Waldemar Gonsiorek

Discovery and characterization of vicriviroc (SCH 417690), a CCR5 antagonist with potent activity against human immunodeficiency virus type 1.

ABSTRACT Inhibiting human immunodeficiency virus type 1 (HIV-1) infection by blocking the host cell coreceptors CCR5 and CXCR4 is an emerging strategy for antiretroviral therapy. Currently, several novel coreceptor inhibitors are being developed in the clinic, and early results have proven promising. In this report, we describe a novel CCR5 antagonist, vicriviroc (formerly SCH-D or SCH 417690), with improved antiviral activity and pharmacokinetic properties compared to those of SCH-C, a previously described CCR5 antagonist. Like SCH-C, vicriviroc binds specifically to the CCR5 receptor and prevents infection of target cells by CCR5-tropic HIV-1 isolates. In antiviral assays, vicriviroc showed potent, broad-spectrum activity against genetically diverse and drug-resistant HIV-1 isolates and was consistently more active than SCH-C in inhibiting viral replication. This compound demonstrated synergistic anti-HIV activity in combination with drugs from all other classes of approved antiretrovirals. Competition binding assays revealed that vicriviroc binds with higher affinity to CCR5 than SCH-C. Functional assays, including inhibition of calcium flux, guanosine 5′-[35S]triphosphate exchange, and chemotaxis, confirmed that vicriviroc acts as a receptor antagonist by inhibiting signaling of CCR5 by chemokines. Finally, vicriviroc demonstrated diminished affinity for the human ether a-go-go related gene transcript ion channel compared to SCH-C, suggesting a reduced potential for cardiac effects. Vicriviroc represents a promising new candidate for the treatment of HIV-1 infection.

Murine CXCR1 Is a Functional Receptor for GCP-2/CXCL6 and Interleukin-8/CXCL8

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPγS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-α, -β, -γ/CXCL1–3, or rat CINC-1–3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

A novel, orally active CXCR1/2 receptor antagonist, sch527123, inhibits neutrophil recruitment, mucus production, and goblet cell hyperplasia in animal models of pulmonary inflammation

Sch527123 [2-hydroxy-N,N-dimethyl-3-[[2-[[1(R)-(5-methyl-2-furanyl)propyl]amino]-3,4-dioxo-1-cyclobuten-1-yl]amino]ben-zamide] is a potent, selective antagonist of the human CXCR1 and CXCR2 receptors (Gonsiorek et al., 2007). Here we describe its pharmacologic properties at rodent CXCR2 and at the CXCR1 and CXCR2 receptors in the cynomolgus monkey, as well as its in vivo activity in models demonstrating prominent pulmonary neutrophilia, goblet cell hyperplasia, and mucus production. Sch527123 bound with high affinity to the CXCR2 receptors of mouse (Kd = 0.20 nM), rat (Kd = 0.20 nM), and cynomolgus monkey (Kd = 0.08 nM) and was a potent antagonist of CXCR2-mediated chemotaxis (IC50 ∼3–6 nM). In contrast, Sch527123 bound to cynomolgus CXCR1 with lesser affinity (Kd = 41 nM) and weakly inhibited cynomolgus CXCR1-mediated chemotaxis (IC50 ∼1000 nM). Oral treatment with Sch527123 blocked pulmonary neutrophilia (ED50 = 1.2 mg/kg) and goblet cell hyperplasia (32–38% inhibition at 1–3 mg/kg) in mice following the intranasal lipopolysaccharide (LPS) administration. In rats, Sch527123 suppressed the pulmonary neutrophilia (ED50 = 1.8 mg/kg) and increase in bronchoalveolar lavage (BAL) mucin content (ED50 =<0.1 mg/kg) induced by intratracheal (i.t.) LPS. Sch527123 also suppressed the pulmonary neutrophilia (ED50 = 1.3 mg/kg), goblet cell hyperplasia (ED50 = 0.7 mg/kg), and increase in BAL mucin content (ED50 = <1 mg/kg) in rats after i.t. administration of vanadium pentoxide. In cynomolgus monkeys, Sch527123 reduced the pulmonary neutrophilia induced by repeat bronchoscopy and lavage (ED50 = 0.3 mg/kg). Therefore, Sch527123 may offer benefit for the treatment of inflammatory lung disorders in which pulmonary neutrophilia and mucus hypersecretion are important components of the underlying disease pathology.

Pharmacological Characterization of Sch527123, a Potent Allosteric CXCR1/CXCR2 Antagonist

In neutrophils, growth-related protein-α (CXCL1) and interleukin-8 (CXCL8), are potent chemoattractants (Cytokine 14:27–36, 2001; Biochemistry 42:2874–2886, 2003) and can stimulate myeloperoxidase release via activation of the G protein-coupled receptors CXCR1 and CXCR2. The role of CXCR1 and CXCR2 in the pathogenesis of inflammatory responses has encouraged the development of small molecule antagonists for these receptors. The data presented herein describe the pharmacology of 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide (Sch527123), a novel antagonist of both CXCR1 and CXCR2. Sch527123 inhibited chemokine binding to (and activation of) these receptors in an insurmountable manner and, as such, is categorized as an allosteric antagonist. Sch527123 inhibited neutrophil chemotaxis and myeloperoxidase release in response to CXCL1 and CXCL8 but had no effect on the response of these cells to C5a or formyl-methionyl-leucyl-phenylalanine. The pharmacological specificity of Sch527123 was confirmed by testing in a diversity profile against a panel of enzymes, channels, and receptors. To measure compound affinity, we characterized [3H]Sch527123 in both equilibrium and nonequilibrium binding analyses. Sch527123 binding to CXCR1 and CXCR2 was both saturable and reversible. Although Sch527123 bound to CXCR1 with good affinity (Kd = 3.9 ± 0.3 nM), the compound is CXCR2-selective (Kd = 0.049 ± 0.004 nM). Taken together, our data show that Sch527123 represents a novel, potent, and specific CXCR2 antagonist with potential therapeutic utility in a variety of inflammatory conditions.

CCR2 and CXCR4 regulate peripheral blood monocyte pharmacodynamics and link to efficacy in experimental autoimmune encephalomyelitis

BackgroundCCR2 plays a key role in regulating monocyte trafficking to sites of inflammation and therefore has been the focus of much interest as a target for inflammatory disease.MethodsHere we examined the effects of CCR2 blockade with a potent small molecule antagonist to determine the pharmacodynamic consequences on the peripheral blood monocyte compartment in the context of acute and chronic inflammatory processes.ResultsWe demonstrate that CCR2 antagonism in vivo led to a rapid decrease in the number of circulating Ly6Chi monocytes and that this decrease was largely due to the CXCR4-dependent sequestration of these cells in the bone marrow, providing pharmacological evidence for a mechanism by which monocyte dynamics are regulated in vivo. CCR2 antagonism led to an accumulation of circulating CCL2 and CCL7 levels in the blood, indicating a role for CCR2 in regulating the levels of its ligands under homeostatic conditions. Finally, we show that the pharmacodynamic changes due to CCR2 antagonism were apparent after chronic dosing in mouse experimental autoimmune encephalomyelitis, a model in which CCR2 blockade demonstrated a dramatic reduction in disease severity, manifest in a reduced accumulation of monocytes and other cells in the CNS.ConclusionCCR2 antagonism in vivo has tractable pharmacodynamic effects that can be used to align target engagement with biologic effects on disease activity.

Pharmacological targeting reveals distinct roles for CXCR2/CXCR1 and CCR2 in a mouse model of arthritis.

Neutrophils and monocytes are abundantly represented in the synovial fluid and tissue in rheumatoid arthritis patients. We therefore explored the effects of small molecule chemokine receptor antagonists to block migration of these cells in anti-collagen antibody-induced arthritis. Targeting neutrophil migration with the CXCR2/CXCR1 antagonist SCH563705 led to a dose-dependent decrease in clinical disease scores and paw thickness measurements and clearly reduced inflammation and bone and cartilage degradation based on histopathology and paw cytokine analyses. In contrast, targeting monocyte migration with the CCR2 antagonist MK0812 had no effect on arthritis disease severity. The pharmacodynamic activities of both SCH563705 and MK0812 were verified by assessing their effects on the peripheral blood monocyte and neutrophil populations. SCH563705 selectively reduced the peripheral blood neutrophil frequency, and caused an elevation in the CXCR2 ligand CXCL1. MK0812 selectively reduced the peripheral blood monocyte frequency, and caused an elevation in the CCR2 ligand CCL2. The much greater impact of CXCR2/CXCR1 antagonism relative to CCR2 antagonism in this model of arthritis highlights the therapeutic potential for targeting CXCR2/CXCR1 in human arthritides.

Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL-60 cells: antagonism of hCCR1 activation by MIP-1β

C‐C chemokine receptor‐1 (CCR1) has been implicated in mediating a variety of inflammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP‐1α/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. We used radioligand binding and [35S]‐GTPγS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3‐hCCR1) to characterize a panel of chemokines (HCC‐1, MIP‐1α, MIP‐1β, MIP‐1δ, MPIF‐1, MCP‐2, MCP‐3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced 125I‐MIP‐1α with a wide range of potencies and, with the exception of MCP‐2, acted as full agonists in stimulating [35S]‐GTPγS exchange. We then assessed the utility of HL‐60 cells cultured with known differentiating agents (PMA, DMSO, dibutyryl‐cAMP or retinoic acid) for investigating CCR1 pharmacology. In [35S]‐GTPγS exchange assays, membranes from cells cultured with retinoic acid (4–6 days) were the most responsive to activation by MIP‐1α and MPIF‐1. FACS analysis and comparative pharmacology confirmed that these activities were mediated by CCR1. Using [35S]‐GTPγS exchange assays, intracellular calcium flux and/or whole cell chemotaxis assays in HL‐60(Rx) cells, we validated that MIP‐1α was the most potent CCR1 ligand (MIP‐1α>MPIF‐1>RANTESMIP‐1β) although the ligands differed in their efficacy as agonists. MPIF‐1 was the more efficacious (MPIF‐1>RANTES=MIP‐1α>>MIP‐1β). 125I‐MIP‐1β binding in Ba/F3‐hCCR1 and HL‐60(Rx) membranes was competitively displaced by MIP‐1α, MPIF‐1 and MIP‐1β. The binding Ki for these chemokines with 125I‐MIP‐1β were essentially identical in the two membrane systems. Lastly, MIP‐1β antagonized [35S]‐GTPγS exchange, Ca2+ flux and chemotaxis in HL‐60(Rx) cells in response to robust agonists such as MIP‐1α, RANTES and MPIF‐1. Based on our results, we propose that MIP‐1β could function as an endogenous inhibitor of CCR1 function.

Characterization of peripheral human cannabinoid receptor (hCB2) expression and pharmacology using a novel radioligand, [35S]Sch225336.

Studies to characterize the endogenous expression and pharmacology of peripheral human cannabinoid receptor (hCB2) have been hampered by the dearth of authentic anti-hCB2 antibodies and the lack of radioligands with CB2 selectivity. We recently described a novel CB2 inverse agonist, N-[1(S)-[4-[[4-methoxy-2-[(4methoxyphenyl)sulfonyl] phenyl]sulfonyl] phenyl]ethyl]methane-sulfonamide (Sch225336), that binds hCB2 with high affinity and excellent selectivity versus hCB1. The precursor primary amine of Sch225336 was prepared and reacted directly with [35S]mesyl chloride (synthesized from commercially obtained [35S]methane sulfonic acid) to generate [35S]Sch225336. [35S]Sch225336 has high specific activity (>1400 Ci/mmol) and affinity for hCB2 (65 pm). Using [35S]Sch225336, we assayed hemopoietic cells and cell lines to quantitate the expression and pharmacology of hCB2. Lastly, we used [35S]Sch225336 for detailed autoradiographic analysis of CB2 in lymphoid tissues. Based on these data, we conclude that [35S]Sch225336 represents a unique radioligand for the study of CB2 endogenously expressed in blood cells and tissues.

Synthesis and structure-activity relationships of heteroaryl substituted-3,4-diamino-3-cyclobut-3-ene-1,2-dione CXCR2/CXCR1 receptor antagonists.

Comprehensive SAR studies were undertaken in the 3,4-diaminocyclobut-3-ene-1,2-dione class of CXCR2/CXCR1 receptor antagonists to explore the role of the heterocycle on chemokine receptor binding affinities, functional activity, as well as oral exposure in rat. The nature of the heterocycle as well as the requisite substitution pattern around the heterocycle was shown to have a dramatic effect on the overall biological profile of this class of compounds. The furyl class, particularly the 4-halo adducts, was found to possess superior binding affinities for both the CXCR2 and CXCR1 receptors, functional activity, as well as oral exposure in rat versus other heterocyclic derivatives.

Pharmacological characterization of human S1P4 using a novel radioligand, [4,5-3H]-dihydrosphingosine-1-phosphate

Sphingosine‐1‐phosphate (S1P) is a bioactive lipid that affects a variety of cellular processes through both its actions as a second messenger and via activation of a family of G protein‐coupled receptors (S1P1–5). The study of S1P receptor pharmacology, particularly S1P4, has been hindered by the lack of high‐affinity radioligands with good specific activity. The studies presented herein characterize [3H]DH‐S1P as a stable, high‐affinity radioligand for S1P4 pharmacology. Using a transfected Ba/F3 cell line selected for high hS1P4 surface expression, we compared the consequences of different cellular backgrounds and commercial sources of sphingophospholipids on S1P4 characterization. The development and subsequent use of the assay described has enabled us to extensively and definitively characterize the pharmacology of the human S1P4 receptor.