers of ΔHepHIF1α−/− mice. This protection was also associated with a reduction in Ly6c+ cell infiltration and decreased fibrosis in livers of ΔHepHIF1α−/− mice. In summary, these results indicate that moderate ethanol exposure leads to hypoxia/HIF1α-mediated signaling in hepatocytes and induction of p53-dependent apoptosis of hepatocytes, resulting in increased hepatic fibrosis during chronic CCl4 exposure.ViewExpand abstractSourceExtensive protein interactions involving cytochrome P450 3A4: A possible contributor to the formation of a metabolosomeArticleOct 2014Ryoichi FujiwaraTomoo ItohCytochrome P450 (CYP) 3A4 is a membrane protein that catalyzes hydroxylation of endogenous and exogenous substrates. Protein–protein interaction is a crucial factor that regulates the function of enzymes. However, protein–protein interactions involving human CYPs have not been fully understood. In this study, extensive protein–protein interactions involving CYP3A4 were determined by a shotgun analysis of immunoprecipitate utilizing anti-CYP3A4 antibody. Our shotgun analysis revealed that 149 proteins were immunoprecipitated with anti-CYP3A4 antibody in human liver microsomes. We further determined that 51 proteins of 149 proteins were specifically immunoprecipitated with the anti-CYP3A4 antibody. Our analysis demonstrated that other CYP isoforms are interacting with CYP3A4, which is in agreement with previous findings. Based on our current and previous findings, we propose that drug-metabolizing enzymes such as CYP3A4 and UDP-glucuronosyltransferase 2B7 form a metabolosome, which is a functional unit of metabolism consisting of multiple metabolism-related proteins.ViewExpand abstractSourceMetabolism and disposition of MM-433593, a selective FAAH-1 inhibitor, in monkeysArticleOct 2014Ali R. BanijamaliJames D. WakefieldAra MermerianRobert W. BusbyMM-433593 is a highly potent and selective inhibitor of fatty acid amide hydrolase-1 (FAAH-1) with potential utility as an orally administered treatment of pain, inflammation, and other disorders. In this study, we investigated the metabolism and pharmacokinetics of MM-433593 in monkeys, and compared plasma and urine metabolites of this compound to the in vitro metabolites produced by monkey hepatocytes. Intravenous administration of MM-433593 to cynomolgus monkeys produced a rapid distribution phase and slower elimination phase with a mean systemic clearance rate of 8–11 mL/min/kg. Absolute oral bioavailability was determined to be 14–21% with maximum plasma concentrations reached ~3 h (Tmax) following a 10 mg/kg oral dose. The average terminal half-life of MM-433593 was 17–20 h, and there were no qualitative sex differences in the metabolite profile of MM-433593. The major site of metabolism was oxidation of the methyl group at the five position of the indole ring, which was confirmed by chromatography and mass spectrometry comparison to a synthesized authentic standard. This metabolite was further oxidized to the corresponding carboxylic acid and/or conjugated with sulfate, glucuronide, or glutathione. In all, 18 metabolites were found in plasma and urine. In vitro incubations of MM-433593 with monkey hepatocytes yielded 13 metabolites, all of which were found in vivo, indicating a good correlation between the in vitro and in vivo metabolism data. A comprehensive pathway for the metabolism of MM-433593 is proposed, including a plausible, five-step biotransformation for the formation of N-acetylcysteine conjugate metabolite (M18) from the hydroxylated parent (M5).ViewExpand abstractSourceEffect of perampanel, a novel AMPA antagonist, on benzodiazepine-resistant status epilepticus in a lithium-pilocarpine rat modelArticleOct 2014Takahisa HanadaKatsutoshi IdoTakashi KosasaThis study assessed the efficacy of diazepam, and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor antagonists perampanel and GYKI52466 in a lithium-pilocarpine status epilepticus (SE) model. SE was induced in rats using lithium chloride, scopolamine methyl bromide, and pilocarpine. Diazepam 10, 20, or 40 mg kg−1, or perampanel 1, 2.5, 5, or 8 mg kg−1 were administered intravenously at 10 or 30 min after seizure onset, and GYKI52466 50 mg kg−1, or combinations of diazepam 2.5–5 mg kg−1 and perampanel 0.5–1 mg kg−1, were administered intravenously at 30 min after seizure onset. Diazepam 20 mg kg−1 terminated seizures (based on electroencephalography and assessment of behavioral seizures) in 2/6 rats at 10 min and 0/6 rats at 30 min (ED50: 10 min, 30 mg kg−1; 30 min, not determined). Perampanel 8 mg kg−1 terminated seizures in 6/6 rats at both 10 and 30 min (ED50: 10 min 1.7 mg kg−1; 30 min, 5.1 mg kg−1). GYKI52466 50 mg kg−1 terminated seizures in 2/4 rats at 30 min. Co-administration of diazepam 5 mg kg−1 and perampanel 1 mg kg−1 terminated seizures in 9/9 rats at 30 min. In conclusion, perampanel and GYKI52466 provided efficacy in a lithium-pilocarpine SE model at 30 min after seizure onset, when SE was refractory to diazepam, supporting the therapeutic potential of AMPA receptor antagonists for refractory SE. The perampanel dose required to terminate seizures was reduced by combination with diazepam, suggesting synergy.ViewExpand abstractSourceDevelopment of a ‘patient information leaflet’ for use following assessment of patients with reported or suspected paracetamol overdose in the UKArticleDec 2014Thomas PotterWui Ling ChanJohn R. H. Archer[...]David M. WoodThe aim of this study was to design an information leaflet for patients with paracetamol overdose based on Medicines and Healthcare products Regulatory Agency guidance and to assess its readability. A two-sided one page information leaflet was designed for patients being discharged from hospital after a paracetamol overdose. Patients presenting with an acute paracetamol overdose, irrespective of whether they were treated or not, were recruited to read the leaflet and then answer a brief structured questionnaire based on the leaflet. The readability of the information leaflet was assessed using the Flesch reading ease score. Thirty patients (15 male, 12 female, 3 not recorded; mean age 38 ± 13.0 years) were recruited, wherein 100% of patients reported the language used was understandable, 96.6% knew which symptoms would require urgent medical review after discharge and 100% of patients knew the liver was affected by paracetamol. The Flesch reading ease score was 67.6 (out of a maximum of 100), equivalent to a UK reading age of 10–11years old. Our information leaflet for all patients being discharged after paracetamol overdose was well received by patients, provided them with the required knowledge and had an appropriate reading age based on UK literacy rates. We would recommend that this leaflet could be used as a template on a national level, localized to individual hospitals, to improve patient knowledge of paracetamol toxicity, and facilitate early medical review in the event of deterioration following discharge from the hospital.ViewExpand abstractSourceSpecies differences in sinusoidal and canalicular efflux transport of mycophenolic acid 7-O-glucuronide in sandwich-cultured hepatocytesArticleApr 2014Kazuhiro TetsukaNicolas GerstKouichi TamuraJeffrey MastersMetabolism and sinusoidal/canalicular efflux of mycophenolic acid (MPA) was investigated using sandwich-cultured hepatocytes (SCHs). After applying MPA to SCHs from humans, wild-type rats, and multidrug resistance-associated protein (Mrp) 2-deficient rats, the MPA metabolites 7-O-glucuronide (MPAG) and acyl glucuronide (AcMPAG) were detected in the intracellular compartment of the SCHs. Sinusoidal efflux of MPAG was detected in all SCH preparations including Mrp2-deficient rat SCHs, whereas canalicular efflux of MPAG was observed in wild-type rat and human SCHs but not in Mrp2-deficient rat SCHs. The ratio of canalicular efflux to net (canalicular plus sinusoidal) efflux was 37 ± 8% in wild-type rat SCHs, while the ratio in human SCHs was significantly lower (20 ± 2%, P 0.05), indicating species differences in the direction of hepatic MPAG transport. This 20% ratio in human SCHs corresponds to a high sinusoidal MPAG efflux (80%) that can in part account for the urine-dominated recovery of MPAG in humans. Both sinusoidal and canalicular MPAG efflux in rat SCHs shows a good correspondence to urinary and biliary recovery of MPAG after MPA dosing. The sinusoidal efflux of AcMPAG in human SCHs was detected from one out of three donors, suggesting donor-to-donor variation. In conclusion, this study demonstrates the predictive value of SCHs for elucidating the interplay of metabolism and efflux transport, in addition to demonstrating a species difference between rat and human in sinusoidal and canalicular efflux of MPAG.ViewExpand abstractSourceA clinical therapeutic protein drug–drug interaction study: coadministration of denosumab and midazolam in postmenopausal women with osteoporosisArticleApr 2014Graham JangAllegra KaufmanEdward Lee[...]Desmond PadhiDrug–disease interactions involving therapeutic proteins that target cytokines and potentially impact cytochrome P450 (CYP) enzymes have been of increased interest to drug regulatory agencies and industry sponsors in recent years. This parallel-group open-label study evaluated the effects of the monoclonal antibody denosumab, an inhibitor of the cytokine RANKL, on the pharmacokinetics of the probe CYP3A4 substrate midazolam in postmenopausal women with osteoporosis. The pharmacokinetics of a 2 mg oral dose of midazolam was evaluated on days 1 and 16. Subjects in Group A received a 60 mg subcutaneous dose of denosumab on day 2, 2 weeks before the second midazolam dose, while subjects in Group B did not. For Group A (n = 17), point estimates for the ratio of least square means for midazolam exposures based on maximum observed plasma concentration (Cmax) and areas under the plasma concentration–time curve (AUCs) on day 16 versus day 1 ranged from 1.02 to 1.04 and 90% confidence intervals were within 0.80–1.25. No period effect was observed for Group B (n = 8). Midazolam and denosumab coadministration was safe and well tolerated. Inhibition of the cytokine RANKL by denosumab does not affect CYP3A4 in postmenopausal women with osteoporosis and will not alter the pharmacokinetics of drugs metabolized by this enzyme. These results are consistent with data suggesting that RANKL does not impact markers of inflammation and represent the first clinical data demonstrating a lack of effect on CYP3A4 of a therapeutic protein that is a cytokine modulator.ViewExpand abstractSourcePharmacological characterization of a novel potent, selective, and orally active phosphodiesterase 10A inhibitor, PDM-042 [(E)-4-(2-(2-(5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)vinyl)-6-(pyrrolidin-1-yl)pyrimidin-4-yl)morpholine] in rats: potentialArticleAug 2016Keita ArakawaShunsuke MaeharaNatsuko Yuge[...]Kazunari NakaoRecently, we identified a novel phosphodiesterase 10A (PDE10A) inhibitor, PDM-042 ((E)-4-(2-(2-(5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)vinyl)-6-(pyrrolidin-1-yl)pyrimidin-4-yl)morpholine). PDM-042 showed potent inhibitory activities for human and rat PDE10A with IC50 values of less than 1 nmol/L and more than 1000-fold selectivity against other phosphodiesterases. Tritiated PDM-042, [3H]PDM-042, had high affinity for membranes prepared from rat striatum with a Kd value of 8.5 nmol/L. The specific binding of [3H]PDM-042 was displaced in a concentration-dependent manner by PDM-042 and another structurally unrelated PDE10A inhibitor, MP-10. In rat studies, PDM-042 showed excellent brain penetration (striatum/plasma ratio = 6.3), occupancy rate (86.6% at a dose of 3 mg/kg), and good oral bioavailability (33%). These data indicate that PDM-042 is a potent, selective, orally active, and brain-penetrable PDE10A inhibitor. In behavioral studies using rat models relevant to schizophrenia, PDM-042 significantly antagonized MK-801-induced hyperlocomotion (0.1–0.3 mg/kg) without affecting spontaneous locomotor activity and attenuated the conditioned avoidance response (CAR) (0.3–1 mg/kg). In tests for adverse effects, PDM-042 had a minimal effect on catalepsy, even at a much higher dose (10 mg/kg) than the minimal effective dose (0.3 mg/kg) in the CAR. Furthermore, PDM-042 had no effect on prolactin release or glucose elevation up to 3 mg/kg, while risperidone increased prolactin release and olanzapine enhanced glucose levels at doses near their efficacious ones in the CAR. Our results suggest that PDM-042 is a good pharmacological tool that can be used to investigate the role of PDE10A and may have therapeutic potential for the treatment of schizophrenia.ViewExpand abstractSourceQuantitative systems toxicology (QST) reproduces species differences in PF‐04895162 liver safety due to combined mitochondrial and bile acid toxicityArticleDec 2019GT GenerauxVinal V. LakhaniYuching Yang[...]Lisl K.M. ShodaMany compounds that appear promising in preclinical species, fail in human clinical trials due to safety concerns. The FDA has strongly encouraged the application of modeling in drug development to improve product safety. This study illustrates how DILIsym, a computational representation of liver injury, was able to reproduce species differences in liver toxicity due to PF‐04895162 (ICA‐105665). PF‐04895162, a drug in development for the treatment of epilepsy, was terminated after transaminase elevations were observed in healthy volunteers (NCT01691274). Liver safety concerns had not been raised in preclinical safety studies. DILIsym, which integrates in vitro data on mechanisms of hepatotoxicity with predicted in vivo liver exposure, reproduced clinical hepatotoxicity and the absence of hepatotoxicity observed in the rat. Simulated differences were multifactorial. Simulated liver exposure was greater in humans than rats. The simulated human hepatotoxicity was demonstrated to be due to the interaction between mitochondrial toxicity and bile acid transporter inhibition; elimination of either mechanism from the simulations abrogated injury. The bile acid contribution occurred despite the fact that the IC50 for bile salt export pump (BSEP) inhibition by PF‐04895162 was higher (311 µmol/L) than that has been generally thought to contribute to hepatotoxicity. Modeling even higher PF‐04895162 liver exposures than were measured in the rat safety studies aggravated mitochondrial toxicity but did not result in rat hepatotoxicity due to insufficient accumulation of cytotoxic bile acid species. This investigative study highlights the potential for combined in vitro and computational screening methods to identify latent hepatotoxic risks and paves the way for similar and prospective studies.View1 RecommendationExpand abstractSourcePhase I study of PF-04895162, a Kv7 channel inhibitor, reveals unexpected hepatotoxicity in healthy subjects, but not rats or monkeys: clinical evidence of disrupted bile acid homeostasisArticleFeb 2019Michael D. AleoJiri AubrechtPaul D Bonin[...]Pinky DuaDuring a randomized Phase 1 clinical trial the drug candidate, PF‐04895162 (ICA‐105665), caused transaminase elevations (≥grade 1) in six of eight healthy subjects treated at 300 mg twice daily for 2‐weeks (NCT01691274). This was unexpected since studies in rats ( 6 months) and cynomolgus monkeys ( 9 months) treated up to 100 mg/kg/day did not identify the liver as a target organ. Mechanistic studies showed PF‐04895162 had low cytotoxic potential in human hepatocytes, but inhibited liver mitochondrial function and bile salt export protein (BSEP) transport. Clinical relevance of these postulated mechanisms of liver injury was explored in three treated subjects that consented to analysis of residual pharmacokinetic plasma samples. Compared to a nonresponder, two subjects with transaminase elevations displayed higher levels of miRNA122 and total/conjugated bile acid species, whereas one demonstrated impaired postprandial clearance of systemic bile acids. Elevated taurine and glycine conjugated to unconjugated bile acid ratios were observed in two subjects, one before the onset of elevated transaminases. Based on the affinity of conjugated bile acid species for transport by BSEP, the profile of plasma conjugated/unconjugated bile acid species was consistent with inhibition of BSEP. These data collectively suggest that the human liver injury by PF‐04895162 was due to alterations in bile acid handling driven by dual BSEP/mitochondrial inhibition, two important risk factors associated with drug‐induced liver injury in humans. Alterations in systemic bile acid composition were more important than total bile acids in the manifestation of clinical liver injury and may be a very early biomarker of BSEP inhibition.ViewExpand abstractSourceCombined treatment with a selective PDE10A inhibitor TAK-063 and either haloperidol or olanzapine at subeffective doses produces potent antipsychotic-like effects without affecting plasma prolactin levels and cataleptic responses in rodentsArticleFeb 2018Kazunori SuzukiAkina HaradaHirobumi Suzuki[...]Haruhide KimuraActivation of indirect pathway medium spiny neurons (MSNs) via promotion of cAMP production is the principal mechanism of action of current antipsychotics with dopamine D2 receptor antagonism. TAK-063 [1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one] is a novel phosphodiesterase 10A inhibitor that activates both direct and indirect pathway MSNs through increasing both cAMP and cGMP levels by inhibition of their degradation. The activation of indirect pathway MSNs through the distinct mechanism of action of these drugs raises the possibility of augmented pharmacological effects by combination therapy. In this study, we evaluated the potential of combination therapy with TAK-063 and current antipsychotics, such as haloperidol or olanzapine after oral administration. Combined treatment with TAK-063 and either haloperidol or olanzapine produced a significant increase in phosphorylation of glutamate receptor subunit 1 in the rat striatum. An electrophysiological study using rat corticostriatal slices showed that TAK-063 enhanced N-methyl-D-aspartic acid receptor-mediated synaptic responses in both direct and indirect pathway MSNs to a similar extent. Further evaluation using pathway-specific markers revealed that coadministration of TAK-063 with haloperidol or olanzapine additively activated the indirect pathway, but not the direct pathway. Combined treatment with TAK-063 and either haloperidol or olanzapine at subeffective doses produced significant effects on methamphetamine- or MK-801-induced hyperactivity in rats and MK-801-induced deficits in prepulse inhibition in mice. TAK-063 at 0.1 mg/kg did not affect plasma prolactin levels and cataleptic response from antipsychotics in rats. Thus, TAK-063 may produce augmented antipsychotic-like activities in combination with antipsychotics without effects on plasma prolactin levels and cataleptic responses in rodents.ViewExpand abstractSourceA Phase 1 first‐in‐human study of the safety, tolerability, and pharmacokinetics of the ROBO2 fusion protein PF‐06730512 in healthy participantsArticleAug 2021Chay Ngee LimConstantino KantaridisIsabelle Huyghe[...]Gabriele E. SonnenbergAbstract Proteinuria associated with podocyte effacement is a hallmark of focal segmental glomerulosclerosis (FSGS). Preclinical studies implicated ROBO2/SLIT2 signaling in the regulation of podocyte adhesion, and inhibition of this pathway is a novel target to slow FSGS disease progression. This first‐in‐human dose‐escalation study evaluated the safety, tolerability, pharmacokinetics, and immunogenicity of PF‐06730512, an Fc fusion protein that targets the ROBO2/SLIT2 pathway, in healthy adults. In this Phase 1, double‐blind, sponsor‐open study, single ascending dose (SAD) cohorts were randomized to receive up to 1000 mg or placebo intravenously (IV); multiple ascending dose (MAD) cohorts were randomized to receive up to 400 mg subcutaneous (SC) doses, 1000 mg IV dose, or matching placebo. Safety evaluations were performed up to 71 (SAD) and 113 (MAD) days after dosing; blood samples were collected to measure serum PF‐06730512 concentrations and antidrug antibodies (ADA) to PF‐06730512. Seventy‐nine participants (SAD, 47; MAD, 32) were enrolled. There were 108 mild (SAD, 46; MAD, 62) and 21 moderate (SAD, 13; MAD, 8) treatment‐emergent adverse events (TEAEs); no deaths, treatment‐related serious AEs, severe TEAEs, or infusion reactions were reported. PF‐06730512 exposure generally increased in an approximately dose‐proportional manner; mean t1/2 ranged from 12–15 days across 50–1000 mg doses. Immunogenicity incidence was low (SAD, 0 ADA+; MAD, 2 ADA+). In conclusion, single IV doses of PF‐06730512 up to 1000 mg and multiple IV and SC dosing up to 1000 and 400 mg, respectively, were safe and well tolerated in healthy participants. Further trials in patients with FSGS are warranted. Clinical trial registration: Clinicaltrials.gov: NCT03146065.ViewExpand abstractSourcePharmacokinetics and pharmacodynamics of the cytolytic anti‐CD38 human monoclonal antibody TAK‐079 in monkey – model assisted preparation for the first in human trialArticleJun 2018Stefan RoepckeNele PlockJosh Yuan[...]Glennda SmithsonWe are studying the fully human, IgG1λ cytolytic monoclonal antibody TAK‐079, which binds CD38. CD38 is expressed on plasma and natural killer (NK) cells constitutively and upregulated on subsets of B and T lymphocytes upon activation. TAK‐079 cross‐reacts with CD38 expressed by cynomolgus monkeys and depletes subsets of NK, B, and T cells. Therefore, safety and function of TAK‐079 was evaluated in this species, prior to clinical development, using bioanalytical, and flow cytometry assays. We pooled the data from eight studies in healthy monkeys (dose range 0.03‐100 mg/kg) and developed mathematical models that describe the pharmacokinetics and the exposure–effect relationship for NK cells, B cells, and T cells. NK cell depletion was identified as the most sensitive pharmacodynamic effect of TAK‐079. It was adequately described with a turnover model (C50 = 27.5 μg/mL on depletion rate) and complete depletion was achieved with an IV dose of 0.3 mg/kg. Intermediate effects on T‐cell counts were described with a direct response model (C50 = 11.9 μg/mL) and on B‐cell counts with a 4‐transit‐compartment model (C50 = 19.8 μg/mL on depletion rate). Our analyses substantiate the observation that NK, B and T cells are cleared by TAK‐079 at different rates and required different time spans to replete the blood compartment. The models were used to simulate pharmacokinetic and cell depletion profiles in humans after applying a straightforward scaling approach for monoclonal antibodies in preparation for the first‐in‐human clinical trial.ViewExpand abstractSourceExploring the overlapping binding sites of ifenprodil and EVT‐101 in GluN2B‐containing NMDA receptors using novel chicken embryo forebrain cultures and molecular modelingArticleJun 2019Marthe Fredheim FjelldalThibaud FreydLinn Evenseth[...]Ragnhild E PaulsenN‐methyl‐d‐aspartate receptors (NMDAR) are widely expressed in the brain. GluN2B subunit‐containing NMDARs has recently attracted significant attention as potential pharmacological targets, with emphasis on the functional properties of allosteric antagonists. We used primary cultures from chicken embryo forebrain (E10), expressing native GluN2B‐containing NMDA receptors as a novel model system. Comparing the inhibition of calcium influx by well‐known GluN2B subunit‐specific allosteric antagonists, the following rank order of potency was found: EVT‐101 (EC50 22 ± 8 nmol/L) Ro 25‐6981 (EC50 60 ± 30 nmol/L) ifenprodil (EC50 100 ± 40 nmol/L) eliprodil (EC50 1300 ± 700 nmol/L), similar to previous observations in rat cortical cultures and cell lines overexpressing chimeric receptors. The less explored Ro 04‐5595 had an EC50 of 186 ± 32 nmol/L. Venturing to explain the differences in potency, binding properties were further studied by in silico docking and molecular dynamics simulations using x‐ray crystal structures of GluN1/GluN2B amino terminal domain. We found that Ro 04‐5595 was predicted to bind the recently discovered EVT‐101 binding site, not the ifenprodil‐binding site. The EVT‐101 binding pocket appears to accommodate more structurally different ligands than the ifenprodil‐binding site, and contains residues essential in ligand interactions necessary for calcium influx inhibition. For the ifenprodil site, the less effective antagonist (eliprodil) fails to interact with key residues, while in the EVT‐101 pocket, difference in potency might be explained by differences in ligand‐receptor interaction patterns.ViewExpand abstractSourcePharmacological inhibition of STAT3 by BP‐1‐102 inhibits intracranial aneurysm formation and rupture in mice through modulating inflammatory responseArticleFeb 2021Zhixian JiangJiaxin HuangLingtong You[...]Bingyu LiAs an inhibitor of STAT3, BP‐1‐102 can regulate the inflammation response caused by vascular smooth muscle cells (VSMCs) by inhibiting the JAK/STAT3/NF‐κB pathway, thereby attenuating the symptoms of intracranial aneurysm (IA). IA mouse model was established by stereotactic injection of elastase to evaluate the effect of BP‐1‐102. The expression levels of smooth muscle markers and matrix metalloproteinases (MMPs) were detected by qRT‐PCR, and the levels of inflammatory factors were detected by ELISA and qRT‐PCR. The protein levels of the NF‐κB signaling pathway factors were examined by Western blot. BP‐1‐102 reduced blood pressure in aneurysm mice, up‐regulated smooth muscle cell markers MHC, SMA, and SM22, and down‐regulated the expression of MMP2 and MMP9 in vascular tissues. At the same time, BP‐1‐102 also down‐regulated the expression levels of inflammatory response factors and the NF‐κB pathway proteins. In the IA model, BP‐1‐102 can reduce the expression of inflammatory factors and MMPs bound to NF‐κB by inhibiting the activation of the JAK/STAT3/NF‐κB pathway proteins, and then restore the vascular wall elastin to reduce blood pressure, thereby treating aneurysm. BP‐1‐102 reduces the production of inflammatory cytokines, as well as inhibits the activation of the JAK/STAT3/NF‐κB signaling. Therefore, it restores the vascular wall elastin to reduce blood pressure, thereby treating aneurysm.ViewExpand abstractSourceLack of cross-resistance to FF-10501, an inhibitor of inosine-5′-monophosphate dehydrogenase, in azacitidine-resistant cell lines selected from SKM-1 and MOLM-13 leukemia cell linesArticleFeb 2016Motohiko MuraseHiroyuki IwamuraKensuke Komatsu[...]Yasuhiro ShimadaResistance to azacitidine is a major issue in the treatments of myelodysplastic syndrome and acute myeloid leukemia, and previous studies suggest that changes in drug metabolism are involved in the resistance. Therefore, drugs with mechanisms resistant or alternative to such metabolic changes have been desired for the treatment of resistant disease. We generated azacitidine-resistant cells derived from SKM-1 and MOLM-13 leukemia cell lines in vitro, analyzed the mechanisms, and examined the impact on the efficacy of other antimetabolic drugs. It appeared that the cell growth-inhibitory effect of azacitidine, expression levels of uridine–cytidine kinase 2, and the concentrations of azacitidine triphosphate were remarkably decreased in the resistant cells compared with those in parent cells. These results were consistent with previous observations that azacitidine resistance is derived from metabolic changes. Cross-resistance of greater than 10-fold (shift in IC50 value) was observed in azacitidine-resistant cells for decitabine and for cytarabine, but not for gemcitabine or the inosine-5′-monophosphate dehydrogenase (IMPDH) inhibitors FF-10501 and mycophenolate mofetil (cross-resistance to 5-fluorouracil was cell line dependent). The IMPDH inhibitors maintained their cell growth-inhibitory activities in the azacitidine-resistant cell lines, in which the levels of adenine phosphoribosyltransferase (which converts FF-10501 to its active form, FF-10501 ribosylmonophosphate [FF-10501RMP]), FF-10501RMP, and the target enzyme, IMPDH, were equivalent to those in the parent cell lines. These results suggest that an IMPDH inhibitor such as FF-10501 could be an alternative therapeutic treatment for leukemia patients with acquired resistance to azacitidine.ViewExpand abstractSourceDSP-1053, a novel serotonin reuptake inhibitor with 5-HT1A partial agonistic activity, displays fast antidepressant effect with minimal undesirable effects in juvenile ratsArticleJun 2015Taro KatoYuji MatsumotoMasanori Yamamoto[...]Kazuki YabuuchiEnhancement of serotonergic neurotransmission has been the main stream of treatment for patients with depression. However, delayed therapeutic onset and undesirable side effects are major drawbacks for conventional serotonin reuptake inhibitors. Here, we show that DSP-1053, a novel serotonin reuptake inhibitor with 5-HT1A partial agonistic activity, displays fast antidepressant efficacy with minimal undesirable effects, especially nausea and emesis in animal models. DSP-1053 bound human serotonin transporter and 5-HT1A receptor with the Ki values of 1.02 ± 0.06 and 5.05 ± 1.07 nmol/L, respectively. This compound inhibited the serotonin transporter with an IC50 value of 2.74 ± 0.41 nmol/L and had an intrinsic activity for 5-HT1A receptors of 70.0 ± 6.3%. In rat microdialysis, DSP-1053, given once at 3 and 10 mg kg−1, dose-dependently increased extracellular 5-HT levels. In the rat forced swimming test, 2-week administration of DSR-1053 (1 mg kg−1) significantly reduced rats immobility time after treatment, whereas paroxetine (3 and 10 mg kg−1) required 3-week administration to reduce rats immobility time. In olfactory bulbectomy model, 1- and 2-week administration of DSP-1053 reduced both of emotional scores and activity in the open field, whereas paroxetine required 2 weeks to show similar beneficial effects. Although single administration of DSP-1053-induced emesis and vomiting in the rat and Suncus murinus, multiple treatment with this compound, but not with paroxetine, decreased the number of vomiting episodes. These results highlight the important role of 5-HT1A receptors in both the efficacy and tolerability of DSP-1053 as a new therapeutic option for the treatment of depression.ViewExpand abstractSourceDirect and cytokine-mediated effects of albumin-fused growth hormone, TV-1106, on CYP enzyme expression in human hepatocytes in vitroArticleJun 2018Maciej CzerwinskiImmaculate AmunomVictor Piryatinsky[...]Merav BassanSome biologics can modulate cytokines that may lead to changes in expression of drug‐metabolizing enzymes and cause drug‐drug interactions (DDI). DDI potential of TV‐1106—an albumin‐fused growth hormone (GH)—was investigated. In this study, human blood was exposed to recombinant human growth hormone (rhGH) or TV‐1106, followed by isolation of the plasma and its application to human hepatocytes. While the treatment of blood with rhGH increased multiple cytokines, treatment of blood with TV‐1106 had no effect on any of the nine cytokines tested. The interleukin (IL)‐6 concentration was higher in the rhGH then in the TV‐1106‐treated plasma (P .05). While rhGH had little or no effect on CYP1A2 or CYP2C19 mRNA but increased CYP3A4 mRNA twofold, TV‐1106 had little or no effect on cytochrome P450 (CYP) mRNAs in hepatocytes. Although the plasma from rhGH‐treated blood lowered CYP1A2 activity, the TV‐1106 plasma had no effect on CYP activities. The CYP1A2 activity was lower in the rhGH‐ then in the TV‐1106‐plasma treated hepatocytes (P .05). The results indicated that fusing GH with albumin made TV‐1106 an unlikely participant of CYP1A2, CYP2C19 or CYP3A4‐facilitated, direct or cytokine‐driven DDI.ViewExpand abstractSourceSubtype selective fluorescent ligands based on ICI 118,551 to study the human β2‐adrenoceptor in CRISPR/Cas9 genome‐edited HEK293T cells at low expression levelsArticleMay 2021Joelle GouldingSarah MistryMark Soave[...]Stephen John HillFluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β2 -adrenoceptor (β2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β2 AR (pKD 7.0) with good selectivity over the β1 AR (pKD 6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; β2 AR pKD 7.48), 9c (ICI 118,551-βAla-βAla-BODIPY-FL-X; β2 AR pKD 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; β2 AR pKD 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; β2 AR pKD 7.42). 9a (ICI 118,551-βAla-βAla-BODIPY-X-630/650) had the highest affinity at recombinant β2 ARs (pKD 7.57), but also exhibited significant binding affinity to the β1 AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular β2 ARs in HEK293 T cell expressing exogenous β2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular β2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T β2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.ViewExpand abstractSourceUltraviolet irradiation increases green fluorescence of dihydrorhodamine (DHR) 123: false-positive results for reactive oxygen species generationArticleApr 2017Pascal DjiadeuDhia AzzouzMeraj Alam Khan[...]Nades PalaniyarDihydrorhodamine (DHR) 123 is a fluorophore commonly used for measuring reactive oxygen species (ROS), often after exposing cells to ultraviolet (UV) irradiation or oxidative burst inducers such as Phorbol 12-myristate 13-acetate (PMA). However, the negative effects of UV irradiation on oxidation of DHR123 itself to green fluorescence rhodamine (R) 123 under different experimental conditions (e.g., different buffers, media, cells, ROS detection techniques) have not been fully appreciated. We determined the effect of UV on DHR123 fluorescence, using a cell-free system, and A549 epithelial cells, NIH/3T3 fibroblast cells, Jurkat T cells, primary human T cells, HL-60 neutrophils and primary human neutrophils. We found that UV irradiation rapidly increases green fluorescence of DHR123 in cell-free solutions. The intensity of green fluorescence increases with increasing amounts of DHR123 and UV exposure. The fluorescence increase was greater in Roswell Park Memorial Institute medium (RPMI) than DMEM media. The presence of DMSO (0–1.25%, v/v) in RPMI further increases the fluorescence signal. Phosphate buffered solution (PBS) and Hanks Balanced Salt Solution (HBSS) generate considerable background signal with DHR123, and increasing DMSO concentration greatly increases the fluorescence signal in these buffers. However, after UV irradiation the amount of DHR123 that remains unoxidized generates sufficient fluorescence signal to measure the ROS produced by H2O2 and peroxidase in vitro or Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated ROS production within HL-60 neutrophils or primary human neutrophils. We conclude that UV irradiation oxidizes DHR123 to generate Rhodamine 123 (R123) green fluorescence signal, and that the R123 present in the culture supernatant could give erroneous results in plate reader assays. However, flow cytometry and fluorescence microscopy reliably detect ROS in cells such as neutrophils. Overall, avoiding false-positive results when detecting ROS using DHR123 requires selection of, agonists, the correct buffers, media, cell types, and measurement techniques.ViewExpand abstractSourceActive transport of rhodamine 123 by the human multidrug transporter P‐glycoprotein involves two independent outer gatesArticleApr 2020Fauzia NasimDiethart SchmidGergely Szakács[...]Thomas StocknerHuman P‐glycoprotein (P‐gp) is a multispecific drug‐efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo‐electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry‐related outer gates. Experiments were aided by availability of the X‐ray structure of a homodimeric eukaryotic homolog of P‐gp from red alga (CmABCB1), which defined the role of an apical tyrosine residue (Y358) in outer gate formation. We mutated analogous tyrosine residues in each half of the human full‐length transporter (Y310, Y953) to alanine. These mutants were introduced in engineered transporters which bind rhodamine 123 in one of two symmetry‐related binding modes only. Outer gate dysfunction was detected by a loss of active transport characteristics, while these mutants retained the ability for outward downhill transport. Our data demonstrate that symmetric tyrosine residues Y310 and Y953 are involved in formation of two distinct symmetry‐related outer gates, which operate contingent on the rhodamine 123 binding mode. Hence, the rotationally symmetric architecture of P‐gp, which determines duality in ATP binding and rhodamine 123 interaction, also forms the basis for the existence of two independently operating outer gates.View1 RecommendationExpand abstractSourceRepeated exposure of house dust mite induces progressive airway inflammation in mice: Differential roles of CCL17 and IL-13ArticleMay 2021Ravi MalaviyaZhao ZhouHolly A Raymond[...]Tadimeti S RaoWe conducted a systematic evaluation of lung inflammation indued by repeated intranasal exposure (for 10 consecutive days) to a human aeroallergen, house dust mite (HDM) in BALB/c mice. Peak influx of neutrophils, monocytes/lymphocytes, and eosinophils was observed in bronchoalveolar lavage (BAL) on days 1, 7 and 11, respectively, and normalized to baseline by day 21. Peak elevations of Th2, myeloid-derived cytokines/chemokines and serum IgE were seen both in BAL and lung tissue homogenates between days 7 and 11, and declined thereafter; however, IL-33 levels remained elevated from day 7 to day 21. Airway hyperreactivity to inhaled methacholine was significantly increased by day 11 and decreased to baseline by day 21. The lung tissue showed perivascular and peribronchial cuffing, epithelial hypertrophy and hyperplasia and goblet cell formation in airways by day 11, and resolution by day 21. Levels of soluble collagen and tissue inhibitors of metalloproteinases (TIMP) also increased reflecting tissue remodeling in the lung. Microarray analysis demonstrated a significant time-dependent up-regulation of several genes including IL-33, CLCA3, CCL17, CD4, CD10, CD27, IL-13, Foxa3, IL-4, IL-10, and CD19, in BAL cells as well as the lung. Pre-treatment of HDM challenged mice with CCL17 and IL-13 antibodies reduced BAL cellularity, airway hyper-responsiveness (AHR), and histopathological changes. Notably, anti-IL-13, but not anti-CCL17 monoclonal antibodies (mAbs) reduced BAL neutrophilia while both mAbs attenuated eosinophilia. These results suggest that CCL17 has an overlapping, yet distinct profile versus IL-13 in the HDM model of pulmonary inflammation and potential for CCL17-based therapeutics in treating Th2 inflammation.ViewExpand abstractSourceIdentifying cytochrome P450s involved in oxidative metabolism of synthetic cannabinoid N‐(adamantan‐1‐yl)‐1‐(5‐fluoropentyl)‐1H‐indole‐3‐carboxamide (STS‐135)ArticleFeb 2020Sabrina JonesAzure YarbroughWilliam E Fantegrossi[...]Ryoichi FujiwaraSynthetic cannabinoids (SCBs), designer drugs marketed as legal alternatives to marijuana, act as ligands to cannabinoid receptors; however, they have increased binding affinity and potency, resulting in toxicity symptoms such as cardiovascular incidents, seizures, and potentially death. N‐(adamantan‐1‐yl)‐1‐(5‐fluoropentyl)‐1H‐indole‐3‐carboxamide (STS‐135) is a third generation SCB. When incubated with hepatocytes, it undergoes oxidation, hydrolysis, and glucuronidation, resulting in 29 metabolites, with monohydroxy STS‐135 (M25) and dihydroxy STS‐135 (M21) being the predominant metabolites. The enzymes responsible for this oxidative metabolism were unknown. Thus, the aim of this study was to identify the cytochrome P450 (P450s or CYPs) enzymes involved in the oxidative metabolism of STS‐135. In this study, STS‐135 was incubated with liver, intestinal, and brain microsomes and recombinant P450s to determine the enzymes involved in its metabolism. Metabolite quantification was carried out using ultra‐performance liquid chromatography. STS‐135 was extensively metabolized in HLMs and HIMs. Screening assays indicated CYP3A4 and CYP3A5 could be responsible for STS‐135’s oxidation. Through incubations with genotyped HLMs, CYP3A4 was identified as the primary oxidative enzyme. Interestingly, CYP2J2, a P450 isoform expressed in cardiovascular tissues, showed high activity towards the formation of M25 with a Km value of 11.4 μmol/L. Thus, it was concluded that STS‐135 was primarily metabolized by CYP3A4 but may have extrahepatic metabolic pathways as well. Upon exposure to STS‐135, individuals with low CYP3A4 activity could retain elevated blood concentration, resulting in toxicity. Additionally, CYP2J2 may aid in protecting against STS‐135‐induced cardiovascular toxicity.View2 RecommendationsExpand abstractSourcePreclinical characterization of AMPA receptor potentiator TAK‐137 as a therapeutic drug for schizophreniaArticleJun 2019Maiko TanakaAkiyoshi KunugiAtsushi Suzuki[...]Haruhide KimuraThe downregulation of the glutamate system may be involved in positive, negative, and cognitive symptoms of schizophrenia. Through enhanced glutamate signaling, the activation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) receptor, an ionotropic glutamate receptor, could be a new therapeutic strategy for schizophrenia. TAK‐137 is a novel AMPA receptor potentiator with minimal agonistic activity; in this study, we used rodents and nonhuman primates to assess its potential as a drug for schizophrenia. At 10 mg kg−1 p.o., TAK‐137 partially inhibited methamphetamine‐induced hyperlocomotion in rats, and at 3, 10, and 30 mg kg−1 p.o., TAK‐137 partially inhibited MK‐801‐induced hyperlocomotion in mice, suggesting weak effects on the positive symptoms of schizophrenia. At 0.1 and 0.3 mg kg−1 p.o., TAK‐137 significantly ameliorated MK‐801‐induced deficits in the social interaction of rats, demonstrating potential improvement of impaired social functioning, which is a negative symptom of schizophrenia. The effects of TAK‐137 were evaluated on multiple cognitive domains—attention, working memory, and cognitive flexibility. TAK‐137 enhanced attention in the five‐choice serial reaction time task in rats at 0.2 mg kg−1 p.o., and improved working memory both in rats and monkeys: 0.2 and 0.6 mg kg−1 p.o. ameliorated MK‐801‐induced deficits in the radial arm maze test in rats, and 0.1 mg kg−1 p.o. improved the performance of ketamine‐treated monkeys in the delayed matching‐to‐sample task. At 0.1 and 1 mg kg−1 p.o., TAK‐137 improved the cognitive flexibility of subchronic phencyclidine‐treated rats in the reversal learning test. Thus, TAK‐137‐type AMPA receptor potentiators with low intrinsic activity may offer new therapies for schizophrenia.ViewExpand abstractSourceUpregulated UCA1 contributes to oxaliplatin resistance of hepatocellular carcinoma through inhibition of miR-138-5p and activation of AKT/mTOR signaling pathwayArticleFeb 2021Guolin HuangLi LiChaoyong Liang[...]Yang JieHepatocellular carcinoma (HCC) inevitably developed oxaliplatin (OXA) resistance after long-term treatment, but the mechanism remains unclear. Here, we found that LncRNA UCA1 was upregulated in most of OXA-resistant HCC tissues and cells (HepG2/OXA and SMMC-7721/OXA). Follow-up analysis and online Kaplan-Meier Plotter revealed that HCC patients with high UCA1 level had a shorter survival compared with those with low expression. Overexpression of UCA1 increased OXA IC50 in HepG2 and SMMC-7721 cells, whereas knockdown of UCA1 decreased OXA IC50 in resistant counterparts. Moreover, dual luciferase reporter assay showed that co-transfection of UCA1-WT plasmid with miR-138-5p mimics enhanced fluorescence signals, whereas co-transfection of UCA1-Mut plasmid and miR-138-5p mimics did not induce any changes. Consistently, UCA1 levels in HepG2/OXA and SMMC-7721/OXA cells were downregulated after transfected with miR-138-5p mimics. UCA1 silencing or transfection of miR-138-5p mmics inhibited the activation of AKT and mTOR in HepG2/OXA and SMMC-7721/OXA cells, whereas UCA1 overexpression increased the phosphorylated AKT and mTOR levels in parental counterparts. Rapamycin or miR-138-5p mimics similarly suppressed the activation of AKT and mTOR, whereas UCA1 overexpression exert opposite roles. Interestingly, administration of rapamycin or miR-138-5p mimics apparently antagonized the effects of UCA1 on AKT and mTOR activation. Besides, depletion of UCA1 triggered more dramatic regression of HepG2 xenografts than that of HepG2/OXA xenografts with OXA treatment and impaired the p-AKT and p-mTOR levels in vivo. In conclusion, our findings provide the evidence that UCA1 may contribute to OXA resistance via miR-138-5p-mediated AK /mTOR activation, suggesting that UCA1 is a potential therapeutic target for HCC.View1 RecommendationExpand abstractSourceMass balance and pharmacokinetics of an oral dose of 14 C-napabucasin in healthy adult male subjectsArticleFeb 2021Xiaoshu DaiMichael D. KarolMatthew Hitron[...]Brandon T GuffordThis phase 1, open-label study assessed14 C-napabucasin absorption, metabolism, and excretion, napabucasin pharmacokinetics, and napabucasin metabolites (primary objectives); safety/tolerability were also evaluated. Eight healthy males (18-45 years) received a single oral 240-mg napabucasin dose containing ~100 μCi14 C-napabucasin. Napabucasin was absorbed and metabolized to dihydro-napabucasin (M1; an active metabolite [12.57-fold less activity than napabucasin]), the sole major circulating metabolite (median time to peak concentration: 2.75 and 2.25 h, respectively). M1 plasma concentration versus time profiles generally mirrored napabucasin; similar arithmetic mean half-lives (7.14 and 7.92 h, respectively) suggest M1 formation was rate limiting. Napabucasin systemic exposure (per Cmax and AUC) was higher than M1. The total radioactivity (TRA) whole blood:plasma ratio (AUClast : 0.376; Cmax : 0.525) indicated circulating drug-related compounds were essentially confined to plasma. Mean TRA recovery was 81.1% (feces, 57.2%; urine, 23.8%; expired air, negligible). Unlabeled napabucasin and M1 recovered in urine accounted for 13.9% and 11.0% of the dose (sum similar to urine TRA recovered); apparent renal clearance was 8.24 and 7.98 L/h. No uniquely human or disproportionate metabolite was quantified. Secondary glucuronide and sulfate conjugates were common urinary metabolites, suggesting napabucasin was mainly cleared by reductive metabolism. All subjects experienced mild treatment-emergent adverse events (TEAEs), the majority related to napabucasin. The most commonly reported TEAEs were gastrointestinal disorders. There were no clinically significant laboratory, vital sign, electrocardiogram, or physical examination changes. Napabucasin was absorbed, metabolized to M1 as the sole major circulating metabolite, and primarily excreted via feces. A single oral 240-mg dose was generally well tolerated.ViewExpand abstractSourceInter‐individual and inter‐regional variations in enteric drug metabolizing enzyme activities: Results with cryopreserved human intestinal mucosal epithelia (CHIM) from the small intestines of 14 donorsArticleOct 2020Albert P LiMing-Chih HoNovera Alam[...]Cornelis E. C. A. HopWe have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12‐inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non‐P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway‐selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12‐inch segments of the proximal small intestine shows that the segments immediately after the first 12‐inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.ViewExpand abstractSource14,15-epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC12 and rat hippocampal neuronal cellsArticleOct 2018Ami OguroTakumi InoueSuguru N KudohSusumu ImaokaPolyunsaturated fatty acids, such as arachidonic acid, are accumulated in brain and induce neuronal differentiation. Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) by cytochrome P450s. In this study, we found that 14,15‐EET and 20‐HETE‐enhanced NGF‐induced rat pheochromocytoma PC12 cell neurite outgrowth even at the concentration of 100 nmol L−1. LC‐MS analysis revealed that 14,15‐EET was effectively produced from arachidonic acid by rat CYP2C11, 2C13, and 2C23, and these P450s were expressed in PC12 cells. An inhibitor of these P450s, ketoconazole, inhibited neurite outgrowth, whereas inhibition of soluble epoxide hydrolase, which hydrolyzes EETs to their corresponding diols enhanced neurite outgrowth. To determine the mechanism of neurite formation enhancement by arachidonic acid metabolites, we focused on transient receptor potential (TRP) channels expressed in PC12 cells. The TRPV4 inhibitor HC067047, but not the TRPV1 inhibitor capsazepine, inhibited the effects of 14,15‐EET on neurite outgrowth of PC12. Furthermore, 14,15‐EET increased the cytosolic calcium ion concentration and this increase was inhibited by HC067047. 14,15‐EET also enhanced neurite outgrowth of primary cultured neuron from rat hippocampus. This study suggests that arachidonic acid metabolites produced by P450 contribute to neurite outgrowth through calcium influx.ViewExpand abstractSourceAbsorption, distribution, metabolism, and excretion of [14C]‐dasotraline in humansArticleFeb 2017Yuluan ChenEstela SkendeJing Lin[...]Seth C. HopkinsDasotraline is a dopamine and norepinephrine reuptake inhibitor, and the early clinical trials show a slow absorption and long elimination half-life. To investigate the absorption, distribution, metabolism, and excretion of dasotraline in humans, a single dose of [14C]-dasotraline was administered to eight healthy male adult volunteers. At 35 days, 90.7% of the dosed radioactivity was recovered in the urine (68.3%) and feces (22.4%). The major metabolic pathways involved were: (1) amine oxidation to form oxime M41 and sequential sulfation to form M42 or glucuronidation to form M43; (2) N-hydroxylation and sequential glucuronidation to form M35; (3) oxidative deamination to form (S)-tetralone; (4) mono-oxidation of (S)-tetralone and sequential glucuronidation to form M31A and M32; and (5) N-acetylation to form (1R,4S)-acetamide M102. A total of 8 metabolites were detected and structurally elucidated with 4 in plasma (M41, M42, M43, and M35), 7 in urine (M41, M42, M43, M31A, M32, M35, and (S)-tetralone), and 3 in feces (M41, (S)-tetralone, and (1R,4S)-acetamide). The 2 most abundant circulating metabolites were sulfate (M42) and glucuronide (M43) conjugates of the oxime of dasotraline, accounting for 60.1% and 15.0% of the total plasma radioactivity, respectively; unchanged dasotraline accounted for 8.59%. The oxime M41 accounted for only 0.62% of the total plasma radioactivity and was detected only at early time points. M35 was a minor glucuronide metabolite, undetectable by radioactivity but identified by mass spectrometry. The results demonstrate that dasotraline was slowly absorbed, and extensively metabolized by oxidation and subsequent phase II conjugations. The findings from this study also demonstrated that metabolism of dasotraline by humans did not produce metabolites that may cause a safety concern.ViewExpand abstractSourceCombined treatment with a selective PDE10A inhibitor TAK-063 and either haloperidol or olanzapine at subeffective doses produces potent antipsychotic-like effects without affecting plasma prolactin levels and cataleptic responses in rodentsArticleFeb 2018Kazunori SuzukiAkina HaradaHirobumi Suzuki[...]Haruhide KimuraActivation of indirect pathway medium spiny neurons (MSNs) via promotion of cAMP production is the principal mechanism of action of current antipsychotics with dopamine D2 receptor antagonism. TAK-063 [1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one] is a novel phosphodiesterase 10A inhibitor that activates both direct and indirect pathway MSNs through increasing both cAMP and cGMP levels by inhibition of their degradation. The activation of indirect pathway MSNs through the distinct mechanism of action of these drugs raises the possibility of augmented pharmacological effects by combination therapy. In this study, we evaluated the potential of combination therapy with TAK-063 and current antipsychotics, such as haloperidol or olanzapine after oral administration. Combined treatment with TAK-063 and either haloperidol or olanzapine produced a significant increase in phosphorylation of glutamate receptor subunit 1 in the rat striatum. An electrophysiological study using rat corticostriatal slices showed that TAK-063 enhanced N-methyl-D-aspartic acid receptor-mediated synaptic responses in both direct and indirect pathway MSNs to a similar extent. Further evaluation using pathway-specific markers revealed that coadministration of TAK-063 with haloperidol or olanzapine additively activated the indirect pathway, but not the direct pathway. Combined treatment with TAK-063 and either haloperidol or olanzapine at subeffective doses produced significant effects on methamphetamine- or MK-801-induced hyperactivity in rats and MK-801-induced deficits in prepulse inhibition in mice. TAK-063 at 0.1 mg/kg did not affect plasma prolactin levels and cataleptic response from antipsychotics in rats. Thus, TAK-063 may produce augmented antipsychotic-like activities in combination with antipsychotics without effects on plasma prolactin levels and cataleptic responses in rodents.ViewExpand abstractSourceA Phase 1 first‐in‐human study of the safety, tolerability, and pharmacokinetics of the ROBO2 fusion protein PF‐06730512 in healthy participantsArticleAug 2021Chay Ngee LimConstantino KantaridisIsabelle Huyghe[...]Gabriele E. SonnenbergAbstract Proteinuria associated with podocyte effacement is a hallmark of focal segmental glomerulosclerosis (FSGS). Preclinical studies implicated ROBO2/SLIT2 signaling in the regulation of podocyte adhesion, and inhibition of this pathway is a novel target to slow FSGS disease progression. This first‐in‐human dose‐escalation study evaluated the safety, tolerability, pharmacokinetics, and immunogenicity of PF‐06730512, an Fc fusion protein that targets the ROBO2/SLIT2 pathway, in healthy adults. In this Phase 1, double‐blind, sponsor‐open study, single ascending dose (SAD) cohorts were randomized to receive up to 1000 mg or placebo intravenously (IV); multiple ascending dose (MAD) cohorts were randomized to receive up to 400 mg subcutaneous (SC) doses, 1000 mg IV dose, or matching placebo. Safety evaluations were performed up to 71 (SAD) and 113 (MAD) days after dosing; blood samples were collected to measure serum PF‐06730512 concentrations and antidrug antibodies (ADA) to PF‐06730512. Seventy‐nine participants (SAD, 47; MAD, 32) were enrolled. There were 108 mild (SAD, 46; MAD, 62) and 21 moderate (SAD, 13; MAD, 8) treatment‐emergent adverse events (TEAEs); no deaths, treatment‐related serious AEs, severe TEAEs, or infusion reactions were reported. PF‐06730512 exposure generally increased in an approximately dose‐proportional manner; mean t1/2 ranged from 12–15 days across 50–1000 mg doses. Immunogenicity incidence was low (SAD, 0 ADA+; MAD, 2 ADA+). In conclusion, single IV doses of PF‐06730512 up to 1000 mg and multiple IV and SC dosing up to 1000 and 400 mg, respectively, were safe and well tolerated in healthy participants. Further trials in patients with FSGS are warranted. Clinical trial registration: Clinicaltrials.gov: NCT03146065.ViewExpand abstractSourcePharmacokinetics and pharmacodynamics of the cytolytic anti‐CD38 human monoclonal antibody TAK‐079 in monkey – model assisted preparation for the first in human trialArticleJun 2018Stefan RoepckeNele PlockJosh Yuan[...]Glennda SmithsonWe are studying the fully human, IgG1λ cytolytic monoclonal antibody TAK‐079, which binds CD38. CD38 is expressed on plasma and natural killer (NK) cells constitutively and upregulated on subsets of B and T lymphocytes upon activation. TAK‐079 cross‐reacts with CD38 expressed by cynomolgus monkeys and depletes subsets of NK, B, and T cells. Therefore, safety and function of TAK‐079 was evaluated in this species, prior to clinical development, using bioanalytical, and flow cytometry assays. We pooled the data from eight studies in healthy monkeys (dose range 0.03‐100 mg/kg) and developed mathematical models that describe the pharmacokinetics and the exposure–effect relationship for NK cells, B cells, and T cells. NK cell depletion was identified as the most sensitive pharmacodynamic effect of TAK‐079. It was adequately described with a turnover model (C50 = 27.5 μg/mL on depletion rate) and complete depletion was achieved with an IV dose of 0.3 mg/kg. Intermediate effects on T‐cell counts were described with a direct response model (C50 = 11.9 μg/mL) and on B‐cell counts with a 4‐transit‐compartment model (C50 = 19.8 μg/mL on depletion rate). Our analyses substantiate the observation that NK, B and T cells are cleared by TAK‐079 at different rates and required different time spans to replete the blood compartment. The models were used to simulate pharmacokinetic and cell depletion profiles in humans after applying a straightforward scaling approach for monoclonal antibodies in preparation for the first‐in‐human clinical trial.ViewExpand abstractSourceExploring the overlapping binding sites of ifenprodil and EVT‐101 in GluN2B‐containing NMDA receptors using novel chicken embryo forebrain cultures and molecular modelingArticleJun 2019Marthe Fredheim FjelldalThibaud FreydLinn Evenseth[...]Ragnhild E PaulsenN‐methyl‐d‐aspartate receptors (NMDAR) are widely expressed in the brain. GluN2B subunit‐containing NMDARs has recently attracted significant attention as potential pharmacological targets, with emphasis on the functional properties of allosteric antagonists. We used primary cultures from chicken embryo forebrain (E10), expressing native GluN2B‐containing NMDA receptors as a novel model system. Comparing the inhibition of calcium influx by well‐known GluN2B subunit‐specific allosteric antagonists, the following rank order of potency was found: EVT‐101 (EC50 22 ± 8 nmol/L) Ro 25‐6981 (EC50 60 ± 30 nmol/L) ifenprodil (EC50 100 ± 40 nmol/L) eliprodil (EC50 1300 ± 700 nmol/L), similar to previous observations in rat cortical cultures and cell lines overexpressing chimeric receptors. The less explored Ro 04‐5595 had an EC50 of 186 ± 32 nmol/L. Venturing to explain the differences in potency, binding properties were further studied by in silico docking and molecular dynamics simulations using x‐ray crystal structures of GluN1/GluN2B amino terminal domain. We found that Ro 04‐5595 was predicted to bind the recently discovered EVT‐101 binding site, not the ifenprodil‐binding site. The EVT‐101 binding pocket appears to accommodate more structurally different ligands than the ifenprodil‐binding site, and contains residues essential in ligand interactions necessary for calcium influx inhibition. For the ifenprodil site, the less effective antagonist (eliprodil) fails to interact with key residues, while in the EVT‐101 pocket, difference in potency might be explained by differences in ligand‐receptor interaction patterns.ViewExpand abstractSourcePharmacological inhibition of STAT3 by BP‐1‐102 inhibits intracranial aneurysm formation and rupture in mice through modulating inflammatory responseArticleFeb 2021Zhixian JiangJiaxin HuangLingtong You[...]Bingyu LiAs an inhibitor of STAT3, BP‐1‐102 can regulate the inflammation response caused by vascular smooth muscle cells (VSMCs) by inhibiting the JAK/STAT3/NF‐κB pathway, thereby attenuating the symptoms of intracranial aneurysm (IA). IA mouse model was established by stereotactic injection of elastase to evaluate the effect of BP‐1‐102. The expression levels of smooth muscle markers and matrix metalloproteinases (MMPs) were detected by qRT‐PCR, and the levels of inflammatory factors were detected by ELISA and qRT‐PCR. The protein levels of the NF‐κB signaling pathway factors were examined by Western blot. BP‐1‐102 reduced blood pressure in aneurysm mice, up‐regulated smooth muscle cell markers MHC, SMA, and SM22, and down‐regulated the expression of MMP2 and MMP9 in vascular tissues. At the same time, BP‐1‐102 also down‐regulated the expression levels of inflammatory response factors and the NF‐κB pathway proteins. In the IA model, BP‐1‐102 can reduce the expression of inflammatory factors and MMPs bound to NF‐κB by inhibiting the activation of the JAK/STAT3/NF‐κB pathway proteins, and then restore the vascular wall elastin to reduce blood pressure, thereby treating aneurysm. BP‐1‐102 reduces the production of inflammatory cytokines, as well as inhibits the activation of the JAK/STAT3/NF‐κB signaling. Therefore, it restores the vascular wall elastin to reduce blood pressure, thereby treating aneurysm.ViewExpand abstractSourceLack of cross-resistance to FF-10501, an inhibitor of inosine-5′-monophosphate dehydrogenase, in azacitidine-resistant cell lines selected from SKM-1 and MOLM-13 leukemia cell linesArticleFeb 2016Motohiko MuraseHiroyuki IwamuraKensuke Komatsu[...]Yasuhiro ShimadaResistance to azacitidine is a major issue in the treatments of myelodysplastic syndrome and acute myeloid leukemia, and previous studies suggest that changes in drug metabolism are involved in the resistance. Therefore, drugs with mechanisms resistant or alternative to such metabolic changes have been desired for the treatment of resistant disease. We generated azacitidine-resistant cells derived from SKM-1 and MOLM-13 leukemia cell lines in vitro, analyzed the mechanisms, and examined the impact on the efficacy of other antimetabolic drugs. It appeared that the cell growth-inhibitory effect of azacitidine, expression levels of uridine–cytidine kinase 2, and the concentrations of azacitidine triphosphate were remarkably decreased in the resistant cells compared with those in parent cells. These results were consistent with previous observations that azacitidine resistance is derived from metabolic changes. Cross-resistance of greater than 10-fold (shift in IC50 value) was observed in azacitidine-resistant cells for decitabine and for cytarabine, but not for gemcitabine or the inosine-5′-monophosphate dehydrogenase (IMPDH) inhibitors FF-10501 and mycophenolate mofetil (cross-resistance to 5-fluorouracil was cell line dependent). The IMPDH inhibitors maintained their cell growth-inhibitory activities in the azacitidine-resistant cell lines, in which the levels of adenine phosphoribosyltransferase (which converts FF-10501 to its active form, FF-10501 ribosylmonophosphate [FF-10501RMP]), FF-10501RMP, and the target enzyme, IMPDH, were equivalent to those in the parent cell lines. These results suggest that an IMPDH inhibitor such as FF-10501 could be an alternative therapeutic treatment for leukemia patients with acquired resistance to azacitidine.ViewExpand abstractSourceDSP-1053, a novel serotonin reuptake inhibitor with 5-HT1A partial agonistic activity, displays fast antidepressant effect with minimal undesirable effects in juvenile ratsArticleJun 2015Taro KatoYuji MatsumotoMasanori Yamamoto[...]Kazuki YabuuchiEnhancement of serotonergic neurotransmission has been the main stream of treatment for patients with depression. However, delayed therapeutic onset and undesirable side effects are major drawbacks for conventional serotonin reuptake inhibitors. Here, we show that DSP-1053, a novel serotonin reuptake inhibitor with 5-HT1A partial agonistic activity, displays fast antidepressant efficacy with minimal undesirable effects, especially nausea and emesis in animal models. DSP-1053 bound human serotonin transporter and 5-HT1A receptor with the Ki values of 1.02 ± 0.06 and 5.05 ± 1.07 nmol/L, respectively. This compound inhibited the serotonin transporter with an IC50 value of 2.74 ± 0.41 nmol/L and had an intrinsic activity for 5-HT1A receptors of 70.0 ± 6.3%. In rat microdialysis, DSP-1053, given once at 3 and 10 mg kg−1, dose-dependently increased extracellular 5-HT levels. In the rat forced swimming test, 2-week administration of DSR-1053 (1 mg kg−1) significantly reduced rats immobility time after treatment, whereas paroxetine (3 and 10 mg kg−1) required 3-week administration to reduce rats immobility time. In olfactory bulbectomy model, 1- and 2-week administration of DSP-1053 reduced both of emotional scores and activity in the open field, whereas paroxetine required 2 weeks to show similar beneficial effects. Although single administration of DSP-1053-induced emesis and vomiting in the rat and Suncus murinus, multiple treatment with this compound, but not with paroxetine, decreased the number of vomiting episodes. These results highlight the important role of 5-HT1A receptors in both the efficacy and tolerability of DSP-1053 as a new therapeutic option for the treatment of depression.ViewExpand abstractSourceDirect and cytokine-mediated effects of albumin-fused growth hormone, TV-1106, on CYP enzyme expression in human hepatocytes in vitroArticleJun 2018Maciej CzerwinskiImmaculate AmunomVictor Piryatinsky[...]Merav BassanSome biologics can modulate cytokines that may lead to changes in expression of drug‐metabolizing enzymes and cause drug‐drug interactions (DDI). DDI potential of TV‐1106—an albumin‐fused growth hormone (GH)—was investigated. In this study, human blood was exposed to recombinant human growth hormone (rhGH) or TV‐1106, followed by isolation of the plasma and its application to human hepatocytes. While the treatment of blood with rhGH increased multiple cytokines, treatment of blood with TV‐1106 had no effect on any of the nine cytokines tested. The interleukin (IL)‐6 concentration was higher in the rhGH then in the TV‐1106‐treated plasma (P .05). While rhGH had little or no effect on CYP1A2 or CYP2C19 mRNA but increased CYP3A4 mRNA twofold, TV‐1106 had little or no effect on cytochrome P450 (CYP) mRNAs in hepatocytes. Although the plasma from rhGH‐treated blood lowered CYP1A2 activity, the TV‐1106 plasma had no effect on CYP activities. The CYP1A2 activity was lower in the rhGH‐ then in the TV‐1106‐plasma treated hepatocytes (P .05). The results indicated that fusing GH with albumin made TV‐1106 an unlikely participant of CYP1A2, CYP2C19 or CYP3A4‐facilitated, direct or cytokine‐driven DDI.ViewExpand abstractSourceSubtype selective fluorescent ligands based on ICI 118,551 to study the human β2‐adrenoceptor in CRISPR/Cas9 genome‐edited HEK293T cells at low expression levelsArticleMay 2021Joelle GouldingSarah MistryMark Soave[...]Stephen John HillFluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β2 -adrenoceptor (β2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β2 AR (pKD 7.0) with good selectivity over the β1 AR (pKD 6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; β2 AR pKD 7.48), 9c (ICI 118,551-βAla-βAla-BODIPY-FL-X; β2 AR pKD 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; β2 AR pKD 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; β2 AR pKD 7.42). 9a (ICI 118,551-βAla-βAla-BODIPY-X-630/650) had the highest affinity at recombinant β2 ARs (pKD 7.57), but also exhibited significant binding affinity to the β1 AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular β2 ARs in HEK293 T cell expressing exogenous β2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular β2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T β2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.ViewExpand abstractSourceUltraviolet irradiation increases green fluorescence of dihydrorhodamine (DHR) 123: false-positive results for reactive oxygen species generationArticleApr 2017Pascal DjiadeuDhia AzzouzMeraj Alam Khan[...]Nades PalaniyarDihydrorhodamine (DHR) 123 is a fluorophore commonly used for measuring reactive oxygen species (ROS), often after exposing cells to ultraviolet (UV) irradiation or oxidative burst inducers such as Phorbol 12-myristate 13-acetate (PMA). However, the negative effects of UV irradiation on oxidation of DHR123 itself to green fluorescence rhodamine (R) 123 under different experimental conditions (e.g., different buffers, media, cells, ROS detection techniques) have not been fully appreciated. We determined the effect of UV on DHR123 fluorescence, using a cell-free system, and A549 epithelial cells, NIH/3T3 fibroblast cells, Jurkat T cells, primary human T cells, HL-60 neutrophils and primary human neutrophils. We found that UV irradiation rapidly increases green fluorescence of DHR123 in cell-free solutions. The intensity of green fluorescence increases with increasing amounts of DHR123 and UV exposure. The fluorescence increase was greater in Roswell Park Memorial Institute medium (RPMI) than DMEM media. The presence of DMSO (0–1.25%, v/v) in RPMI further increases the fluorescence signal. Phosphate buffered solution (PBS) and Hanks Balanced Salt Solution (HBSS) generate considerable background signal with DHR123, and increasing DMSO concentration greatly increases the fluorescence signal in these buffers. However, after UV irradiation the amount of DHR123 that remains unoxidized generates sufficient fluorescence signal to measure the ROS produced by H2O2 and peroxidase in vitro or Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated ROS production within HL-60 neutrophils or primary human neutrophils. We conclude that UV irradiation oxidizes DHR123 to generate Rhodamine 123 (R123) green fluorescence signal, and that the R123 present in the culture supernatant could give erroneous results in plate reader assays. However, flow cytometry and fluorescence microscopy reliably detect ROS in cells such as neutrophils. Overall, avoiding false-positive results when detecting ROS using DHR123 requires selection of, agonists, the correct buffers, media, cell types, and measurement techniques.ViewExpand abstractSourceActive transport of rhodamine 123 by the human multidrug transporter P‐glycoprotein involves two independent outer gatesArticleApr 2020Fauzia NasimDiethart SchmidGergely Szakács[...]Thomas StocknerHuman P‐glycoprotein (P‐gp) is a multispecific drug‐efflux transporter, which plays an important role in drug resistance and drug disposition. Recent cryo‐electron microscopy structures confirmed its rotationally symmetric architecture, which allows dual interaction with ATP and substrates. We here report the existence of two distinct, symmetry‐related outer gates. Experiments were aided by availability of the X‐ray structure of a homodimeric eukaryotic homolog of P‐gp from red alga (CmABCB1), which defined the role of an apical tyrosine residue (Y358) in outer gate formation. We mutated analogous tyrosine residues in each half of the human full‐length transporter (Y310, Y953) to alanine. These mutants were introduced in engineered transporters which bind rhodamine 123 in one of two symmetry‐related binding modes only. Outer gate dysfunction was detected by a loss of active transport characteristics, while these mutants retained the ability for outward downhill transport. Our data demonstrate that symmetric tyrosine residues Y310 and Y953 are involved in formation of two distinct symmetry‐related outer gates, which operate contingent on the rhodamine 123 binding mode. Hence, the rotationally symmetric architecture of P‐gp, which determines duality in ATP binding and rhodamine 123 interaction, also forms the basis for the existence of two independently operating outer gates.View1 RecommendationExpand abstractSourceRepeated exposure of house dust mite induces progressive airway inflammation in mice: Differential roles of CCL17 and IL-13ArticleMay 2021Ravi MalaviyaZhao ZhouHolly A Raymond[...]Tadimeti S RaoWe conducted a systematic evaluation of lung inflammation indued by repeated intranasal exposure (for 10 consecutive days) to a human aeroallergen, house dust mite (HDM) in BALB/c mice. Peak influx of neutrophils, monocytes/lymphocytes, and eosinophils was observed in bronchoalveolar lavage (BAL) on days 1, 7 and 11, respectively, and normalized to baseline by day 21. Peak elevations of Th2, myeloid-derived cytokines/chemokines and serum IgE were seen both in BAL and lung tissue homogenates between days 7 and 11, and declined thereafter; however, IL-33 levels remained elevated from day 7 to day 21. Airway hyperreactivity to inhaled methacholine was significantly increased by day 11 and decreased to baseline by day 21. The lung tissue showed perivascular and peribronchial cuffing, epithelial hypertrophy and hyperplasia and goblet cell formation in airways by day 11, and resolution by day 21. Levels of soluble collagen and tissue inhibitors of metalloproteinases (TIMP) also increased reflecting tissue remodeling in the lung. Microarray analysis demonstrated a significant time-dependent up-regulation of several genes including IL-33, CLCA3, CCL17, CD4, CD10, CD27, IL-13, Foxa3, IL-4, IL-10, and CD19, in BAL cells as well as the lung. Pre-treatment of HDM challenged mice with CCL17 and IL-13 antibodies reduced BAL cellularity, airway hyper-responsiveness (AHR), and histopathological changes. Notably, anti-IL-13, but not anti-CCL17 monoclonal antibodies (mAbs) reduced BAL neutrophilia while both mAbs attenuated eosinophilia. These results suggest that CCL17 has an overlapping, yet distinct profile versus IL-13 in the HDM model of pulmonary inflammation and potential for CCL17-based therapeutics in treating Th2 inflammation.ViewExpand abstractSourceIdentifying cytochrome P450s involved in oxidative metabolism of synthetic cannabinoid N‐(adamantan‐1‐yl)‐1‐(5‐fluoropentyl)‐1H‐indole‐3‐carboxamide (STS‐135)ArticleFeb 2020Sabrina JonesAzure YarbroughWilliam E Fantegrossi[...]Ryoichi FujiwaraSynthetic cannabinoids (SCBs), designer drugs marketed as legal alternatives to marijuana, act as ligands to cannabinoid receptors; however, they have increased binding affinity and potency, resulting in toxicity symptoms such as cardiovascular incidents, seizures, and potentially death. N‐(adamantan‐1‐yl)‐1‐(5‐fluoropentyl)‐1H‐indole‐3‐carboxamide (STS‐135) is a third generation SCB. When incubated with hepatocytes, it undergoes oxidation, hydrolysis, and glucuronidation, resulting in 29 metabolites, with monohydroxy STS‐135 (M25) and dihydroxy STS‐135 (M21) being the predominant metabolites. The enzymes responsible for this oxidative metabolism were unknown. Thus, the aim of this study was to identify the cytochrome P450 (P450s or CYPs) enzymes involved in the oxidative metabolism of STS‐135. In this study, STS‐135 was incubated with liver, intestinal, and brain microsomes and recombinant P450s to determine the enzymes involved in its metabolism. Metabolite quantification was carried out using ultra‐performance liquid chromatography. STS‐135 was extensively metabolized in HLMs and HIMs. Screening assays indicated CYP3A4 and CYP3A5 could be responsible for STS‐135’s oxidation. Through incubations with genotyped HLMs, CYP3A4 was identified as the primary oxidative enzyme. Interestingly, CYP2J2, a P450 isoform expressed in cardiovascular tissues, showed high activity towards the formation of M25 with a Km value of 11.4 μmol/L. Thus, it was concluded that STS‐135 was primarily metabolized by CYP3A4 but may have extrahepatic metabolic pathways as well. Upon exposure to STS‐135, individuals with low CYP3A4 activity could retain elevated blood concentration, resulting in toxicity. Additionally, CYP2J2 may aid in protecting against STS‐135‐induced cardiovascular toxicity.View2 RecommendationsExpand abstractSourcePreclinical characterization of AMPA receptor potentiator TAK‐137 as a therapeutic drug for schizophreniaArticleJun 2019Maiko TanakaAkiyoshi KunugiAtsushi Suzuki[...]Haruhide KimuraThe downregulation of the glutamate system may be involved in positive, negative, and cognitive symptoms of schizophrenia. Through enhanced glutamate signaling, the activation of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) receptor, an ionotropic glutamate receptor, could be a new therapeutic strategy for schizophrenia. TAK‐137 is a novel AMPA receptor potentiator with minimal agonistic activity; in this study, we used rodents and nonhuman primates to assess its potential as a drug for schizophrenia. At 10 mg kg−1 p.o., TAK‐137 partially inhibited methamphetamine‐induced hyperlocomotion in rats, and at 3, 10, and 30 mg kg−1 p.o., TAK‐137 partially inhibited MK‐801‐induced hyperlocomotion in mice, suggesting weak effects on the positive symptoms of schizophrenia. At 0.1 and 0.3 mg kg−1 p.o., TAK‐137 significantly ameliorated MK‐801‐induced deficits in the social interaction of rats, demonstrating potential improvement of impaired social functioning, which is a negative symptom of schizophrenia. The effects of TAK‐137 were evaluated on multiple cognitive domains—attention, working memory, and cognitive flexibility. TAK‐137 enhanced attention in the five‐choice serial reaction time task in rats at 0.2 mg kg−1 p.o., and improved working memory both in rats and monkeys: 0.2 and 0.6 mg kg−1 p.o. ameliorated MK‐801‐induced deficits in the radial arm maze test in rats, and 0.1 mg kg−1 p.o. improved the performance of ketamine‐treated monkeys in the delayed matching‐to‐sample task. At 0.1 and 1 mg kg−1 p.o., TAK‐137 improved the cognitive flexibility of subchronic phencyclidine‐treated rats in the reversal learning test. Thus, TAK‐137‐type AMPA receptor potentiators with low intrinsic activity may offer new therapies for schizophrenia.ViewExpand abstractSourceUpregulated UCA1 contributes to oxaliplatin resistance of hepatocellular carcinoma through inhibition of miR-138-5p and activation of AKT/mTOR signaling pathwayArticleFeb 2021Guolin HuangLi LiChaoyong Liang[...]Yang JieHepatocellular carcinoma (HCC) inevitably developed oxaliplatin (OXA) resistance after long-term treatment, but the mechanism remains unclear. Here, we found that LncRNA UCA1 was upregulated in most of OXA-resistant HCC tissues and cells (HepG2/OXA and SMMC-7721/OXA). Follow-up analysis and online Kaplan-Meier Plotter revealed that HCC patients with high UCA1 level had a shorter survival compared with those with low expression. Overexpression of UCA1 increased OXA IC50 in HepG2 and SMMC-7721 cells, whereas knockdown of UCA1 decreased OXA IC50 in resistant counterparts. Moreover, dual luciferase reporter assay showed that co-transfection of UCA1-WT plasmid with miR-138-5p mimics enhanced fluorescence signals, whereas co-transfection of UCA1-Mut plasmid and miR-138-5p mimics did not induce any changes. Consistently, UCA1 levels in HepG2/OXA and SMMC-7721/OXA cells were downregulated after transfected with miR-138-5p mimics. UCA1 silencing or transfection of miR-138-5p mmics inhibited the activation of AKT and mTOR in HepG2/OXA and SMMC-7721/OXA cells, whereas UCA1 overexpression increased the phosphorylated AKT and mTOR levels in parental counterparts. Rapamycin or miR-138-5p mimics similarly suppressed the activation of AKT and mTOR, whereas UCA1 overexpression exert opposite roles. Interestingly, administration of rapamycin or miR-138-5p mimics apparently antagonized the effects of UCA1 on AKT and mTOR activation. Besides, depletion of UCA1 triggered more dramatic regression of HepG2 xenografts than that of HepG2/OXA xenografts with OXA treatment and impaired the p-AKT and p-mTOR levels in vivo. In conclusion, our findings provide the evidence that UCA1 may contribute to OXA resistance via miR-138-5p-mediated AK /mTOR activation, suggesting that UCA1 is a potential therapeutic target for HCC.View1 RecommendationExpand abstractSourceMass balance and pharmacokinetics of an oral dose of 14 C-napabucasin in healthy adult male subjectsArticleFeb 2021Xiaoshu DaiMichael D. KarolMatthew Hitron[...]Brandon T GuffordThis phase 1, open-label study assessed14 C-napabucasin absorption, metabolism, and excretion, napabucasin pharmacokinetics, and napabucasin metabolites (primary objectives); safety/tolerability were also evaluated. Eight healthy males (18-45 years) received a single oral 240-mg napabucasin dose containing ~100 μCi14 C-napabucasin. Napabucasin was absorbed and metabolized to dihydro-napabucasin (M1; an active metabolite [12.57-fold less activity than napabucasin]), the sole major circulating metabolite (median time to peak concentration: 2.75 and 2.25 h, respectively). M1 plasma concentration versus time profiles generally mirrored napabucasin; similar arithmetic mean half-lives (7.14 and 7.92 h, respectively) suggest M1 formation was rate limiting. Napabucasin systemic exposure (per Cmax and AUC) was higher than M1. The total radioactivity (TRA) whole blood:plasma ratio (AUClast : 0.376; Cmax : 0.525) indicated circulating drug-related compounds were essentially confined to plasma. Mean TRA recovery was 81.1% (feces, 57.2%; urine, 23.8%; expired air, negligible). Unlabeled napabucasin and M1 recovered in urine accounted for 13.9% and 11.0% of the dose (sum similar to urine TRA recovered); apparent renal clearance was 8.24 and 7.98 L/h. No uniquely human or disproportionate metabolite was quantified. Secondary glucuronide and sulfate conjugates were common urinary metabolites, suggesting napabucasin was mainly cleared by reductive metabolism. All subjects experienced mild treatment-emergent adverse events (TEAEs), the majority related to napabucasin. The most commonly reported TEAEs were gastrointestinal disorders. There were no clinically significant laboratory, vital sign, electrocardiogram, or physical examination changes. Napabucasin was absorbed, metabolized to M1 as the sole major circulating metabolite, and primarily excreted via feces. A single oral 240-mg dose was generally well tolerated.ViewExpand abstractSourceInter‐individual and inter‐regional variations in enteric drug metabolizing enzyme activities: Results with cryopreserved human intestinal mucosal epithelia (CHIM) from the small intestines of 14 donorsArticleOct 2020Albert P LiMing-Chih HoNovera Alam[...]Cornelis E. C. A. HopWe have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12‐inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non‐P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway‐selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12‐inch segments of the proximal small intestine shows that the segments immediately after the first 12‐inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.ViewExpand abstractSource14,15-epoxyeicosatrienoic acid produced by cytochrome P450s enhances neurite outgrowth of PC12 and rat hippocampal neuronal cellsArticleOct 2018Ami OguroTakumi InoueSuguru N KudohSusumu ImaokaPolyunsaturated fatty acids, such as arachidonic acid, are accumulated in brain and induce neuronal differentiation. Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) by cytochrome P450s. In this study, we found that 14,15‐EET and 20‐HETE‐enhanced NGF‐induced rat pheochromocytoma PC12 cell neurite outgrowth even at the concentration of 100 nmol L−1. LC‐MS analysis revealed that 14,15‐EET was effectively produced from arachidonic acid by rat CYP2C11, 2C13, and 2C23, and these P450s were expressed in PC12 cells. An inhibitor of these P450s, ketoconazole, inhibited neurite outgrowth, whereas inhibition of soluble epoxide hydrolase, which hydrolyzes EETs to their corresponding diols enhanced neurite outgrowth. To determine the mechanism of neurite formation enhancement by arachidonic acid metabolites, we focused on transient receptor potential (TRP) channels expressed in PC12 cells. The TRPV4 inhibitor HC067047, but not the TRPV1 inhibitor capsazepine, inhibited the effects of 14,15‐EET on neurite outgrowth of PC12. Furthermore, 14,15‐EET increased the cytosolic calcium ion concentration and this increase was inhibited by HC067047. 14,15‐EET also enhanced neurite outgrowth of primary cultured neuron from rat hippocampus. This study suggests that arachidonic acid metabolites produced by P450 contribute to neurite outgrowth through calcium influx.ViewExpand abstractSourceAbsorption, distribution, metabolism, and excretion of [14C]‐dasotraline in humansArticleFeb 2017Yuluan ChenEstela SkendeJing Lin[...]Seth C. HopkinsDasotraline is a dopamine and norepinephrine reuptake inhibitor, and the early clinical trials show a slow absorption and long elimination half-life. To investigate the absorption, distribution, metabolism, and excretion of dasotraline in humans, a single dose of [14C]-dasotraline was administered to eight healthy male adult volunteers. At 35 days, 90.7% of the dosed radioactivity was recovered in the urine (68.3%) and feces (22.4%). The major metabolic pathways involved were: (1) amine oxidation to form oxime M41 and sequential sulfation to form M42 or glucuronidation to form M43; (2) N-hydroxylation and sequential glucuronidation to form M35; (3) oxidative deamination to form (S)-tetralone; (4) mono-oxidation of (S)-tetralone and sequential glucuronidation to form M31A and M32; and (5) N-acetylation to form (1R,4S)-acetamide M102. A total of 8 metabolites were detected and structurally elucidated with 4 in plasma (M41, M42, M43, and M35), 7 in urine (M41, M42, M43, M31A, M32, M35, and (S)-tetralone), and 3 in feces (M41, (S)-tetralone, and (1R,4S)-acetamide). The 2 most abundant circulating metabolites were sulfate (M42) and glucuronide (M43) conjugates of the oxime of dasotraline, accounting for 60.1% and 15.0% of the total plasma radioactivity, respectively; unchanged dasotraline accounted for 8.59%. The oxime M41 accounted for only 0.62% of the total plasma radioactivity and was detected only at early time points. M35 was a minor glucuronide metabolite, undetectable by radioactivity but identified by mass spectrometry. The results demonstrate that dasotraline was slowly absorbed, and extensively metabolized by oxidation and subsequent phase II conjugations. The findings from this study also demonstrated that metabolism of dasotraline by humans did not produce metabolites that may cause a safety concern.ViewExpand abstractSourceNeutrophil fluorescence in clozapine users is attributable to a 14kDa secretable proteinArticleOct 2020Sera A. J. de WithWai Hong ManCoen Maas[...]Tamar TakClozapine is the only antipsychotic agent with demonstrated efficacy in refractory schizophrenia. However, use of clozapine is hampered by its adverse effects, including potentially fatal agranulocytosis. Recently, we showed an association between neutrophil autofluorescence and clozapine use. In this study, we evaluated the subcellular localization of clozapine‐associated fluorescence and tried to elucidate its source. Neutrophils of clozapine users were analyzed with fluorescence microscopy to determine the emission spectrum and localization of the fluorescence signal. Next, these neutrophils were stimulated with different degranulation agents to determine the localization of fluorescence. Lastly, isolated neutrophil lysates of clozapine users were separated by SDS‐PAGE and evaluated. Clozapine‐associated fluorescence ranged from 420 nm to 720 nm, peaking at 500‐550 nm. Fluorescence was localized in a large number of small loci, suggesting granular localization of the signal. Neutrophil degranulation induced by Cytochalasin B/fMLF reduced fluorescence, whereas platelet‐activating factor (PAF)/fMLF induced degranulation did not, indicating that the fluorescence originates from a secretable substance in azurophilic granules. SDS‐PAGE of isolated neutrophil lysates revealed a fluorescent 14kDa band, suggesting that neutrophil fluorescence is likely to be originated from a 14kDa protein/peptide fragment. We conclude that clozapine‐associated fluorescence in neutrophils is originating from a 14kDa soluble protein (fragment) present in azurophilic granules of neutrophils. This protein could be an autofluorescent protein already present in the cell and upregulated by clozapine, or a protein altered by clozapine to express fluorescence. Future studies should further explore the identity of this protein and its potential role in the pathophysiology of clozapine‐induced agranulocytosis.ViewExpand abstractSourceIn vitro metabolism of exemestane by hepatic cytochrome P450s: impact of nonsynonymous polymorphisms on formation of the active metabolite 17 β -dihydroexemestaneArticleJun 2017Amity PetersonZuping XiaGang ChenPhilip LazarusExemestane (EXE) is an endocrine therapy commonly used by postmenopausal women with hormone-responsive breast cancer due to its potency in inhibiting aromatase-catalyzed estrogen synthesis. Preliminary in vitro studies sought to identify phase I EXE metabolites and hepatic cytochrome P450s (CYP450s) that participate in EXE biotransformation. Phase I metabolites were identified by incubating EXE with HEK293-overexpressed CYP450s. CYP450s 1A2, 2C8, 2C9, 2C19, 2D6, 3A4, and 3A5 produce 17β-dihydroexemestane (17β-DHE), an active major metabolite, as well as two inactive metabolites. 17β-DHE formation in pooled human liver microsomes subjected to isoform-specific CYP450 inhibition was also monitored using tandem mass spectrometry. 17β-DHE production in human liver microsomes was unaffected by isoform-specific inhibition of CYP450s 2A6, 2B6, and 2E1 but decreased 12–39% following inhibition of drug-metabolizing enzymes from CYP450 subfamilies 1A, 2C, 2D, and 3A. These results suggest that redundancy exists in the EXE metabolic pathway with multiple hepatic CYP450s catalyzing 17β-DHE formation in vitro. To further expand the knowledge of phase I EXE metabolism, the impact of CYP450 genetic variation on 17β-DHE formation was assessed via enzyme kinetic parameters. Affinity for EXE substrate and enzyme catalytic velocity were calculated for hepatic wild-type CYP450s and their common nonsynonymous variants by monitoring the reduction of EXE to 17β-DHE. Several functional polymorphisms in xenobiotic-metabolizing CYP450s 1A2, 2C8, 2C9, and 2D6 resulted in deviant enzymatic activity relative to wild-type enzyme. Thus, it is possible that functional polymorphisms in EXE-metabolizing CYP450s contribute to inter-individual variability in patient outcomes by mediating overall exposure to the drug and its active metabolite, 17β-DHE.ViewExpand abstract12345Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed.Discover the world s research20+ million members135+ million publications700k+ research projectsJoin for freeCompanyAbout usNewsCareersSupportHelp CenterBusiness solutionsAdvertisingRecruiting© 2008-2021 ResearchGate GmbH. 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