Mouse IL-2/CD25 Fusion Protein Induces Regulatory T Cell Expansion and Immune Suppression in Preclinical Models of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is associated with an IL-2-deficient state, with regulatory T cells (Tregs) showing diminished immune regulatory capacity. A low dose of IL-2 has shown encouraging clinical benefits in SLE patients; however, its clinical utility is limited because of the requirement of daily injections and the observation of increase in proinflammatory cytokines and in non-Tregs. We recently showed that a fusion protein of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, was effective in treating diabetes in NOD mice by selectively inducing Treg expansion. In this report, we show that mouse IL-2 (mIL-2)/CD25 at doses up to 0.5 mg/kg twice a week induced a robust Treg expansion without showing signs of increase in the numbers of NK, CD4+Foxp3-, or CD8+ T cells or significant increase in proinflammatory cytokines. In both NZB × NZW and MRL/lpr mice, mIL-2/CD25 at 0.2-0.4 mg/kg twice a week demonstrated efficacy in inducing Treg expansion, CD25 upregulation, and inhibiting lupus nephritis based on the levels of proteinuria, autoantibody titers, and kidney histology scores. mIL-2/CD25 was effective even when treatment was initiated at the time when NZB × NZW mice already showed signs of advanced disease. Furthermore, we show coadministration of prednisolone, which SLE patients commonly take, did not interfere with the ability of mIL-2/CD25 to expand Tregs. The prednisolone and mIL-2/CD25 combination treatment results in improvements in most of the efficacy readouts relative to either monotherapy alone. Taken together, our results support further evaluation of IL-2/CD25 in the clinic for treating immune-mediated diseases such as SLE.

Persistent IL-2 Receptor Signaling by IL-2/CD25 Fusion Protein Controls Diabetes in NOD Mice by Multiple Mechanisms.

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2-based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies.

IL-2/CD25: A Long-Acting Fusion Protein That Promotes Immune Tolerance by Selectively Targeting the IL-2 Receptor on Regulatory T Cells.

Low-dose IL-2 represents an immunotherapy to selectively expand regulatory T cells (Tregs) to promote tolerance in patients with autoimmunity. In this article, we show that a fusion protein (FP) of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, has greater in vivo efficacy than rIL-2 at Treg expansion and control of autoimmunity. Biochemical and functional studies support a model in which IL-2 interacts with CD25 in the context of this FP in trans to form inactive head-to-tail dimers that slowly dissociate into an active monomer. In vitro, IL-2/CD25 has low sp. act. However, in vivo IL-2/CD25 is long lived to persistently and selectively stimulate Tregs. In female NOD mice, IL-2/CD25 administration increased Tregs within the pancreas and reduced the instance of spontaneous diabetes. Thus, IL-2/CD25 represents a distinct class of IL-2 FPs with the potential for clinical development for use in autoimmunity or other disorders of an overactive immune response.

A novel function of CXCL10 in mediating monocyte production of proinflammatory cytokines.

IFN-γ-inducible protein 10 (CXCL10), a chemokine that is abundantly secreted in response to inflammatory stimuli, has been implicated in the pathogenesis of multiple inflammatory diseases, such as inflammatory bowel disease. Whereas CXCL10 is traditionally recognized for recruiting pathogenic T cells to inflamed sites, its nonchemotactic role during inflammation remains poorly defined. In this report, we identified a novel function of CXCL10 in the regulation of the inflammatory potential of human monocytes to produce cytokines. We found that CXCL10 was necessary and sufficient for IFN-γ-primed human monocytes to induce a robust production of proinflammatory cytokines, such as IL-12 and IL-23. CXCL10-induced monocyte production of these cytokines depended on CXCR3 receptor engagement as well as on the Iκ B kinase and p38 MAPK signaling pathways. By using an innate-mediated murine colitis model, we demonstrated that anti-CXCL10 Ab treatment robustly suppressed the local production of myeloid-derived inflammatory cytokines and intestinal tissue damage. Together, our data unravel a previously unappreciated role of CXCL10 in the amplification of myeloid cell-mediated inflammatory responses. Targeting CXCL10 is therefore an attractive approach to treating inflammatory diseases that are driven by innate and adaptive immunity.

Discovery of indazole aldosterone synthase (CYP11B2) inhibitors as potential treatments for hypertension.

We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.

Discovery of Spirocyclic Aldosterone Synthase Inhibitors as Potential Treatments for Resistant Hypertension.

Herein we report the discovery and hit-to-lead optimization of a series of spirocyclic piperidine aldosterone synthase (CYP11B2) inhibitors. Compounds from this series display potent CYP11B2 inhibition, good selectivity versus related CYP enzymes, and lead-like physical and pharmacokinetic properties.

Investigation of piperazine benzamides as human ß3 adrenergic receptor agonists for the treatment of overactive bladder.

The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human ß3-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established ß3 potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.

Imidazopyridyl compounds as aldosterone synthase inhibitors.

The inhibition of aldosterone synthase (CYP11B2) may be an effective treatment of hypertension and heart failure, among other ailments. Previously reported benzimidazole CYP11B2 inhibitors led the way for bioisosteric imidazopyridines that are both potent and selective over CYP11B1.

Discovery of benzamides as potent human ß3 adrenergic receptor agonists.

The paper will describe the synthesis and SAR studies that led to the discovery of benzamide (reverse amide) as potent and selective human ß3-adrenergic receptor agonist. Based on conformationally restricted pyrrolidine scaffold we discovered earlier, pyrrolidine benzoic acid intermediate 22 was synthesized. From library synthesis and further optimization efforts, several structurally diverse reverse amides such as 24c and 24i were found to have excellent human ß3-adrenergic potency and good selectivity over the ß1 and ß2 receptors. In addition to human ß1, ß2, ß3 and hERG data, PK of selected compounds will be described.

Discovery of Vibegron: A Potent and Selective ß3 Adrenergic Receptor Agonist for the Treatment of Overactive Bladder.

The discovery of vibegron, a potent and selective human ß3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical ß3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived ß3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.

Discovery of Triazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Discovery of Benzimidazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Dual Inhibition of Interleukin-23 and Interleukin-17 Offers Superior Efficacy in Mouse Models of Autoimmunity.

Therapies targeting either interleukin (IL)-23 or IL-17 have shown promise in treating T helper 17 (Th17)-driven autoimmune diseases. Although IL-23 is a critical driver of IL-17, recognition of nonredundant and independent functions of IL-23 and IL-17 has prompted the notion that dual inhibition of both IL-23 and IL-17 could offer even greater efficacy for treating autoimmune diseases relative to targeting either cytokine alone. To test this hypothesis, we generated selective inhibitors of IL-23 and IL-17 and tested the effect of either treatment alone compared with their combination in vitro and in vivo. In vitro, using a novel culture system of murine Th17 cells and NIH/3T3 fibroblasts, we showed that inhibition of both IL-23 and IL-17 completely suppressed IL-23-dependent IL-22 production from Th17 cells and cooperatively blocked IL-17-dependent IL-6 secretion from the NIH/3T3 cells to levels below either inhibitor alone. In vivo, in the imiquimod induced skin inflammation model, and in the myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis model, we demonstrated that dual inhibition of IL-17 and IL-23 was more efficacious in reducing disease than targeting either cytokine alone. Together, these data support the hypothesis that neutralization of both IL-23 and IL-17 may provide enhanced benefit against Th17 mediated autoimmunity and provide a basis for a therapeutic strategy aimed at dual targeting IL-23 and IL-17.

Rhesus monkey model for concurrent analyses of in vivo selectivity, pharmacokinetics and pharmacodynamics of aldosterone synthase inhibitors.

INTRODUCTION: In vivo profiles of aldosterone synthase inhibitors (ASIs) have been investigated utilizing various rodent models. Due to lack of CYP17 activity, rodents produce corticosterone rather than cortisol as that of humans, which raised concern to their effectiveness in translational pharmacological characterization of ASI. METHODS: A rhesus monkey model that combines a low sodium diet with adrenocorticotropin (ACTH) treatment was developed. Plasma concentrations of steroid metabolites associated with reactions catalyzed by CYP11B2 and CYP11B1 were measured concurrently by a UPLC/MS method. RESULTS: Plasma concentration of aldosterone in regular diet fed rhesus monkeys was low at 109pg/mL. Aldosterone concentrations were increased to 252pg/mL when animals were maintained on a low sodium diet for 3weeks, and to 300pg/mL with ACTH treatment at 0.3mg/kg. The combination of low sodium diet with ACTH treatment further increased plasma concentration of aldosterone to 730pg/mL and other steroid metabolites at various levels. Intravenous administration of ASI, fadrozole (0.001-1mg/kg) or LCI699 (0.003-3mg/kg), led to dose-dependent reductions in aldosterone and 18-hydroxycorticosterone, increases in 11-deoxycorticosterone and 11-deoxycortisol, and bell-shaped changes in cortisol and corticosterone. In vivo selectivity of CYP11B2/CYP11B1 for fadrazole was 26-fold and LCI-699 was 27-fold, which was consistent with relative selectivity using in vitro values from recombinant cells transfected with rhesus monkey CYP11B2 and CYP11B1. DISCUSSION: This model enables concurrent characterization of pharmacokinetics, pharmacodynamics and selectivity of CYP11B2 over CYP11B1 inhibition in the same animal. It may be used as a translational model for pharmacological characterization of ASI.

Design, synthesis, and evaluation of conformationally restricted acetanilides as potent and selective ß3 adrenergic receptor agonists for the treatment of overactive bladder.

A series of conformationally restricted acetanilides were synthesized and evaluated as ß3-adrenergic receptor agonists (ß3-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine ß3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent ß3-AR mediated responses in a rat bladder hyperactivity model.

Synthesis and immunological activities of novel Toll-like receptor 7 and 8 agonists.

Single-stranded oligoribonucleotides (ORNs) stimulate innate immune responses through TLR7 and TLR8. Specific linkages and chemical modifications incorporated into synthetic ORN can greatly enhance nuclease stability, selectivity, and potency. In the present study, we have synthesized 15 ORN containing different sequence compositions and chemical modifications and studied their TLR7- and TLR8-mediated immune response profiles in HEK293 cells expressing human TLR7 or TLR8, human PBMCs, mDCs and pDCs, non-human primate (NHP) PBMCs, and in vivo in mice and NHPs. Based on the results obtained, eight of the ORNs containing specific chemical modifications induced immune responses through both TLR7 and TLR8, including activation of NF-κB in TLR7- and TLR8-transfected cell lines; induction of IFN-α, IL-6, TNF-α, IL-12, and IP-10 in human PBMCs; IFN-α induction in human pDCs; CD80 upregulation in human pDCs and mDCs; IL-12 induction following acute administration in mice; IFN-α, IP-10, IL-6, and IL-12 induction in NHP PBMCs; and IFN-α, IP-10, and IL-6 induction following acute administration in NHPs. Seven of the ORNs show selectivity for TLR8-induced responses; they specifically activate only TLR8-transfected cell lines, induce cytokines other than IFN-α in human and NHP PBMCs, activate mDCs more than pDCs, and do not induce IL-12 acutely in mice, consistent with the lack of functional TLR8 in mice. The novel TLR8-selective ORNs also induce cytokines other than IFN-α acutely in NHPs. In conclusion, we have designed and synthesized novel ORNs with varying sequence compositions and chemical modifications, which selectively act as agonists of TLR8 or dual agonists of TLR7 and TLR8.

Design of a novel pyrrolidine scaffold utilized in the discovery of potent and selective human ß3 adrenergic receptor agonists.

A novel class of human ß(3)-adrenergic receptor agonists was designed in effort to improve selectivity and metabolic stability versus previous disclosed ß(3)-AR agonists. As observed, many of the ß(3)-AR agonists seem to need the acyclic ethanolamine core for agonist activity. We have synthesized derivatives that constrained this moiety by introduction of a pyrrolidine. This unique modification maintains human ß(3) functional potency with improved selectivity versus ancillary targets and also eliminates the possibility of the same oxidative metabolites formed from cleavage of the N-C bond of the ethanolamine. Compound 39 exhibited excellent functional ß(3) agonist potency across species with good pharmacokinetic properties in rat, dog, and rhesus monkeys. Early de-risking of this novel pyrrolidine core (44) via full AMES study supports further research into various new ß(3)-AR agonists containing the pyrrolidine moiety.

Dual targeting of CCR2 and CX3CR1 in an arterial injury model of vascular inflammation.

OBJECTIVES: The chemokine receptors CCR2 and CX3CR1 are important in the development of coronary artery disease. The purpose of this study is to analyze the effect of a novel CCR2 inhibitor in conjunction with CX3CR1 deletion on vascular inflammation. METHODS: The novel CCR2 antagonist MRL-677 was characterized using an in vivo model of monocyte migration. To determine the relative roles of CCR2 and CX3CR1 in vascular remodeling, normal or CX3CR1 deficient mice were treated with MRL-677. After 14 days, the level of intimal hyperplasia in the artery was visualized by paraffin sectioning and histology of the hind limbs. RESULTS: MRL-677 is a CCR2 antagonist that is effective in blocking macrophage trafficking in a peritoneal thioglycollate model. Intimal hyperplasia resulting from vascular injury was also assessed in mice. Based on the whole-blood potency of MRL-677, sufficient drug levels were maintained for the entire 14 day experimental period to afford good coverage of mCCR2 with MRL-677. Blocking CCR2 with MRL-677 resulted in a 56% decrease in the vascular injury response (n = 9, p < 0.05) in normal animals. Mice in which both CCR2 and CX3CR1 pathways were targeted (CX3CR1 KO mice given MRL-677) had an 88% decrease in the injury response (n = 6, p = 0.009). CONCLUSION: In this study we have shown that blocking CCR2 with a low molecular weight antagonist ameliorates the inflammatory response to vascular injury. The protective effect of CCR2 blockade is increased in the presence of CX3CR1 deficiency suggesting that CX3CR1 and CCR2 have non-redundant functions in the progression of vascular inflammation.

CCR2 antagonists.

Inhibition of CCR2 has been considered as a target for multiple therapeutic diseases including autoimmune disease, atherosclerosis, pain, and metabolic disease, based in part on the critical role this receptor plays on monocyte migration. Numerous companies have reported programs to identify CCR2 antagonists. Common challenges to the development of CCR2 agents have included poor activity at the rodent receptor and selectivity for both other chemokine receptors and ion channels. This review summarizes the rationale for targeting CCR2 in disease, the recent progress in the identification of potent and select CCR2 antagonists, and the current status of clinical trials for CCR2 agents.

Synthesis and immunological activities of novel agonists of toll-like receptor 9.

Novel agonists of TLR9 with two 5'-ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-alpha production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-alpha and IP-10) in rhesus macaques after intra-muscular administration.