HK1020873B - Compositions comprising antifungal agent and acetate buffer - Google Patents

Description

Composition comprising an antifungal agent and an acetate buffer

Background

The present invention relates to compositions for the treatment and/or prevention of fungal infections.

There is an increasing need for new antifungal agents that are effective against conditional fungal infections caused by, for example, candida, aspergillus, cryptococcus and pneumocystis carinii. Current treatments such as amphotericin B and fluconazole are inadequate due to toxicity and resistance options. The compositions of the present invention are considered to be both safe and fungicidal.

The compositions of the present invention contain a compound which is useful as an antibacterial, particularly as an antifungal agent or as an antiprotozoal agent. As an antifungal agent, it can be used for controlling filamentous fungi and yeasts. Particularly suitable for use in the treatment of fungal infections in mammals, particularly those caused by candida species such as candida albicans, candida tropicalis, candida krusei, c. In particular, the compositions contain candida strains that have been found to be effective against amphotericin B and fluconazole-resistant. The composition is also useful for treating and/or preventing Pneumocystis carinii pneumonia, which is particularly susceptible to infection in immunocompromised patients such as those suffering from AIDS.

The compositions of the present invention are safe, stable, reproducible, lyophilized formulations that are particularly useful for administering antifungal agents to patients in need of such agents.

Summary of the invention

The present invention relates to a pharmaceutical composition comprising

a) A pharmaceutically effective amount of a compound having the formula:

b) a pharmaceutically acceptable amount of an effective acetate buffer to provide a pH of between about 4 and 7; and

c) a pharmaceutically acceptable amount of an excipient such as sucrose/mannitol to form a lyophilized cake.

In a particular embodiment, the composition of the invention comprises about 5-200mg/ml of Compound I or a pharmaceutically acceptable salt thereof, about 12.5mM-200mM acetate buffer, about 10-200mg/ml bulking agent and water. Preferably, the compositions of the present invention comprise about 30-50mg/ml of Compound I or a pharmaceutically acceptable salt thereof, about 20-60mM acetate buffer, about 30-70mg/ml of a bulking sugar or a combination of bulking sugars effective to form a lyophilized cake and water.

In a more specific embodiment, the formulation is prepared as a solution of 42mg/ml Compound I, 30mg/ml sucrose, 20mg/ml mannitol, 1.5mg/ml (25mM) acetic acid, adjusted to a pH of about 6 with sodium hydroxide. The solution was then filled into vials having a volume of 1.25ml and lyophilized. The lyophilized cake thus prepared contained 52.5mg of compound I, 62.5mg of sugar and 31.25 μ M of acetate buffer per vial. The lyophilized cake was then reconstituted by dilution with 21ml of diluent. 20ml was aspirated from the dilution and transferred to a 200ml infusion bag. The patient is then infused with a solution containing about 0.25mg/ml of Compound I, about 0.03mM or 0.15mM acetate buffer, about 0.3mg/ml bulking agent to prepare a composition having a pH of about 5-7.

Detailed description of the invention

The formulations of the present invention may improve the chemical stability of the pharmaceutical composition. The stability has the advantage that the shelf life of the pharmaceutical product can be extended. Previous formulations of tartrate buffers contained pharmaceutically significant amounts of unwanted degradation products. Formulations buffered with acetate may result in less degradants and more stable formulations. An extended shelf life of the pharmaceutical composition may provide significant economic advantages.

It has been found that compounds of the formula and pharmaceutically acceptable salts thereof are significantly more stable on storage when formulated in the presence of acetate buffer.

Compound I is claimed and described in U.S. patent No. 5378804. The preparation method is disclosed in the above patent and U.S. Pat. No. 5552521 (published by 9/3/1996).

The compound itself is highly labile and can degrade in a variety of ways including, but not limited to, hydrolysis, dimerization, and oxidation. However, this instability has previously been overcome using a means of lyophilizing the compound in a tartrate buffered formulation. However, the formulation is relatively stable while undergoing degradation at a relatively high rate.

Providing an acetate buffer instead, the lyophilized product is more stable and contains less unwanted degradants while extending the shelf life of the composition. Which makes it attractive as a commercial product.

The present invention also relates to a method of treating fungal infections caused by candida, aspergillus, and pneumocystis carinii comprising administering to a patient in need of such treatment a composition comprising a compound of formula I in an amount effective to treat the fungal infection. The invention further relates to a method for the prophylaxis of Pneumocystis carinii infections in a patient, which method comprises administering a prophylactic amount of a compound of formula I.

The acetate buffered formulations of the present invention include an effective amount of acetate to provide a pharmaceutically acceptable pH, e.g., to provide a pH environment in the range of 5 to 8, preferably about 6 to about 7. To provide an effective amount of acetate buffer to achieve the desired pH, appropriate amounts of sodium acetate and acetic acid or appropriate amounts of acetic acid and sodium hydroxide can be used. The buffer is generally present in the range of about 12.5mM to about 200mM, preferably about 20 to 60mM, more preferably about 25 to 50 mM.

Excipients such as bulking agents, i.e., excipient sugars, are used to provide an aesthetically pleasing, suitable lyophilized cake, a solid diluent for the active ingredient, and a suitable absorbent for moisture. Sugars useful in the present invention include sucrose, lactose, mannitol, or a combination thereof. It has been found that sucrose and mannitol provide a more stable formulation and form the composition into a pharmaceutically elegant cake. The excipient is generally present in an amount of about 10-200mg/ml, preferably about 30-70mg/ml, more preferably about 40-60 mg/ml.

The composition is not limited to the active ingredient, acetate buffer and filler, but may also include other pharmaceutically acceptable diluents, excipients or carriers. The formulation is suitable for long term storage in glass containers commonly used in the pharmaceutical industry, for example, in concentrated form in borosilicate glass type I containers of standard USP.

The compositions of the present invention are generally prepared according to the following steps:

1) dissolving a combination of bulking agents or drugs in water;

2) adding acetic acid and adjusting the pH to about 3.7 (if necessary);

3) adding compound I and dissolving it by adjusting the pH to about 5 to about 6 with a base;

4) the solution thus prepared was filtered and filled into freeze-dried vials and frozen at-50 ℃;

5) freeze-drying the frozen preparation at-20 deg.C, and second drying at 15 deg.C (2 days for complete cycle); and

6) the freeze-dried vials were sealed and stored at about 5 ℃.

The lyophilized formulation of the composition may be diluted with a suitable diluent at the time of administration to obtain a final concentration, e.g., about 5.0mg/ml, which is suitable for transfer into an infusion bag for use by a patient in need of the active ingredient.

The term "pharmaceutically acceptable salts" refers to non-toxic salts of the active ingredient, including mono-, di-or tri-acid forms, which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Pharmaceutically acceptable salts of salts suitable as acid addition salts and as anions providing quaternary salts are those derived from the following acids: such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, maleic acid, citric acid, acetic acid, tartaric acid, succinic acid, oxalic acid, malic acid, glutamic acid, pamoic acid, and the like, and include other acids related to the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science (66, 2 (1977)).

The term "pharmaceutically effective amount" refers to an amount of an active ingredient that can produce a biological or therapeutic response in a tissue, system, or animal that is being investigated by a researcher or clinician.

The compositions of the present invention may be administered to a patient in need of treatment and/or prevention of a fungal infection. They may be used in the treatment of candida species such as candida albicans, candida tropicalis, candida krusei, c. They may also be used in the treatment and/or prophylaxis of pneumocystis carinii pneumonia, which is particularly susceptible to infection in immunocompromised patients, such as those suffering from AIDS.

The dosage of using the composition of the present invention is selected according to various factors including type, species, age, weight, sex and medical condition of the patient; the severity of the disease being treated; the route of administration; renal and hepatic function of the patient; the particular active ingredient or salt thereof used. The ordinarily skilled physician can readily determine and prescribe the pharmaceutically effective amount required to prevent the development of an antagonistic or arresting disease.

For intravenous administration, the most preferred dosage of the active ingredient is from about 1.67 to about 33 μ g/kg/min, and the infusion rate is about 200 ml/hour. To administer this amount of active ingredient, the composition of the invention should contain 0.025-0.50mg/ml of active ingredient (based on a 50kg body weight patient).

The active ingredient, compound I, is generally prepared according to the following:

first compound II of the formula:

reduction to give compound III of the formula:

this is subsequently converted into compound IV of the formula:

it is then stereoselectively converted into compound I by replacing the phenylthio group.

In another method, compound II is reacted with thiophenol to provide compound IV-a of the formula:

subsequently, compound I is reacted withV-a is reduced to compound IV of the formula:

it is then stereoselectively converted into compound I by replacement with phenylthio.

Preparation of Compound I

a) Synthesis and isolation of Compound III

Compound II (15.9g, 89% area purity, 3.4% water (by weight), 0.0128mol) was added to anhydrous THF (0.64L) and the suspension was dried to < 10% water (mol) by refluxing over 3A molecular sieves. The mixture was reconstituted to original volume by the addition of additional anhydrous THF and the suspension was cooled to below 4 ℃ using an ice/water/methanol bath.

Anhydrous BH was added over 10 minutes₃·SMe₂(10.91g, 0.144mol), the reaction mixture was maintained at 0-4 ℃. The reaction was monitored by HPLC until the ratio of starting material to product was 1: 1 indicating the end of the reaction (3.5 h). At 4 hours, the mixture was cooled to-12 ℃ and the reaction was quenched slowly with 2N HCl (0.036L). The solution was diluted to 1.14L with water. The analytical yield of compound III was 6.60g (47%).

The quenched solution was diluted to 4L and loaded onto a media-pressure column of LiChroprep RP-C18 adsorbent (158 g). After loading, the column was washed with 1.2L of water, the amine was eluted with 1.9L of 1: 4 (vol.) acetonitrile: water, and then the amine was eluted with 3.8L of 1: 3 (vol.) acetonitrile: water.

The rich fractions (cuts) (> 80% area) were combined and diluted with water to 1: 7.3 (vol) acetonitrile: water (ca. 1.7L). The compound was applied to the same column as above, and the column was washed with 0.57L of water. The desired compound was eluted with 0.57L of methanol. The rich fractions (> 85% area) were combined, rotary evaporated and concentrated under static high vacuum to give 6.81g (87% pure by weight, 6.8% water by weight) containing 5.92g of compound III (where R1 is dimethyltridecyl) hydrochloride isolated in 43% yield.

b) Preparation of phenyl sulfide (Compound IV)

Compound III (5.80g, 0.00533mol) was added to 0.23L of anhydrous acetonitrile, cooled to-5 ℃ at which time thiophenol (3.10g, 0.028mol) was added. TFA (36g, 24.5ml, 0.308mol) was added over at least 20 minutes to maintain the temperature of the reaction mixture below 0 ℃. The reaction was maintained at-10 ℃ -0 ℃ until HPLC analysis showed < 3% (area) of starting material (3.75 h). At this point, cooled water (0.56L) was added slowly (1h) while cooling the reaction mixture maintaining the temperature below 5 ℃. The analytical yield d of the alpha-and beta-phenyl sulfide adduct as trifluoroacetate was 4.82g (71%).

The solution was applied to the same column as described in step a, the column was washed with water (0.57L), and then the adsorbed organic compound was eluted with methanol (0.5L). Rotary evaporation and static high vacuum concentration of the rich fraction. 7.20g (purity 57% by weight, 5.1% by weight of water)) of crude phenyl sulfide trifluoroacetate are obtained as amorphous foamy solid. The yield of phenyl sulfide from the correct separation procedure was 4.10g (61%) as a 93: 7 diastereomer mixture of alpha-and beta-aminal.

c) Conversion of Compound IV to Compound I-1

Crude phenyl sulfide triflate (8.4g crude, 57% pure by weight, 0.00377mol) was added to ethylenediamine (24ml) with stirring at room temperature. The resulting solution was stirred for 1.5 hours for complete replacement, then methanol (40ml) was added followed by acetic acid (45ml), maintaining the temperature below 25 ℃ with ice bath cooling. Resulting in a thick slurry. Water (160ml) was added to dissolve the slurry and the aqueous layer was extracted by gentle shaking with hexane (75 ml). The hexane layer was back extracted with water (40ml) and the combined aqueous layers were filtered through a porous medium in a sintered glass funnel and then purified by preparative HPLC using a 50mm diameter C18 column using 22% acetonitrile/78% aqueous 0.15% acetic acid as eluent. The enriched fraction was freeze-dried to give 4.2g of 85% by weight of pure compound I-1 as the diacetate salt in 78% yield from the isolation step.

d) Crystal of Compound I-1

The above solid (2.3g) was dissolved in ethanol (25ml), followed by addition of water (2.7 ml). The solution was filtered through a sintered glass funnel to remove foreign substances. To the filtrate was added acetic acid (0.14ml), followed by slow addition (1.75h) of ethyl acetate (14 ml). The solution was seeded and aged for 1 h. The remainder of the ethyl acetate (32ml) was added over 5h and left for a further 1 h. The crystallized solid was collected on a sintered glass funnel and washed with ethanol/ethyl acetate/water (6 ml/9ml/0.5ml, respectively). The wet cake was dried under a stream of nitrogen to give 1.91g (1.75g analytical, 88% recovery) of the diacetate salt of compound I-1.

Example 1

Preparation of formulation 1 the amount of Compound I42 mg/ml sucrose 30mg/ml mannitol 20mg/ml acetic acid 1.5mg/ml sodium hydroxide was adjusted to pH 5-6.2

The filling volume is 0.875ml-1.8ml

In general, a 25ml volumetric flask is charged with 0.75g of sucrose and 0.5g of mannitol, about 17.5ml of water, 0.5ml of 75mg/ml acetic acid solution and 42mg/ml of Compound I. The solution was mixed and the pH of the solution was adjusted to 6 with 1M sodium hydroxide. The volume was adjusted with water and the pH was ensured. The solution was filtered through a Millex-GV syringe filter and filled into 10ml tubular glass vials in an amount of 1.75ml per vial. The bottle was partially stoppered with a freeze-dried plug and freeze-dried to give a solid freeze-dried cake at the bottom of the bottle.

The lyophilized preparation was diluted with 10.5ml, 10ml was aspirated, and diluted to 200ml to a final concentration of 0.25mg/ml before administration to patients.

Additional formulations containing the following components (each prepared as a solution at a concentration of 40-42mg/ml active ingredient) were prepared according to the above method:

TABLE 1

Examples	Buffer solution	Candy(s)
			2	Tartrate 50mM (7.5mg/ml)	Is free of
3	Tartrate 50mM (7.5mg/ml)	Lactose (30mg/ml) mannitol (20mg/ml)
			4	Tartrate 50mM (7.5mg/ml)	Mannitol (50mg/ml)
5	Acetate 25mM (1.5mg/ml)	Lactose (30mg/ml) mannitol (20mg/ml)
			6	Tartrate 50mM (7.5mg/ml)	Sucrose (50mg/ml)
7	Acetate 25mM (1.5mg/ml)	Sucrose (50mg/ml)
			8	Acetate salt 50mM (3.0mg/ml)	Lactose (30mg/mnl) mannitol (20mg/ml)

The formulations were stored as a freeze-dried at 5 ℃ and tested for stability every about 4 weeks. Stability and formation of degradants were determined by gradient HPLC using standard methods known to those skilled in the art.

Formulations 1, 5, 7 and 8 were surprisingly found to be significantly more stable than the other formulations and showed significantly less unwanted degradants.

Sequence listing (1) general information (i) applicants: nerrkar, Maneesh

Hunke，William A

Kaufman, MichaelJ (ii) title of the invention: antifungal composition (iii) number of sequences: 1(iv) communication address:

(A) the addressee: elliott Korsen

(B) Street: box 2000, 126 e lincoln Ave.

(C) City: rahway

(D) State: NJ

(E) The state is as follows: united states of America

(F) And (3) post code: 07065(v) computer readable form:

(A) media type: flexible disk

(B) A computer: IBMPC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patent in Release #1.0, version #1.30(vi) data of the present application

(A) Application No.:

(B) submission date:

(C) and (4) classification: (viii) attorney/attorney profiles

(A) Name: korsen, Elliott

(B) Registration number: 32705

(C) Reference/profile No.: 19636(ix) telecommunications data

(A) Telephone: 908-594-5493

(B) Faxing: 908-594-4720(2) SEQ ID NO: 1, information: (i) sequence characteristics:

(A) length: 6 amino acids

(B) Type (2): amino acids

(C) Chain type: is unknown

(D) Topology: cyclic (ii) molecular type: peptide (iii) hypothesis: (iii) none (iv) antisense strand: none (xi) order description: SEQ ID NO: 1Xaa Thr Xaa Xaa Xaa Xaa 15

Claims (21)

1. A pharmaceutical composition for intravenous administration to a patient comprising

a) A pharmaceutically effective amount of a compound having the formula:

b) a pharmaceutically acceptable amount of an excipient effective to form a lyophilized cake; and

c) a pharmaceutically acceptable amount of acetate buffer effective to achieve a pharmaceutically acceptable pH, which is 4-7.

2. The pharmaceutical composition of claim 1 comprising

A pharmaceutically acceptable amount of compound I;

the excipient is a filler or a combination of fillers in the range of 10-200 mg/ml; and

a pharmaceutically acceptable amount of acetate buffer effective to obtain a pH between 4 and 7.

3. The composition of claim 2, comprising 5-200mg/ml of compound I or a pharmaceutically acceptable salt thereof, 12.5mM-200mM acetate buffer, 10-200mg/ml of bulking agent, and water.

4. The composition of claim 3, wherein said pharmaceutically acceptable salt is an acid addition salt.

5. The composition of claim 3, comprising 30-50mg/ml of compound I or a pharmaceutically acceptable salt thereof, 20-60mM acetate buffer, 30-70mg/ml of a bulking sugar or a combination of bulking sugars effective to form a lyophilized cake and water.

6. The composition of claim 5, comprising 42mg/ml of Compound I or a pharmaceutically acceptable salt thereof, 25mM acetate buffer, 30mg/ml sucrose, 20mg/ml mannitol and water.

7. The composition of claim 5, comprising 42mg/ml of Compound I or a pharmaceutically acceptable salt thereof, 50mM acetate buffer, 30mg/ml sucrose, 20mg/ml mannitol and water.

8. Use of a composition according to claim 1 in the manufacture of a medicament for the treatment and/or prevention of a fungal infection in a mammal.

9. The use of claim 8, wherein the mammal is a human.

10. The use of claim 8 or 9, wherein the fungal infection is an infection caused by candida spp.

11. The use of claim 8, wherein the fungal infection is an infection caused by Aspergillus.

12. The use of claim 8, wherein the fungal infection is an infection caused by Pneumocystis carinii.

13. Use of a composition according to claim 6 in the manufacture of a medicament for the treatment and/or prevention of fungal infection in a mammal.

14. The use of claim 13, wherein the fungal infection is an infection caused by candida.

15. The use of claim 13, wherein the fungal infection is an infection caused by aspergillus.

16. The use of claim 13, wherein the fungal infection is an infection caused by Pneumocystis carinii.

17. Use of a composition according to claim 7 in the manufacture of a medicament for the treatment and/or prevention of fungal infection in a mammal.

18. The use of claim 17, wherein the fungal infection is an infection caused by candida.

19. The use of claim 17, wherein the fungal infection is an infection caused by aspergillus.

20. The use of claim 17, wherein the fungal infection is an infection caused by Pneumocystis carinii.

21. A process for preparing a pharmaceutical composition comprising a compound having the formula or a pharmaceutically acceptable salt thereof, the process comprising the steps of:

a) dissolving 30-70mg/ml of filler or drug combination in water;

b) adding 20-60mM acetic acid and adjusting pH to 3.7;

c) adding and dissolving 30-50mg/ml of a compound of formula I and then adjusting the pH to 5-6 with a base;

d) filtering the solution containing the compound of formula I and filling the lyophilization flask;

e) freezing the filled lyophilization flask containing the filtered solution to-50 ℃;

f) lyophilizing said filled lyophilization vial with a freezing solution at a temperature of about 15 ℃ prior to-20 ℃ to obtain a pharmaceutical composition comprising a compound of formula I; and

g) the stoppered lyophilized vials containing the pharmaceutical compositions of the compound of formula I were stored at 5 ℃.