SHR-A1811 (antibody-drug conjugate) in advanced HER2-mutant non-small cell lung cancer: a multicenter, open-label, phase 1/2 study.

A dose-escalation and expansion, phase 1/2 study (ClinicalTrials.gov, NCT04818333) was conducted to assess the novel antibody-drug conjugate SHR-A1811 in pretreated HER2-altered advanced non-small cell lung cancer (NSCLC). Here, we report results from the phase 1 portion. Patients who had previously failed or were intolerant to platinum-based chemotherapy were enrolled and received SHR-A1811 intravenously at doses of 3.2 to 8.0 mg/kg every 3 weeks. Dose escalation followed a Bayesian logistic regression model that included overdose control, with subsequent selection of tolerable levels for dose expansion. Overall, 63 patients were enrolled, including 43 receiving a recommended dose for expansion of 4.8 mg/kg. All patients had HER2-mutant disease. Dose-limiting toxicity occurred in one patient in the 8.0 mg/kg dose cohort. Grade ≥ 3 treatment-related adverse events occurred in 29 (46.0%) patients. One patient in the 6.4 mg/kg cohort died due to interstitial lung disease. As of April 11, 2023, the 4.8 mg/kg cohort showed an objective response rate of 41.9% (95% CI 27.0-57.9), and a disease control rate of 95.3% (95% CI 84.2-99.4). The median duration of response was 13.7 months, with 13 of 18 responses ongoing. The median progression-free survival was 8.4 months (95% CI 7.1-15.0). SHR-A1811 demonstrated favourable safety and clinically meaningful efficacy in pretreated advanced HER2-mutant NSCLC.

Safety, Efficacy, and Pharmacokinetics of SHR-A1811, a Human Epidermal Growth Factor Receptor 2-Directed Antibody-Drug Conjugate, in Human Epidermal Growth Factor Receptor 2-Expressing or Mutated Advanced Solid Tumors: A Global Phase I Trial.

PURPOSE: SHR-A1811 is an antibody-drug conjugate composed of an anti-human epidermal growth factor receptor 2 (HER2) antibody trastuzumab, a cleavable linker, and a topoisomerase I inhibitor payload. We assessed the safety, tolerability, antitumor activity, and pharmacokinetics of SHR-A1811 in heavily pretreated HER2-expressing or mutated advanced solid tumors. METHODS: This global, multi-center, first-in-human, phase I trial was conducted at 33 centers. Patients who had HER2-expressing or mutated unresectable, advanced, or metastatic solid tumors and were refractory or intolerant to standard therapies were enrolled. SHR-A1811 was administered intravenously at doses ranging from 1.0 to 8.0 mg/kg once every 3 weeks. The primary end points were dose-limiting toxicity, safety, and the recommended phase II dose. RESULTS: From September 7, 2020, to February 27, 2023, 307 patients who had undergone a median of three (IQR, 2-5) previous treatment regimens in the metastatic setting received SHR-A1811 treatment. As of data cutoff (February 28, 2023), one patient from the 6.4 mg/kg group experienced dose-limiting toxicities (pancytopenia and colitis). The most common grade 3 or higher adverse events (AEs) included decreased neutrophil count (119 [38.8%]) and decreased WBC count (70 [22.8%]). Interstitial lung disease occurred in only eight (2.6%) patients. Serious AEs and deaths occurred in 70 (22.8%) and 13 (4.2%) patients, respectively. SHR-A1811 led to objective responses in 59.9% (184/307) of all patients, 76.3% (90/118) of HER2-positive breast cancer, 60.4% (55/91) of HER2 low-expressing breast cancer, and 45.9% (39/85 with evaluable tumor responses) of the 98 nonbreast tumors. CONCLUSION: SHR-A1811 exhibited acceptable tolerability, promising antitumor activity, and a favorable pharmacokinetic profile in heavily pretreated advanced solid tumors. The recommended phase II dose of 4.8 or 6.4 mg/kg was selected for various tumor types.

Prediction of pyrotinib exposure based on physiologically-based pharmacokinetic model and endogenous biomarker.

Pyrotinib, a novel irreversible epidermal growth factor receptor dual tyrosine kinase inhibitor, is mainly (about 90%) eliminated through cytochrome P450 (CYP) 3A mediated metabolism in vivo. Meanwhile, genotype is a key factor affecting pyrotinib clearance and 4ß-hydroxycholesterol is an endogenous biomarker of CYP3A activity that can indirectly reflect the possible pyrotinib exposure. Thus, it is necessary to evaluate the clinical drug-drug interactions (DDI) between CYP3A perpetrators and pyrotinib, understand potential exposure in specific populations including liver impairment and geriatric populations, and explore the possible relationships among pyrotinib exposure, genotypes and endogenous biomarker. Physiologically-based pharmacokinetic (PBPK) model can be used to replace prospective DDI studies and evaluate external and internal factors that may influence system exposure. Herein, a basic PBPK model was firstly developed to evaluate the potential risk of pyrotinib coadministration with strong inhibitor and guide the clinical trial design. Subsequently, the mechanistic PBPK model was established and used to quantitatively estimate the potential DDI risk for other CYP3A modulators, understand the potential exposure of specific populations, including liver impairment and geriatric populations. Meanwhile, the possible relationships among pyrotinib exposure, genotypes and endogenous biomarker were explored. With the help of PBPK model, the DDI clinical trial of pyrotinib coadministration with strong inhibitor has been successfully completed, some DDI clinical trials may be waived based on the predicted results and clinical trials in specific populations can be reasonably designed. Moreover, the mutant genotypes of CYP3A4*18A and CYP3A5*3 were likely to have a limited influence on pyrotinib clearance, and the genotype-independent linear correlation coefficient between endogenous biomarker and system exposure was larger than 0.6. Therefore, based on the reliable predicted results and the linear correlations between pyrotinib exposure and endogenous biomarker, dosage adjustment of pyrotinib can be designed for clinical practice.

Population pharmacokinetic modeling of pyrotinib in patients with HER2-positive advanced or metastatic breast cancer.

OBJECTIVE: Pyrotinib, a new oral irreversible pan-ErbB tyrosine kinase inhibitor (TKI), has been approved in China for the treatment of HER2-positive advanced or metastatic breast cancer. This study aimed to conduct a population pharmacokinetics (PK) analysis of pyrotinib and to evaluate the impact of patient characteristics on pyrotinib's PK. METHOD: A total of 1152 samples, provided by 59 adult female patients from two phase I clinical trials, were analyzed by nonlinear mixed-effects modeling. Monte Carlo simulation was conducted to assess impact of covariates on the exposure to pyrotinib. RESULTS: The PK of pyrotinib was adequately described by a one-compartment model with first-order absorption and elimination. Patient's age and total protein levels could affect pyrotinib's apparent volume of distribution, and concomitant use of montmorillonite could decrease the bioavailability of pyrotinib by 50.3%. No PK interactions were observed between capecitabine and pyrotinib. CONCLUSION: In this study, a population PK model of pyrotinib was developed to determine the influence of patient characteristics on the PK of pyrotinib. While patient age and total protein levels can significantly affect the apparent distribution volume of pyrotinib, the magnitude of the impact was limited, thus no dosage adjustment was recommended. Furthermore, concomitant use of montmorillonite for diarrhea needs to be taken with precaution.

Physiologically based pharmacokinetic-pharmacodynamic modeling for prediction of vonoprazan pharmacokinetics and its inhibition on gastric acid secretion following intravenous/oral administration to rats, dogs and humans.

Vonoprazan is characterized as having a long-lasting antisecretory effect on gastric acid. In this study we developed a physiologically based pharmacokinetic (PBPK)-pharmacodynamic (PD) model linking to stomach to simultaneously predict vonoprazan pharmacokinetics and its antisecretory effects following administration to rats, dogs, and humans based on in vitro parameters. The vonoprazan disposition in the stomach was illustrated using a limited-membrane model. In vitro metabolic and transport parameters were derived from hepatic microsomes and Caco-2 cells, respectively. We found the most predicted plasma concentrations and pharmacokinetic parameters of vonoprazan in rats, dogs and humans were within twofold errors of the observed data. Free vonoprazan concentrations (fu × C2) in the stomach were simulated and linked to the antisecretory effects of the drug (I) (increases in pH or acid output) using the fomula dI/dt = k × fu × C2 × (Imax - I) - kd × I. The vonoprazan dissociation rate constant kd (0.00246 min-1) and inhibition index KI (35 nM) for H+/K+-ATPase were obtained from literatures. The vonoprazan-H+/K+-ATPase binding rate constant k was 0.07028 min-1· µM-1 using ratio of kd to KI. The predicted antisecretory effects were consistent with the observations following intravenous administration to rats (0.7 and 1.0 mg/kg), oral administration to dogs (0.3 and 1.0 mg/kg) and oral single dose or multidose to humans (20, 30, and 40 mg). Simulations showed that vonoprazan concentrations in stomach were 1000-fold higher than those in the plasma at 24 h following administration to human. Vonoprazan pharmacokinetics and its antisecretory effects may be predicted from in vitro data using the PBPK-PD model of the stomach. These findings may highlight 24-h antisecretory effects of vonoprazan in humans following single-dose or the sustained inhibition throughout each 24-h dosing interval during multidose administration.

Short-chain fatty acids oppositely altered expressions and functions of intestinal cytochrome P4503A and P-glycoprotein and affected pharmacokinetics of verapamil following oral administration to rats.

OBJECTIVES: To investigate effects of short-chain fatty acids (SCFAs) on expressions and functions of intestinal cytochrome P4503A (Cyp3a) and P-glycoprotein (P-gp). To develop a semi-physiologically based pharmacokinetic (semi-PBPK) model for assessing their contributions. METHODS: Verapamil pharmacokinetics was investigated following oral administration to rats receiving water containing 150 mm SCFAs for 3 weeks. Cyp3a activities in intestinal and liver mircosomes were assessed by norverapamil formation. In-situ single-pass perfusion was used to evaluate intestinal transport of verapamil and P-gp function. Functions and expressions of Cyp3a and P-gp were measured in mouse primary enterocytes following 48-h exposure to SCFAs. Contributions of intestinal P-gp and Cyp3a to verapamil pharmacokinetics were assessed using a semi-PBPK model. KEY FINDINGS: Short-chain fatty acids significantly increased oral plasma exposures of verapamil and norverapamil. SCFAs upregulated Cyp3a activity and expression, but downregulated P-gp function and expression in rat intestine, which were repeated in mouse primary enterocytes. PBPK simulation demonstrated contribution of intestinal Cyp3a to oral plasma verapamil exposure was minor, and the increased oral plasma verapamil exposure was mainly attributed to downregulation of intestinal P-gp. CONCLUSIONS: Short-chain fatty acids oppositely regulated functions and expressions of intestinal Cyp3a and P-gp. The downregulation of P-gp mainly contributed to the increased oral plasma verapamil exposure by SCFAs.

Prediction of Atorvastatin Pharmacokinetics in High-Fat Diet and Low-Dose Streptozotocin-Induced Diabetic Rats Using a Semiphysiologically Based Pharmacokinetic Model Involving Both Enzymes and Transporters.

Atorvastatin is a substrate of cytochrome P450 3a (CYP3a), organic anion-transporting polypeptides (OATPs), breast cancer-resistance protein (BCRP), and P-glycoprotein (P-gp). We aimed to develop a semiphysiologically based pharmacokinetic (semi-PBPK) model involving both enzyme and transporters for predicting the contributions of altered function and expression of CYP3a and transporters to atorvastatin transport in diabetic rats by combining high-fat diet feeding and low-dose streptozotocin injection. Atorvastatin metabolism and transport parameters comes from in situ intestinal perfusion, primary hepatocytes, and intestinal or hepatic microsomes. We estimated the expressions and functions of these proteins and their contributions. Diabetes increased the expression of hepatic CYP3a, OATP1b2, and P-gp but decreased the expression of intestinal CYP3a, OATP1a5, and P-gp. The expression and function of intestinal BCRP were significantly decreased in 10-day diabetic rats but increased in 22-day diabetic rats. Based on alterations in CYP3a and transporters by diabetes, the developed semi-PBPK model was successfully used to predict atorvastatin pharmacokinetics after oral and intravenous doses to rats. Contributions to oral atorvastatin PK were intestinal OATP1a5 < intestinal P-gp < intestinal CYP3a < hepatic CYP3a < hepatic OATP1b2 < intestinal BRCP. Contributions of decreased expression and function of intestinal CYP3a and P-gp by diabetes to oral atorvastatin plasma exposure were almost attenuated by increased expression and function of hepatic CYP3a and OATP1b2. Opposite alterations in oral plasma atorvastatin exposure in 10- and 22-day diabetic rats may be explained by altered intestinal BCRP. In conclusion, the altered atorvastatin pharmacokinetics by diabetes was the synergistic effects of altered intestinal or hepatic CYP3a and transporters and could be predicted using the developed semi-PBPK.

Simultaneously predict pharmacokinetic interaction of rifampicin with oral versus intravenous substrates of cytochrome P450 3A/P­glycoprotein to healthy human using a semi-physiologically based pharmacokinetic model involving both enzyme and transporter turnover.

Several reports demonstrated that rifampicin affected pharmacokinetics of victim drugs following oral more than intravenous administration. We aimed to establish a semi-physiologically based pharmacokinetic (semi-PBPK) model involving both enzyme and transporter turnover to simultaneously predict pharmacokinetic interaction of rifampicin with oral versus intravenous substrates of cytochrome P450 (CYP) 3A4/P­glycoprotein (P-GP) in human. Rifampicin was chosen as the CYP3A /P-GP inducer. Thirteen victim drugs including P-GP substrates (digoxin and talinolol), CYP3A substrates (alfentanil, midazolam, nifedipine, ondansetron and oxycodone), dual substrates of CYP3A/P-GP (quinidine, cyclosporine A, tacrolimus and verapamil) and complex substrates (S-ketamine and tramadol) were chosen to investigate drug-drug interactions (DDIs) with rifampicin. Corresponding parameters were cited from literatures. Before and after multi-dose of oral rifampicin, the pharmacokinetic profiles of victim drugs for oral or intravenous administration to human were predicted using the semi-PBPK model and compared with the observed values. Contribution of both CYP3A and P-GP induction in intestine and liver by rifampicin to pharmacokinetic profiles of victim drugs was investigated. The predicted pharmacokinetic profiles of drugs before and after rifampicin administration accorded with the observations. The predicted pharmacokinetic parameters and DDIs were successful, whose fold-errors were within 2. It was consistent with observations that the DDIs of rifampicin with oral victim drugs were larger than those with intravenous victim drugs. DDIs of rifampicin with CYP3A or P-GP substrates following oral versus intravenous administration to human were successfully predicted using the developed semi-PBPK model.

Impairment of Intestinal Monocarboxylate Transporter 6 Function and Expression in Diabetic Rats Induced by Combination of High-Fat Diet and Low Dose of Streptozocin: Involvement of Butyrate-Peroxisome Proliferator-Activated Receptor-γ Activation.

Generally, diabetes remarkably alters the expression and function of intestinal drug transporters. Nateglinide and bumetanide are substrates of monocarboxylate transporter 6 (MCT6). We investigated whether diabetes down-regulated the function and expression of intestinal MCT6 and the possible mechanism in diabetic rats induced by a combination of high-fat diet and low-dose streptozocin. Our results indicated that diabetes significantly decreased the oral plasma exposure of nateglinide. The plasma peak concentration and area under curve in diabetic rats were 16.9% and 28.2% of control rats, respectively. Diabetes significantly decreased the protein and mRNA expressions of intestinal MCT6 and oligopeptide transporter 1 (PEPT1) but up-regulated peroxisome proliferator-activated receptor γ (PPARγ) protein level. Single-pass intestinal perfusion demonstrated that diabetes prominently decreased the absorption of nateglinide and bumetanide. The MCT6 inhibitor bumetanide, but not PEPT1 inhibitor glycylsarcosine, significantly inhibited intestinal absorption of nateglinide in rats. Coadministration with bumetanide remarkably decreased the oral plasma exposure of nateglinide in rats. High concentrations of butyrate were detected in the intestine of diabetic rats. In Caco-2 cells (a human colorectal adenocarcinoma cell line), bumetanide and MCT6 knockdown remarkably inhibited the uptake of nateglinide. Butyrate down-regulated the function and expression of MCT6 in a concentration-dependent manner but increased PPARγ expression. The decreased expressions of MCT6 by PPARγ agonist troglitazone or butyrate were reversed by both PPARγ knockdown and PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Four weeks of butyrate treatment significantly decreased the oral plasma concentrations of nateglinide in rats, accompanied by significantly higher intestinal PPARγ and lower MCT6 protein levels. In conclusion, diabetes impaired the expression and function of intestinal MCT6 partly via butyrate-mediated PPARγ activation, decreasing the oral plasma exposure of nateglinide.

Characteristics of ß-oxidative and reductive metabolism on the acyl side chain of cinnamic acid and its analogues in rats.

Cinnamic acid and its analogues (pyragrel and ozagrel) undergo chain-shortened (ß-oxidative) and reductive metabolism on acyl side chain. In this study, we characterized the ß-oxidative and reductive metabolism on acyl side chain of cinnamic acid and its analogues using primary rat hepatocytes, hepatic mitochondrial, and microsomal systems. A compartmental model including parent compounds and metabolites was developed to characterize in vivo ß-oxidative and reductive metabolism following an intravenous dose of parent compounds to rats. The fitted total in vivo clearance values were further compared with the in vitro values predicted by the well-stirred model. We showed that hepatic microsomal CYP450s did not catalyze ß-oxidative or reductive metabolism of the three compounds. Similar to ß-oxidation of fatty acids, ß-oxidative metabolism on their acyl side chain occurred mainly in mitochondria, which was highly dependent on ATP, CoA and NAD+. Fatty acids and NADH inhibited the ß-oxidative metabolism. Reductive metabolism occurred in both mitochondria and microsomes. Reduction in mitochondria was ATP-, CoA-, and NAD(P)H-dependent and reversible, which was suppressed by enoyl reductase inhibitor triclosan. Reduction in microsomes was ATP-, CoA-, and NADPH-dependent but little affected by triclosan. Both plasma concentrations of ß-oxidative metabolites and reductive metabolites were successfully fitted using the compartmental model. The estimated total in vivo clearance values were consistent with those predicted from hepatocytes and organelles, implicating significance of in vitro kinetics. These findings demonstrate the roles of hepatic mitochondria and microsomes in ß-oxidative and reductive metabolism on acyl side chain of cinnamic acid and its analogues along with their metabolic characteristics.

Increase in P-glycoprotein levels in the blood-brain barrier of partial portal vein ligation /chronic hyperammonemia rats is medicated by ammonia/reactive oxygen species/ERK1/2 activation: In vitro and in vivo studies.

Liver failure altered P-glycoprotein (P-gp) function and expression at blood-brain barrier (BBB), partly owing to hyperammonemia. We aimed to examine the effects of partial portal vein ligation (PVL) plus chronic hyperammonemia (CHA) on P-gp function and expression at rat BBB. Experimental rats included sham-operation (SH), PVL, CHA and PVL+CHA. The PVL+CHA rats were developed by ammonia-containing diet for 2 weeks after operation. The brain-to-plasma concentration ratios (Kp) and apparent unidirectional influx constants (Kin) of rhodamine123 and sodium fluorescein were measured to assess function of P-gp and BBB integrity, respectively. Human cerebral microvascular endothelial cells (HCMEC/D3) were used to assess effects of ammonia on P-gp expression and function. It was found that PVL+CHA significantly decreased Kp and Kin of rhodamine123 without affecting brain distribution of fluorescein. The P-gp expressions in membrane protein in cortex and hippocampus were significantly increased in CHA and PVL +CHA rats, especially in PVL + CHA rats, while remarkably increased phosphorylated ERK1/2 was only found in PVL +CHA rats. Expressions of tight junction proteins claudin-5 and occluding in rat brain remained unchanged. In vitro data showed that NH4Cl increased reactive oxygen species, membrane expression and function of P-gp as well as phosphorylated ERK1/2 levels in HCMEC/D3. The NH4Cl-induced alterations were reversed by reactive oxygen species scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. In conclusion, PVL+CHA increased function and membrane translocation of P-gp at rat BBB partly via ammonia. Reactive oxygen species/ERK1/2 pathway activation may be one of the reasons that ammonia upregulated P-gp expression and function at BBB.

Predicting Antitumor Effect of Deoxypodophyllotoxin in NCI-H460 Tumor-Bearing Mice on the Basis of In Vitro Pharmacodynamics and a Physiologically Based Pharmacokinetic-Pharmacodynamic Model.

Antitumor evaluation in tumor-bearing mouse is time- and energy-consuming. We aimed to investigate whether in vivo antitumor efficacy could be predicted on the basis of in vitro pharmacodynamics using deoxypodophyllotoxin (DPT), an antitumor candidate in development, as a model compound. Proliferation kinetics of monolayer-cultivated NCI-H460 cells under various DPT concentrations were quantitatively investigated and expressed as calibration curves. Koch two-phase natural growth model combined with sigmoid Emax model, i.e., dM/dt = 2λ0λ1M/(λ1 + 2λ0M) - Emax C γ /(EC50γ + C γ )·M, was introduced to describe cell proliferation (M) against time under DPT treatment (C). Estimated in vitro pharmacodynamic parameters were: EC50, 8.97 nM; Emax , 0.820 day-1, and γ, 7.13. A physiologically based pharmacokinetic model including tumor compartment was introduced to predict DPT disposition in plasma, tumor tissue, and main normal tissues of NCI-H460 tumor-bearing mice following a single dose. The in vivo pharmacodynamic model and parameters were assumed the same as the in vitro ones, and linked with simulated tumor pharmacokinetic profiles by a physiologically based pharmacokinetic (PBPK) model to build a PBPK-pharmacodynamic (PBPK-PD) model. After natural growth parameters (λ0 and λ1) were estimated, the objective in this study was to predict with the PBPK-PD model the tumor growth in NCI-H460 tumor-bearing mice during multidose DPT treatment, a use of the model similar to what others have reported. In our work, the model was successfully applied to predict tumor growth in SGC-7901 tumor-bearing mice. The resulting data indicated that in vivo antitumor efficacy might be predicted on the basis of in vitro cytotoxic assays via a PBPK-PD model approach. We demonstrated that the approach is reasonable and applicable and may facilitate and accelerate anticancer candidate screening and dose regimen design in the drug discovery process.

Acute liver failure enhances oral plasma exposure of zidovudine in rats by downregulation of hepatic UGT2B7 and intestinal P-gp.

HIV infection is often associated with liver failure, which alters the pharmacokinetics of many drugs. In this study we investigated whether acute liver failure (ALF) altered the pharmacokinetics of the first-line anti-HIV agent zidovudine (AZT), a P-gp/BCRP substrate, in rats. ALF was induced in rats by injecting thioacetamide (TAA, 300 mg·kg-1·d-1, ip) for 2 days. On the second day after the last injection of TAA, the pharmacokinetics of AZT was investigated following both oral (20 mg/kg) and intravenous (10 mg/kg) administration. ALF significantly increased the plasma concentrations of AZT after both oral and intravenous doses of AZT, but without affecting the urinary excretion of AZT. AZT metabolism was studied in rat hepatic microsomes in vitro, which revealed that hepatic UGT2B7 was the main enzyme responsible for the formation of AZT O-glucuronide (GAZT); ALF markedly impaired AZT metabolism in hepatic microsomes, which was associated with the significantly decreased hepatic UGT2B7 expression. Intestinal absorption of AZT was further studied in rats via in situ single-pass intestinal perfusion. Intestinal P-gp function and intestinal integrity were assessed with rhodamine 123 and FD-70, respectively. We found that ALF significantly downregulated intestinal P-gp expression, and had a smaller effect on intestinal BCRP. Further studies showed that ALF significantly increased the intestinal absorption of both rhodamine 123 and AZT without altering intestinal integrity, thus confirming an impairment of intestinal P-gp function. In conclusion, ALF significantly increases the oral plasma exposure of AZT in rats, a result partly attributed to the impaired function and expression of hepatic UGT2B7 and intestinal P-gp.

Prediction of Deoxypodophyllotoxin Disposition in Mouse, Rat, Monkey, and Dog by Physiologically Based Pharmacokinetic Model and the Extrapolation to Human.

Deoxypodophyllotoxin (DPT) is a potential anti-tumor candidate prior to its clinical phase. The aim of the study was to develop a physiologically based pharmacokinetic (PBPK) model consisting of 13 tissue compartments to predict DPT disposition in mouse, rat, monkey, and dog based on in vitro and in silico inputs. Since large interspecies difference was found in unbound fraction of DPT in plasma, we assumed that Kt:pl,u (unbound tissue-to-plasma concentration ratio) was identical across species. The predictions of our model were then validated by in vivo data of corresponding preclinical species, along with visual predictive checks. Reasonable matches were found between observed and predicted plasma concentrations and pharmacokinetic parameters in all four animal species. The prediction in the related seven tissues of mouse was also desirable. We also attempted to predict human pharmacokinetic profile by both the developed PBPK model and interspecies allometric scaling across mouse, rat and monkey, while dog was excluded from the scaling. The two approaches reached similar results. We hope the study will help in the efficacy and safety assessment of DPT in future clinical studies and provide a reference to the preclinical screening of similar compounds by PBPK model.

Interspecies differences in metabolism of deoxypodophyllotoxin in hepatic microsomes from human, monkey, rat, mouse and dog.

Deoxypodophyllotoxin (DPT) is a natural lignan product which has drawn much attention due to its pharmacological properties including antitumor effect. The purpose of this study was to investigate interspecies differences in metabolism of DPT in hepatic microsomes from human (HLM), cynomolgus monkey (CyLM), rat (RLM), mouse (MLM) and dog (DLM). Incubation of DPT with hepatic microsomes from five species in the presence of NADPH resulted in formation of seven metabolites, five of which were compared with the synthetic standards. M2 was the most abundant metabolite in microsomes from all species. Rank order of intrinsic clearance for M2 formation was RLM > CyLM > MLM > HLM > DLM. In HLM, sulfaphenazole showed the strongest inhibition effect on M2 formation, but neither ticlopidine nor ketoconazole inhibited M2 formation in HLM. Results from cDNA-expressed human CYP450s experiments showed that clearance of M2 formation was much higher in CYP2C9 and CYP2C19 than that in CYP3A4. Contributions of the three CYP450 isoforms to M2 formation in HLM were estimated using relative activity factor (RAF) method or correction by amount of CYP450 isoforms in HLM. M2 formation in HLM was mainly attributed to CYP2C9, followed by CYP2C19. Involvement of CYP3A4 was minor.

Acute liver failure impairs function and expression of breast cancer-resistant protein (BCRP) at rat blood-brain barrier partly via ammonia-ROS-ERK1/2 activation.

We once reported that P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) were oppositely regulated at the blood-brain barrier (BBB) of thioacetamide-induced acute liver failure (ALF) rats. This study aimed to investigate whether ALF affected function and expression of breast cancer-resistant protein (BCRP) at the BBB of rats and the role of ammonia in the regulation. ALF rats were developed by intraperitoneal (i.p.) injection of thioacetamide (300 mg/kg) for 2 days. Hyperammonemic rats were developed by NH4 Ac (i.p. 4.5 mmol/kg). BCRP function and expression were measured by brain distribution of specific substrates (prazosin and methotrexate) and western blot, respectively. MDCK-BCRP cells and primarily cultured rat brain microvessel endothelial cells (rBMECs) were employed to investigate possible mechanisms through which ammonia regulated BCRP function and expression. The results showed that both ALF and hyperammonemia significantly weakened function and expression of BCRP in the brain of rats. The function and expression of BCRP in MDCK-BCRP cells and rBMECs were strikingly decreased after exposure to NH4 Cl and H2 O2 , accompanied by remarkable increases in the levels of phosphorylated ERK1/2 and reactive oxygen species (ROS). The altered BCRP expression and function by ammonia and H2 O2 were restored by ROS scavenger N-acetylcysteine and ERK1/2 inhibitor U0126. Markedly increased levels of ERK1/2 phosphorylation and ROS were found in the brains of ALF rats and hyperammonemic rats. All above results indicated ALF down-regulated expression and function of BCRP at BBB of rats partly via hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia impaired BCRP expression and function at the BBB. The present study showed that the expression and function of breast cancer resistant protein (BCRP) at blood-brain barrier (BBB) of thioacetamide-induced ALF rats were down-regulated which partly attribute to hyperammonemia. Activation of ROS-mediated ERK1/2 phosphorylation may be one of the reasons that ammonia suppressed BCRP expression and function. Impaired BCRP at BBB might enhanced pharmacological/toxic effects of corresponding substrates on CNS.

Ciprofloxacin blocked enterohepatic circulation of diclofenac and alleviated NSAID-induced enteropathy in rats partly by inhibiting intestinal ß-glucuronidase activity.

AIM: Diclofenac is a non-steroidal anti-inflammatory drug (NSAID), which may cause serious intestinal adverse reactions (enteropathy). In this study we investigated whether co-administration of ciprofloxacin affected the pharmacokinetics of diclofenac and diclofenac-induced enteropathy in rats. METHODS: The pharmacokinetics of diclofenac was assessed in rats after receiving diclofenac (10 mg/kg, ig, or 5 mg/kg, iv), with or without ciprofloxacin (20 mg/kg, ig) co-administered. After receiving 6 oral doses or 15 intravenous doses of diclofenac, the rats were sacrificed, and small intestine was removed to examine diclofenac-induced enteropathy. ß-Glucuronidase activity in intestinal content, bovine liver and E coli was evaluated. RESULTS: Following oral or intravenous administration, the pharmacokinetic profile of diclofenac displayed typical enterohepatic circulation, and co-administration of ciprofloxacin abolished the enterohepatic circulation, resulted in significant reduction in the plasma content of diclofenac. In control rats, ß-glucuronidase activity in small intestinal content was region-dependent: proximal intestine

In vitro metabolism of l-corydalmine, a potent analgesic drug, in human, cynomolgus monkey, beagle dog, rat and mouse liver microsomes.

l-Corydalmine (l-CDL) was under development as an oral analgesic agent, exhibiting potent analgesic activity in preclinical models. The objective of this study was to compare metabolic profiles of l-CDL in liver microsomes from mouse, rat, monkey, dog and human. Six metabolites (M1-M6) were identified using LC-Q/TOF in liver microsomes from the five species. The metabolism of l-CDL included O-demethylation (M1-3) and hydroxylation (M4-6). The desmethyl metabolites were the major ones among the five species, which accounted for more than 84%. Data from chemical inhibition in human liver microsomes (HLM) and human recombinant CYP450s demonstrated that CYP2D6 exhibited strong catalytic activity towards M1 and M2 formations, while CYP2C9 and CYP2C19 also catalyzed M2 formation. Formations of M3 and hydroxyl metabolites (M4 and M5) were mainly catalyzed by CYP3A4. Further studies showed that M1 and M2 were main metabolites in HLM. The kinetics of M1 and M2 formations in HLM and recombinant CYP450s were also investigated. The results showed that M1 and M2 formations in HLM and recombinant CYP2D6 characterized biphasic kinetics, whereas sigmoid Vmax model was better used to fit M2 formation by recombinant CYP2C9 and CYP2C19. The contributions of CYP2D6 to M1 and M2 formations in HLM were estimated to be 75.3% and 50.7%, respectively. However, the contributions of CYP2C9 and CYP2C19 to M2 formation were only 5.0% and 4.1%, respectively. All these data indicated that M1 and M2 were main metabolites in HLM, and CYP2D6 was the primary enzyme responsible for their formations.

Involvement of pregnane X receptor in the impaired glucose utilization induced by atorvastatin in hepatocytes.

Accumulating evidences demonstrated that statins impaired glucose utilization. This study was aimed to investigate whether PXR was involved in the atorvastatin-impaired glucose utilization. Rifampicin/PCN served as PXR activator control. Glucose utilization, glucose uptake, protein levels of GLUT2, GCK, PDK2, PEPCK1 and G6Pase in HepG2 cells were measured. PXR inhibitors, PXR overexpression and PXR siRNA were applied to verify the role of PXR in atorvastatin-impaired glucose utilization in cells. Hypercholesterolemia rats induced by high fat diet feeding, orally received atorvastatin (5 and 10 mg/kg), pravastatin (10 mg/kg) for 14 days, or intraperitoneally received PCN (35 mg/kg) for 4 days. Results showed that glucose utilization was markedly inhibited by atorvastatin, simvastatin, pitavastatin, lovastatin and rifampicin. Neither rosuvastatin nor pravastatin showed the similar effect. Atorvastatin and pravastatin were selected for the following study. Atorvastatin and rifampicin significantly inhibited glucose uptake and down-regulated GLUT2 and GCK expressions. Similarly, overexpressed PXR significantly down-regulated GLUT2 and GCK expressions and impaired glucose utilization. Ketoconazole and resveratrol attenuated the impaired glucose utilization by atorvastatin and rifampicin in both parental and overexpressed PXR cells. PXR knockdown significantly up-regulated GLUT2 and GCK proteins and abolished the decreased glucose consumption and uptake by atorvastatin and rifampicin. Animal experiments showed that atorvastatin and PCN significantly elicited postprandial hyperglycemia, leading to increase in glucose AUC. Expressions of GLUT2 and GCK in rat livers were markedly down-regulated by atorvastatin and PCN. In conclusion, atorvastatin impaired glucose utilization in hepatocytes via repressing GLUT2 and GCK expressions, which may be partly due to PXR activation.

Paroxetine decreased plasma exposure of glyburide partly via inhibiting intestinal absorption in rats.

Accumulating evidences have shown that diabetes is often accompanied with depression, thus it is possible that oral antidiabetic agent glyburide and antidepressive agent paroxetine are co-administered in diabetic patients. The aim of this study was to assess interactions between glyburide and paroxetine in rats. Effect of paroxetine on pharmacokinetics of orally administered glyburide was investigated. Effect of naringin (NAR), an inhibitor of rat intestinal organic anion transporting polypeptides 1a5 (Oatp1a5), on pharmacokinetics of glyburide was also studied. The results showed that co-administration of paroxetine markedly reduced plasma exposure and prolonged Tmax of glyburide, accompanied by significant increase in fecal excretion of glyburide. Co-administration of naringin also significantly decreased plasma exposure of glyburide. Data from intestinal perfusion experiments showed that both paroxetine and naringin significantly inhibited intestinal absorption of glyburide. Caco-2 cells were used to investigate whether paroxetine and naringin affected intestinal transport of glyburide and fexofenadine (a substrate of Oatp1a5). The results showed that both paroxetine and naringin greatly inhibited absorption of glyburide and fexofenadine. All results gave a conclusion that co-administration of paroxetine decreased plasma exposure of glyburide in rats via inhibiting intestinal absorption of glyburide, which may partly be attributed to the inhibition of intestinal Oatp1a5 activity.