Major Clearance Pathway Research Articles

Lenacapavir Exhibits Atropisomerism-Mechanistic Pharmacokinetics and Disposition Studies of Lenacapavir Reveal Intestinal Excretion as a Major Clearance Pathway.

Lenacapavir (LEN), a long-acting injectable, is the first approved human immunodeficiency virus type 1 capsid inhibitor and one of a few Food and Drug Administration-approved drugs that exhibit atropisomerism. LEN exists as a mixture of two class 2 atropisomers that interconvert at a fast rate (half-life < 2 hours) with a ratio that is stable over time and unaffected by enzymes or binding to proteins in plasma. LEN exhibits low systemic clearance (CL) in nonclinical species and humans; however, in all species, the observed CL was higher than the in vitro predicted CL. The volume of distribution was moderate in nonclinical species and consistent with the tissue distribution observed by whole-body autoradiography in rats. LEN does not distribute to brain, consistent with being a P-glycoprotein (P-gp) substrate. Mechanistic drug disposition studies with [14C]LEN in intravenously dosed bile duct-cannulated rats and dogs showed a substantial amount of unchanged LEN (31%-60% of dose) excreted in feces, indicating that intestinal excretion (IE) was a major clearance pathway for LEN in both species. Coadministration of oral elacridar, a P-gp inhibitor, in rats decreased CL and IE of LEN. Renal excretion was < 1% of dose in both species. In plasma, almost all radioactivity was unchanged LEN. Low levels of metabolites in excreta included LEN conjugates with glutathione, pentose, and glucuronic acid, which were consistent with metabolites formed in vitro in Hμrel hepatocyte cocultures and those observed in human. Our studies highlight the importance of IE for efflux substrates that are highly metabolically stable compounds with slow elimination rates. SIGNIFICANCE STATEMENT: LEN is a long-acting injectable that exists as conformationally stable atropisomers. Due to an atropisomeric interconversion rate that significantly exceeds the in vivo elimination rate, the atropisomer ratio of LEN remains constant in circulation. The disposition of LEN highlights that intestinal excretion has a substantial part in the elimination of compounds that are metabolically highly stable and efflux transporter substrates.

Physiologically Based Pharmacokinetic Modeling of the Drug-Drug Interaction Between CYP3A4 Substrate Glasdegib and Moderate CYP3A4 Inducers in Lieu of a Clinical Study.

Glasdegib (DAURISMO) is a hedgehog pathway inhibitor approved for the treatment of acute myeloid leukemia (AML). Cytochrome P450 3A4 (CYP3A4) has been identified as a major metabolism and clearance pathway for glasdegib. The role of CYP3A4 in the clearance of glasdegib has been confirmed with clinical drug-drug interaction (DDI) studies following the coadministration of glasdegib with the strong CYP3A4 inhibitor ketoconazole and the strong inducer rifampin. To evaluate potential drug interactions with CYP3A4 modulators, the coadministration of glasdegib with a moderate CYP3A4 inducer, efavirenz, was evaluated using physiologically based pharmacokinetic (PBPK) modeling using the Simcyp simulator. The glasdegib compound file was developed using measured physicochemical properties, data from human intravenous and oral pharmacokinetics, absorption, distribution, metabolism, and excretion studies, and invitro reaction phenotyping results. The modeling assumptions, model parameters, and assignments of fractional CYP3A4 metabolism were verified using results from clinical pharmacokinetics (PK) and DDI studies with ketoconazole and rifampin. The verified glasdegib and efavirenz compound files, the latter of which was available in the Simcyp simulator, were used to estimate the potential impact of efavirenz on the PK of glasdegib. PBPK modeling predicted a glasdegib area under the concentration-time curve ratio of 0.45 and maximum plasma concentration ratio of 0.75 following coadministration with efavirenz. The PBPK results, in lieu of a formal clinical study, informed the drug label, with the recommendation to double the clinical dose of glasdegib when administered in conjunction with a moderate CYP3A4 inducer, followed by a resumption of the original dose 7days post-discontinuation.

A phase I, single-center, open-label study to investigate the absorption, distribution, metabolism and excretion of encorafenib following a single oral dose of 100 mg [14 C] encorafenib in healthy male subjects.

Encorafenib is a novel kinase inhibitor of BRAF V600E as well as wild-type BRAF and CRAF and has received approval, in combination with binimetinib, to treat BRAF V600E or V600K mutation-positive unresectable or metastatic melanoma or in combination with cetuximab to treat BRAF V600E mutation-positive colorectal cancer. The absorption, distribution, metabolism and excretion (ADME) of encorafenib was studied by administering [14 C] encorafenib (100 mg containing 90 μCi of radiolabeled material) to 4 healthy male subjects (NCT01436656). Following a single oral 100-mg dose of [14 C] encorafenib to healthy male subjects, the overall recovery of radioactivity in the excreta was ≥93.9% in all four subjects, indicating that good mass balance was achieved. An equal mean of 47.2% for the radioactivity dose was eliminated in the feces and urine. The percentage of the dose eliminated in the feces (5.0%) and urine (1.8%) as unchanged encorafenib was minor. Metabolism was found to be the major clearance pathway (~88% of the recovered radioactive dose) for encorafenib in humans and is predominantly mediated through N-dealkylation of the isopropyl carbamic acid methyl ester to form the primary phase 1 direct metabolite M42.5 (LHY746). Oral absorption was estimated from the radioactive dose recovered in the urine (47.2%) and the total radioactive dose recovered in the feces as metabolites (39%). Based on these values and the assumptions that encorafenib and its metabolites are stable in feces, the fraction of oral absorption was estimated to be at least ~86%.

TFEB regulates cellular labile iron and prevents ferroptosis in a TfR1-dependent manner

Autophagy is a major clearance pathway for misfolded α-synuclein which promotes ferroptosis through NCOA4-mediated ferritin degradation. The regulation of these two processes to achieve improved neuroprotection in Parkinson's disease (PD) must be elucidated. Transcription factor EB (TFEB) is a master regulator of both autophagy and lysosome biogenesis, and lysosomes are important cellular iron storage organelles; however, the role of TFEB in ferroptosis and iron metabolism remains unclear. In this study, TFEB overexpression promoted the clearance of misfolded α-synuclein and prevented ferroptosis and iron overload. TFEB overexpression up-regulated transferrin receptor 1 (TfR1) synthesis and increased the localization of TfR1 in the lysosome, facilitating lysosomal iron import and transient lysosomal iron storage. TFEB overexpression increased the levels of cellular iron-safe storage proteins (both ferritin light and heavy chains). These functions in iron metabolism maintain the cellular labile iron at a low level and electrical activity, even under iron overload conditions. Notably, lower levels of cellular labile iron and the upregulation of ferritin light and heavy chains were reversed after TfR1 knockdown in cells overexpressing TFEB, indicating that TFEB regulates cellular labile iron and suppresses ferroptosis in a TfR1 dependent manner. Taken together, this evidence of the regulation of iron metabolism enriches our understanding of the function of TFEB. In addition, TFEB overexpression protects against ferroptosis and iron overload and provides a new direction and perspective for autophagy regulation in PD.

Absorption, Metabolism, and Excretion of Taselisib (GDC-0032), a Potent β-Sparing PI3K Inhibitor in Rats, Dogs, and Humans.

Taselisib (also known as GDC-0032) is a potent and selective phosphoinositide 3-kinase (PI3K) inhibitor that displays greater selectivity for mutant PI3Kα than wild-type PI3Kα To better understand the absorption, distribution, metabolism, and excretion properties of taselisib, mass balance studies were conducted following single oral doses of [14C]taselisib in rats, dogs, and humans. Absolute bioavailability (ABA) of taselisib in humans was determined by oral administration of taselisib at the therapeutic dose followed by intravenous dosing of [14C]taselisib as a microtracer. The ABA in humans was 57.4%. Absorption of taselisib was rapid in rats and dogs and moderately slow in humans. The recovery of radioactivity in excreta was high (>96%) in the three species where feces was the major route of excretion. Taselisib was the major circulating component in the three species with no metabolite accounting for >10% of the total drug-derived material. The fraction absorbed of taselisib was 35.9% in rats and 71.4% in dogs. In rats, absorbed drug underwent moderate to extensive metabolism and biliary excretion of taselisib was minor. In dog, biliary excretion and metabolism were major clearance pathways. In humans, 84.2% of the dose was recovered as the parent drug in excreta indicating that metabolism played a minor role in the drug's clearance. Major metabolism pathways were oxidation and amide hydrolysis in the three species while methylation was another prominent metabolism pathway in dogs. The site of methylation was identified on the triazole moiety. In vitro experiments characterized that the N-methylation was dog-specific and likely mediated by a thiol methyltransferase. SIGNIFICANCE STATEMENT: This study provides a comprehensive description of the absorption, distribution, and metabolism and pharmacokinetic properties of taselisib in preclinical species and humans. This study demonstrated the importance of oral bioavailability results for understanding taselisib's clearance pathways. The study also describes the identification and characterization of a unique dog-specific N-methylation metabolite of taselisib and the enzyme mediating N-methylation in vitro.

Mass balance, metabolic disposition, and pharmacokinetics of a novel selective inhibitor of PI3Kδ [14C] SHC014748M in healthy Chinese subjects following oral administration.

SHC014748M is a potent, novel selective PI3Kδ isoform inhibitor and is proposed for the treatment of non-Hodgkin lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma. This study investigated the pharmacokinetics, mass balance, metabolism and excretion of SHC014748M in Chinese male subjects following a single oral dose of 150mg (100μCi) [14C] SHC014748M. Six healthy Chinese male subjects administrated an oral suspension of 150mg (100μCi) [14C] SHC014748M and the samples of blood, urine and feces were collected for measuring. Liquid chromatography-tandem mass spectrometry and liquid scintillation counter were utilized to obtain mass balance and the pharmacokinetic data. The median Tmax for [14C]-radioactivity was 1.6 ± 0.5h after the oral administration of [14C] SHC014748M and the mean Cmax was 3863 ± 354ngEq./mL in plasma, while the mean Cmax, t1/2 values and AUC0-∞ values for total radioactivity in whole blood were 2466 ± 518ngEq./mL, 32.2 ± 30.5h and 66,236 ± 44,232h * ng Eq./mL, respectively. Fecal excretion was proposed as the predominant elimination route, accounting for a mean of 90.68 ± 11.38% of the administered dose, whereas the mean urine excretion was 6.00 ± 1.48% within 336h post-dose. The proposed major metabolic pathway of [14C] SHC014748M in the human body were as follows: (I) monooxidation, (II) glucuronide acid conjugation, and (III) monoxide-hydrogenation. SHC014748M was absorbed, metabolized and excreted with unchanged SHC014748M as its main circulating component in plasma following oral administration. In addition, it was speculated that fecal excretion was the principal excretion pathway; meanwhile, monohydroxy, glucuronide conjugation, oxygen, and hydrogenation were the major clearance pathways of SHC014748M through urine and/or feces. The trial registration number: CTR20202505.

Absorption, metabolism and excretion of once-weekly somapacitan, a long-acting growth hormone derivative, after single subcutaneous dosing in human subjects

Somapacitan is a reversible albumin-binding growth hormone (GH) derivative in clinical development for once-weekly administration in patients with adult GH deficiency (AGHD) and children with GH deficiency (GHD). To date, the use of somapacitan in AGHD or severe AGHD has been approved in the USA and Japan, respectively. This study (ClinicalTrials.gov, NCT02962440) investigated the absorption, metabolism and excretion, as well as the pharmacokinetics (PK), of tritium-labelled somapacitan ([3H]-somapacitan). Seven healthy males received a single subcutaneous dose of 6 mg somapacitan containing [3H]-somapacitan 20 MBq. Blood, serum, plasma, urine, faeces, and expired air were collected for radioactivity assessment. Metabolites were identified and quantified in plasma and urine collected. The PK of plasma components were determined, and the radioactive peaks of the most abundant plasma metabolites and urine metabolites were selected for analysis. Twenty-eight days after dosing, 94.0% of the administered dose was recovered as [3H]-somapacitan-related material, most of which was excreted in urine (80.9%); 12.9% was excreted in faeces, and an insignificant amount (0.2%) was exhaled in expired air. PK properties of [3H]-somapacitan-related material appeared to be consistent across plasma, serum and blood. Three abundant plasma metabolites (P1, M1 and M1B) and two abundant urine metabolites (M4 and M5) were identified. The total exposure of intact somapacitan accounted for 59% of the total exposure of all somapacitan-related material, P1 accounted for 21% and M1 plus M1B accounted for 12%. M4 and M5 were the most abundant urine metabolites and accounted for 37% and 8% of the dosed [3H]-somapacitan radioactivity, respectively. No intact somapacitan was found in excreta. Two subjects had six adverse events (AEs); all were mild in severity and unlikely to be related to trial product. The majority of dosed [3H]-somapacitan (94%) was recovered as excreted metabolites. Urine was the major route for excretion of somapacitan metabolites, followed by faeces, and exhalation in expired air was negligible. The low molecular weights of identified urine metabolites demonstrate that somapacitan was extensively degraded to small residual fragments that were excreted (fully biodegradable). The extensive metabolic degradation and full elimination of metabolites in excreta were the major clearance pathways of somapacitan and the key elements in its biological fate. A single dose of 6 mg somapacitan (containing [3H]-somapacitan) in healthy male subjects was well tolerated with no unexpected safety issues identified.

Physiologically Based Pharmacokinetic (PBPK) Modeling for Betrixaban and the Impact of P-Glycoprotein Inhibition on Betrixaban Pharmacokinetics

Introduction: Betrixaban is a factor Xa inhibitor indicated for the prophylaxis of venous thromboembolism (VTE) in adult patients hospitalized for an acute medical illness. A physiologically based pharmacokinetic (PBPK) model for betrixaban provided the mechanistic understanding of the contribution of P-glycoprotein (P-gp), as well as CYP3A4, to the disposition of betrixaban, and predictions of the effects of drug-drug interactions mediated by P-gp and/or CYP3A4. The PBPK model specifically evaluated contribution of the interaction with the P-gp transporter to the disposition of betrixaban, as well as the effects of co-administration of P-gp inhibitors (ketoconazole, verapamil, clarithromycin) on betrixaban pharmacokinetics (PK). Further, in vitro and clinical studies confirmed the lack of a significant interaction of betrixaban with CYP3A4.Methods: The base PBPK model was developed using physiochemical data, in vitro metabolism andtransport data generated using recombinant CYP enzymes and human hepatocytes, and relevant clinical data (eg, observed renal clearance). The model was verified by comparing model predictions with PK data derived from single oral doses of betrixaban (from 80 to 550 mg) in healthy volunteers. The combined contribution of intestinal and hepatic P-gp transport to the disposition of betrixaban was verified by comparing model predictions to the observed ketoconazole and verapamil clinical drug-drug interaction data from healthy volunteers. The verified PBPK model was prospectively employed to predict the impact of a strong P-gp inhibitor (clarithromycin) and a moderate P-gp inhibitor (diltiazem) on betrixaban PK, and to predict the impact of food intake on betrixaban PK and compare it against the observed data in phase 3 studies. The Simcyp Population-Based Simulator (v15 release 1) was used for all simulations.Results: The major clearance pathway for betrixaban was biliary clearance (assigned to hepatic P-gp) as scaled from uptake data generated in human hepatocytes, accounting for ~80% of clearance after IV administration (CLIV). CYP3A4 played virtually no role in the disposition of betrixaban. The base PBPK model incorporating an intestinal P-gp component within the ADAM model resulted in simulations that predicted concentrations very close to the clinically observed pharmacokinetic non-linearity arising from multiple oral dosing of betrixaban in the dose range of 80 to 550 mg in healthy volunteers. The model predicted that the inhibition of intestinal and hepatic P-gp transport of betrixaban would result in the geometric mean area under the curve (AUC) and maximal concentration (Cmax) ratios due to concurrent ketoconazole treatment of 2.4 and 2.5, respectively, which were consistent with the observed ratios of 2.1 and 2.3. The predicted betrixaban geometric mean AUC and Cmax ratios due to concurrent verapamil treatment were 2.3 and 3.1, respectively, compared with the observed ratios of 3.0 and 4.7. The model qualitatively predicted the negative impact of food intake on betrixaban exposure (Figure). The predicted AUC and Cmax for patients treated with betrixaban in the fed state in the phase 3 study were within 1.5-fold of the observed values. In the final application of the PBPK model to predict the impact of the strong and moderate P-gp inhibitors, the predicted AUC and Cmax ratios due to clarithromycin treatment were 3.76 and 3.11, respectively, and due to diltiazem treatment were 1.1 and 1.1, respectively.Conclusions: The PBPK model for betrixaban adequately predicted the clinically observed PK non-linearity and accounted for the observed drug-drug interaction effects due to co-administration of betrixaban with ketoconazole and verapamil in healthy volunteers. The PBPK model did not predict a role for CYP3A4 in the clearance of betrixaban and accurately predicted a negative food effect. Intestinal and hepatic P-gp were identified as major contributors to the observed non-linearity in the kinetics of betrixaban and observed drug-drug interaction effects with P-gp inhibitors. [Display omitted] DisclosuresLeeds:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Ke:Portola Pharmaceuticals, Inc.: Consultancy. Rowland Yeo:Portola Pharmaceuticals, Inc.: Consultancy. Curnutte:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

Weight of Evidence and Human Relevance Evaluation of the Benfluralin Mode of Action in Rats (Part II): Thyroid carcinogenesis

Benfluralin is an herbicide of the dinitroaniline class used to control grasses and weeds. In a 2 year dietary study in rats, benfluralin increased incidences of thyroid follicular adenoma and carcinoma at high dietary concentrations (≥2500 ppm). The benfluralin toxicology database suggests the mode of action (MOA) is initiated by induction of liver metabolizing enzymes, particularly thyroid hormone specific UGTs, a major pathway for T4 clearance in rats. As reported with phenobarbital, this effect triggers negative feedback regulation, increasing thyroid stimulating hormone (TSH) release into circulating blood. When sustained over time, this leads to thyroid changes such as follicular hypertrophy, hyperplasia and thyroid follicular tumors with chronic exposures. The described MOA was previously established in rat studies with various chemical activators of xenobiotic receptors in the liver. It is generally considered as non-relevant in humans, due to differences between humans and rats in T4 turnover and susceptibility to this carcinogenic MOA. A structured methodology based on the IPCS/MOA/Human Relevance framework was used in the evaluation of available benfluralin data, and the conclusion was determined that the carcinogenic potential of benfluralin in the thyroid is not relevant in humans.

H63D variant of the homeostatic iron regulator (HFE) gene alters α-synuclein expression, aggregation, and toxicity.

Pathological features of Parkinson's disease include the formation of Lewy bodies containing α-synuclein and the accumulation of iron in the substantia nigra. Previous studies have suggested that iron accumulation contributes to the Parkinson's disease pathology through reactive oxygen species production and accelerated α-synuclein aggregation. This study examines the effects of commonly occurring H63D variant of the homeostatic iron regulatory (HFE) gene on α-synuclein pathology in cell culture and animal models. H63D HFE expression in SH-SY5Y cells lowered endogenous α-synuclein levels and significantly decreased pre-formed fibril-induced α-synuclein aggregation. H63D HFE cells were also protected from pre-formed fibril-induced apoptosis. Autophagic flux, a major pathway for α-synuclein clearance, was increased in H63D HFE cells. Expression of REDD1 was elevated and rapamycin treatment was unable to further induce autophagy, indicating mTORC1 inhibition as the main mechanism of autophagy induction. Moreover, siRNA knockdown of REDD1 in H63D HFE cells decreased autophagic flux and increased the sensitivity to PFF-mediated toxicity. While iron chelator (deferiprone) treatment rescued WT HFE cells from pre-formed fibril toxicity, it exacerbated or was unable to rescue H63D HFE cells. In the in vivo pre-formed fibril intracranial injection model, H67D Hfe (mouse homolog of the human H63D HFE variant) C57BL/6J×129 mice showed less α-synuclein aggregation and less decline in motor function compared to WT Hfe. Collectively, this study suggests that H63D HFE variant modifies α-synuclein pathology through the induction of autophagy and has the potential to impact the pathogenesis and treatment response in Parkinson's disease.

P66 - Evaluation of species differences in major clearance pathways between humans and animals

P66 - Evaluation of species differences in major clearance pathways between humans and animals

Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross-linking Mass Spectrometry (QXL-MS)

Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of age-related neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions.

A Novel Unified Approach to Predict Human Hepatic Clearance for Both Enzyme- and Transporter-Mediated Mechanisms Using Suspended Human Hepatocytes.

The accurate prediction of human pharmacokinetics is critically important in modern drug discovery since it drives both pharmacological and toxicological effects. Although significant progress has been made in predicting drug disposition by hepatic drug-metabolizing enzymes, predicting transporter-mediated clearance is still highly uncertain. Furthermore, different approaches are often used to predict clearance with and without transporter involvement, hence the major clearance pathway for a compound must first be determined to know which approach to use. As a result of these challenges, a novel unified method has been developed using cryopreserved suspended human hepatocytes to predict human hepatic clearance for both enzyme- and transporter-mediated mechanisms. This method hypothesizes that, once in vitro metabolic stability is scaled by partition coefficients between hepatocytes and buffer with 4% bovine serum albumin, in vivo clearance can be better predicted. With this method, good in vitro-in vivo correlation of human hepatic clearance has been obtained for a set of 32 structurally diverse compounds, including such transporters as organic anion-transporting polypeptide substrates. The clearance predictions for most compounds are within 3-fold of observed values. This is the first time that multiple compounds result in good in vitro-in vivo extrapolation using an entirely "bottom-up" approach without any empirical scaling factor when transporter-mediated clearance is involved. Potential exceptions are compounds with significant biliary and/or extra-hepatic clearance. The method offers an alternative approach to more accurately predict human hepatic clearance when multiple complex mechanisms are involved.

Impaired autophagic and mitochondrial functions are partially restored by ERT in Gaucher and Fabry diseases.

The major cellular clearance pathway for organelle and unwanted proteins is the autophagy-lysosome pathway (ALP). Lysosomes not only house proteolytic enzymes, but also traffic organelles, sense nutrients, and repair mitochondria. Mitophagy is initiated by damaged mitochondria, which is ultimately degraded by the ALP to compensate for ATP loss. While both systems are dynamic and respond to continuous cellular stressors, most studies are derived from animal models or cell based systems, which do not provide complete real time data about cellular processes involved in the progression of lysosomal storage diseases in patients. Gaucher and Fabry diseases are rare sphingolipid disorders due to the deficiency of the lysosomal enzymes; glucocerebrosidase and α-galactosidase A with resultant lysosomal dysfunction. Little is known about ALP pathology and mitochondrial function in patients with Gaucher and Fabry diseases, and the effects of enzyme replacement therapy (ERT). Studying blood mononuclear cells (PBMCs) from patients, we provide in vivo evidence, that regulation of ALP is defective. In PBMCs derived from Gaucher patients, we report a decreased number of autophagic vacuoles with increased cytoplasmic localization of LC3A/B, accompanied by lysosome accumulation. For both Gaucher and Fabry diseases, the level of the autophagy marker, Beclin1, was elevated and ubiquitin binding protein, SQSTM1/p62, was decreased. mTOR inhibition did not activate autophagy and led to ATP inhibition in PBMCs. Lysosomal abnormalities, independent of the type of the accumulated substrate suppress not only autophagy, but also mitochondrial function and mTOR signaling pathways. ERT partially restored ALP function, LC3-II accumulation and decreased LC3-I/LC3-II ratios. Levels of lysosomal (LAMP1), autophagy (LC3), and mitochondrial markers, (Tfam), normalized after ERT infusion. In conclusion, there is mTOR pathway dysfunction in sphingolipidoses, as observed in both PBMCs derived from patients with Gaucher and Fabry diseases, which leads to impaired autophagy and mitochondrial stress. ERT partially improves ALP function.

In Silico Prediction of Major Clearance Pathways of Drugs among 9 Routes with Two-Step Support Vector Machines.

The clearance pathways of drugs are critical elements for understanding the pharmacokinetics of drugs. We previously developed in silico systems to predict the five clearance pathway using a rectangular method and a support vector machine (SVM). In this study, we improved our classification system by increasing the number of clearance pathways available for our prediction (CYP1A2, CYP2C8, CYP2C19, and UDP-glucuronosyl transferases (UGTs)) and by accepting multiple major pathways. Using the four default descriptors (charge, molecular weight, logD at pH7.0, and unbound fraction in plasma), three kinds of SVM-based predictors based on traditional single-step approach or two-step focusing approaches with subset or partition clustering were developed. The two-step approach with subset clustering resulted in the highest prediction performance. The feature-selection of additional descriptors based on a greedy algorithm was employed to further improve the predictability. The prediction accuracy for each pathway was increased to more than 0.83 with the exception of CYP2C19 and UGTs pathways, whose accuracies were below 0.7. Prediction performance of CYP1A2, CYP3A4 and renal excretion pathways were found to be acceptable using external dataset. We successfully constructed a novel SVM-based predictor for the multiple major clearance pathways based on chemical structures.

Biotransformation of Ilaprazole in Human Liver Microsomes and Human: Role of CYP3A4 in Ilaprazole Clearance and Drug-Drug Interaction.

Ilaprazole is a new proton pump inhibitor and is currently marketed in China and South Korea for the treatment of gastric and duodenal ulcer. Ilaprazole has a favorable long half-life and minimal pharmacokinetic variability associated with CYP2C19 polymorphism. Sulfoxide oxidation of ilaprazole is catalyzed mainly by CYP3A4. Thus, it has been widely accepted that CYP3A4 plays a major role in the clearance of ilaprazole in humans. However, absorption, distribution, metabolism, and excretion data of radiolabeled ilaprazole in humans are not available. The primary goal of this study was to determine if sulfoxide oxidation is a major metabolic pathway of ilaprazole in humans. Metabolite profiles of ilaprazole, ilaprazole sulfide, and ilaprazole sulfone in human liver microsomes (HLMs) were characterized and quantitively analyzed by liquid chromatography (LC)/UV/high-resolution mass spectrometry (HRMS). Moreover, metabolites of ilaprazole in human urine and feces were detected and identified by LC-HRMS. The results revealed that sulfoxide reduction to ilaprazole sulfide rather than sulfoxide oxidation was the major biotransformation pathway in HLMs. Sulfoxide reduction also occurred in HLMs without NADPH or in deactivated HLMs. Ilaprazole sulfide and its multiple oxidative metabolites were major drug-related components in human urine and feces, where there were no ilaprazole sulfone and its metabolites. A small amount of the parent drug was found in feces. Thus, we propose that nonenzymatic sulfoxide reduction rather than CYP3A4-medidated sulfoxide oxidation is the major metabolic clearance pathway of ilaprazole in humans. Consequently, it is predicted that ilaprazole has no significant drug-drug interaction via CYP3A4 inhibition or induction by a coadministered drug.

PHB2 interacts with LC3 and SQSTM1 is required for bile acids-induced mitophagy in cholestatic liver

Mitophagy is a major pathway for clearance of injured mitochondria. However, whether mitophagy is involved in the cholestasis-induced damages of hepatic mitochondria remains unknown. We here aimed to investigate the molecular links between cholestasis and hepatic mitophagy. We show that mitophagy is increased significantly in livers of biliary atresia (BA) that is cholestatic disease in infants. The mitochondrial-toxicity bile acids treatment increases the activities of mitophagy in hepatocytes. Mechanistically, we find that the prohibitin 2 (PHB2) is crucial for cholestasis-mediated mitophagy in vitro. On the one hand, PHB2 binds the autophagosomal membrane-associated protein LC3 upon injured mitochondria via an LC3-interaction region domain. On the other hand, PHB2 forms a ternary protein complex with sequestosome 1 (SQSTM1) and LC3, leading to loading of LC3 onto the damaged mitochondria. Altogether, our study suggests that PHB2 is required for cholestasis-induced mitophagy via LC3 onto the injured mitochondria.

Serotonin and Norepinephrine Reuptake Inhibitors.

This chapter covers antidepressants that fall into the class of serotonin (5-HT) and norepinephrine (NE) reuptake inhibitors. That is, they bind to the 5-HT and NE transporters with varying levels of potency and binding affinity ratios. Unlike the selective serotonin (5-HT) reuptake inhibitors (SSRIs), most of these antidepressants have an ascending rather than a flat dose-response curve. The chapter provides a brief review of the chemistry, pharmacology, metabolism, safety and adverse effects, clinical use, and therapeutic indications of each antidepressant. Venlafaxine, a phenylethylamine, is a relatively weak 5-HT and weaker NE uptake inhibitor with a 30-fold difference in binding of the two transporters. Therefore, the drug has a clear dose progression, with low doses predominantly binding to the 5-HT transporter and more binding of the NE transporter as the dose ascends. Venlafaxine is metabolized to the active metabolite O-desmethylvenlafaxine (ODV; desvenlafaxine) by CYP2D6, and it therefore is subject to significant inter-individual variation in blood levels and response dependent on variations in CYP2D6 metabolism. The half-life of venlafaxine is short at about 5h, with the ODV metabolite being 12h. Both parent compound and metabolite have low protein binding and neither inhibit CYP enzymes. Therefore, both venlafaxine and desvenlafaxine are potential options if drug-drug interactions are a concern, although venlafaxine may be subject to drug-drug interactions with CYP2D6 inhibitors. At low doses, the adverse effect profile is similar to an SSRI with nausea, diarrhea, fatigue or somnolence, and sexual side effects, while venlafaxine at higher doses can produce mild increases in blood pressure, diaphoresis, tachycardia, tremors, and anxiety. A disadvantage of venlafaxine relative to the SSRIs is the potential for dose-dependent blood pressure elevation, most likely due to the NE reuptake inhibition caused by higher doses; however, this adverse effect is infrequently observed at doses below 225mg per day. Venlafaxine also has a number of potential advantages over the SSRIs, including an ascending dose-antidepressant response curve, with possibly greater overall efficacy at higher doses. Venlafaxine is approved for MDD as well as generalized anxiety disorder, social anxiety disorder, and panic disorder. Desvenlafaxine is the primary metabolite of venlafaxine, and it is also a relatively low-potency 5-HT and NE uptake inhibitor. Like venlafaxine it has a favorable drug-drug interaction profile. It is subject to CYP3A4 metabolism, and it is therefore vulnerable to enzyme inhibition or induction. However, the primary metabolic pathway is direct conjugation. It is approved in the narrow dose range of 50-100mg per day. Duloxetine is a more potent 5-HT and NE reuptake inhibitor with a more balanced profile of binding at about 10:1 for 5HT and NE transporter binding. It is also a moderate inhibitor of CYP2D6, so that modest dose reductions and careful monitoring will be needed when prescribing duloxetine in combination with drugs that are preferentially metabolized by CYP2D6. The most common side effects identified in clinical trials are nausea, dry mouth, dizziness, constipation, insomnia, asthenia, and hypertension, consistent with its mechanisms of action. Clinical trials to date have demonstrated rates of response and remission in patients with major depression that are comparable to other marketed antidepressants reviewed in this book. In addition to approval for MDD, duloxetine is approved for diabetic peripheral neuropathic pain, fibromyalgia, and musculoskeletal pain. Milnacipran is marketed as an antidepressant in some countries, but not in the USA. It is approved in the USA and some other countries as a treatment for fibromyalgia. It has few pharmacokinetic and pharmacodynamic interactions with other drugs. Milnacipran has a half-life of about 10h and therefore needs to be administered twice per day. It is metabolized by CYP3A4, but the major pathway for clearance is direct conjugation and renal elimination. As with other drugs in this class, dysuria is a common, troublesome, and dose-dependent adverse effect (occurring in up to 7% of patients). High-dose milnacipran has been reported to cause blood pressure and pulse elevations. Levomilnacipran is the levorotary enantiomer of milnacipran, and it is pharmacologically very similar to the racemic compound, although the side effects may be milder within the approved dosing range. As with other NE uptake inhibitors, it may increase blood pressure and pulse, although it appears to do so less than some other medications. All medications in the class can cause serotonin syndrome when combined with MAOIs.

Assessing Autophagic Activity and Aggregate Formation of Mutant Huntingtin in Mammalian Cells.

The accumulation of mutant aggregate-prone proteins is a hallmark of the majority of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Autophagy, a cytosolic bulk degradation system, is the major clearance pathway for several aggregate-prone proteins, such as mutant huntingtin. The autophagosome-associated protein LC3-II is a specific marker of autophagic flux within cells, whereas aggregate formation of mutant huntingtin represents a good readout for studying autophagy modulation. Here we describe the method of assessing autophagic flux using LC3-II western blotting and substrate clearance by expressing the N-terminal fragment of huntingtin (htt exon 1) containing an expanded polyglutamine tract in mammalian cells.

Systemic clearance and brain distribution of carbazole-based cyanine compounds as Alzheimer\u2019s disease drug candidates

SLM and SLOH, two analogues of carbazole-based cyanine compounds, have been shown to inhibit β-amyloid peptide aggregation in vitro and in Alzheimer’s disease model mice, which could be potentially developed into drugs for disease treatment. To pave the way for further pharmacokinetics-pharmacodynamics study, we set to investigate these compounds’ systemic clearance pathways and their brain exposure. We found that they generally exhibited relatively low plasma clearance which comprised of hepatic clearance and biliary clearance. Phase I oxidative metabolites for SLM and for SLOH upon microsomes incubation were identified, and the metabolism by CYP3A4 were found to be the major (>70%) hepatic clearance pathway, while the efflux by P-gp and BCRP located in the canalicular membrane of hepatocytes led to high biliary clearance. The permeation of SLM and SLOH through the brain endothelium was affected by the efflux transporters (P-gp and BCRP) and influx transporter (OATP2B1). The unbound interstitial fluid to plasma ratio (Kpuu,brain) was 8.10 for SLOH and 11.0 for SLM, which favored brain entry and were several folds higher than that in wild-type mice. Taken together, these carbazole compounds displayed low plasma clearance and high brain permeability, which entitle further development.