ANTIBIOTIC FIIRV 104/18 COMPLEX AND THE ISOLATED INDIVIDUAL FACTORS THEREOF

Abstract

An antibiotic FIIRV 104/18 complex comprising factors F793, F795, F797, F813 and F683, of formula (I), wherein R represents a glycosidic radical as defined in the specification, the isolated individual factors, a method for their production by cultivation of Dactylosporangium FIIRV sp. 104/18 DSM 32068 under aerobic conditions, and pharmaceutical formulations containing them as active ingredient and their use in the treatment of bacterial infections, caused by bacteria of at least one of the genera Enterococci, Streptococci, Staphylococci and Moraxella, including bacteria of multi resistant strains, in particular Staphylococcus spp., Streptococcus spp, and Enterococcus spp. resistant to methicillin and/or vancomycin.

##STR00001##

Claims

1-6. (canceled)

7. A process for the preparation of an antibiotic FIIRV 104/18 complex of formula (I), ##STR00009## comprising factors F793, F795, F797, F813 and F683 wherein R represents ##STR00010## or one or more of the isolated individual factors: factor F793 of formula ##STR00011## factor F795 of formula ##STR00012## factor F797 of formula ##STR00013## factor F813 of formula ##STR00014## or factor F683 of formula ##STR00015## the process comprising (a) cultivating Dactylosporangium FIIRV sp. 104/18 DSM 32068, or a variant or mutant thereof maintaining the ability to produce the antibiotic of formula (I), under aerobic conditions, in an aqueous nutrient medium containing usable sources of carbon, nitrogen and inorganic salts; (b) isolating the resulting antibiotic of formula (I) from the whole fermentation broth, or from the separated mycelium or from the filtered or centrifuged fermentation broth; (c) purifying the isolated antibiotic of formula (I).

8. The process according to claim 7, wherein the strain Dactylosporangium FIIRV sp. 104/18 DSM 32068 is pre-cultured.

9. The process according to claim 7, wherein the isolation of the antibiotic FIIRV 104/18 complex of claim 1 is carried out by centrifuging or filtering the fermentation broth and recovering the antibiotic from the supernatant or filtered broth according to at least one technique selected from the group consisting of: extraction with a water-immiscible solvent, precipitation by adding a non-solvent or by changing the pH of the solution, absorption chromatography, partition chromatography, reverse phase partition chromatography, ion exchange chromatography and molecular exclusion chromatography, or a combination of two or more of said techniques.

10. The process according to claim 7, wherein the isolation of the antibiotic FIIRV 104/18 complex of claim 1 is carried out by separating the mycelium from the supernatant of the fermentation broth and extracting the mycelium with a water-miscible solvent whereby, after the removal of the spent mycelium, a water-miscible solution containing the crude antibiotic is obtained, which is processed to recover the antibiotic FIIRV 104/18 complex by means of at least one technique selected from the group consisting of: extraction with a water-miscible solvent, precipitation by adding a non-solvent or by changing the pH of the solution, absorption chromatography, partition chromatography, reverse phase partition chromatography, ion exchange chromatography and molecular exclusion chromatography, or a combination of two or more of said techniques.

11. The process according to claim 7, wherein the isolation of the antibiotic FIIRV 104/18 complex is carried out by separating the mycelium from the supernatant of the fermentation broth and extracting the mycelium with a water-immiscible solvent whereby, after the removal of the spent mycelium, a solution containing the crude antibiotic is obtained, which is subjected to further purification steps.

12. The process according to claim 7, wherein the isolation of the antibiotic FIIRV 104/18 complex is carried out on the whole microbial culture by adding an absorbing resin, maintaining the obtained mixture under stirring until the antibiotic product is almost entirely bound to the resin, separating the resin and mycelium by filtration or centrifugation and submitting said separated material to extraction with a water-miscible solvent, thus obtaining a solution of the antibiotic FIIRV 104/18 complex from which the product is recovered by at least one of the techniques selected from the group consisting of extraction with a water-miscible solvent, precipitation by adding a non-solvent or by changing the pH of the solution, absorption chromatography, partition chromatography, reverse phase partition chromatography, ion exchange chromatography, molecular exclusion chromatography and a combination thereof.

13. The process according to claim 7, wherein the individual factors F793, F795, F797, F813 and F683 are separated from the antibiotic FIIRV 104/18 complex by using a preparative HLPC method.

14. A pharmaceutical composition comprising the antibiotic FIIRV 104/18 complex or an isolated individual factor thereof as defined in claim 7 or a combination of one or more factors in any proportion and further comprising a pharmaceutical acceptable carrier.

15. (canceled)

16. The pharmaceutical composition according to claim 14, wherein the composition is in orally, topically, or parenterally administrable form.

17. The pharmaceutical composition according to claim 16, wherein the composition is in the form of a capsule, a tablet, a lozenge, a liquid solution, a liquid suspension, an aqueous solution, an aqueous suspension, an oily solution, an oily suspension, a hydrophobic base ointment, a hydrophobic base cream, a hydrophobic base lotion, a hydrophobic base paint, a hydrophobic base powder, a hydrophilic base ointment, a hydrophilic base cream, a hydrophilic base lotion, a hydrophilic base paint, and a hydrophilic base powder.

18.-21. (canceled)

22. The pharmaceutical composition according to claim 14, wherein the dosage range is comprised between 1 and 40 mg of active ingredient per Kg of body weight.

23. A method for treating a bacterial infection comprising administering to a patient in need thereof an effective amount of antibiotic FIIRV 104/18 complex or one or more of the individual factors thereof as defined in claim 7.

24. The method for treating a bacterial infection according to claim 23, wherein the bacterial infection is caused by bacteria of at least one of the genera selected from the group consisting of: Enterococci, Streptococci, Staphylococci and Moraxella.

25. The method for treating a bacterial infection according to claim 24, wherein the bacterial infection is caused by bacteria selected from the multi resistant strain of Staphylococcus spp., Streptococcus spp., and Enterococcus spp.

26. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0099] FIG. 1 shows the LC-MS (Liquid Chromatography-Mass Spectrometry) analysis of the factor F795.

[0100] FIG. 2 shows the full-scan low resolution spectrum of factor F795. The adducts ions are identified and assigned.

[0101] FIG. 3 represents the UV-visible spectrum of factor F795 dissolved in CH.sub.3CN:NH.sub.4COOH 1:1.

[0102] FIG. 4 represents .sup.1H-NMR spectrum of factor F795 recorded in CDCl.sub.3 at 300? K on a Bruker Avance 500 MHz spectrometer.

[0103] FIG. 5 represents the .sup.13C-NMR spectrum of factor F795 recorded in CDCl.sub.3 at 300? K on a Bruker Avance 500 MHz spectrometer.

[0104] FIG. 6 represents the overall stereostructure of factor F795 structure.

[0105] FIG. 7 shows the LC-MS (Liquid Chromatography-Mass Spectrometry) analysis of the Factor of F797.

[0106] FIG. 8 shows the full-scan low resolution spectrum of factor F797. The adducts ions are identified and assigned.

[0107] FIG. 9 represents the UV spectrum of factors F795, F797, F793, F683 dissolved in CH.sub.3CN:NH.sub.4COOH 1:1.

[0108] FIG. 10 shows the full-scan low resolution spectrum of factor F683. The adducts ions are identified and assigned.

[0109] FIG. 11 shows the full-scan low resolution spectrum of factor F793. The adducts ions are identified and assigned.

[0110] FIG. 12 shows the full-scan low resolution spectrum of antibiotic F813. The adducts ions are identified and assigned.

EXAMPLES

[0111] The Examples set forth below are for illustrative purposes only and are not intended to limit, in any way, the scope of the present invention.

Example 1

Analytical Methods and Materials

[0112] Unless otherwise noted, reagents and solvents are used as received from commercial suppliers. Proton nuclear magnetic resonance (NMR) spectra are obtained on Bruker Avance spectrometer at 500 MHz. Spectra are given in ppm (?) and coupling constants, J, are reported in Hertz. Tetramethylsilane (TMS) is used as an internal standard.

[0113] Mass spectra are collected using as LC-MS instrument a Thermo Scientific LTQ-xl instrument (Thermo Fisher, Scientific Inc. CA, USA) fitted with a ion trap electrospray ionization (ESI) source.

[0114] Accurate mass measurements are collected using a ESI-FT-ICR Solarix instrument (Bruker Daltonics, Germany, GmbH). HPLC analyses are obtained using a Luna C18(2) column (250?4.6 mm, Phenomenex, Torrance, Calif.) or a Symmetry C18 column 250?4.6 mm, (Waters; Milford Mass., USA) with Thermo Scientific Accela PDA detector or UV detection at 223 nm using a standard solvent gradient program where Phase A (A) is 10 mM ammonium formate pH 4.5, and Phase B (B) is CH.sub.3CN. Here below are reported the solvent gradient program of the two methods used (Method 1 or Method 2).

Method 1:

[0115]

TABLE-US-00004 Time Flow (min) (mL/min) % A % B 0 1.0 75 25 35 1.0 10 90 40 1.0 10 90 45 1.0 75 25

Method 2:

[0116]

TABLE-US-00005 Time Flow (min) (mL/min) % A % B 0 1.0 60 40 3 1.0 60 40 15 1.0 45 55 17.5 1.0 10 90 20.5 1.0 10 90 21 1.0 60 40

[0117] The preparative HPLC are carried out by using a Shimadzu AP-20 liquid chromatograph (Shimadzu Corporation, Japan). UV data are acquired at 254 nm and 360 nm and the below reported solvent gradient program is used: (A) is 20 mM ammonium formate pH 4.5, and B is CH.sub.3CN.

Preparative Method:

[0118]

TABLE-US-00006 Time Flow (min) (mL/min) % A % B 0 8.0 65 35 2 20.0 65 35 8 20.0 65 35 14 20.0 58 42 19 20.0 58 42 36 20.0 45 55 40 20.0 30 70 42 20.0 10 90 45 20.0 10 90 48 20.0 65 35

Example 2

Fermentation Method of Dactylosporangium FIIRV Sp. 104/18 DSM 32068

[0119] Dactylosporangium FIIRV sp. 104/18 DSM 32068 strain is maintained on oatmeal agar (ISP medium 3) plates for 3-4 weeks at 28? C. The microbial content of one plate is scraped with 5 mL sterile water and inoculated into 500 mL Erlenmeyer flasks containing 100 mL of seed medium (AF/MS) which is composed of (g/L): 20 g dextrose monohydrate, 2 g yeast extract, 8 g soybean meal, 1 g of NaCl and 4 g of CaCO.sub.3. The medium is prepared in deionized-water and pH adjusted to 7.3 prior to sterilization at 121? C. for 20 minutes. The inoculated flasks are grown at 28? C., on a rotatory shaker operating at 200 rpm. After 96-120 hours, 4% of this culture is inoculated into a second series of flasks containing the same fermentation medium and grown for another 14 days under the same conditions. The production of antibiotic FIIRV 104/18 complex is monitored by HPLC as previously described, after extraction of the mycelium with the 2 mL ethanol per gram of mycelium. The extraction is performed at room temperature under stirring for two hours.

Example 3

Recovery of Antibiotic FIIRV 104/18 Complex

[0120] The fermentation broth described in Example 2 is centrifuged to obtain 8 L of supernatant and 2 L of mycelium. Antibiotic FIIRV 104/18 complex is found both in the supernatant (A) and in the mycelium (B) and these fractions are processed separately. Alternatively, the whole strain culture can be added with adsorbing resin and processed as in method (C).

(A) To the supernatant of the centrifuged broth 300 mL of Diaion HP-20 polystyrenic resin are added; the mixture is stirred 2 hours at room temperature and then the resin is recovered, washed with 1 L ethanol: water 1:3 (v/v) and then eluted twice with 1 L ethanol stirring overnight at room temperature. The pooled eluted fractions containing antibiotic FIIRV 104/18 complex are concentrated to small volume on a rotary evaporator and then diluted with 500 mL water. The resulting suspension is extracted with 2?500 mL of ethyl acetate. The ethyl acetate extract is washed with an equal volume of water followed by an equal volume of saturated solution of NaCl. The ethyl acetate extract is further dried by passing over a bed of anhydrous sodium sulfate. Antibiotic FIIRV 104/18 complex is then crystallized from the ethyl acetate solution by evaporation under vacuum to small volume and/or addition of excess petroleum ether to give precipitation, and then recovered by filtration.
(B) 250 mL of acetonitrile are first added to 1.2 kg of centrifuged biomass (mycelium) and allowed to contact for 30 min at room temperature, then 2.4 L of ethyl acetate are added and the suspension is further stirred for 16 h at room temperature. The liquid containing water, acetonitrile and ethyl acetate is removed and concentrated to small volume on a rotary evaporator and then the resulting suspension is washed with 2 equal volumes of water to remove acetonitrile and then washed with an equal volume of a saturated NaCl solution. The washed ethyl acetate extract is then further dried with anhydrous sodium sulfate and the crude complex of factors is obtained after evaporation under vacuum.
(C) To 10 L of strain culture Diaion HP-20 polystyrenic resin (ratio 1:40 w/v) is added at the end of the fermentation period to adsorb the secreted secondary metabolites. The culture and resin are shaken at 215 r.p.m. for two additional hours. The resin and cell mass are collected by centrifugation or filtration through paper disks, and the residue is washed with deionized water to remove salts. The resin and cell mass are then resuspended and stirred with 2.5 L of ethanol for 2-4 hours at room temperature. The ethanol extract is centrifuged or filtered, the solid bulk is washed several times with 500 mL of ethanol and finally stirred with 500 mL of ethyl acetate for 30 minutes at room temperature, then filtered. Finally, the organic solvents are removed under vacuum to yield a crude extract. This is then processed as described in (B). The ethyl acetate extract is washed with an equal volume of water followed by an equal volume of saturated solution of NaCl. The ethyl acetate extract is further dried by passing over a bed of anhydrous sodium sulfate, then evaporated under vacuum.

Example 4

Recovery of Antibiotic FIIRV 104/18 Individual Factors: F793, F795, F797, F813 and F683

[0121] The individual factors may be separated from the antibiotic FIIRV 104/18 complex, prepared according to the procedure described in Example 3, by using the preparative HPLC method described in Example 1 (phase A: 20 mM ammonium formate pH 4.5, phase B: CH.sub.3CN). The separation is performed by using a column (250?21.2 mm) Phenomenex Luna-C18(2) (Phenomenex, Torrance Calif.). The antibiotic FIIRV 104/18 complex is dissolved in DMSO:H.sub.2O:CH.sub.3CN 1:1:1 (v/v). In these conditions, factors F683 and F813 co-elute with 40% of phase B and show a rt of 13.0 min; pure factor F797 elutes with 42% phase B at rt 17.5 min, factor F795 elutes with 48% phase B at 27.5 min and factor F793 elutes with 50% phase B at 30.5 min. The eluted fractions containing pure individual antibiotic FIIRV 104/18 factors F797, F795 and F793 are collected and concentrated under vacuum to aqueous phase. The water solutions are extracted with ethyl acetate and concentrated to minimal volume; petroleum ether is added and pure factors are precipitated and filtered. From 1.2 kg of mycelium, 175.5 mg of factor F795, 128.5 mg of factor F797 and 8.7 mg of factor F793 are recovered.

[0122] Factors F683 and F813 are separated and purified from the above reported mixed fractions by preparative HPLC using a Phenomenex Luna Pheny Hexyl 5? (250?10 mm) column. A sample of antibiotic FIIRV 104/18 F683 and F813 mixture is dissolved in DMSO:H.sub.2O:CH.sub.3CN 1:1:1 (v/v) and a 25 minutes linear gradient elution from 25% to 40% of phase B at 8 mL flow rate is performed. Phase B is CH.sub.3CN. Phase A is ammonium formate 20 mM pH4.5. Pure factor F683 elutes at 16.5 min rt with 36% phase B, and pure factor F813 elutes at 17.5 min rt with 37% phase B. The eluted fractions containing pure antibiotic FIIRV 104/18 factors F683 and F813 are collected and concentrated to aqueous phase under vacuum. Pure factors are obtained by extracting the aqueous phase with ethyl acetate, concentrating to minimal volume said extract, adding petroleum ether thereto and recovering the precipitated product by filtration. From 1.2 kg of mycelium, 11.1 mg of pure F683 and 2.7 mg of F813 are recovered.