Shashi K. Ramaiah

Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis

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.

Role of neutrophils in the pathogenesis of acute inflammatory liver injury.

Polymorphonuclear leukocytes (neutrophils) are essential in the defense against invading microorganisms, tissue trauma or any inciting inflammatory signals. Hepatic infiltration of neutrophils is an acute response to recent or ongoing liver injury, hepatic stress or unknown systemic inflammatory signals. Once neutrophils reach the liver, they can cause mild-to-severe tissue damage and consequent liver failure. For neutrophils to appear in the liver, neutrophils have to undergo systemic activation (priming) by inflammatory mediators such as cytokines, chemokines, complement factors, immune complexes, opsonized particles and other biologically active molecules, e.g., platelet activating factor. Neutrophils accumulated in the hepatic microvasculature (sinusoids and postsinusoidal venules) can extravasate (transmigrate) into the hepatic parenchyma if they receive a signal from distressed cells. Transmigration can be mediated by a chemokine gradient established towards the hepatic parenchyma and generally involves orchestration by adhesion molecules on neutrophils (β2 integrins) and on endothelial cells (intracellular adhesion molecules, ICAM-1). After transmigration, neutrophils adhere to distressed hepatocytes through their β2 integrins and ICAM-1 expressed on hepatocytes. Neutrophil contact with hepatocytes mediate oxidative killing of hepatocytes by initiation of respiratory burst and neutrophil degranulation leading to hepatocellular oncotic necrosis. Neutrophil-mediated liver injury has been demonstrated in a variety of diseases and chemical/drug toxicities. Relevant examples are discussed in this review.

Betulinic Acid Inhibits Prostate Cancer Growth through Inhibition of Specificity Protein Transcription Factors

Betulinic acid is a pentacyclic triterpene natural product initially identified as a melanoma-specific cytotoxic agent that exhibits low toxicity in animal models. Subsequent studies show that betulinic acid induces apoptosis and antiangiogenic responses in tumors derived from multiple tissues; however, the underlying mechanism of action is unknown. Using LNCaP prostate cancer cells as a model, we now show that betulinic acid decreases expression of vascular endothelial growth (VEGF) and the antiapoptotic protein survivin. The mechanism of these betulinic acid-induced antiangiogenic and proapoptotic responses in both LNCaP cells and in tumors is due to activation of selective proteasome-dependent degradation of the transcription factors specificity protein 1 (Sp1), Sp3, and Sp4, which regulate VEGF and survivin expression. Thus, betulinic acid acts as a novel anticancer agent through targeted degradation of Sp proteins that are highly overexpressed in tumors.

Mechanisms of Immune-Mediated Liver Injury

Hepatic inflammation is a common finding during a variety of liver diseases including drug-induced liver toxicity. The inflammatory phenotype can be attributed to the innate immune response generated by Kupffer cells, monocytes, neutrophils, and lymphocytes. The adaptive immune system is also influenced by the innate immune response leading to liver damage. This review summarizes recent advances in specific mechanisms of immune-mediated hepatotoxicity and its application to drug-induced liver injury. Basic mechanisms of activation of lymphocytes, macrophages, and neutrophils and their unique mechanisms of recruitment into the liver vasculature are discussed. In particular, the role of adhesion molecules and various inflammatory mediators in this process are explored. In addition, the authors describe mechanisms of liver cell damage by these inflammatory cells and critically evaluate the functional significance of each cell type for predictive and idiosyncratic drug-induced liver injury. It is expected that continued advances in our understanding of immune mechanisms of liver injury will lead to an earlier detection of the hepatotoxic potential of drugs under development and to an earlier identification of susceptible individuals at risk for predictive and idiosyncratic drug toxicities.

Higher neutrophil infiltration mediated by osteopontin is a likely contributing factor to the increased susceptibility of females to alcoholic liver disease

Alcoholic liver disease (ALD) is a major public health problem in the United States and women are known to be more susceptible to ALD. However, the precise mechanism for increased susceptibility of females to ALD is not completely understood. The present study is based on the hypothesis that induction of osteopontin (OPN), a matricellular protein, is the likely contributing factor for higher neutrophil recruitment in females during alcoholic steatohepatitis (ASH). ASH was induced in male and female Sprague‐Dawley rats by feeding them a Lieber‐DeCarli diet containing ethanol (EtOH) for 6 weeks, followed by a single injection of lipopolysaccharide (LPS, 10 mg/kg, ip). Liver injury, measured by plasma transaminase elevations and confirmed by haematoxylin and eosin‐stained liver sections, revealed ∼25‐fold higher liver injury in the female ASH model compared with the males. Although steatosis, necrosis, and neutrophil infiltration were evident in both male and female rats, hepatic neutrophilic necrotic foci were noted as early as 2 h after LPS injection in the EtOH‐treated female rats. Hepatic neutrophil infiltration correlated with higher expression of cleaved (cOPN) and uncleaved OPN in the EtOH + LPS‐treated female rats compared with the males. OPN secretion was localized predominantly in the biliary epithelium and females had significantly higher OPN mRNA than their male counterparts in the ASH model. The ability of OPN to attract neutrophils was further confirmed in vivo, in a peritonitis rat model, and by neutralizing OPN (nOPN) antibody experiments. Hepatic neutrophil infiltration was largely inhibited (∼50%) by nOPN antibody. Flow cytometry experiments revealed OPN‐mediated up‐regulation of the CD11b neutrophil adhesion molecule. In conclusion, these data suggest that higher hepatic expression of OPN is the likely reason for higher and early hepatic neutrophil infiltration making females more susceptible to ALD during ASH. Copyright

Early-phase alcoholic liver disease: an update on animal models, pathology, and pathogenesis.

Alcoholic liver disease (ALD) remains to be one of the most common etiology of liver disease and is a major cause of morbidity and mortality worldwide. The pathologic stages of ALD comprises of steatosis, steatohepatitis, and fibrosis/cirrhosis. Steatosis and steatohepatitis represents the early phase of ALD and are precursor stages for fibrosis/cirrhosis. Numerous research efforts have been directed at recognizing cofactors interacting with alcohol in the pathogenesis of steatosis and steatohepatitis. This review will elucidate the constellation of complex pathogenesis, available animal models, and microscopic pathologic findings mostly in the early-phase of ALD. The role of endotoxin, reactive oxygen species, alcohol metabolism, and cytokines are discussed. Understanding the mechanisms of early-phase ALD should provide insight into the development of therapeutic strategies and thereby decrease the morbidity and mortality associated with ALD.

Liver Proteome Analysis in a Rodent Model of Alcoholic Steatosis

Alcoholic steatosis (AS) is the initial pathology associated with early stage alcoholic liver disease (ALD) and is characterized by the accumulation of fat in the liver. AS is considered clinically benign because it is reversible, and the progression of AS to alcoholic steatohepatitis (ASH) is a key step in the development of ALD. A two-dimensional gel electrophoresis (2DE)-mass spectrometry (MS) proteomic approach was used to investigate the protein expression pattern underlying AS, as the first step toward determining liver tissue biomarkers for early stage ALD. Several proteins involved in fatty acid and amino acid metabolism were up-regulated in 3- and 6-week ethanol-fed rats relative to isocaloric controls, which suggest a higher energy demand upon chronic exposure to ethanol. In addition, the expression of two proteins associated with alcohol-induced oxidative stress, peroxiredoxin 6 (PRDX6) and aldehyde dehydrogenase 2 (ALDH2), was down-regulated in ethanol fed rats, and suggests an increase in reactive oxygen species and oxidative stress. To investigate if irreversible protein modification arising from oxidative stress during AS impacts protein levels, the extent of carbonylated proteins in the ethanol and isocaloric groups was identified using mass spectrometry. The detection of modified proteins involved in antioxidant functions further supports the notion that oxidative modification of these proteins leads to protein turnover during AS. In addition, the carbonylation of betaine-homocysteine S-methyltransferase, a protein implicated in fatty liver development, in 3-week and 6-week ethanol exposed samples suggests that this protein could be a marker for early stage AS.

Enhancing the utility of alanine aminotransferase as a reference standard biomarker for drug-induced liver injury

Drug-induced liver injury (DILI) is the most frequent cause of discontinuation of new chemical entities during development. DILI can either be intrinsic/predictable or an idiosyncratic type. These two forms of DILI are contrasted in their manifestation and diagnosis. Even with regulatory guidance (FDA, 2009), there is still a gap in our ability to identify predictable DILI, both specifically and sensitively. Alanine aminotransferase (ALT) is the principal reference standard biomarker to diagnose DILI, yet its current application in preclinical to clinical translation for decision-making purposes has imperfections: (1) analytical ALT assay uniformity across industry would be aided by common analytical processes; (2) assessment of ALT toxicological performance in a large preclinical analysis would help to establish a true threshold of elevation for predictable DILI and improve translational use across various stages of pharmaceutical development and finally, (3) clinical evaluation of ALT elevations prospectively and retrospectively is recommended to define and manage variations in clinical study subjects including rising body mass index (BMI) range and ALT upper limit of normal (ULN) in the broader population over time. The emergence of new hepatotoxicity biomarkers necessitates a parallel and equivalent assessment to the aminotransferases in a regulatory qualification model.

Selective Inhibition of BTK Prevents Murine Lupus and Antibody-Mediated Glomerulonephritis

Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton’s tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.

Hepatic neutrophil infiltration in the pathogenesis of alcohol-induced liver injury.

ABSTRACT The histopathologic evidence of the presence of neutrophils within the liver parenchyma is a prominent feature of alcoholic hepatitis in both experimental animals and chronic human alcoholics. However, the precise mechanisms by which neutrophils infiltrate the liver and cause liver injury still remain to be fully elucidated. For neutrophils to infiltrate the liver, they have to undergo systemic activation (priming) by proinflammatory cytokines, chemokines, complement factors, and other biologically active molecules (e.g., platelet-activating factor). Neutrophils accumulated in the hepatic microvasculature (sinusoids and postsinusoidal venules) can extravasate (transmigrate) into the hepatic parenchyma if they receive appropriate signals from previously sensitized or distressed cells. Transmigration can be mediated by a chemokine gradient established toward the hepatic parenchyma and generally involves the interaction between adhesion molecules on neutrophils (β2 integrins) and on endothelial cells (intercellular adhesion molecules [ICAM-1]). Following transmigration, neutrophils adhere to sensitized hepatocytes through their β2 integrins and ICAM-1 expressed on hepatocytes and mediate killing of hepatocytes mostly by oxidant stress and proteases. These neutrophilic events during chronic alcohol ingestion, based mostly on experiments with rodent models, will be emphasized in this review.