Utility of animal models for identification of potential therapeutics for rheumatoid arthritis.

Animal models of rheumatoid arthritis (RA) are widely used for testing potential new therapies for RA. However, the question of which animal model is most predictive of therapeutic efficacy in human RA commonly arises in data evaluation. A retrospective review of the animal models used to evaluate approved, pending RA therapies, and compounds that were discontinued during phase II or III clinical trials found that the three most commonly used models were adjuvant-induced arthritis (AIA) in rats and collagen-induced arthritis (CIA) in rats and mice. Limited data were found for more recently developed genetically modified animal models. Examination of the efficacy of various compounds in these animal models revealed that a compound's therapeutic efficacy, rather than prophylactic efficacy, in AIA and CIA models was more predictive of clinical efficacy in human RA than data from either model alone.

Design, synthesis, and analysis of a polyethelene glycol-modified (PEGylated) small molecule inhibitor of integrin {alpha}4{beta}1 with improved pharmaceutical properties.

Integrin alpha4beta1 plays an important role in inflammatory processes by regulating the migration of leukocytes into inflamed tissues. Previously, we identified BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid], a highly selective and potent (K(D) of 9 pM) small molecule inhibitor of alpha4beta1. Although BIO5192 is efficacious in various animal models of inflammatory disease, high doses and daily treatment of the compound are needed to achieve a therapeutic effect because of its relatively short serum half-life. To address this issue, polyethylene glycol modification (PEGylation) was used as an approach to improve systemic exposure. BIO5192 was PEGylated by a targeted approach in which derivatizable amino groups were incorporated into the molecule. Two sites were identified that could be modified, and from these, five PEGylated compounds were synthesized and characterized. One compound, 2a-PEG (K(D) of 19 pM), was selected for in vivo studies. The pharmacokinetic and pharmacodynamic properties of 2a-PEG were dramatically improved relative to the unmodified compound. The PEGylated compound was efficacious in a rat model of experimental autoimmune encephalomyelitis at a 30-fold lower molar dose than the parent compound and required only a once-a-week dosing regimen compared with a daily treatment for BIO5192. Compound 2a-PEG was highly selective for alpha4beta1. These studies demonstrate the feasibility of PEGylation of alpha4beta1-targeted small molecules with retention of activity in vitro and in vivo. 2a-PEG, and related compounds, will be valuable reagents for assessing alpha4beta1 biology and may provide a new therapeutic approach to treatment of human inflammatory diseases.

An assessment of the mechanistic differences between two integrin alpha 4 beta 1 inhibitors, the monoclonal antibody TA-2 and the small molecule BIO5192, in rat experimental autoimmune encephalomyelitis.

Integrin alpha 4 beta 1 plays an important role in inflammatory processes by regulating the migration of lymphocytes into inflamed tissues. Here we evaluated the biochemical, pharmacological, and pharmacodynamic properties and efficacy in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, of two types of alpha 4 beta 1 inhibitors, the anti-rat alpha 4 monoclonal antibody TA-2 and the small molecule inhibitor BIO5192 [2(S)-[[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino]-4-[4-methyl-2(S)-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid]. TA-2 has been extensively studied in rats and provides a benchmark for assessing function. BIO5192 is a highly selective and potent (KD of <10 pM) inhibitor of alpha 4 beta 1. Dosing regimens were identified for both inhibitors, which provided full receptor occupancy during the duration of the study. Both inhibitors induced leukocytosis, an effect that was used as a pharmacodynamic marker of activity, and both were efficacious in the EAE model. Treatment with TA-2 caused a decrease in alpha 4 integrin expression on the cell surface, which resulted from internalization of alpha 4 integrin/TA-2 complexes. In contrast, BIO5192 did not modulate cell surface alpha 4 beta 1. Our results with BIO5192 indicate that alpha 4 beta 7 does not play a role in this model and that blockade of alpha 4 beta 1/ligand interactions without down-modulation is sufficient for efficacy in rat EAE. BIO5192 is highly selective and binds with high affinity to alpha 4 beta 1 from four of four species tested. These studies demonstrate that BIO5192, a novel, potent, and selective inhibitor of alpha 4 beta 1 integrin, will be a valuable reagent for assessing alpha 4 beta 1 biology and may provide a new therapeutic for treatment of human inflammatory diseases.

Discordant effects of anti-VLA-4 treatment before and after onset of relapsing experimental autoimmune encephalomyelitis.

Initial migration of encephalitogenic T cells to the central nervous system (CNS) in relapsing experimental autoimmune encephalomyelitis (R-EAE), an animal model of multiple sclerosis (MS), depends on the interaction of the alpha4 integrin (VLA-4) expressed on activated T cells with VCAM-1 expressed on activated cerebrovascular endothelial cells. Alternate homing mechanisms may be employed by infiltrating inflammatory cells after disease onset. We thus compared the ability of anti-VLA-4 to regulate proteolipid protein (PLP) 139-151-induced R-EAE when administered either before or after disease onset. Preclinical administration of anti-VLA-4 either to naive recipients of primed encephalitogenic T cells or to mice 1 week after peptide priming, i.e., before clinical disease onset, inhibited the onset and severity of clinical disease. In contrast, Ab treatment either at the peak of acute disease or during remission exacerbated disease relapses and increased the accumulation of CD4(+) T cells in the CNS. Most significantly, anti-VLA-4 treatment either before or during ongoing R-EAE enhanced Th1 responses to both the priming peptide and endogenous myelin epitopes released secondary to acute tissue damage. Collectively, these results suggest that treatment with anti-VLA-4 Ab has multiple effects on the immune system and may be problematic in treating established autoimmune diseases such as MS.

Regulation of inflammation by collagen-binding integrins alpha1beta1 and alpha2beta1 in models of hypersensitivity and arthritis.

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

The effect of leukotriene synthesis inhibitors in models of acute and chronic inflammation.

OBJECTIVE: To assess the efficacy of leukotriene synthesis inhibitors, alone and in combination with a nonsteroidal antiinflammatory drug, as potential treatments for rheumatoid arthritis (RA), using the mouse air pouch model and the collagen-induced arthritis (CIA) model. METHODS: Two selective leukotriene synthesis inhibitors, Bay x 1005 and Bay y 1015, were compared with zileuton in terms of their ability to decrease exudate volume, cell infiltration, and leukotriene B4 (LTB4) production in response to zymosan injection in the mouse air pouch model. The mouse CIA model was used to assess the effect of leukotriene synthesis inhibitors in a model of chronic inflammation. Bay y 1015 and Bay x 1005, and the cyclooxygenase inhibitor naproxen, were evaluated individually and in combination, for their antiarthritic potency in the mouse CIA model. RESULTS: The results indicate that neither zileuton, Bay x 1005, nor Bay y 1015 inhibited exudate production. All 3 compounds decreased LTB4 levels in be air pouch, with Bay y 1015 being the most effective. Cell infiltration was significantly decreased with Bay x 1005, but the degree of this decrease did not appear to correlate with LTB4 levels. No inhibition of arthritis was observed with any compound administered alone. In contrast, a significant inhibition of CIA was observed in animals that received both naproxen and either Bay y 1015 or Bay x 1005. CONCLUSION: Inhibitors of both cyclooxygenase and leukotriene synthesis in combination may be a more effective treatment of RA than either class of inhibitors alone.

Expression of HLA-B27 in transgenic mice is controlled by gene(s) mapping between H-2D and H-2L loci.

The level of HLA-B27 transgene expression on the cell surface is dependent on the host H-2 haplotype. Mice homozygous for the H-2b, H-2f, H-2s, H-2p, H-2r, and H-2k haplotypes express B27 at high levels. An intermediate level of B27 expression is observed in H-2v mice whereas low levels of B27 are expressed in H-2q and H-2d mice. The decreased expression of B27 maps to the D region of the major histocompatibility complex. Recombinant strain B10.RKDB (DdLb) mapped the low expression gene centromeric to H-2L. In order to determine the low expression within the H-2D region, the B27 transgene was introduced into B10.D2-H-2dm1 and BALB/c-H-2dm2 mice. Expression of B27 in both of these strains was high indicating that neither H-2Dd nor H-2Ld is responsible for the low expression. This maps the effect between the H-2D and H-2L loci. In addition, introduction of human beta 2-microglobulin (beta 2m) into B10.D2-B27 transgenic mice caused a marked enhancement of B27 expression on the cell surface suggesting that the defect in B27 expression in certain haplotypes is due to an inability of B27 to associate with endogenous mouse beta 2m. We propose that gene(s) mapping between D and L (either D2, D3, D4, or some as yet unidentified gene) may be involved in class I assembly by helping association of beta 2m with class I. This putative molecule, designated "Assembly Enhancer (AE)" might have a negative influence in the association between human class I and mouse beta 2m.

Expression of HLA-B27 in transgenic mice is controlled by the H-2D gene(s).

The HLA-B27 transgene was introduced into mice of the B10 background with various H-2 haplotypes. High levels of B27 antigen were detected on the surface of peripheral-blood lymphocytes from mice homozygous for the H-2b, H-2f, H-2s, H-2p, H-2r and H-2k haplotypes. In contrast, cell-surface expression of B27 was decreased in mice homozygous for the H-2d, H-2q and H-2v haplotypes. Only minimal levels of B27 could be detected on cells from B10.RKDB (KkSkDdLb) mice, indicating that the H-2D region of the H-2d haplotype appears to interfere with B27 expression. To try to determine the specific area of the H-2D genes affecting B27 expression, the B27 transgene was introduced into B10.D2-dml and BALB/c-dm2 mice. Expression was normal in the dm2 animals which have only one functional gene, Dd, in the H-2D region. High levels of B27 expression were observed in the dml animals. These mice also have only one functional H-2D gene, but it is a hybrid gene formed by the fusion of the 5' part of the Dd gene and the 3' part of the Ld gene. Thus, low expression of B27 is controlled by gene(s) mapping between H-2D and H-2L.