METABOLIC AND PHARMACOKINETIC STUDIES OF BENZTROPINE AND TRIHEXYPHENIDYL

Benz trop ine (3-diphenylmethoxytropine) and trihexyphenidy1(1-cyclohexyl-phenyl-3-piperidinopropan-1-ol) are synthetic anticholinergic drugs used in the treatment of Parkinson's disease and neuroleptic-induced Parkinsonism. Although the drugs have been used clinically for many years, their pharmacokinetics have not been previously determined. This seems to be due to the absence of sensitive and specific methods for assaying the drugs in biological fluids. Therefore, it was an analytical challenge to develop very sensitive and specific methods to study pharmacokinetics for benztropine and trihexyphenidyl. Radioimmunoassay (RIA) as an alternative analytical procedure to chemical analyses was thought to provide the ultrasensitivity required for trace analysis of these drugs. The assessment of specificity in RIA and development of drug specific antibodies require knowledge of the metabolic profile of the drug(s) in question. Therefore, a systematic investigation of the metabolism of each benztropine and trihexyphenidyl was undertaken. Following oral administration of benztropine, phase-I metabolites, benztropine N-oxide, N-desmethylbenztropine, tropine, 4'-hydroxybenztropine, N-desmethy1-4'- hydroxybenztropine, 4 ' -hydroxybenztropine N-oxide and methoxy- 4'-hydroxybenztropine, together with unmetabolized ivbenztropine, were isolated and identified in rat urine and bile by GC-electron impact mass spectrometry i(EI GC/MS), micro-column LC-electrospray mass spectrometry (ES LC/MS) and HPLC followed by mass analysis. In addition, four intact glucuronides corresponding to the hydroxylated metabolites of benztropine were also identified by HPLC and ES/MS analysis. The same metabolites were also identified in human plasma and urine. These results provided the first systematic report of the metabolic profile of benztropine in rats and humans. 1-Hydroxycyclohexyl-phenyl-3-piperidinopropan-l-ol (hydroxytrihexyphenidyl) and trihexyphenidyl N-oxide were detected in human plasma and rat bile after oral dosing of trihexyphenidyl by direct comparison with authentic samples. 1-Keto-cyclohexyl-phenyl-3-piperidinopropan-l-ol (keto- trihexyphenidyl), 1-keto-hydroxycyclohexyl-pheny1-3- piperidinopropan-l-ol (keto- hydroxytrihexyphenidyl), 1- dihydroxycyclohexyl-phenyl-3-piperidinopropan-l-ol (trihexyphenidyl with two hydroxyl groups), 1-cyclohexyl-l-pheny1-1-hydroxyl propanal and 1-cyclohexyl-l-pheny1-1-hydroxyl propanoic acid were also identified by using EI GC/MS, ES LC/MS and HPLC. In addition, the LC/MS with positive and negative ES ionizations combined with a HPLC system was successfully employed in the analysis of the intact glucuronides and sulfates of hydroxylated metabolites. This is the first comprehensive report on metabolism of trihexyphenidyl in rats and humans beyond previous work which identified hydroxylated trihexyphenidyl in human urine. Two haptens, benztropine hemisuccinate and trihexyphenidyl hemisuccinate, were synthesized for the development of RIA procedures for benztropine and trihexyphenidyl, respectively. The haptens were coupled to bovine serum albumin or porcine thyroglobulin to obtain the respective hapten-protein conjugates. Polyclonal antibodies to each hapten-protein conjugate were raised in New Zealand white rabbits and the titers of the developed antisera were determined by evaluating their binding to the iappropriate, tritiated analyte. The respective antiserum, which had the highest titer and minimal cross-reactivities to major metabolites and the antipsychotic drugs such as fluphenazine, flupenthixol, chlorpromazine and haloperidol, was selected for the development of RIAs. The developed RIAs for benztropine and trihexyphenidyl were sensitive enough that they can quantitate down to 31 and 7.8 pg, respectively, using a 200 gL plasma sample. Each of the developed RIA procedures was then applied to the analysis of steady state plasma samples obtained from patients undergoing treatment with benztropine or trihexyphenidyl and plasma samples obtained from healthy volunteers after administration of a single 4-mg oral dose of vibenztropine or trihexyphenidyl. The concentration vs. time curves obtained indicated that benztropine may undergo enterohepatic recycling. The pharmacokinetic parameters for each drug in human volunteers were also estimated based on either non-compartmental model or compartmental model analyses. The RIAs developed in this work are by far the most sensitive methods for the analysis of benztropine and trihexyphenidyl. They are the only methods 1 capable of investigating the pharmacokinetics of these drugs in humans after low single oral doses

Transcriptomics and metabolomics analyses provide insights into resistance genes of tree ferns

As ancient organisms, tree ferns play a crucial role as an evolutionary bridge between lower and higher plant species, providing various utilitarian benefits. However, they face challenges such as overexploitation, climate change, adverse environmental conditions, and insect pests, resulting in conservation concerns. In this study, we provide an overview of metabolic and transcriptomic resources of leaves in two typical tree ferns, A. spinulosa and A. metteniana, and explore the resistance genes for the first time. The landscape of metabolome showed that the compound skimmin may hold medicinal significance. A total of 111 differentially accumulated metabolites (DAMs) were detected, with pathway enrichment analysis highlighting 14 significantly enriched pathways, including 2-oxocarboxylic acid metabolism possibly associated with environmental adaptations. A total of 14,639 differentially expressed genes (DEGs) were found, among which 606 were resistance (R) genes. We identified BAM1 as a significantly differentially expressed R gene, which is one of the core genes within the R gene interaction network. Both the maximum-likelihood phylogenetic tree and the PPI network revealed a close relationship between BAM1, FLS2, and TMK. Moreover, BAM1 showed a significant positive correlation with neochlorogenic acid and kaempferol-7-O-glucoside. These metabolites, known for their antioxidant and anti-inflammatory properties, likely play a crucial role in the defense response of tree ferns. This research provides valuable insights into the metabolic and transcriptomic differences between A. spinulosa and A. metteniana, enhancing our understanding of resistance genes in tree ferns

A Hybrid Wavelet de-noising and Rank-Set Pair Analysis approach for forecasting hydro-meteorological time series

Accurate, fast forecasting of hydro-meteorological time series is presently a major challenge in drought and flood mitigation. This paper proposes a hybrid approach, wavelet de-noising (WD) and Rank-Set Pair Analysis (RSPA), that takes full advantage of a combination of the two approaches to improve forecasts of hydro-meteorological time series. WD allows decomposition and reconstruction of a time series by the wavelet transform, and hence separation of the noise from the original series. RSPA, a more reliable and efficient version of Set Pair Analysis, is integrated with WD to form the hybrid WD-RSPA approach. Two types of hydro-meteorological data sets with different characteristics and different levels of human influences at some representative stations are used to illustrate the WD-RSPA approach. The approach is also compared to three other generic methods: the conventional Auto Regressive Integrated Moving Average (ARIMA) method, Artificial Neural Networks (ANNs) (BP-error Back Propagation, MLP-Multilayer Perceptron and RBF-Radial Basis Function), and RSPA alone. Nine error metrics are used to evaluate the model performance. Compared to three other generic methods, the results generated by WD-REPA model presented invariably smaller error measures which means the forecasting capability of the WD-REPA model is better than other models. The results show that WD-RSPA is accurate, feasible, and effective. In particular, WD-RSPA is found to be the best among the various generic methods compared in this paper, even when the extreme events are included within a time series

Analysis of proliferative activity in oral gingival epithelium in immunosuppressive medication induced gingival overgrowth

BACKGROUND: Drug-induced gingival overgrowth is a frequent adverse effect associated principally with administration of the immunosuppressive drug cyclosporin A and also certain antiepileptic and antihypertensive drugs. It is characterized by a marked increase in the thickness of the epithelial layer and accumulation of excessive amounts of connective tissue. The mechanism by which the drugs cause gingival overgrowth is not yet understood. The purpose of this study was to compare proliferative activity of normal human gingiva and in cyclosporine A-induced gingival overgrowth. METHODS: Gingival samples were collected from 12 generally healthy individuals and 22 Cyclosporin A-medicated renal transplant recipients. Expression of proliferating cell nuclear antigen was evaluated in formalin-fixed, paraffin-embedded gingival samples using an immunoperoxidase technique and a monoclonal antibody for this antigen. RESULTS: There were differences between the Cyclosporin A group and control group in regard to proliferating cell nuclear antigen and epithelial thickness. In addition, the degree of stromal inflammation was higher in the Cyclosporin A group when compared with the control group. CONCLUSION: The results suggest that the increased epithelial thickness observed in Cyclosporin A-induced gingival overgrowth is associated with increased proliferative activity in keratinocytes

IMI – Oral Biopharmaceutics Tools project – Evaluation of Bottom-up PBPK Prediction Success Part 2: An Introduction to the Simulation Exercise and Overview of Results

Orally administered drugs are subject to a number of barriers impacting bioavailability (F-oral), causing challenges during drug and formulation development. Physiologically-based pharmacokinetic (PBPK) modelling can help during drug and formulation development by providing quantitative predictions through a systems approach. The performance of three available PBPK software packages (GI-Sim, Simcyp (R), and GastroPlus (TM)) were evaluated by comparing simulated and observed pharmacokinetic (PK) parameters. Since the availability of input parameters was heterogeneous and highly variable, caution is required when interpreting the results of this exercise. Additionally, this prospective simulation exercise may not be representative of prospective modelling in industry, as API information was limited to sparse details. 43 active pharmaceutical ingredients (APIs) from the OrBiTo database were selected for the exercise. Over 4000 simulation output files were generated, representing over 2550 study arm-institution-software combinations and approximately 600 human clinical study arms simulated with overlap. 84% of the simulated study arms represented administration of immediate release formulations, 11% prolonged or delayed release, and 5% intravenous (i.v.). Higher percentages of i.v. predicted area under the curve (AUC) were within two-fold of observed (52.9%) compared to per oral (p.o.) (37.2%), however, F-oral and relative AUC (F-rel) between p.o. formulations and solutions were generally well predicted (64.7% and 75.0%). Predictive performance declined progressing from i.v. to solution and immediate release tablet, indicating the compounding error with each layer of complexity. Overall performance was comparable to previous large-scale evaluations. A general overprediction of AUC was observed with average fold error (AFE) of 1.56 over all simulations. AFE ranged from 0.0361 to 64.0 across the 43 APIs, with 25 showing overpredictions. Discrepancies between software packages were observed for a few APIs, the largest being 606, 171, and 81.7-fold differences in AFE between SimCYP and GI-Sim, however average performance was relatively consistent across the three software platforms.</p

IMI – Oral biopharmaceutics tools project – Evaluation of bottom-up PBPK prediction success part 3: Identifying gaps in system parameters by analysing In Silico performance across different compound classes

Three Physiologically Based Pharmacokinetic software packages (GI-Sim, Simcyp (R) Simulator, and GastroPlus (TM)) were evaluated as part of the Innovative Medicine Initiative Oral Biopharmaceutics Tools project (OrBiTo) during a blinded "bottom-up" anticipation of human pharmacokinetics. After data analysis of the predicted vs. measured pharmacokinetics parameters, it was found that oral bioavailability (F-oral) was underpredicted for compounds with low permeability, suggesting improper estimates of intestinal surface area, colonic absorption and/or lack of intestinal transporter information. Foralwas also underpredicted for acidic compounds, suggesting overestimation of impact of ionisation on permeation, lack of information on intestinal transporters, or underestimation of solubilisation of weak acids due to less than optimal intestinal model pH settings or underestimation of bile micelle contribution. F-oral was overpredicted for weak bases, suggesting inadequate models for precipitation or lack of in vitro precipitation information to build informed models. Relative bioavailability was underpredicted for both high logP compounds as well as poorly water-soluble compounds, suggesting inadequate models for solubility/dissolution, underperforming bile enhancement models and/or lack of biorelevant solubility measurements. These results indicate areas for improvement in model software, modelling approaches, and generation of applicable input data. However, caution is required when interpreting the impact of drug-specific properties in this exercise, as the availability of input parameters was heterogeneous and highly variable, and the modellers generally used the data "as is" in this blinded bottom-up prediction approach.</p

METABOLIC AND PHARMACOKINETIC STUDIES OF BENZTROPINE AND TRIHEXYPHENIDYL

Benz trop ine (3-diphenylmethoxytropine) and trihexyphenidy1(1-cyclohexyl-phenyl-3-piperidinopropan-1-ol) are synthetic anticholinergic drugs used in the treatment of Parkinson's disease and neuroleptic-induced Parkinsonism. Although the drugs have been used clinically for many years, their pharmacokinetics have not been previously determined. This seems to be due to the absence of sensitive and specific methods for assaying the drugs in biological fluids. Therefore, it was an analytical challenge to develop very sensitive and specific methods to study pharmacokinetics for benztropine and trihexyphenidyl. Radioimmunoassay (RIA) as an alternative analytical procedure to chemical analyses was thought to provide the ultrasensitivity required for trace analysis of these drugs. The assessment of specificity in RIA and development of drug specific antibodies require knowledge of the metabolic profile of the drug(s) in question. Therefore, a systematic investigation of the metabolism of each benztropine and trihexyphenidyl was undertaken. Following oral administration of benztropine, phase-I metabolites, benztropine N-oxide, N-desmethylbenztropine, tropine, 4'-hydroxybenztropine, N-desmethy1-4'- hydroxybenztropine, 4 ' -hydroxybenztropine N-oxide and methoxy- 4'-hydroxybenztropine, together with unmetabolized ivbenztropine, were isolated and identified in rat urine and bile by GC-electron impact mass spectrometry i(EI GC/MS), micro-column LC-electrospray mass spectrometry (ES LC/MS) and HPLC followed by mass analysis. In addition, four intact glucuronides corresponding to the hydroxylated metabolites of benztropine were also identified by HPLC and ES/MS analysis. The same metabolites were also identified in human plasma and urine. These results provided the first systematic report of the metabolic profile of benztropine in rats and humans. 1-Hydroxycyclohexyl-phenyl-3-piperidinopropan-l-ol (hydroxytrihexyphenidyl) and trihexyphenidyl N-oxide were detected in human plasma and rat bile after oral dosing of trihexyphenidyl by direct comparison with authentic samples. 1-Keto-cyclohexyl-phenyl-3-piperidinopropan-l-ol (keto- trihexyphenidyl), 1-keto-hydroxycyclohexyl-pheny1-3- piperidinopropan-l-ol (keto- hydroxytrihexyphenidyl), 1- dihydroxycyclohexyl-phenyl-3-piperidinopropan-l-ol (trihexyphenidyl with two hydroxyl groups), 1-cyclohexyl-l-pheny1-1-hydroxyl propanal and 1-cyclohexyl-l-pheny1-1-hydroxyl propanoic acid were also identified by using EI GC/MS, ES LC/MS and HPLC. In addition, the LC/MS with positive and negative ES ionizations combined with a HPLC system was successfully employed in the analysis of the intact glucuronides and sulfates of hydroxylated metabolites. This is the first comprehensive report on metabolism of trihexyphenidyl in rats and humans beyond previous work which identified hydroxylated trihexyphenidyl in human urine. Two haptens, benztropine hemisuccinate and trihexyphenidyl hemisuccinate, were synthesized for the development of RIA procedures for benztropine and trihexyphenidyl, respectively. The haptens were coupled to bovine serum albumin or porcine thyroglobulin to obtain the respective hapten-protein conjugates. Polyclonal antibodies to each hapten-protein conjugate were raised in New Zealand white rabbits and the titers of the developed antisera were determined by evaluating their binding to the iappropriate, tritiated analyte. The respective antiserum, which had the highest titer and minimal cross-reactivities to major metabolites and the antipsychotic drugs such as fluphenazine, flupenthixol, chlorpromazine and haloperidol, was selected for the development of RIAs. The developed RIAs for benztropine and trihexyphenidyl were sensitive enough that they can quantitate down to 31 and 7.8 pg, respectively, using a 200 gL plasma sample. Each of the developed RIA procedures was then applied to the analysis of steady state plasma samples obtained from patients undergoing treatment with benztropine or trihexyphenidyl and plasma samples obtained from healthy volunteers after administration of a single 4-mg oral dose of vibenztropine or trihexyphenidyl. The concentration vs. time curves obtained indicated that benztropine may undergo enterohepatic recycling. The pharmacokinetic parameters for each drug in human volunteers were also estimated based on either non-compartmental model or compartmental model analyses. The RIAs developed in this work are by far the most sensitive methods for the analysis of benztropine and trihexyphenidyl. They are the only methods 1 capable of investigating the pharmacokinetics of these drugs in humans after low single oral doses

Abstract 5466: Efficacy of ASC60, an oral fatty acid synthase inhibitor, in two tumor mouse models

Abstract Background: Fatty acid synthase (FASN) is a key enzyme controlling endogenous lipid biosynthesis and overexpressed in many cancers. Studies have shown that FASN inhibition can disrupt lipid synthesis in tumor cells and disrupt tumor-associated signal transduction, suggesting FASN inhibition can serve as a potential treatment for cancer. Here we report the in vivo efficacy data of ASC60, a small molecular FASN inhibitor, either as a single agent or in combination with mouse programmed cell death-1 (mPD-1) antibody in two tumor mouse models. Methods: In the MC38 mouse model, mice were inoculated subcutaneously with MC38 cells. When the average tumor volume reached approximately 62 mm3, mice were treated with ASC60 (60, and 100 mg/kg) or vehicle once a day (QD) for 3 weeks in combination mPD-1 antibody twice a week (1 mg/kg, BIW) for 2 weeks. In patient derived orthotopic xenograft (PDOX) brain metastasis mouse model, luciferase labelled breast cancer cells (LD1-2009-362541-Luc) were transplanted into the brain of nude mice. Tumor volumes were estimated by the luciferase reporter signal. When the average signal reached 4.12X107 photon/sec, mice were treated with ASC60 (20, 60, and 100 mg/kg) or vehicle QD for 4 weeks. In both mouse models, body weights and tumor volumes were measured regularly, and tumor growth inhibitions (TGI) of different groups were compared. Results: All animals showed a gradually increase in body weight during the study and no significant difference of body weight or body weight change was found among different groups. In the MC38 colon cancer mouse model, mice treated with ASC60 (60 or 100 mg/kg) in combination with mPD-1 antibody showed higher TGI values compared to those treated with mPD-1 alone (91.86%, 99.88% vs 89.84%) on Day 13, indicating combination with ASC60 would enhance the antitumor activity of mPD-1 antibody. In PDOX mouse model, mice treated with ASC60 (20, 60, and 100 mg/kg) showed dose dependent TGI values of 10.83%, 36.95% and 49.21% on Day 13 and of 6.46%, 21.27% and 30.47% on Day 27, respectively. ASC60 also showed a significant improvement in life span (51.5 days in ASC60 60 mg/kg vs 39 days in vehicle control). Conclusion: Previous in vitro testing showed that inhibition of palmitate synthesis by ASC60 was 137-fold lower in mouse than in human (IC50: 2.05 µM in mouse vs 0.015 µM in human). However, results of the present studies showed that ASC60 could suppress tumor growth and/or expand lifespan in the two tumor mouse models either alone or in combination with mPD-1 antibody. ASC60 will possibly demonstrate better efficacies in human. Citation Format: Jinzi J. Wu, Handan He. Efficacy of ASC60, an oral fatty acid synthase inhibitor, in two tumor mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5466.</jats:p

Abstract 5529: <i>In vivo</i> efficacy evaluation of ASC61, an oral PD-L1 inhibitor, in two tumor mouse models

Abstract Background: ASC61 is an oral inhibitor prodrug of programmed cell death-ligand 1 (PD-L1). ASC61 is converted to its pharmacologically active metabolite ASC61-A in the plasma. In vitro studies showed that ASC61-A could activate PD-L1 dimerization and membrane PD-L1 protein internalization, interfere PD-1/PD-L1 interaction and enhance T-cell activation. Here we report the in vivo efficacy of ASC61 in two tumor mouse models. ASC61 was found to have the best antitumor activity among all test drugs in the syngeneic tumor mouse model, BALB/c mice bearing CT-26-hPD-L1 tumors. Then a humanized mouse model, PD-1/PD-L1 dKI HuGEMM bearing hPD-L1 MC38 tumor, was used to further validate the therapeutic efficacy of ASC61. Methods: In the BALB/c mouse model, mice were inoculated subcutaneously with CT-26-hPD-L1 cells. When the average tumor volume reached approximately 69 mm3, mice were randomly assigned and treated twice a day (BID) with vehicle, reference drug (Atezolizumab), ASC61, GLC01-589 or GLC01-633 for 19 days. In the PD-1/PD-L1 dKI HuGEMM mouse model, MC38 hPD-L1 cells were subcutaneously inoculated in the right rear flank region of mice. Once the mean tumor size reached approximately 78.3 mm3, mice were randomized and treated BID for 16 days with vehicle, Atezolizumab, ASC61 (50 or 100 mg/kg). In both models, body weights and tumor volumes were measured regularly, and tumor growth inhibitions (TGI) of different groups were compared. Results: All mice showed a gradually increase in body weight during the study and no significant difference of body weight or body weight change was found among different groups in both mouse models. In the BALB/c mouse model, ASC61 50 mg/kg showed significantly inhibitory effects on the tumor growth with the best TGI value of 52.9% (p &amp;lt; 0.05) on Day 19, even better than that of the reference drug (40.77%). In the PD-1/PD-L1 dKI HuGEMM mouse model, Group 2 to 4 showed TGI values of 69.62%, 33.62% and 63.15% on Day 16, respectively. Atezolizumab 5 mg/kg BIW and ASC61 100 mg/kg BID produced similar significant antitumor efficacies (both p &amp;lt; 0.001). Survival analysis showed significant differences between Group 1 and Group 2 to 4 (p = 0.0002, 0.0338 and 0.0027, respectively). Conclusion: Results of this study show that ASC61 is well tolerated in both mouse models, and ASC61 has comparable antitumor efficacies as the FDA-approved PD-L1 antibody, Atezolizumab. ASC61 is an oral PD-L1 inhibitor, and thus will have better patient compliance than injective PD-1/PD-L1 antibody. ASC61 first in human clinical trial will begin soon. Citation Format: Jinzi J. Wu, Handan He. In vivo efficacy evaluation of ASC61, an oral PD-L1 inhibitor, in two tumor mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5529.</jats:p

Isoflurane Preconditioning Protects the Myocardium Against Ischemia and Reperfusion Injury by Upregulating GRM1 Expression

Background:Reduction in myocardial I/R injury has become the key to the therapy of ischemic cardiovascular disease. Isoflurane (ISO) preconditioning can mimic the major potent protective mechanisms and attenuate ischemia injury. Nevertheless, the mechanisms involved in the cardioprotective effects afforded by isoflurane preconditioning have never been evaluated systematically.Methods:Mice were randomly divided into an ISO preconditioning group and control group. The size of the infarcted region was measured, and comparisons between ISO preconditioning and control animals were made. The metabotropic glutamate receptor type 1(GRM1) expression levels in all groups were determined by quantitative PCR. GRM1 protein expression and DNA damage relative protein γ-H2AX were measured by western blot analysis. The oxidative stress was detected by immunofluorescence after staining with the Dihydroethidium (DHE).Results:ISO preconditioning significantly reduced the IR induced infarct volumes and reversed the GRM1 protein expression level in I/R induced myocardial injury. Moreover, ISO preconditioning has a protective effect in reducing the I/R induced DNA damage and oxidative stress.Conclusion:The results of the present study have demonstrated that the expression of GRM1 provides a protective role in ISO preconditioning against I/R-induced myocardial infarction by reducing the oxidative stress and DNA damage.</jats:sec