Polymyxin antibiotic; Colistin A sulfate hydrate targets the Gram-negative bacterial cell membrane. Its mechanism involves binding to lipopolysaccharides and phospholipids, competitively displacing divalent cations (calcium and magnesium) . This action disrupts membrane integrity, leading to leakage of intracellular contents and rapid bacterial cell death.

Colistin A sulfate hydrate exhibits potent concentration-dependent bactericidal activity. MIC values for multidrug-resistant E. coli and P. aeruginosa are 0.25 and 2 μg/mL, respectively . It significantly reduces MDR E. coli and P. aeruginosa counts within 2 hours at 2× MIC, with complete killing observed by 8 hours (E. coli) and 4 hours (P. aeruginosa) at 4× MIC . Colistin effectively mediates in vitro activity against various clinical isolates, with MIC₉₀ values of 1 μg/mL for A. baumannii, 3 μg/mL for P. aeruginosa, and 16 μg/mL for Stenotrophomonas maltophilia . Colistin-based combinations (e.g., with amikacin or levofloxacin) demonstrate synergistic biofilm eradication against P. aeruginosa; 2 mg/L colistin combined with 32 mg/L amikacin or 4-8 mg/L levofloxacin can kill biofilm-embedded bacteria within 24 hours .

In a murine thigh infection model, a colistin loading dosage regimen (Day 1: 50 mg/kg q12h, Days 2-3: 25 mg/kg q12h) resulted in a 5-6 log₁₀ CFU/mL reduction for both P. aeruginosa strains with colistin MICs of 0.5 and 1 μg/mL . The same reduction was observed for the 1 μg/mL MIC strain only with the loading dosage, showing the importance of high initial drug exposure . In a porcine biofilm infection model, colistin combined with amikacin or levofloxacin shortened biofilm eradication time compared to monotherapy. For colistin-resistant P. aeruginosa (CRPAO1) biofilm, the 6-day eradication rate for the colistin + amikacin regimen was 90%, compared to 10% for amikacin alone and 40% for colistin alone .

Traditional cell-free enzyme/receptor binding assays (such as SPR or ITC) are not standard for colistin A sulfate hydrate, as its primary mechanism of action is membrane disruption independent of a specific enzyme. However, protein binding studies have been conducted via ultrafiltration: using colistin sulfate at concentrations approximating clinical levels, the unbound fraction (fu) in perfusate was determined for both colistin A (fu=0.42) and colistin B (fu=0.60), which are slightly higher than values reported for rat plasma (0.36 and 0.52, respectively) . In an isolated perfused rat kidney model, renal clearance (CLR) of colistin A and B was low (<0.05 mL/min) and clearance ratios (CR) were significantly <1 (indicating net reabsorption) . Less than 10% of the drug eliminated from the perfusate was recovered in urine, suggesting significant renal accumulation .

Cell-based assays are not the standard method for evaluating colistin A's antibacterial activity; instead, standardized microbiological methods are employed. The broth microdilution method determines MIC and MBC: serial two-fold dilutions of colistin are prepared in a 96-well plate and inoculated with a standardized bacterial suspension (~5×10⁵ CFU/mL). After 16-20 hours of incubation at 35±2°C, the MIC is the lowest concentration with no visible bacterial growth . Time-kill curve assays assess bactericidal activity over time: bacteria are exposed to various colistin concentrations (e.g., 0.5×, 2×, 4× MIC), and aliquots are plated at intervals (0, 1, 2, 4, 8, 12, 24 h) for colony counting .

Murine Thigh Infection Model: Specific-pathogen-free female ICR mice are rendered neutropenic by cyclophosphamide injections (150 mg/kg and 100 mg/kg) . Thigh infections are established by intramuscular injection of 0.1 mL of mid-log-phase P. aeruginosa bacterial suspension (~10⁷ CFU/mL). Treatment begins 2 hours post-infection. Colistin is administered subcutaneously at a loading dosage (Day 1: 50 mg/kg q12h) followed by maintenance dosing (Days 2-3: 25 mg/kg q12h). At specified time points, mice are euthanized, thigh muscles are harvested, homogenized, serially diluted, and plated onto Mueller-Hinton agar for CFU enumeration . Isolated Perfused Rat Kidney Model: Kidneys are isolated from male Sprague-Dawley rats and perfused with a recirculating modified Krebs-Henseleit buffer at 37°C. Colistin sulfate (2 μg/mL) is added to the perfusate. Urine and perfusate samples are collected over 120 minutes to determine CLR, CR, and %TR (tubular reabsorption). Transport inhibitors (tetraethylammonium for organic cation transporters, glycylglycine for peptide transporters) are co-perfused to elucidate reabsorption mechanisms .

Colistin A exhibits poor oral absorption (less than 1%), making it suitable only for topical or intravenous administration for systemic infections . Following IV administration in healthy subjects (at a dose equivalent to 0.452 mg/kg), PK parameters include: Cmax of 1.08 ± 0.18 mg/L, AUC₀-₁₂h of 4.73 ± 0.89 h·mg/L, terminal half-life of 3.65 ± 0.55 h, Vd of 16.82 ± 2.70 L, and total body clearance of 3.24 ± 0.51 L/h . In cerebrospinal fluid (CSF), colistin levels are significantly lower (Cmax ~90 ng/mL) . Less than 1% of an IV dose is recovered unchanged in urine within 24 hours (cumulative urinary recovery 0.9 ± 0.7%), indicating predominant non-renal elimination pathways . The drug undergoes extensive net tubular reabsorption in the kidney (>90%), leading to significant renal accumulation but minimal urinary excretion . No accumulation is observed after multiple doses of 10,000 units/kg q12h for 7 days .

Colistin 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 colistin 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 colistin 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 colistin 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 for kidneys; 300 µg/kg for eggs; and 50 µg/kg for cow and sheep milk. The recommended MRLs result in a total daily intake (TMDI) of 229 µg (55% of the acceptable daily intake). Calculated upper limits for daily intake (EDIs) range from 4% (chicken) to 9% (cattle) of the EDI. The EDI of 56.9 µg (14% of the EDI) was calculated based on the highest median among the tissues and edible species.

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Colistin A is classified as a nephrotoxic agent. In the isolated perfused rat kidney model, colistin exhibited extensive net tubular reabsorption (>90%) and significant renal accumulation, with less than 10% of the eliminated drug recovered in urine . The organic cation transporter (OCT) and peptide transporter (PEPT) systems are implicated in colistin's renal accumulation, as co-administration of tetraethylammonium (an OCT inhibitor) and glycylglycine (a PEPT inhibitor) significantly increased colistin's clearance ratio . However, a recent clinical trial in healthy subjects reported no nephrotoxicity with colistin sulfate IV administration . Genetic toxicology studies indicate colistin does not exhibit significant genotoxic activity or carcinogenic structural warnings . No tumors or precancerous lesions were observed in 26-week repeat-dose studies in rats . The primary toxicological concern for risk assessment is disruption of the gut colonization barrier through toxicity to the gut microbiota, with E. coli being the most susceptible (MIC₅₀ = 1 μg/mL for 50% of strains) .

[1]. Resurgence of Colistin: A Review of Resistance, Toxicity, Pharmacodynamics, and Dosing. Pharmacotherapy. 2010 Dec; 30(12): 1279-1291.

Colistin, a polymyxin antibiotic, was discovered in the late 1940s for the treatment of Gram-negative bacterial infections. After several years of clinical use, its use declined due to reports of significant nephrotoxicity and neurotoxicity. In recent years, colistin has regained attention as a last-line treatment for infections caused by multidrug-resistant bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. The high morbidity and mortality rates of these Gram-negative pathogens necessitate antibiotics that can cover them, making colistin a crucial treatment option. Unfortunately, case reports indicate that resistance to colistin has emerged in all three bacteria. While the exact mechanisms of colistin resistance are not fully understood, the PmrA-PmrB and PhoP-PhoQ gene regulatory systems are hypothesized to play a role. Because colistin is a last resort, its dosage must be optimized; furthermore, inadequate dosage is associated with the development of resistance. However, due to the lack of pharmacokinetic and pharmacodynamic studies and the lack of standardized dosage units, it is difficult to develop optimal dosing regimens and specific dosing guidelines for colistin. In critically ill patients who may experience multiple organ failure, renal insufficiency may alter the pharmacokinetics of colistin. Therefore, for these patients, the dosage must be adjusted to achieve the best efficacy and minimum toxicity. Regarding the toxicity of colistin, most studies have shown that nephrotoxicity is reversible and occurs less frequently than previously expected, while neurotoxicity is rare. Further research is needed to fully understand the impact of the two regulatory systems on resistance, and the colistin dosage required to inhibit and overcome these resistance patterns. [1]

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Colistin A sulfate hydrate (PubChem CID: 24825758) has a molecular formula of C₅₃H₁₀₄N₁₆O₁₈S and a molecular weight of 1,285.55 g/mol . The compound comprises multiple components, the primary being colistin A (polymyxin E1) and colistin B (polymyxin E2) . For storage, the powder form is stable for up to 3 years at -20°C or 2 years at 4°C; solutions are stable for up to 6 months at -80°C or 1 month at -20°C . While colistin use declined historically due to nephrotoxicity concerns, it has been revived as a last-line treatment for MDR Gram-negative infections given the limited antibiotic pipeline . Unlike the prodrug colistin methanesulfonate (CMS), which requires slow in vivo conversion to active colistin, colistin sulfate achieves clinically relevant concentrations more quickly and avoids the unpredictable conversion rates associated with CMS (only 1.4% to ~30% conversion in humans) . The product is for research use only, not for human or veterinary use . A major ongoing challenge is the emergence of plasmid-mediated mobilized colistin resistance (mcr) genes, which threaten the long-term utility of this last-resort antibiotic class.

C53H104N16O18S

1285.55

1168.7655

24825758

{(+)-6-methyloctanoyl}-{Dab}-Thr-{Dab}-{Dab}-{Dab}-{D-Leu}-Leu-{Dab}-{Dab}-Thr (Lactam bridge:Dab-4-Thr-10)

{(+)-6-methyloctanoyl}-{Dab}T{Dab}{Dab}{Dab}{D-Leu}L{Dab}{Dab}T (Lactam bridge:Dab-4-Thr-10)

Typically exists as solid at room temperature

-2.8

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

XDJYMJULXQKGMM-HHAJOKTESA-N

InChI=1S/C53H100N16O13/c1-9-30(6)12-10-11-13-41(72)60-33(14-20-54)48(77)69-43(32(8)71)53(82)65-36(17-23-57)45(74)64-38-19-25-59-52(81)42(31(7)70)68-49(78)37(18-24-58)62-44(73)34(15-21-55)63-50(79)39(26-28(2)3)67-51(80)40(27-29(4)5)66-46(75)35(16-22-56)61-47(38)76/h28-40,42-43,70-71H,9-27,54-58H2,1-8H3,(H,59,81)(H,60,72)(H,61,76)(H,62,73)(H,63,79)(H,64,74)(H,65,82)(H,66,75)(H,67,80)(H,68,78)(H,69,77)/t30-,31+,32+,33-,34-,35-,36-,37-,38-,39-,40+,42-,43-/m0/s1

(6S)-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]-6-methyloctanamide

Colistin A; 7722-44-3; UNII-500HI50Z9H; 500HI50Z9H; COLISTIN A [MI]; BRN 0604503; COLISTIN A [WHO-DD]; ...; Colistin A Sulfate Hydrate (~90%);

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.)