Assessment Of Drug Effects Research Articles

Towards Measuring Brain Function on Groups of People in the Real World

In three studies, EEGs from three groups of participants were recorded during progressively more real world situations after drinking alcoholic beverages that brought breath alcohol contents near the limit for driving in California 30 minutes after drinking. A simple equation that measured neurophysiological effects of alcohol in the first group of 15 participants performing repetitive cognitive tasks was applied to a second group of 15 operating an automobile driving simulator, and to a third group of 10 ambulatory people recorded simultaneously during a cocktail party. The equation derived from the first group quantified alcohol’s effect by combining measures of higher frequency (beta) and lower frequency (theta) power into a single score. It produced an Area Under the Receiver Operator Characteristic Curve of .73 (p<.05; 67% sensitivity in recognizing alcohol and 87% specificity in recognizing placebo). Applying the same equation to the second group operating the driving simulator, AUC was .95, (p<.0001; 93% sensitivity and 73% specificity), while for the cocktail party group AUC was .87 (p<.01; 80% sensitivity and 80% specificity). EEG scores were significantly related to breath alcohol content in all studies. Some individuals differed markedly from the overall response evident in their respective groups. The feasibility of measuring the neurophysiological effect of a psychoactive substance from an entire group of ambulatory people at a cocktail party suggests that future studies may be able to fruitfully apply brain function measures derived under rigorously controlled laboratory conditions to assess drug effects on groups of people interacting in real world situations.

C-Myc induced compensated cardiac hypertrophy increases free fatty acid utilization for the citric acid cycle

The protooncogene C-Myc (Myc) regulates cardiac hypertrophy. Myc promotes compensated cardiac function, suggesting that the operative mechanisms differ from those leading to heart failure. Myc regulation of substrate metabolism is a reasonable target, as Myc alters metabolism in other tissues. We hypothesize that Myc induced shifts in substrate utilization signal and promote compensated hypertrophy. We used cardiac specific Myc-inducible C57/BL6 male mice between 4–6months old that develop hypertrophy with tamoxifen (tam) injections. Isolated working hearts and 13Carbon (13C)-NMR were used to measure function and fractional contributions (Fc) to the citric acid cycle by using perfusate containing 13C-labeled free fatty acids, acetoacetate, lactate, unlabeled glucose and insulin. Studies were performed at pre-hypertrophy (3-days tam, 3dMyc), established hypertrophy (7-days tam, 7dMyc) or vehicle control (Cont). Non-transgenic siblings (NTG) received 7-days tam or vehicle to assess drug effect. Hypertrophy was assessed by echocardiograms and heart weights. Western blots were performed on key metabolic enzymes. Hypertrophy occurred in 7dMyc only. Cardiac function did not differ between groups. Tam alone did not affect substrate contributions in NTG. Substrate utilization was not significantly altered in 3dMyc versus Cont. The free fatty acid FC was significantly greater in 7dMyc versus Cont with decreased unlabeled Fc, which is predominately exogenous glucose. Free fatty acid flux to the citric acid cycle increased while lactate flux was diminished in 7dMyc compared to Cont. Total protein levels of a panel of key metabolic enzymes were unchanged; however total protein O-GlcNAcylation was increased in 7dMyc. Substrate utilization changes for the citric acid cycle did not precede hypertrophy; therefore they are not the primary signal for cardiac growth in this model. Free fatty acid utilization and oxidation increase at established hypertrophy. Understanding the mechanisms whereby this change maintained compensated function could provide useful information for developing metabolic therapies to treat heart failure. The molecular signaling for this metabolic change may occur through O-GlcNAcylation. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".

Beta-Adrenoreceptor Agonism Influences Retinal Responses to Hypoxia in a Model of Retinopathy of Prematurity

In a mouse model of oxygen-induced retinopathy (OIR), a well-established model of retinopathy of prematurity (ROP), blocking beta-adrenoreceptors (β-ARs), and, in particular, β2-ARs, counteracts retinal responses to hypoxia. In the present work, we determined the effects of the β-AR agonist isoproterenol on retinal angiogenesis and β-AR signaling to better clarify the role of sympathetic transmission in ROP. Isoproterenol was administered subcutaneously. Protein kinase A activity was determined by a colorimetric assay to assess drug effectiveness. Blood pressure and heart-to-body weight ratio were measured. Vascular endothelial growth factor (VEGF) and norepinephrine were measured with ELISA. Retinal neovascularization was assessed by CD31 immunohistochemistry. β-AR-coupled adenylyl cyclase (AC) activity was measured with a competition assay. β-ARs, G-protein-coupled receptor kinase (GRK)2, and β-arrestins were determined by Western blot. Association of β-arrestins with β2-ARs was assessed by immunoprecipitation. Isoproterenol-induced modulation of protein kinase A activity suggests that the drug was effective at the receptor level. Isoproterenol did not affect cardiovascular parameters, but decreased retinal levels of VEGF and reduced pathogenic neovascularization, likely through an influence on sympathetic transmission. In fact, isoproterenol downregulated β2-AR expression, recovered the hypoxia-induced increase in β-AR-coupled AC activity, and increased GRK2 and β-arrestins, which promote β-AR desensitization through the uncoupling of G-protein-coupled receptors from G proteins. Immunoprecipitation studies demonstrated that β-AR desensitization involved β2-ARs. Our findings suggest that hypoxia-induced retinal neovascularization depends at least in part on increased sympathetic transmission, as reduction of sympathetic drive by agonist-induced β2-AR desensitization inhibits some of the hallmarks of OIR.

Iloperidone (Fanapt®), a novel atypical antipsychotic, is a potent HERG blocker and delays cardiac ventricular repolarization at clinically relevant concentration

QT interval prolongation on the electrocardiogram (ECG) has extensively been reported with iloperidone, a novel antipsychotic drug. The objective of the present study was to evaluate the effects of iloperidone on cardiac ventricular repolarization at three different levels; in vitro, ex vivo and in vivo. (1) In vitro level: whole-cell patch-clamp experiments were performed on HERG-transfected HEK293 cells exposed to iloperidone 0.01–1μmol/L (n=35cells, total) to assess drug effect on HERG current. (2) Ex vivo level: Langendorff retroperfusion experiments were performed on isolated hearts from male Hartley guinea pigs (n=7) exposed to iloperidone 100nmol/L with/without chromanol 293B 10μmol/L to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization (MAPD90). (3) In vivo level: ECG recordings using wireless cardiac telemetry were performed in guinea pigs (n=5) implanted with radio transmitters and treated with a single oral gavage dose of iloperidone 3mg/kg. (1) Patch-clamp experiments revealed an estimated IC50 for iloperidone on HERG current of 161±20nmol/L. (2) While pacing the hearts at stimulation cycle lengths of 200 or 250ms, or during natural sinus rhythm (no external pacing), iloperidone 100nmol/L prolonged MAPD90 by respectively 9.2±0.9, 11.2±1.6 and 21.4±2.3ms. After adding chromanol 293B, MAPD90 was further prolonged by 7.3±3.3, 11.5±2.3 and 29.2±6.7ms, respectively. (3) Iloperidone 3mg/kg p.o. caused a maximal 42.7±10.2ms prolongation of corrected QT interval (QTcF), 40min after administration. Iloperidone prolongs the QT interval, the cardiac action potentials and is a potent HERG blocker. Patients are at increased risk of cardiac proarrhythmia during iloperidone treatment, as this drug possesses significant cardiac repolarization-delaying properties at clinically relevant concentration.

Stability criteria for the outcomes of statistical tests to assess drug effectiveness with a single study

At least two adequate and well-controlled clinical studies are usually required to support effectiveness of a certain treatment. In some circumstances, however, a single study providing strong results may be sufficient. Some statistical stability criteria for assessing whether a single study provides very persuasive results are known. A new criterion is introduced, and it is based on the conservative estimation of the reproducibility probability in addition to the possibility of performing statistical tests by referring directly to the reproducibility probability estimate. These stability criteria are compared numerically and conceptually. This work aims to help both regulatory agencies and pharmaceutical companies to decide if the results of a single study may be sufficient to establish effectiveness.

Galantamine (Reminyl®) delays cardiac ventricular repolarization and prolongs the QT interval by blocking the HERG current

Galantamine is a reversible inhibitor of acetylcholinesterase and an allosteric-potentiating ligand of the nicotinic acetylcholine receptors. It is used for treating mild-to-moderate Alzheimer's disease. Interestingly, QT interval prolongation on the electrocardiogram (ECG), malignant ventricular arrhythmias and syncope have been reported with galantamine. Our objective was to evaluate the effects of galantamine on cardiac ventricular repolarization. Three sets of experiments were undertaken: 1) Whole cell patch-clamp experiments: HERG- or KCNQ1+KCNE1-transfected cells were exposed to galantamine 0.1–1000μmol/l (n=25 cells, total) to assess drug effect on HERG and KCNQ1+KCNE1 currents. 2) Langendorff perfusion experiments: Isolated hearts from male Hartley guinea pigs (n=9) were exposed to galantamine 1μmol/l to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization (MAPD90). 3) Cardiac telemetry experiments: Guinea pigs (n=7) implanted with wireless transmitters were injected a single intraperitoneal (i.p.) dose of galantamine 3mg/kg and 24h ECG recordings were made. 1) The estimated IC50 for galantamine on HERG current was 760.2μmol/l. Moreover, galantamine 10μmol/l had a small inhibiting effect on KCNQ1+KCNE1 current (12.17±2.19% inhibition, n=10 cells). 2) While pacing at cycle lengths of 150, 200 or 250ms, galantamine 1μmol/l prolonged MAPD90 by respectively 5.1±1.6ms, 9.4±1.9ms and 12.1±2.1ms. 3) Galantamine 3mg/kgi.p. caused a maximal 11.9±2.7ms prolongation of the corrected QT (QTc). Galantamine is a weak HERG blocker. This contributes to its mild QT-prolonging effect. Patients could be at risk of cardiac proarrhythmia during drug overdosage or interactions involving cytochrome 2D6 drug-metabolizing enzyme.

Modelling of Mouse Experimental Colitis by Global Property Screens: A Holistic Approach to Assess Drug Effects in Inflammatory Bowel Disease

Preclinical disease models play an important role in the establishment of new treatment paradigms, identification of biomarkers and assessment of drug efficacy and safety. However, the accuracy of these models in context of the human disease are sometimes questioned, e.g. due to trials failing to confirm efficacy in humans. We suggest that one reason behind this gap in predictability may relate to how the preclinical data is analyzed and interpreted. In the present paper, we introduce a holistic approach to analyze and illustrate data in context of one of the most commonly used colitis models, i.e. the mouse dextran sulphate sodium (DSS) colitis model. Diseased mice were followed over time along disease progression and by use of tool pharmacological compounds activating nuclear hormone receptors, respectively. A new multivariate statistics approach was applied including principal component analysis (PCA) with treatment prediction subsequent to establishing the principal component analysis model. Thus, several studies could be overlaid and compared to each other in a new, comprehensive and holistic way. This method, named mouse colitis global property screening, appears applicable not only to any animal modelling series of studies but also to human clinical studies. The prerequisites for the study set up and calculations are delineated and examples of new learnings from the global property screening will be presented.

Preliminary Web-Based Measures Development for GHB: Expectancies, Functions, and Withdrawal

Background: Much of what is understood regarding gamma hydroxybutyrate (GHB) treatment is based on hospital case studies for overdose and withdrawal, and there are currently no measures developed specifically for GHB or its analogs (e.g., gamma butyrolactone and 1,4-butanediol) to assess drug effect expectancies, reasons for starting use, withdrawal effects, and knowledge and opinions about use. Objectives: This pilot study (N = 61) was conducted to begin measures development to assess experiences, functions of use, and opinions regarding use as indicated by respondents taking a Web-based survey. Methods: Minimum average partial correlation and parallel analysis procedures are employed to create scales. Results: Scales were developed to assess expectancies, reasons for use, withdrawal, and knowledge/opinions of use with median α = .79 and that account for 8.69–24.17% of the variance. Conclusion: Scales have relatively good psychometric properties and replication is needed. Scientific Significance: GHB-specific measures may greatly assist in furthering our understanding of protective and risk factors for use, and withdrawal phenomena.

Simulation of Clinical Outcome for Pomalidomide Plus Low-Dose Dexamethasone in Patients with Refractory Multiple Myeloma Based on Week 8 M-Protein Response

Abstract 1881 Background:Drug-disease modeling and simulation has recently been proposed to support drug development decisions in patients with liquid and solid tumors (Claret et al J Clin Oncol, 27:4103-4108, 2010; Wang et al. Clin Pharmacol Ther, 86:167-174, 2009). We hypothesized that similar modeling based on a decrease in M-protein may be applied in multiple myeloma. The objective was to use a modeling and simulation approach to leverage available lenalidomide multiple myeloma data and support pomalidomide development decisions by, 1) developing a drug-independent link between tumor burden reduction (as assessed by change from baseline in serum M-Protein) and survival and PFS and 2) simulating expected survival and PFS based on interim M-protein data of the phase 1/2 trial (CC-4047-MM02, NCT00833833) of pomalidomide (POM) in patients with relapsed and refractory multiple myeloma who have received at least 2 prior therapies including lenalidomide (LEN) and bortezomib. Methods:M-Protein measurements were modeled as a function of time from 704 patients included in two phase 3 clinical studies of LEN plus dexamethasone (DEX) vs. DEX (MM010, Dimopoulos, NEJM 357, 2123–2132, 2007 and MM009, Weber, NEJM 357, 2133–2142, 2007). Models were developed for survival and PFS times as a function of change in M-protein level at end-of-cycle 2 (week 8) from baseline as well as other prognostic factors. Interim M-protein data from the ongoing MM-002 POM study (217 patients) were modeled to simulate clinical endpoints. Results:Based on lenalidomide Phase 3 data, week-8 change in M-Protein (p < 0.00001), ECOG performance status (p < 0.0009), baseline albumin, hemoglobin and creatinine levels (p < 0.01) were significant independent predictors of survival when week-8 change in M-Protein (p < 0.00001) and baseline hemoglobin (p < 0.001) were significant independent predictors of PFS. Observed survival and PFS distributions over 100 weeks in lenalidomide studies and difference between the two treatments (LEN + DEX vs. DEX) were consistent with the 95% confidence intervals (CI) of the models. Model predictions (95% PIs) of median survival and PFS based on week-8 change in M-Protein following treatment with POM and POM + DEX are presented in the table below. The recently disclosed observed medians and 95% CIs for all endpoints and all arms from MM-002 are included in the model.SimulatedObservedSurvivalMedian (week)95% PIMedian (week)Pom67.845.8–101.359.3Pom + DEX78.353.5–116.162.6PFSPom16.59.7–27.710.7Pom + DEX22.514.6–34.316.6 Conclusions:Change in M-protein level as a continuous longitudinal biomarker appears to be a potential indicator to assess drug effect in multiple myeloma studies using modeling and simulation based on early interim data. PFS predicted by the model and the actual PFS are similar and thus may support end-of-Phase 2 decision (Bruno et al Clin Pharmacol Ther 86:136-138, 2009). Current simulations indicate encouraging results for POM in a refractory multiple myeloma patient population who have received prior treatment that includes lenalidomide and bortezomib but these exploratory methods require confirmation as part of ongoing studies. Disclosures:Bruno:Pharsight: Employment; Celgene Corporation: Research Funding. Off Label Use: Pomalidomide is not an FDA licensed product for multiple myeloma treatment. Jonsson:Pharsight: Employment; Celgene Corporation: Research Funding. Zaki:Celgene Corporation: Employment. Jacques:Celgene Corporation: Employment. Swern:Celgene Corporation: Employment. Richardson:Millennium Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Celgene Corporation: Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees. Claret:Celgene Corporation: Consultancy, Research Funding; Pharsight: Employment.

Abstract A106: Investigation of 5-fluorodeoxycytidine with tetrahydrouracil as a demethylation regimen in solid tumors.

Abstract Methylation-induced silencing of gene promoters is widely documented in numerous cancer types, and the anti-tumor activity of agents that target this phenomenon has established DNA methyltransferase (DNMT1) as a valid drug target. We have investigated the combination of 5-fluorodeoxycytidine with Tetrahydrouridine (FdCyd + THU) in vitro and in vivo using continuous exposures up to 12 weeks. Supplementation of the medium with Deoxythymidine (10 μM) was required to prevent FdCyd cytotoxicity and allow sufficient time for demethylation to occur. Decitabine was used as the positive control throughout the evaluation. The bladder cancer line EJ6 was sensitive to FdCyd + THU, as evidenced by an increase in cell doubling time, a decrease in LINE1 methylation, and an initial decrease in total DNMT1 concentrations, determined with a newly-developed, quantitative two-site immunoassay. The triple negative breast cancer line MDA-MB-231 was resistant to the regimen, as assessed by all three criteria. The mechanism of this combination regimen in tumor cells isolated from paracentesis specimens from patients enrolled on an NCI-supported Phase 1 clinical trial of FdCyd + THU; and was confirmed by finding changes in both DNMT1 levels and LINE1 methylation. Upregulation of expression of p16 (INK4a), but not GSTP1 or RASSF1, was observed in EJ6 cells following FdCyd + THU by Western blotting. An immunofluorescence assay for p16 expression in circulating tumor cells (CTCs) has been developed and implemented in the Phase 1 trial. The ongoing Phase 2 trial of this regimen will include measurements of DNMT1 and LINE1 methylation in tumor biopsies and p16 in CTCs to directly assess drug effect on the DNMT1 target. This Research has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E and NCI RAID Project #266 Grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A106.

Analysis of Missing Mechanism in IVUS Imaging Clinical Trials with Missing Covariates

Intravascular ultrasound (IVUS) is a clinical imaging procedure used to assess drug effects on the progression of coronary atherosclerosis in clinical trials. It is an invasive medical procedure of measuring coronary artery atheroma (plaque) volume, and leads to high missing rates (often over 30%). This paper uses an IVUS Phase II clinical trial data to explore the missing mechanism of IVUS endpoint, the percent atheroma volume (PAV). We proposed a moving-window method to examine the relationship between continuous covariates such as lipid endpoint and the probability of missing IVUS values, which provides a general approach for missing mechanism exploration. The moving-window method is more intuitive and provides a fuller picture about the relationship. In the example, some covariates such as lipid measures also have high missing rates after 12 months because of compliance issues probably caused by fatigue of blood drawing. We found that if the method of last observation carried forward (LOCF) is used to impute the lipid endpoints, it leads to biologically unexplainable results. Using the multiple imputation approach for the missing covariates results in a more reasonable conclusion about the IVUS missing mechanism. Age, race, and baseline PAV are identified as key potential contributors to the probability of missing IVUS endpoint. This finding can be used to reduce missing values in future IVUS trials by setting up appropriate inclusion and exclusion criteria at the trial design stages.

Tizanidine (Zanaflex)

Tizanidine (Zanaflex) is a centrally acting imidazoline muscle relaxant that is structurally similar to clonidine (α(2)-adrenergic agonist) but not to other myorelaxants such as baclofen or benzodiazepines. Interestingly, cardiac arrhythmias and QT interval prolongation have been reported with tizanidine. To evaluate the effects of tizanidine on cardiac ventricular repolarization. (1) Whole-cell patch-clamp experiments: HERG- or KCNQ1+KCNE1-transfected cells were exposed to tizanidine 0.1-100 µmol/L (n = 29 cells, total) to assess drug effect on the rapid (I(Kr)) and slow (I(Ks)) components of the delayed rectifier potassium current. (2) Langendorff retroperfusion experiments: isolated hearts from male Hartley guinea pigs (n = 6) were exposed to tizanidine 1 µmol/L to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization (MAPD(90)). (3) In vivo wireless cardiac telemetry experiments: guinea pigs (n = 6) implanted with radio transmitters were injected a single intraperitoneal (ip) dose of tizanidine 0.25 mg/kg and 24 hours electrocardiography (ECG) recordings were made. (1) Patch-clamp experiments revealed an estimated IC(50) for tizanidine on I(Kr) above 100 µmol/L. Moreover, tizanidine 1 µmol/L had hardly any effect on I(Ks) (5.23% ± 4.54% inhibition, n = 5 cells). (2) While pacing the hearts at stimulation cycle lengths of 200 or 250 ms, tizanidine 1 µmol/L prolonged MAPD(90) by 8.22 ± 2.03 (6.7%) and 11.70 ± 3.08 ms (8.5%), respectively (both P < .05 vs baseline). (3) Tizanidine 0.25 mg/kg ip caused a maximal 11.93 ± 1.49 ms prolongation of corrected QT interval (QTc), 90 minutes after injection. Tizanidine prolongs the QT interval by blocking I(Kr). Patients could be at risk of cardiac proarrhythmia during impaired drug elimination, such as in case of CYP1A2 inhibition during drug interactions.

Reliability, validity and sensitivity of Montgomery-Asberg depression rating scale for patients with current major depression disorder

Objective To examine the reliability,validity and sensitivity of Montgomery-Asberg Depression Rating Scale (MADRS) for patients with current major depression disorder (MDD). Methods One hundred and twenty-two current MDD (DSM-Ⅳ) patients were administered with MADRS, Hamilton Rating Scale for Depression-17 item version (HAMD) and Clinical Global Impression of Severity (CGI-S) at baseline, 12 patients were selected to complete rater agreement test,and 47 patients receiving antidepressant treatment were followed up at 2,4,6 and 8 week and administered with MADRS and HAMD. Correlation analysis, reliability analysis and effect size (ES) calculation were used to determine the reliability,validity and sensitivity to changes during drug treatment. Results Intra rater reliability for MADRS was 0. 954. Baseline item-total score correlations were between 0. 445 and 0. 770 (P ＜ 0. 01 ), and the average correlation was 0. 629. The Cronbach α coefficient was 0. 847. The criterion related validity with HAMD and CGI-S was 0. 853 and 0. 672 (P＜0.01) ,respectively. The re-test reliability for MADRS at 2,4,6 and 8 week was 0. 737 ,0. 651,0. 543 and 0. 524 (P＜0. 01 ) ,respectively.MADRS had higher ES than HAMD when taken as clinical endpoint outcome measurement (0.41 vs 0.40,0.87 vs 0. 72,1.14 vs 0. 88,1.20 vs 0. 96 for 2nd,4th,6th and 8th week, respectively). Conclusion MADRS has good reliability and validity for patients with MDD. It is more sensitive to assess drug effect than HAMD. Key words: Montgomery-asberg depression rating scale ; Major depression disorder ; Reliability ; Validity ; Sensitivity ;

Genetic models of sensorimotor gating: relevance to neuropsychiatric disorders.

Sensorimotor gating, or the ability of a sensory event to suppress a motor response, can be measured operationally via prepulse inhibition (PPI) of the startle response. PPI is deficient in schizophrenia patients as well as other neuropsychiatric disorders, can be measured across species, and has been used widely as a translational tool in preclinical neuropharmacological and genetic research. First developed to assess drug effects in pharmacological and developmental models, PPI has become one of the standard behavioral measures in genetic models of schizophrenia and other neuropsychiatric disorders that exhibit PPI deficits. In this chapter we review the literature on genetic models of sensorimotor gating and discuss the utility of PPI as a tool in phenotyping mutant mouse models. We highlight the approaches to genetic mouse models of neuropsychiatric disease, discuss some of the important caveats to these approaches, and provide a comprehensive table covering the more recent genetic models that have evaluated PPI.

Phosphatidylcholine metabolic transformation and progression signature as a pharmacodynamic biomarker.

Phosphatidylcholine metabolic transformation and progression signature as a pharmacodynamic biomarker.

The statistics of phase 0 trials

The PD-driven phase 0 trial is a new form, designed to be a first-in-man study, often of a new agent, conducted to assess drug effect on a molecular target, by means of a pharmacodynamic (PD) assay, in a very small number (10-15) of patients. Such a study is meant to be a proof of principle trial to determine whether the agent yields the PD effect predicted by pre-clinical studies. The dosage is meant to be pharmacologically active, but is neither toxic nor likely to yield clinical benefit. Such a trial may be used to serve as a very early test of an agent's biologic effect, allowing for early weeding out of ineffective agents, or as an early means of determining the most promising of competing analogue agents. This manuscript will present designs for such PD-driven studies that are statistically efficient and rigorous, focusing on non-comparative trials. The phase 0 trial promises to become an increasingly important tool for facilitating and speeding the development of new therapeutic agents, particularly in oncology.

In Vitro Cardiac Repolarization Assays: Guinea Pig Papillary Muscles Vs. Canine Purkinje Fibers

Delayed or accelerated cardiac repolarization is potentially proarrhythmic. In this study, we compared a guinea pig papillary muscle APD (GPPM-APD) assay to a canine Purkinje fiber APD (CPF-APD) assay in assessing drug effects on cardiac repolarization. Papillary muscles (right ventricle) and Purkinje fibers were stimulated at 0.5 Hz, action potentials were recorded using microelectrode techniques, repolarization assessed using APD30, 50, 90 values, and the calculated triangulation parameter, APD30-90. Eight compounds (6 positives, 2 negatives) were tested in both preparations. Percent changes in APD90 values obtained with the GPPM-APD assay were less than those obtained in the CPF-APD assay (table). Repolarization parameters in the GPPM-APD assay exhibited a rank sensitivity order of APD30 < APD50 ≈ APD90 < APD30-90 in detecting effects of the five hERG blockers on repolarization. APD90 was the most sensitive parameter in detecting effects of the hERG activator (A-935142.0). These results suggest the GPPM-APD assay and the CPF-APD assay are valuable in assessing drug effects on cardiac repolarization with comparable effects in both assays.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Dog left ventricular midmyocardial myocytes for assessment of drug‐induced delayed repolarization: short‐term variability and proarrhythmic potential

Evaluation of the potential for delayed ventricular repolarization and proarrhythmia by new drugs is essential. We investigated if dog left ventricular midmyocardial myocytes (LVMMs) that can be used as a preclinical model to assess drug effects on action potential duration (APD) and whether in these cells, short-term variability (STV) or triangulation could predict proarrhythmic potential. Beagle LVMMs and Purkinje fibres (PFs) were used to record APs. Effects of six reference drugs were assessed on APD at 50% (APD(50)) and 90% (APD(90)) of repolarization, STV(APD), triangulation (ratio APD(90)/APD(50)) and incidence of early afterdepolarizations (EADs) at 1 and 0.5 Hz. LVMMs provided stable recordings of AP, which were not affected by four sequential additions of dimethyl sulphoxide. Effects of dofetilide, d-sotalol, cisapride, pinacidil and diltiazem, but not of terfenadine, on APD in LVMMs were found to be comparable with those recorded in PFs. LVMMs, but not PFs, exhibited a proarrhythmic response to I(Kr) blockers. Incidence of EADs was not related to differences in AP prolongation or triangulation, but corresponded to beat-to-beat variability of repolarization, here quantified as STV of APD. LVMMs provide a suitable preclinical model to assess the effects of new drugs on APD and also yield additional information about putative indicators of proarrhythmia that add value to an integrated QT/TdP risk assessment. Our findings support the concept that increased STV(APD) may predict drug-induced proarrhythmia.

Toward a Personalized Pharmacotherapy of Respiratory Diseases

Toward a Personalized Pharmacotherapy of Respiratory Diseases

Development of multi-electrode array screening for anticonvulsants in acute rat brain slices

The acute hippocampal brain slice preparation is an important in vitro screening tool for potential anticonvulsants. Application of 4-aminopyridine (4-AP) or removal of external Mg 2+ ions induces epileptiform bursting in slices which is analogous to electrical brain activity seen in status epilepticus states. We have developed these epileptiform models for use with multi-electrode arrays (MEAs), allowing recording across the hippocampal slice surface from 59 points. We present validation of this novel approach and analyses using two anticonvulsants, felbamate and phenobarbital, the effects of which have already been assessed in these models using conventional extracellular recordings. In addition to assessing drug effects on commonly described parameters (duration, amplitude and frequency), we describe novel methods using the MEA to assess burst propagation speeds and the underlying frequencies that contribute to the epileptiform activity seen. Contour plots are also used as a method of illustrating burst activity. Finally, we describe hitherto unreported properties of epileptiform bursting induced by 100 μM 4-AP or removal of external Mg 2+ ions. Specifically, we observed decreases over time in burst amplitude and increase over time in burst frequency in the absence of additional pharmacological interventions. These MEA methods enhance the depth, quality and range of data that can be derived from the hippocampal slice preparation compared to conventional extracellular recordings. It may also uncover additional modes of action that contribute to anti-epileptiform drug effects.