Complement component 5 (C5)

With an IC50 value of 474.5 pM, zilucoplan (RA101495; 1-1000 nM; 30 min) inhibits the increase in C5a plasma levels in human whole blood caused by lipopolysaccharides. When zilucoplan is used at a concentration of 1 nM, C5a plasma levels are reduced by 65.7% [2]. By preventing C5 from being broken down by C5 convertase into C5a and C5b and attaching to preformed C5b to sterically block interaction with C6, zilucoplan binds to complement component 5 (C5) and prevents the downstream assembly of the membrane attack complex (MAC; C5b-9)[1].

In C5-deficient mice treated with human complement, zilucoplan (RA101495; 10 mg/kg; SC; daily, for 6 d) inhibits the development of immune-mediated necrotizing myopathy (IMNM)[1]. In C57BL/6 mice, zilucoplan (10 mg/kg; SC; daily, for 6 d) shows protective effects on the prevention of myopathy[1].

Animal/Disease Models: C57BL/10SnJ C5-deficient (C5def) mice with anti-HMGCR+ IMNM IgG xenografts[1]
Doses: 10 mg/kg
Route of Administration: subcutaneous (sc) injection; daily, for 6 days
Experimental Results: Prevented muscle strength loss in C5def mice with less complement deposition on myofibres and consequently less necrosis/regeneration.

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Animal/Disease Models: C57BL/6 mice with anti-HMGCR+ IMNM IgG xenografts[1]
Doses: 10 mg/kg
Route of Administration: subcutaneous (sc) injection; daily, for 6 days
Experimental Results: Prevented muscle weakness and decreased regenerated myofibres. diminished necrotic cells as well as regenerating cells expressing foetal myosin.

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.

Zilucoplan is a synthetic macrocyclic peptide composed of 15 amino acids, with the molecular formula C172H278N24O55. Its sodium salt is used to treat adult generalized myasthenia gravis (a disease that causes muscle weakness and fatigue), in which the immune system produces antibodies against acetylcholine receptors. It is a complement component 5 inhibitor and an immunosuppressant. It is a macrocyclic compound, homocyclic peptide, and polyether. It is the conjugate acid of zilucoplan(4-). Zilucoplan is a synthetic macrocyclic peptide composed of 15 amino acids. It is a complement inhibitor that blocks the activation of C5, a complement protein involved in initiating an inflammatory response in the innate immune system. On October 17, 2023, zilucoplan received its first FDA approval for the treatment of generalized myasthenia gravis. On December 4, 2023, the European Medicines Agency (EMA) approved zilucoplan as adjunctive therapy for the same disease. Zilucoplan is a complement inhibitor. The mechanism of action of zilucoplan is as a complement inhibitor. Zilucoplan is a synthetic macrocyclic peptide that inhibits terminal complement protein C5, exhibiting potential anti-inflammatory and cytoprotective effects. After subcutaneous injection, the complement inhibitor Zilucoplan binds to a unique site on terminal complement protein C5, thereby blocking the cleavage of C5 into C5a and C5b and inhibiting the assembly of the C5b-dependent membrane attack complex (MAC). Zilucoplan also inhibits the interaction between C5b and C6, further blocking MAC assembly. This can prevent MAC-mediated erythrocyte lysis and destruction that occurs in complement-mediated diseases such as paroxysmal nocturnal hemoglobinuria (PNH), generalized myasthenia gravis (gMG), and lupus nephritis (LN). C5 is a complement pathway protein highly expressed in the liver. Drug Indications Zilucoplan is indicated for adult patients with generalized myasthenia gravis (gMG) as approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), as a primary treatment or adjunctive therapy to standard treatment, for patients with positive anti-acetylcholine receptor (AChR) antibodies. Treatment of Myasthenia Gravis Mechanism of Action The complement system is part of the innate immune system and plays a crucial role in responding to inflammatory responses to pathogens. Activation pathways of the complement system include the cleavage of complement protein C5 by C5 convertase, generating potent anaphylatoxins C5a and C5b. C5 cleavage also recruits C6, C7, C8, and C9. C5b binds to C6 to form the terminal complement complex C5b9, a hydrophilic pore that spans the cell membrane. C5b9 leads to the influx of water and ions, ultimately resulting in osmotic lysis of target cells. The terminal complement cascade is closely associated with the pathophysiology of a variety of inflammatory and autoimmune diseases, including myasthenia gravis. Myasthenia gravis is an autoimmune disease characterized by the presence of pathogenic autoantibodies that bind to the acetylcholine receptor (AChR). The membrane attack complex (MAC) that accumulates on the postsynaptic plasma membrane of the neuromuscular junction leads to muscle weakness and damage. The exact mechanism by which zilucoplan treats myasthenia gravis is not fully elucidated. Zilucoplan binds with high affinity to complement protein C5, inhibiting its cleavage into C5a and C5b, thereby preventing the formation of C5b9.

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Pharmacodynamics
In clinical trials involving adult patients with anti-AChR antibody-positive myasthenia gravis (gMG), zilucoplan improved the signs and symptoms of myasthenia gravis, including muscle weakness. In vitro studies showed that it blocks the activation of clinical variants of C5.
Zilucoplan inhibits complement in a dose-dependent manner: in clinical trials, zilucoplan achieved a complement inhibition rate of 97.5% by the end of the first week and maintained it throughout the 12-week treatment period.

C172H278N24O55

3562.17557096481

3560.972

1841136-73-9

Zilucoplan TFA

133083018

White to off-white solid powder

4.8

28

57

142

251

6980

16

O=C([C@H](C1CCCCC1)NC([C@@H]1CCCN1C([C@H](CC1C=CC(=CC=1)O)NC([C@H](CCC(=O)O)NC([C@H](CC1=CNC2C1=CC=CN=2)NC([C@H](CC1C=CC(=CC=1)O)NC([C@H](C(C)(C)C)NC([C@H](CC(=O)O)N(C)C([C@@H]1CC(NCCCC[C@@H](C(N[C@H](C(N[C@@H](CCC(=O)O)C(N[C@@H](CCCNC(=N)N)C(N[C@@H](CC2C=CC=CC=2)C(N1)=O)=O)=O)=O)C(C)C)=O)NC(C)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)N[C@H](C(=O)O)CCCCNC(CCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCNC(CC[C@@H](C(=O)O)NC(CCCCCCCCCCCCCCC)=O)=O)=O

JDXCOXKBIGBZSK-PSNKNOTQSA-N

InChI=1S/C172H278N24O55/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/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)/t131-,132-,133-,134-,135-,136-,137-,138-,139-,140-,141-,142-,143-,151-,152-,153+/m0/s1

(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

Zilbrysq; Zilucoplan; 1841136-73-9; YG391PK0CC; zilucoplanum; RA101495;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO: ~100 mg/mL (28.1 mM)

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.)