Characterization of Pharmacologic and Pharmacokinetic Properties of CCX168, a Potent and Selective Orally Administered Complement 5a Receptor Inhibitor, Based on Preclinical Evaluation and Randomized Phase 1 Clinical Study

Abstract

The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773.

Fig 2. The Complement Cascade Showing the Point of Intervention of CCX168.

The complement cascade can be activated by the classical, lectin, or alternative pathways and leads to the formation of C3a, C3b, C5b-9 (terminal complement complex) and C5a. During the amplification loop, full length C3 is cleaved to form C3a and C3b; C3aR is the G protein coupled expressed receptor that responds to C3a, while C3b covalently binds to foreign surfaces and aids in phagocytosis and clearance. C5 convertase is formed during the amplification loop, leading to the cleavage of full length C5 at a specific arginine-leucine bond to form C5a and C5b. C5b associates with complement components C6, C7, C8, and C9 to form the terminal complement complex or membrane attack complex (MAC), typically on the surface of pathogenic bacterial cells. C5aR (CD88) is the G protein coupled receptor expressed by innate immune cells, such as neutrophils, that responds to C5a (12 kDa), a potent pro-inflammatory mediator [2,11]. C5a rapidly induces expression of adhesion molecules on the cell surface of innate immune cells, such as neutrophils, and induces the directed migration, or chemotaxis, of these cells. C5a also mediates inflammation by stimulating vascular permeability, neutrophil degranulation, and release of lysosomal proteases and oxidative free radicals [8]. C5a is a transient molecule, being rapidly degraded of its carboxy terminal arginine [12], and hence losing about 10-fold activity on C5aR, and then being internalized and degraded via C5L2, a 7 trans-membrane receptor that has an anti-inflammatory role [–17]. CCX168 is a small molecule antagonist of C5aR that selectively and competitively binds to this receptor.

Fig 3. In vitro Characterization of CCX168 Using C5aR-Expressing U937 Cells.

(A) CCX168 inhibition of [¹²⁵I]-C5a binding to U937 cells with a potency (IC₅₀ value) of 0.1 nM; each data point is the mean of 4 replicates ± standard error; study repeated 5 separate times, representative experiment shown. (B) Effects of vehicle control (■) and 1 nM (○) or 10 nM (●) CCX168 on C5a-mediated chemotaxis of U937 cells in buffer; each data point is the mean of 8 replicates ± standard error; study repeated 5 separate times, representative experiment shown. (C) C5a-mediated chemotaxis of U937 cells in the absence (●) or presence (■) of 1 μM CCX168, as well as following 1 μM CCX168 / 3x wash (□) and 1 μM CCX168 / 6x wash (Δ) treatments; each data point is the mean of 8 replicates ± standard error; study repeated 2 separate times, representative experiment shown. (D) C5a-induced intracellular calcium release in U937 cells, as measured by FLIPR, in the presence of vehicle control (●) and various concentrations of CCX168, 1 nM (○), 10 nM (■), or 100 nM (▲); each data point is the mean of 4 replicates ± standard error; study repeated 2 separate times, representative experiment shown. (E) C5a-mediated chemotaxis of U937 cells in 100% human plasma in the presence of vehicle control (■) and 1 nM (○) or 10 nM (●) CCX168; each data point is the mean of 8 replicates ± standard error; study repeated 2 separate times, representative experiment shown. (F) Inhibition by CCX168 of U937 cell chemotaxis towards 0.1 nM C5a in the presence (□) or absence (●) of α1-acid glycoprotein (AGP, 5 mg/mL) in buffer containing 5% human serum albumin (HSA); each data point is the mean of 8 replicates ± standard error; study repeated 2 separate times, representative experiment shown.

Fig 4. In vitro Characterization of CCX168 Using Freshly-Isolated Human Neutrophils or Human Whole Blood.

(A) Sequential intracellular calcium release in human neutrophils in response to C5a (100 nM), CXCL8 (100 nM) or ionomycin (1 μg/mL), in the presence (blue line) or absence (black line) of CCX168 (10 μM). CCX168 blocked calcium release induced by C5a but not by the CXCR1 ligand CXCL8. (B) Binding of [¹²⁵I]-C5a to human neutrophils in the presence of a range of concentrations of CCX168. CCX168 inhibited [¹²⁵I]-C5a binding with a potency (IC₅₀ value) of 0.2 nM. Each data point represents the mean of 4 replicates ± standard error, and the experiment was repeated 2 separate times. (C) C5a-induced chemotaxis of leukocytes in human whole blood, in the presence of vehicle control (■), and 25 nM (○) or 250 nM (●) CCX168. CCX168 inhibited leukocyte chemotaxis in a dose-dependent manner. Each data point represents the mean of 8 replicates ± standard error, and the experiment was repeated 5 separate times. (D) C5a-induced upregulation of CD11b on the surface of neutrophils in human whole blood in the presence of vehicle control (○) or 50 nM CCX168 (●). CCX168 inhibited CD11b upregulation. Each data point represents the mean of 4 replicates ± standard error, and the experiment was repeated 4 separate times. (E) C5a-induced oxidative burst in isolated human neutrophils in the presence of vehicle control (red histogram) or CCX168 (100 nM, blue histogram). The empty histograms represent untreated neutrophils (i.e., no C5a) in the presence of vehicle control (solid black line) or CCX168 (dotted black line). CCX168 blocked the C5a-induced oxidative burst but did not affect untreated neutrophils. The experiment was repeated 3 times. (F) Chemotaxis of leukocytes in human whole blood towards synovial fluid in the presence of vehicle control or CCX168 (100 nM). Experiments using synovial fluid from a patient with rheumatoid arthritis (RA) or osteoarthritis (OA) are shown. CCX168 inhibited leukocyte chemotaxis induced by each of these samples. Each bar represents the mean of 8 replicates ± standard error, and the experiment was repeated two times. **p<0.01 based on Student’s t-test.

Fig 5. Biological Effects of CCX168 on Transgenic Human C5aR Knock-in Mice.

(A) C5a-induced upregulation of CD11b on the surface of neutrophils in whole blood from human C5aR knock-in mice, after addition of vehicle (■), 10 nM (○), or 100 nM (●) CCX168. CCX168 inhibited CD11b upregulation in a dose-dependent manner. Each data point represents the mean of 3 replicates ± standard error; the study was repeated 3 separate times. (B) C5a-induced upregulation of CD11b on the surface of neutrophils in whole blood from human C5aR knock-in mice 1 hour after oral dosing with vehicle (●), 0.075 mg/kg (□), or 0.15 mg/kg (■) CCX168. Neutrophil CD11b upregulation was diminished in blood from CCX168-treated mice. Plasma concentrations of CCX168 are indicated. (C) Schematic of the C5a-induced leukopenia study in human C5aR knock-in mice. One hour following oral administration of CCX168, C5a (20 μg/kg) was administered intravenously, with blood drawn immediately before and 1 minute after C5a injection. Blood samples were analyzed for leukocyte numbers. (D) Effect of CCX168 in the C5a-induced leukopenia study in hC5aR knock-in mice. The percent change in the number of leukocytes in the blood sample collected after C5a injection, relative to the sample collected prior to C5a injection, is shown for each group (4 mice per group). Above each bar is the average concentration of CCX168 in the pre-injection blood samples. CCX168 inhibited the depletion of blood leukocytes caused by intravenous administration of C5a. *p<0.05, **p<0.01 based on Student’s t-test.

Fig 6. Biological Effects of CCX168 in Cynomolgus Monkeys.

(A) In vitro C5a-induced chemotaxis of freshly-isolated neutrophils from cynomolgus monkeys, conducted in 100% cynomolgus plasma containing vehicle (□) or 50 nM CCX168 (●). CCX168 inhibited neutrophil chemotaxis (A₂ = 3.0 nM). Each data point represents the mean of 8 replicates ± standard error, and the study was repeated 2 separate times. (B) In vivo effect of CCX168 in the C5a-induced neutropenia model in monkeys. The percent change in the number of neutrophils in the blood collected after C5a injection, relative to the sample collected prior to C5a injection, is shown (4 monkeys per group). Above each bar is the average concentration of CCX168 in the pre-injection blood samples. CCX168 inhibited, in a dose-dependent manner, the depletion of blood neutrophils caused by intravenous administration of C5a. **p<0.01 based on Student’s t-test.

Fig 1. Subject Disposition for Phase 1 Clinical Study.

Fig 7. Pharmacokinetic Profile of CCX168 in Healthy Human Volunteers.

Plasma concentration versus time profiles of CCX168 after (A) single oral doses of 1 mg (◊), 3 mg (∇), 10 mg (Δ), 30 mg (○), or 100 mg (□) CCX168 and (B) after receiving CCX168 once daily at 1 mg (◊), 3 mg (∇), or 10 mg (Δ), or twice daily at 30 mg (○) or 50 mg (□) for 7 days. The data points represent the mean ± standard error.

Fig 8. Pharmacodynamic Profile of CCX168 in Healthy Human Volunteers.

Pharmacologic inhibition of C5aR by CCX168 as measured with an on-site, whole-blood ex vivo assay of C5a activity. (A) C5a-induced upregulation of CD11b on the surface of neutrophils in whole blood collected in the placebo group (●), and at 2 hours (♦) and 24 hours (◊) after a single dose of 10 mg CCX168, at 2 hours (▲) and 12 hours (Δ) after a single dose of 30 mg CCX168, and 2 hours (■) and 12 hours (□) after a single dose of 100 mg CCX168. CD11b upregulation was diminished in blood from subjects dosed with CCX168. (B) C5a-induced upregulation of CD11b on the surface of neutrophils in whole blood collected at 2 hours (●) or 12 hours (○) after the last dose of a 7-day regimen of placebo, or 2 hours (▲) or 12 hours (Δ) after the last dose of a 7-day regimen of 30 mg CCX168 given twice daily. CD11b upregulation was diminished in both blood samples from CCX168 subjects with a 10-fold decrease in C5a potency exhibited in the 12-hour samples.