Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells.

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.

Effect of pterostilbene on in vitro drug metabolizing enzyme activity.

Pterostilbene is a natural polyphenol compound found in small berries that is related to resveratrol, but has better bioavailability and a longer half-life. The purpose of this study was to assess the potential inhibitory effect of pterostilbene on in vitro drug metabolism. The effect of pterostilbene on cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzyme activities were studied using the enzyme-selective substrates amodiaquine (CYP2C8), midazolam (CYP3A4), estradiol (UGT1A1), serotonin (UGT1A6) and mycophenolic acid (UGT1A8/9/10). The IC50 value was used to express the strength of inhibition. Further, a volume per dose index (VDI) was used to estimate the potential for in vivo interactions. Pterostilbene significantly inhibited CYP2C8 and UGT1A6 activities. The IC50 (mean ±â€¯SE) values for CYP2C8 and UGT1A6 inhibition were 3.0 ±â€¯0.4 µM and 15.1 ±â€¯2.8 µM, respectively; the VDI exceeded the predefined threshold of 5 L/dose for both CYP2C8 and UGT1A6, suggesting a potential for interaction in vivo. Pterostilbene did not inhibit the metabolism of the other enzyme-selective substrates. The results of this study indicate that pterostilbene inhibits CYP2C8 and UTG1A6 activity in vitro and may inhibit metabolism by these enzymes in vivo. Clinical studies are warranted to evaluate the in vivo relevance of these interactions.

Salivary clearance of caffeine in children.

Background: Measurement of salivary caffeine (1,3,7-trimethylpurine-2,6-dione or 1,3,7-trimethylxanthine) clearance can, in principle, be used to assess hepatic function, diagnose chronic hepatic disease and conduct investigations of substrates of hepatic cytochrome P450 (CYP) isozymes in children, without recourse to venepuncture. However, little is known about childhood sexual dimorphism of hepatic CYP isoforms. Furthermore, the association, if any, between salivary caffeine clearance and age in children has not hitherto been established. The aims of this study were to assess whether salivary caffeine clearance differs between boys and girls and whether it varies with age during childhood. Methods: Following at least 24 h' abstinence from dietary caffeine, nine boys (mean (standard error) age 9.6 (1.1) y) and eight girls (mean age 11.0 (1.2) y), none of whom was a smoker or suffered from chronic hepatic disease, ingested an oral caffeine dose titrated by body mass, namely 3 mg kg-1. Salivary samples collected two and 14 h later underwent spectrophotometric caffeine analysis. Results: The boys and the girls were age matched. The mean caffeine clearance in the boys was 2.47 (0.33) mL min-1 kg-1, while that in the girls was 2.20 (0.31) mL min-1 kg-1 (p = 0.56). The salivary caffeine clearance was negatively correlated with age (r = -0.59, p = 0.01). Conclusion: Stratification by sex appears to be unnecessary when considering childhood salivary caffeine clearance or when conducting investigations in children of CYP1A2 and xanthine oxidase substrates. Furthermore, childhood salivary caffeine clearance is negatively correlated with age.

Modification of ginsenoside saponin composition via the CRISPR/Cas9-mediated knockout of protopanaxadiol 6-hydroxylase gene in Panax ginseng.

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

Role of cytochrome P450 for vitamin D metabolisms in patients with neurodegenerative disorders.

Introduction: We previously reported lower serum 25-hydroxyvitamin D concentrations in patients with Alzheimer's disease (AD), Parkinson's disease (PD) and Multiple system atrophy (MSA) compared to healthy controls (HC), whereas 1,25-di-hydroxyvitamin D levels were solely lower in MSA patients. We investigate serum concentrations of P450 involved in Vitamin D(VD) hydroxylation to clarify the responsible hydroxylase for the low serum concentrations of VD metabolites. Methods: A total of 79 individuals were enrolled including 20 HC, 20 AD, 19 PD and 20 MSA patients. The serum concentrations of P450 involved in VD hydroxylation were assayed by ELISA. The data were analyzed by the nonparametric Kruskal-Wallis test between groups. Results: Though CYP2R1 and CYP27A1 mediate 25-hydroxylation for VD, CYP2R1 is the main hydroxylase, and CYP27A1 is also involved in VD synthesis. CYP2R1 concentrations showed no differences among groups, while lower CYP27A1 concentrations were found in PD (p < 0.05) and MSA (p < 0.005) compared to HC and differences between AD and MSA (p < 0.05), however no differences between PD and MSA. CYP27B1 is the main 1α-hydroxylase for 25-hydroxyvitamin D and showed differences between HC and PD (p < 0.05), between HC and MSA (p < 0.005) and between PD and MSA (p = 0.055). CYP24A1, which inactivate 1,25-di-hydroxyvitamin D, showed no differences among groups. Conclusions: CYP27A1 might affect VD synthesis and cause low 25-hydroxyvitamin D levels in AD, PD and MSA patients. Low 1,25-di-hydroxyvitamin D levels in MSA patients might be caused by impaired feedback mediated by CYP27B1.

Generation of new inhibitors of selected cytochrome P450 subtypes- In silico study.

Physicochemical and pharmacokinetic compound profile has crucial impact on compound potency to become a future drug. Ligands with desired activity profile cannot be used for treatment if they are characterized by unfavourable physicochemical or ADMET properties. In the study, we consider metabolic stability and focus on selected subtypes of cytochrome P450 - proteins, which take part in the first phase of compound transformations in the organism. We develop a protocol for generation of new potential inhibitors of selected cytochrome isoforms. Its subsequent stages are composed of generation and assessment of new derivatives of known cytochrome inhibitors, docking and evaluation of the compound possible inhibition on the basis of the obtained ligand-protein complexes. Besides the library of new potential agents inhibiting particular cytochrome subtypes, we also prepare a graph neural network that predicts the change in activity for all modifications of the starting molecule. In addition, we perform a systematic statistical study on the influence of particular substitutions on the potential inhibition properties of generated compounds (both mono- and di-substitutions are considered), provide explanations of the inhibitory predictions and prepare an on-line visualization platform enabling manual inspection of the results. The developed methodology can greatly support the design of new cytochrome P450 inhibitors with the overarching goal of generation of new metabolically stable compounds. It enables instant evaluation of possible compound-cytochrome interactions and selection of ligands with the highest potential of possessing desired biological activity.

The pathophysiology of rapid fluctuations in mental status associated with olanzapine: A case report.

Background: Olanzapine toxicity is reported to be a rare but specific phenomenon characterized by rapid fluctuations between somnolence and agitation, which has been referred to as "agitation despite sedation." A similar phenomenon is observed as an adverse reaction of the long-acting injectable olanzapine formulation, which has been referred to as "delirium/sedation syndrome." Case presentation: This case report describes a 48-year-old man diagnosed with schizophrenia who experienced rapid fluctuations between somnolence and agitation during a cross-titration of olanzapine to clozapine. The patient had normal serum levels of both medications and the symptoms resolved with the discontinuation of olanzapine. Conclusion: Rapid fluctuations in mental status between somnolence and agitation are not clearly described among other antipsychotics, and it is possible that this phenomenon may be specific to olanzapine. The findings of this case report suggested that this phenomenon was likely the result of the oversaturation of (H1) and (M1) receptors.

Unsymmetrical aromatic disulfides as SARS-CoV-2 Mpro inhibitors: Molecular docking, molecular dynamics, and ADME scoring investigations.

COVID-19 pandemic caused by very severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) agent is an ongoing major global health concern. The disease has caused more than 452 million affected cases and more than 6 million death worldwide. Hence, there is an urgency to search for possible medications and drug treatments. There are no approved drugs available to treat COVID-19 yet, although several vaccine candidates are already available and some of them are listed for emergency use by the world health organization (WHO). Identifying a potential drug candidate may make a significant contribution to control the expansion of COVID-19. The in vitro biological activity of asymmetric disulfides against coronavirus through the inhibition of SARS-CoV-2 main protease (Mpro) protein was reported. Due to the lack of convincing evidence those asymmetric disulfides have favorable pharmacological properties for the clinical treatment of Coronavirus, in silico evaluation should be performed to assess the potential of these compounds to inhibit the SARS-CoV-2 Mpro. In this context, we report herein the molecular docking for a series of 40 unsymmetrical aromatic disulfides as SARS-CoV-2 Mpro inhibitor. The optimal binding features of disulfides within the binding pocket of SARS-CoV-2 endoribonuclease protein (Protein Data Bank [PDB]: 6LU7) was described. Studied compounds were ranked for potential effectiveness, and those have shown high molecular docking scores were proposed as novel drug candidates against SARS-CoV-2. Moreover, the outcomes of drug similarity and ADME (Absorption, Distribution, Metabolism, and Excretion) analyses have may have the effectiveness of acting as medicines, and would be of interest as promising starting point for designing compounds against SARS-CoV-2. Finally, the stability of these three compounds in the complex with Mpro was validated through molecular dynamics (MD) simulation, in which they displayed stable trajectory and molecular properties with a consistent interaction profile.

A practical strategy to develop isoform-selective near-infrared fluorescent probes for human cytochrome P450 enzymes.

Currently, the development of selective fluorescent probes toward targeted enzymes is still a great challenge, due to the existence of numerous isoenzymes that share similar catalytic capacity. Herein, a double-filtering strategy was established to effectively develop isoenzyme-specific fluorescent probe(s) for cytochrome P450 (CYP) which are key enzymes involving in metabolism of endogenous substances and drugs. In the first-stage of our filtering approach, near-infrared (NIR) fluorophores with alkoxyl group were prepared for the screening of CYP-activated fluorescent substrates using a CYPs-dependent incubation system. In the second stage of our filtering approach, these candidates were further screened using reverse protein-ligand docking to effectively determine CYP isoenzyme-specific probe(s). Using our double-filtering approach, probes S9 and S10 were successfully developed for the real-time and selective detection of CYP2C9 and CYP2J2, respectively, to facilitate high-throughput screening and assessment of CYP2C9-mediated clinical drug interaction risks and CYP2J2-associated disease diagnosis. These observations suggest that our strategy could be used to develop the isoform-specific probes for CYPs.

Effect of silymarin on blood coagulation profile and osmotic fragility in carbon tetrachloride induced hepatotoxicity in male Wistar rats.

Reports about the impact of Carbon tetrachloride (CCl4) hepatotoxicity on coagulation profile have been inconsistent. Multiple investigators have however demonstrated the effectiveness of silymarin in the resolution of anomalies induced by CCl4, although the effect of silymarin on the impact of CCl4 hepatotoxicity, especially coagulation profile and osmotic fragility have not been investigated. The liver, the primary site for the secretion of coagulation proteins, can become impaired in CCl4 hepatotoxicity, and silymarin reportedly increases hepatic protein synthesis as part of its hepatoprotective mechanism. This study assessed the effect of silymarin on blood coagulation profile and erythrocyte osmotic fragility in CCl4 induced hepatotoxicity in rats. Twenty male Wistar rats were allocated into four groups (n = 5) at random, namely: Control, CCl4 given CCl4 (1 ml/kg) administered intraperitoneally twice a week, Silymarin (S) given silymarin (100 mg/kg/day) orally, and S+CCl4 given silymarin (100 mg/kg/day) orally and (1 ml/kg) CCl4 one hour after, intraperitoneally twice a week for a duration of four weeks. Results showed protraction of activated partial thromboplastin time and thrombin time, increased erythrocyte osmotic fragility, liver damage, dyslipidemia, oxidative stress and lipid peroxidation in rats given CCl4. Silymarin attenuated most of these effects as observed from comparison between CCl4 and S+CCl4 rats. The findings of this study suggests that pretreatment with silymarin attenuated disruption in coagulation profile and erythrocyte osmotic fragility in CCl4 induced hepatotoxicity in Wistar rats.

Inhibitory effects of Thai herbal extracts on the cytochrome P450 3A-mediated the metabolism of gefitinib, lapatinib and sorafenib.

Herbal products are widely used in cancer patients via co-administration with chemotherapy. Previous studies have demonstrated that pharmacokinetic interactions between herbs and anticancer drugs exist due to inhibition of drug-metabolizing enzymes, particularly cytochrome P450s (CYPs). The aim of this study was to determine the inhibitory effects of Andrographis paniculata, Curcuma zedoaria, Ganoderma lucidum, Murdannia loriformis and Ventilago denticulata extracts on the metabolism of gefitinib, lapatinib and sorafenib. The activities of CYP3A in human liver microsome on the metabolism of gefitinib, lapatinib and sorafenib in the absence and presence of Thai herbal extracts were assayed using high-performance liquid chromatography analysis. Curcuma zedoaria extract potently inhibited CYP3A-mediated lapatinib and sorafenib metabolism with IC50 values of 4.18 ± 3.20 and 7.59 ± 1.23 µg/mL, respectively, while the metabolism of gefitinib was strongly inhibited by Murdannia loriformis and Ventilago denticulata extracts with IC50 values of 7.53 ± 2.87 and 7.06 ± 1.23 µg/mL, respectively. Andrographis paniculata and Ganoderma lucidum extracts had less effect on the metabolism of the tested anticancers (IC50 values >10 µg/mL). In addition, kinetic analysis of the ability of Curcuma zedoaria extract to inhibit CYP3A-mediated metabolism of anticancer drugs was best described by the noncompetitive and competitive inhibition models with Ki values of 20.08 and 11.55 µg/mL for the metabolism of gefitinib and sorafenib, respectively. The present study demonstrated that there were potential pharmacokinetic interactions between tyrosine kinase inhibitors and herbal extracts.

A Phase 1 Dose-Escalation Study of the Cardiac Myosin Inhibitor Aficamten in Healthy Participants.

This phase 1, randomized, double-blind, placebo-controlled study of aficamten (formerly CK-3773274) in healthy adults identified a pharmacologically active range of doses and exposures. At doses that were pharmacologically active (single doses of ≤50 mg or daily dosing of ≤10 mg for 14 or 17 days), aficamten appeared to be safe and well tolerated. Adverse events were generally mild and no more frequent than with placebo. Pharmacokinetic assessments showed dose proportionality over the range of single doses administered, and pharmacokinetics were not affected by administration with food or in otherwise healthy individuals with a cytochrome P450 2D6 poor metabolizer phenotype. (A Single and Multiple Ascending Dose Study of CK-3773274 in Health Adult Subjects; NCT03767855).

Understanding the physiological functions of the host xenobiotic-sensing nuclear receptors PXR and CAR on the gut microbiome using genetically modified mice.

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

Pathogenesis of Alcohol-Associated Liver Disease.

Excessive alcohol consumption is a global healthcare problem with enormous social, economic, and clinical consequences. While chronic, heavy alcohol consumption causes structural damage and/or disrupts normal organ function in virtually every tissue of the body, the liver sustains the greatest damage. This is primarily because the liver is the first to see alcohol absorbed from the gastrointestinal tract via the portal circulation and second, because the liver is the principal site of ethanol metabolism. Alcohol-induced damage remains one of the most prevalent disorders of the liver and a leading cause of death or transplantation from liver disease. Despite extensive research on the pathophysiology of this disease, there are still no targeted therapies available. Given the multifactorial mechanisms for alcohol-associated liver disease pathogenesis, it is conceivable that a multitherapeutic regimen is needed to treat different stages in the spectrum of this disease.

Health effects and known pathology associated with the use of E-cigarettes.

In recent years, new nicotine delivery methods have emerged, and many users are choosing electronic cigarettes (e-cigarettes) over traditional tobacco cigarettes. E-cigarette use is very popular among adolescents, with more than 3.5 million currently using these products in the US. Despite the increased prevalence of e-cigarette use, there is limited knowledge regarding the health impact of e-cigarettes on the general population. Based on published findings by others, E-cigarette is associated with lung injury outbreak, which increased health and safety concerns related to consuming this product. Different components of e-cigarettes, including food-safe liquid solvents and flavorings, can cause health issues related to pneumonia, pulmonary injury, and bronchiolitis. In addition, e-cigarettes contain alarmingly high levels of carcinogens and toxicants that may have long-lasting effects on other organ systems, including the development of neurological manifestations, lung cancer, cardiovascular disorders, and tooth decay. Despite the well- documented potential for harm, e-cigarettes do not appear to increase susceptibility to SARS-CoV- 2 infection. Furthermore, some studies have found that e-cigarette users experience improvements in lung health and minimal adverse effects. Therefore, more studies are needed to provide a definitive conclusion on the long-term safety of e-cigarettes. The purpose of this review is to inform the readers about the possible health-risks associated with the use of e-cigarettes, especially among the group of young and young-adults, from a molecular biology point of view.

Time effect of rutaecarpine on caffeine pharmacokinetics in rats.

Rutaecarpine is reported as a potent inducer of CYP1A2 enzyme in rats. There are natural herbal supplements containing rutaecarpine that are designed to enhance the CYP1A2-dependent removal of caffeine from blood so that people can have coffee later in the day without causing sleep interference. This study aimed to determine the minimum amount of time needed from oral rutaecarpine administration until the observed effect of rutaecarpine on caffeine pharmacokinetics (PK) in 15 male Sprague-Dawley rats. PK parameters for caffeine and its metabolites in the control and rutaecarpine groups were calculated using WinNonlin®. Results showed that orally administered rutaecarpine at 100 mg/kg dose as early as 3 h before oral caffeine administration significantly decreased the oral systemic exposure and mean residence time of caffeine and its metabolites due to decreased caffeine bioavailability (by up to 75%) and increased clearance. The systemic exposure of caffeine and its metabolites were also decreased when caffeine was given intravenously, though this effect was less pronounced than when caffeine was given orally. Although plasma level of rutaecarpine was undetectable (less than 10 ng/mL), rutaecarpine still induced hepatic CYP1A2 activity. Results from 7-methoxyresorufin O-demethylation activity, which is specific to CYP1A2, showed that 3 h after one rutaecarpine oral dose, CYP1A2 activity in rat liver tissue was increased by 3- fold. This finding suggested that rutaecarpine effectively induced CYP1A2 activity in the liver.

Predicting the potential toxicity of 26 components in Cassiae semen using in silico and in vitro approaches.

Cassiae semen are dried and ripe seeds of Cassia obtusifolia L. or Cassia tora L. (Fabaceae) and have been made into roasted tea or used as a traditional medicine in Asian countries. However, it was reported to result in liver and renal toxicity. The components of Cassiae semen that induce hepatotoxicity or nephrotoxicity remain unknown. In the present study, we evaluate the potential toxicity of 26 newly isolated compounds from Cassiae semen using quantitative structure-activity relationship (QSAR) methods and co-culture of hepatic and renal cell approaches, and we aim to illustrate the relationship between the structural characteristics and cytotoxicity by general linear models (GLMs). Both the QSAR models and co-culture of hepatic and renal cell systems predicted that 6 compounds were potentially hepatotoxic, 10 compounds were potentially nephrotoxic, and specific anthraquinones and anthraquinone-glucosides were potential toxicants in Cassiae semen. Specific groups such as -OH and -OCH3 at the R1, R2, R3, and R7 positions influenced the cytotoxicity.

Can remdesivir and its parent nucleoside GS-441524 be potential oral drugs? An in vitro and in vivo DMPK assessment.

Remdesivir (RDV) is the only US Food and Drug Administration (FDA)-approved drug for treating COVID-19. However, RDV can only be given by intravenous route, and there is a pressing medical need for oral antivirals. Significant evidence suggests that the role of the parent nucleoside GS-441524 in the clinical outcomes of RDV could be largely underestimated. We performed an in vitro and in vivo drug metabolism and pharmacokinetics (DMPK) assessment to examine the potential of RDV, and particularly GS-441524, as oral drugs. In our in vitro assessments, RDV exhibited prohibitively low stability in human liver microsomes (HLMs, t 1/2 = âˆ¼1 min), with the primary CYP-mediated metabolism being the mono-oxidation likely on the phosphoramidate moiety. This observation is poorly aligned with any potential oral use of RDV, though in the presence of cobicistat, the microsomal stability was drastically boosted to the level observed without enzyme cofactor NADPH. Conversely, GS-441524 showed excellent metabolic stability in human plasma and HLMs. In further in vivo studies in CD-1 mice, GS-441524 displayed a favorable oral bioavailability of 57%. Importantly, GS-441524 produced adequate drug exposure in the mice plasma and lung, and was effectively converted to the active triphosphate, suggesting that it could be a promising oral antiviral drug for treating COVID-19.

Unlocking the Mechanisms of Cutaneous Adverse Drug Reactions: Activation of the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway by EGFR Inhibitors Triggers Keratinocyte Differentiation and Polarization of Epidermal Immune Responses.

EGFR inhibitors used in oncology therapy modify the keratinocyte differentiation processes, impairing proper skin barrier formation and leading to cutaneous adverse drug reactions. To uncover the molecular signatures associated with cutaneous adverse drug reactions, we applied phosphoproteomic and transcriptomic assays on reconstructed human epidermis tissues exposed to a therapeutically relevant concentration of afatinib, a second-generation EGFR inhibitor. After drug exposure, we observed activation of the phosphatidylinositol 3-kinase/protein kinase B pathway associated with an increased expression of gene families involved in keratinocyte differentiation, senescence, oxidative stress, and alterations in the epidermal immune-related markers. Furthermore, our results show that afatinib may interfere with vitamin D3 metabolism, acting via CYP27A1 and CYP24A1 to regulate calcium concentration through the phosphatidylinositol 3-kinase/protein kinase B pathway. Consequently, basal layer keratinocytes switch from a pro-proliferating to a prodifferentiative program, characterized by upregulation of biomarkers associated with increased keratinization, cornification, T helper type 2 response, and decreased innate immunity. Such effects may increase skin susceptibility to cutaneous penetration of irritants and pathogens. Taken together, these findings demonstrate a molecular mechanism of EGFR inhibitor-induced cutaneous adverse drug reactions.

TGR5 controls bile acid composition and gallbladder function to protect the liver from bile acid overload.

BACKGROUND & AIMS: As the composition of the bile acid (BA) pool has a major impact on liver pathophysiology, we studied its regulation by the BA receptor Takeda G protein coupled receptor (TGR5), which promotes hepatoprotection against BA overload. METHODS: Wild-type, total and hepatocyte-specific TGR5-knockout, and TGR5-overexpressing mice were used in: partial (66%) and 89% extended hepatectomies (EHs) upon normal, ursodeoxycholic acid (UDCA)- or cholestyramine (CT)-enriched diet, bile duct ligation (BDL), cholic acid (CA)-enriched diet, and TGR5 agonist (RO) treatments. We thereby studied the impact of TGR5 on: BA composition, liver injury, regeneration and survival. We also performed analyses on the gut microbiota (GM) and gallbladder (GB). Liver BA composition was analysed in patients undergoing major hepatectomy. RESULTS: The TGR5-KO hyperhydrophobic BA composition was not directly related to altered BA synthesis, nor to TGR5-KO GM dysbiosis, as supported by hepatocyte-specific KO mice and co-housing experiments, respectively. The TGR5-dependent control of GB dilatation was crucial for BA composition, as determined by experiments including RO treatment and/or cholecystectomy. The poor TGR5-KO post-EH survival rate, related to exacerbated peribiliary necrosis and BA overload, was improved by shifting BAs toward a less toxic composition (CT treatment). After either BDL or a CA-enriched diet with or without cholecystectomy, we found that GB dilatation had strong TGR5-dependent hepatoprotective properties. In patients, a more hydrophobic liver BA composition was correlated with an unfavourable outcome after hepatectomy. CONCLUSIONS: BA composition is crucial for hepatoprotection in mice and humans. We indicate TGR5 as a key regulator of BA profile and thereby as a potential hepatoprotective target under BA overload conditions. LAY SUMMARY: Through multiple in vivo experimental approaches in mice, together with a patient study, this work brings some new light on the relationships between biliary homeostasis, gallbladder function, and liver protection. We showed that hepatic bile acid composition is crucial for optimal liver repair, not only in mice, but also in human patients undergoing major hepatectomy.