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
Very poor absorption from gastrointestinal tract.
/COLISTIMETHATE SODIUM/...IS EXCRETED IN DOG URINE TO MUCH GREATER EXTENT THAN SIMPLE SULFATE FORM, & THEORETICALLY SHOULD BE BETTER IN URINARY INFECTIONS.
DRUG PASSES FROM MATERNAL TO FETAL CIRCULATION. PREMATURE INFANTS INJECTED WITH 1 MG/KG DO NOT DEVELOP EFFECTIVE PLASMA CONCN OF ANTIBIOTIC; WITH DOSE OF 2 MG/KG, PEAK VALUE OF ABOUT 5 UG/ML IS REACHED IN 30 MIN. /COLISTIMETHATE/
PLASMA CONCN ARE HIGHER IN PERSONS WITH RENAL INSUFFICIENCY & ARE RELATED TO DEGREE OF RENAL DYSFUNCTION. COLISTIMETHATE IS EXCRETED MAINLY BY GLOMERULAR FILTRATION. URINE CONCN EXCEED 200 UG/ML DURING FIRST 2 HR AFTER USUAL IM DOSE. /COLISTIMETHATE/
DRUG IS EXCRETED MORE RAPIDLY IN CHILDREN THAN IN ADULTS. COLISTIMETHATE DOES NOT GAIN ACCESS TO CEREBROSPINAL FLUID, EVEN WHEN MENINGES ARE INFLAMED. /COLISTIMETHATE/
For more Absorption, Distribution and Excretion (Complete) data for COLISTIN (8 total), please visit the HSDB record page.

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Metabolism / Metabolites
As 80% of the dose can be recovered unchanged in the urine, and there is no biliary excretion, it can be assumed that the remaining drug is inactivated in the tissues, however the mechanism is unknown.
...hydrolyzed in vivo to colistin and possibly other metabolites with fewer substituted amino groups...
AFTER IV INJECTION RABBITS, 75% DOSE EXCRETED IN URINE UNCHANGED. SMALL AMT FOUND IN BILE. COLISTIN-N-GLUCURONIDE FOUND IN URINE (1.7% OF DOSE) & BILE (6.7% OF DOSE). /SODIUM COLISTIN METHANE SULFONATE/

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Biological Half-Life
5 hours
IM INJECTION OF 150 MG OF COLISTIMETHATE IN ADULTS PRODUCE PEAK PLASMA CONCN OF 6 UG/ML @ 2 HR; THIS DECLINES WITH HALF-TIME OF 2 HR. SAME QUANTITY GIVEN IV YIELDS MAXIMAL PLASMA CONCN OF 18 UG/ML; THIS FALLS TO ABOUT 0.4 UG/ML @ 12 HR. /COLISTIMETHATE/

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Objectives: To determine the disposition of colistin methanesulphonate (CMS) and colistin following intravenous (iv) administration of CMS in rats.
Methods: Five rats received a single iv bolus of 15 mg/kg CMS. Plasma concentrations of CMS and of colistin formed by the hydrolysis of CMS were determined by HPLC. The pharmacokinetic parameters of CMS and colistin were calculated using non-compartmental analysis.
Results: Total body clearance, volume of distribution at steady state and terminal half-life of CMS averaged 11.7 mL/min/kg, 299 mL/kg and 23.6 min, respectively. The mean terminal half-life of colistin was 55.7 min. Approximately 60% of the dose was eliminated via the urine in 24 h and presented as a mixture of CMS and colistin.
Conclusions: Colistin appeared in plasma soon after administration of CMS, indicating rapid conversion of CMS into colistin. CMS had a shorter terminal half-life than did colistin, indicating that the disposition of the colistin generated from CMS was rate-limited by its elimination. Most of the dose was recovered in urine, half in the form of colistin. The high percentage of colistin recovered in urine was believed to be formed by hydrolysis of CMS in the bladder and in the collection vessel, and/or conversion from CMS in the kidney.[2]

Colistin has no significant genotoxic activity or structural alerts for carcinogenicity, is poorly absorbed from the gastrointestinal tract, and no neoplastic or preneoplastic lesions were observed in 26-week studies in rats given repeated oral or parenteral doses. As such, the Committee concluded that colistin compounds are unlikely to be carcinogenic. The relevant endpoint for risk assessment was determined to be disruption of the colonization barrier in the colon via toxicity to gut flora, the most sensitive organism of which was E. coli, with a minimum inhibitory concentration for 50% of strains (MIC50) of 1 µg colistin base/ml in an in vitro study. The Committee established an ADI of 0–7 µg/kg bw/d (420 µg/p/d for 60-kg adult) on the basis of the MIC50 for E. coli converted to an upper bound ADI value using the following formula: ADI (µg/kg bw/d) = (MIC50 (1 µg/ml) x mass of colonic contents (220 g))/(bioavailable fraction of dose (0.5) x safety factor (1) x bodyweight (60 kg)). The Committee recommended MRLs, measured as colistin A+B, in cattle, sheep, goats, pigs, chickens, turkeys and rabbits of 150 µg/kg liver, muscle and fat (including skin+fat, where applicable), 200 µg/kg in kidney, 300 µg/kg in hens’ eggs, and 50 µg/kg in cows’ and sheep’s milk. The MRLs recommended would result in a TMDI of 229 µg (55% ADI). The calculated EDI values represent 4% (for chickens) to 9% (for cattle) of the upper bound of the ADI. The EDI of 56.9 µg (14% ADI) is calculated using the highest median values from among the tissues and food-producing species.

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Interactions
COLISTIMETHATE...MAY INTERACT...WITH CARBENICILLIN. /COLISTIMETHATE/
COLISTIN METHANESULFONATE REDUCED BY MORE THAN 20% THE AMT OF URINARY SALICYLAMIDE GLUCURONIDE FORMED IN CHILDREN FROM 20 MG/KG DOSE SALICYLAMIDE. /COLISTIN METHANESULFONATE/
Since nephrotoxic and/or neurotoxic effects may be additive, concurrent or sequential use of colistimethate sodium and other drugs with similar toxic potentials (e.g., aminoglycosides, amphotericin B, capreomycin, methoxyflurane, polymyxin B sulfate, vancomycin) should be avoided, if possible. /Colistimethate sodium/

[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 polypeptide antibiotic from Bacillus colistinus. It is composed of Polymyxins E1 and E2 (or Colistins A, B, and C) which act as detergents on cell membranes. Colistin is less toxic than Polymyxin B, but otherwise similar; the methanesulfonate is used orally.
Colistin has been reported in Rhizopus arrhizus with data available.
Cyclic polypeptide antibiotic from Bacillus colistinus. It is composed of Polymyxins E1 and E2 (or Colistins A, B, and C) which act as detergents on cell membranes. Colistin is less toxic than Polymyxin B, but otherwise similar; the methanesulfonate is used orally.
See also: Colistimethate Sodium (annotation moved to).

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Drug Indication
For the treatment of acute or chronic infections due to sensitive strains of certain gram-negative bacilli, particularly Pseudomonas aeruginosa.

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Mechanism of Action
Colistin is a surface active agent which penetrates into and disrupts the bacterial cell membrane. Colistin is polycationic and has both hydrophobic and lipophilic moieties. It interacts with the bacterial cytoplasmic membrane, changing its permeability. This effect is bactericidal. There is also evidence that polymyxins enter the cell and precipitate cytoplasmic components, primarily ribosomes.
POLYMYXIN B IS SURFACE-ACTIVE AGENT... CONTAINING LIPOPHILIC & LIPOPHOBIC GROUPS SEPARATED WITHIN MOLECULE. /POLYMYXIN B/
PERMEABILITY OF THE BACTERIAL MEMBRANE CHANGES IMMEDIATELY ON CONTACT WITH DRUG. SENSITIVITY TO POLYMYXIN B APPARENTLY IS RELATED TO THE PHOSPHOLIPID CONTENT OF THE CELL WALL-MEMBRANE COMPLEX. /POLYMYXIN B/
Colistin acts like a cationic detergent and binds to and damages the bacterial cytoplasmic membrane of susceptible bacteria. Damage to the bacterial cytoplasmic membrane alters the osmotic barrier of the membrane and causes leakage of essential intracellular metabolites and nucleosides.

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Therapeutic Uses
Antibiotics, Peptide
THERAPEUTIC INDICATIONS FOR COLISTIN ARE ESSENTIALLY SAME AS THOSE FOR POLYMYXIN B. INFECTIONS OF CERTAIN TYPES CAUSED BY PSEUD AERUGINOSA ARE ESPECIALLY SUSCEPTIBLE.
PRIMARY USE OF POLYMYXIN B IS FOR TREATMENT OF INFECTIONS CAUSED BY GRAM-NEGATIVE BACTERIA, ESPECIALLY PSEUDOMONAS. ...IS EFFECTIVE IN TREATMENT OF URINARY TRACT INFECTIONS CAUSED BY PSEUDOMONAS OR OTHER GRAM-NEGATIVE BACILLI RESISTANT TO OTHER ANTIMICROBIAL AGENTS... /POLYMYXIN B/
/POLYMYXIN B & COLISTIMETHATE SODIUM/...RECOMMENDED FOR TREATING PERITONITIS & PNEUMONIA, BUT SOME AUTHORITIES QUESTION THEIR EFFECTIVENESS FOR THESE PURPOSES. /COLISTIMETHATE SODIUM/
For more Therapeutic Uses (Complete) data for COLISTIN (11 total), please visit the HSDB record page.

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Drug Warnings
ADVERSE REACTIONS TO COLISTIMETHATE HAVE BEEN NOTED IN 20% OF PT GIVEN THE DRUG; THEY ARE GENERALLY REVERSIBLE... /COLISTIMETHATE/
COLISTIMETHATE SODIUM SHOULD NOT BE ADMIN INTRATHECALLY. /COLISTIMETHATE SODIUM/
...NOT INDICATED FOR INFECTIONS CAUSED BY PROTEUS OR NEISSERIA SPECIES. /SODIUM COLISTIMETHATE/
POSSIBLE EMBRYOTOXIC & TERATOGENIC EFFECTS HAVE BEEN REPORTED WHEN COLISTIMETHATE SODIUM WAS ADMINISTERED TO PREGNANT RABBITS. /NA COLISTIMETHATE/
For more Drug Warnings (Complete) data for COLISTIN (13 total), please visit the HSDB record page.

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Pharmacodynamics
Colistin is a polymyxin antibiotic agent. Polymyxins are cationic polypeptides that disrupt the bacterial cell membrane through a detergentlike mechanism. With the development of less toxic agents, such as extended-spectrum penicillins and cephalosporins, parenteral polymyxin use was largely abandoned, except for the treatment of multidrug-resistant pulmonary infections in patients with cystic fibrosis. More recently, however, 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 the revived use of the 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.)