Topoisomerase II ( IC50 = 36.7 μM ); Quinolone

With IC50 values of 13.8 μg/ml and 0.109 μg/ml, respectively, gatifloxacin sesquihydrate targets HeLa cell topoisomerase II, Escherichia coli NIHJ JC-2 DNA gyrase, and Staphylococcus aureus MS5935 topoisomerase IV. as well as 265 μg/ml[1]. With MIC values of 0.05 μg/ml, 0.0063 μg/ml, and 122 μg/ml, gatifloxacin sesquihydrate targets three different types of bacteria: Escherichia coli NIHJ JC-2 DNA gyrase, Staphylococcus aureus MS5935 topoisomerase IV, and HeLa cell topoisomerase II[1]. Sesquihydrate gatifloxacin demonstrates antibacterial activity against step one, step two, step three, and step four mutants as well as wild-type strains (MS5935, MS5952, MR5867, and MR6009), with MIC values ranging from 0.05 to 0.10 μg/ml, 0.20 μg/ml, 1.56 to 3.13 μg/ml, 1.56 to 6.25 μg/ml, and 50 to 200 μg/ml, in that order. The most effective treatment for the second- and third-step mutants (MS5952, MR5867, and MR6009) was gatifloxacin sesquihydrate, with the exception of the strain MS5935's second-step mutant [2]. The NorA transformant NY12 is effectively inhibited by gatifloxacin sesquihydrate (MIC: 0.39 μg/ml) [2]. Insulin content was considerably reduced to 60% on day 1 and by 20 μM and 100 μM gatifloxacin sesquihydrate on day 3, respectively, after a 72-hour exposure to the drug. The percentage of content dropped further to 50.1% and 44.7%[3].

Gatifloxacin sesquihydrate (subcutaneous injection; 100 mg/kg; 3 times daily; 30 days) significantly reduced the amount of lesions on the footpads of mice with Nocardia brasiliensis [4].
Gatifloxacin at 100 mg/kg maintained plasma levels over the MIC of N. brasiliensis HUJEG-1 (0.25 μg/ml) for more than 4 h, reaching a maximum concentration in serum of 18 μg/ml (Fig. 1). Linezolid at 25 mg/kg also kept concentrations above the MIC (0.12 μg/ml) for more than 4 h, with a maximum concentration in serum of 50 μg/ml. Given these results, we decided to use gatifloxacin at 100 mg/kg three times daily, injected subcutaneously, and linezolid also three times per day at 25 mg/kg. In Fig. 2, the effect of gatifloxacin on the development of the lesions is shown. The animals showed a decrease in the number of lesions, comparable to the effect of linezolid. When the results were analyzed with the one-way analysis of variance test, both treatments, either with linezolid or with gatifloxacin, were statistically significant with a P value of 0.001 compared with the group of animals injected with saline. [4]

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Effect of gatifloxacin withdrawal on insulin secretion and islet insulin content [3]
Mouse pancreatic islets were cultured in the presence of 20 or 100 μM gatifloxacin for one day, washed thoroughly with gatifloxacin-free RPMI medium, and cultured for an additional two days in the gatifloxacin-free medium. Glucose-induced insulin secretion was greatly decreased by gatifloxacin treatment, but recovered after removal of gatifloxacin from the culture medium (Fig. 5A). Islet insulin content was decreased by gatifloxacin similarly, while frequently recovering by withdrawal of gatifloxacin in the 20 μM gatifloxacin group (to 77% of control (0 μM Gatifloxacin)) and not at all in the 100 μM gatifloxacin group (Fig. 5B). Since culture in the presence of gatifloxacin lowers islet insulin content, insulin secretion was expressed as % content, and insulin release from islets cultured with both 20 μM gatifloxacin and 100 μM gatifloxacin showed almost complete recovery upon discontinuation of the drug (Fig. 5C).

The bacterial enzymes DNA gyrase and topoisomerase IV are inhibited by the antibiotic gatifloxacin, which belongs to the fourth generation fluoroquinolone family.

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Enzyme assay. [1]
The decatenation activity of the reconstituted topoisomerase IV was determined by the method of Peng and Marians with minor modifications. The reactions were analyzed by electrophoresis, and DNA quantification in agarose gels was carried out after ethidium bromide staining. The brightness of the bands corresponding to decatenated monomers of kinetoplast DNA was determined by densitometric analysis with FMBIO II Multi-View. The supercoiling activity of DNA gyrase was determined by the method of Gellert et al. with minor modifications. Analysis was performed as described for the topoisomerase IV assay. The relaxation activity of topoisomerase II was determined by the method of Oomori et al. The inhibitory effect of each quinolone on type II topoisomerase was assayed by determining the concentration required to inhibit 50% of the enzyme reaction (IC50). Selectivity was determined by dividing the IC50 for HeLa cell topoisomerase II by the IC50 for bacterial type II topoisomerase.

Antimicrobial Like other members of the fourth-generation fluoroquinolone family of antibiotics, gatifloxacin inhibits the bacterial enzymes DNA gyrase and topoisomerase IV. When it came to the second-step mutants (grlA gyrA; gatifloxacin MIC range, 1.56 to 3.13 microg/ml) and the third-step mutants (grlA gyrA grlA; gatifloxacin MIC range, 1.56 to 6.25 microg/ml), gatifloxacin exhibited activity comparable to that of tosufloxacin and more potent than those of norfloxacin, ofloxacin, ciprofloxacin, and sparfloxacin. These results suggest that gatifloxacin has the most potent inhibitory activity against singly mutated topo IV and singly mutated DNA gyrase among the quinolones studied. In the case of Pseudomonas aeruginosa-infected corneal ulcers, ophthalmic gatifloxacin 0.3% is at least as effective as ciprofloxacin when given less frequently. Fluorescein retention scores showed a trend favoring gatifloxacin.

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Analysis of mouse insulin-2 mRNA from cultured islets and MIN6 cells [3]
After groups of 50 islets were cultured with or without gatifloxacin for 3 days, poly(A)+ RNAs were isolated using a Poly(A)Pure kit and first strand cDNAs were synthesized by SuperScript™ II Reverse Transcriptase system according to the manufacturer's instructions. TaqMan™ quantitative polymerase chain reaction (PCR) assay for mouse Insulin-2 (mIns-2) was performed using forward and reverse mIns-2-specific primers and probes in an ABI PRISM™ 7000 Sequence Detection System. The results are expressed as the ratio of mIns-2 mRNA to mouse Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA. MIN6 cells were cultured in Dulbecco's Minimal Essential Medium supplemented with 25 mM glucose and 13% fetal bovine serum with or without gatifloxacin for 3 days. Total RNA (10 μg) prepared with TRIzol reagent was used for Northern blot analysis. Mouse β-actin mRNA was used for standardization. Insulin promoter activity was evaluated in MIN6 cells transfected with the human insulin promoter-luciferase reporter gene and cultured for three days with or without 100 μM gatifloxacin, using Dual-Luciferase Reporter Assay System according to manufacture's instructions. Mean values of luciferase activity relative to the gatifloxacin-untreated control were calculated from duplicate wells.

Animal/Disease Models: Female balb/c (Bagg ALBino) mouse, infected with Nocardia brasiliensis in the right hind footpad [4]
Doses: 100 mg/kg
Route of Administration: subcutaneous injection; 3 times a day; 30-day
Experimental Results: diminished mouse damage of production.

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Isolation of mouse pancreatic islets and islet culture [3]
Pancreatic islets were isolated from fed male C57Bl/6 mice aged 12–16 weeks by collagenase digestion method. For short term exposure, fresh islets were used. For long term exposure, islets were cultured with or without gatifloxacin in RPMI medium containing 10% fetal bovine serum and 11.1 mM glucose, and used after the indicated culture periods for subsequent experiments. In some experiments (Fig. 5), the islets were cultured in the presence of 20 or 100 μM gatifloxacin for one day, washed with gatifloxacin-free RPMI medium three times to remove remaining gatifloxacin in the culture medium, and then cultured for additional two days in gatifloxacin-free medium.

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For the animal assays, we utilized Nocardia brasiliensis HUJEG-1, which has been utilized in previous studies. The MICs of this strain, determined by the broth microdilution method, are 0.25 μg/ml for gatifloxacin and 0.12 μg/ml for linezolid. For the determination of the plasma levels of gatifloxacin and linezolid, several doses of these compounds were used. Linezolid was used at 10 mg/kg body weight, 25 mg/kg, and 50 mg/kg, and gatifloxacin at 50 mg/kg, 75 mg/kg, and 100 mg/kg. Eight- to 12-week-old female BALB/c mice were injected subcutaneously with the antimicrobials. For each dose tested, 27 mice were utilized; 24 were injected with the selected dose, and 3 mice were not injected to represent time zero. Next, 500-μl blood samples were taken from the infraorbital sinus of each mouse, which previously had undergone general anesthesia with ethylic ether. The samples were taken from groups of three mice each at the following time intervals: 0 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 10 h. After sample collection, the plastic tubes containing the blood were centrifuged and the plasma was separated and frozen at −70°C. Plasma concentrations were determined by using a previously validated high performance liquid chromatography method. For the therapeutic assays, 8- to 12-week-old female BALB/c mice were inoculated with 20 mg of Nocardia brasiliensis in the right hind footpad. Seven days later, the therapeutic assay was started. Groups of 15 mice each were used. One group was injected subcutaneously in the back with 0.1 ml of pyrogen-free saline; the rest were treated with either gatifloxacin at 100 mg/kg or linezolid at 25 mg/kg. All the treatments, including the saline solution, were given subcutaneously on the back three times per day during a 4-week period. The development of lesions in the footpad of the animals was scored by two independent readers as described previously. This system classifies the lesions from those animals presenting absolutely no lesions or inflammation as negative or zero and the lesions from animals presenting severe lesions extending above the metatarsal bones as 4+. Differences among the therapeutic groups were analyzed using the analysis of variance test and confirmed with the Dunnet analysis. [4]

Absorption, Distribution and Excretion
After oral administration, gatifloxacin is well absorbed in the gastrointestinal tract, with an absolute bioavailability of 96%. Metabolisms/Metabolites Gatifloxacin undergoes limited biotransformation in the human body; less than 1% of the dose is excreted in the urine as ethylenediamine and methylethylenediamine metabolites. Half-life: 7-14 hours

Toxicity Summary
Gatifloxacin's bactericidal effect stems from its inhibition of topoisomerase II (DNA gyrase) and topoisomerase IV, both essential enzymes for bacterial DNA replication, transcription, repair, and recombination. Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no clinical information regarding the use of gatifloxacin during lactation. Traditionally, fluoroquinolones are not used in infants due to concerns about adverse effects on developing joints. However, recent studies suggest the risk is minimal. Calcium in breast milk may prevent the absorption of small amounts of fluoroquinolones in breast milk, but there is currently insufficient data to confirm or refute this claim. Lactating women may use gatifloxacin, but monitoring for potential impacts on the infant's gut microbiota, such as diarrhea or candidiasis (thrush, diaper rash), is necessary. However, it is generally preferable to use other medications with known safety information. The risk to a breastfeeding infant from the mother's use of gatifloxacin-containing ear drops or eye drops is negligible. After using eye drops, to significantly reduce the amount of medication entering breast milk, press the tear duct at the corner of the eye for at least 1 minute, then blot away any excess medication with absorbent tissue.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding rate
20%

[1]. Inhibitory activities of gatifloxacin (AM-1155), a newly developed fluoroquinolone, against bacterial and mammalian type II topoisomerases.Antimicrob Agents Chemother. 1998 Oct;42(10):2678-81.

[2]. Antibacterial activity of gatifloxacin (AM-1155, CG5501, BMS-206584), a newly developed fluoroquinolone, against sequentially acquired quinolone-resistant mutants and the norA transformant of Staphylococcus aureus. Antimicrob Agents Chemother. 1998 Aug;42(8):1917-22.

[3]. Gatifloxacin acutely stimulates insulin secretion and chronically suppresses insulin biosynthesis. Eur J Pharmacol. 2006 Dec 28;553(1-3):67-72.

[4]. In vivo therapeutic effect of gatifloxacin on BALB/c mice infected with Nocardia brasiliensis. Antimicrob Agents Chemother. 2008 Apr;52(4):1549-50.

Gatifloxacin sesquihydrate belongs to the quinoline class of compounds. Gatifloxacin is a synthetic 8-methoxyfluoroquinolone compound with antibacterial activity against a variety of Gram-negative and Gram-positive bacteria. Gatifloxacin exerts its effects by inhibiting DNA gyrase (an enzyme involved in DNA replication, transcription, and repair) and topoisomerase IV (an enzyme involved in the allocation of chromosomal DNA during bacterial cell division). Gatifloxacin is a fluoroquinolone antibacterial agent and a DNA topoisomerase II inhibitor, used as an ophthalmic solution to treat bacterial conjunctivitis. See also: Gatifloxacin (note moved to). Gatifloxacin is a monocarboxylic acid, chemically named 4-oxo-1,4-dihydroquinoline-3-carboxylic acid, with a cyclopropyl group substituted at the nitrogen atom, and fluorine, 3-methylpiperazin-1-yl, and methoxy groups substituted at positions 6, 7, and 8, respectively. Gatifloxacin is a fourth-generation fluoroquinolone antibiotic. Like other drugs in its class, it inhibits bacterial topoisomerase type II. It has a dual action of anti-infection, inhibition of DNA topoisomerase (ATP hydrolase), and antibacterial activity. Gatifloxacin is a quinoline monocarboxylic acid, N-arylpiperazine, organofluorine compound, quinolone compound, and also a quinolone antibiotic. Gatifloxacin is an antibiotic belonging to the fourth-generation fluoroquinolone class. Its mechanism of action is the inhibition of bacterial DNA gyrase and topoisomerase IV. In 1999, Bristol-Myers Squibb first marketed it under the brand name Tequin® for the treatment of respiratory infections. Gatifloxacin is available in tablets and various aqueous solutions for intravenous injection. It is also marketed as eye drops under the brand name Zymar®, sold by Allergan. Due to the high incidence of reported adverse events related to gatifloxacin-related glycemic abnormalities, and the higher probability of hyperglycemia and hypoglycemia in patients taking gatifloxacin compared to those taking macrolide antibiotics, the U.S. Food and Drug Administration (FDA) revoked approval for non-ophthalmic drug products containing gatifloxacin. Anhydrous gatifloxacin is a quinolone antibacterial drug. Gatifloxacin is a synthetic 8-methoxyfluoroquinolone compound with antibacterial activity against a variety of Gram-negative and Gram-positive bacteria. Gatifloxacin works by inhibiting DNA gyrase (an enzyme involved in DNA replication, transcription, and repair) and topoisomerase IV (an enzyme involved in the allocation of chromosomal DNA during bacterial cell division). Gatifloxacin is a fourth-generation fluoroquinolone antibiotic; like other drugs in its class, it inhibits bacterial DNA gyrase and topoisomerase IV. Bristol-Myers Squibb launched gatifloxacin in 1999 under the brand name Tequin® for the treatment of respiratory infections. The company licensed the drug from Kyorin Pharmaceutical Co., Ltd. of Japan. Allergan produces an eye drop formulation called Zymar®. Gatifloxacin is available in tablets and various aqueous solutions for intravenous injection. [Wikipedia]
A fluoroquinolone antibacterial agent and DNA topoisomerase II inhibitor, used as an ophthalmic solution for the treatment of bacterial conjunctivitis.

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Drug Indications
For the treatment of bronchitis, sinusitis, community-acquired pneumonia, and skin infections (abscesses, wounds) caused by Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, and Streptococcus pyogenes.
FDA Label
Mechanism of Action
Gatifloxacin's bactericidal action stems from its inhibition of topoisomerase II (DNA gyrase) and topoisomerase IV, two enzymes essential for bacterial DNA replication, transcription, repair, and degradation.
Pharmacodynamics
Gatifloxacin is a synthetic broad-spectrum 8-methoxyfluoroquinolone antibacterial drug, available orally or intravenously. It has bactericidal activity by binding to an enzyme called DNA gyrase, blocking bacterial DNA replication. DNA gyrase unwinds the DNA double helix, thus preventing DNA replication into two double helices. Notably, this drug has an affinity for bacterial DNA gyrase that is 100 times greater than that for mammalian DNA gyrase. Gatifloxacin is a broad-spectrum antibiotic effective against both Gram-positive and Gram-negative bacteria. This product is intended only for the treatment or prophylaxis of infections confirmed or highly suspected to be caused by bacteria. We determined the inhibitory activities of gatifloxacin against Staphylococcus aureus topoisomerase IV, Escherichia coli DNA gyrase, and HeLa cell topoisomerase II, and compared them with the inhibitory activities of several quinolone drugs. The inhibitory activities of quinolones against these type II topoisomerases were significantly correlated with their antibacterial activity or cytotoxicity (correlation coefficients [r] = 0.926 for Staphylococcus aureus, r = 0.972 for Escherichia coli, and r = 0.648 for HeLa cells). Gatifloxacin exhibited potent inhibitory activity against bacterial type II topoisomerases (IC50 for Staphylococcus aureus topoisomerase IV was 13.8 μg/ml; IC50 for Escherichia coli DNA gyrase was 0.109 μg/ml), but among the quinolones tested, it showed the lowest activity against HeLa cell topoisomerase II (IC50 265 μg/ml). There was a significant correlation between the inhibitory activities of quinolone drugs on Staphylococcus aureus topoisomerase IV and Escherichia coli DNA gyrase (r = 0.969). However, their inhibitory activity on HeLa cell topoisomerase II was not correlated with their inhibitory activity on either of these two bacterial enzymes. Gatifloxacin had an IC50 value of 19 against HeLa cell topoisomerase II, which was more than 2400 times higher than its IC50 values against Staphylococcus aureus topoisomerase IV and Escherichia coli DNA gyrase. These ratios are higher than those of other quinolone drugs, indicating that gatifloxacin has higher selectivity for bacterial type II topoisomerases. [1]

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Through sequential screening of four Staphylococcus aureus strains with multiple fluoroquinolone drugs, mutants of the first to fourth steps were obtained, and alternating mutations in the grlA and gyrA genes were observed. The increase in gatifloxacin MIC accompanying these mutation steps indicates that the main targets of gatifloxacin in wild-type and the first, second, and third step mutants are wild-type topoisomerase IV, wild-type DNA gyrase, single mutant topoisomerase IV, and single mutant DNA gyrase, respectively. Gatifloxacin's activity against the second step mutant (grlA gyrA; gatifloxacin MIC range 1.56 to 3.13 μg/ml) is comparable to tosufloxacin and superior to norfloxacin, ofloxacin, ciprofloxacin, and sparfloxacin; its activity against the third step mutant (grlA gyrA grlA; gatifloxacin MIC range 1.56 to 6.25 μg/ml) is the strongest, indicating that among the quinolones tested, gatifloxacin has the strongest inhibitory activity against single mutant topoisomerase IV and single mutant DNA gyrase. In addition, gatifloxacin screened out resistant mutants from wild-type and step-2 mutants at a low frequency. Gatifloxacin had potent antimicrobial activity against Staphylococcus aureus strain NY12 overexpressing NorA (norA transformant) (MIC 0.39 μg/ml), but slightly lower than its antimicrobial activity against the parental strain SA113. The increase in MIC value of quinolone drugs tested against NY12 strain was negatively correlated with the hydrophobicity of quinolone drugs (correlation coefficient -0.93; P < 0.01). Therefore, the slight decrease in the antimicrobial activity of gatifloxacin can be attributed to its high hydrophobicity. These properties of gatifloxacin may explain its good antimicrobial activity against clinical isolates of quinolone-resistant Staphylococcus aureus carrying grlA, gyrA and/or norA mutations. [2]

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Gatifloxacin can cause hypoglycemia and hyperglycemia in diabetic and non-diabetic patients. Recent reports indicate that gatifloxacin stimulates insulin secretion by inhibiting ATP-sensitive potassium channels (K(ATP) channels) in pancreatic β-cells. Gatifloxacin-induced hypoglycemia is associated with co-administration of sulfonylureas and typically occurs immediately after administration. We found that gatifloxacin acutely stimulates insulin secretion in mouse islets, while glibenclamide has an additive effect on gatifloxacin-induced insulin secretion. On the other hand, gatifloxacin-induced hyperglycemia usually takes several days to develop. We also demonstrated that long-term use of gatifloxacin reduces islet insulin levels by inhibiting insulin biosynthesis, a process that may be related to gatifloxacin-induced hyperglycemia. Furthermore, discontinuation of gatifloxacin improves insulin secretion response. These data elucidate the different mechanisms by which gatifloxacin induces hyperglycemia and hypoglycemia and suggest close monitoring of blood glucose levels during gatifloxacin administration, especially in elderly patients with renal insufficiency, undiagnosed diabetes, or other metabolic disorders. These findings have significant implications for clinical practice due to the increased risk of potentially life-threatening glycemic abnormalities during gatifloxacin treatment. [3] In this study, we evaluated the effect of gatifloxacin on the progression of Nocardia brasiliensis infection in experimental mice, using linezolid as a control. Gatifloxacin was administered subcutaneously at a dose of 100 mg/kg body weight every 8 hours for 4 weeks. This compound was as effective as linezolid in reducing lesion formation. [4]

C38H50F2N6O11

804.834

804.35

C, 56.71; H, 6.26; F, 4.72; N, 10.44; O, 21.87

180200-66-2

Gatifloxacin;112811-59-3; Gatifloxacin hydrochloride;121577-32-0; 121577-32-0; 316819-28-0 (mesylate); 180200-66-2 (sesquihydrate); 404858-36-2 (hemihydrate); 1190043-25-4; 1189946-71-1

5282384

Typically exists as solid at room temperature

1.386 g/cm3

607.8ºC at 760 mmHg

321.4ºC

6.43E-23mmHg at 25°C

4.555

7

19

8

57

653

0

C1=C4C(=C(OC)C(=C1F)N2CC(NCC2)C)N(C3CC3)C=C(C4=O)C(O)=O.C5=C8C(=C(OC)C(=C5F)N6CC(NCC6)C)N(C7CC7)C=C(C8=O)C(O)=O.O.O.O

RMJMZKDEVNTXHE-UHFFFAOYSA-N

InChI=1S/2C19H22FN3O4.3H2O/c2*1-10-8-22(6-5-21-10)16-14(20)7-12-15(18(16)27-2)23(11-3-4-11)9-13(17(12)24)19(25)26;;;/h2*7,9-11,21H,3-6,8H2,1-2H3,(H,25,26);3*1H2

1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic acid;trihydrate

Gatifloxacin sesquihydrate; 180200-66-2; 1-Cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid sesquihydrate; Gatifloxacin hydrate; Gatifloxacin [USAN]; L4618BD7KJ; 3-Quinolinecarboxylic acid, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-1-piperazinyl)-4-oxo-, hydrate (2:3); Gatifloxacin (USAN);

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.)