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.

Effectiveness of systemic family therapy versus treatment as usual for young people after self-harm: a pragmatic, phase 3, multicentre, randomised controlled trial.

BACKGROUND: Self-harm in adolescents is common and repetition occurs in a high proportion of these cases. Scarce evidence exists for effectiveness of interventions to reduce self-harm. METHODS: This pragmatic, multicentre, randomised, controlled trial of family therapy versus treatment as usual was done at 40 UK Child and Adolescent Mental Health Services (CAMHS) centres. We recruited young people aged 11-17 years who had self-harmed at least twice and presented to CAMHS after self-harm. Participants were randomly assigned (1:1) to receive manualised family therapy delivered by trained and supervised family therapists or treatment as usual by local CAMHS. Participants and therapists were aware of treatment allocation; researchers were masked. The primary outcome was hospital attendance for repetition of self-harm in the 18 months after group assignment. Primary and safety analyses were done in the intention-to-treat population. The trial is registered at the ISRCTN registry, number ISRCTN59793150. FINDINGS: Between Nov 23, 2009, and Dec 31, 2013, 3554 young people were screened and 832 eligible young people consented to participation and were randomly assigned to receive family therapy (n=415) or treatment as usual (n=417). Primary outcome data were available for 795 (96%) participants. Numbers of hospital attendances for repeat self-harm events were not significantly different between the groups (118 [28%] in the family therapy group vs 103 [25%] in the treatment as usual group; hazard ratio 1·14 [95% CI 0·87-1·49] p=0·33). Similar numbers of adverse events occurred in both groups (787 in the family therapy group vs 847 in the treatment as usual group). INTERPRETATION: For adolescents referred to CAMHS after self-harm, having self-harmed at least once before, our family therapy intervention conferred no benefits over treatment as usual in reducing subsequent hospital attendance for self-harm. Clinicians are therefore still unable to recommend a clear, evidence-based intervention to reduce repeated self-harm in adolescents. FUNDING: National Institute for Health Research Health Technology Assessment programme.

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.

Negative inotropic effect of a CB2 agonist A-955840 in isolated rabbit ventricular myocytes is independent of CB1 and CB2 receptors.

A-955840, a selective CB2 agonist, has been shown to elicit concentration-dependent decreases in cardiac contractility in the anesthetized dog (decreased maximal velocity of left ventricular pressure development [LV dP/dt max]). However, it is unknown whether this represents a direct effect or a response dependent on other factors (such as autonomic tone and neurohumoral factors) present in vivo. This study examined if A-955840 had a direct effect on contractility of isolated cardiac myocytes, and if so to determine the potential mechanisms. Contractility was assessed in vitro using percent changes in maximal shortening velocity of sarcomeres (dL/dt max) and fractional shortening of sarcomere length (FS) in rabbit left ventricular myocytes. L-type calcium current in myocytes was recorded using wholecell voltage-clamp techniques. A-955840 reduced dL/dt max and FS in a reversible and concentration-dependent manner with an IC50 of 11.4 µg/mL (based on dL/dt max) which is similar to the estimated IC50 value of 9.8 µg/mL based on the effects of A-955840 on LV dP/dt max in anesthetized dogs. A-955840 (4.1 µg/mL) reduced myocyte contractility (%FS) to a similar extent in the absence and presence of a CB2 antagonist, SR-2 (24.0 ± 3.4 vs 23.1 ± 3.0 %, n=5) or a CB1 antagonist, Rimonabant (18.8 ± 2.3 vs 19.8 ± 2.7 %, n=5). A-955840 (4.1 µg/mL) also reduced L-type calcium current of rabbit ventricular myocytes (1.05 ± 0.11 vs 0.70 ± 0.12 nA, n=5, P < 0.01). These results suggest that A-955840 exerts direct negative inotropic effects on isolated rabbit ventricular myocytes, which is mediated by neither CB2 nor CB1 receptors, and consistent with off-target negative inotropy mediated by inhibition of the cardiac L-type calcium current.

A novel secretagogue increases cardiac contractility by enhancement of L-type Ca2+ current.

N'1-(3,3,6,8-tetramethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yliden)-2-cyanoethanohydrazide (TTYC) increases secretion of glucagon-like peptide-1 and intracellular Ca(2+) concentration in GLUTag cells. The purpose of the present study was to examine if TTYC exerts positive inotropic effects on isolated rabbit ventricular myocytes and in vivo heart in anesthetized rats, and if so to further define the potential mechanism of action. Contractility was assessed in vitro using changes in fractional shortening (FS) of myocyte sarcomere length and in vivo using changes in the velocity of left ventricular pressure. Changes in L-type Ca(2+) current of ventricular myocytes were evaluated using whole-cell voltage-clamp techniques. TTYC increased FS of myocyte sarcomere length in a concentration-dependent manner. The positive inotropic effect was not abrogated by beta-adrenergic blockade (propranolol) or protein kinase A inhibition. TTYC enhanced peak L-type Ca(2+) current in a voltage-dependent manner (current amplitudes increased by 4.0-fold at -10 mV and 1.5-fold at +10 mV). Voltage-dependence of steady-state activation of L-type Ca(2+) current was shifted by 15 mV in the negative direction. Inactivation time course of the L-type Ca(2+) currents at voltages of -10 to 20 mV was significantly slowed by 0.3 microM TTYC. In vivo studies demonstrated that TTYC increased cardiac contractility in a dose-dependent manner. In conclusion, TTYC is a novel L-type Ca(2+) current activator with positive cardiac inotropic effects. Negative shifting of the voltage-dependence of L-type Ca(2+) current activation and reduced inactivation are two mechanisms responsible for the enhanced L-type Ca(2+) current that contribute to the positive inotropic effects.

Effect of hot versus cold climates on power output, muscle activation, and perceived fatigue during a dynamic 100-km cycling trial.

The purpose of this study was to examine the influence of environmental temperature on power output, muscle activation, body temperature, and perceived physical strain during a dynamic self-paced 100-km cycling trial. Nine endurance-trained male cyclists (mean + or - s: age 31 + or - 6 years; VO(2max) 62.1 + or - 8.5 ml x kg(-1) x min(-1)) completed two 100-km experimental trials, interspersed with five 1-km and four 4-km high-intensity epochs, in hot (34 degrees C) and cold (10 degrees C) environments. Measurements consisted of power output, rectal and skin temperature, muscle activation of vastus lateralis, biceps femoris and soleus, ratings of perceived exertion, thermal sensation and pain intensity in the quadriceps. Power output and muscle activation of the biceps femoris and soleus were lower in the hot trial (22 km; P < 0.05) prior to significant (P < 0.05) differences in rectal temperature [38.8 degrees C (cold) vs. 39.1 degrees C (hot)] at 42 km. Muscle activation of the vastus lateralis, biceps femoris, and soleus was significantly (P < 0.001) correlated with power output and thermal sensation (r > 0.68) but not with perceived pain or exertion. Thus, a hyperthermic-induced anticipatory reduction of muscle activation may have occurred during the hot exercise trials only. Fatigue and pacing during prolonged dynamic exercise in the cold appears to be influenced by factors dissociated from hyperthermic-induced stress.

Temporal and kinetic analysis of unilateral jumping in the vertical, horizontal, and lateral directions.

The aims of this study were to: (1) assess the reliability of various kinetic and temporal variables for unilateral vertical, horizontal, and lateral countermovement jumps; (2) determine whether there are differences in vertical ground reaction force production between the three types of jumps; (3) quantify the magnitude of asymmetry between limbs for variables that were established as reliable in a healthy population and whether asymmetries were consistent across jumps of different direction; and (4) establish the best kinetic predictor(s) of jump performance in the vertical, horizontal, and lateral planes of motion. Thirty team sport athletes performed three trials of the various countermovement jumps on both legs on two separate occasions. Eccentric and concentric peak force and concentric peak power were the only variables with acceptable reliability (coefficient of variation = 3.3-15.1%; intra-class correlation coefficient = 0.70-0.96). Eccentric and concentric peak vertical ground reaction force (14-16%) and concentric peak power (45-51%) were significantly (P < 0.01) greater in the vertical countermovement jump than in the horizontal countermovement jump and lateral countermovement jump, but no significant difference was found between the latter two jumps. No significant leg asymmetries (-2.1% to 9.3%) were found in any of the kinetic variables but significant differences were observed in jump height and distance. The best single predictors of vertical countermovement jump, horizontal countermovement jump, and lateral countermovement jump performance were concentric peak vertical power/body weight (79%), horizontal concentric peak power/body weight (42.6%), and eccentric peak vertical ground reaction force/body weight (14.9%) respectively. These findings are discussed in relation to monitoring and developing direction-specific jump performance.

Duration of Loxosceles reclusa venom detection by ELISA from swabs.

BACKGROUND: Diagnosis of Loxosceles reclusa envenomations is currently based upon clinical presentation. An enzyme-linked immunosorbent assay (ELISA) can detect surface Loxosceles venom at the envenomation site, allowing diagnostic confirmation. The length of time that venom on the skin is recoverable non-invasively is unknown. MATERIALS AND METHODS: To investigate duration of recoverable venom antigen, whole venom and fractionated sphingomyelinase D venom aliquots were injected subcutaneously in New Zealand White rabbits. Cotton and Dacron swabs were compared for venom recovery over a 21-day period using a surface swab technique. RESULTS: Significant amounts of Loxosceles reclusa antigen were found on the surface of rabbit skin after experimental injection of whole venom and sphingomyelinase D. The duration of recoverable antigen using this experimental model appears to be at least two weeks and as long as 21 days in some cases. CONCLUSIONS: Because the duration of the recoverable antigen is seen to be at least two weeks, the ELISA venom test appears capable of detecting venom on most patients presenting with Loxosceles envenomations. This detection system will allow the physician more accurate determination of whether the lesion is from a brown recluse spider or some other agent that can cause this type of necrotic ulcer.

Validation of an EMG-driven, graphically based isometric musculoskeletal model of the cervical spine.

EMG-driven musculoskeletal modeling is a method in which loading on the active and passive structures of the cervical spine may be investigated. A model of the cervical spine exists; however, it has yet to be criterion validated. Furthermore, neck muscle morphometry in this model was derived from elderly cadavers, threatening model validity. Therefore, the overall aim of this study was to modify and criterion validate this preexisting graphically based musculoskeletal model of the cervical spine. Five male subjects with no neck pain participated in this study. The study consisted of three parts. First, subject-specific neck muscle morphometry data were derived by using magnetic resonance imaging. Second, EMG drive for the model was generated from both surface (Drive 1: N=5) and surface and deep muscles (Drive 2: N=3). Finally, to criterion validate the modified model, net moments predicted by the model were compared against net moments measured by an isokinetic dynamometer in both maximal and submaximal isometric contractions with the head in the neutral posture, 20 deg of flexion, and 35 deg of extension. Neck muscle physiological cross sectional area values were greater in this study when compared to previously reported data. Predictions of neck torque by the model were better in flexion (18.2% coefficient of variation (CV)) when compared to extension (28.5% CV) and using indwelling EMG did not enhance model predictions. There were, however, large variations in predictions when all the contractions were compared. It is our belief that further work needs to be done to improve the validity of the modified EMG-driven neck model examined in this study. A number of factors could potentially improve the model with the most promising probably being optimizing various modeling parameters by using methods established by previous researchers investigating other joints of the body.

Effect of starting stance on initial sprint performance.

The effect of different starting stances from a standing position on short sprint times and the subsequent variability in times was investigated in this study. A dual-beam timing light system was used to measure 5- and 10-m times for 3 different standing starts commonly found in the sporting environment: parallel (feet parallel to the start line), split (lead left foot on start line, right leg back), and false (initial parallel start, right leg drops back to split start when movement initiated). The parallel start was found to be significantly (alpha < 0.05) slower than the other 2 stances for both the 5- ( approximately 8.3%) and the 10-m (approximately 5.9%) distances. Within the trial, variation of the different starting stances was equally consistent; however, there was less variability for the 10-m distance (CV = 1.16-1.67%) than the 5-m distance (CV = 1.43-2.15%) for each start for both men and women. The split and false start seem to offer the best option as a movement strategy for minimizing short-distance sprint times. However, the benefits of these 2 starts are less clear if total movement time is the variable of interest.

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.

Examination of EMG normalisation methods for the study of the posterior and posterolateral neck muscles in healthy controls.

The purpose of this study was to examine the reliability of normalisation methods used in the study of the posterior and posterolateral neck muscles in a group of healthy controls. Six asymptomatic male subjects performed a total of 12 maximum voluntary isometric contractions (MVIC) and 60%-submaximal isometric contractions (60%-MVIC) against the torque arm of an isokinetic dynamometer whilst surface and intramuscular electromyography (EMG) was recorded unilaterally from representative posterior and posterolateral locations. Reliability was calculated using intra-class correlation coefficient (ICC), relative standard error of measurement (%SEM) and relative coefficient of variation (%CV). Maximal torque output was found to be highly reliable in the directions of extension and right lateral bending when the first of three MVIC contractions was excluded. When averaged across contraction direction, high reliability was found for both surface (MVIC: ICC=0.986, %SEM=7.5, %CV=9.2; 60%-MVIC: ICC=0.975, %SEM=10, %CV=13.7) and intramuscular (MVIC: ICC=0.910, %SEM=20, %CV=19.1; 60%-MVIC: ICC=0.952, %SEM=16.5, %CV=13.5) electrodes. Intramuscular electrodes displayed the least reliability in right lateral bending. The use of visual feedback markedly increased the reliability of 60%-MVIC contractions.

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.