Cyclophilin D; phosphatase activity of protein phosphatase 2B (PP2B/calcineurin)

In T cells, cyclophilin and cyclosporin A can bind [1]. By creating the Cyclophilin-Cyclosporin A complex, cyclosporin A inhibits calcineurin [2]. With an IC50 value of 7 nM, cyclosporin A inhibits calcineurin in activated cells [3]. Cyclosporin A prevents NF-AT from moving to the nucleus [4]. With an IC50 of 39 nM, cyclosporin A inhibits mitochondrial MTP opening [5].

When administered parenterally or orally to mice, rats, and guinea pigs, cyclosporine A exerts immunosuppressive effects [6]. In organ transplantation, cyclosporine A can be given to stop organ rejection [7].

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The mouse model of pleurisy induced by carrageenan is characterized by a significant enhancement of cell migration due to neutrophils 4 h after pleurisy induction. Forty-eight hours after pleurisy induction, a significant increase in cell migration due to mononuclear cells occurs. Recently, studies in our laboratory have demonstrated that cyclosporine A (CsA) inhibits leukocyte migration in the pleural cavity and lungs in the mouse model of pleurisy induced by carrageenan. In the present work we evaluated whether CsA was able to downregulate CD11a/CD18 adhesion molecule in the lungs, as well as TNFalpha and IL-1 beta levels in the fluid leakage of the pleural cavity in this model. Our results showed that CsA significantly decreased CD11a/CD18 in the lungs, as well as TNFalpha and IL-1 beta levels in the fluid leakage of the pleural cavity 4 h and 48 h after pleurisy induction. It is our hypothesis that the inhibitory effect elicited by CsA upon these adhesion molecules may be also be attributed to the downregulation of TNFalpha and IL-1 beta cytokines[8].

Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 X 10(-7) moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH2-terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56 degrees C and at pH 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism[1].

The immunosuppressive agents cyclosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilins and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cyclophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation[3].

Experimental protocol.
Pleurisy caused by carrageenan (0.1%, i.p.)33 exhibits a biphasic response (4 h and 48 h). Thus, both interval-points were chosen to analyse the CD11a/CD18 adhesion molecule in the lungs, as well as TNFα and IL-1β levels in the fluid leakage of the pleural cavity. Doses of cyclosporin A were chosen as previously established.22,23 Briefly, the doses of 1 mg/kg and 2 mg/kg (1 h and 0.5 h before) of cyclosporine (i.p.) were effective in significantly inhibiting neutrophil and mononuclear influxes to the pleural cavity, 4 h and 48 h after pleurisy induction.

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Dissolved in 0.5% solution of tragacanth; 45 mg/kg; oral gavage

Rat

Absorption, Distribution and Excretion
The absorption of cyclosporine occurs mainly in the intestine. Absorption of cyclosporine is highly variable with a peak bioavailability of 30% sometimes occurring 1-8 hours after administration with a second peak observed in certain patients. The absorption of cyclosporine from the GI tract has been found to be incomplete, likely due to first pass effects. Cmax in both the blood and plasma occurs at approximately 3.5 hours post-dose. The Cmax of a 0.1% cyclosporine ophthalmic emulsion is 0.67 ng/mL after instilling one drop four times daily. A note on erratic absorption During chronic administration, the absorption of Sandimmune Soft Gelatin Capsules and Oral Solution have been observed to be erratic, according to Novartis prescribing information. Those being administered the soft gelatin capsules or oral solution over the long term should be regularly monitored by testing cyclosporine blood concentrations and adjusting the dose accordingly. When compared with the other oral forms of Sandimmune, Neoral capsules and solution have a higher rate of absorption that results in a higher Tmax and a 59% higher Cmax with a 29 % higher bioavailability.
After sulfate conjugation, cyclosporine remains in the bile where it is broken down to the original compound and then re-absorbed into the circulation. Cyclosporine excretion is primarily biliary with only 3-6% of the dose (including the parent drug and metabolites) excreted in the urine while 90% of the administered dose is eliminated in the bile. From the excreted proportion, under 1% of the dose is excreted as unchanged cyclosporine.
The distribution of cyclosporine in the blood consists of 33%-47% in plasma, 4%-9% in the lymphocytes, 5%-12% in the granulocytes, and 41%-58% in the erythrocytes. The reported volume of distribution of cyclosporine ranges from 4-8 L/kg. It concentrates mainly in leucocyte-rich tissues as well as tissues that contain high amounts of fat because it is highly lipophilic. Cyclosporine, in the eye drop formulation, crosses the blood-retinal barrier.
Cyclosporin shows a linear clearance profile that ranges from 0.38 to 3 Lxh/kg, however, there is substantial inter- patient variability. A 250 mg dose of cyclosporine in the oral soft gelatin capsule of a lipid micro-emulsion formulation shows an approximate clearance of 22.5 L/h.
Following oral admin of cyclosporine, the time to peak blood concns is 1.5-2.0 hr. Admin with food both delays & decreases absorption. High & low fat meals consumed within 30 min of admin decr the AUC by approx 13% & the max concn by 33%. This makes it imperative to individualize dosage regimens for outpatients. Cyclosporine is distributed extensively outside the vascular compartment. After iv dosing, the steady-state volume of distribution has been reported to be as high as 3-5 liters/kg in solid-organ transplant recipients. Only 0.1% of cyclosporine is excreted unchanged in urine. ... Cyclosporine & its metabolites are excreted principally through the bile into the feces, with only approx 6% being excreted in the urine. Cyclosporine also is excreted in human milk.
... Absorption of cyclosporine is incomplete following oral admin. The extent of absorption depends upon several variables, including the individual patient & formulation used. The elimination of cyclosporine form the blood is generally biphasic, with a terminal half-life of 5-18 hr. After iv infusion, clearance is approx 5-7 ml/min/kg in adult recipients of renal transplants, but results differ by age & patient populations. For example, clearance is slower in cardiac transplant patients & more rapid in children. The relationship between admin dose & the area under the plasma concn-vs-time curve is linear within the therapeutic range, but the intersubject variability is so large that individual monitoring is required.
Clinicians can administer cyclosporine by continuous iv infusion during the first few days after transplantation, then orally by twice-daily doses, to achieve plasma cyclosporine concns (measured by HPLC) of 75-150 ng/ml (equivalent to whole blood cyclosporine concns of 300-600 ng/ml measured by radioimmunoassay). It appears safe to maintain a trough plasma cyclosporine concn of about 75-150 ng/ml; however, this does not necessarily guarantee safety from nephrotoxicity. Because of preferential distribution of cyclosporine & its metabolites into red blood cells, blood levels are generally higher than plasma levels. When blood cyclosporine levels are 300-600 ng/ml by radioimmunoassay, cerebrospinal fluid levels range from 10-50 ng/ml. The apparent volume of distribution in children under 10 yr of age is about 35 l/kg, & in adults, 4.7 l/kg.
The elimination half-life of an oral cyclosporine dose of 350 mg is 8.9 hr; after a 1400 mg dose, the half-life is 11.9 hr. Elimination occurs predominantly by metab in the liver to form 18-25 metabolites. Metabolites of cyclosporine possess little immunosuppressive activity. Cyclosporine is extensively metabolized in the liver by cytochrome P450IIIA oxidase; however, neurotoxicity & possibly nephrotoxoicity usually correlate with raised blood levels of cyclosporine metabolites. Only 0.1% of a dose ix s excreted unchanged.
For more Absorption, Distribution and Excretion (Complete) data for CYCLOSPORIN A (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Cyclosporine is metabolized in the intestine and the liver by CYP450 enzymes, predominantly CYP3A4 with contributions from CYP3A5. The involvement of CYP3A7 is not clearly established. Cyclosporine undergoes several metabolic pathways and about 25 different metabolites have been identified. One of its main active metabolites, AM1, demonstrates only 10-20% activity when compared to the parent drug, according to some studies. The 3 primary metabolites are M1, M9, and M4N, which are produced from oxidation at the 1-beta, 9-gamma, and 4-N-demethylated positions, respectively.
Cyclosporine is extensively metabolized in the liver by the cytochrome-P450 3A (CYP3A) enzyme system & to a lesser degree by the GI tract & kidneys. At least 25 metabolites have been identified in human bile, feces, blood, & urine. Although the cyclic peptide structure of cyclosporine is relatively resistant to metab, the side chains are extensively metabolized. All of the metabolites have both reduced biological activity & toxicity compared to the parent drug.

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Biological Half-Life
The half-life of cyclosporine is biphasic and very variable on different conditions but it is reported in general to last 19 hours. Prescribing information also states a terminal half-life of approximately 19 hours, but with a range between 10 to 27 hours.

Interactions
Cyclosporine interacts with a wide variety of commonly used drugs, & close attention must be paid to drug interactions. Any drug that affects microsomal enzymes, especially the CYP3A system, may affect cyclosporine blood concns. Substances that inhibit this enzyme can decr cyclosporine metab & incr blood concns. These include calcium channel blockers (e.g., verapamil, nicardipine), antifungal agents (e.g., fluconazole, ketoconazole), antibiotics (e.g., erythromycin), glucocorticoids (e.g., methylprednisolone), HIV-protease inhibitors (e.g., indinavir), & other drugs (e.g., allupurinol & metoclopramide). In addition, grapefruit & grapefruit juice block the CYP3A system & incr cyclosporine blood concns & thus should be avoided by patients receiving the drug. In contrast, drugs that induce CYP3A activity can incr cyclosporine metab & decr blood concns. Drugs that can decr cyclosporine concns in this manner include antibiotics (e.g., nafcillin & refampin), anticonvulsants (e.g., phenobarbital, phenytoin), & other drugs (e.g., octreotide, ticlopidine). In general, close monitoring of cyclosporine blood levels & the levels of other drugs is required when such combinations are used. Interactions between cyclosporine & sirolimus have led to the recommendation that admin of the two drugs be separated by time. Sirolimus aggravate cyclosporine-induced renal dysfunction, while cyclosporine increases sirolimus-induced hyperlipemia & myelosuppression. Other cyclosporine-drug interactions of concern include additive nephrotoxicity when coadministered with nonsteroidal antiinflammatory drugs & other drugs that cause renal dysfunction; elevation in methotrexate levels when the two drugs are coadministered; & reduced clearance of other drugs, including prednisolone, digoxin, & lovastatin.
Cyclosporine increases the volume of distribution, half-life, & renally eliminated fraction of digoxin. Cyclosporine potentiates vecuronium blockade & prolongs recovery time. Drugs with clinically established effects on cyclosporine metab are/: calcium channel blockers, Diltiazepam, Nifedipine, Verapamil, Ceftriaxone, Erythromycin, Norfloxacin, Ketoconazole, Fluconazole, Ciprofloxacin, Josamycin, Methyltestosterone, Omeprazole, Sulindac, Sex hormones, Corticosteroids, Metolazone, Acetazolamide, Alcohol, Cimetidine, Danazol, Imipenem/cilastin, Itraconazole, Oral contraceptives, Pristinamycin- Incr blood cyclosporine levels; Sulfadimidine, Phenytoin, Phenobarbital, Primidone, Carbamazepine, Rifampin, Ethambutol, Isoniazid, Quinine, Griseofulvin, Rifamycin, Warfarin, Chlorambucil- Decr blood cyclosporine levels/. /From table/
/Concurrent use with allopurinol, androgens, bromocriptine, cimetidine, clarithromycin, danazol, diltiazem, erythromycin, estrogens, fluconazole, HIV protease inhibitors, itraconazole, ketoconazole, metoclopramide, miconazole, nefazodone, nicardipine, verapamil/ may increase blood concentrations of cyclosporine by inhibiting cytochrome p450 3A enzymes, and may increase the risk of hepatotoxicity and nephrotoxicity; because of its similarity to ketoconazole, miconazole may be expected to have the same effect; although concurrent use of HIV protease inhibitors and cyclosporine have not been studied, HIV protease inhibitors are known to inhibit cytochrome p450 3A enzymes; frequent monitoring of blood cyclosporine concentrations and hepatic and renal function may be needed if these drugs are used concurrently with cyclosporine.
Concurrent use of NSAIDs, especially indomethacin, with cyclosporine may increase the risk of renal failure; concurrent administration with cyclosporine may also result in hyperkalemia; additive decreases in renal function have been reported with concurrent use of diclofenac or naproxen with cyclosporine.
For more Interactions (Complete) data for CYCLOSPORIN A (20 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 1489 mg/kg
LD50 Rat ip 147 mg/kg
LD50 Rat sc 286 mg/kg
LD50 Rat iv 24 mg/kg
For more Non-Human Toxicity Values (Complete) data for CYCLOSPORIN A (7 total), please visit the HSDB record page.

[1]. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science. 1984 Nov 2;226(4674):544-7.

[2]. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991 Aug 23;66(4):807-15.

[3]. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3686-90.

[4]. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature. 1991 Aug 29;352(6338):803-7.

[5]. Interactions of cyclophilin with the mitochondrial inner membrane and regulation of the permeability transition pore, and cyclosporin A-sensitive channel. J Biol Chem. 1996 Jan 26;271(4):2185-92.

[6]. Effects of the new anti-lymphocytic peptide cyclosporin A in animals. Immunology. 1977 Jun;32(6):1017-25.

[7]. Randomised trial comparing FK506 and cyclosporin in prevention of liver allograft rejection. European FK506 Multicentre Liver Study Group. Lancet, 1994, 344(8920), 423-428.

[8]. Cyclosporin A inhibits CD11a/CD18 adhesion molecules due to inhibition of TNFalpha and IL-1 beta levels in the mouse model of pleurisy induced by carrageenan. Cell Adh Migr. 2008 Oct-Dec;2(4):231-5.

[9]. Increased cyclosporine bioavailability induced by experimental nephrotic syndrome in rats. Can J Physiol Pharmacol. 2007 May;85(5):502-6.

[10]. Lysyl oxidase inhibitors attenuate cyclosporin A-induced nephropathy in mouse. Sci Rep. 2021 Jun 14;11(1):12437.

Therapeutic Uses
Clinical indications for cyclosporine are kidney, liver, heart, & other organ transplantation; rheumatoid arthritis; & psoriasis. ... Cyclosporine usually is used in combination with other agents, especially glucocorticoids & either azathioprine or mycophenolate mofetil &, most recently, sirolimus. ... In rheumatoid arthritis, cyclosporine is used in cases of severe disease that have not responded to methotrexate. Cyclosporine can be used in combination with methotrexate, but the levels of both drugs must be monitored closely. In psoriasis, cyclosporine is indicated for treatment of adult nonimmunocompromised patients with severe & disabling disease who have failed other systemic therapies. Because of its mechanism of action, there is a theoretical bases for the use of cyclosporine in a variety of other T-cell-mediated diseases. Cyclosporine has been reported to be effective in Behcet's acute ocular syndrome, endogenous uveitis, atopic dermatitis, inflammatory bowel disease, & nephrotic syndrome when standard therapies have failed.
For prevention of allograft rejection in adults and children ... .
Cyclosporine is indicated, usually in combination with corticosteroids, for prevention of rejection of renal, hepatic, and cardiac transplants (allografts). /Included in US product labeling/
Cyclosporine is also indicated for prevention of rejection of heart-lung and pancreatic transplants. /NOT included in US product labeling/
For more Therapeutic Uses (Complete) data for CYCLOSPORIN A (13 total), please visit the HSDB record page.

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Drug Warnings
Non-PVC containers & administration sets should be used to administer cyclosporine solns. ... Use of glass containers & tubing that does not contain DEHP to administer cyclosporine was recommended.
Cyclosporine is distributed into breast milk. Mothers taking cyclosporine should not breast-feed their babies, because of the potential risk of serious adverse effects (e.g., hypertension, nephrotoxicity, malignancy) in the infant.
Appropriate studies performed to date in pediatric patients receiving cyclosporine for organ transplantation have not demonstrated pediatrics-specific problems that would limit the usefulness of cyclosporine in children. Cyclosporine has been used in pediatric patients 1 year of age and older receiving organ transplantations. Pediatric patients have increased clearance of cyclosporine as compared with adult patients. The safety and efficacy of cyclosporine to treat psoriasis and rheumatoid arthritis in pediatric patients have not been established.
Geriatric patients were included in the clinical trials of cyclosporine to treat rheumatoid arthritis. Geriatric patients were more likely to experience hypertension and increases in serum creatinine concentrations than were younger adult patients.
For more Drug Warnings (Complete) data for CYCLOSPORIN A (30 total), please visit the HSDB record page.

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Pharmacodynamics
Cyclosporine exerts potent immunosuppressive actions on T cells, thereby prolonging survival following organ and bone marrow transplants. This drug prevents and controls serious immune-mediated reactions including allograft rejection, graft versus host disease, and inflammatory autoimmune disease. Some notable effects of cyclosporine are hypertrichosis, gingival hyperplasia, and hyperlipidemia. There is also some debate about this drug causing nephrotoxicity.

C62H111N11O12

1202.61

1201.841

C, 61.92; H, 9.30; N, 12.81; O, 15.96

59865-13-3

Cyclosporin A acetate-d4;Cyclosporin A-13C2,d4;Cyclosporin A-d4;Cyclosporin A-d3;222295-76-3

5284373

Forms white prismatic crystals from acetone

1.0±0.1 g/cm3

1293.8±65.0 °C at 760 mmHg

148-151°C

736.3±34.3 °C

0.0±0.6 mmHg at 25°C

1.468

3.35

5

12

15

85

2330

12

O([H])[C@]([H])([C@]([H])(C([H])([H])[H])C([H])([H])/C(/[H])=C(\[H])/C([H])([H])[H])[C@@]1([H])C(N([H])[C@]([H])(C(N(C([H])([H])[H])C([H])([H])C(N(C([H])([H])[H])[C@]([H])(C(N([H])[C@]([H])(C(N(C([H])([H])[H])[C@]([H])(C(N([H])[C@@]([H])(C([H])([H])[H])C(N([H])[C@]([H])(C([H])([H])[H])C(N(C([H])([H])[H])[C@@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])C(N(C([H])([H])[H])[C@]([H])(C(N(C([H])([H])[H])[C@]([H])(C(N1C([H])([H])[H])=O)C([H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O)=O)=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O)C([H])([H])C([H])([H])[H])=O

PMATZTZNYRCHOR-CGLBZJNRSA-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

(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-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

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)

Solubility in Formulation 1: 2.62 mg/mL (2.18 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (1.73 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 2.08 mg/mL (1.73 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (1.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 2% DMSO +30%PEG 300 +5% Tween 80 +ddH2O: 5mg/mL

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Solubility in Formulation 6: 20 mg/mL (16.63 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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