Josephine Pelletier

Reduction in the severity of graft-versus-host disease and increased survival in allogenic mice by treatment with monoclonal antibodies to cell adhesion antigens LFA-1 alpha and MALA-2.

Bone marrow transplantation is a therapeutic treatment for many life-threatening hematologic disorders, especially leukemia and certain immune deficiency diseases. However, acute graft-versus-host disease is often associated with bone marrow transplantation. In mice, allogeneic GVHD appears to be mediated by both host natural killer cells and donor T cells. In vitro and in vivo experiments demonstrate that treatment with either YN1/1.7 or M17/4.2 mabs is immunomodulatory and inhibits both the mixed lymphocyte reaction and natural killer cell activity. In addition, utilizing an allogeneic model of acute, lethal GVHD with C57B1/6 mice as donors and sublethally irradiated BDFi mice as recipients, treatment of host mice with anti-LFA-lα (Ml7/ 4.2) or anti-MALA-2 (YN1/1.7) mabs at a dose of 10 mg/kg/day for 10 days significantly reduced GVHD and enhanced survival. Mabs to lymphocyte adhesion molecules such as LFA-lα and MALA-2 may provide a useful therapy for the treatment of GVHD.

Pyrazinoindolone inhibitors of MAPKAP-K2

Optimization of pyrazinoindolone inhibitors of MAPKAP-K2 (MK2) provides a reasonable balance of cellular potency and physicochemical properties. Mechanistic studies support the inhibition of MK2 which is responsible for the sub-micromolar cellular efficacy.

Monoclonal antibody to MALA-2 (ICAM-1) reduces acute autoimmune nephritis in kdkd mice

Hereditary tubulointerstitial nephritis is a prominent cause of renal failure in humans. A variety of animal models utilizing immunologically induced nephritis have been developed. The kdkd congenic variant of the CBA/Ca mouse has normal kidneys at birth but develops progressive, lethal autoimmune nephritis beginning at approximately Week 8. The destruction of renal tubular epithelium in mediated by a population of antigen-specific, H-2Kk-restricted, Lyt-2+, L3T4- T cells. The present experiments demonstrate that systemic treatment with anti-ICAM-1 monoclonal antibody reduces kidney disease in kdkd mice. Anti-ICAM-1 mab localizes to inflammatory sites in the kidney and effects a significant reduction in leukocyte infiltration. Concomitantly, urine protein levels of anti-ICAM-1-treated mice are significantly reduced. The use of anti-adhesion molecule monoclonal antibodies that alter leukocyte activity and/or trafficking may be useful therapies for certain autoimmune disorders.

Nonsteroidal Dissociated Glucocorticoid Agonists Containing Azaindoles as Steroid A-Ring Mimetics

Syntheses and structure-activity relationships (SAR) of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain azaindole moieties as A-ring mimetics and display various degrees of in vitro dissociation between gene transrepression and transactivation. Collagen induced arthritis studies in mouse have demonstrated that in vitro dissociated compounds (R)-16 and (R)-37 have steroid-like anti-inflammatory properties with improved metabolic side effect profiles, such as a reduced increase in body fat and serum insulin levels, compared to steroids.

A GPBAR1 (TGR5) Small Molecule Agonist Shows Specific Inhibitory Effects on Myeloid Cell Activation In Vitro and Reduces Experimental Autoimmune Encephalitis (EAE) In Vivo

GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.

Non-steroidal dissociated glucocorticoid agonists: indoles as A-ring mimetics and function-regulating pharmacophores

We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound (R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.

Discovery of a potent and dissociated non-steroidal glucocorticoid receptor agonist containing an alkyl carbinol pharmacophore.

Synthesis and structure-activity relationship (SAR) of a series of alkyl and cycloalkyl containing non-steroidal dissociated glucocorticoid receptor (GR) agonists is reported. This series of compounds was identified as part of an effort to replace the CF3 group in a scaffold represented by 1a. The study culminated in the identification of compound 14, a t-butyl containing derivative, which has shown potent activity for GR, selectivity against the progesterone receptor (PR) and the mineralocorticoid receptor (MR), in vitro anti-inflammatory activity in an IL-6 transrepression assay, and dissociation in a MMTV transactivation counter-screen. In a collagen-induced arthritis mouse model, 14 displayed prednisolone-like efficacy, and lower impact on body fat and free fatty acids than prednisolone at an equivalent anti-inflammatory dose.

CCR1 Plays a Critical Role in Modulating Pain through Hematopoietic and Non-Hematopoietic Cells

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1−/− animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freunds adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1−/− mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

BTK inhibition ameliorates kidney disease in spontaneous lupus nephritis

Lupus nephritis is a common disease manifestation of SLE, in which immune complex deposition and macrophage activation are important contributors to disease pathogenesis. Brutons tyrosine kinase (BTK) plays an important role in both B cell and FcgammaR mediated myeloid cell activation. In the current study, we examined the efficacy of BI-BTK-1, a recently described irreversible BTK inhibitor, in the classical NZB × NZW F1 (NZB/W) and MRL/lpr spontaneous mouse models of SLE. NZB/W mice were randomly assigned to a treatment (0.3 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg) or control group and began treatment at 22 weeks of age. The experimental setup was similar in MRL/lpr mice, but with a single treated (10 mg/kg, beginning at 8-9 weeks of age) and control group. A separate experiment was performed in the MRL/lpr strain to assess the ability of BI-BTK-1 to reverse established kidney disease. Early treatment with BI-BTK-1 significantly protected NZB/W and MRL/lpr mice from the development of proteinuria, correlating with significant renal histological protection, decreased anti-DNA titers, and increased survival in both strains. BI-BTK-1 treated mice displayed a significant decrease in nephritis-associated inflammatory mediators (e.g. LCN2 and IL-6) in the kidney, combined with a significant inhibition of immune cell infiltration and accumulation. Importantly, BI-BTK-1 treatment resulted in the reversal of established kidney disease. BTK inhibition significantly reduced total B cell numbers and all B cell subsets (immature, transitional, follicular, marginal zone, and class switched) in the spleen of NZB/W mice. Overall, the significant efficacy of BI-BTK-1 in ameliorating multiple pathological endpoints associated with kidney disease in two distinct murine models of spontaneous lupus nephritis provides a strong rationale for BTK inhibition as a promising treatment approach for lupus nephritis.