Complement component 5 (C5)

Zilucoplan (RA101495; 1-1000 nM; 30 min) inhibits the increase in C5a plasma levels in human whole blood caused by lipopolysaccharides with an IC50 value of 474.5 pM. When zilucoplan is used at a concentration of 1 nM, C5a plasma levels are reduced by 65.7% [2]. Zilucoplan binds to complement component 5 (C5) and inhibits the cleavage of C5 by the C5 convertase into C5a and C5b. This prevents C5 from forming membrane pores and consequent cell death. Additionally, Zilucoplan blocks the downstream assembly of the membrane attack complex (MAC; C5b-9)[1].

Zilucoplan (RA101495; 10 mg/kg; SC; daily, for 6 d) supplemented with human complement impedes the development of immune-mediated necrotizing myopathy (IMNM) in C5-deficient mice[1]. For six days, Zilucoplan (10 mg/kg; SC; daily) protects against myopathy in C57BL/6 mice[1].

Purified immunoglobulin G (IgG) from an anti-HMGCR+ IMNM patient was co-injected intraperitoneally with human complement in C57BL/6, C5-deficient B10 (C5def) and Rag2 deficient (Rag2-/-) mice. Zilucoplan was administered subcutaneously in a preventive or interventional paradigm, either injected daily throughout the duration of the experiment in C57BL/6 and C5def mice or 8 days after disease induction in Rag2-/- mice. [1]

Absorption, Distribution and Excretion
Following single or multiple daily subcutaneous injections of 0.3 mg/kg zilucoplan, the time to peak plasma concentration (Tmax) ranges from 3 to 6 hours. In healthy subjects, daily subcutaneous injections of 0.3 mg/kg zilucoplan for 14 days resulted in an approximately 3-fold increase in both peak plasma concentration and exposure (AUCtau). Following repeated daily subcutaneous injections of 0.3 mg/kg zilucoplan, plasma concentrations of zilucoplan stabilized, reaching steady-state trough concentrations over 4 to 12 weeks of treatment. Less than 1% of zilucoplan and its metabolites are excreted in urine and feces. In a population pharmacokinetic analysis of adult patients with myasthenia gravis, the steady-state mean volume of distribution was 3.51 L. No further information is available. Metabolisms/Metabolites Zilucoplan is expected to be degraded into small peptides and amino acids via catabolic pathways. RA103488 and RA102758 are the two major metabolites detected in plasma. RA103488 is generated by CYP4F2-mediated metabolism and has pharmacological activity comparable to its parent compound; however, due to the much lower concentration of RA103488 compared to zilopran, its contribution to the pharmacological action of zilopran is expected to be small. RA102758 is formed by protease-mediated degradation and has no pharmacological activity. The AUC values of both metabolites are approximately 10% of the AUC value of the parent drug.

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Biological Half-Life
The mean plasma terminal half-life of zilopran is approximately 172 hours (7 to 8 days).

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
It is expected that zilopran will be degraded into small peptides and amino acids via catabolic pathways in the maternal circulation and infant gastrointestinal tract. It is unlikely to be absorbed by breastfed infants and will not have adverse effects on them. No special precautions are required.
◉ Effects on Breastfed Infants
No relevant published information was found as of the revision date.
◉ Effects on Lactation and Breast Milk
No relevant published information was found as of the revision date.

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Protein Binding
Zipran and its two major metabolites, RA103488 and RA102758, bind to plasma proteins at a rate exceeding 99%.

[1]. Prevention of Anti-HMGCR Immune-Mediated Necrotising Myopathy by C5 Complement Inhibition in a Humanised Mouse Model. Biomedicines. 2022 Aug 20;10(8):2036.

[2]. Chemical synthesis and characterisation of the complement C5 inhibitory peptide zilucoplan. Amino Acids. 2021 Jan;53(1):143-147.

Introduction: Immune-mediated necrotizing myopathy (IMNM) is associated with pathogenic anti-signal recognition granule (SRP) or 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibodies, at least in part, through activation of the classical complement pathway. We evaluated the efficacy of the investigational drug zilucoplan (a macrocyclic peptide inhibitor of complement component 5 (C5)) using a humanized IMNM mouse model. Methods: Purified immunoglobulin G (IgG) from anti-HMGCR-positive IMNM patients was co-intraperitoneally injected with human complement into C57BL/6, C5-deficient B10 (C5def), and Rag2-deficient (Rag2-/-) mice. Zilucoplan was administered subcutaneously daily in C57BL/6 and C5def mice as a prophylactic or interventional regimen, and on day 8 post-disease in Rag2-/- mice. Results: Prophylactic administration of ziroblan prevented muscle strength decline in C5-deficient mice (anti-HMGCR+ group vs. anti-HMGCR++ ziroblan group: p = 0.0289; control group vs. anti-HMGCR++ ziroblan group: p = 0.4634) and wild-type C57BL/6 mice (anti-HMGCR+ group vs. anti-HMGCR++ ziroblan group: p = 0.0002; control group vs. anti-HMGCR++ ziroblan group: p = 0.0939), while also reducing C5b-9 deposition on muscle fibers and the number of regenerated muscle fibers. Following disease induction, ziroblan intervention in Rag2-/- mice reduced complement deposition and the number of regenerated muscle fibers in muscle, but to a lesser extent. In the latter case, C5 inhibitors did not significantly improve muscle strength. Conclusion: Early administration of zilopran to a humanized immune-mediated necrotizing myopathy (IMNM) mouse model can prevent the occurrence of myopathy at both the clinical and histological levels. [1] Zilopran, a complement component C5 inhibitory peptide, is currently in a phase III clinical trial for myasthenia gravis (MG). Although its clinical development has entered the late stage, there are no detailed reports on its chemical synthesis in the literature. This study describes a chemical synthesis method for zilopran and verifies that the synthesized compound can block the generation of C5a induced by lipopolysaccharide (LPS) in human blood. [2]

C172H278N24O55.XC2HF3O2

3562.18 (free base)

3674.96565

Zilucoplan;1841136-73-9

172915692

Typically exists as solid at room temperature

29

62

142

258

7060

16

CCCCCCCCCCCCCCCC(=O)N[C@@H](CCC(=O)NCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCC(=O)NCCCC[C@@H](C(=O)O)NC(=O)[C@H](C1CCCCC1)NC(=O)[C@@H]2CCCN2C(=O)[C@H](CC3=CC=C(C=C3)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC4=CNC5=C4C=CC=N5)NC(=O)[C@H](CC6=CC=C(C=C6)O)NC(=O)[C@H](C(C)(C)C)NC(=O)[C@H](CC(=O)O)N(C)C(=O)[C@@H]7CC(=O)NCCCC[C@@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N7)CC8=CC=CC=C8)CCCNC(=N)N)CCC(=O)O)C(C)C)NC(=O)C)C(=O)O.C(=O)(C(F)(F)F)O

InChI=1S/C172H278N24O55.C2HF3O2/c1-9-10-11-12-13-14-15-16-17-18-19-20-27-44-146(202)182-136(170(226)227)53-56-144(200)177-64-67-229-69-71-231-73-75-233-77-79-235-81-83-237-85-87-239-89-91-241-93-95-243-97-99-245-101-103-247-105-107-249-109-111-251-113-112-250-110-108-248-106-104-246-102-100-244-98-96-242-94-92-240-90-88-238-86-84-236-82-80-234-78-76-232-74-72-230-70-68-228-66-59-145(201)175-60-31-29-41-135(169(224)225)186-165(220)152(126-37-25-22-26-38-126)193-162(217)142-43-34-65-196(142)168(223)140(116-125-47-51-129(199)52-48-125)190-157(212)133(54-57-148(204)205)184-161(216)139(117-127-120-180-154-130(127)39-32-62-178-154)188-159(214)138(115-124-45-49-128(198)50-46-124)189-166(221)153(172(5,6)7)194-163(218)143(119-150(208)209)195(8)167(222)141-118-147(203)176-61-30-28-40-131(181-122(4)197)158(213)192-151(121(2)3)164(219)185-134(55-58-149(206)207)156(211)183-132(42-33-63-179-171(173)174)155(210)187-137(160(215)191-141)114-123-35-23-21-24-36-123;3-2(4,5)1(6)7/h21,23-24,32,35-36,39,45-52,62,120-121,126,131-143,151-153,198-199H,9-20,22,25-31,33-34,37-38,40-44,53-61,63-119H2,1-8H3,(H,175,201)(H,176,203)(H,177,200)(H,178,180)(H,181,197)(H,182,202)(H,183,211)(H,184,216)(H,185,219)(H,186,220)(H,187,210)(H,188,214)(H,189,221)(H,190,212)(H,191,215)(H,192,213)(H,193,217)(H,194,218)(H,204,205)(H,206,207)(H,208,209)(H,224,225)(H,226,227)(H4,173,174,179);(H,6,7)/t131-,132-,133-,134-,135-,136-,137-,138-,139-,140-,141-,142-,143-,151-,152-,153+;/m0./s1

IFJJUMZQZVMOIJ-VTHJDBHLSA-N

(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,5S,8S,11S,14S,22S)-22-acetamido-11-benzyl-8-(3-carbamimidamidopropyl)-5-(2-carboxyethyl)-3,6,9,12,16,23-hexaoxo-2-propan-2-yl-1,4,7,10,13,17-hexazacyclotricosane-14-carbonyl]-methylamino]-3-carboxypropanoyl]amino]-3,3-dimethylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]pyrrolidine-2-carbonyl]amino]-2-cyclohexylacetyl]amino]-6-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(hexadecanoylamino)butanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]hexanoic acid;2,2,2-trifluoroacetic acid

Zilbrysq; ZILUCOPLAN TFA; AT46982

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.)