Pub Date : 2023-03-01DOI: 10.1016/j.vascn.2023.107253
David Holdsworth , Derek D. Best , Katarina Haist , Kyle O'Donohue , Anson Phillips , Matthew M. Abernathy , Brian Roche , Derek J. Leishman
The number of animals used in a nonhuman primate (NHP) in vivo QTc assessment conducted as part of the safety pharmacology (SP) studies on a potential new drug is relatively small (4–8 subjects). The number is much smaller than the number of healthy volunteers in a conventional thorough QT (TQT) study (40–60 volunteers). How is it possible that such small studies could offer an equivalent sensitivity in an integrated nonclinical and clinical cardiac repolarization risk assessment?
This study provided the opportunity to empirically demonstrate in a large number of NHPs the performance of a nonclinical evaluation at a similar size to a TQT study.
By contrasting an analysis mimicking the sampling and aggregation of QTc interval data in a manner which is TQT-like with a more conventional SP-like analysis it was demonstrated that the SP-like analysis was more sensitive. In prospective power calculations 80% power at p = 0.05 can be achieved for a 5 ms QTc change with only n = 8 NHPs using the SP-like analysis and in a group of only 4 NHPs 80% power to detect 10 ms could be achieved. By contrast groups of 24 NHPs would be required to achieve 80% power to detect 5 ms using the TQT-like sampling and aggregation approach.
Overall, this study has demonstrated that smaller safety pharmacology in vivo QTc assessments using all the available data in larger data aggregates can achieve sensitivity comparable to a human TQT study.
作为潜在新药安全药理学(SP)研究的一部分,用于非人灵长类动物(NHP)体内QTc评估的动物数量相对较少(4-8个受试者)。这个数字远远小于传统的全面QT (TQT)研究中健康志愿者的数量(40-60名志愿者)。这么小的研究怎么可能在综合的非临床和临床心脏复极风险评估中提供同等的敏感性?本研究提供了一个机会,在大量的NHPs中实证证明了与TQT研究规模相似的非临床评估的表现。通过对比以类似tqt的方式模拟QTc区间数据的采样和聚合的分析,与更传统的类似sp的分析相比,表明类似sp的分析更敏感。在前瞻性功率计算中,使用类似sp的分析,在只有n = 8个NHPs的情况下,对于5 ms QTc变化,在p = 0.05时可以获得80%的功率,而在只有4个NHPs的情况下,可以获得80%的功率来检测10 ms。相比之下,使用类似tqt的采样和聚合方法,需要24个NHPs组才能达到80%的功率来检测5 ms。总的来说,这项研究表明,使用所有可用数据进行更大数据集的体内QTc安全性药理学评估可以达到与人类TQT研究相当的灵敏度。
{"title":"Comparison of validity of standard nonclinical group size selection versus standard clinical group sizes for nonhuman primate QTc prolongation evaluation","authors":"David Holdsworth , Derek D. Best , Katarina Haist , Kyle O'Donohue , Anson Phillips , Matthew M. Abernathy , Brian Roche , Derek J. Leishman","doi":"10.1016/j.vascn.2023.107253","DOIUrl":"10.1016/j.vascn.2023.107253","url":null,"abstract":"<div><p>The number of animals used in a nonhuman primate (NHP) in vivo QTc assessment conducted as part of the safety pharmacology (SP) studies on a potential new drug is relatively small (4–8 subjects). The number is much smaller than the number of healthy volunteers in a conventional thorough QT (TQT) study (40–60 volunteers). How is it possible that such small studies could offer an equivalent sensitivity in an integrated nonclinical and clinical cardiac repolarization risk assessment?</p><p>This study provided the opportunity to empirically demonstrate in a large number of NHPs the performance of a nonclinical evaluation at a similar size to a TQT study.</p><p>By contrasting an analysis mimicking the sampling and aggregation of QTc interval data in a manner which is TQT-like with a more conventional SP-like analysis it was demonstrated that the SP-like analysis was more sensitive. In prospective power calculations 80% power at <em>p</em> = 0.05 can be achieved for a 5 ms QTc change with only <em>n</em> = 8 NHPs using the SP-like analysis and in a group of only 4 NHPs 80% power to detect 10 ms could be achieved. By contrast groups of 24 NHPs would be required to achieve 80% power to detect 5 ms using the TQT-like sampling and aggregation approach.</p><p>Overall, this study has demonstrated that smaller safety pharmacology in vivo QTc assessments using all the available data in larger data aggregates can achieve sensitivity comparable to a human TQT study.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9121097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Y. Koshman, Aimee L. Bielinski, Brandan M. Bird, J. R. Green, K. Kowalkowski, Lai-Zhang Jie, P. K. Mahalingaiah, James W. Sawicki, N. Talaty, Amanda S. Wilsey, Mark T. Zafiratos, T. V. Van Vleet
INTRODUCTION Secondary pharmacology profiling is routinely applied in pharmaceutical drug discovery to investigate the pharmaceutical effects of a drug at molecular targets distinct from (off-target) the intended therapeutic molecular target (on-target). Data from a randomized, placebo-controlled clinical trial, the APPROVe (Adenomatous Polyp Prevention on VIOXX, rofecoxib) trial, raised significant concerns about COX-2 inhibition as a primary or secondary target, shaping the screening and decision-making processes of some pharmaceutical companies. COX-2 is often included in off-target screens due to cardiovascular (CV) safety concerns about secondary interactions with this target. Several potential mechanisms of COX-2-mediated myocardial infarctions have been considered including, effects on platelet stickiness/aggregation, vasal tone and blood pressure, and endothelial cell activation. In the present study, we focused on each of these mechanisms as potential effects of COX-2 inhibitors, to find evidence of mechanism using various in vitro and in vivo preclinical models. METHODS Compounds tested in the study, with a range of COX-2 selectivity, included rofecoxib, celecoxib, etodolac, and meloxicam. Compounds were screened for inhibition of COX-2 vs COX-1 enzymatic activity, ex vivo platelet aggregation (using whole blood from multiple species), ex vivo canine femoral vascular ring model, in vitro human endothelial cell activation (with and without COX-2 induction), and in vivo cardiovascular assessment (anesthetized dog). RESULTS The COX-2 binding assessment generally confirmed the COX-2 selectivity previously reported. COX-2 inhibitors did not have effects on platelet function (spontaneous aggregation or inhibition of aggregation), cardiovascular parameters (mean arterial pressure, heart rate, and left ventricular contractility), or endothelial cell activation. However, rofecoxib uniquely produced an endothelial mediated constriction response in canine femoral arteries. CONCLUSION Our data suggest that rofecoxib-related cardiovascular events in humans are not predicted by COX-2 potency or selectivity. In addition, the vascular ring model suggested possible adverse cardiovascular effects by COX-2 inhibitors, although these effects were not seen in vivo studies. These results may also suggest that COX-2 inhibition alone is not responsible for rofecoxib-mediated adverse cardiovascular outcomes.
{"title":"Disconnect between COX-2 selective inhibition and cardiovascular risk in preclinical models.","authors":"Y. Koshman, Aimee L. Bielinski, Brandan M. Bird, J. R. Green, K. Kowalkowski, Lai-Zhang Jie, P. K. Mahalingaiah, James W. Sawicki, N. Talaty, Amanda S. Wilsey, Mark T. Zafiratos, T. V. Van Vleet","doi":"10.2139/ssrn.4173740","DOIUrl":"https://doi.org/10.2139/ssrn.4173740","url":null,"abstract":"INTRODUCTION\u0000Secondary pharmacology profiling is routinely applied in pharmaceutical drug discovery to investigate the pharmaceutical effects of a drug at molecular targets distinct from (off-target) the intended therapeutic molecular target (on-target). Data from a randomized, placebo-controlled clinical trial, the APPROVe (Adenomatous Polyp Prevention on VIOXX, rofecoxib) trial, raised significant concerns about COX-2 inhibition as a primary or secondary target, shaping the screening and decision-making processes of some pharmaceutical companies. COX-2 is often included in off-target screens due to cardiovascular (CV) safety concerns about secondary interactions with this target. Several potential mechanisms of COX-2-mediated myocardial infarctions have been considered including, effects on platelet stickiness/aggregation, vasal tone and blood pressure, and endothelial cell activation. In the present study, we focused on each of these mechanisms as potential effects of COX-2 inhibitors, to find evidence of mechanism using various in vitro and in vivo preclinical models.\u0000\u0000\u0000METHODS\u0000Compounds tested in the study, with a range of COX-2 selectivity, included rofecoxib, celecoxib, etodolac, and meloxicam. Compounds were screened for inhibition of COX-2 vs COX-1 enzymatic activity, ex vivo platelet aggregation (using whole blood from multiple species), ex vivo canine femoral vascular ring model, in vitro human endothelial cell activation (with and without COX-2 induction), and in vivo cardiovascular assessment (anesthetized dog).\u0000\u0000\u0000RESULTS\u0000The COX-2 binding assessment generally confirmed the COX-2 selectivity previously reported. COX-2 inhibitors did not have effects on platelet function (spontaneous aggregation or inhibition of aggregation), cardiovascular parameters (mean arterial pressure, heart rate, and left ventricular contractility), or endothelial cell activation. However, rofecoxib uniquely produced an endothelial mediated constriction response in canine femoral arteries.\u0000\u0000\u0000CONCLUSION\u0000Our data suggest that rofecoxib-related cardiovascular events in humans are not predicted by COX-2 potency or selectivity. In addition, the vascular ring model suggested possible adverse cardiovascular effects by COX-2 inhibitors, although these effects were not seen in vivo studies. These results may also suggest that COX-2 inhibition alone is not responsible for rofecoxib-mediated adverse cardiovascular outcomes.","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49477363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1016/j.vascn.2022.107231
Madhura R.J. , Varsha A. , Anirban Chakraborthy , Mohana Kumar B. , Veena Shetty A. , Murali Badanthadka
Protein malnutrition continues to be a major global issue. A stable animal model to address protein malnutrition and its effect on various disease conditions is necessary. In the present study, we have formulated and standardized a low protein diet (LPD) to develop a protein malnutrition model using Balb/C mice. Healthy male Balb/C mice were weaned and exposed to LPD combinations while another group exposed to normal diet (18% protein). Animal survival, change in body weight, body mass index (BMI), biochemical parameters, antioxidant status, and liver histopathology were used to confirm the development of malnourished mice model (marasmic-kwashiorkor). Mice receiving 10% protein diet showed moderate weight gain, higher BMI, and no mortality compared to the 6% protein group. The former group showed remarkable differences in BMI, biochemical and antioxidant parameters. Further, histopathological changes against the normal group at weeks 20 and 30 confirmed the development of protein malnutrition in mice on 10% protein diet. The study confirms the development of a stable, economical, reproducible, and clinically relevant protein malnutrition model using the formulated 10% protein diet. Further, the model can be used for short and long-term studies to investigate the pathophysiology of malnutrition in any disease/condition.
{"title":"Protein malnutrition in BALB/C mice: A model mimicking clinical scenario of marasmic-kwashiorkor malnutrition","authors":"Madhura R.J. , Varsha A. , Anirban Chakraborthy , Mohana Kumar B. , Veena Shetty A. , Murali Badanthadka","doi":"10.1016/j.vascn.2022.107231","DOIUrl":"10.1016/j.vascn.2022.107231","url":null,"abstract":"<div><p>Protein malnutrition continues to be a major global issue. A stable animal model to address protein malnutrition and its effect on various disease conditions is necessary. In the present study, we have formulated and standardized a low protein diet (LPD) to develop a protein malnutrition model using Balb/C mice. Healthy male Balb/C mice were weaned and exposed to LPD combinations while another group exposed to normal diet (18% protein). Animal survival, change in body weight, body mass index (BMI), biochemical parameters, antioxidant status, and liver histopathology were used to confirm the development of malnourished mice model (marasmic-kwashiorkor). Mice receiving 10% protein diet showed moderate weight gain, higher BMI, and no mortality compared to the 6% protein group. The former group showed remarkable differences in BMI, biochemical and antioxidant parameters. Further, histopathological changes against the normal group at weeks 20 and 30 confirmed the development of protein malnutrition in mice on 10% protein diet. The study confirms the development of a stable, economical, reproducible, and clinically relevant protein malnutrition model using the formulated 10% protein diet. Further, the model can be used for short and long-term studies to investigate the pathophysiology of malnutrition in any disease/condition.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10808694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1016/j.vascn.2022.107204
Rosemary V. Santos , Siddhartha Bhatt , Stephen Foote , Donna Church , Ricardo Fernandes , Jan Bernal , Laura Singer
Social housing of laboratory rabbits is encouraged and thought to improve animal welfare due to the social nature of this species. However, there is limited published information comparing the physiologic and cardiovascular (CV) effects of paired and single housed adult female rabbits in commonly used laboratory caging. This study describes measurement of heart rate, systolic blood pressure, activity level, body temperature and pairing methods in four female New Zealand White rabbits that were previously implanted with M10 cardiovascular telemetry devices. Data was collected in single housed rabbits having no history of social housing while they were undisturbed in the home cage, during restraint, intramuscular injections and intravenous blood collection. The same animals were then placed in compatible pairs and housed in conventional Allentown caging. As expected, we found increased activity in paired rabbits but no significant differences in body temperatures, and CV parameters in single and paired rabbits undergoing the same procedures. These data suggest that paired rabbits can be used for safety pharmacology studies with minimal impact to data, while supporting improved animal welfare.
{"title":"Method of measuring effects of study procedures in single and pair housed New Zealand White rabbits (Oryctolagus cuniculus)","authors":"Rosemary V. Santos , Siddhartha Bhatt , Stephen Foote , Donna Church , Ricardo Fernandes , Jan Bernal , Laura Singer","doi":"10.1016/j.vascn.2022.107204","DOIUrl":"10.1016/j.vascn.2022.107204","url":null,"abstract":"<div><p>Social housing of laboratory rabbits is encouraged and thought to improve animal welfare due to the social nature of this species. However, there is limited published information comparing the physiologic and cardiovascular (CV) effects of paired and single housed adult female rabbits in commonly used laboratory caging. This study describes measurement of heart rate, systolic blood pressure, activity level, body temperature and pairing methods in four female New Zealand White rabbits that were previously implanted with M10 cardiovascular telemetry devices. Data was collected in single housed rabbits having no history of social housing while they were undisturbed in the home cage, during restraint, intramuscular injections and intravenous blood collection. The same animals were then placed in compatible pairs and housed in conventional Allentown caging. As expected, we found increased activity in paired rabbits but no significant differences in body temperatures, and CV parameters in single and paired rabbits undergoing the same procedures. These data suggest that paired rabbits can be used for safety pharmacology studies with minimal impact to data, while supporting improved animal welfare.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10464413","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1016/j.vascn.2022.107239
Yuji Mano
Denileukin Diftitox (DD), comprising fragments of diphtheria toxin (DT) and interleukin-2 (IL2), was developed for the treatment of lymphoma and has been approved for marketing in Japan. Toxicological evaluation including pharmacokinetics and immunogenicity in preclinical animals is important for drug development and thus the assays of DD and anti-drug antibody (ADA) were developed by electrochemiluminescence (ECL) detection. For the DD assay, ruthenium-labeled anti-DT Ab and biotinylated anti-IL2 Ab were mixed with serum samples and the mixture was captured by streptavidin-coated wells for ECL detection. For the ADA assay, signals of immuno-complex of biotinylated DD, ruthenium-labeled DD, and ADA, bound to streptavidin plate were determined. DD was quantifiable from 10 ng/mL. Accuracy and precision of quality control samples were within ±20% and 20%, respectively, and stability of DD in rat serum was successfully assessed. Precision of positive control samples of ADA was within the acceptance criteria and cut point values for ADA detection in the screening and confirmatory assay were determined by statistical analysis. Drug-induced ADA was detected by screening assay followed by confirmatory assay. The developed method was successfully applied to assess pharmacokinetics and immunogenicity to support toxicity studies in rats.
{"title":"An electrochemiluminescence assay for quantification of Denileukin Diftitox and its anti-drug antibodies in rat serum","authors":"Yuji Mano","doi":"10.1016/j.vascn.2022.107239","DOIUrl":"10.1016/j.vascn.2022.107239","url":null,"abstract":"<div><p>Denileukin Diftitox (DD), comprising fragments of diphtheria toxin (DT) and interleukin-2 (IL2), was developed for the treatment of lymphoma and has been approved for marketing in Japan. Toxicological evaluation including pharmacokinetics and immunogenicity in preclinical animals is important for drug development and thus the assays of DD and anti-drug antibody (ADA) were developed by electrochemiluminescence (ECL) detection. For the DD assay, ruthenium-labeled anti-DT Ab and biotinylated anti-IL2 Ab were mixed with serum samples and the mixture was captured by streptavidin-coated wells for ECL detection. For the ADA assay, signals of immuno-complex of biotinylated DD, ruthenium-labeled DD, and ADA, bound to streptavidin plate were determined. DD was quantifiable from 10 ng/mL. Accuracy and precision of quality control samples were within ±20% and 20%, respectively, and stability of DD in rat serum was successfully assessed. Precision of positive control samples of ADA was within the acceptance criteria and cut point values for ADA detection in the screening and confirmatory assay were determined by statistical analysis. Drug-induced ADA was detected by screening assay followed by confirmatory assay. The developed method was successfully applied to assess pharmacokinetics and immunogenicity to support toxicity studies in rats.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10451571","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1016/j.vascn.2022.107208
Cheryl Tyszkiewicz , Vincent Bernardo , Siddhartha Bhatt , Susan Goody , Todd Wisialowski
Compound-mediated locomotion changes, conducted via open field infrared photobeam breaks, are an important common component of neurological assessments conducted in safety pharmacology studies. In addition to open field locomotor activity assessments, activity data (derived from changes in signal strength) from cardiovascular (CV) telemetry studies can also be an alternative method potentially used to assess locomotor effects. However, comparisons of these two methods have not been extensively characterized. The goal of this work was to compare these two methodologies to assess activity in rats using reference compounds known to have central nervous system (CNS)-stimulant (preladenant) or CNS-depressant (chlorpromazine) effects. Open field activity was conducted using the Kinder Scientific Motor Monitor system and data were collected for 30 min at each drug's expected time of maximum plasma exposure (Tmax). Telemetry-based CV assessment data were continuously acquired using DSI radiotelemetry instrumented animals for 24 h postdose (HPD). Drugs were administered during the lights-on period for both study types. Administration of preladenant caused increases in activity within 0.5–2 HPD for both methods. While administration of chlorpromazine caused decreases in activity in the infrared beam-based open field assessment (1.0–1.5 HPD), there was no effect on telemetry-derived activity during a similar time period. However, telemetry-derived decreases in activity were observed during the lights-off period (16–20 HPD), suggesting CNS-depressant compounds may be mischaracterized if the optimal dose administration time is not selected based on the light/dark cycle and pharmacokinetics. Overall, these results suggest that telemetry-based activity assessment is capable of detecting CNS-stimulant effects of compounds.
{"title":"A comparison of activity data generated from cardiovascular telemetry studies - With quantitative open field locomotor studies in Wistar Han rats","authors":"Cheryl Tyszkiewicz , Vincent Bernardo , Siddhartha Bhatt , Susan Goody , Todd Wisialowski","doi":"10.1016/j.vascn.2022.107208","DOIUrl":"10.1016/j.vascn.2022.107208","url":null,"abstract":"<div><p>Compound-mediated locomotion changes, conducted via open field infrared photobeam breaks, are an important common component of neurological assessments conducted in safety pharmacology studies. In addition to open field locomotor activity assessments, activity data (derived from changes in signal strength) from cardiovascular (CV) telemetry studies can also be an alternative method potentially used to assess locomotor effects. However, comparisons of these two methods have not been extensively characterized. The goal of this work was to compare these two methodologies to assess activity in rats using reference compounds known to have central nervous system (CNS)-stimulant (preladenant) or CNS-depressant (chlorpromazine) effects. Open field activity was conducted using the Kinder Scientific Motor Monitor system and data were collected for 30 min at each drug's expected time of maximum plasma exposure (Tmax). Telemetry-based CV assessment data were continuously acquired using DSI radiotelemetry instrumented animals for 24 h postdose (HPD). Drugs were administered during the lights-on period for both study types. Administration of preladenant caused increases in activity within 0.5–2 HPD for both methods. While administration of chlorpromazine caused decreases in activity in the infrared beam-based open field assessment (1.0–1.5 HPD), there was no effect on telemetry-derived activity during a similar time period. However, telemetry-derived decreases in activity were observed during the lights-off period (16–20 HPD), suggesting CNS-depressant compounds may be mischaracterized if the optimal dose administration time is not selected based on the light/dark cycle and pharmacokinetics. Overall, these results suggest that telemetry-based activity assessment is capable of detecting CNS-stimulant effects of compounds.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10516671","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1016/j.vascn.2022.107209
Prabha Rajput, Sairam Krishnamurthy
Mitochondria is an essential organelle; it produces 95% of the adenine triphosphate (ATP) of cells, their dysfunction is related to the pathogenesis of multiple diseases, such as diabetes mellitus, cardiovascular and neurological disorders. Various pharmacologic agents are known to target mitochondrial function. Moreover, the toxic side effects of multiple drugs used to treat diseases are related to the impairment of mitochondrial function. Thus, there is a need to develop a method to evaluate the effect of pharmacologic agents for their potential and side effects to identify effective mitochondrial-modulating agents. Therefore, the objective of this study was to develop and validate an ex-vivo method for studying the effect of pharmacologic agents on mitochondrial function and rescue of dysfunction. Dimethyl sulfoxide (DMSO) concentrations that drugs were soluble in and maintained mitochondrial function were determined. Metformin (MET) is a known mitochondrial complex-1 inhibitor tested for its ability to compromise mitochondrion function. Coenzyme Q10 (Q10) and Resveratrol (RSV), which are known to enhance mitochondrial function, were added alone and dose-dependent, tested for the ability to rescue metformin-induced mitochondrial dysfunction. Ex-vivo liver and brain mitochondrial function was assessed using an oxytherm Clark-type oxygen electrode. DMSO was found to be toxic above 10% and drugs insoluble below 5%. The addition of 0.5 mg/ml MET decreased liver and brain mitochondrial respiratory control rate (RCR). At the same time, Q10 improved RCR in normal mitochondria and a concentration-dependent manner in MET-induced dysfunctional mitochondria. RSV was added in the last step of the experiment to confirm that compromised function is due to MET. Hence this method can be used to screen pharmacological agents for their potential therapeutics or toxic effect on mitochondria.
{"title":"Standardization and validation of novel ex-vivo method for mitochondrial bioenergetics using mitochondrial modulators","authors":"Prabha Rajput, Sairam Krishnamurthy","doi":"10.1016/j.vascn.2022.107209","DOIUrl":"10.1016/j.vascn.2022.107209","url":null,"abstract":"<div><p>Mitochondria is an essential organelle; it produces 95% of the adenine triphosphate (ATP) of cells, their dysfunction is related to the pathogenesis of multiple diseases, such as diabetes mellitus, cardiovascular and neurological disorders. Various pharmacologic agents are known to target mitochondrial function. Moreover, the toxic side effects of multiple drugs used to treat diseases are related to the impairment of mitochondrial function. Thus, there is a need to develop a method to evaluate the effect of pharmacologic agents for their potential and side effects to identify effective mitochondrial-modulating agents. Therefore, the objective of this study was to develop and validate an <em>ex-vivo</em> method for studying the effect of pharmacologic agents on mitochondrial function and rescue of dysfunction. Dimethyl sulfoxide (DMSO) concentrations that drugs were soluble in and maintained mitochondrial function were determined. Metformin (MET) is a known mitochondrial complex-1 inhibitor tested for its ability to compromise mitochondrion function. Coenzyme Q10 (Q10) and Resveratrol (RSV), which are known to enhance mitochondrial function, were added alone and dose-dependent, tested for the ability to rescue metformin-induced mitochondrial dysfunction. <em>Ex-vivo</em> liver and brain mitochondrial function was assessed using an oxytherm Clark-type oxygen electrode. DMSO was found to be toxic above 10% and drugs insoluble below 5%. The addition of 0.5 mg/ml MET decreased liver and brain mitochondrial respiratory control rate (RCR). At the same time, Q10 improved RCR in normal mitochondria and a concentration-dependent manner in MET-induced dysfunctional mitochondria. RSV was added in the last step of the experiment to confirm that compromised function is due to MET. Hence this method can be used to screen pharmacological agents for their potential therapeutics or toxic effect on mitochondria.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10451020","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1016/j.vascn.2022.107207
Sude Oflu , Sezin Erarpat , Buse Tuğba Zaman , Ömer Tahir Günkara , Sezgin Bakırdere , Fatma Turak
In this study, an analytical method with high accuracy and precision was developed for the determination of methamphetamine in human urine and serum samples by gas chromatography–mass spectrometry (GC–MS). A simultaneous derivatization and spray assisted droplet formation-liquid phase microextraction (SADF-LPME) method was proposed to derivatize and preconcentrate target analyte. Quadruple isotope dilution (ID4) was used to provide high accuracy and precision for methamphetamine determination in the samples. After the optimization studies for the derivatization and microextraction parameters, limit of detection (LOD) and limit of quantitation (LOQ) for the developed SADF-LPME method were found to be 48.0 and 159.9 μg/kg, respectively. Recovery studies were implemented to verify the applicability and accuracy of the developed method for human urine and serum samples. The SADF-LPME method gave low percent recovery results (30.5–61.0%) for the spiked urine and serum samples showing that it failed to minimize or eliminate matrix effects for the analyte. Hence, methamphetamine acetamide-d3 was synthesized and purified in our research laboratory to be used as methamphetamine isotopic analogue in the ID4 method. When the SADF-LPME method was combined with ID4, the percent recovery values for urine and serum samples were calculated as 99.7–100.0% and 99.4–100.2%, respectively. These results demonstrated the applicability and accuracy of the proposed method for urine and serum samples.
{"title":"Combination of quadrupole isotope dilution mass spectrometry with simultaneous derivatization and spray assisted droplet formation-liquid phase microextraction for the determination of methamphetamine in human urine and serum samples by gas chromatography mass spectrometry","authors":"Sude Oflu , Sezin Erarpat , Buse Tuğba Zaman , Ömer Tahir Günkara , Sezgin Bakırdere , Fatma Turak","doi":"10.1016/j.vascn.2022.107207","DOIUrl":"10.1016/j.vascn.2022.107207","url":null,"abstract":"<div><p>In this study, an analytical method with high accuracy and precision was developed for the determination of methamphetamine in human urine and serum samples by gas chromatography–mass spectrometry (GC–MS). A simultaneous derivatization and spray assisted droplet formation-liquid phase microextraction (SADF-LPME) method was proposed to derivatize and preconcentrate target analyte. Quadruple isotope dilution (ID<sup>4</sup>) was used to provide high accuracy and precision for methamphetamine determination in the samples. After the optimization studies for the derivatization and microextraction parameters, limit of detection (LOD) and limit of quantitation (LOQ) for the developed SADF-LPME method were found to be 48.0 and 159.9 μg/kg, respectively. Recovery studies were implemented to verify the applicability and accuracy of the developed method for human urine and serum samples. The SADF-LPME method gave low percent recovery results (30.5–61.0%) for the spiked urine and serum samples showing that it failed to minimize or eliminate matrix effects for the analyte. Hence, methamphetamine acetamide-<em>d3</em> was synthesized and purified in our research laboratory to be used as methamphetamine isotopic analogue in the ID<sup>4</sup> method. When the SADF-LPME method was combined with ID<sup>4</sup>, the percent recovery values for urine and serum samples were calculated as 99.7–100.0% and 99.4–100.2%, respectively. These results demonstrated the applicability and accuracy of the proposed method for urine and serum samples.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10825857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01DOI: 10.1016/j.vascn.2022.107210
Tingting Liu , Yongliang Tong , Jinli Gao , Wenjing Fang , Junliang Wu , Xionghua Peng , Xiaotian Fan , Xiaoqiang Chen , Jianhua Sun , Sheldon Cao , Zhaohui Li , Likun Gong , Qiuping Qin , Dianwen Ju
Immunogenicity has been a major concern in the safety evaluation of therapeutic proteins. The assessment of the unwanted immunogenicity of the therapeutic proteins performed in animals prior to clinical trials has been a regulatory requirement. In preclinical studies of therapeutic proteins, cynomolgus monkeys are usually the most relevant animal species. ZV0203, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently bound to a cytotoxic drug (Duo-5), possesses a novel format of antibody drug conjugates. In this study, we reported the development, validation, and application of a bridging enzyme-linked immunosorbent assay for the detection of antibodies against ZV0203 in cynomolgus monkey serum. Drug interference at low positive control (18.0 ng/mL) and high positive control (130 ng/mL) of anti-ZV0203 antibodies was not observed when ZV0203 concentration is below 1.74 μg/mL and 1.49 μg/mL, respectively. In addition, no interference was found from mouse IgG1, but interference was observed with human IgG1. No effect of hemolysis was found on the analysis results of the testing samples present in 100% pooled rabbit serum containing 2% (V/V) erythrocyte hemolysates. Besides, spiked anti-ZV0203 antibody in rabbit serum was stable after 5 freeze/thaw cycles. The results showed that the method is suitable for the detection of anti-ZV0203 antibodies in cynomolgus monkey serum. The assay was also successfully applied in the repeated dose study of ZV0203.
{"title":"Development of a bridging ELISA for detection of antibodies against ZV0203 in cynomolgus monkey serum","authors":"Tingting Liu , Yongliang Tong , Jinli Gao , Wenjing Fang , Junliang Wu , Xionghua Peng , Xiaotian Fan , Xiaoqiang Chen , Jianhua Sun , Sheldon Cao , Zhaohui Li , Likun Gong , Qiuping Qin , Dianwen Ju","doi":"10.1016/j.vascn.2022.107210","DOIUrl":"10.1016/j.vascn.2022.107210","url":null,"abstract":"<div><p>Immunogenicity has been a major concern in the safety evaluation of therapeutic proteins. The assessment of the unwanted immunogenicity of the therapeutic proteins performed in animals prior to clinical trials has been a regulatory requirement. In preclinical studies of therapeutic proteins, cynomolgus monkeys are usually the most relevant animal species. ZV0203, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently bound to a cytotoxic drug (Duo-5), possesses a novel format of antibody drug conjugates. In this study, we reported the development, validation, and application of a bridging enzyme-linked immunosorbent assay for the detection of antibodies against ZV0203 in cynomolgus monkey serum. Drug interference at low positive control (18.0 ng/mL) and high positive control (130 ng/mL) of anti-ZV0203 antibodies was not observed when ZV0203 concentration is below 1.74 μg/mL and 1.49 μg/mL, respectively. In addition, no interference was found from mouse IgG1, but interference was observed with human IgG1. No effect of hemolysis was found on the analysis results of the testing samples present in 100% pooled rabbit serum containing 2% (<em>V</em>/V) erythrocyte hemolysates. Besides, spiked anti-ZV0203 antibody in rabbit serum was stable after 5 freeze/thaw cycles. The results showed that the method is suitable for the detection of anti-ZV0203 antibodies in cynomolgus monkey serum. The assay was also successfully applied in the repeated dose study of ZV0203.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10451023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Histamine is strongly associated with the onset of allergic conjunctivitis. The most recent cloned histamine H4 receptor antagonist is highly expected as a new therapeutic drug candidate. As a model for a therapeutic drug targeting the histamine H4 receptor, a mouse model in which conjunctivitis symptoms are induced by instilling 4-methylhistamine, a histamine H4 receptor agonist, has been reported. However, the affinity of the H4 receptor for histamine varies in species, and it is known that the histamine binding affinity for the guinea pig H4 receptor is closer to that for human receptor than mice receptor. In this paper, we investigated a possibility that a guinea pig model would become a drug efficacy evaluation model with higher evaluation accuracy than the mouse model.
As a result, hyperemia was observed in the conjunctivae and iris of guinea pigs after instillation of 4-methylhistamine and specifically suppressed by the histamine H4 receptor antagonist. Unlikely to the previously reported mouse model, however, none of edema, increased vascular permeability or scratching behavior was observed, suggesting that there may be differences between mice and guinea pigs not only in the binding affinity of histamine to the H4 receptor but also in the biological reaction to 4-methylhistamine. Although the symptoms of allergic conjunctivitis do not appear comprehensively in the guinea pig model, results of this study indicated a possibility that this model can be used as a simple screening model in the early stages of drug development.
{"title":"Characteristics of histamine H4 receptor agonist-induced allergic conjunctivitis model in Guinea pigs","authors":"Hidemi Mochizuki , Susumu Suyama , So-Young Youm , Pil-Su Ho , Akihito Shimoi","doi":"10.1016/j.vascn.2022.107203","DOIUrl":"https://doi.org/10.1016/j.vascn.2022.107203","url":null,"abstract":"<div><p>Histamine is strongly associated with the onset of allergic conjunctivitis. The most recent cloned histamine H<sub>4</sub> receptor antagonist is highly expected as a new therapeutic drug candidate. As a model for a therapeutic drug targeting the histamine H<sub>4</sub> receptor, a mouse model in which conjunctivitis symptoms are induced by instilling 4-methylhistamine, a histamine H<sub>4</sub> receptor agonist, has been reported. However, the affinity of the H<sub>4</sub> receptor for histamine varies in species, and it is known that the histamine binding affinity for the guinea pig H<sub>4</sub> receptor is closer to that for human receptor than mice receptor. In this paper, we investigated a possibility that a guinea pig model would become a drug efficacy evaluation model with higher evaluation accuracy than the mouse model.</p><p>As a result, hyperemia was observed in the conjunctivae and iris of guinea pigs after instillation of 4-methylhistamine and specifically suppressed by the histamine H<sub>4</sub> receptor antagonist. Unlikely to the previously reported mouse model, however, none of edema, increased vascular permeability or scratching behavior was observed, suggesting that there may be differences between mice and guinea pigs not only in the binding affinity of histamine to the H<sub>4</sub> receptor but also in the biological reaction to 4-methylhistamine. Although the symptoms of allergic conjunctivitis do not appear comprehensively in the guinea pig model, results of this study indicated a possibility that this model can be used as a simple screening model in the early stages of drug development.</p></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49871297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}