BackgroundThe Janus kinase (JAK) family of tyrosine kinases includes JAK1, JAK2, JAK3 and TYK2, and is required for signaling through Type I and Type II cytokine receptors. CP-690,550 is a potent and selective JAK inhibitor currently in clinical trials for rheumatoid arthritis (RA) and other autoimmune disease indications. In RA trials, dose-dependent decreases in neutrophil counts (PBNC) were observed with CP-690,550 treatment. These studies were undertaken to better understand the relationship between JAK selectivity and PBNC decreases observed with CP-690,550 treatment.MethodsPotency and selectivity of CP-690,550 for mouse, rat and human JAKs was evaluated in a panel of in vitro assays. The effect of CP-690,550 on granulopoiesis from progenitor cells was also assessed in vitro using colony forming assays. In vivo the potency of orally administered CP-690,550 on arthritis (paw edema), plasma cytokines, PBNC and bone marrow differentials were evaluated in the rat adjuvant-induced arthritis (AIA) model.ResultsCP-690,550 potently inhibited signaling through JAK1 and JAK3 with 5-100 fold selectivity over JAK2 in cellular assays, despite inhibiting all four JAK isoforms with nM potency in in vitro enzyme assays. Dose-dependent inhibition of paw edema was observed in vivo with CP-690,550 treatment. Plasma cytokines (IL-6 and IL-17), PBNC, and bone marrow myeloid progenitor cells were elevated in the context of AIA disease. At efficacious exposures, CP-690,550 returned all of these parameters to pre-disease levels. The plasma concentration of CP-690,550 at efficacious doses was above the in vitro whole blood IC50 of JAK1 and JAK3 inhibition, but not that of JAK2.ConclusionResults from this investigation suggest that CP-690,550 is a potent inhibitor of JAK1 and JAK3 with potentially reduced cellular potency for JAK2. In rat AIA, as in the case of human RA, PBNC were decreased at efficacious exposures of CP-690,550. Inflammatory end points were similarly reduced, as judged by attenuation of paw edema and cytokines IL-6 and IL-17. Plasma concentration at these exposures was consistent with inhibition of JAK1 and JAK3 but not JAK2. Decreases in PBNC following CP-690,550 treatment may thus be related to attenuation of inflammation and are likely not due to suppression of granulopoiesis through JAK2 inhibition.

Toxicity is a leading cause of attrition at all stages of the drug development process. The majority of safety-related attrition occurs preclinically, suggesting that approaches to identify 'predictable' preclinical safety liabilities earlier in the drug development process could lead to the design and/or selection of better drug candidates that have increased probabilities of becoming marketed drugs. In this Review, we discuss how the early application of preclinical safety assessment--both new molecular technologies as well as more established approaches such as standard repeat-dose rodent toxicology studies--can identify predictable safety issues earlier in the testing paradigm. The earlier identification of dose-limiting toxicities will provide chemists and toxicologists the opportunity to characterize the dose-limiting toxicities, determine structure-toxicity relationships and minimize or circumvent adverse safety liabilities.

Cyclooxygenase (COX) exists in 2 related but unique isoforms: one is constitutive (COX-1) and functions in normal cell physiology, and the other is inducible (COX-2) and is expressed in response to inflammatory stimuli. Nonsteroidal antiinflammatory drugs (NSAIDs) cause renal toxicity following inhibition of renal cyclooxygenases. Humans and animals exhibit differences in susceptibility to NSAID-related renal toxicity, which may be associated with differences in expression of 1 or both isoforms of COX in the kidney. In this study, we evaluated COX-1 and COX-2 expression in the kidneys of mixed-breed dogs, Sprague-Dawley rats, cynomolgus monkeys, and humans. In addition, the effect of volume depletion on renal COX expression was investigated in rats, dogs, and monkeys. COX expression was evaluated using 1 or more of the following procedures: reverse transcriptase polymerase chain reaction, in situ hybridization, and immunohistochemistry. We demonstrated that both COX isoforms are expressed in the kidneys of all species examined, with differences in the localization and level of basal expression. COX-1 is expressed at high levels in the collecting ducts and renal vasculature of all species and in a small number of papillary interstitial cells in rats, monkeys, and humans. Basal levels of COX-2 are present in the maculae densa, thick ascending limbs, and papillary interstitial cells in rats and dogs and in glomerular podocytes and small blood vessels in monkeys and humans. COX-2 expression is markedly increased in volume-depleted rats and dogs but not monkeys. These results indicate that significant interspecies differences exist in the presence and distribution of COX isoforms, which may help explain the difference in species susceptibility to NSAID-related renal toxicity.

SummaryPopulations of murine peritoneal B-1 and splenic B-2 cells, highly purified by negative selection techniques, were used to demonstrate that B-1 cells completely fail to enter cell cycle in response to surface immunoglobulin M (slgM) crosslinking without any decrease in cell number or viability. This failure of B-1 cell responsiveness appears to represent a specific defect in sIgM-derived signaling inasmuch as stimulation to enter S phase occurs normally in response to activated and fixed T cells, and to lipopolysaccharide (LPS). The level at which slgM signaling fails was determined by evaluating the nuclear expression of the transcription factor complex, NF-KB, whose slgMmediated induction in B-2 cells is dependent on protein kinase C (PKC) activation but is independent of protein synthesis. There was no induction of nuclear NF-KB in B-1 cells stimulated by slgM crosslinking, although NF-KB was stimulated by phorbol myristate acetate and by LPS. In contrast, NF-IcB was induced in B-2 cells by all three stimuli. Thus, in B-1 cells, the slgM-mediated induction of a transcription factor that is substantially stimulated by anti-IgM in B-2 cells is blocked. However, all slgM-derived signaling in B-1 cells was not impaired inasmuch as anti-IgM increased I-A antigen expression. These results strongly suggest that slgM receptor-mediated signaling in B-1 cells is interrupted early in the signal transduction pathway, at a point proximal to the activation of PKC. These results further demonstrate that transcription factor induction can be used to analyze the level at which receptor-mediated signaling is blocked.