Complement 5a receptor (IC50: 0.1 nM)

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.
CCX168 displaced [125I]-C5a binding to C5aR on a human myeloid cell line (U937) with a potency (IC50 value) of 0.1 nM (Fig 3A). Two measures of CCX168 potency were used, the IC50 (50% inhibition of a half-maximal agonist concentration), and the dose ratio or A2 (the concentration of CCX168 that produces a 2-fold right-shift in C5a activity) (Fig 3B and 3C). CCX168 inhibits C5a-mediated chemotaxis of U937 cells with a potency (A2) of 0.2 nM. Addition of CCX168 to U937 cells in a calcium mobilization assay inhibited C5a with a potency (A2) of 0.1 nM (Fig 3D). CCX168 inhibits chemotaxis of U937 cells in 100% human plasma (Fig 3E) with no loss of effect in the presence of α1-acid glycoprotein (Fig 3F). Furthermore, CCX168 did not display any agonist activities with any of the assays used (cytoplasmic calcium flux, chemotaxis, or CD11b upregulation). CCX168 is selective for C5aR, with no activity (IC50 >5,000 nM) measured with the C5aR-related receptors C5L2, C3aR, ChemR23, GPR1, and FPR1, a panel of 18 of the chemokine receptors, a panel of 54 pharmacologically relevant receptors, and the cytochrome P450 enzymes 1A2, 2C9, 2C19, 2D6, 3A4 (details in S1 and S2 Tables). In addition, CCX168 did not inhibit the hERG potassium ion channel as measured in a patch clamp assay (IC50 <5000 nM).[1]
CCX168 competitively and selectively blocked C5a-induced calcium mobilization in purified human neutrophils, with an IC50 value of 0.2 nM (Fig 4A). Similar results were obtained using purified human blood monocytes. CCX168 also inhibited binding of [125I]-C5a to C5aR on human neutrophils with an IC50 of 0.2 nM (Fig 4B). Moreover, CCX168 inhibited C5a-mediated chemotaxis of neutrophils in freshly-collected human blood with an A2 of 1.7 nM (Fig 4C). CCX168 inhibited C5a-induced increases in the level of integrin CD11b on neutrophils in human whole blood with an A2 of 3.0 nM (Fig 4D). In addition, CCX168 inhibited C5a-induced release of reactive-oxygen species from isolated neutrophils, and was able to completely block respiratory burst in these neutrophils (Fig 4E). Synovial fluid samples taken from subjects with rheumatoid arthritis (RA) or osteoarthritis (OA) induced chemotaxis of human blood leukocytes [24]; CCX168 significantly reduced this response, suggesting that both types of synovial samples contain active C5a (Fig 4F).

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[1].

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Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO–induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN[2].

In vitro assays Chemotaxis, calcium mobilization, and radioligand binding assays were conducted as previously described. The respiratory burst assay was conducted as described. The ability of CCX168 to affect C5a-mediated migration was determined by quantifying the extent of the rightward shift in the concentration curves. This was expressed as an “A” value. For example, an A2 value indicates the concentration of CCX168 that results in a two-fold rightward shift of the dose-response curve for a C5a-mediated effect, and correlates with 50% receptor occupancy by a competitive antagonist such as CCX168. Potency calculations (A2) from functional assays were made as described using the following equation[1].

Human U937 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and with dibutyryl cAMP (0.5 mM) added to the cells 18 hours before use. THP-1, HEK293, MOLT4, Baf3 and MDA-MB435 cells were obtained from ATCC and grown according to their recommendations. L1.2 cells were licensed from Dr. Eugene Butcher. Activated human T lymphocytes were cultured as described. All blood was collected into EDTA as an anti-coagulant. Human whole blood was collected from healthy volunteers and used within two hours. Neutrophils were isolated from human whole blood using standard density gradient separation methods. Cynomolgus monkey whole blood was from the California National Primate Research Center and was used within four hours of collection[1].

For in vivo assays, CCX168 was formulated in PEG-400/solutol-HS-15 (70:30)
C5a-induced leukopenia in human C5aR knock-in mice Human C5aR knock-in mice were dosed with vehicle (PEG-400/solutol-HS15 70:30, 5 mL/kg) or CCX168 by oral gavage. One hour after dosing, C5a (20 μg/kg, 0.1 mL dose volume) was injected intravenously and blood samples collected from retro-orbital eye bleeds. Blood leukocyte levels were analyzed by flow cytometry. C5a-induced neutropenia in cynomolgus monkeys All experiments performed in cynomolgus monkeys were performed at Covance Research Products with the approval of Covance Research Products Animal Care and Use Committee and in compliance with the Guide for the Care and Use of Laboratory Animals essentially as previously described[1].

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Humanization of Mice with Human C5aR[2]
Standard homologous recombination techniques were used to create mice with the murine C5a receptor replaced with the human C5a receptor. These mice had a mixed genetic background of 129S6 and C57BL/6. In addition to standard confirmation by genotyping, the effectiveness of replacement of the mC5aR with the hC5aR was tested by determining leukocyte expression of mC5aR versus hC5aR on peripheral blood leukocytes by flow cytometry, and by measuring CCX168 suppression of human C5a-induced chemotaxis of thioglycollate-induced peritoneal leukocytes from mC5aR versus hC5aR mice. CCX168 was formulated in polyethylene glycol 400/Solutol (70/30). Response to human C5a of hC5aR knock-in mouse leukocytes was tested in vitro using a previously described chemotaxis assay.22 In brief, migration of cells from the upper to the lower ChemoTX chamber in response to different concentrations of human C5a was determined by adding CyQUANT solution to each lower chamber and measuring the intensity of fluorescence (Migration Signal) of the DNA-binding fluorescent CyQUANT after 120 minutes, which is a relative measure of cell numbers. In vitro, human C5aR responds equally well to murine C5a and human C5a (data not shown), which is in accord with previously reported results.23 The cross-reactivity of CCX168 has been tested against a panel of over 20 chemotactic receptor (including CCR1–10, CXCR1–7, C5L2, C3aR, and ChemR23) and has at least four orders of magnitude less reactivity versus C5aR (data not shown). According to use of a previously described method,24 the effect on in vivo chemotaxis of oral pretreatment 2 hours before intraperitoneal thioglycollate injection with vehicle or a single dose of 30 mg/kg of CCX168 on cell count in peritoneal lavage was measured 24 hours after intraperitoneal injection of thioglycollate. CCX168 effects on C5a-induced leukopenia was studied in hC5aR knock-in mice 1 hour after oral administration of CCX168 by comparing leukocyte counts in blood drawn 1 minute before and 1 minute after intravenous administration of C5a (20 μg/kg).

[]1]. 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. PLoS One. 2016 Oct 21;11(10):e0164646.
[2]. C5a receptor (CD88) blockade protects against MPO-ANCA GN. J Am Soc Nephrol. 2014 Feb;25(2):225-31.

Anti-neutrophil cytoplasmic (auto)antibody (ANCA)-associated vasculitis (AAV) is a rare (estimated incidence of 3 cases per 100,000 per year) form of "pauci-immune" systemic small-vessel vasculitis typified by the presence of ANCAs in the serum. The full spectrum of AAV includes granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA), and drug-induced AAV. AAV may be associated with necrotizing and crescentic glomerulonephritis (NCGN). Despite complex pathophysiology, studies over the past ~2 decades have identified a key role for the alternative complement pathway and, in particular, the interaction between the anaphylatoxin fragment C5a and its cognate C5aR receptor in AAV. Avacopan (formerly CCX168) is an allosteric C5aR antagonist indicated for use in AAV. Avacopan was granted FDA approval on October 8, 2021, and is currently marketed under the name TAVNEOS by ChemoCentryx, Inc. On January 19, 2022, the European Commission approved avacopan for the treatment of adult patients with severe, active granulomatosis polyangiitis (GPA) or microscopic polyangiitis (MPA) - the two main forms of ANCA-associated vasculitis - in combination with [rituximab] or [cyclophosphamide]. Avacopan was approved by Health Canada on April 20, 2022.

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Avacopan is a Complement 5a Receptor Antagonist. The mechanism of action of avacopan is as a Complement 5a Receptor Antagonist, and Cytochrome P450 3A4 Inhibitor. Avacopan is indicated for the adjunctive treatment of adult patients with severe active anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (granulomatosis with polyangiitis and microscopic polyangiitis; GPA/MPA) in combination with standard therapy including glucocorticoids. Avacopan does not eliminate the need for glucocorticoids. In Europe, avacopan is approved for the treatment of adults with severe, active granulomatosis polyangiitis (GPA) or microscopic polyangiitis (MPA) in combination with rituximab or cyclophosphamide. Tavneos, in combination with a rituximab or cyclophosphamide regimen, is indicated for the treatment of adult patients with severe, active granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA). Treatment of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis.

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Avacopan is a complement 5a receptor (C5aR) antagonist that blocks C5a-induced upregulation of C11b (integrin alpha M) on neutrophils and inhibits C5a-mediated neutrophil activation and migration. Avacopan has been associated with hypersensitivity reactions, including angioedema, and hepatotoxicity, as evidenced by elevated liver transaminases. Likely due to its effect on the complement pathway, avacopan has also been associated with hepatitis B virus reactivation and serious infections, which should be monitored for as appropriate.

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Biological Half-Life: A single 30 mg dose of avacopan given with food to healthy subjects resulted in mean elimination half-lives of 97.6 and 55.6 hours for avacopan and its M1 metabolite, respectively.

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Anti-neutrophil cytoplasmic (auto)antibody (ANCA)-associated vasculitis (AAV) is considered a "pauci-immune" form of systemic small-vessel vasculitis accompanied by the presence of ANCAs in the serum. The full spectrum of AAV includes granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA), and drug-induced AAV. AAV may be associated with necrotizing and crescentic glomerulonephritis (NCGN). Of the various known ANCAs, the major antigens are myeloperoxidase (MPO) and proteinase 3 (PR3/myeloblastin). The pathophysiology giving rise to AAV is complex, though a working model has been proposed. An initial trigger, such as infection, causes differentiation of naive T cells into TH17 helper T cells that induce the release from macrophages of pro-inflammatory cytokines (e.g., TNF-α and IL-1β), which prime neutrophils. Concurrently, the anaphylatoxin C5a is produced through activation of the alternative complement pathway, which also primes neutrophils through binding to the C5a receptor (C5aR; CD88). Primed neutrophils undergo physiological changes, including upregulating the display of ANCA antigens on their surface. Circulating ANCAs bind to displayed ANCA antigens on the surface of neutrophils; simultaneously, the Fc region of these ANCAs is recognized by Fcγ receptors on other neutrophils, resulting in excessive neutrophil activation. Activated neutrophils form NETs (neutrophil extracellular traps), which induce tissue damage and vasculitis. MPO/PR3 in NETs induces further ANCA production through dendritic cell- and CD4+ T cell-mediated activation of B cells, further exacerbating the condition. A role for complement was not initially considered in AAV due to a lack of excessive complement or immunoglobulin deposition in AAV lesions. However, extensive molecular studies confirmed a significant role for the alternative complement pathway, acting through C3 and C5, in the pathogenesis of AAV. The C5a fragment, generated by C5 cleavage, can bind to both the C5aR and C5a-like receptor (C5L2) on the surface of neutrophils; C5aR binding is associated with AAV while C5L2 binding has a protective effect. As the alternative complement pathway is self-sustaining in the absence of down-regulation by specific proteins, it is likely a significant driver of AAV. Furthermore, neutrophils activated by C5a release reactive oxygen species, properdin, and other molecules that stimulate the complement pathway leading to the production of more C5a in a vicious cycle. Avacopan (CCX168) is a specific C5aR receptor allosteric antagonist that inhibits C5a-mediated neutrophil activation both _in vitro_ and _in vivo_. By inhibiting the C5a/C5aR axis, avacopan should have minimal effects on the formation of the membrane attack complex (which includes C5b) and therefore little effect on the innate immune response in treated patients.

C33H35F4N3O2

581.66

581.2665

C, 68.14; H, 6.07; F, 13.07; N, 7.22; O, 5.50

1346623-17-3

1346623-17-3;

49841217

Typically exists as solids (or liquids in special cases) at room temperature

8.13

61.4

FC1=C([H])C([H])=C([H])C(C([H])([H])[H])=C1C(N1C([H])([H])C([H])([H])C([H])([H])[C@]([H])(C(N([H])C2C([H])=C([H])C(C([H])([H])[H])=C(C(F)(F)F)C=2[H])=O)[C@@]1([H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C1([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])=O

PUKBOVABABRILL-YZNIXAGQSA-N

InChI=1S/C33H35F4N3O2/c1-20-12-15-25(19-27(20)33(35,36)37)39-31(41)26-10-6-18-40(32(42)29-21(2)7-5-11-28(29)34)30(26)22-13-16-24(17-14-22)38-23-8-3-4-9-23/h5,7,11-17,19,23,26,30,38H,3-4,6,8-10,18H2,1-2H3,(H,39,41)/t26-,30-/m0/s1

(2R,3S)-2-[4-(cyclopentylamino)phenyl]-1-(2-fluoro-6-methylbenzoyl)-N-[4-methyl-3-(trifluoromethyl)phenyl]piperidine-3-carboxamide

CCX-168; Tavneos; CCX168; CCX 168. Avacopan

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)