Zucker Lean Rats With Hepatic Steatosis Recapitulate Asymptomatic Metabolic Syndrome and Exhibit Greater Sensitivity to Drug-Induced Liver Injury Compared With Standard Nonclinical Sprague-Dawley Rat Model.

Fatty liver disease is a potential risk factor for drug-induced liver injury (DILI). Despite advances in nonclinical in vitro and in vivo models to assess liver injury during drug development, the pharmaceutical industry is still plagued by idiosyncratic DILI. Here, we tested the hypothesis that certain features of asymptomatic metabolic syndrome (namely hepatic steatosis) increase the risk for DILI in certain phenotypes of the human population. Comparison of the Zucker Lean (ZL) and Zucker Fatty rats fed a high fat diet (HFD) revealed that HFD-fed ZL rats developed mild hepatic steatosis with compensatory hyperinsulinemia without increases in liver enzymes. We then challenged steatotic HFD-fed ZL rats and Sprague-Dawley (SD) rats fed normal chow, a nonclinical model widely used in the pharmaceutical industry, with acetaminophen overdose to induce liver injury. Observations in HFD-fed ZL rats included increased liver injury enzymes and greater incidence and severity of hepatic necrosis compared with similarly treated SD rats. The HFD-fed ZL rats also had disproportionately higher hepatic drug accumulation, which was linked with abnormal hepatocellular efflux transporter distribution. Here, we identify ZL rats with HFD-induced hepatic steatosis as a more sensitive nonclinical in vivo test system for modeling DILI compared with SD rats fed normal chow.

A precision therapy against cancers driven by KIT/PDGFRA mutations.

Targeting oncogenic kinase drivers with small-molecule inhibitors can have marked therapeutic benefit, especially when administered to an appropriate genomically defined patient population. Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in various mechanisms of kinase activation. Therapeutic benefit to patients can best be optimized through an in-depth understanding of the disease-driving mutations combined with the ability to match these insights to tailored highly selective drugs. This rationale is presented for BLU-285, a clinical stage inhibitor of oncogenic KIT and PDGFRA alterations, including activation loop mutants that are ineffectively treated by current therapies. BLU-285, designed to preferentially interact with the active conformation of KIT and PDGFRA, potently inhibits activation loop mutants KIT D816V and PDGFRA D842V with subnanomolar potency and also inhibits other well-characterized disease-driving KIT mutants both in vitro and in vivo in preclinical models. Early clinical evaluation of BLU-285 in a phase 1 study has demonstrated marked activity in patients with diseases associated with KIT (aggressive systemic mastocytosis and gastrointestinal stromal tumor) and PDGFRA (gastrointestinal stromal tumor) activation loop mutations.

Nerve growth factor inhibition with tanezumab influences weight-bearing and subsequent cartilage damage in the rat medial meniscal tear model.

OBJECTIVE: To investigate whether the effects of nerve growth factor (NGF) inhibition with tanezumab on rats with medial meniscal tear (MMT) effectively model rapidly progressive osteoarthritis (RPOA) observed in clinical trials. METHODS: Male Lewis rats underwent MMT surgery and were treated weekly with tanezumab (0.1, 1 or 10 mg/kg), isotype control or vehicle for 7, 14 or 28 days. Gait deficiency was measured to assess weight-bearing on the operated limb. Joint damage was assessed via histopathology. A second arm, delayed onset of treatment (starting 3-8 weeks after MMT surgery) was used to control for analgesia early in the disease process. A third arm, mid-tibial amputation, evaluated the dependency of the model on weight-bearing. RESULTS: Gait deficiency in untreated rats was present 3-7 days after MMT surgery, with a return to normal weight-bearing by days 14-28. Prophylactic treatment with tanezumab prevented gait deficiency and resulted in more severe cartilage damage. When onset of treatment with tanezumab was delayed to 3-8 weeks after MMT surgery, there was no increase in cartilage damage. Mid-tibial amputation completely prevented cartilage damage in untreated MMT rats. CONCLUSIONS: These data suggest that analgesia due to NGF inhibition during the acute injury phase is responsible for increased voluntary weight-bearing and subsequent cartilage damage in the rat MMT model. This model failed to replicate the hypotrophic bone response observed in tanezumab-treated patients with RPOA.

Intrathecal delivery of frataxin mRNA encapsulated in lipid nanoparticles to dorsal root ganglia as a potential therapeutic for Friedreich's ataxia.

In Friedreich's ataxia (FRDA) patients, diminished frataxin (FXN) in sensory neurons is thought to yield the predominant pathology associated with disease. In this study, we demonstrate successful usage of RNA transcript therapy (RTT) as an exogenous human FXN supplementation strategy in vitro and in vivo, specifically to dorsal root ganglia (DRG). Initially, 293 T cells were transfected with codon optimized human FXN mRNA, which was translated to yield FXN protein. Importantly, FXN was rapidly processed into the mature functional form of FXN (mFXN). Next, FXN mRNA, in the form of lipid nanoparticles (LNPs), was administered intravenously in adult mice. Examination of liver homogenates demonstrated efficient FXN LNP uptake in hepatocytes and revealed that the mitochondrial maturation machinery had efficiently processed all FXN protein to mFXN in ~24 h in vivo. Remarkably, greater than 50% mFXN protein derived from LNPs was detected seven days after intravenous administration of FXN LNPs, suggesting that the half-life of mFXN in vivo exceeds one week. Moreover, when FXN LNPs were delivered by intrathecal administration, we detected recombinant human FXN protein in DRG. These observations provide the first demonstration that RTT can be used for the delivery of therapeutic mRNA to DRG.

In vivo imaging implicates CCR2(+) monocytes as regulators of neutrophil recruitment during arthritis.

The infiltration of neutrophils and monocytes is a prominent feature of inflammatory diseases including human rheumatoid arthritis. Understanding how neutrophil recruitment is regulated during pathogenesis is crucial for developing anti-inflammatory therapies. We optimized the K/B×N serum-induced mouse arthritis model to study neutrophil trafficking dynamics in vivo using two-photon microscopy. Arthritogenic serum was injected subcutaneously into one hind footpad to induce a local arthritis with robust neutrophil recruitment. Using this approach, we showed that the depletion of monocytes with clodronate liposomes impaired neutrophil recruitment specifically at the transendothelial migration step. The depletion of CCR2(+) monocytes with the monoclonal antibody MC-21 reproduced these effects, implicating CCR2(+) monocytes as key regulators of neutrophil extravasation during arthritis initiation. However, monocyte depletion did not prevent neutrophil extravasation in response to bacterial challenge. These findings suggest that anti-inflammatory therapies targeting monocytes may act in part through antagonizing neutrophil extravasation at sites of aseptic inflammation.

JAK inhibition with tofacitinib suppresses arthritic joint structural damage through decreased RANKL production.

OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.

Enhanced GITR/GITRL interactions augment IL-27 expression and induce IL-10-producing Tr-1 like cells.

The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.

Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550).

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-ß. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.

Th17 cells can provide B cell help in autoantibody induced arthritis.

K/BxN mice develop a spontaneous destructive arthritis driven by T cell dependent anti-glucose-6-phosphate isomerase (GPI) antibody production. In this study, a modified version of the K/BxN model, the KRN-cell transfer model (KRN-CTM), was established to determine the contribution of Th17 cells in the development of chronic arthritis. The transfer of naive KRN T cells into B6.TCR.Cα(-/-)H-2(b/g7) T cell deficient mice induced arthritis by day 10 of transfer. Arthritis progressively developed for a period of up to 14 days following T cell transfer, thereafter the disease severity declined, but did not resolve. Both IL-17A and IFNγ were detected in the recovered T cells from the popliteal lymph nodes and ankles. The transfer of KRN Th17 polarized KRN CD4(+) T cells expressing IL-17A and IFNγ induced arthritis in all B6.TCR.Cα(-/-)H-2(b/g7) mice however the transfer of Th1 polarized KRN CD4(+) T cells expressing IFNγ alone induced disease in only 2/3 of the mice and disease induction was delayed compared to Th17 transfers. Th17 polarized KRN/T-bet(-/-) cells induced arthritis in all mice and surprisingly, IFNγ was produced demonstrating that T-bet expression is not critical for arthritis induction, regardless of the cytokine expression. Neutralization of IFNγ in KRN Th17 transfers resulted in earlier onset of disease while the neutralization of IL-17A delayed disease development. Consistent with K/BxN mice, naive KRN T cell transfers and Th17 polarized KRN/T-bet(-/-) transfers induced anti-GPI IgG(1) dominant responses while KRN Th17 cells induced high levels of IgG(2b). These data demonstrate that Th17 cells can participate in the production of autoantibodies that can induce arthritis.

Characterization of the KRN cell transfer model of rheumatoid arthritis (KRN-CTM), a chronic yet synchronized version of the K/BxN mouse.

In this study, a chronic yet synchronized version of the K/BxN mouse, the KRN-cell transfer model (KRN-CTM), was developed and extensively characterized. The transfer of purified splenic KRN T cells into T cell-deficient B6.TCR.Calpha(-/-)H-2(b/g7) mice induced anti-glucose 6-phosphate isomerase antibody-dependent chronic arthritis in 100% of the mice with uniform onset of disease 7 days after T cell transfer. Cellular infiltrations were assessed by whole-ankle transcript microarray, cytokine and chemokine levels, and microscopic and immunohistochemical analyses 7 through 42 days after T cell transfer. Transcripts identified an influx of monocytes/macrophages and neutrophils into the ankles and identified temporal progression of cartilage damage and bone resorption. In both serum and ankle tissue there was a significant elevation in interleukin-6, whereas macrophage inflammatory protein-1 alpha and monocyte chemotactic protein-1 were only elevated in tissue. Microscopic and immunohistochemical analyses revealed a time course for edema, synovial hypertrophy and hyperplasia, infiltration of F4/80-positive monocytes/macrophages and myeloperoxidase-positive neutrophils, destruction of articular cartilage, pannus invasion, bone resorption, extra-articular fibroplasia, and joint ankylosis. The KRN cell transfer model replicates many features of chronic rheumatoid arthritis in humans in a synchronized manner and lends itself to manipulation of adoptively transferred T cells and characterizing specific genes and T cell subsets responsible for rheumatoid arthritis pathogenesis and progression.

Optical tomographic imaging discriminates between disease-modifying anti-rheumatic drug (DMARD) and non-DMARD efficacy in collagen antibody-induced arthritis.

INTRODUCTION: Standard measurements used to assess murine models of rheumatoid arthritis, notably paw thickness and clinical score, do not align well with certain aspects of disease severity as assessed by histopathology. We tested the hypothesis that non-invasive optical tomographic imaging of molecular biomarkers of inflammation and bone turnover would provide a superior quantitative readout and would discriminate between a disease-modifying anti-rheumatic drug (DMARD) and a non-DMARD treatment. METHODS: Using two protease-activated near-infrared fluorescence imaging agents to detect inflammation-associated cathepsin and matrix metalloprotease activity, and a third agent to detect bone turnover, we quantified fluorescence in paws of mice with collagen antibody-induced arthritis. Fluorescence molecular tomographic (FMT) imaging results, which provided deep tissue detection and quantitative readouts in absolute picomoles of agent fluorescence per paw, were compared with paw swelling, clinical scores, a panel of plasma biomarkers, and histopathology to discriminate between steroid (prednisolone), DMARD (p38 mitogen-activated protein kinase (MAPK) inhibitor) and non-DMARD (celecoxib, cyclooxygenase-2 (COX-2) inhibitor) treatments. RESULTS: Paw thickness, clinical score, and plasma biomarkers failed to discriminate well between a p38 MAPK inhibitor and a COX-2 inhibitor. In contrast, FMT quantification using near-infrared agents to detect protease activity or bone resorption yielded a clear discrimination between the different classes of therapeutics. FMT results agreed well with inflammation scores, and both imaging and histopathology provided clearer discrimination between treatments as compared with paw swelling, clinical score, and serum biomarker readouts. CONCLUSIONS: Non-invasive optical tomographic imaging offers a unique approach to monitoring disease pathogenesis and correlates with histopathology assessment of joint inflammation and bone resorption. The specific use of optical tomography allowed accurate three-dimensional imaging, quantitation in picomoles rather than intensity or relative fluorescence, and, for the first time, showed that non-invasive imaging assessment can predict the pathologist's histology inflammation scoring and discriminate DMARD from non-DMARD activity.

14-deoxyandrographolide as a platelet activating factor antagonist in bovine neutrophils.

14-Deoxyandrographolide (14-DAP) is a labdane diterpene isolated from Andrographis paniculata with previously reported calcium channel blocking activity. Its potential platelet activating factor (PAF) antagonistic activity in bovine neutrophils was assessed. 14-DAP, in concentrations between 10-100 microM, reduced the extracellular acidification rate and the intracellular alkalinization in a dose-dependent manner. In addition, 14-DAP reduced PAF-induced calcium flux in the presence of extracellular calcium, and tyrosine phosphorylation of a 44 kDa protein corresponding to the MAPK(ERK1). However, 14-DAP reduced the 3H-PAF binding with a Ki of 7.8 x 10 (- 9)M, and a Hill slope of 0.63, suggesting that there is more than one binding site for 14-DAP. We concluded that 14-DAP is an effective antagonist of PAF-mediated processes in bovine neutrophils, probably by virtue of its calcium channel blocking property.

Platelet-activating factor increases pH(i) in bovine neutrophils through the PI3K-ERK1/2 pathway.

1. Platelet-activating factor (PAF) is known to stimulate a variety of neutrophil activities, including chemotaxis, phagocytosis, degranulation, reactive oxygen species production and intracellular pH increase. The purpose of this study was to investigate the effect of PAF on pH((i)), specifically if these changes in pH are the result of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway activation in bovine neutrophils. 2. PAF caused intracellular alkalinization in 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester-loaded bovine neutrophils. This phenomenon seems to be mediated by amiloride-sensitive Na(+)/H(+) exchange, and is inhibited by WEB2086 (a selective PAF receptor antagonist), genistein (a tyrosine kinase inhibitor), wortmannin and LY294002 (PI3K inhibitors), and PD98059 and UO126 (MEK inhibitors). 3. PAF 100 nm induced an increase in tyrosine phosphorylation of proteins 62, 44 and 21 kDa with a maximum response at 2 min of incubation. 4. Unlike human neutrophils, bovine neutrophils are strongly stimulated by PAF via phosphorylation of ERK1/2 (extracellular-signal-regulated protein kinase) with an EC(50) of 30 and 13 nm, respectively. 5. PAF MAPK activation was also inhibited by WEB2086, pertussis toxin (PTX), genistein, wortmannin, LY294002, PD98059 and UO126 in bovine neutrophils. The ERK1/2 activation is dependent on PI3K pathway, because protein kinase B was phosphorylated by PAF and inhibited by wortmannin and LY294002, but not by U0126. 6. Our results suggest that PAF induces intracellular alkalinization via PI3K-MAPK activation. This effect is upstream regulated by PAF receptor, PTX-sensitive G protein, tyrosine kinase, PI3K and MEK1/2 in bovine neutrophils.

Effect of 14-deoxyandrographolide on calcium-mediated rat uterine smooth muscle contractility.

In this study, the effect of 14-deoxyandrographolide (14-DAP) on calcium channel-dependent rat uterine smooth muscle contraction was evaluated. Using a tissue bath preparation, 14-DAP was able to reduce the contractile response to 0.3 and 3.0 mm of CaCl(2), with an IC(50) of 1.24 +/- 0.23 x 10(-5) m and 5.94 +/- 0.29 x 10(-5) m, respectively. 14-DAP shifted the CaCl(2) cumulative dose response curve to the right, increasing the EC(50) from 2.08 +/- 0.20 x 10(-4) m to 4.22 +/- 0.22 x 10(-4) m (5 micrometer 14-DAP) and 2.5 +/- 1.0 x 10(-3) m (50 micrometer 14-DAP). In order to determine if 14-DAP had any effect on intracellular calcium, the relaxant response to 14-DAP following contraction by oxytocin, PGF(2alpha) and vanadate in Ca(+2)-free solution was compared with that of isoproterenol and phenylbutazone. While isoproterenol and phenylbutazone relaxed the smooth muscle in a dose-dependent manner, 14-DAP did not have any effect on either the oxytocin, PGF(2alpha) or vanadate-induced smooth muscle contraction. Based on these data, it appears that 14-DAP is an uterine smooth muscle relaxant which produces a selective blockade of voltage operated calcium channels.