ACE/angiotensin-converting enzyme

It has been demonstrated that in individuals with hypertension, captopril (SQ 14225) has a similar morbidity and effectiveness to diuretics and beta-blockers. It has been demonstrated that captopril slows the advancement of diabetic nephropathy, but enalapril and lisinopril stop the disease's progression in patients with normoalbuminuric diabetes [4]. The solution contains equimolar ratios of captopril in both its cis and trans states, with the enzyme exclusively choosing the trans form of the compound. The enzyme and its substrate binding base exhibit structural and stereoelectronic complementarity [5].

Captopril, an ACE inhibitor, antagonizes the effect of the RAAS. The RAAS is a homeostatic mechanism for regulating hemodynamics, water and electrolyte balance. During sympathetic stimulation or when renal blood pressure or blood flow is reduced, renin is released from the granular cells of the juxtaglomerular apparatus in the kidneys. In the blood stream, renin cleaves circulating angiotensinogen to ATI, which is subsequently cleaved to ATII by ACE. ATII increases blood pressure using a number of mechanisms. First, it stimulates the secretion of aldosterone from the adrenal cortex. Aldosterone travels to the distal convoluted tubule (DCT) and collecting tubule of nephrons where it increases sodium and water reabsorption by increasing the number of sodium channels and sodium-potassium ATPases on cell membranes. Second, ATII stimulates the secretion of vasopressin (also known as antidiuretic hormone or ADH) from the posterior pituitary gland. ADH stimulates further water reabsorption from the kidneys via insertion of aquaporin-2 channels on the apical surface of cells of the DCT and collecting tubules. Third, ATII increases blood pressure through direct arterial vasoconstriction. Stimulation of the Type 1 ATII receptor on vascular smooth muscle cells leads to a cascade of events resulting in myocyte contraction and vasoconstriction. In addition to these major effects, ATII induces the thirst response via stimulation of hypothalamic neurons. ACE inhibitors inhibit the rapid conversion of ATI to ATII and antagonize RAAS-induced increases in blood pressure. ACE (also known as kininase II) is also involved in the enzymatic deactivation of bradykinin, a vasodilator. Inhibiting the deactivation of bradykinin increases bradykinin levels and may sustain its effects by causing increased vasodilation and decreased blood pressure.

ACE Inhibition Assay[1]
The inhibition of ACE activity by various concentrations of EA and CP as well as their IC50 values were measured using a spectrophotometric method with HHL as substrate as described by Chang et al. with modification. Briefly, a 20 mM sodium borate buffer containing 0.3 M NaCl (pH 8.3) was used for the preparation of EA, CP, ACE, and substrate HHL solutions. The ACE-catalyzed reaction was performed for 30 min at 37 °C in test tubes of the following compositions: 100 μL of EA or CP, 100 μL of ACE solution (40 mU/mL), and 100 μL of HHL (15 mM) solutions (A1); 100 μL of EA or CP solution and 200 μL of borate buffer (A2); 100 μL of borate buffer, 100 μL of ACE solution, and 100 μL of HHL solution (A3); and 300 μL of borate buffer (A4). The enzymatic reaction was stopped by adding 3 mL of alkaline solution of OPA solution (pH 12.0). The absorbance of each reaction was measured at 390 nm using a Beckman DU-640, after incubation for 20 min at 25 °C. Inhibition of ACE by EA or CP was calculated using the following equation: inhibition (%) = [1 – (A1 – A2)/(A3 – A4)] × 100. The IC50 value of ACE activity was calculated by the equation IC50 = (50 – b)/m derived from a linear regression graph of ACE activation, where b is the intercept and m is the slope of the equation.

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Determination of Kinetic Parameters of ACE Inhibition[1]
Kinetic parameters of Vmax and Km values were determined according to the Michaelis–Menten kinetic model. The reaction rate for the formation of l-histidyl-l-leucine from HHL by ACE (40 mU/mL) was determined by the above-mentioned method with EA (0.091 μM) or CP (0.00625 μM) and without EA or CP to get the saturation curves and then plotted against HHL concentrations (0.94, 1.85, 3.75, 7.50, 15 mM). The Lineweaver–Burk plot was derived using the saturation curves to determine the type of inhibition. Kinetic parameters (Km and Vmax) were calculated using MS Excel.

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The inhibitory activity of captopril, a thiol-containing competitive inhibitor of the angiotensin-converting enzyme, ACE, against esterase activity of carbonic anhydrase, CA was investigated. This small molecule, as well as enalapril, was selected in order to represents both thiol and carboxylate, as two well-known metal binding functional groups of metalloprotein inhibitors. Since captopril, has also been observed to inhibit other metalloenzymes such as tyrosinase and metallo-beta lactamase through binding to the catalytic metal ions and regarding CA as a zinc-containing metallo-enzyme, in the current study, we set out to determine whether captopril/enalapril inhibit CA esterase activity of the purified human CA II or not? Then, we revealed the inhibitors' potencies (IC50, Ki and Kdiss values) and also mode of inhibition. Our results also showed that enalapril is more potent CA inhibitor than captopril. Since enalapril represents no sulfhydryl moiety, thus carboxylate group may have a determinant role in inhibiting of CA esterase activity, the conclusion confirmed by molecular docking studies. Additionally, since CA inhibitory potencies of captopril/enalapril were much lower than those of classic sulfonamide drugs, the findings of the current study may explain why these drugs exhibit no effective CA inhibition at the concentrations reached in vivo and also may shed light on the way of generating new class of inhibitors that will discriminately inhibit various CA isoforms[2].

The angiotensin converting enzyme (ACE) inhibitors are widely used in the management of essential hypertension, stable chronic heart failure, myocardial infarction (MI) and diabetic nephropathy. There is an increasing number of new agents to add to the nine ACE inhibitors (benazepril, cilazapril, delapril, fosinopril, lisinopril, pentopril, perindopril, quinapril and ramipril) reviewed in this journal in 1990. The pharmacokinetic properties of five newer ACE inhibitors (trandolapril, moexipril, spirapril, temocapril and imidapril) are reviewed in this update. All of these new agents are characterised by having a carboxyl functional groups and requiring hepatic activation to form pharmacologically active metabolites. They achieve peak plasma concentrations at similar times (t(max)) to those of established agents. Three of these agents (trandolapril, moexipril and imidapril) require dosage reductions in patients with renal impairment. Dosage reductions of moexipril and temocapril are recommended for elderly patients, and dosages of moexipril should be lower in patients who are hepatically impaired. Moexipril should be taken 1 hour before meals, whereas other ACE inhibitors can be taken without regard to meals. The pharmacokinetics of warfarin are not altered by concomitant administration with trandolapril or moexipril. Although imidapril and spirapril have no effect on digoxin pharmacokinetics, the area under the concentration-time curve of imidapril and the peak plasma concentration of the active metabolite imidaprilat are decreased when imidapril is given together with digoxin. Although six ACE inhibitors (captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril) have been approved for use in heart failure by the US Food and Drug Administration, an overview of 32 clinical trials of ACE inhibitors in heart failure showed that no significant heterogeneity in mortality was found among enalapril, ramipril, quinapril, captopril, lisinopril, benazepril, perindopril and cilazapril. Initiation of therapy with captopril, ramipril, and trandolapril at least 3 days after an acute MI resulted in all-cause mortality risk reductions of 18 to 27%. Captopril has been shown to have similar morbidity and mortality benefits to those of diuretics and beta-blockers in hypertensive patients. Captopril has been shown to delay the progression of diabetic nephropathy, and enalapril and lisinopril prevent the development of nephropathy in normoalbuminuric patients with diabetes. ACE inhibitors are generally characterised by flat dose-response curves. Lisinopril is the only ACE inhibitor that exhibits a linear dose-response curve. Despite the fact that most ACE inhibitors are recommended for once-daily administration, only fosinopril, ramipril, and trandolapril have trough-to-peak effect ratios in excess of 50%[5].

Absorption, Distribution and Excretion
60-75% in fasting individuals; food decreases absorption by 25-40% (some evidence indicates that this is not clinically significant)
The drug /captopril/ is metabolized and renally excreted. More than 95% of a dose is excreted renally, both as unchanged (45-50%) drug and as metabolites.
In dogs, approximately 75% of an oral dose is absorbed but food in the GI tract reduces bioavailability by 30-40%. It is distributed to most tissues (not the CNS) and is 40% bound to plasma proteins in dogs.
Approximately 60-75% of an oral dose of captopril is rapidly absorbed from the GI tract in fasting healthy individuals or hypertensive patients. Food may decrease absorption of captopril by up to 25-40%, although there is some evidence that this effect is not clinically important. Following oral administration of a single 100-mg dose of captopril in fasting healthy individuals in one study, average peak blood drug concentrations of 800 ng/mL were attained in 1 hour.
/MILK/ Concentrations of captopril in human milk are approximately one percent of those in maternal blood.
For more Absorption, Distribution and Excretion (Complete) data for Captopril (7 total), please visit the HSDB record page.

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Metabolism / Metabolites
Hepatic. Major metabolites are captopril-cysteine disulfide and the disulfide dimer of captopril. Metabolites may undergo reversible interconversion.
About half the absorbed dose of captopril is rapidly metabolized, mainly to captopril-cysteine disulfide and the disulfide dimer of captopril. In vitro studies suggest that captopril and its metabolites may undergo reversible interconversions. It has been suggested that the drug may be more extensively metabolized in patients with renal impairment than in patients with normal renal function.

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Biological Half-Life
2 hours
A 43 year old patient with mild heart failure attempted suicide by ingesting between 5000 and 7500 mg of captopril. Blood pressure oscillated around 100-120/50-75 mm Hg and pulse rate showed no tendency to accelerate (75-100/min). ... The calculated half-life of captopril was 4.4 hr. ...
The half life of captopril is about 2.8 hours in dogs ... .
The elimination half-life of unchanged captopril appears to be less than 2 hours in patients with normal renal function. The elimination half-life of captopril and its metabolites is correlated with creatinine clearance and increases to about 20-40 hours in patients with creatinine clearances less than 20 mL/minute and as long as 6.5 days in anuric patients.

Toxicity Summary
IDENTIFICATION AND USE: Captopril is angiotensin-converting enzyme (ACE) inhibitor and antihypertensive agent. HUMAN STUDIES: Captopril prevents the conversion of angiotensin I to angiotensin II (a potent vasoconstrictor) by competing with the physiologic substrate (angiotensin I) for the active site of ACE. The affinity of the drug for ACE is approximately 30,000 times greater than that of angiotensin I. Inhibition of ACE results in decreased plasma angiotensin II concentrations and, consequently, blood pressure may be reduced in part through decreased vasoconstriction. Captopril-induced bone marrow suppression is rare, except in certain high-risk patient populations. Severe exfoliative rashes have also been associated with captopril. A 57 year old male with mild impairment of renal function secondary to diabetic glomerulosclerosis developed acute renal failure associated with a generalized desquamative skin rash and peripheral eosinophilia shortly after initiation of antihypertensive therapy with captopril. Cholestatic jaundice is a rare complication associated with the use captopril. The severity of the disease may range from cholestasis on liver histology to overt fulminant hepatic failure. A case of a 75 year old male who committed suicide by taking an overdose of captopril was reported. He took approximately ninety 12.5 mg captopril tablets. Captopril administration over 3 months or more generates red blood cells zinc depletion. Hypogeusia (a reduced ability to taste things) is a reported side effect of captopril. Linkage of hypogeusia to zinc deficiency has been suggested. The fetal toxicity of captopril in the 2nd and 3rd trimesters is similar to other ACE inhibitors. The use of this drug during the 2nd and 3rd trimesters may cause teratogenicity and severe fetal and neonatal toxicity. Fetal toxic effects may include anuria, oligohydramnios, fetal hypocalvaria, intrauterine growth restriction, prematurity, and patent ductus arteriosus. Still birth or neonatal death may occur. Anuria-associated oligohydramnios may product fetal limb contractures, craniofacial deformation, and pulmonary hypoplasia. Sever anuria and hypotension, which is resistant to both pressor agents and volume expansion, may occur in the newborn following in utero exposure. Newborn renal function and blood pressure should be closely monitored. Mutagenicity studies with captopril in fixed combination with hydrochlorothiazide have not been conducted, but the effects of the individual components in a 2:1 ratio have been studied. Captopril/hydrochlorothiazide did not exhibit mutagenic or clastogenic potential in a sister chromatid exchange test in human lymphocytes. In a cytogenetics assay in human lymphocytes exposed to captopril/hydrochlorothiazide concentrations of 5, 25, and 50 ug/mL (total concentration of both drugs) with metabolic activation, chromosomal abnormalities were not observed consistently. When such aberrations were observed, no concentration response was noted. ANIMAL STUDIES: No evidence of carcinogenesis was observed in rats or mice receiving captopril dosages of 50-1350 mg/kg daily for 2 years. Reproduction studies in hamsters and rats using large doses of captopril have not revealed evidence of teratogenic effects. However, the drug was embryocidal and was associated with a low incidence of craniofacial malformations in rabbits, probably as a result of the marked decrease in blood pressure caused by the drug in this species. Reduction in neonatal survival occurred in the offspring of rats receiving captopril continuously during gestation and lactation, and an increased incidence of stillbirths has reportedly occurred in ewes. Mutagenicity studies with captopril in fixed combination with hydrochlorothiazide have not been conducted, but the effects of the individual components in a 2:1 ratio have been studied. Captopril/hydrochlorothiazide did not exhibit mutagenic or clastogenic potential in vitro with or without metabolic activation in a bacterial reverse mutation (Ames) assays using Salmonella, a forward mutation assay in Saccharomyces pombe, and a gene conversion assay using Saccharomyces cerevisiae. Captopril and hydrochlorothiazide in a 2:1 ratio were not genotoxic in the in vivo mouse micronucleus test at an oral dose of 2,500 mg/kg (total concentration of both drugs). ECOTOXICITY STUDIES: Captopril induces oxidative stress in C. carpio.

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Hepatotoxicity
Captopril, like other ACE inhibitors, has been associated with a low rate of serum aminotransferase elevations (
Likelihood score: B (likely but rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Because of the low levels of captopril in breastmilk, amounts ingested by the infant are small and would not be expected to cause any adverse effects in breastfed infants.
◉ Effects in Breastfed Infants
In one report of 12 mothers, several continued to breastfeed their infants while taking captopril 100 mg three times daily. No adverse effects were seen in the infants.[1]
A woman was diagnosed with Cushing's disease during pregnancy. Postpartum she took metyrapone 250 mg 3 times daily, bisoprolol 10 mg twice daily, and captopril 12.5 mg twice daily. She breastfed her preterm infant about 50% milk and 50% formula. At 5 weeks postpartum, the infant's pediatric team found his growth and development to be appropriate.[3]
◉ Effects on Lactation and Breastmilk
In a series of controlled studies reported in one paper, captopril had no effect on the circadian rhythm of prolactin, the response to prolactin-stimulating drugs or serum prolactin in patients with prolactin-secreting tumors.[4]
In a study of young hypertensive males, captopril 25 mg orally markedly decreased serum prolactin at 90 minutes after the dose compared to placebo.[5] The maternal prolactin level in a mother with established lactation may not affect her ability to breastfeed.
In one report, 1 woman out of 12 subjects was unable to produce enough milk for the study while taking captopril 100 mg 3 times daily even though she had been successfully breastfeeding for 6 months.[1] It is not known if this decrease was an effect of captopril.

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Protein Binding
25-30% bound to plasma proteins, primarily albumin

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Interactions
The case of a 70 yr old hypertensive man who presented with a 2 month history of 3 nodular angiomatous lesions on the left arm following treatment with 75 mg/day of oral captopril (Lopril) and 200 mg/day of oral acebutolol hydrochloride (Sectral) for 6 yr is reported. The patient had no history of drug product transfusion, iv drugs, or opportunistic infections. Clinical and histopathological findings were typical of Kaposi's sarcoma. Captopril was stopped, and one month later, the Kaposi's sarcoma lesions had begun to disappear; after 3 months, no lesions were seen. Biopsy showed residual features of Kaposi's sarcoma. However, it is not known whether captopril alone or an interaction with acebutolol hydrochloride resulted in Kaposi's sarcoma.
Concomitant oral administration of captopril and antacids may decrease the rate and extent of GI absorption of captopril. Oral administration of a single, 50-mg dose of captopril 15 minutes after an oral dose of an antacid containing magnesium carbonate and aluminum and magnesium hydroxides resulted in a 40-45% decrease in captopril bioavailability, and a delay and decrease in peak serum concentrations of the drug. However, there is some evidence that this potential interaction may not be clinically important, but additional study is necessary.
Neuropathy reportedly developed in 2 patients receiving captopril and cimetidine. However, further documentation of this potential interaction is necessary.
Initiation of captopril therapy has been associated with unexplained hypoglycemia in several diabetic patients whose diabetes had been controlled with insulin or oral antidiabetic agents. Testing in these patients indicated that captopril may increase insulin sensitivity; the mechanism of this effect is not known. The risk of precipitating hypoglycemia should be considered when captopril therapy is initiated in diabetic patients.
For more Interactions (Complete) data for Captopril (22 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 4245 mg/kg
LD50 Mouse oral 2500 mg/kg
LD50 Mouse iv 663 mg/kg
LD50 Rat iv 554 mg/kg
For more Non-Human Toxicity Values (Complete) data for Captopril (6 total), please visit the HSDB record page.

[1]. Eritadenine from Edible Mushrooms Inhibits Activity of Angiotensin Converting Enzyme in Vitro. J Agric Food Chem. 2016;64(11):2263-2268.

[2]. Captopril/enalapril inhibit promiscuous esterase activity of carbonic anhydrase at micromolar concentrations: An in vitro study. Chem Biol Interact. 2017;265:24-35.

[3]. Simplified captopril analogues as NDM-1 inhibitors. Bioorg Med Chem Lett. 2014;24(1):386-389.

[4]. The molecular basis for the selection of captopril cis and trans conformations by angiotensin I converting enzyme. Bioorg Med Chem Lett, 2006. 16(19): p. 5084-7.

[5]. Song, J.C. and C.M. White, Clinical pharmacokinetics and selective pharmacodynamics of new angiotensin converting enzyme inhibitors: an update. Clin Pharmacokinet, 2002. 41(3): p. 207-24.

Therapeutic Uses
Angiotensin-Converting Enzyme Inhibitors; Antihypertensive Agents
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. Captopril is included in the database.
Captopril tablets are indicated for the treatment of hypertension. ... Captopril tablets are effective alone and in combination with other antihypertensive agents, especially thiazide-type diuretics. The blood pressure lowering effects of captopril and thiazides are approximately additive. /Included in US product label/
Captopril tablets are indicated in the treatment of congestive heart failure usually in combination with diuretics and digitalis. The beneficial effect of captopril in heart failure does not require the presence of digitalis, however, most controlled clinical trial experience with captopril has been in patients receiving digitalis, as well as diuretic treatment. /Included in US product label/
For more Therapeutic Uses (Complete) data for Captopril (11 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ WARNING: FETAL TOXICITY. When pregnancy is detected, discontinue captopril tablets as soon as possible. Drugs that act directly on the renin-angiotensin system can cause injury and death to the developing fetus.
Captopril is generally well tolerated in most patients; however, serious adverse effects (e.g., neutropenia, agranulocytosis, proteinuria, aplastic anemia) have been reported rarely, mainly in patients with renal impairment (especially those with collagen vascular disease). Captopril- induced adverse effects are often alleviated by dosage reduction, occasionally disappear despite continued treatment and without dosage reduction, and are usually reversible following discontinuance of the drug. The most common adverse effects of captopril are rash and loss of taste perception. Adverse effects requiring discontinuance of captopril therapy occur in about 4-12% of patients.
Captopril is contraindicated in patients with known hypersensitivity to the drug or to another angiotension-converting enzyme inhibitor (eg, those who experienced angioedema during therapy with another angiotension-converting enzyme inhibitor).
Patients receiving captopril should be warned not to interrupt or discontinue therapy unless instructed by their physician. Patients with congestive heart failure receiving captopril should be cautioned against rapid increases in physical activity.
For more Drug Warnings (Complete) data for Captopril (32 total), please visit the HSDB record page.

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Pharmacodynamics
Captopril, an ACE inhibitor, antagonizes the effect of the RAAS. The RAAS is a homeostatic mechanism for regulating hemodynamics, water and electrolyte balance. During sympathetic stimulation or when renal blood pressure or blood flow is reduced, renin is released from the granular cells of the juxtaglomerular apparatus in the kidneys. In the blood stream, renin cleaves circulating angiotensinogen to ATI, which is subsequently cleaved to ATII by ACE. ATII increases blood pressure using a number of mechanisms. First, it stimulates the secretion of aldosterone from the adrenal cortex. Aldosterone travels to the distal convoluted tubule (DCT) and collecting tubule of nephrons where it increases sodium and water reabsorption by increasing the number of sodium channels and sodium-potassium ATPases on cell membranes. Second, ATII stimulates the secretion of vasopressin (also known as antidiuretic hormone or ADH) from the posterior pituitary gland. ADH stimulates further water reabsorption from the kidneys via insertion of aquaporin-2 channels on the apical surface of cells of the DCT and collecting tubules. Third, ATII increases blood pressure through direct arterial vasoconstriction. Stimulation of the Type 1 ATII receptor on vascular smooth muscle cells leads to a cascade of events resulting in myocyte contraction and vasoconstriction. In addition to these major effects, ATII induces the thirst response via stimulation of hypothalamic neurons. ACE inhibitors inhibit the rapid conversion of ATI to ATII and antagonize RAAS-induced increases in blood pressure. ACE (also known as kininase II) is also involved in the enzymatic deactivation of bradykinin, a vasodilator. Inhibiting the deactivation of bradykinin increases bradykinin levels and may sustain its effects by causing increased vasodilation and decreased blood pressure.

C9H15NO3S

217.29

217.077

C, 49.75; H, 6.96; N, 6.45; O, 22.09; S, 14.76

62571-86-2

Captopril hydrochloride;198342-23-3;Captopril-d3;1356383-38-4

44093

White to off-white, crystalline powder
Crystals from ethyl acetate/hexane

1.3±0.1 g/cm3

427.0±40.0 °C at 760 mmHg

104-108 °C(lit.)

212.1±27.3 °C

0.0±2.2 mmHg at 25°C

1.551

0.27

2

4

3

14

244

2

S([H])C([H])([H])[C@@]([H])(C([H])([H])[H])C(N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(=O)O[H])=O

FAKRSMQSSFJEIM-BQBZGAKWSA-N

InChI=1S/C9H15NO3S/c1-6(5-14)8(11)10-4-2-3-7(10)9(12)13/h6-7,14H,2-5H2,1H3,(H,12,13)/t6-,7-/m0/s1

(2S)-1-[(2S)-2-methyl-3-sulfanylpropanoyl]pyrrolidine-2-carboxylic acid

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (11.51 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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 2: ≥ 2.5 mg/mL (11.51 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 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 3: ≥ 2.5 mg/mL (11.51 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 32.5 mg/mL (149.57 mM) in PBS (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.)