Development and validation of a sensitive UPLC-MS/MS analytical method for GFH009 in rat plasma and its application to toxicokinetics studies.

GFH009 is a potent, highly selective, small molecule that targets and inhibits the activity of the CDK9/cyclin T1 regulatory complex of P-TEFb. This study aimed to develop and validate a highly selective and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for precise quantification of GFH009 in rat plasma. This method was subsequently employed for conducting toxicokinetic studies of GFH009 in rats. Plasma was prepared using a simple protein precipitation method by acetonitrile. Chromatographic separation of the analytes was achieved on a BEH C18 analytical column with a rapid 3.0 min run time and a flow rate of 0.5 ml/min. The calibration curves for plasma samples exhibited excellent linearity over a wide concentration range of 1.0-1,000 ng/ml for GFH009. Intra- and inter-day accuracies were within 92.7-105.7%, and precisions were no more than 6.7%. Furthermore, the analyte demonstrated stability under four different storage conditions, with variations of <15.0%. This study pioneers a methodological innovation by introducing a highly reliable, specific and sensitive analytical method for GFH009 in rat plasma. The successful application of this method in toxicokinetic studies further underscores its significance, offering valuable insights for the methodology of clinical pharmacokinetic research.

Enhancing drug-drug Interaction Prediction by Integrating Physiologically-Based Pharmacokinetic Model with Fraction Metabolized by CYP3A4.

BACKGROUND: Enhancing the precision of drug-drug interaction (DDI) prediction is essential for improving drug safety and efficacy. The aim is to identify the most effective fraction metabolized by CY3A4 (fm) for improving DDI prediction using physiologically based pharmacokinetic (PBPK) models. RESEARCH DESIGN AND METHODS: The fm values were determined for 33 approved drugs using a human liver microsome for in vitro measurements and the ADMET Predictor software for in silico predictions. Subsequently, these fm values were integrated into PBPK models using the GastroPlus platform. The PBPK models, combined with a ketoconazole model, were utilized to predict AUCR (AUCcombo with ketoconazole/AUCdosing alone), and the accuracy of these predictions was evaluated by comparison with observed AUCR. RESULTS: The integration of in vitro fm method demonstrates superior performance compared to the in silico fm method and fm of 100% method. Under the Guest-limits criteria, the integration of in vitro fm achieves an accuracy of 76%, while the in silico fm and fm of 100% methods achieve accuracies of 67% and 58%, respectively. CONCLUSIONS: Our study highlights the importance of in vitro fm data to improve the accuracy of predicting DDIs and demonstrates the promising potential of in silico fm in predicting DDIs.

Mechanism-Based Pharmacokinetic Model for the Deglycosylation Kinetics of 20(S)-Ginsenosides Rh2.

Aim: The 20(S)-ginsenoside Rh2 (Rh2) is being developed as a new antitumor drug. However, to date, little is known about the kinetics of its deglycosylation metabolite (protopanoxadiol) (PPD) following Rh2 administration. The aim of this work was to 1) simultaneously characterise the pharmacokinetics of Rh2 and PPD following intravenous and oral Rh2 administration, 2) develop and validate a mechanism-based pharmacokinetic model to describe the deglycosylation kinetics and 3) predict the percentage of Rh2 entering the systemic circulation in PPD form. Methods: Plasma samples were collected from rats after the I.V. or P.O. administration of Rh2. The plasma Rh2 and PPD concentrations were determined using HPLC-MS. The transformation from Rh2 to PPD, its absorption, and elimination were integrated into the mechanism based pharmacokinetic model to describe the pharmacokinetics of Rh2 and PPD simultaneously at 10 mg/kg. The concentration data collected following a 20 mg/kg dose of Rh2 was used for model validation. Results: Following Rh2 administration, PPD exhibited high exposure and atypical double peaks. The model described the abnormal kinetics well and was further validated using external data. A total of 11% of the administered Rh2 was predicted to be transformed into PPD and enter the systemic circulation after I.V. administration, and a total of 20% of Rh2 was predicted to be absorbed into the systemic circulation in PPD form after P.O. administration of Rh2. Conclusion: The developed model provides a useful tool to quantitatively study the deglycosylation kinetics of Rh2 and thus, provides a valuable resource for future pharmacokinetic studies of glycosides with similar deglycosylation metabolism.

Predicting the Drug-Drug Interaction Mediated by CYP3A4 Inhibition: Method Development and Performance Evaluation.

The prediction of drug-drug interactions (DDIs) plays critical roles for the estimation of DDI risk caused by inhibition of CYP3A4. The aim of this paper is to develop a physiologically based pharmacokinetic (PBPK)-DDI model for prediction of the DDI co-administrated with ketoconazole in humans and evaluate the predictive performance of the model. The pharmacokinetic and biopharmaceutical properties of 35 approved drugs, as victims, were collected for the development of a PBPK model, which were linked to the PBPK model of ketoconazole for the DDI prediction. The PBPK model of victims and ketoconazole were validated by matching actual in vivo pharmacokinetic data. The predicted results of DDI were compared with actual data to evaluate the predictive performance. The percentage of predicted ratio of AUC (AUCR), Cmax (CmaxR), and Tmax (TmaxR) was 75%, 69%, and 91%, respectively, which were within the twofold threshold (range, 0.5-2.0×) of the observed values. Only 3% of the predicted AUCRs are obviously underestimated. After integration of the reported fraction of metabolism (fm) into the PBPK-DDI model for limited four cases, the model-predicted AUCRs were improved from the twofold range of the observed AUCRs to the 90% confidence interval. The developed method could reasonably predict drug-drug interaction with a low risk of underestimation. The present accuracy of the prediction was improved compared with that of static mechanistic models. The evaluation of predictive performance increases the confidence using the model to evaluate the risk of DDIs co-administrated with ketoconazole before the in vivo DDI study.

Preclinical pharmacokinetics, disposition, and translational pharmacokinetic/pharmacodynamic modeling of savolitinib, a novel selective cMet inhibitor.

Savolitinib is a novel small-molecule selective cMet inhibitor. This work characterized its pharmacokinetics in preclinical phase, established the preclinical relationships between PK, cMet modulation and anti-tumor efficacy. In vitro and in vivo animal studies were performed for PK characterization. Savolitinib showed good absorption, moderate tissue distribution, low to intermediate clearance, and low accumulation. Hepatic oxidative metabolism followed by urinary and biliary excretions was the major elimination pathway. Based on preclinical PK data, human PK profiles were predicted using empirical methods. Pharmacodynamic studies for evaluating cMet inhibition and anti-tumor efficacy were conducted in nude mice bearing Hs746t xenograft. PK/PD models were built to link the PD measurements to nude mouse PK. The established integrated preclinical PK/PD model contained a two-compartment non-linear PK model, a biomarker link model and a tumor growth transit model. The IC50 of cMet inhibition and the concentration achieving half of the maximal Hs746t tumor reduction by savolitinib were equal to 12.5 and 3.7 nM (free drug), respectively. Based on the predicted human PK data, as well as the established PK/PD model in nude mouse, the human PD (cMet inhibition) profiles were also simulated. This research supported clinical development of savolitinib. Understanding the preclinical PK/PD relationship of savolitinib provides translational insights into the cMet-targeted drug development.

Evaluation of Generic Methods to Predict Human Pharmacokinetics Using Physiologically Based Pharmacokinetic Model for Early Drug Discovery of Tyrosine Kinase Inhibitors.

BACKGROUND: Requirements for predicting human pharmacokinetics in drug discovery are increasing. Developing different methods of human pharmacokinetic prediction will facilitate lead optimization, candidate nomination, and dosing regimens before clinical trials at various early drug discovery stages. OBJECTIVES: To develop and validate generic methods of human pharmacokinetic prediction to meet the requirements in early drug discovery. METHODS: The physiologically based pharmacokinetic (PBPK) model implemented in Gastroplus™ was used for human pharmacokinetic predictions. The absorption, distribution, metabolism, and excretion properties of drugs in humans predicted from molecular structure and extrapolated from tested preclinical data were used as inputs in the PBPK model. The approaches were validated by comparison of the predicted pharmacokinetic parameters with actual pharmacokinetic parameters of 15 marketed small-molecule compounds approved by the US Food and Drug Administration. Based on the validation and reported approaches, we proposed a strategy for human pharmacokinetic prediction at different drug discovery stages. RESULTS: Obvious underestimation of exposure (< 1/3 of actual exposure) was not observed using in silico prediction as inputs, which may reduce the probability of missing the potential compounds with predicted false low exposure. The simulated human pharmacokinetic results using tested data as inputs were superior to those obtained via in silico prediction. Both methods similarly predicted the multiphasic shape of pharmacokinetic profiles. CONCLUSION: These generic PBPK approaches of full in silico prediction or perdition using a combination of tested in vivo and in vitro data were validated and proved useful for human pharmacokinetic predictions.

Correction to: Evaluation of Generic Methods to Predict Human Pharmacokinetics Using Physiologically Based Pharmacokinetic Model for Early Drug Discovery of Tyrosine Kinase Inhibitors.

The original version of this article unfortunately contained a mistake. Conflict of interest statement was incorrect. The corrected COI statement is given below.

Preclinical pharmacokinetics and disposition of a novel selective VEGFR inhibitor fruquintinib (HMPL-013) and the prediction of its human pharmacokinetics.

PURPOSE: This study evaluated the preclinical pharmacokinetics (PK) and disposition of fruquintinib (HMPL-013), a small molecule vascular endothelial growth factor receptors inhibitor. METHODS: In vitro and in vivo PK/ADME assays were conducted. Allometry and PK modeling/simulation were conducted to predict human PK parameters and the time course profiles. RESULTS: HMPL-013 has high permeability without efflux. It shows moderate oral bioavailability of 42-53 % and Tmax < 4 h in mouse, rat, dog and monkey, with exposure-dose linearity proved in rats and dogs. No significant food effect is on dog PK. HMPL-013 has moderately high tissue distribution. It majorly distributes in gastrointestinal tract, liver, kidney, adrenal and adipose. The plasma protein binding fraction is 88-95 % in mouse, rat, dog and human, invariable up to 10 µM. The in vivo clearance of HMPL-013 is low, consistent with the in vitro scaling. Three major oxidative metabolites were identified in liver microsomes of mouse, rat, dog, monkey and human. Dog is mostly similar to human regarding in vitro metabolism. Demethylation, hydroxylation and sequential glucuronidation are the major in vivo metabolic reactions. Direct urinary and biliary excretion of HMPL-013 is negligible. Metabolizing to M1 (demethylation), sequentially glucuronidating, followed by biliary excretion, and to a less extent, by urinary excretion, are important elimination pathways for HMPL-013 in rats. HMPL-013 has low risk of drug-drug interaction. It is predicted to have favorable human PK properties and low efficacious dose. CONCLUSION: HMPL-013 demonstrates good preclinical PK and enables successful human PK and dose projection. It is valuable for further clinical development.

Metabonomic profiling of liver metabolites by gas chromatography-mass spectrometry and its application to characterizing hyperlipidemia.

The measurement of metabolites in tissues is of great importance in metabonomic research in the biomedical sciences, providing more relevant information than is available from systemic biofluids. The liver is the most important organ/tissue for most biochemical reactions, and the metabolites in the liver are of great interest to scientists. To develop an optimized extraction method and comprehensive profiling technique for liver metabolites, organic solvents of various compositions were designed using design of experiments to extract metabolites from the liver, and the metabolites were profiled by gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). The resolved peak areas were processed by principle components analysis, partial least-squares projections to latent structures, and discriminant analysis. The results suggest the highest extraction efficiency was for methanol-water, which maximized the majority of GC/TOF-MS responses. The optimal solvent was applied to extract metabolites in liver of hyperlipidemia hamster and the control. The GC/TOF-MS profiles of liver metabolites showed obvious differences between hyperlipidemic hamsters and controls. A comparison of liver and serum data from the same animals identified common biomarkers and presented complementary information. Our results suggest that liver metabonomics is a valuable technique and that the combined analysis of systematic biofluids and local tissues is meaningful and complementary, recovering more comprehensive metabonomic data than either analysis alone.

Metabonomic characterization of early atherosclerosis in hamsters with induced cholesterol.

Atherosclerosis is a complicated and multifactorial disease, induced not only by genotype, but also, even more importantly, by environmental factors. Study on the metabolic perturbation of endogenous compounds may offer deeper insight into development of atherosclerosis. Gas chromatography/mass spectrometry (GC/MS)-based metabonomics was used to profile a metabolic fingerprint of serum obtained from hamsters with induced cholesterol. The deconvoluted GC/MS data were processed by multivariate statistical analysis tools, such as principal component analysis (PCA) and partial least squares projection to latent structure and discriminant analysis (PLS-DA). For the first time we showed a time-dependent development of the model animal from normal to hypercholesterolaemia, and further to early atherosclerosis. Twenty-one compounds were identified as markers involved in the development to atherosclerosis. Identification of the compounds suggests that amino acid metabolism and fatty acid oxidation are significantly perturbed following cholesterol overloading. The data provide novel information to approach the pathophysiological processes of the hypercholesterolaemia and atherosclerosis disease continuum.

Organic solvent extraction and metabonomic profiling of the metabolites in erythrocytes.

We used erythrocytes as the model tissue to evaluate an optimal solution for the extraction of intracellular metabolites and time-dependent variation of the metabolome in living cells. Projection to latent structure (PLS) of the GC/MS and LC/MS data suggested that the most efficient solution for the extraction of metabolites from wet erythrocytes (50 mg) could be a methanol-chloroform-water mixture (950 microL, 700:200:50, v/v/v). PLS-discriminant analysis (DA) clearly profiled a time-dependent alternation of metabolic phenotype of erythrocytes. Identification of the metabolites showed that the process was characterized by accumulating of metabolic products and depleting of nutritious substances in erythrocytes during incubation.

Determination of 20(S)-ginsenoside Rh1 and its aglycone 20(S)-protopanaxatriol in rat plasma by sensitive LC-APCI-MS method and its application to pharmacokinetic study.

Liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method has been developed for the measurement of the concentrations of 20(S)-ginsenoside Rh1 and its aglycone 20(S)-protopanaxatriol in rat plasma with panaxatriol as internal standard. The method involved single liquid-liquid extraction of both 20(S)-ginsenoside Rh1 and 20(S)-protopanaxatriol from plasma samples with n-butanol. The limit of quantification (LOQ) was 5 ng mL(-1) for both compounds. The method was validated within the linear range 5-2000 ng.mL(-1) for both compounds. The correlation coefficient for the calibration regression line was 0.999 or better. Intra-day and inter-day accuracy were better than 15%. The method has been successfully used for the pharmacokinetic studies in rats. After intravenous administrations, the mean retention times of 20(S)-ginsenoside Rh1 and 20(S)-protopanaxatriol were 17.1 +/- 2.0 min and 3.46 +/- 0.33 h, respectively.

Sensitive determination of 20(S)-protopanaxadiol in rat plasma using HPLC-APCI-MS: application of pharmacokinetic study in rats.

20(S)-Protopanaxadiol (PPD), the main metabolite of protopanoxadiol type ginsenosides (e.g. Rg3 and Rh2), is a very promising anti-cancer drug candidate. To evaluate the pharmacokinetic property of PPD, we reported a reliable, sensitive and simple method utilizing liquid chromatography (HPLC)-atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) to determine PPD. PPD and the internal standard, panoxadiol (PD) were extracted from plasma with acetic ether, separated on a C18 reverse column, and then analyzed by APCI-MS. Targeting fragment ion at m/z 425 for both PPD and PD was monitored in selected-ion monitoring (SIM) mode. PPD can be quantitatively determined at the concentration as low as 1 ng/mL using 200 microL plasma. And the sensitive method showed excellent linearity over a range from 1 to 1000 ng/mL, high recovery, accuracy and precision at the concentrations of 2.5, 100.0 and 1000.0 ng/mL, respectively. The method was successfully applied to pharmacokinetic study of PPD in rats. Pharmacokinetic parameters were calculated and absolute bioavailability of PPD was 36.8+/-12.4%, at least ten times higher than that of Rg3 and Rh2, indicating its good absorption in gastrointestinal tract. It was further suggested that PPD be a promising anti-cancer candidate and probably responsible for the observed pharmacological activity of Rg3 and Rh2.

Gas chromatography/time-of-flight mass spectrometry based metabonomic approach to differentiating hypertension- and age-related metabolic variation in spontaneously hypertensive rats.

Metabonomics is a systematic approach to the study of in vivo metabolic profiles and therefore allows deep insight into and a better understanding of the pathogenesis of disease. To characterize the development of hypertension, a hypertensive animal model, the spontaneously hypertensive rat (SHR), and its normotensive control, the Wistar Kyoto (WKY) rat, were investigated and their blood plasma analyzed using the high-throughput metabolomic tool, gas chromatography/time-of-flight mass spectrometry (GC/TOFMS). A total of 187 peaks were quantitatively determined after deconvolution, and 78 of them were identified. Principal components analysis (PCA) and projection to latent structure partial least-squares discriminant analysis (PLS-DA) were used to process the GC/TOFMS data. The resulting mathematical models were further validated by cross-validation. Plasma compositional differences of many identified compounds showed hypertension-related variation between SHR and WKY rats, and age-related changes from 10 to 18 weeks for both the SHR and WKY rats. These compositional changes involved compounds such as hexadecanoic acid, linoleic acid, oleic acid, stearic acid, 3-hydroxybutyric acid, citric acid, threonic acid, tyrosine, tryptophan, threonine, phenylalanine, serine, ornithine, methionine, 3-hydroxyproline, creatinine, erythrose, myo-inositol, D-methylglucopyranoside, tocopherol, sitosterol, and nonesterified cholesterol. Significantly elevated free fatty acids (FFA) were observed in SHR relative to those in WKY rats, and their levels increased as the SHR aged from 10 to 18 weeks. The close correlation between FFA and hypertension suggests that FFA are potential biomarker candidates for hypertension and they may play an important role in the development of hypertension in SHR. It is also indicated that GC/TOFMS-based metabonomics is a powerful approach to identifying potential biomarkers and investigating the pathological processes of hypertension and the physiological developments of aging.

Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry.

Sediment in urine may contain low-molecular-weight compounds that should be included in the analysis. To date, no systematic investigation has addressed this issue. We investigated three primary factors that influence the extraction efficiency of metabolites during preparation of urine samples for metabolomic research: centrifugation, pH, and extraction solvents. Obtained with the use of gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) technique and principal component analysis (PCA), our results indicate that (1) conventional centrifugation causes an apparent loss of some metabolites, indicating that urine samples for metabolomic research should not be centrifuged before procedures are undertaken to recover the metabolites; (2) pH adjustment has a large impact on the recovery of metabolites and is therefore not encouraged; (3) with design of experiment analysis, methanol and water yield the optimal extraction efficiency. Differences between rat and human urine were observed and are discussed. Ninety-nine metabolites identified in rat and human urine are presented. An efficient protocol is proposed for the pretreatment of urine samples.

Simultaneous determination of GFA and its active metabolites in human plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies.

A sensitive and rapid liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for simultaneous quantification of guanfu base A (GFA) and its metabolites guanfu base I (GFI) and guanfu alcohol-amine (AA) in human plasma with phenoprolamine hydrochloride (DDPH) as the internal standard. The analytes were extracted from human plasma by using liquid-liquid extraction with ethyl acetate and the LC separation was performed on a Diamonsil C(18) analytical column (150 mm x 2.1 mm i.d., 5 microm). The MS acquisition was performed in selected ion monitoring (SIM) mode of positive ions. Analysis was carried out in SIM mode at m/z 430.25 for GFA [M+H](+), m/z 388.25 for GFI [M+H](+), m/z 346.25 for AA [M+H](+) and m/z 344.20 for the IS DDPH [M+H](+). The calibration curves were linear over the range of 50-5000 ng/mL for GFA and 5-1000 ng/mL for GFI and AA, with coefficients of correlation above 0.999. The lower limit of quantification for GFA was 1 ng/mL, while for GFI and AA were both 5 ng/mL. The intra- and inter-day precisions (CV) of analysis were within 9%, and the accuracy ranged from 91% to 108%. The overall recoveries for GFA, GFI and AA were about 94.2%, 87.8% and 80.6%, respectively. The total LC-MS run-time was only 5.5 min. This quantitation method was successfully applied to the simultaneous determination of GFA and its metabolites in human plasma for the metabolic study and pharmacokinetic evaluation.

Pharmacokinetic study with N-Ile1-Thr2-63-desulfato-r-hirudin in rabbits by means of bioassay.

AIM: To study the pharmacokinetic (PK) properties in rabbits treated with N-Ile(1)-Thr(2)-63-desulfato-r-hirudin (rH) newly developed in China by means of bioassay in order to provide preclinical experiment basis for its development as a novel anticoagulant agent. METHODS: rH plasma concentration was determined using bioassay based on ex vivo antithrombin activity of rH. Normal rabbits received iv rH 4.0, 2.0 and 1.0 mg/kg or sc rH 2.0 mg/kg, respectively. The rabbits with acute severe renal failure were given iv rH 2.0 mg/kg. RESULTS: The bioassay described in this paper met requirements for study of PK in rabbits. The major PK parameters after iv dosing were as follows: t(1/2beta) 58.4-59 min. V(d) 0.09-0.12 L/kg, CL 0.0035-0.0040 L/(kg.min); AUC were proportional to the doses, t(1/2) and CL did not change significantly with the doses. The sc bioavailability reached 94%. The rabbits suffering from acute severe renal failure presented 11-fold longer t(1/2beta) and 13-fold greater AUC than normal healthy rabbits. CONCLUSION: rH exhibited rapid elimination, distribution was only limited to extracellular space and good absorption from sc site. The excretion of rH by kidneys played a very important role in the elimination of rH. The PK of rH could be described by the two- and one-compartment model after iv and sc dosing, respectively, and followed linear kinetics.