Off-target pharmacological activity at various kinases: Potential functional and pathological side effects.

In drug discovery, during the lead optimization and candidate characterization stages, novel small molecules are frequently evaluated in a battery of in vitro pharmacology assays to identify potential unintended, off-target interactions with various receptors, transporters, ion channels, and enzymes, including kinases. Furthermore, these screening panels may also provide utility at later stages of development to provide a mechanistic understanding of unexpected safety findings. Here, we present a compendium of the most likely functional and pathological outcomes associated with interaction(s) to a panel of 95 kinases based on an extensive curation of the scientific literature. This panel of kinases was designed by AbbVie based on safety-related data extracted from the literature, as well as from over 20 years of institutional knowledge generated from discovery efforts. For each kinase, the scientific literature was reviewed using online databases and the most often reported functional and pathological effects were summarized. This work should serve as a practical guide for small molecule drug discovery scientists and clinical investigators to predict and/or interpret adverse effects related to pharmacological interactions with these kinases.

Disconnect between COX-2 selective inhibition and cardiovascular risk in preclinical models.

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.

Ventricular rate adaptation: a novel, rapid, cellular-based in-vitro assay to identify proarrhythmic and torsadogenic compounds.

INTRODUCTION: Delayed cardiac repolarization is an established risk factor for proarrhythmia and Torsades-de-Pointes (TdeP) that is typically measured in vitro during slow, regular stimulation. We have developed an alternative, novel, and rapid cellular-based approach for predicting drug-induced proarrhythmia that detects changes in electrical refractoriness based on mechanical responses (measured optically) during increasingly rapid trains of stimulation interspersed with pauses (mimicking the clinically observed short-long-short (SLS) stimulation sequence associated with the TdeP initiation). METHODS: Acutely isolated rabbit ventricular myocytes were superfused and electrically stimulated using an accelerating pacing protocol (APP) consisting of 12 consecutive pacing segments (10 beats per segment) with incrementally faster cycle lengths; trains were separated by pauses to identify loss of stimulus capture as well as to mimic clinically observed SLS sequences. Drug effects were evaluated based on a myocyte's ability to contract during progressively faster pacing segments (rate-adaptation); the earliest rate during which the myocyte fails to respond (longest cycle length with incomplete capture (CLIC)) was used to quantify electrophysiologic effects. RESULTS: Torsadogenic drugs known to delay repolarization during slow stimulation prolonged CLIC and dramatically limited the ability to respond to progressively rapid stimulation. The recognized proarrhythmic compounds E-4031, cisapride, grepafloxacin, and haloperidol rapidly prolonged CLIC at and above therapeutic concentrations in a concentration-dependent manner, while negative controls (captopril, indomethacin, and loratidine) do not affect rate-adaptation. DISCUSSION: Ventricular rate adaptation represents a novel approach for rapidly detecting drugs with torsadogenic risk using rapid rhythms that are typically not employed when evaluating proarrhythmic risk. This method is well suited for detecting and avoiding potential cardiac liabilities early in drug discovery ("frontloading") prior to final selection of candidate drugs.

The effects of plasma proteins on delayed repolarization in vitro with cisapride, risperidone, and D, L-sotalol.

INTRODUCTION: Drug-induced long QT syndrome (LQTS) has been linked to arrhythmias (including Torsades de Pointes and sudden cardiac death), and has led to an increased awareness of the potential risk of delayed repolarization in vitro and in vivo. However, in vitro assessments of delayed repolarization have not been fully predictive of in vivo effects. METHODS: To define the extent to which plasma protein binding (ppb) contributes to such disparities in repolarization studies, we compared drug-induced prolongation of the canine Purkinje fiber action potential duration (APD(90)) in vitro during superfusion with 100% Tyrode's solution (Tyrodes), canine plasma [50% plasma/50% Tyrodes] and a 5% solution of recombinant human serum albumin in Tyrodes (HSA). Drugs evaluated included cisapride (>98% ppb), risperidone (90% ppb), and d, l-sotalol (negligible ppb). Effects on APD were monitored using standard microelectrode techniques under physiologic conditions and temperature ([K(+)]=4 mM, 37 degrees C) during slow stimulation (2 s basic cycle length). RESULTS: The effects of cisapride and risperidone on Purkinje fiber APD(90) were significantly attenuated in the presence of plasma proteins. However, with cisapride, the extent of reduction with plasma proteins was significantly less than predicted based on calculated free drug levels. DISCUSSION: We conclude that while plasma protein binding does reduce APD prolongation seen with bound drugs, this effect is not well correlated with the calculated plasma protein binding or expected clinical free fraction. Because of the complex drug interactions that occur in plasma, the electrophysiological effects seen with bound drugs are not well correlated with the calculated free fraction and thus caution should be exercised when assigning a predictive safety window. Thus, the canine Purkinje fiber assay is useful for defining the modulation of delayed repolarization due to plasma protein binding of novel therapeutic agents.

Maintenance of remission in Crohn's disease: current and emerging therapeutic options.

Crohn's disease is a chronic inflammatory bowel disorder with a relapsing and remitting course. Once remission is achieved, the main aim of the management of Crohn's disease is maintenance of that remission. Significant advances have been made into understanding the aetiology and pathogenesis of inflammatory bowel disease. With these advances in understanding come increasing numbers of new agents and therapies, aimed both at active disease and the subsequent maintenance of remission in Crohn's disease. Current therapeutic strategies in maintaining remission in Crohn's disease include 5-aminosalicylates (e.g. sulfasalazine, mesalazine), thiopurines (e.g. azathioprine, 6-mercaptopurine [mercaptopurine]), methotrexate and infliximab. The 5-aminosalicylates appear to have efficacy limited to either surgically induced remission and/or limited small bowel Crohn's disease. The immunomodulators now have an established role in Crohn's maintenance. Azathioprine and 6-mercaptopurine are effective in chronic active disease and corticosteroid-dependent Crohn's disease. Methotrexate has similar indications, although it appears to be an alternative in patients who are intolerant of, or resistant to, the thiopurines. The most recent breakthrough has been in the field of biological therapy for maintenance of remission in Crohn's disease. Treatment of patients with the anti-tumour necrosis factor (TNF)-alpha antibody infliximab has been shown already to be effective in inducing remission. Recent studies have now confirmed a role for infliximab in delaying relapse and maintaining remission in patients responsive to infliximab induction therapy. However, results with soluble TNF alpha receptors have been disappointing. A number of other biological and nonbiological agents have shown potential, though trials of the 'newer' biological agents have thus far been disappointing, in the maintenance of remission in Crohn's disease. The evidence for theses agents is currently limited, in many cases to treating active disease; however, these data are discussed in this article in order to provide an overview of future potential therapies. The aim of this review is to provide clinicians with an insight into current and emerging therapeutic agents for the maintenance of remission of Crohn's disease.