Clofazimine pharmacokinetics in HIV-infected adults with diarrhea: Implications of diarrheal disease on absorption of orally administered therapeutics.

Oral drug absorption kinetics are usually established in populations with a properly functioning gastrointestinal tract. However, many diseases and therapeutics can alter gastrointestinal physiology and cause diarrhea. The extent of diarrhea-associated impact on drug pharmacokinetics has not been quantitatively described. To address this knowledge gap, we used a population pharmacokinetic modeling approach with data collected in a phase IIa study of matched human immunodeficiency virus (HIV)-infected adults with/without cryptosporidiosis and diarrhea to examine diarrhea-associated impact on oral clofazimine pharmacokinetics. A population pharmacokinetic model was developed with 428 plasma samples from 23 HIV-infected adults with/without Cryptosporidium infection using nonlinear mixed-effects modeling. Covariates describing cryptosporidiosis-associated diarrhea severity (e.g., number of diarrhea episodes, diarrhea grade) or HIV infection (e.g., viral load, CD4+ T cell count) were evaluated. A two-compartment model with lag time and first-order absorption and elimination best fit the data. Maximum diarrhea grade over the study duration was found to be associated with a more than sixfold reduction in clofazimine bioavailability. Apparent clofazimine clearance, intercompartmental clearance, central volume of distribution, and peripheral volume of distribution were 3.71 L/h, 18.2 L/h (interindividual variability [IIV] 45.0%), 473 L (IIV 3.46%), and 3434 L, respectively. The absorption rate constant was 0.625 h-1 (IIV 149%) and absorption lag time was 1.83 h. In conclusion, the maximum diarrhea grade observed for the duration of oral clofazimine administration was associated with a significant reduction in clofazimine bioavailability. Our results highlight the importance of studying disease impacts on oral therapeutic pharmacokinetics to inform dose optimization and maximize the chance of treatment success.

Hollow-fibre system model of tuberculosis reproducibility and performance specifications for best practice in drug and combination therapy development.

BACKGROUND: The hollow-fibre system model of tuberculosis (HFS-TB) has been endorsed by regulators; however, application of HFS-TB requires a thorough understanding of intra- and inter-team variability, statistical power and quality controls. METHODS: Three teams evaluated regimens matching those in the Rapid Evaluation of Moxifloxacin in Tuberculosis (REMoxTB) study, plus two high-dose rifampicin/pyrazinamide/moxifloxacin regimens, administered daily for up to 28 or 56 days against Mycobacterium tuberculosis (Mtb) under log-phase growth, intracellular growth or semidormant growth under acidic conditions. Target inoculum and pharmacokinetic parameters were pre-specified, and the accuracy and bias at achieving these calculated using percent coefficient of variation (%CV) at each sampling point and two-way analysis of variance (ANOVA). RESULTS: A total of 10 530 individual drug concentrations, and 1026 individual cfu counts were measured. The accuracy in achieving intended inoculum was >98%, and >88% for pharmacokinetic exposures. The 95% CI for the bias crossed zero in all cases. ANOVA revealed that the team effect accounted for <1% of variation in log10 cfu/mL at each timepoint. The %CV in kill slopes for each regimen and different Mtb metabolic populations was 5.10% (95% CI: 3.36%-6.85%). All REMoxTB arms exhibited nearly identical kill slopes whereas high dose regimens were 33% faster. Sample size analysis revealed that at least three replicate HFS-TB units are needed to identify >20% difference in slope, with a power of >99%. CONCLUSIONS: HFS-TB is a highly tractable tool for choosing combination regimens with little variability between teams, and between replicates.

Sputum lipoarabinomannan (LAM) as a biomarker to determine sputum mycobacterial load: exploratory and model-based analyses of integrated data from four cohorts.

BACKGROUND: Despite the high global disease burden of tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb) infection, novel treatments remain an urgent medical need. Development efforts continue to be hampered by the reliance on culture-based methods, which often take weeks to obtain due to the slow growth rate of Mtb. The availability of a "real-time" measure of treatment efficacy could accelerate TB drug development. Sputum lipoarabinomannan (LAM; an Mtb cell wall glycolipid) has promise as a pharmacodynamic biomarker of mycobacterial sputum load. METHODS: The present analysis evaluates LAM as a surrogate for Mtb burden in the sputum samples from 4 cohorts of a total of 776 participants. These include those from 2 cohorts of 558 non-TB and TB participants prior to the initiation of treatment (558 sputum samples), 1 cohort of 178 TB patients under a 14-day bactericidal activity trial with various mono- or multi-TB drug therapies, and 1 cohort of 40 TB patients with data from the first 56-day treatment of a standard 4-drug regimen. RESULTS: Regression analysis demonstrated that LAM was a predictor of colony-forming unit (CFU)/mL values obtained from the 14-day treatment cohort, with well-estimated model parameters (relative standard error ≤ 22.2%). Moreover, no changes in the relationship between LAM and CFU/mL were observed across the different treatments, suggesting that sputum LAM can be used to reasonably estimate the CFU/mL in the presence of treatment. The integrated analysis showed that sputum LAM also appears to be as good a predictor of time to Mycobacteria Growth Incubator Tube (MGIT) positivity as CFU/mL. As a binary readout, sputum LAM positivity is a strong predictor of solid media or MGIT culture positivity with an area-under-the-curve value of 0.979 and 0.976, respectively, from receiver-operator curve analysis. CONCLUSIONS: Our results indicate that sputum LAM performs as a pharmacodynamic biomarker for rapid measurement of Mtb burden in sputum, and thereby may enable more efficient early phase clinical trial designs (e.g., adaptive designs) to compare candidate anti-TB regimens and streamline dose selection for use in pivotal trials. Trial registration NexGen EBA study (NCT02371681).

Model-Based Meta-Analysis of Relapsing Mouse Model Studies from the Critical Path to Tuberculosis Drug Regimens Initiative Database.

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains a leading infectious disease-related cause of death worldwide, necessitating the development of new and improved treatment regimens. Nonclinical evaluation of candidate drug combinations via the relapsing mouse model (RMM) is an important step in regimen development, through which candidate regimens that provide the greatest decrease in the probability of relapse following treatment in mice may be identified for further development. Although RMM studies are a critical tool to evaluate regimen efficacy, making comprehensive "apples to apples" comparisons of regimen performance in the RMM has been a challenge in large part due to the need to evaluate and adjust for variability across studies arising from differences in design and execution. To address this knowledge gap, we performed a model-based meta-analysis on data for 17 unique regimens obtained from a total of 1592 mice across 28 RMM studies. Specifically, a mixed-effects logistic regression model was developed that described the treatment duration-dependent probability of relapse for each regimen and identified relevant covariates contributing to interstudy variability. Using the model, covariate-normalized metrics of interest, namely, treatment duration required to reach 50% and 10% relapse probability, were derived and used to compare relative regimen performance. Overall, the model-based meta-analysis approach presented herein enabled cross-study comparison of efficacy in the RMM and provided a framework whereby data from emerging studies may be analyzed in the context of historical data to aid in selecting candidate drug combinations for clinical evaluation as TB drug regimens.

The potential deployment of a pan-tuberculosis drug regimen in India: A modelling analysis.

There is increasing interest in future, highly-potent 'pan-TB' regimens against tuberculosis (TB), that may be equally effective in both drug-susceptible and rifampicin-resistant (RR) forms of TB. Taking the example of India, the country with the world's largest burden of TB, we show that adoption of these regimens could be: (i) epidemiologically impactful, and (ii) cost-saving to the national TB programme, even if the regimen itself is more costly than current TB treatment. Mathematical modelling suggests that deployment of a pan-TB regimen in 2022 would reduce the annual incidence of TB in 2030 by 23.9% [95% Bayesian credible intervals [CrI] 17.6-30.8%] if used to treat all TB cases, and by 2.30% [95% CrI 1.57-3.48%] if used to treat only RR-TB. Notably, with a regimen costing less than USD 359 (95% CrI 287-441), treating all diagnosed TB cases with the pan-TB regimen yielded greater cost-savings than treating just those diagnosed with RR-TB. One limitation of our approach is that it does not capture the risk of resistance to the new regimen. We discuss ways in which this risk could be mitigated using modern adherence support mechanisms, as well as drug sensitivity testing at the point of TB diagnosis, to prevent new resistant forms from becoming established. A combination of such approaches would be important for maximising the useful lifetime of any future regimen.

Daily Dosing for Bedaquiline in Patients with Tuberculosis.

The bedaquiline regimen for the treatment of multidrug-resistant tuberculosis (MDR-TB) in adults is a loading dose of 400 mg QD for 2 weeks followed by 200 mg thrice weekly (TIW) for 22 weeks. Most TB antibiotics administered with bedaquiline are given QD. Using pharmacokinetic simulations, we explored alternative QD bedaquiline regimens and determined that 200 mg QD for 8 weeks followed by 100 mg QD provides comparable exposures to the approved regimen. This simpler regimen is under clinical evaluation.

Publisher Correction: A patient-level pooled analysis of treatment-shortening regimens for drug-susceptible pulmonary tuberculosis.

The version of this article originally published was not open access. This article should have been open access. The error has been fixed, and the article is now open access.

A patient-level pooled analysis of treatment-shortening regimens for drug-susceptible pulmonary tuberculosis.

Tuberculosis kills more people than any other infectious disease. Three pivotal trials testing 4-month regimens failed to meet non-inferiority margins; however, approximately four-fifths of participants were cured. Through a pooled analysis of patient-level data with external validation, we identify populations eligible for 4-month treatment, define phenotypes that are hard to treat and evaluate the impact of adherence and dosing strategy on outcomes. In 3,405 participants included in analyses, baseline smear grade of 3+ relative to <2+, HIV seropositivity and adherence of ≤90% were significant risk factors for unfavorable outcome. Four-month regimens were non-inferior in participants with minimal disease defined by <2+ sputum smear grade or non-cavitary disease. A hard-to-treat phenotype, defined by high smear grades and cavitation, may require durations >6 months to cure all. Regimen duration can be selected in order to improve outcomes, providing a stratified medicine approach as an alternative to the 'one-size-fits-all' treatment currently used worldwide.

Silver-Catalyzed tert-Butyl 3-Oxopent-4-ynoate π-Cyclizations: Controlling the Ring Size-Hydroxypyrone or Pulvinone Formation-by Counterion and Additive Optimization.

tert-Butyl 2,5-diaryl-3-oxopent-4-ynoates, obtained from arylacetylenes and the acid chloride of tert-butyl 2-phenylmalonate, represent strongly enolized ß-ketoesters. Their C≡C bonds were activated by Ag(I) salts so that de- tert-butylating π-cyclizations occurred. The latter followed a 6- endo- dig mode giving 3,6-diaryl-4-hydroxy-2-pyrones, or a 5- exo- dig mode giving ( Z)-configured 2-aryl-4-(arylmethylidene)tetronic acids ("pulvinones"). Perfectly selective pyrone formations were induced by AgSbF6 in methanol and equally selective pulvinone formations by Ag2CO3 and DABCO in acetonitrile.

Evaluating the safety, tolerability, pharmacokinetics and efficacy of clofazimine in cryptosporidiosis (CRYPTOFAZ): study protocol for a randomized controlled trial.

BACKGROUND: Cryptosporidium infection and diarrhea (cryptosporidiosis) is a life-threatening infection in persons with HIV and also in children of 6-18 months of age in the developing world. To date, only nitazoxanide is licensed for treatment of cryptosporidiosis, and only in persons after the first year of life and with healthy immune systems. Clofazimine (CFZ: Lamprene®), an established drug that has been used for leprosy for more than 50 years, recently has been described as effective against Cryptosporidium in vitro and in mouse infections. The efficacy and pharmacokinetics of CFZ in vivo, in HIV-infected patients with cryptosporidial diarrhea are not known. METHODS: CRYPTOFAZ includes a randomized, double-blind, placebo-controlled study of the safety, tolerability and Cryptosporidium inhibitory activity of orally administered CFZ in subjects with HIV infection and chronic diarrhea with Cryptosporidium. An additional open label aspect of the study will compare the pharmacokinetics (PK) of orally administered CFZ in HIV-infected individuals with and without Cryptosporidium-associated diarrhea. The study will recruit a total of 66 subjects. Study participants will be given either CFZ or a placebo for 5 days while in hospital and will be followed up after discharge. Cryptosporidium will be diagnosed by quantitative PCR as the definitive test and by stool ELISA, which will also be used to quantify the shedding of Cryptosporidium in stool. PK will be studied on plasma and stool samples. Primary endpoints include reduction in the number of Cryptosporidium shed in stools over a 5-day period and compared to placebo recipients and the PK of CFZ in plasma assessed by area under the curve, peak plasma concentration, and half-life (T ½) determined after the last dose. DISCUSSION: This study provides an opportunity to explore a possible treatment option for HIV-infected patients with cryptosporidial diarrhea, who, as of now in Malawi and most of sub-Saharan Africa, do not have a definitive treatment apart from supportive care. The strength of this study lies in it being a randomized, double-blind, placebo-controlled trial. If shown to be effective and safe, the findings will also lay a foundation for a future study of the use of CFZ in children 6-18 months of age. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03341767 . Registered on 14 November 2017.

Towards Bridging Translational Gap in Cardiotoxicity Prediction: an Application of Progressive Cardiac Risk Assessment Strategy in TdP Risk Assessment of Moxifloxacin.

Drug-induced cardiac arrhythmia, especially occurrence of torsade de pointes (TdP), has been a leading cause of attrition and post-approval re-labeling and withdrawal of many drugs. TdP is a multifactorial event, reflecting more than just drug-induced cardiac ion channel inhibition and QT interval prolongation. This presents a translational gap in extrapolating pre-clinical and clinical cardiac safety assessment to estimate TdP risk reliably, especially when the drug of interest is used in combination with other QT-prolonging drugs for treatment of diseases such as tuberculosis. A multi-scale mechanistic modeling framework consisting of physiologically based pharmacokinetics (PBPK) simulations of clinically relevant drug exposures combined with Quantitative Systems Toxicology (QST) models of cardiac electro-physiology could bridge this gap. We illustrate this PBPK-QST approach in cardiac risk assessment as exemplified by moxifloxacin, an anti-tuberculosis drug with abundant clinical cardiac safety data. PBPK simulations of moxifloxacin concentrations (systemic circulation and estimated in heart tissue) were linked with in vitro measurements of cardiac ion channel inhibition to predict the magnitude of QT prolongation in healthy individuals. Predictions closely reproduced the clinically observed QT interval prolongation, but no arrhythmia was observed, even at ×10 exposure. However, the same exposure levels in presence of physiological risk factors, e.g., hypokalemia and tachycardia, led to arrhythmic event in simulations, consistent with reported moxifloxacin-related TdP events. Application of a progressive PBPK-QST cardiac risk assessment paradigm starting in early development could guide drug development decisions and later define a clinical "safe space" for post-approval risk management to identify high-risk clinical scenarios.

Quantitative approach for cardiac risk assessment and interpretation in tuberculosis drug development.

Cardiotoxicity is among the top drug safety concerns, and is of specific interest in tuberculosis, where this is a known or potential adverse event of current and emerging treatment regimens. As there is a need for a tool, beyond the QT interval, to quantify cardiotoxicity early in drug development, an empirical decision tree based classifier was developed to predict the risk of Torsades de pointes (TdP). The cardiac risk algorithm was developed using pseudo-electrocardiogram (ECG) outputs derived from cardiac myocyte electromechanical model simulations of increasing concentrations of 96 reference compounds which represented a range of clinical TdP risk. The algorithm correctly classified 89% of reference compounds with moderate sensitivity and high specificity (71 and 96%, respectively) as well as 10 out of 12 external validation compounds and the anti-TB drugs moxifloxacin and bedaquiline. The cardiac risk algorithm is suitable to help inform early drug development decisions in TB and will evolve with the addition of emerging data.

Month 2 culture status and treatment duration as predictors of recurrence in pulmonary tuberculosis: model validation and update.

BACKGROUND: New regimens capable of shortening tuberculosis treatment without increasing the risk of recurrence are urgently needed. A 2013 meta-regression analysis, using data from trials published from 1973 to 1997 involving 7793 patients, identified 2-month sputum culture status and treatment duration as independent predictors of recurrence. The resulting model predicted that if a new 4-month regimen reduced the proportion of patients positive at month 2 to 1%, it would reduce to 10% the risk of a relapse rate >10% in a trial with 680 subjects per arm. The 1% target was far lower than anticipated. METHODS: Data from the 8 arms of 3 recent unsuccessful phase 3 treatment-shortening trials of fluoroquinolone-substituted regimens (REMox, OFLOTUB, and RIFAQUIN) were used to assess and refine the accuracy of the 2013 meta-regression model. The updated model was then tested using data from a treatment shortening trial reported in 2009 by Johnson et al. FINDINGS: The proportions of patients with recurrence as predicted by the 2013 model were highly correlated with observed proportions as reported in the literature (R2 = 0.86). Using the previously proposed threshold of 10% recurrences as the maximum likely considered acceptable by tuberculosis control programs, the original model correctly identified all 4 six-month regimens as satisfactory, and 3 of 4 four-month regimens as unsatisfactory (sensitivity = 100%, specificity = 75%, PPV = 80%, and NPV = 100%). A revision of the regression model based on the full dataset of 66 regimens and 11181 patients resulted in only minimal changes to its predictions. A test of the revised model using data from the treatment shortening trial of Johnson et al found the reported relapse rates in both arms to be consistent with predictions. INTERPRETATION: Meta-regression modeling of recurrence based on month 2 culture status and regimen duration can inform the design of future phase 3 tuberculosis clinical trials.

Analysis of spontaneous hippocampal activity allows sensitive detection of acetylcholine-mediated effects.

INTRODUCTION: Excitation of Acetylcholine-mediated (Ach) transmission (especially if irreversible) may pose life-threatening adverse events by increasing neuronal network activity. Unfortunately, adequate detection of this liability during early drug development is hampered, because published ex vivo electrophysiological models are very insensitive to this regard. For example, Eserine, which reversibly inhibits acetylcholinesterase (AchE) in the double digit nM range, affects electrically evoked potentials in hippocampal slices only at ≥10µM. Here, a significantly more sensitive method for detecting Ach-mediated alternations is presented by analyzing spontaneous neuronal network activity in hippocampal slices. METHODS: The microelectrode array (MEA) technique with an 8×8 electrode grid was applied to analyze evoked and spontaneous extracellular field recordings in parallel from acute rat hippocampal slices. For evoked potentials, the Schaffer collateral CA3-CA1 pathway was electrically stimulated and the resulting field potential analyzed at the CA1 pyramidal layer. Spontaneous spike activity was detected as negative inflections from the 100Hz high pass filtered signal. Spike frequency was analyzed within the whole CA1 region. RESULTS: Modification of Ach-mediated neuronal transmission via carbachol, Eserine, or Diisopropylfluorophosphate (DFP) does not induce any effects on evoked field potentials at physiologically relevant concentrations. Similar to previous reports, subtle effects were detectable at very high concentrations. By contrast, spontaneous spike frequency was already increased within the expected concentration range. Eserine-induced effects can also be reversed by atropine and washout. On the contrary, effects by the irreversible AchE-blocker DFP could not be washed out. DISCUSSION: Compared to evoked field potentials, spontaneous spike activity in the hippocampal CA1 region appears to be a significantly more sensitive parameter for functional electrophysiological analysis of drug induced Ach-mediated effects. This finding may supplement existing models for detection and prediction of drug-related adverse effects like seizure liability already during early development stages.

Synthetic Aß oligomers (Aß(1-42) globulomer) modulate presynaptic calcium currents: prevention of Aß-induced synaptic deficits by calcium channel blockers.

Alzheimer's disease is accompanied by increased brain levels of soluble amyloid-ß (Aß) oligomers. It has been suggested that oligomers directly impair synaptic function, thereby causing cognitive deficits in Alzheimer's disease patients. Recently, it has been shown that synthetic Aß oligomers directly modulate P/Q-type calcium channels, possibly leading to excitotoxic cascades and subsequent synaptic decline. Using whole-cell recordings we studied the modulation of recombinant presynaptic calcium channels in HEK293 cells after application of a stable Aß oligomer preparation (Aß1-42 globulomer). Aß globulomer shifted the half-activation voltage of P/Q-type and N-type calcium channels to more hyperpolarized values (by 11.5 and 7.5 mV). Application of non-aggregated Aß peptides had no effect. We then analyzed the potential of calcium channel blockers to prevent Aß globulomer-induced synaptic decline in hippocampal slice cultures. Specific block of P/Q-type or N-type calcium channels with peptide toxins completely reversed Aß globulomer-induced deficits in glutamatergic neurotransmission. Two state-dependent low molecular weight P/Q-type and N-type calcium channel blockers also protected neurons from Aß-induced alterations. On the contrary, inhibition of L-type calcium channels failed to reverse the deficit. Our data show that Aß globulomer directly modulates recombinant P/Q-type and N-type calcium channels in HEK293 cells. Block of presynaptic calcium channels with both state-dependent and state-independent modulators can reverse Aß-induced functional deficits in synaptic transmission. These findings indicate that presynaptic calcium channel blockers may be a therapeutic strategy for the treatment of Alzheimer's disease.

Establishment of a secondary screening assay for P/Q-type calcium channel blockers.

Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically overactive channels. We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steady-state inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast, inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch clamp recordings. Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and--if necessary--a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable for the generation of assays in other ion channel programs.

The effects of aldosterone synthase inhibition on aldosterone and cortisol in patients with hypertension: a phase II, randomized, double-blind, placebo-controlled, multicenter study.

Blockade of the renin-angiotensin-aldosterone system (RAAS) is an established method to lower blood pressure in patients with hypertension. Aldosterone, the end product of the RAAS cascade, acts by increasing salt reabsorption in the kidney and catecholamine release from the adrenal medulla. Currently available aldosterone inhibitors have the disadvantage of increasing circulating aldosterone and thus may lead to aldosterone breakthrough. Aldosterone synthase inhibition (ASI) is a novel approach to suppressing the RAAS. Due to homology between the enzymes responsible for aldosterone synthesis (CYP11B2) and cortisol synthesis (CYP11B1), the blockade of aldosterone synthesis may also suppress cortisol release. The authors evaluated the effect of the novel ASI LCI699 on the cortisol response to adrenocorticotropic hormone (ACTH) stimulation in patients with hypertension in order to find the maximally tolerated dose (MTD) in this patient population. Among the 63 patients evaluated, there was a dose- and time-dependent effect of LCI699 on both aldosterone and ACTH-stimulated cortisol. Based on exposure-response analysis, the MTD was estimated to be 1.30 mg once daily with a 90% prediction interval of 0.88 mg once daily to 1.81 mg once daily. No patients required intervention for adrenal insufficiency. LCI699 was well tolerated with no serious adverse events.

Development and validation of a fluorescence-based HTS assay for the identification of P/Q-type calcium channel blockers.

Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is evidence that migraine, Alzheimer's disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of disease symptoms. Although the medical need is high, there are no such compounds on the market. Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type calcium channel under control of a tetracycline (Tet) promoter. The accessory ß1.1 and α2δ1 subunits were co-expressed constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 µM. Hit-to-lead (HTL) efforts identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized. An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp analysis at inactivated channel state. The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological diseases.

DG-041 inhibits the EP3 prostanoid receptor--a new target for inhibition of platelet function in atherothrombotic disease.

Receptors for prostanoids on platelets include the EP3 receptor for which the natural agonist is the inflammatory mediator prostaglandin E(2) (PGE(2)) produced in atherosclerotic plaques. EP3 is implicated in atherothrombosis and an EP3 antagonist might provide atherosclerotic lesion-specific antithrombotic therapy. DG-041 (2,3-dichlorothiophene-5-sulfonic acid, 3-[1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-indol-7-yl]acryloylamide) is a direct-acting EP3 antagonist currently being evaluated in Phase 2 clinical trials. We have examined the contributions of EP3 to platelet function using the selective EP3 agonist sulprostone and also PGE(2), and determined the effects of DG-041 on these. Studies were in human platelet-rich plasma or whole blood and included aggregometry and flow cytometry. Sulprostone enhanced aggregation induced by primary agonists including collagen, TRAP, platelet activating factor, U46619, serotonin and adenosine diphosphate, and enhanced P-selectin expression and platelet-leukocyte conjugate formation. It inhibited adenylate cyclase (measured by vasodilator-stimulated phosphoprotein phosphorylation) and enhanced Ca(2+) mobilization. It potentiated platelet function even in the presence of aspirin and/or AR-C69931 (a P2Y(12) antagonist). DG-041 antagonized the effects of sulprostone on platelet function. The effect of PGE(2) on platelet aggregation depended on the nature of the agonist and the concentration of PGE(2) used as a consequence of both pro-aggregatory effects via EP3 and anti-aggregatory effects via other receptors. DG-041 potentiated the protective effects of PGE(2) on platelet aggregation by inhibiting the pro-aggregatory effect via EP3 stimulation. DG-041 remained effective in the presence of a P2Y(12) antagonist and aspirin. DG-041 warrants continued investigation as a potential agent for the treatment of atherothrombosis without inducing unwanted bleeding risk.