β-lactam
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].

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].

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Cilastatin potently inhibits imipenem hydrolysis by purified porcine Renal Membrane Dipeptidase (MDP) with an IC50 of 0.3 ± 0.01 µM. [1]
Cilastatin inhibits imipenem hydrolysis by the purified bacterial metallo-β-lactamase CphA with an IC50 of 178 ± 11 µM, indicating it is substantially less potent against the bacterial enzyme compared to the mammalian MDP. [1]
At a high concentration (10 mM), Cilastatin partially inhibits (approximately 60%) imipenem hydrolysis by a partially purified metallo-β-lactamase from Bacteroides fragilis. [1]
Cilastatin (at concentrations up to 10 mM) does not inhibit the L1 metallo-β-lactamase from Xanthomonas maltophilia when using imipenem or nitrocefin as the substrate. [1]
Cilastatin itself is not hydrolyzed by the tested bacterial metallo-β-lactamases. [1]
Cilastatin (at 0.3 mM) completely inhibits the hydrolysis of the characteristic MDP substrates Gly-D-Phe and glycyldehydrophenylalanine (Gly-dh-Phe) by purified MDP, as shown by HPLC analysis. [1]

In a mouse model of systemic infection (female mice, strain CD-1, 20 g), Imipenem plus Cilastatin can shield mice against infections with S. aureus, E. coli, and P. aeruginosa[3].

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Cilastatin (in combination with imipenem) has been shown in previous animal studies to reduce vancomycin-induced nephrotoxicity, as indicated by decreased serum BUN and creatinine levels in rabbits and rats. [2]

Spectrophotometric Assay for Imipenem Hydrolysis: The activity of purified MDP or CphA enzyme against imipenem was assessed spectrophotometrically. The enzyme was incubated in Tris-HCl buffer (pH 8.0) with 0.1 mM imipenem as the substrate. The hydrolysis of imipenem was monitored by measuring the decrease in absorbance at 299 nm (A299). To determine inhibition, Cilastatin was added to the reaction mixture at various final concentrations ranging from 1.0 nM to 0.01 M. The rate of absorbance change in the presence of inhibitor was compared to the uninhibited control to calculate percent inhibition and IC50 values. [1]
HPLC-based Assay for Dipeptide/Dehydropeptide Hydrolysis: Purified MDP or CphA enzyme was incubated in Tris-HCl buffer (pH 8.0) with either 3 mM Gly-D-Phe or 1 mM glycyldehydrophenylalanine (Gly-dh-Phe) as substrates. Reactions were carried out in the presence or absence of 0.3 mM Cilastatin. After incubation at 37°C, reactions were terminated by boiling. Precipitated material was removed by centrifugation. The products of hydrolysis (D-Phe from Gly-D-Phe or phenylpyruvate from Gly-dh-Phe) were separated and quantified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 column. A linear gradient of acetonitrile in phosphoric acid was used for elution, and product detection was performed by measuring absorbance at 214 nm. [1]

Porcine renal proximal tubular epithelial cells (RPTECs) were cultured and treated with vancomycin (0.6, 3, and 6 mg/mL) with or without cilastatin (200 µg/mL) for 24 hours. Cell morphology was observed using phase-contrast microscopy. [2]
Cell detachment was quantified by collecting detached cells and analyzing them via flow cytometry. [2]
Apoptosis was assessed by DAPI staining of nuclei to observe nuclear condensation and fragmentation, and by quantifying nucleosomal DNA fragmentation using an ELISA kit. [2]
Necrosis was evaluated by measuring lactate dehydrogenase (LDH) release into the culture medium after 24 and 48 hours. [2]
Early and late apoptosis were analyzed by flow cytometry using annexin V and propidium iodide staining. [2]
Cell viability was measured using the MTT assay, and mitochondrial function was assessed in real-time by monitoring MTT reduction at 570 nm. [2]
Long-term recovery was evaluated by colony-forming unit assay after 7 days of growth in drug-free medium. [2]
Intracellular vancomycin accumulation was measured using fluorescence polarization immunoassay (TDX) after 24 hours of treatment. [2]

Female CD-1 mice (weighing 20 ± 2 g) were used. [3]
Mice were infected intraperitoneally with a bacterial suspension in Trypticase soy broth or 5% hog gastric mucin. [3]
Antibiotics (imipenem plus cilastatin as the comparator combination) were administered subcutaneously in 0.5 ml of 0.2% aqueous agar, starting 0.5 hours after infection. [3]
Four to five dose levels were tested, with 5 mice per dose level, and survival was monitored for 7 days to determine the median effective dose (ED₅₀) by probit analysis. [3]

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.

Protein Binding
It has been reported that cilastatin has a plasma protein binding rate of 35-40%. At the concentration used (200 µg/mL), cilastatin did not produce cytotoxic or necrotic effects on renal tubular epithelial cells (RPTEC). [2] Cilastatin does not interfere with the bactericidal activity of vancomycin. [2]

[1]. 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]. Protective Effects of Cilastatin Against Vancomycin-Induced Nephrotoxicity. Biomed Res Int. 2015;2015:704382.

[3]. 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 often used in combination with imipenem to block the metabolism of imipenem. The development of cilastatin is based on the structural similarity between the hydrolyzable bond and the dehydropeptide in imipenem, which is a substrate of the renal membrane dipeptidase (MDP). Its main clinical use is as an inhibitor of this renal enzyme, preventing the degradation of the carbapenem antibiotic imipenem in vivo, thereby protecting its antibacterial activity. [1] This study shows that cilastatin can also inhibit certain bacterial metallo-β-lactamases (especially CphA), but its inhibitory efficacy is much lower than that of mammalian MDP. This suggests that although mammalian and bacterial zinc metalloenzymes lack significant similarity in amino acid sequence, they still share some common mechanistic features. [1] Cilastatin has a narrow spectrum of inhibition against bacterial metallo-β-lactamases; it can inhibit CphA and partially inhibit the enzyme of Bacteroides fragilis, but cannot inhibit the enzyme of Stenotrophomonas maltophilia L1. [1]
This study suggests that cilastatin or its analogues could serve as lead compounds for the development of novel bacterial metallo-β-lactamase inhibitors and may help differentiate between different subclasses of these enzymes. [1]

C16H26N2O5S

358.45

358.156

C, 53.61; H, 7.31; N, 7.82; O, 22.32; S, 8.95

82009-34-5

Cilastatin sodium;81129-83-1;Cilastatin-15N,d3;2738376-83-3

6435415

White to off-white solid powder

1.275 g/cm3

655.5ºC at 760 mmHg

156-158ºC

1.569

2.523

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

DHSUYTOATWAVLW-WFVMDLQDSA-N

InChI=1S/C16H26N2O5S/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)/b12-6-/t10-,11+/m1/s1

2-Heptenoic acid, 7-((2-amino-2-carboxyethyl)thio)-2-(((2,2-dimethylcyclopropyl)carbonyl)amino)-, (R-(R*,S*-(Z)))-

MK 791; M K-791; MK0791; MK791;Cilastatin; MK 0791; MK-0791;

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)

DMSO : 72~100 mg/mL (200.86~278.98 mM )
1M NaOH : ~100 mg/mL (~278.98 mM)
H2O : 7~12.5 mg/mL (~34.87 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (7.67 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 27.5 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 (6.97 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 (6.97 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: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.75 mg/mL (7.67 mM)

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Solubility in Formulation 5: 20 mg/mL (55.80 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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