DNA gyrase

Sitafloxacin (DU6859a) monohydrate exhibits antibacterial activity against wild-type ATCC 49619, gyrA mutant SP39, and parC mutant 1026523 streptococcus pneumoniae stran, with MIC values of 0.03, 0.12, and 0.06 mg/L, respectively[1]. For quinolone-susceptible strains of Streptococcus pneumoniae, sitafloxacin (DU6859a) monohydrate has antibacterial activity with MIC values of 0.03 mg/L for the EG 00218 strain and 0.03 mg/L for the EG 00093 strain, respectively[1]. DNA gyrase and topoisomerase IV (TopoIV) are inhibited by sitafloxacin (DU6859a) monohydrate, with >IC50s of 4.38 and 3.12 mg/L, respectively[1].

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DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC(50) ratios against DNA gyrase and topoisomerase IV. Conclusions: DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants [1].

Sitafloxacin monohydrate (DU6859a; 12.5–100 mg/kg; ir; daily for 4 weeks; female BALB/c mice) possesses antibacterial properties. M. There was no sign of footpad swelling and ulcerans cells could be separated from the infected footpads[2].

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Efficacy of a new fluoroquinolone, sitafloxacin (DU-6859a), against Mycobacterium ulcerans was evaluated in vivo using the mouse footpad system. The growth of M. ulcerans in mouse footpads was completely inhibited when mice were fed with sitafloxacin at a dose of 25 mg/kg body weight per day; on the other hand similar effects were observed with ofloxacin at a dose of 100 mg/kg body weight per day. In the presence of rifampin, the above dose of sitafloxacin could be reduced by 75% to achieve total inhibition, while, under similar circumstances, the dose of ofloxacin could be reduced by only 50%. Either used singly or in combination with rifampin, the effects of sitafloxacin were bactericidal. The results suggest that sitafloxacin should be evaluated as a chemotherapeutic agent against M. ulcerans infection [2].

Determination of inhibitory activities against DNA gyrase and topoisomerase IV from S. pneumoniae [1]
DNA gyrase supercoiling and TopoIV decatenation assays were carried out as described previously22,23 with minor modifications. For the DNA gyrase supercoiling assay, the reaction mixture contained 40 mM Tris–HCl (pH 7.5), 30 mM KCl, 5 mM MgCl2, 1 mM spermidine, 4 mM ATP, 1 mM dithiothreitol (DTT) and 20 µg of BSA per mL. One unit of each purified GyrA and GyrB was incubated with 0.1 µg of relaxed pBR322 plasmid DNA for 60 min at 37°C. For the TopoIV decatenation assay, the reaction mixture contained 40 mM Tris–HCl (pH 7.5), 20 mM KCl, 5 mM MgCl2, 0.5 mM ATP, 1 mM DTT and 50 µg of BSA per mL. The decatenation activity was performed using 1 U of each purified ParC and ParE as well as 0.4 µg of catenated kinetoplast DNA (k-DNA) for 60 min at 37°C.

Bacterial strains and antimicrobial susceptibility testing [1]
Two quinolone-susceptible strains of S. pneumoniae (EG00093 and EG00218), which did not harbour mutations in the QRDRs of the gyrA, gyrB, parC and parE genes, and two first-step parC mutants (60 and 1026523) were used for mutant selection experiments. In 2002, EG00093 and EG00218 were selected as representative strains of quinolone-susceptible S. pneumoniae from a surveillance collection in Japan. Strain 60 harbouring a single QRDR mutation in parC (Ser-79→Tyr) and isolate SP39 with a gyrA mutation (Ser-81→Phe) are both mutants of ATCC 49619. Strain 1026523 is a clinical isolate harbouring a combination of mutations within the QRDRs of parC (Ser-79→Phe) and parE (Ile-460→Val), which was obtained from the GLOBAL surveillance study in 2003 (Focus Bio-Inova Inc., Herndon, VA, USA). The determination of MICs was performed at least in duplicate, according to a standard agar dilution method.20

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Determination of mutant selection frequency and MPC [1]
The determination of mutant selection frequency and MPC experiments were performed concurrently. The method for measuring the mutation frequency and MPC was a modification of that described previously.21 Each isolate was grown for 12 h on heart infusion agar containing 5% sheep blood at 35°C. Several colonies were then suspended in sterile PBS at a turbidity equivalent to that of a 0.5 McFarland standard (1 × 108 cfu/mL). The suspension (20 mL) was divided equally into four flasks each containing 500 mL of fresh BHIY broth (total volume of 2 L) and incubated for an additional 6 h without shaking. Cultures were then concentrated 10- to 30-fold by centrifugation (5000 g for 30 min at 20°C) to yield a concentration of 1010–1011 cfu/mL. Aliquots of 100 µL of the bacterial suspension (containing 109–1010 cfu) were spread onto BHI agar plates containing 10% horse-defibrinated blood with multiples of the MICs for each quinolone. The plates containing quinolones were incubated at 35°C for 72 h, and the antibiotic-free plates were incubated at 35°C for 16–24 h. The frequency of mutant selection was calculated as the ratio of the number of resistant colonies at 72 h to the number of cfu plated. The MPC of each quinolone was determined as the lowest concentration that prevented the growth of resistant colonies when more than 1010 bacteria were spread on agar plates and incubated for 72 h at 35°C.

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PCR amplification of QRDRs and DNA sequence analysis [1]
The presence of mutations in the QRDRs of the gyrA, gyrB, parC and parE genes was investigated by PCR. The primer sequences used to amplify the gyrA QRDR were as follows: SPGA3, 5′-GTCAATCTGACAAAGGAGATGAAGG-3′ (position 25 to 49) and SPGA6, 5′-CAATCTCTGTACGAGAACGTAGGAC-3′ (position 715 to 739). For the amplification of the gyrB QRDR, SPGB3: 5′-TTACCAATCGCCTCTTCAGTGAAGC-3′ (position 1070 to 1094) and SPGB4: 5′-CTTCCAACCTTGACACCATAGATTGG-3′ (position 1621 to 1646) were used; for the amplification of the parC QRDR, SPPC1: 5′-GGCTTTGTATCTTATGTCTAACATTC-3′ (position −14 to 27) and SPPC6: 5′-AAACTGCAGCATCTATGACCTCAGC-3′ (position 548 to 573) and for the amplification of the parE QRDR, SPPE3: 5′-AGTTGTGGATGGAATAGTGGCT-3′ (position 1061 to 1084) and SPPE4: 5′-GGACATCTTGTAAGAGGTGGGAG-3′ (position 1615 to 1638). Amplification was performed in a total volume of 50 µL containing 1 µL of template DNA, 4 µL of each deoxynucleoside triphosphate (2.5 mM each), 5 µL of 10× Ex Taq DNA polymerase buffer, 1 µL of each primer (10 pmol/µL) and 0.25 µL of Ex Taq DNA polymerase (5 U/μL). Amplification conditions were 94°C for 2 min, 35 cycles of 98°C for 20 s, 62°C for 2 min and 72°C for 3 min, with a final extension of 3 min at 68°C. DNA sequencing of PCR products was carried out with an ABI PRISM 3100 Avant Genetic Analyzer in accordance with the manufacturer's instructions.

Animal/Disease Models: BALB/c female mice [2]
Doses: 12.5, 25, 50 and 100 mg/kg
Route of Administration: po (oral gavage); one time/day for 4 weeks
Experimental Results: Inhibition of Mycobacterium ulcerans and Mycobacterium ulcerans Cell growth.

[1]. Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae. J Antimicrob Chemother. 2008 Jul;62(1):98-104.

[2]. Activities of sitafloxacin (DU-6859a), either singly or in combination with rifampin, against Mycobacterium ulcerans infection in mice. J Chemother. 2003 Feb;15(1):47-52.

Sitafloxacin is a member of quinolines, a quinolone antibiotic and a fluoroquinolone antibiotic.

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Objectives: DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones. Methods: MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC(50)) values. [1]

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In conclusion, the balanced dual-targeting activity of DC-159a and sitafloxacin against DNA gyrase and TopoIV might provide a greater potential to minimize the development of quinolone resistance. DC-159a and sitafloxacin could be potential therapeutic options for pneumococcal pneumonia caused by quinolone-susceptible and less quinolone-susceptible isolates. [1]

C19H18CLF2N3O3

409.8178

409.1

127254-12-0

Sitafloxacin hydrate;163253-35-8;Sitafloxacin hydrochloride;346607-37-2;Sitafloxacin monohydrate;163253-37-0

461399

Typically exists as solid at room temperature

1.6±0.1 g/cm3

629.2±55.0 °C at 760 mmHg

334.3±31.5 °C

0.0±1.9 mmHg at 25°C

1.699

0.87

2

8

3

28

761

3

O=C(C1=CN([C@H]2[C@@H](F)C2)C3=C(C=C(F)C(N(C[C@H]4N)CC54CC5)=C3Cl)C1=O)O

PNUZDKCDAWUEGK-CYZMBNFOSA-N

InChI=1S/C19H18ClF2N3O3/c20-14-15-8(17(26)9(18(27)28)5-25(15)12-4-10(12)21)3-11(22)16(14)24-6-13(23)19(7-24)1-2-19/h3,5,10,12-13H,1-2,4,6-7,23H2,(H,27,28)/t10-,12+,13+/m0/s1 SMILES

7-((S)-7-amino-5-azaspiro[2.4]heptan-5-yl)-8-chloro-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

Sitafloxacin; Gracevit; Sitafloxacin anhydrous; DU 6859; DU-6859-a; DU 6859-A; Sitafloxacin [INN]; DU 6859; DU-6859a; DU 6859A; 3GJC60U4Q8

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.)