Dehydropeptidase; leukotriene D4 dipeptidase
The primary target of Cilastatin sodium is dehydropeptidase I (DPH-I), a renal enzyme that metabolizes imipenem. By inhibiting this enzyme, cilastatin prevents the degradation of imipenem in the kidneys, increasing its urinary concentration and prolonging its half-life. This allows imipenem to be used effectively for the treatment of urinary tract infections and other serious infections.

Without lessening the antimicrobial effect of Vancomycin, ciplastatin (200 μg/mL; 24 hours; RPTECs) treatment prevents Vancomycin-induced proximal tubule apoptosis and boosts cell viability[2].
In vitro, cilastatin sodium has been shown to inhibit dehydropeptidase I activity. The compound's ability to inhibit the enzyme has been confirmed in enzymatic assays using purified DPH-I or kidney homogenates. The compound does not have direct antibacterial activity but enhances the efficacy of imipenem.

Ipenem and cilastatin together protected mice against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa infection in a mouse model of systemic infection (female mice, CD-1 strain, 20 g) [3].
In vivo, cilastatin sodium is used in combination with imipenem for the treatment of serious infections. The combination is effective against a wide range of Gram-positive and Gram-negative bacteria, including anaerobes. Cilastatin prevents the renal metabolism of imipenem, allowing for effective treatment of infections, including those of the urinary tract.

In vitro enzyme or receptor binding (non-cell) assays for cilastatin sodium involve measuring its inhibition of dehydropeptidase I activity. The assay uses a purified DPH-I enzyme or kidney homogenates and a substrate, such as imipenem or a synthetic substrate. The enzymatic activity is monitored by measuring the degradation of the substrate. The compound is incubated with the enzyme and substrate at varying concentrations, and the IC50 is determined.

Cell Line: Renal proximal tubular epithelial cells (RPTECs)
Concentration: 200 μg/mL
Incubation Time: 24 hours
Result: Significantly ameliorated Vancomycin-induced nuclear apoptosis.
In vitro cell-based assays for cilastatin sodium are not typically performed, as it does not have direct antibacterial activity. Its effects are evaluated in combination with imipenem using standard antimicrobial susceptibility testing methods. Bacterial cultures are grown in the presence of imipenem and cilastatin, and the minimum inhibitory concentration (MIC) is determined.

In vivo animal experiments for cilastatin sodium are conducted using animal models of infection. The compound is administered intravenously in combination with imipenem, and its efficacy is evaluated by measuring the reduction in bacterial load in tissues. The compound's effects on the pharmacokinetics of imipenem are also assessed.

Absorption, Distribution and Excretion
According to the official label of the U.S. Food and Drug Administration (FDA), approximately 70% of cilastatin is excreted in the urine, but published literature reports excretion rates as high as 98%. The volume of distribution of cilastatin is 14.6–20.1 liters. The total clearance of cilastatin is 0.2 liters/hour/kg, and the renal clearance is 0.10–0.16 liters/hour/kg. Biological Half-Life The half-life of cilastatin is approximately 1 hour.
Pharmacokinetic (PK) properties of cilastatin sodium indicate that it is administered intravenously in combination with imipenem. The compound has a molecular weight of 380.39 and a molecular formula of C16H25N2NaO5S. It is a solid at room temperature. Cilastatin is eliminated renally, and its half-life is similar to that of imipenem. Dose adjustments are required in patients with renal impairment.

Protein Binding
It has been reported that cilastatin has a plasma protein binding rate of 35-40%.
Toxicology (toxicology) data for cilastatin sodium indicate that it is generally well-tolerated. Common side effects include nausea, vomiting, and diarrhea. The compound may also cause hypersensitivity reactions, including rash and anaphylaxis. It should be used with caution in patients with a history of hypersensitivity to penicillins or other beta-lactam antibiotics.

[1]. Keynan S, et al. The renal membrane dipeptidase (dehydropeptidase I) inhibitor, cilastatin, inhibits the bacterialmetallo-beta-lactamase enzyme CphA. Antimicrob Agents Chemother. 1995 Jul;39(7):1629-31.
[2]. S Keynan, et al. The Renal Membrane Dipeptidase (Dehydropeptidase I) Inhibitor, Cilastatin, Inhibits the Bacterial Metallo-Beta-Lactamase Enzyme CphA. Antimicrob Agents Chemother. 1995 Jul;39(7):1629-31.
[3]. Blanca Humanes, et al. Protective Effects of Cilastatin Against Vancomycin-Induced Nephrotoxicity. Biomed Res Int. 2015;2015:704382.
[4]. P J Petersen, et al. In Vitro and in Vivo Activities of LJC10,627, a New Carbapenem With Stability to Dehydropeptidase I. Antimicrob Agents Chemother. 1991 Jan;35(1):203-7.

Cilastatin is a thioether formed by the oxidative coupling reaction of the sulfhydryl group of L-cysteine with the 7-position of (2Z)-2-({[(1S)-2,2-dimethylcyclopropyl]carbonyl}amino)hept-2-enoic acid. It is an inhibitor of dehydropeptidase I (membrane dipeptidase, 3.4.13.19), an enzyme located at the brush border of the renal tubules responsible for degrading the antibiotic imipenem. Therefore, cilastatin (in its sodium form) in combination with imipenem prolongs the antibacterial activity of imipenem by preventing its metabolism in the kidneys to inactive and potentially nephrotoxic products. Cilastatin also acts as a leukotriene D4 dipeptidase inhibitor, preventing the metabolism of leukotriene D4 to leukotriene E4. It is a protease inhibitor, EC 3.4.13.19 (membrane dipeptidase) inhibitor, exogenous substance, and environmental pollutant. It is a non-protein L-α-amino acid, L-cysteine derivative, organosulfur compound, and carboxamide. It is the conjugate acid of cilastatin (1-). Cilastatin is an inhibitor of renal dehydropeptidase, an enzyme responsible for the metabolism of thiamethoxam β-lactam antibiotics and the conversion of leukotriene D4 to leukotriene E4. Since the antibiotic imipenem is one of the antibiotics that can be hydrolyzed by dehydropeptidase, cilastatin is used in combination with imipenem to inhibit its metabolism. The first combination formulation containing these two drugs was approved by the FDA in November 1985 under the brand name Primaxin, marketed by Merck. A newer triplet formulation was approved in July 2019 under the brand name Recarbrio, which also contains [relebactam]. Cilastatin is a renal dehydropeptidase inhibitor. The mechanism of action of cilastatin is as a dipeptidase inhibitor. Cilastatin has been reported to be used in cattle and honeybees, and relevant data are available.
Cilastatin is a renal dehydropeptidase-1 and leukotriene D4 dipeptidase inhibitor. Because the antibiotic imipenem is hydrolyzed by dehydropeptidase-1 located at the brush border of the renal tubules, cilastatin is often used in combination with imipenem to enhance its efficacy. This drug also inhibits the metabolism of leukotriene D4 to leukotriene E4.
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Drug Indications

Cilastatin can be used in combination with imipenem, alone or without rebactam, to treat bacterial infections, including respiratory, skin, bone, gynecological, urinary tract, and intra-abdominal infections, as well as sepsis and endocarditis.
FDA Label
Mechanism of Action

Cilastatin is a renal dehydropeptidase-1 inhibitor. Because the antibiotic imipenem is hydrolyzed by dehydropeptidase-1 located at the brush border of the renal tubules, cilastatin is used in combination with imipenem to block the metabolism of imipenem.

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Pharmacodynamics
Cilastatin is a compound that inhibits human dehydropeptidase. Renal dehydropeptidase degrades the antibiotic imipenem. Therefore, cilastatin must be used in combination with intravenous imipenem to protect it from dehydropeptidase degradation and prolong its antibacterial effect. However, cilastatin itself does not have antibacterial activity. Increasing renal excretion of unmetabolized imipenem appears to prevent proximal tubular necrosis induced by high doses of imipenem.

Other information: Cilastatin sodium is a renal dehydropeptidase I inhibitor used in combination with imipenem. It is also known as MK-0791. The compound is available as a sterile powder for injection in combination with imipenem. Its CAS number is 81129-83-1.

C16H25N2NAO5S

380.4348

380.138

C, 50.51; H, 6.62; N, 7.36; Na, 6.04; O, 21.03; S, 8.43

81129-83-1

Cilastatin;82009-34-5

6435415

Solid powder

655.5ºC at 760 mmHg

350.2ºC

1.189

4

7

11

24

519

2

C([C@H]1CC1(C)C)(=O)N/C(/C(=O)O)=C\CCCCSC[C@H](N)C(=O)O.[Na]

QXPBTTUOVWMPJN-QBNHLFMHSA-M

InChI=1S/C16H26N2O5S.Na/c1-16(2)8-10(16)13(19)18-12(15(22)23)6-4-3-5-7-24-9-11(17)14(20)21/h6,10-11H,3-5,7-9,17H2,1-2H3,(H,18,19)(H,20,21)(H,22,23)/q+1/p-1/b12-6-/t10-,11+/m1./s1

sodium S-((Z)-6-carboxy-6-((S)-2,2-dimethylcyclopropane-1-carboxamido)hex-5-en-1-yl)-L-cysteinate

Cilastatin Monosodium Salt ; MK 0791; MK0791; M K-0791; MK791; MK-791; MK-791; Cilastatin sodium; Recarbrio;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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 : ~72 mg/mL ( ~200.86 mM )
Water : 7~100 mg/mL(~262.86 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.)