Discovery of Allosteric, Potent, Subtype Selective, and Peripherally Restricted TrkA Kinase Inhibitors.

Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein, we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious, and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound 23.

Discovery of Potent, Selective, and Peripherally Restricted Pan-Trk Kinase Inhibitors for the Treatment of Pain.

Hormones of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), are known to activate the family of Tropomyosin receptor kinases (TrkA, TrkB, and TrkC). Moreover, inhibition of the TrkA kinase pathway in pain has been clinically validated by the NGF antibody tanezumab, leading to significant interest in the development of small molecule inhibitors of TrkA. Furthermore, Trk inhibitors having an acceptable safety profile will require minimal brain availability. Herein, we discuss the discovery of two potent, selective, peripherally restricted, efficacious, and well-tolerated series of pan-Trk inhibitors which successfully delivered three candidate quality compounds 10b, 13b, and 19. All three compounds are predicted to possess low metabolic clearance in human that does not proceed via aldehyde oxidase-catalyzed reactions, thus addressing the potential clearance prediction liability associated with our current pan-Trk development candidate PF-06273340.

The Discovery of a Potent, Selective, and Peripherally Restricted Pan-Trk Inhibitor (PF-06273340) for the Treatment of Pain.

The neurotrophin family of growth factors, comprised of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), is implicated in the physiology of chronic pain. Given the clinical efficacy of anti-NGF monoclonal antibody (mAb) therapies, there is significant interest in the development of small molecule modulators of neurotrophin activity. Neurotrophins signal through the tropomyosin related kinase (Trk) family of tyrosine kinase receptors, hence Trk kinase inhibition represents a potentially "druggable" point of intervention. To deliver the safety profile required for chronic, nonlife threatening pain indications, highly kinase-selective Trk inhibitors with minimal brain availability are sought. Herein we describe how the use of SBDD, 2D QSAR models, and matched molecular pair data in compound design enabled the delivery of the highly potent, kinase-selective, and peripherally restricted clinical candidate PF-06273340.

Recommendations for safety pharmacology evaluations of oligonucleotide-based therapeutics.

This document was prepared by the Safety Pharmacology Subcommittee of the Oligonucleotide Safety Working Group (OSWG), a group of industry and regulatory scientists involved in the development and regulation of therapeutic oligonucleotides. The mission of the Subcommittee was to develop scientific recommendations for the industry regarding the appropriate scope and strategies for safety pharmacology evaluations of oligonucleotides (ONs). These recommendations are the consensus opinion of the Subcommittee and do not necessarily reflect the current expectations of regulatory authorities. 1) Safety pharmacology testing, as described in the International Conference on Harmonisation (ICH) S7 guidance, is as applicable to ONs as it is to small molecule drugs and biotherapeutics. 2) Study design considerations for ONs are similar to those for other classes of drugs. In general, as with other therapeutics, studies should evaluate the drug product administered via the clinical route. Species selection should ideally consider relevance of the model with regard to the endpoints of interest, pharmacological responsiveness, and continuity with the nonclinical development program. 3) Evaluation of potential effects in the core battery (cardiovascular, central nervous, and respiratory systems) is recommended. In general: a. In vitro human ether-a-go-go-related gene (hERG) testing does not provide any specific value and is not warranted. b. Emphasis should be placed on in vivo evaluation of cardiovascular function, typically in nonhuman primates (NHPs). c. Due to the low level of concern, neurologic and respiratory function can be assessed concurrently with cardiovascular safety pharmacology evaluation in NHPs, within repeat-dose toxicity studies, or as stand-alone studies. In the latter case, rodents are most commonly used. 4) Other dedicated safety pharmacology studies, beyond the core battery, may have limited value for ONs. Although ONs can accumulate in the kidney and liver, evaluation of functional changes in these organs, as well as gastrointestinal (GI) and unintended "pro-inflammatory" effects, may be best evaluated during repeat-dose toxicity studies. Broad receptor- or ligand-binding profiling has not historically been informative for most ON subclasses, but may have value for investigative purposes.

Reducing safety-related drug attrition: the use of in vitro pharmacological profiling.

In vitro pharmacological profiling is increasingly being used earlier in the drug discovery process to identify undesirable off-target activity profiles that could hinder or halt the development of candidate drugs or even lead to market withdrawal if discovered after a drug is approved. Here, for the first time, the rationale, strategies and methodologies for in vitro pharmacological profiling at four major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis and Pfizer) are presented and illustrated with examples of their impact on the drug discovery process. We hope that this will enable other companies and academic institutions to benefit from this knowledge and consider joining us in our collaborative knowledge sharing.

Zebrafish assays as early safety pharmacology screens: paradigm shift or red herring?

The recent flurry of interest in the potential use of the zebrafish (Danio rerio) in Drug Discovery has also led to the development of a range of assays purported to be useful as early screens in safety pharmacology. The purpose of this commentary is to take stock of the available zebrafish assays in the context of alternative mammalian cell-based assays, and of the validation outcomes to date. In addition, we report the results of a recent survey of the membership of the Safety Pharmacology Society regarding their views on zebrafish assays. The survey data indicate that the preferred way forward would be a collaborative effort between the pharmaceutical/biotechnology industry (as potential/eventual customers), and the zebrafish contract research companies (as suppliers), alongside expert input from academia and regulatory authorities.

Zebrafish based assays for the assessment of cardiac, visual and gut function--potential safety screens for early drug discovery.

INTRODUCTION: Safety pharmacology is integral to the non-clinical safety assessment of new chemical entities prior to first administration to humans. The zebrafish is a well established model organism that has been shown to be relevant to the study of human diseases. The potential role of zebrafish in safety pharmacology was evaluated using reference compounds in three models assessing cardiac, visual and intestinal function. METHODS: Compound toxicity was first established in zebrafish to determine the non toxic concentration of a blinded set of 16 compounds. In the cardiac assay, zebrafish larvae at 3 days post fertilisation (d.p.f.) were exposed to compounds for 3 h before measurement of the atrial and ventricular rates. To investigate visual function, the optomotor response was assessed in 8 d.p.f. larvae following a 5 day compound exposure. In the intestinal function assay, the number of gut contractions was measured in 7 d.p.f. larvae after a 1 h compound exposure. Finally, compound uptake was determined for 9 of the 16 compounds to measure the concentration of compound absorbed by the zebrafish larvae. RESULTS: Seven compounds out of nine produced an expected effect that was statistically significant in the cardiac and visual functions assays. In the gut contraction assay, six out of ten compounds showed a statistically significant effect that was also the expected result whilst two displayed anticipated but non-significant effects. The compound uptake method was used to determine larval tissue concentrations and allowed the identification of false negatives when compound was poorly absorbed into the zebrafish. DISCUSSION: Overall, results generated in three zebrafish larvae assays demonstrated a good correlation between the effects of compounds in zebrafish and the data available from other in vivo models or known clinical adverse effects. These results suggest that for the cardiac, intestinal and visual function, zebrafish assays have the potential to predict adverse drug effects and supports their possible role in early safety assessment of novel compounds.

A role for the middle C terminus of G-protein-activated inward rectifier potassium channels in regulating gating.

We have used sulfhydryl-modifying reagents to investigate the regulation of G-protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increased the current by approximately 17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism.

High-throughput screening for ion channel modulators.

Ion channels present a group of targets for major clinical indications, which have been difficult to address due to the lack of suitable rapid but biologically significant methodologies. To address the need for increased throughput in primary screening, the authors have set up a Beckman/Sagian core system to fully automate functional fluorescence-based assays that measure ion channel function. They apply voltage-sensitive fluorescent probes, and the activity of channels is monitored using Aurora's Voltage/Ion Probe Reader (VIPR). The system provides a platform for fully automated high-throughput screening as well as pharmacological characterization of ion channel modulators. The application of voltage-sensitive fluorescence dyes coupled with fluorescence resonance energy transfer is the basis of robust assays, which can be adapted to the study of a variety of ion channels to screen for both inhibitors and activators of voltage-gated and other ion channels.