Synthesis and in vitro evaluation of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)-pteridines as dual immunosuppressive and anti-inflammatory agents.

Screening of a pteridine-based compound library led to the identification of compounds exhibiting immunosuppressive as well as anti-inflammatory activity. Optimization afforded a series of 2-amino-4-N-piperazinyl-6-(3,4-dimethoxyphenyl)pteridine analogues. The most potent congeners in this series displayed low nM IC(50) values in the Mixed Lymphocyte Reaction (MLR) assay. In addition, these compounds also have potent anti-inflammatory activity as measured in the Tumor Necrosis Factor (TNF) assay.

Anti-inflammatory activity of a pteridine derivative (4AZA2096) alleviates TNBS-induced colitis in mice.

Elevated production of tumor necrosis factor (TNF) plays a central role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis and Crohn's disease. Naturally occurring pteridine analogs have been reported to have potent immunomodulatory activity, especially on TNF production. The aim of this study is to identify small molecule TNF inhibitiors derived from pteridine and to prove their in vivo efficacy in an inflammatory model. A focused chemical library based on the pteridine scaffold was screened in vitro on lipopolysaccharide (LPS)-induced TNF production in peripheral blood mononuclear cells (PBMC). One synthetic pteridine analog (4AZA2096), shown to have strong inhibitory activity, was selected and tested for its efficacy to treat trinitrobenzenesulfonate (TNBS)-induced colitis in mice, a model of Crohn's disease. Colitis was induced by rectal administration of 1 mg TNBS in 50% ethanol after presensitization via the skin. The synthetic pteridine analog 4AZA2096 was shown to potently inhibit LPS-induced TNF production in vitro. Colitic mice treated with 4AZA2096 orally (20 mg/kg/day) recovered more rapidly and, histologically, had a reduction of inflammatory lesions, less edema, a reduction of goblet cell loss, and reduced wall thickness. Cell infiltration in the colon, especially infiltration of neutrophils, as shown by myeloperoxidase (MPO) activity, was reduced in 4AZA2096-treated animals. Intralesional TNF production was lower in mice of the treated groups, whereas interleukin-18 (IL-18) and interferon-gamma (IFN-gamma) mRNA were not affected. Treatment had no effect on anti-TNBS antibody production, arguing against generalized immunosuppression. In conclusion, we identified a pteridine derivative, 4AZA2096, with strong inhibitory activity on TNF production and a remission- inducing effect in TNBS colitis, supporting further preclinical and clinical development of this novel TNF inhibitor for treatment of inflammatory diseases.

Development of mouse-specific contraceptive vaccines: infertility in mice immunized with peptide and polyepitope antigens.

Mouse-specific immunocontraceptive peptides have been identified in mouse proteins with key roles in reproduction from sequence comparisons to other species and tested for efficacy as immunocontraceptive antigens. Peptides were derived from granulocyte-macrophage colony-stimulating factor (GMCSF), the placental 27 kDa heat-shock protein (HSP), leukemia inhibitory factor receptor (LIFR), oviduct glycoprotein (OGP), proliferin (PLF), prolactin (PRL), sperm protein SP56 and mouse zona pellucida subunits 1 and 3 (ZP1, ZP3). Fertility of female BALB/c mice was reduced after immunization with several peptides either conjugated to a carrier protein or in the form of recombinant polyepitopes. The most effective conjugated peptides (SP56, GMCSF and PRL) induced peptide-specific serum antibodies and reduced fertility by 50%. Fertility of mice was also reduced after immunization with polyepitope antigens containing up to five different peptides fused to maltose-binding protein, but antibodies were not produced against all the encoded peptides. The most effective polyepitope antigen (containing PLF, SP56, ZP1 and ZP3 peptides) reduced fertility by 50% but induced only SP56 and ZP1 antibodies. We demonstrate that lack of antibody response to a given peptide epitope (ZP3) can be overcome if repeated copies are used in the polyepitope antigen construct, but the effect varies between mouse strains. We conclude that infertility induced in mice with a range of peptide-based vaccines is dependent on antigen formulation and genetic factors but does not necessarily correlate with peptide-specific antibody levels. In light of these results, strategies to improve the efficacy of peptide-based antifertility vaccines are discussed.