LEVOCARRIMYCIN, PHARMACEUTICAL COMPOSITIONS, PREPARATION METHODS AND USES THEREOF

• US 20130150316A1
• Filed: 05/25/2011
• Published: 06/13/2013
• Est. Priority Date: 05/25/2010
• Status: Active Grant

• Current Assignee: Shenyang Fuyang Pharmaceutical Technology Co., Ltd.
• Original Assignee: Shenyang Toglian Group Company Limited
• Inventors: Jiang, Yang, Hao, Yuyou
• Granted Patent: US 9,089,586 B2
• Time in Patent Office: Days
• US Class Current: 514/30
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/7048 having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin , digitoxin or digoxin
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: C07H 17/08 Hetero rings containing eight or more ring members, e.g. erythromycins
• CPC Class Codes: C12P 19/62 the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Abstract

The present invention relates to levocarrimycin, its pharmaceutical compositions, preparation methods and application. Levocarrimycin is a mixture of isovalerylspiramycin III, II and I as main components and contains some isobutyrylspiramycin III and II, butyrylspiramycin III and II, propionylspiramycin III and II, as well as acetylspiramycin III and II, among which, the content of isovalerylspiramycin III is no less than 30 wt %, the total content of isovalerylspiramycin III, II and I is no less than 60 wt %, and the content of acylspiramycin is 80-98 wt %. Specific optical rotation of said levocarrimycin is [α]_D=−52°˜−57° in the solution of 0.02 g/ml chloroform at temperature of 25° C. The present invention also relates to the crystalline compound of isovalerylspiramycin III, II or I in levocarrimycin, and pharmaceutical compositions containing the said levocarrimycin. In present invention, the active components in levocarrimycin or its pharmaceutical compositions have optical activity and excellent anti-infective effect.

Claims

1-21. -21. (canceled)

22. A levocarrimycin, wherein said levocarrimycin is a mixture of isovalerylspiramycin III, II and I as main components and contains certain isobutyrylspiramycin III and II, butyrylspiramycin III and II, propionylspiramycin III and II, as well as acetylspiramycin III and II, among them the content of the isovalerylspiramycin III is not less than 30 wt %, the total content of isovalerylspiramycin III, II and I is no less than 60 wt %, and the content of acylspiramycin is 80-98 wt %, preferably 85˜

98 wt %, further preferably 90˜

98 wt % and most preferably 95˜

98 wt %;

the specific optical rotation of said levocarrimycin is [α

]_D=−

52°

˜

−

57°

in the solution of 0.02 g/ml chloroform at temperature of 25°

C., preferably, −

54°

˜

−

56° and

further preferably, −

55°

.

23. The levocarrimycin according to claim 22, wherein said levocarrimycin also contains spiramycin III and other components, among them the content of spiramycin III is no more than 1.0%, and the total content of other components is 2.0˜

19 wt %, preferably 2.0˜

14.0 wt %, further preferably 2.0˜

9.0 wt % and most preferably 2.0˜

4.0 wt %.

24. The levocarrimycin according to claim 23, wherein said melting point of said levocarrimycin is 112˜

122°

C., preferably 114˜

120°

C. and further preferably 116˜

118°

C.

25. The levocarrimycin according to claim 22, wherein said isovalerylspiramycin III is a crystal compound of levoisovalerylspiramycin III, the isovalerylspiramycin II is a crystal compound of levoisovalerylspiramycin II, or the isovalerylspiramycin I is a crystal compound of levoisovalerylspiramycin I;

when the isovalerylspiramycin III is a crystal compound with III of levoisovalerylspiramycin, said crystal compound measured by the X-ray powder diffraction with Cu—

Ka ray has characteristic peaks of 2θ

at 8.0°

, 10.0°

, 11.2°

, 11.7°

, 16.4°

, 19.1°

, 19.6°

, 20.0°

, 21.4°

, 22.9°

, 23.6° and

29.4°

;

when the isovalerylspiramycin II is a crystal compound with II of levoisovalerylspiramycin, said crystal compound measured by the X-ray powder diffraction with Cu—

Ka ray has characteristic peaks of 2θ

at 10.0°

, 11.6°

, 16.4°

, 17.3°

, 19.1°

, 21.2°

, 22.1°

, 22.7°

, 26.4°

, 26.9°

, 27.5° and

31.5°

;

when the isovalerylspiramycin I is a crystal compound with I of levoisovalerylspiramycin, said crystal compound measured by the X-ray powder diffraction with Cu—

Ka ray has characteristic peaks of 2θ

at 7.6°

, 8.0°

, 10.0°

, 11.4°

, 16.4°

, 17.0°

, 17.5°

, 17.9°

, 19.5°

, 22.7°

, 23.7° and

24.4°

.

26. A pharmaceutical composition of levocarrimycin, wherein said pharmaceutical composition of levocarrimycin contains the levocarrimycin according to claim 22 and pharmaceutically acceptable carrier.

27. The pharmaceutical composition of levocarrimycin according to claim 26, wherein the content of said levocarrimycin is 10˜

90 wt % of the pharmaceutical composition, preferably, 25˜

75 wt %, and further preferably, 40˜

60 wt %.

28. The pharmaceutical composition of levocarrimycin according to claim 27, wherein the pharmaceutical composition of levocarrimycin exists in preparations applicable for pharmaceutical purpose, and the preparations are liquid, solid, semisolid, or gas preparations, said liquid preparations comprise injection, infusion solution, solution, mixture, sirup, tincture, colloid, aromatic water, glycerite, colloid solution, mucilage, suspension, or emulsion;

said solid preparations comprise power injection, lyophilized powder injection, tablet, capsule, powder, granula, pill, sublimed preparation, or pellicle;

the semisolid preparations comprise ointment, plaster, suppository, extract or gel; and

the gas preparations comprise aerosol or spray.

29. The pharmaceutical composition of levocarrimycin according to claim 26, wherein the content of said levocarrimycin is 10˜

1500 mg per unit formulation, preferably, 100˜

1000 mg per unit formulation and further preferably, 200˜

500 mg per unit dosage form.

30. A preparation method for levocarrimycin according to claim 22, which includes culture, fermentation and extraction process, wherein said culture and fermentation is:

culture the cloned fungal strains WSP-195 produced by spiramycin containing 4″

-isovaleryl transferase gene on an slant culture-medium, inoculate it in seed medium, then inoculate it in fermentation medium after culturing, and control the fermentation process by pH regulator;

control the pH at 6.0˜

9.0, preferably, 6.0˜

8.0, and further preferably, 6.0˜

7.5;

the variation curves for pH with time show three continuous phases, the first phase satisfies formula y₁=k₁x₁+6.0, where 0.02275≦

k₁≦

0.1364, 0<

x₁≦

22;

the second phase satisfies y₂=k₂x₂+b₂, where −

0.0735≦

k₂≦

0, 6.5<

b₂≦

10.62, 22≦

x₂≦

56; and

the third phase satisfies formula y₃=k₃x₃+b₃, where 0<

k₃≦

0.0078, 6.06≦

b₃<

6.5, 56≦

x₃≦

120.

31. The preparation method according to claim 30, wherein said pH regulator is at least one or combination of glucose, citric acid, acetic acid, hydrochloric acid, ammonia, sodium hydroxide and potassium hydroxide, preferably, one or combination of glucose, citric acid, acetic acid and ammonia;

further preferably, one or combination of glucose and ammonia.

32. The preparation method according to claim 30, wherein said extraction process is:

process the fermentation liquor with aluminum sulfate to obtain the filtrate, adjust pH to 8.5˜

9.0, extract with butyl acetate, wash the butyl acetate extract with non-saline water and 1% NaH₂PO₄ respectively, then extract with water of pH 2.0˜

2.5 to obtain aqueous extract, adjust the pH to 4.5˜

5.5, volatilize and eliminate the residual butyl acetate to obtain hydrous extract, filter and adjust the pH to 8.5˜

9.0, obtain precipitate, wash precipitate with purified water and dry it to obtain levocarrimycin.

33. The preparation method according to claim 30, wherein said slant culture-medium contains 2% soybean cake meal, 1% glucose, 3% starch, 0.5% CaCO₃, 0.4% NaCl and 2% agar.

34. The preparation method according to claim 30, wherein said seed medium contains 1.5% soybean cake meal, 3.0% starch, 0.4% NaCl, 0.5% CaCO₃, 0.3% peptone and 0.05% KH₂PO₄.

35. The preparation method according to claim 30, wherein said fermentation medium contains 0.5% glucose, 6.0% starch, 0.5% yeast powder, 2.0% fish meal, 0.6% NH₄NO₃, 1.0% NaCl, 0.5% CaCO₃, 0.05% KH₂PO₄, 0.1% MgSO₄, 0.5% soybean oil and 0.02% defoaming agent.

36. The preparation method according to claim 30, wherein the culture on the slant culture-medium lasts for 8˜

15 days at temperature of 28˜

38°

C.;

the culture on the seed medium lasts for 40˜

80 hours at temperature of 25˜

30°

C.; and

the fermentation on the fermentation medium lasts for 72˜

120 hours at temperature of 26˜

30°

C.

37. The preparation method according to claim 30, wherein said preparation method also comprises the following steps:

a) Separate and purify the levocarrimycin to obtain levoisovalerylspiramycin I, II or III;

b) Recrystallize the levoisovalerylspiramycin I, II or III to obtain the crystal compound of levoisovalerylspiramycin I, II or III;

c) Eliminate the acetonitrile in the residual levocarrimycin after separating and purifying levoisovalerylspiramycin I, II or III in step a) through rotary evaporation, then extract with 1 time amount of ethyl acetate, and eliminate the ethyl acetate in the extract through rotary evaporation to obtain paste sample;

re-dissolve the obtained sample with petroleum ether, and eliminate the petroleum ether through rotary evaporation to obtain the levocarrimycin;

d) Mix the crystal compound of levoisovalerylspiramycin I, II or III obtained in step b) with the levocarrimycin obtained in step c) to obtain the levocarrimycin, in which, the isovalerylspiramycin I, II or III is the crystal compound of levoisovalerylspiramycin I, II or III.

38. The preparation method according to claim 37, wherein said separation and purification in step a) is:

Purify the levocarrimycin obtained in the preliminary separation with a preparative high performance liquid chromatography, prepare chromatographic column with ODS, use acetonitrile and ammonium acetate buffer solution as the mobile phase in a gradient elute;

record the separated UV spectrogram through UV detection, and collect the target peaks of levoisovalerylspiramycin I, II or III components;

Chromatographic column;

ODS preparative chromatographic column;

Mobile phase;

acetonitrile (A), 100 mM ammonium acetate solution (B);

Gradient condition;

adopting linear gradient for 0˜

60 min, A is 25%-65%; and

61˜

90 min, A is 65%˜

90%;

Flow velocity;

260 mL/min;

Injection volume;

10 mL;

Sampling concentration;

0.5 g/mL;

Measurement wavelength;

231 nm;

Way of collecting;

collection via UV triggering;

Collect the sample of levoisovalerylspiramycin I according to the retention time 44.759 min of levoisovalerylspiramycin I;

or collect the sample of isovalerylspiramycin II according to the retention time 43.34 min of isovalerylspiramycin II;

or collect the sample of levoisovalerylspiramycin III according to the retention time 48.009 of levoisovalerylspiramycin III;

then eliminate acetonitrile through rotary evaporation, extract with 1 time amount of ethyl acetate, and eliminate the ethyl acetate in the extract through rotary evaporation to obtain paste sample;

re-dissolve the obtained sample with petroleum ether, and eliminate the petroleum ether through rotary evaporation to obtain the white solid powder of levoisovalerylspiramycin I, II or III.

39. The preparation method according to claim 38, wherein

when the isovalerylspiramycin I is a crystal compound of levoisovalerylspiramycin I, the crystal compound is obtained through the following recrystallization process:

dissolve the white solid powder of levoisovalerylspiramycin I in the mixed solvent of absolute methanol, absolute ethyl alcohol and anhydrous acetone, then add pure water while stirring, after that, reduce the temperature to 5°

C.˜

15°

C. while stirring continuously, to obtain the crystal compound of the levoisovalerylspiramycin I, in which the volume ratio of ethyl acetate to absolute ethyl alcohol to anhydrous acetone in the mixed solvent is 1;

0.1˜

10;

0.5˜

1, preferably, 1;

2˜

8;

0.8˜

1;

when the isovalerylspiramycin II is a crystal compound of levoisovalerylspiramycin II, the crystal compound is obtained through the following recrystallization process;

dissolve the white solid powder of levoisovalerylspiramycin II in the mixed solvent of absolute methanol, absolute ethyl alcohol and anhydrous acetone, then add pure water while stirring, after that, reduce the temperature to 5°

C.˜

15°

C. while stirring continuously, to obtain the crystal compound of the levoisovalerylspiramycin II, in which the volume ratio of absolute methanol to absolute ethyl alcohol to anhydrous acetone in the mixed solvent is 1;

0.1˜

10;

0.5˜

1, preferably, 1;

2˜

8;

0.8˜

1;

when the isovalerylspiramycin III is a crystal compound of levoisovalerylspiramycin III, the crystal compound is obtained through the following recrystallization process;

dissolve the white solid powder of levoisovalerylspiramycin III in the mixed solvent of absolute methanol, absolute ethyl alcohol and anhydrous acetone, then add pure water while stirring, after that, reduce the temperature to 5°

C.˜

15°

C. while stirring continuously, to obtain the crystal compound of the levoisovalerylspiramycin III, in which the volume ratio of absolute methanol to absolute ethyl alcohol to anhydrous acetone in the mixed solvent is 1;

0.1˜

10;

0.5˜

1, preferably, 1;

2˜

8;

0.8˜

1.