Immunosuppressant

With an IC50 of 25 nM and at least higher than that of Ppia-/- splenocytes 10 times, cyclosporin (0-2500 nM; Ppia-/- cells) inhibits the proliferation of Ppia+/+ or Ppia+/- splenocytes to a similar extent[1]. Ppia-/-cell gene expression is inhibited by cyclosporine (0-750 nM) [1].

Cyclosporine is an immunosuppressive drug that is widely used to prevent organ transplant rejection. Known intracellular ligands for cyclosporine include the cyclophilins, a large family of phylogenetically conserved proteins that potentially regulate protein folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits calcineurin, a serine/threonine phosphatase that is activated by TCR engagement. Amino acids within the cyclophilins that are critical for binding to cyclosporine have been identified. Most of these residues are highly conserved within the 15 mammalian cyclophilins, suggesting that many are potential targets for the drug. We examined the effects of cyclosporine on immune cells and mice lacking Ppia, the gene encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced proliferation and signal transduction by Ppia(-/-) CD4(+) T cells were resistant to cyclosporine, an effect that was attributable to diminished calcineurin inhibition. Immunosuppressive doses of cyclosporine failed to block the responses of Ppia(-/-) mice to allogeneic challenge. Rag2(-/-) mice reconstituted with Ppia(-/-) splenocytes were also cyclosporine resistant, indicating that this property is intrinsic to Ppia(-/-) immune cells. Thus, among multiple potential ligands, CypA is the primary mediator of immunosuppression by cyclosporine[1].

Absorption, Distribution and Excretion
Cyclosporine is primarily absorbed in the intestine. Individual differences in cyclosporine absorption are significant, with peak bioavailability reaching up to 30%, sometimes occurring 1-8 hours after administration; some patients may experience a second peak. Absorption of cyclosporine in the gastrointestinal tract is incomplete, possibly due to the first-pass effect. Peak plasma concentrations (Cmax) in blood and plasma occur approximately 3.5 hours after administration. A 0.1% cyclosporine ophthalmic emulsion, administered as one drop four times daily, has a peak plasma concentration (Cmax) of 0.67 ng/mL. Note on Absorption Instability: According to Novartis' prescribing information, absorption may be unstable with prolonged use of Santiamine soft capsules and oral solutions. Patients taking soft capsules or oral solutions long-term should have their cyclosporine plasma concentrations monitored regularly and the dosage adjusted accordingly. Compared to other oral formulations of Santiamine, Neoral capsules and solutions exhibit higher absorption rates, resulting in a longer time to peak concentration (Tmax), a higher peak plasma concentration (Cmax) (59% higher), and 29% higher bioavailability. After sulfate conjugation, cyclosporine remains in bile, where it is broken down into its original form and then reabsorbed back into the bloodstream. Cyclosporine is primarily excreted via bile; only 3-6% of the dose (including the original drug and metabolites) is excreted in urine, while 90% of the administered dose is excreted via bile. Of this excretion, less than 1% is excreted unchanged. The distribution of cyclosporine in the blood is as follows: plasma 33%-47%, lymphocytes 4%-9%, granulocytes 5%-12%, and erythrocytes 41%-58%. The reported volume of distribution for cyclosporine is 4-8 L/kg. Due to its high lipophilicity, cyclosporine mainly accumulates in tissues rich in leukocytes and those with high fat content. Ophthalmic drops of cyclosporine can cross the blood-retinal barrier. The clearance of cyclosporine is linear, ranging from 0.38 to 3 L·h/kg, but varies significantly among patients. The clearance of 250 mg of cyclosporine in oral soft capsules (lipid microemulsion form) is approximately 22.5 L/h. Cyclosporine is primarily metabolized in the intestine and liver by CYP450 enzymes, with CYP3A4 playing a major role and CYP3A5 also contributing. The involvement of CYP3A7 is unclear. Cyclosporine has multiple metabolic pathways, and approximately 25 different metabolites have been identified. Some studies suggest that one of its main active metabolites, AM1, has only 10-20% of the activity of the parent drug. The three main metabolites are M1, M9, and M4N, produced by oxidation of the 1-β, 9-γ, and 4-N-demethylation sites, respectively. It is primarily metabolized in the liver via the cytochrome P450 3A enzyme system. Minor metabolism also occurs in the gastrointestinal tract and kidneys. The potency of the metabolites is significantly lower than that of the parent compound. The main metabolites (M1, M9, and M4N) are produced by oxidation of the 1-β, 9-γ, and 4-N-demethylation sites, respectively. Excretion pathway: Primarily excreted via bile; only 6% of the dose (parent drug and its metabolites) is excreted in the urine. Only 0.1% of the dose is excreted unchanged in the urine. Half-life: Biphasic and highly variable, approximately 7 hours (range 7 to 19 hours) in children and approximately 19 hours (range 10 to 27 hours) in adults.

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Biological Half-Life
Cyclosporine has a biphasic half-life, which varies greatly under different conditions, but it has been reported to typically last 19 hours. Prescribing information also indicates that its terminal half-life is approximately 19 hours, but ranges from 10 to 27 hours.

Toxicity Summary
Cyclosporine binds to cyclic proteins. This complex subsequently inhibits calcineurin, which is normally responsible for activating the transcription of interleukin-2. Cyclosporine also inhibits lymphokine production and interleukin release. Its exact mechanism of action in ophthalmic applications is unclear. Cyclosporine emulsions are considered as partial immunomodulators for the treatment of patients with suppressed tear secretion due to ocular inflammation associated with dry keratoconjunctivitis. Toxicity Data
The oral LD50 for mice is 2329 mg/kg, for rats it is 1480 mg/kg, and for rabbits it is >1000 mg/kg. The IV LD50 for mice is 148 mg/kg, for rats it is 104 mg/kg, and for rabbits it is 46 mg/kg.

[1]. Cyclophilin A-deficient mice are resistant to immunosuppression by cyclosporine. J Immunol. 2005 May 15;174(10):6030-8.

Pharmacodynamics
Cyclosporine has a potent immunosuppressive effect on T cells, thereby prolonging survival after organ and bone marrow transplantation. This drug can prevent and control severe immune-mediated reactions, including allogeneic transplant rejection, graft-versus-host disease, and inflammatory autoimmune diseases. Some significant side effects of cyclosporine include hirsutism, gingival hyperplasia, and hyperlipidemia. Furthermore, there is some controversy regarding whether this drug can cause nephrotoxicity.

C62H111N11O12

1202.61

1201.841

79217-60-0

59865-13-3 (Cyclosporine A)

6435893

White to light yellow solid powder

1.0±0.1 g/cm3

1293.8±65.0 °C at 760 mmHg

148-151ºC

736.3±34.3 °C

0.0±0.6 mmHg at 25°C

1.468

3.35

5

12

15

85

2330

2

CCC1C(=O)N(CC(=O)N(C(C(=O)NC(C(=O)N(C(C(=O)NC(C(=O)NC(C(=O)N(C(C(=O)N(C(C(=O)N(C(C(=O)N(C(C(=O)N1)[C@@H]([C@H](C)C/C=C/C)O)C)C(C)C)C)CC(C)C)C)CC(C)C)C)C)C)CC(C)C)C)C(C)C)CC(C)C)C)C

PMATZTZNYRCHOR-KMSBSJHKSA-N

InChI=1S/C62H111N11O12/c1-25-27-28-40(15)52(75)51-56(79)65-43(26-2)58(81)67(18)33-48(74)68(19)44(29-34(3)4)55(78)66-49(38(11)12)61(84)69(20)45(30-35(5)6)54(77)63-41(16)53(76)64-42(17)57(80)70(21)46(31-36(7)8)59(82)71(22)47(32-37(9)10)60(83)72(23)50(39(13)14)62(85)73(51)24/h25,27,34-47,49-52,75H,26,28-33H2,1-24H3,(H,63,77)(H,64,76)(H,65,79)(H,66,78)/b27-25+/t40-,41?,42?,43?,44?,45?,46?,47?,49?,50?,51?,52-/m1/s1

30-ethyl-33-[(E,1R,2R)-1-hydroxy-2-methylhex-4-enyl]-1,4,7,10,12,15,19,25,28-nonamethyl-6,9,18,24-tetrakis(2-methylpropyl)-3,21-di(propan-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecone

cyclosporine; 79217-60-0; Restasis; SangCyA; 30-ethyl-33-[(E,1R,2R)-1-hydroxy-2-methylhex-4-enyl]-1,4,7,10,12,15,19,25,28-nonamethyl-6,9,18,24-tetrakis(2-methylpropyl)-3,21-di(propan-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecone; SCHEMBL4331439; SCHEMBL4454089; PMATZTZNYRCHOR-KMSBSJHKSA-N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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.)