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Scaffold hopping and structure-based drug design were employed to identify substituted 4-aminoquinolines and 4-aminonaphthyridines as potent, small molecule inhibitors of tumor necrosis factor alpha (TNFα). Structure-activity relationships in both the quinoline and naphthyridine series leading to the identification of compound 42 with excellent potency and pharmacokinetic profile are discussed. X-ray co-crystal structure analysis and ultracentrifugation experiments clearly demonstrate that these inhibitors distort the TNFα trimer upon binding, leading to aberrant signaling when the trimer binds to TNF receptor 1 (TNFR1). Pharmacokinetic-pharmacodynamic activity of compound 42 in a TNF-induced IL-6 mouse model and in vivo activity in a collagen antibody-induced arthritis model, where it showed biologic-like in vivo efficacy, will be discussed.

As demonstrated in preclinical animal models, the disruption of PI3Kδ expression or its activity leads to a decrease in inflammatory and immune responses. Therefore, inhibition of PI3Kδ may provide an alternative treatment for autoimmune diseases, such as RA, SLE, and respiratory ailments. Herein, we disclose the identification of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent, selective and orally bioavailable PI3Kδ inhibitors. The lead compound demonstrated efficacy in an in vivo mouse KLH model., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

LFA-1 appears to play a central role in normal immune responses to foreign Ags. In autoimmune or inflammatory diseases, there is increased expression of LFA-1 and/or its counterligand, ICAM-1. Others have demonstrated that the targeted disruption of LFA-1:ICAM interactions, either by gene deletion or Ab treatment in mice, results in reduced leukocyte trafficking, inflammatory responses, and inhibition of inflammatory arthritis in the K/BxN serum transfer model. However, there has been little success in finding a small-molecule LFA-1 antagonist that can similarly impact rodent models of arthritis. In this paper, we present the first reported example of an LFA-1 small-molecule antagonist, BMS-587101, that is efficacious in preclinical disease models. In vitro, BMS-587101 inhibited LFA-1-mediated adhesion of T cells to endothelial cells, T cell proliferation, and Th1 cytokine production. Because BMS-587101 exhibits in vitro potency, cross-reactivity, and oral bioavailability in rodents, we evaluated the impact of oral administration of this compound in two different models of arthritis: Ab-induced arthritis and collagen-induced arthritis. Significant impact of BMS-587101 on clinical score in both models was observed, with inhibition comparable or better than anti-mouse LFA-1 Ab. In addition, BMS-587101 significantly reduced cytokine mRNA levels in the joints of Ab-induced arthritis animals as compared with those receiving vehicle alone. In paws taken from the collagen-induced arthritis study, the bones of vehicle-treated mice had extensive inflammation and bone destruction, whereas treatment with BMS-587101 resulted in marked protection. These findings support the potential use of an LFA-1 small-molecule antagonist in rheumatoid arthritis, with the capacity for disease modification.

We have previously shown that inhibitors of IkappaB kinase beta (IKKbeta), including 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline (BMS-345541), are efficacious against experimental arthritis in rodents. In our efforts to identify an analog as a clinical candidate for the treatment of autoimmune and inflammatory disorders, we have discovered the potent and highly selective IKKbeta inhibitor 2-methoxy-N-((6-(1-methyl-4-(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin-7-yl)pyridin-2-yl)methyl)acetamide (BMS-066). Investigations of its pharmacology in rodent models of experimental arthritis showed that BMS-066 at doses of 5 and 10 mg/kg once daily was effective at protecting rats against adjuvant-induced arthritis, despite showing only weak inhibition at 10 mg/kg against a pharmacodymanic model of tumor necrosis factor alpha production in rats challenged with lipopolysaccharide. The duration of exposure in rats indicated that just 6 to 9 h of coverage per day of the concentration necessary to inhibit IKKbeta by 50% in vivo was necessary for protection against arthritis. Similar findings were observed in the mouse collagen-induced arthritis model, with efficacy observed at a dose providing only 6 h of coverage per day of the concentration necessary to inhibit IKKbeta by 50%. This finding probably results from the cumulative effect on multiple cellular mechanisms that contribute to autoimmunity and joint destruction, because BMS-066 was shown to inhibit a broad spectrum of activities such as T cell proliferation, B cell function, cytokine and interleukin secretion from monocytes, T(H)17 cell function and regulation, and osteoclastogenesis. Thus, only partial and transient inhibition of IKKbeta is sufficient to yield dramatic benefit in vivo, and this understanding will be important in the clinical development of IKKbeta inhibitors.

Dasatinib is an oral small molecule inhibitor of Abl and Src family tyrosine kinases (SFK), including p56(Lck) (Lck). Given the central importance of Lck in transmitting signals from the T-cell receptor (TCR) signaling complex and the potent ability of dasatinib to inhibit Lck activity, we hypothesized this agent could provide a novel route of immunomodulation via targeted inhibition of antigen-induced signaling. Herein, we show that dasatinib inhibits TCR-mediated signal transduction, cellular proliferation, cytokine production, and in vivo T-cell responses. However, dasatinib-mediated inhibition does not induce apoptosis because the effect is reversible or may be overcome by signals bypassing the TCR, such as phorbol ester. Signal transduction and proliferative responses via IL-2 remain essentially unperturbed, suggesting that dasatinib displays specificity for TCR signaling. In addition, dasatinib combined with cyclosporine A or rapamycin led to a much more potent inhibition of T-cell activation, suggesting that targeted inhibition of Lck could be a useful adjunct for enhanced immunomodulation. In combination with currently available immunomodulatory agents, SFK inhibition could potentially increase immunomodulatory efficacy while minimizing toxicity of individual agents.

The biological activities of parathyroid hormone (PTH) on bone are quite complex as demonstrated by its catabolic and anabolic activities on the skeleton. Although there have been many reports describing genes that are regulated by PTH in osteoblast cells, the goal of this study was to utilize a well-established in vivo PTH anabolic treatment regimen to identify genes that mediate bone anabolic effects of PTH. We identified a gene we named PTH anabolic induced gene in bone (PAIGB) that has been reported as brain and acute leukemia cytoplasmic (BAALC). Therefore, using the latter nomenclature, we have discovered that BAALC is a PTH-regulated gene whose mRNA expression was selectively induced in rat tibiae nearly 100-fold (maximal) by a PTH 1-34 anabolic treatment regimen in a time-dependent manner. Although BAALC is broadly expressed, PTH did not regulate BAALC expression in other PTH receptor expressing tissues and we find that the regulation of BAALC protein by PTH in vivo is confined to mature osteoblasts. Further in vitro studies using rat UMR-106 osteoblastic cells show that PTH 1-34 rapidly induces BAALC mRNA expression maximally by 4 h while the protein was induced by 8 h. In addition to being regulated by PTH 1-34, BAALC expression can also be induced by other bone forming factors including PGE(2) and 1,25 dihydroxy vitamin D(3). We determined that BAALC is regulated by PTH predominantly through the cAMP/PKA pathway. Finally, we demonstrate in MC3T3-E1 osteoblastic cells that BAALC overexpression regulates markers of osteoblast differentiation, including downregulating alkaline phosphatase and osteocalcin expression while inducing osteopontin expression. We also demonstrate that these transcriptional responses mediated by BAALC are similar to the responses elicited by PTH 1-34. These data, showing BAALC overexpression can mimic the effect of PTH on markers of osteoblast differentiation, along with the observations that BAALC is induced selectively with a bone anabolic treatment regimen of PTH (not a catabolic treatment regimen), suggest that BAALC may be an important mediator of the PTH anabolic action on bone cell function., ((c) 2006 Wiley-Liss, Inc.)

Background: We previously demonstrated in vitro and in vivo that an IkappaB kinase (IKK) inhibitor blocks cytokine production and suppresses immune responses. These results indicate that a potent IKK inhibitor may have the potential of being a novel therapeutic agent for the prevention of graft rejection., Methods: The IKK inhibitor BMS-345541 was tested in mice for its ability to inhibit anti-CD3-induced interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha production and T-cell proliferation in an in vivo mixed lymphocyte reaction. BMS-345541 was further tested for its ability to suppress graft rejection in a murine nonvascularized heterotopic cardiac allograft model. BMS-345541 was tested as a single agent and in combination with other immunomodulators for inhibition of T-cell proliferation and graft rejection in vivo., Results: BMS-345541 suppressed, in a dose-dependent manner, the production of both IL-2 and TNF-alpha in mice stimulated with an injection of anti-CD3 antibody. Approximately 70% inhibition of both IL-2 and TNF were observed at a dose of 100 mg/kg. When BMS-345541 was administered at 100 mg/kg as a single agent, in vivo T-cell proliferation was not inhibited. However, when combined with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin (200 microg), a synergistic antiproliferative effect was observed, resulting in 77% inhibition of CD4+ T-cell proliferation. In the murine heterotopic heart transplant model, BMS-345541 did not prolong graft survival when administered at 50 mg/kg as a single agent. However, when administered with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin or cyclosporine A (15 mg/kg), graft survival was significantly increased compared with either agent alone., Conclusions: These results indicate that inhibition of IKK may serve as novel adjunctive therapy for the prevention of graft rejection.

1. Systemic and coronary haemodynamics were measured in 6 Yorkshire swine and 6 Yucatan miniature swine under isoflurane anaesthesia to investigate the influence of cocaine following its intravenous administration at 1, 3 and 7 mg kg-1. 2. Cocaine in Yorkshire swine decreased mean arterial pressure and rate pressure product (systolic pressure x heart rate), suggesting a cardiac depressant effect, whereas cocaine in Yucatan miniature swine increased these parameters, consistent with a hyperadrenergic state. 3. Cocaine in both Yorkshire swine and Yucatan miniature swine decreased coronary blood flow and coronary flow reserve, and increased coronary vascular resistance. 4. A modest generalized epicardial coronary artery constriction was observed by angiography, without evidence of focal spasm. 5. Our results confirm a marked vasoconstrictor effect of cocaine on the coronary arterial circulation, predominantly distal to the epicardial coronary arteries, but also indicate important differences in the systemic cardiovascular responses to the drug between two closely related strains of animals within the same species. Due to the similarities between the swine and human coronary arterial vasculature, we suggest that vasoconstriction in the coronary microcirculation may produce cardiac toxicity in man.

The purpose of the present study was to determine the maximal coronary flow reserve (CFR) before and after the administration of successive cocaine doses (0.1, 0.5, 3, and 7 mg/kg IV) for 2 minutes at 10-minute intervals in eight miniature swine. CFR was assessed by the administration of adenosine (0.03, 0.3, and 3 mg IC). Hemodynamic and flow measurements were performed 3 minutes after each dose. Coronary flow (CF) was measured with a Doppler-tipped wire in the proximal left anterior descending coronary artery (LAD). Also, microvessels were dissected, and vessel diameters were measured by a videoelectronic dimension analyzer. In vivo, LAD CF increased fourfold, CFR increased twofold, and coronary vascular resistance (CVR) decreased fourfold after the administration of adenosine. In contrast, LAD CF decreased threefold, CFR decreased onefold, and CVR increased sixfold 3 minutes after the administration of cocaine. Adenosine (3 mg) was repeated 4 minutes after the administration of cocaine, and LAD CF increased 1.4-fold, CVR increased 2.5-fold, and CFR decreased onefold. Thus, adenosine partially reversed the potent cocaine constrictor effect. In vitro, 10(-9) mol/L cocaine decreased the diameter of the coronary microvessels from 129 +/- 12 to 127 +/- 12 microns, and 10(-4) mol/L cocaine decreased coronary microvessel diameter to 114 +/- 15 microns (P < .05). In conclusion, cocaine in vivo decreases CFR, and consistent with the in vivo effect, cocaine in vitro produced constriction of vessels < 200 microns. These results indicate that cocaine can produce profound microvascular spasm. This may contribute to the ischemia/infarction reported in patients who abuse cocaine and who are subsequently found to have normal epicardial coronary arteries.