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Title:
AMPHOTERICIN B SALT
Document Type and Number:
WIPO Patent Application WO/2007/063335
Kind Code:
A2
Abstract:
The invention provides a salt of amphotericin B and meglumine in which the molar ratio of amphotericin B to meglumine is about 1:1.

Inventors:
KLAVENESS JO (NO)
Application Number:
PCT/GB2006/004514
Publication Date:
June 07, 2007
Filing Date:
December 04, 2006
Export Citation:
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Assignee:
DRUG DISCOVERY LAB AS (NO)
COCKBAIN JULIAN (GB)
KLAVENESS JO (NO)
International Classes:
C07H17/08; A61K31/133; A61K31/13; A61K9/00
Foreign References:
GB1430886A1976-04-07
US4002741A1977-01-11
US5942495A1999-08-24
Attorney, Agent or Firm:
COCKBAIN, Julian et al. (St Bride's House 10 Salisbury Square, London EC4Y 8JD, GB)
Download PDF:
Claims:

Claims:

1. A salt of amphotericin B and meglumine in which the molar ratio of amphotericin B to meglumine is about 1:1.

2. A salt as claimed in claim 1 which in solution in water at 50mg amphotericin/mL at 25 0 C has a pH below 9.0.

3. A composition of matter consisting essentially of a salt of amphotericin B and meglumine in which the molar ratio of amphotericin B to meglumine is about 1:1.

4. An injectable pharmaceutical composition comprising an aqueous solution containing dissolved therein amphotericin B and meglumine, or ions thereof, in a molar ratio of about 1:1.

5. An isotonic composition as claimed in claim 4 having an amphotericin B content of 90 to 140 mg/mL.

6. The use of a salt or composition of matter as claimed in any one of claims 1 to 3 for the manufacture of an injectable medicament for use in the treatment by parenteral injection of systemic fungal infection or other systemic infections responsive to amphotericin B.

7. A process for the preparation of a salt of amphotericin B and meglumine which comprises mixing amphotericin B and meglumine in solution in a solvent in approximately equimolar amounts followed by solvent removal or precipitation.

8. A method of treatment of a human or non-human mammalian subject having a systemic infection responsive to amphotericin B which method comprises parenterally injecting said subject with an effective amount of an aqueous solution containing dissolved therein amphotericin B and meglumine, or ions thereof, in a

molar ratio of about 1:1.

9. A method as claimed in claim 8 wherein said subject is HIV-positive.

10. A method as claimed in claim 8 wherein said infection is leishmaniasis.

Description:

Amphotericin B Salt

This invention relates to a salt of amphotericin B and meglumine, pharmaceutical compositions containing said salt and methods of treatment using said salt.

Amphotericin B, also known as fungizone, is a macrolide polyene antibiotic produced by Streptomycetes nodosus M4575 having a formula C 47 H 73 NOi 7 and the molecular weight 924 Da. It is insoluble in water except at strongly acid or base pH values and even then has a solubility of about 0.1 mg/mL. It has broad-spectrum fungicidal activity and may for example be used in the treatment of fungal, helminthic and protozoal infections associated with Candida, Crγptococcus, Aspergillus, Ajellomyces, Fusarium, Coccidiodes, Leishmania, Schistosomia, Torulopsis, Giardia, Trichomonas, and Entamoeba species. In particular it has been used in treatment of opportunistic infections of AIDS patients, of leishmaniasis and of other patients with compromised immune systems. It is indeed the current gold standard for the treatment of systemic fungal infection.

Due to its poor water-solubility, amphotericin B is generally administered as a colloidal formulation, e.g. as a complex with cholesteryl sulphate, or in a lipid or liposomal formulation. Injection of the liposomal or lipid formulation is the preferred treatment for the developing world disease leishmaniasis, which affects an estimated 1.5 million new victims per year globally, and is referred to for example by Sundar et al Annals of Internal Medicine 127:133-137(1997). Such formulations may however cost 10 to 30 times the simple colloidal formulation and in practice are thus inaccessible to the majority of potential patients.

The problem of the water-solubility of amphotericin B was addressed by Kulbakh et al in US-A-4002 741 and US-A-4007166 who proposed the use of a complex of meglumine and amphotericin B which they termed amphoglucamine (CAS 58722- 78-4). Kulbakh et al {supra) prepared amphoglucamine by reacting meglumine with amphotericin B in a molar ratio of at least 3 : 1 to yield a product containing

amphotericin B and meglumine in a molar ratio of at least 1:3. This excess of meglume was considered necessary in order to produce a water soluble material, e.g to avoid precipitation out of amphotericin B.

Meglumine, N-methylglucamine, is a secondary amine polyol which has the formula C 7 Hi 7 NO 5 and a molecular weight of 195 Da. It is known to form salts with acids and complexes with metals and has for example been used as the counterion for charged metal complexes used as injectable contrast agents in MR.imaging (e.g. Magnevist® from Schering AG).

We have now produced a novel salt of amphotericin B and meglumine, which does not contain any significant molar excess of meglune and yet which is water-soluble and suitable for parenteral administration for the treatment of systemic fungal infection and other systemic infections responsive to amphotericin B, in particular opportunist fungal infection of AIDS patients and leishmaniasis, especially visceral leishmaniasis.

Thus viewed from one aspect the invention provides a salt of amphotericin B and meglumine in which the molar ratio of amphotericin B to meglumine is about 1 : 1 (e.g. 1:1 to 1:1.1).

The novel salt, hereinafter "Amphotericin B meglumate", has a mole ratio of 1 : 1 ; however, it may be presented with a slight excess of meglumine, or less advantageously a slight excess of amphotericin B.

The salt of the invention has an activity (ED/mg) against Candida albicans of from 500 to 650, especially 550 to 620, as opposed to the value of 750 for amphoglucamine.

Moreover, the salt of the invention forms significantly less alkaline aqueous solutions than does amphoglucamine, e.g. at a concentration of 50mg amphotericin B/mL in water at 25 0 C, the pH is below 9.0, typically about 8.6, as opposed to the

value of 9.3 for amphoglucamine. This makes the salt more suitable for parenteral injection than amphoglucamine.

Viewed from a further aspect the invention provides a composition of matter consisting essentially of a salt of amphotericin B and meglumine in which the molar ratio of amphotericin B to meglumine is about 1 : 1 (e.g. 1 : 1 to 1 : 1 , 1).

Viewed from a still further aspect the invention provides an injectable pharmaceutical composition comprising an aqueous solution containing dissolved therein amphotericin B and meglumine, or ions thereof, in a molar ratio of about 1 : 1 (e.g. 1:1 to 1:1.1).

Viewed from a still further aspect the invention provides the use of a salt of amphotericin B and meglumine in which the molar ratio of amphotericin B to meglumine is about 1:1 (e.g. 1 :1 to 1:1.1) for the manufacture of an injectable medicament for use in the treatment by parenteral injection of systemic fungal infection or other systemic infections responsive to amphotericin B.

Viewed from a yet still further aspect the invention provides a method of treatment of a human or non-human mammalian subject having a systemic infection responsive to amphotericin B (e.g. a fungal or protozoal infection) which method comprises parenterally injecting said subject with an effective amount of an aqueous solution containing dissolved therein amphotericin B and meglumine, or ions thereof, in a molar ratio of about 1 :1 (e.g. 1:1 to 1:1.1).

The injection solution used in the method of the invention should be sterile and preferably pyrogen- free and may be prepared using the salt in sterile form, a form which is preferred. The solution may if desired contain further components beyond water and the amphotericin B meglumate complex, e.g. physiologically tolerable osmolality adjusting agents (for example to render the solution isotonic, such as sodium chloride for example), preservatives, buffers, further active agents, etc. Typically the solutions may contain up to 0.9% wt. of sodium chloride as an

osmolality adjuster.

The injection solution according to the present invention is preferably isotonic. A solution of amphoglucamine is always hypertonic if the concentration of amphotericin is above 60 to 70 mg per ml (calculated as the 1 :1 salt with meglumine) based on the fact that this complex contains 3 moles excess of meglumine. The present 1 :1 salt can be formulated as an isotonic solution in concentrations up to 150 mg per ml, and is preferably presented in such a solution at an amphotericin B concentration of 90 to 140 mg/mL.

The amphotericin B: meglumine salt of and used according to the present invention is not identical to amphoglucamine which, as discussed above, contains amphotericin B and meglumine in a mole ratio of less than 1:3. The amphoglucamine product is hygroscopic while the amphotericin B meglumate salt of the present invention is not hygroscopic. Further, the amphoglucamine is, as it is described in the prior art, a complex with an undefined excess of meglumine. A product with an undefined amount of a compontent like in amphoglucamine is not a well-defined product as the 1 :1 salt is , from a regulatory point of view. Moreover the use of amphoglucamine in aqueous solution for parenteral injection has not been proposed.

The aqueous solutions of the salt used according to the invention will typically contain the salt at a concentration of 0.5 to 150 mg/mL, preferably 1 to 120 mg/mL, especially 50 to 80 mg/mL. Doses of up to 50 mg amphotericin B for an adult human are preferred. These compositions may be administered at amphotericin B dosages typical for the current lipid or liposomal formulations of amphotericin B, e.g. 0.1 to 8 mg/kg bodyweight/day, preferably 1 to 5 mg/kg/day. The treatment may typically be carried out for up to 80 days.

The salt according to the invention may if desired be stabilized by autoclaving. It is thought that the salt is more stable to exposure to autoclaving temperatures than the prior art amphoglucamine complex.

Administration maybe by infusion or routine injection, e.g. bolus injection, for example intravascular, intramuscular, or subcutaneous. Infusion, e.g. over 1 to 2 hours, is preferred.

The amphotericin B megluminate salt according to the invention may be prepared in solution in solvents such as DMSO, DMF, N-methylpyrrolidone (NMP) and/or alcohols (e.g. Ci -6 alkanols such as methanol) or mixtures thereof. The use of NMP and/or alcohols is especially preferred as extensive work-up to remove solvents such as DMSO or DMF is avoided. The reaction is effected by using approximately equimolar amounts of amphotericin B and meglumine. The salt can be recovered by addition of an ether (e.g. diethyl ether) or a ketone (e.g. acetone) to cause precipitation; however it is preferably recovered by solvent removal, e.g. by evaporation or freeze drying. Thus viewed from a further aspect the invention provides a process for the preparation of a salt of amphotericin B and meglumine which comprises mixing amphotericin B and meglumine in solution in a solvent in approximately equimolar amounts, e.g. a molar ratio of 1 :1 to 1 :1.1, followed by solvent removal or precipitation. The resultant salt is substantially non-hygroscopic, unlike amphoglucamine.

The invention will now be illustrated further with reference to the following non- limiting Examples.

Example 1

Synthesis of Amphotericin B meglumate (1 :1)

A solution of N-methyl-D-glucamine (117 mg, 0.6 mmol) in water (1.5 mL) was added to a stirred solution of amphotericin B (462 mg, 0.50 mmol) in dimethyl sulfoxide (15 mL) heated to 5O 0 C. After 15 minutes, the mixture was filtered through a #3 glass-sinter funnel; the filtrate was poured into a mixture of diethyl ether (45 mL) and acetone (105 mL). The mixture was kept in a refrigerator to cool and filtered through a #3 glass-sinter funnel. The gummy residue was washed with acetone and dried over silica gel at room temperature and 8 mm Hg pressure for two

hours. The resulting brown solid was dissolved in de-ionized water (20 mL) to give a non-turbid amber solution that was freeze-dried to give 0.3O g (54%) of a fluffy yellow solid.

1 H NMR (300 MHz) of the product in DMSO-J 6 showed a 1 : 1 ratio between the N- methyl group of N-methyl-D-glucamine at 2.53 ppm and the methyl group of amphotericin B at 0.90 ppm

Example 2

Solubility of Amphotericin B meglumate in water Amphotericin B meglumate (50 mg) (from Example 1) was dissolved in distilled water (1 ml). The solution was a clear yellow solution without any turbidity.

Example 3

Hygroscopisity of freeze-dried Amphotericin B meglumate Amphotericin B meglumate (16.4 mg) (from Example 1) was kept open in a vial at room temperature (23-24°C) and ambient humidity (34-38%) in the dark for several days. The sample did not gain any appreciable weight. The product was not hygroscopic.

Example 4

Stability of Amphotericin B meglumate in solution

Aqueous solutions of amphotericin B meglumate (5 mg/mL) were kept at room temperature and at 4O 0 C for 11 days in the dark. No sign of degradation was observed in these samples (visual inspection and HPLC analysis).

Example 5

Pharmaceutical composition

350 mg Amphotericin B meglumate (1:1) (produced according to Example 1) is dissolved in 70 mL water for injections and filled under sterile conditions into a vial which is then sealed.

Example 6 Antifungal activity

MIC values {Candida albicans) and {Candida kruseϊ) for amphotericin B meglumate and amphotericin B in water and DMSO was determined in vitro using standard methods.

Results: MIC {Candida albicans) for amphotericin B in DMSO: 0.25 microgram pr ml MIC {Candida kruseϊ) for amphotericin B in DMSO : 1.0 microgram pr. ml

MIC {Candida albicans) for amphotericin B meglumate in DMSO : 0.25 microgram amphotericin B pr. ml

MIC {Candida krusei) for amphotericin B meglumate in DMSO : 1.0 microgram amphotericin B pr. ml

MIC {Candida albicans) for amphotericin B meglumate in water : 0.25 microgram amphotericin B pr. ml

MIC {Candida krusei) for amphotericin B meglumate in water: 1.0 microgram amphotericin B pr. ml MIC values for amphotericin B in water were not determined since amphotericin B is not soluble in water.

Example 7

Synthesis of Amphotericin B meglumate (1 :1) in NMP A solution of N-methyl-D-glucamine (98 mg,0.5 mmol) in water (15 ml) was added in one portion to a solution of amphotericin B (462 mg,0.5 mmol) in N- methylpyrrolidone (15 ml). The solution was stirred for 5 minutes.

The mixture was freeze-dried over night. The residue was dissolved in water (70 ml) and centrifuged for 10 minutes at 4000 rpm. The aqeuous solution was feeze-dried. The title compound was isolated as a yellow solid material. Yield 0.54 g (96 %)

Example 8 pH in aqeuous solution of Amphotericin B meglumate (1:1) Amphotericin B meglumate (prepared in Example 7) was dissolved in water ( 50 mg amphotericin per ml). The pH of the solution at 25 0 C was 8.6.

Example 9 pH in aqeuous solution of Amphoglucamine

Amphotericin meglumine complex (1 :3) was prepared according to US 4,002,741. The complex was dissolved in water (50 mg amphotericin B per ml). The pH in the solution was 9.3 at 25°C.