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.

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

Subject Disposition for Phase 1 Clinical Study.

<p>Subject Disposition for Phase 1 Clinical Study.</p

Subject Disposition for Phase 1 Clinical Study.

<p>Subject Disposition for Phase 1 Clinical Study.</p

<i>In vitro</i> Characterization of CCX168 Using Freshly-Isolated Human Neutrophils or Human Whole Blood.

<p>(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.</p

<i>In vitro</i> Characterization of CCX168 Using C5aR-Expressing U937 Cells.

<p>(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.</p

Biological Effects of CCX168 in Cynomolgus Monkeys.

<p>(A) <i>In vitro</i> 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) <i>In vivo</i> 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.</p

Pharmacokinetic Profile of CCX168 in Healthy Human Volunteers.

<p>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.</p