Bacterial cell wall synthessis; lipopolysaccharides and phospholipids in the outer cell membrane of gram-negative bacteria

Colistins work like detergents to kill gram-negative bacteria. This mechanism includes competitively displacing divalent cations (calcium and magnesium) from the negatively charged phosphate groups of membrane lipids, as well as interaction with the lipopolysaccharides and phospholipids of the outer membrane through electrostatic interference[4]. Colistin, also known as polymyxin E, is a good treatment for infections brought on by gram-negative bacteria that are resistant to many drugs because of its quick bacterial killing, limited spectrum of action, and concomitant delayed development of resistance. Commercially, colistin comes in two forms: colistin methanesulfonate (sodium) for parenteral use and colistin (sulfate), primarily for topical use[4].

Colistin (polymyxin E) (16 mg/kg/day, i.p.) was found to enrich cell cycle arrest genes, indicating that cellular stress or injury caused by colistin inhibits the progression of the cell cycle via p53. [1]. Blood urea nitrogen (BUN), creatinine, and pathological evidence of acute tubular necrosis and apoptosis are all elevated when colonistin (16 mg/kg/day, intraperitoneally) is administered [1].

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Colistin (polymixin E) is an antibiotic prescribed with resurging frequency for multidrug resistant gram negative bacterial infections. It is associated with nephrotoxicity in humans in up to 55% of cases. Little is known regarding genes involved in colistin nephrotoxicity. A murine model of colistin-mediated kidney injury was developed. C57/BL6 mice were administered saline or colistin at a dose of 16 mg/kg/day in 2 divided intraperitoneal doses and killed after either 3 or 15 days of colistin. After 15 days, mice exposed to colistin had elevated blood urea nitrogen (BUN), creatinine, and pathologic evidence of acute tubular necrosis and apoptosis. After 3 days, mice had neither BUN elevation nor substantial pathologic injury; however, urinary neutrophil gelatinase-associated lipocalin was elevated (P = 0.017). An Illumina gene expression array was performed on kidney RNA harvested 72 h after first colistin dose to identify differentially expressed genes early in drug treatment. Array data revealed 21 differentially expressed genes (false discovery rate < 0.1) between control and colistin-exposed mice, including LGALS3 and CCNB1. The gene signature was significantly enriched for genes involved in cell cycle proliferation. RT-PCR, immunoblot, and immunostaining validated the relevance of key genes and proteins. This murine model offers insights into the potential mechanism of colistin-mediated nephrotoxicity. Further studies will determine whether the identified genes play a causative or protective role in colistin-induced nephrotoxicity[3].

MIC determination.[4]
MICs were determined by both broth macrodilution and microdilution in cation-adjusted Mueller-Hinton broth ccording to NCCLS standards. Strains were considered resistant to colistin and colistin methanesulfonate if the MICs were ≥32 mg/liter.

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Time-kill kinetics.[4]
The time-kill kinetics of four strains, ATCC 27853 and three clinical isolates, two of which were mucoid, were examined. The clinical isolates were selected in order to have a range of MICs within the susceptible category. The MICs of colistin and colistin methanesulfonate, respectively, for the four strains were as follows: ATCC 27853, 4 and 16 mg/liter; 18982, 4 and 8 mg/liter; 19056, 1 and 8 mg/liter; and 20223, 4 and 16 mg/liter. Colistin and colistin methanesulfonate were added to a logarithmic-phase broth culture of approximately 106 CFU/ml to yield concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32, and 64 times the MIC for the strain under study. Subcultures for viable counts were performed on nutrient agar at 0, 5, 10, 15, 20, 25, 30, 45, and 60 min and 2, 3, 4, and 24 h after antibiotic addition. Viable counts were determined after 24 h of incubation of subcultures at 37°C.

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PAE.[4]
The in vitro PAE was determined by the standard in vitro method for two of the three clinical strains noted above and the ATCC strain with both agents. For each experiment, P. aeruginosa (≈106 CFU/ml) in logarithmic phase growth was exposed for 15 min (for colistin) or 1 h (for colistin methanesulfonate) in Mueller-Hinton broth to the antibiotics at concentrations of 0.5, 1, 2, 4, 8, and 16 times the MIC. Fifteen minutes of exposure was used for colistin due to its very rapid bactericidal effect, to ensure that there were adequate numbers of bacteria for sampling at the end of the exposure interval. Antibiotic was removed by twice centrifuging at 3,000 × g for 10 min, decanting the supernatant, and resuspending in prewarmed broth. Viable counts were performed at 0, 1, 2, 3, 4, 5, 6, and 24 h on nutrient agar. A growth control was performed in the same fashion but without exposure to antibiotic. The colonies were counted after 24 h of incubation at 37°C. PAE was determined by comparing regrowth of treated and growth control cultures, using the standard formula of the time for the control culture to increase 10-fold subtracted from the time for the treated culture to do the same.

Animal/Disease Models: C57/BL6 black mouse [1]
Doses: 16 mg/kg/day
Route of Administration: 16 mg/kg/day, ip
Experimental Results: Apoptosis, necrosis and PCNA staining were detected in mice.

Absorption, Distribution and Excretion
Gastrointestinal absorption is extremely poor. Cyclic mesylate is excreted in dog urine at a much higher rate than the simple sulfate form, theoretically suggesting better efficacy against urinary tract infections. The drug can cross from the maternal circulation into the fetal circulation. In premature infants, an injection of 1 mg/kg body weight does not achieve an effective plasma antibiotic concentration; an injection of 2 mg/kg body weight reaches a peak of approximately 5 μg/mL within 30 minutes. Cyclic mesylate: Plasma concentrations are higher in patients with renal impairment and are correlated with the degree of renal impairment. Cyclic mesylate is primarily excreted via glomerular filtration. Urinary concentrations exceed 200 μg/mL within 2 hours after a commonly administered intramuscular dose. Cyclic mesylate is excreted faster in children than in adults. Even in cases of meningitis, Cyclic mesylate does not enter the cerebrospinal fluid. /Cyclic Sodium Mesylate/
For more complete data on the absorption, distribution, and excretion of Cyclic (8 types in total), please visit the HSDB record page.

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Metabolism/Metabolites
Since 80% of the dose is excreted unchanged in the urine and not in the bile, it can be inferred that the remaining drug is inactivated in tissues, but the mechanism is unclear.
...hydrolyzed in vivo to Cyclic, and possibly other less amino-substituted metabolites...
After intravenous injection in rabbits, 75% of the dose was excreted unchanged in the urine. Small amounts were found in the bile. Cyclic-N-glucuronide (1.7% of the dose) was found in the urine, and Cyclic-N-glucuronide (6.7% of the dose) was found in the bile. /Cyclic Sodium Mesylate/

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Biological Half-Life
5 hours
After intramuscular injection of 150 mg Cyclic sodium mesylate in adults, the peak plasma concentration reached 6 μg/ml after 2 hours; the half-life was 2 hours. After intravenous injection of the same dose, the peak plasma concentration reached 18 μg/ml; after 12 hours, it decreased to approximately 0.4 μg/ml. /Cyclic Sodium Mesylate/

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Objective: To determine the in vivo distribution of CMS and Cyclic after intravenous injection of Cyclic sodium mesylate (CMS) in rats.
Methods: Five rats were given a single intravenous injection of 15 mg/kg CMS. The concentrations of CMS and its hydrolysis product Cyclic in plasma were determined by high-performance liquid chromatography (HPLC). Pharmacokinetic parameters of CMS and Cyclic were calculated using a non-compartmental model analysis. Results: The mean systemic clearance, steady-state volume of distribution, and terminal half-life of CMS were 11.7 mL/min/kg, 299 mL/kg, and 23.6 min, respectively. The mean terminal half-life of Cyclic was 55.7 min. Approximately 60% of the administered dose was excreted in the urine within 24 hours as a mixture of CMS and Cyclic. Conclusion: Cyclic appeared in the plasma shortly after CMS administration, indicating that CMS is rapidly converted to Cyclic. The terminal half-life of CMS is shorter than that of Cyclic, indicating that the clearance rate of Cyclic generated from CMS is limited by its own clearance rate. Most of the administered dose was excreted in the urine, half of which was in the form of Cyclic. A high proportion of Cyclic was recovered in the urine, believed to be formed by the hydrolysis of CMS in the bladder and collecting ducts, and/or converted from CMS in the kidneys. [2]

Cyclic did not exhibit significant genotoxic activity or carcinogenic structural warnings, was poorly absorbed in the gastrointestinal tract, and no tumors or precancerous lesions were observed in repeated oral or parenteral administration studies in rats over a period of 26 weeks. Therefore, the Committee concluded that Cyclic compounds are unlikely to be carcinogenic. The relevant endpoint for risk assessment was determined to be disruption of the colonic colonization barrier through toxicity to the gut microbiota, with Escherichia coli being the most susceptible. In vitro studies have shown that the minimum inhibitory concentration (MIC50) against 50% of E. coli strains is 1 µg/ml Cyclic base. Based on the MIC50 value of E. coli, the Committee converted the upper limit of ADI to 0–7 µg/kg body weight/day (420 µg/p/d for a 60 kg adult) using the following formula: ADI (µg/kg bw/d) = (MIC50 (1 µg/ml) × colonic contents mass (220 g)) / (bioavailability (0.5) × safety factor (1) × body weight (60 kg)). The committee recommends maximum residue limits (MRLs) of 150 µg/kg for Cyclic A+B in the liver, muscle, and fat (including applicable skin and fat) of cattle, sheep, goats, pigs, chickens, turkeys, and rabbits; 200 µg/kg in the kidneys; 300 µg/kg in eggs; and 50 µg/kg in cow and sheep milk. The recommended MRLs result in a total daily intake (TMDI) of 229 µg (55% of the acceptable daily intake). The calculated acceptable daily intake (EDI) is equivalent to 4% (chicken) to 9% (cattle) of the upper limit of the daily intake. The acceptable daily intake of 56.9 µg (14% of the daily intake) was calculated using the highest median value among tissue and food-producing species.
Interactions
Cyclic mesylate may interact with carbenicillin. /Cyclic Mesylate/
Cyclic mesylate can reduce the amount of salicylamide glucuronide formed in the urine by more than 20% after children take 20 mg/kg of salicylamide.
Because the nephrotoxicity and/or neurotoxicity may be cumulative, the simultaneous or sequential use of Cyclic sodium mesylate and other drugs with similar toxic potential (e.g., aminoglycosides, amphotericin B, capreomycin, methoxyflurane, polymyxin B sulfate, vancomycin) should be avoided as much as possible.

[1]. Colistin: an update on the antibiotic of the 21st century. Expert Rev Anti Infect Ther. 2012 Aug;10(8):917-34.
[2]. Pharmacokinetics of colistin methanesulphonate and colistin in rats following an intravenous dose of colistin methanesulphonate.
[3]. Cell cycle arrest in a model of colistin nephrotoxicity. Physiol Genomics. 2013 Oct 1;45(19):877-88.
[4]. In vitro pharmacodynamic properties of colistin and colistin methanesulfonate againstPseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob Agents Chemother. 2001 Mar;45(3):781-5.

Cyclic is a cyclic polypeptide antibiotic derived from Bacillus myxomyces. It consists of polymyxins E1 and E2 (or Cyclic A, B, and C), which act as detergents for cell membranes. Cyclic is less toxic than polymyxin B, but similar in other respects; mesylate preparations are available orally. Cyclic has also been reported in Rhizopus fungi, and relevant data exist. See also: Cyclic mesylate sodium (note moved to). Drug Indications Used to treat acute or chronic infections caused by susceptible strains of certain Gram-negative bacilli, particularly Pseudomonas aeruginosa.

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Mechanism of Action
Cyclic is a surfactant that penetrates and disrupts bacterial cell membranes. Cyclic is a polycation with both hydrophobic and lipophilic moieties. It interacts with the bacterial cytoplasmic membrane, altering its permeability. This action has a bactericidal effect. There is also evidence that polymyxins can enter cells and precipitate cytoplasmic components (primarily ribosomes).
Polymyxin B is a surfactant…containing separate lipophilic and lipophobic groups within the molecule.
Polymyxin B: The permeability of the bacterial membrane changes immediately upon contact with the drug. Sensitivity to polymyxin B is clearly related to the phospholipid content of the cell wall-membrane complex. Polymyxin B: Cyclic acts like a cationic detergent, binding to and disrupting the structure of the cytoplasmic membrane of susceptible bacteria. Damage to the bacterial cytoplasmic membrane alters the membrane's permeability barrier, leading to the leakage of essential metabolites and nucleosides from the cell.

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Therapeutic Uses
Antibiotics, Peptides
The therapeutic indications for Cyclic are essentially the same as for polymyxin B. Certain infections caused by Pseudomonas aeruginosa are particularly sensitive.
Polymyxin B is primarily used to treat Gram-negative bacterial infections, especially Pseudomonas aeruginosa infections. …Effective against urinary tract infections caused by Pseudomonas aeruginosa or other Gram-negative bacilli resistant to other antimicrobials…/Polymyxin B/
/Polymyxin B and Cyclic Formate/…Recommended for the treatment of peritonitis and pneumonia, but some authorities have questioned its efficacy in treating these conditions. /Cyclic Formate/
For more complete data on the therapeutic uses of Cyclic (11 in total), please visit the HSDB record page.

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Drug Warnings
Adverse reactions occur in 20% of patients treated with Cyclic formate; they are usually reversible…/Cyclic Mesylate/
Cyclic mesylate should not be administered intrathecally. /Cyclic Sodium Mesylate/
...Not suitable for infections caused by Proteus or Neisseria species. /Cyclic Sodium Mesylate/
There have been reports of embryotoxicity and teratogenicity in pregnant rabbits. /Cyclic Sodium Mesylate/
For more complete data on drug warnings for Cyclics (13 in total), please visit the HSDB record page.

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Pharmacodynamics
Cyclic is a polymyxin antibiotic. Polymyxins are cationic polypeptides that disrupt bacterial cell membranes through a detergent-like mechanism. With the advent of less toxic drugs (such as broad-spectrum penicillins and cephalosporins), the use of parenteral polymyxins has largely ceased, except for their use in treating multidrug-resistant lung infections in patients with cystic fibrosis. However, in recent years, the emergence of multidrug-resistant Gram-negative bacteria (such as Pseudomonas aeruginosa and Acinetobacter baumannii) and the lack of new antimicrobial agents have led to a resurgence in the use of polymyxins.

C52H98N16O13

1155.43392

1154.75

1066-17-7

Colistin sulfate;1264-72-8

5311054

Typically exists as solid at room temperature

1.25g/cm3

1536.8ºC at 760mmHg

200-220 °C
200 - 220 °C

883.3ºC

0mmHg at 25°C

1.573

1.535

18

18

28

81

2050

12

CCC(CCCC(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H]1CCNC(=O)[C@]([H])([C@H](O)C)NC(=O)[C@H](CCN)NC(=O)[C@H](CCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@H](CCN)NC1=O)=O)CCN)=O)[C@H](O)C)=O)CCN)=O)C

YKQOSKADJPQZHB-QNPLFGSASA-N

InChI=1S/C52H98N16O13/c1-9-29(6)11-10-12-40(71)59-32(13-19-53)47(76)68-42(31(8)70)52(81)64-35(16-22-56)44(73)63-37-18-24-58-51(80)41(30(7)69)67-48(77)36(17-23-57)61-43(72)33(14-20-54)62-49(78)38(25-27(2)3)66-50(79)39(26-28(4)5)65-45(74)34(15-21-55)60-46(37)75/h27-39,41-42,69-70H,9-26,53-57H2,1-8H3,(H,58,80)(H,59,71)(H,60,75)(H,61,72)(H,62,78)(H,63,73)(H,64,81)(H,65,74)(H,66,79)(H,67,77)(H,68,76)/t29?,30-,31-,32+,33+,34+,35+,36+,37+,38+,39-,41+,42+/m1/s1

N-[(2S)-4-amino-1-[[(2S,3R)-1-[[(2S)-4-amino-1-oxo-1-[[(3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-3-[(1R)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxobutan-2-yl]-5-methylheptanamide

colistin; 1066-17-7; N-[(2S)-4-amino-1-[[(2S,3R)-1-[[(2S)-4-amino-1-oxo-1-[[(3S,6S,9S,12S,15R,18S,21S)-6,9,18-tris(2-aminoethyl)-3-[(1R)-1-hydroxyethyl]-12,15-bis(2-methylpropyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclotricos-21-yl]amino]butan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxobutan-2-yl]-5-methylheptanamide; Colobreathe; Promixin; Colistin,(S); CHEMBL499783; SCHEMBL1979092;

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)

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.)