WO2000047187A1

WO2000047187A1 - Serum albumin-based parenteral formulations of polyene macrolides

Info

Publication number: WO2000047187A1
Application number: PCT/CA2000/000146
Authority: WO
Inventors: Glen S. Kwon; Bong K. Yoo
Original assignee: Kinetana Inc.
Priority date: 1999-02-11
Filing date: 2000-02-10
Publication date: 2000-08-17
Also published as: AU2530700A

Abstract

The present invention provides a method for producing a polyene macrolide-human serum albumin solid dispersion having increased solubility over polyene macrolide alone or polyene macrolide mixed with human serum albumin. It also shows no loss in antifungal activity. The solid dispersion is produced by dissolving the polyene macrolide in a cosolvent; mixing in the human serum albumin solution; and freeze-drying the solution to form a solid dispersion. The solid dispersion is reconstituted prior to administration. Prior to freeze-drying, the composition may be incubated at elevated temperatures.

Description

SERUM ALBUMIN-BASED PARENTERAL FORMULATIONS OF POLYENE MACROLIDES

The present invention is directed to the field of polyene macrolides and. in particular, to polyene macrolides having antifungal activity.

Polyene macrolides such as amphotericin B and nystatin are key drugs for treating systemic fungal diseases. Such diseases are common with immunocompromised patients. These drugs act at a membrane level binding sterols and forming pores that lead to cell death. The selectivity of amphotericin B and nystatin for fungal cells over mammalian cells is due to preferred binding of ergosterol, the primary fungal sterol, over the mammalian sterol cholesterol. However, the selectivity of the drugs is poor and therefore the drugs are highly toxic. The second major drawback of the drugs is poor water solubility. As a result of these drawbacks, amphotericin B administered to patients results in side effects such as nephrotoxicity and cardiotoxicity.

Alternate strategies have been investigated to increase the solubility and specificity, for example, as shown in U.S. Patent No. 5,178,875, U.S. Patent No. 4,822,777, and U.S. Patent No. 5,389,373. One example is liposomal formulations, such as through phospholipid vesicles. Liposomes have a number of disadvantages. They are normally difficult to prepare reproducibly in bulk and can be unstable. Their size instability when stored in an aqueous medium is a major drawback to using such formulations. Typically, amphotericin B-liposome formulations which have an initial size distribution between 200-300 nm will spontaneously

form large liposome structures of up to several microns on long-term storage in an aqueous medium. Liposomes with sizes greater than about 1-2 microns are generally more toxic than smaller liposomes when administered parenterally.

While it is possible to produce an amphotericin B emulsion system by the admixture of commercial fat emulsion products with the commercial solubilized system of amphotericin B, this system is unstable. It produces a precipitate of the drug after this admixture and has poor stability if stored for more than a few hours. As well, the amphotericin B is not intercalated at the oil-water interface in the additive formulation.

Amphotericin B and nystatin easily form aggregates in water and aggregated species of the drugs have been shown to be responsible for their toxicity toward mammalian cells in vitro and in vivo. On the other hand, substantially or fully deaggregated or monomeric species of the drugs have been shown to be less toxic or even nontoxic for mammalian cells but still retain activity toward fungal cells. Ways of deaggregating these drugs are needed as a means of dissociating their toxicity. Detergents have been used to deaggregate amphotericin B. However, extremely high levels of detergents are required. At these high levels, they are too toxic for parenteral administration. The parenteral route of administration is used for polyene macrolides to treat systemic fungal disease since the drugs lack absorption through the oral

route.

Brezis in "Reduced Amphotericin Toxicity in an Albumin Vehicle", Journal of Drug Targeting, 1993, Vol 1, pp.185-189, discloses the use of bovine serum albumin in combination with a commercial solubilized system of amphotericin B. The result was a finding that the toxicity of amphotericin B may be reduced if combined with an albumin

vehicle. However, even though the toxicity of amphotericin B was reduced, the results showed that it was not reduced to clinically useful levels. As well, the resulting composition retained insufficient solubility to be clinically useful. Although Brezis indicates that the toxicity and solubility of amphotericin B may be improved using this method, they still remain at levels which made albumin a clinically insignificant vehicle of administration.

There therefore is a need for a means of producing a solid dispersion of a polyene macrolide to decrease its toxicity and improve its solubility while retaining its antifungal activity.

The present invention provides a composition containing polyene macrolides which retains the antifungal activity of the polyene macrolides but reduces its toxicity and improves its solubility while retaining the same or greater efficacy.

The present invention provides for a composition comprising polyene macrolide and serum albumin. The composition of the present invention is prepared in such a manner as to provide for an improved polyene macrolide composition which is a solid dispersion.

The present invention also provides for a method of preparing the polyene macrolide- serum albumin solid dispersion comprising the steps of combining the polyene macrolide with serum albumin and freeze-drying the polyene macrolide-serum albumin composition. The solid dispersion is reconstituted with a sterile aqueous vehicle prior to administration.

The present invention will be better understood with reference to the following figures

which illustrate preferred embodiments of the present invention and in which

Figure 1 shows the results of X-ray diffraction of nystatin, human albumin serum (HSA), and solid dispersions of nystatin and HSA; and

Figure 2 is a graph showing the hemolysis of pure nystatin and a solid dispersion of

nystatin and HSA. The present applicant has found that serum albumin can be used in combination with polyene macrolides to improve their solubility including amphotericin B and nystatin. It has also been found that human serum albumin can be used to obtain similar results as with bovine serum albumin. The resulting solid dispersion of human serum albumin and a polyene macrolide shows a lower toxicity, improved solubility, and deaggregation in vitro without any loss or a significant loss of antibiotic activity towards fungal cells.

It has also been found that by using a specific method to prepare the solid dispersions, the resulting polyene macrolide-serum albumin solid dispersions have improved solubility and decreased toxicity with no (significant) loss in antifungal activity when compared with the polyene macrolide alone or with the polyene macrolide simply mixed with serum albumin. Further, the compositions of the present invention are less aggregated as compared to polyene macrolides alone or with polyene macrolides simply mixed with serum albumin.

The method of the present invention comprises the steps of dissolving the polyene macrolide in a cosolvent and mixing it with serum albumin; freeze-drying the resulting composition; and reconstituting the composition under suitable conditions.

The polyene macrolide is dissolved in a cosolvent and then mixed with human serum albumin (HSA). The polyene macrolide is preferably pre-solubilized and can therefore bind the HSA more readily. It may be dissolved in any suitable cosolvent and examples of suitable cosolvents are dimethylacetamide and dimethylsulfoxide. The predissolution of the polyene macrolide also increased the ability of the polyene macrolide and the serum albumin to interact. The dissolved polyene macrolide is added to an aqueous solution of HSA.

The resulting composition is freeze-dried in accordance with well known industry

techniques, for example, as described in Peter Cameron ed., Good Pharmaceutical Freeze- Drying Practice, (Illinois; Interpharm Press, Inc., 1997) which is incorporated by reference. The composition may be freeze-dried at a low temperature with or without excipients such as a cryoprotectant. Preferably the temperature is below -40 °C. The water freezes first and may be pulled off with a high vacuum resulting in the concentration of the product. Other known methods of evaporation may be used including spray drying the composition at elevated temperatures.

After freeze drying, the solid dispersion is present in the amorphous state. The polyene macrolide in its natural state is present in both amorphous and crystalline form. After completing the steps of the present invention, no significant amount of a crystalline product appears to be present. The amorphous state provides one rationale for the increased solubility due to the lack of crystal energy in amorphous forms of drugs. In the solid dispersion of the present invention, the amorphous form of nystatin, for example, readily undergoes dissolution as compared with the crystalline form which undergoes slow dissolution.

The freeze-dried composition can be easily reconstituted with a sterile aqueous vehicle. After reconstitution, the composition reaches levels required for use in therapy. By using this method, the resulting composition is less toxic with no (significant) loss of antifungal activity and may result in an increase in antifungal activity. The method also provides for little or no loss of composition during the process of manufacture. The present method therefore provides an increase in the therapeutic index of polyene macrolides.

The method of the present invention may also include the additional step of incubating the polyene macrolide-serum albumin composition prior to the step of freeze-

drying. This incubation allows for the ready deaggregation of the polyene macrolide and binding to the serum albumin. The polyene macrolide and serum albumin composition is incubated at elevated temperatures at neutral or alkaline pH for '/_ - 1 hour. Preferably, the incubation temperature is above 25 °C. This incubation step also increases the ability of the polyene macrolide and the serum albumin to interact and to ensure that the full amount of each component in the aqueous solution is used thereby reducing or eliminating loss of composition.

The composition of the present invention includes a polyene macrolide such as amphotericin B and nystatin. It also includes serum albumin, and preferably human serum albumin. It may optionally include a cryoprotectant such as mannitol and an antioxidant such as butylated hydroxyanisole.

Polyene macrolides includes those antibiotics having large lactone rings connected to sugar moieties and having a number of double bonds. Variations in both the lactone rings and the sugar moieties are known and result in a large number of polyene macrolides. They include such polyene antibiotics as the tetraenes such as nystatin, pentaenes such as aliomycin, methylpentaenes such as filipin, carbonylpentaenes such as mycoticin, hexaenes such as cryptocidine, carbonylhexaenes such as dermostatin, and heptaenes such as amphotericin B. More preferably, the polyene macrolides used in the present invention are nystatin or amphotericin B. Most preferably, the polyene macrolide is nystatin.

The polyene macrolides may be purchased in commercially available clinical formulations. However, it has been found that the results of experiments using clinical formulations of amphotericin B with serum albumin are poor. Such formulations were used by Brezis. It is believed that the poor results are due to the presence of sodium deoxycholate in the clinical formulation. When sodium deoxycholate is present in solution, it inhibits Solubilization by serum albumin thus reducing the amount of polyene macrolide present in solution from the polyene macrolide-serum albumin solid dispersion. While some results are achieved with the present invention when small amounts of sodium deoxycholate are present, it is preferred that no or chemically insignificantly amounts are present in solution for best results.

The polyene macrolide-HSA composition made in accordance with the present invention has increased solubility, reduced toxicity, and is deaggregated when compared with the drug alone or when the drug is simply mixed with HSA. It is also stable at refrigerated conditions for an extended period of time. The antifungal activity of the resulting compositions is the same as or improved over the drug alone or the drug simply mixed with HSA.

The polyene macrolide-human serum albumin formulation may be administered in number of well known manners but is preferably administered parenterally for example by continuous intravenous infusion or by injection which may be intravenous, subcutaneous, or intramuscular. Sustained release preparations may be used. The daily dose will be determined by a skilled person with reference to the patient, drug and the disease. The formulations taught by the present invention may be used to treat a variety of fungal infections in animals and in humans.

Example 1

Deaggregation of amphotericin B by bovine serum albumin - Amphotericin B was

dissolved in dimethyl sulfoxide (DMSO). Various concentrations of amphotericin B were prepared by dilution with 1% or 4% bovine serum albumin (BSA) in isotonic phosphate buffer solution (PBS) at pH 7.4. Fatty acid content of BSA was varied by adding ethanolic solution of lauric acid into BSA solution. Final solutions were incubated at 37°C or 50°C and followed by UN/VIS spectroscopy. The absorbance ratio of Peak I (348 nm) to Peak IV (409 nm) which is an index of aggregation was measured as a function of time. The effect of DOC on the deaggregation of amphotericin B was also monitored in isotonic PBS at pH 7.4.

The results showed that deaggregation of amphotericin B by serum albumin at any studied concentrations of BSA appeared to be slower than deaggregation by a high concentration of DOC. At high concentrations of DOC, monomeric amphotericin B was formed immediately on mixing. Rate constants (k) were obtained ln[(l+R(A² _<X,-A² ₀)/(A'₀- A² _=o)]/[(RA²„-A¹„)/(A¹ ₀-A'_∞)] versus time where R=A'/A²,. A¹, and A² _t were the absorbances of amphotericin B at wavelengths 348 and 409 at time "t" respectively. The more fatty acid bound BSA, the larger the k values measured as initial rates. At 50°C and 37°C, k was 3.4xl0^"3 and 1.7xl0'³ min^"1 respectively. High concentrations of BSA can increase the rate of the deaggregation process. Therefore, serum albumin can deaggregate amphotericin B into a monomeric form and highly deaggregated form which are nontoxic but active against fungal

cells.

Example 2

Preparation of solid dispersion of nystatin (Νvs) and human serum albumin (ΗSA) - Νys (5.0 mg) was dissolved in 0.50 ml dimethylacetamide (DMAC) and 0.35 ml of this solution was added to 10 ml of 5% HSA in isotonic phosphate buffer solution (PBS) at pH

7.4. This solution had a mole ratio of Νys to HSA of 1 :2. The solution of Νys and HSA was

incubated at 37°C for 30 minutes, frozen in dry-ice acetone and dried under vacuum. Solubility of Nvstatin of reconstituted solid dispersion of Nystatin and Human Serum Albumin - A solid dispersion of Nys and HSA (2.0 g) was added to 0.80 ml of distilled water. This mixture was shaken for 5 minutes and filtered through 0.45 μm syringe filter. The filtrate (0.030 ml) was added to 2.0 ml of 50% methanol: 50% DMF. Precipitated HSA was removed by ultracentrifugation. Supernatant containing Nys (0.50 ml) was added to 3.0 ml of 50% methanol: 50% DMF, and the drug was assayed by measurement of absorbance at 307 nm by UV/VIS spectroscopy.

The solubility of Nys prepared as a solid dispersion with serum albumin was about 1,800 μg/ml. Nys added as a solid to an aqueous solution of 5% HSA and equilibrated at 37°C for 30 minutes has a solubility of 240μg/ml. The increase in solubility is due to the method used to prepare the solid dispersion. In the solid dispersion, Nys is present in an amorphous state, which readily undergoes dissolution. On the other hand, Nys as a solid in an aqueous solution undergoes slow dissolution due to its crystalline state.

Example 3

X-rav Diffraction of Nvstatin. Human Serum Albumin and Solid Dispersion of

Nvstatin and Human Serum Albumin

X-ray diffraction showed that human serum albumin was in an amorphous form and Nys was in a crystalline form. However, a solid dispersion of Nys and human serum albumin showed an amorphous form which had higher solubility than a crystalline form of Nys. Example 4

Hemolvsis of Nvstatin and of a Solid Dispersion of Nvstatin and Human Serum Albumin

Whole blood was centrifuged at 2000 rpms and a supernatant pipetted off. Red blood cells (RBC) were diluted with isotonic PBS, pH 7.4. An appropriate amount of RBC was added to each tube. To study hemolysis, varied levels of Nys from a solid dispersion of Nys and HSA was added to each tube containing RBC. As a control, Nys (20 mg) was dissolved in 0.50 ml of DMAC, and varied amounts were added to RBC. Each tube was incubated in a water bath at 37° C for 30 minutes. Unlyzed RBC was removed by centrifugation for 30 seconds. Supernatant was collected and analyzed for hemoglobin by measurement of absorbance at 576 nm by UVNIS spectroscopy. The percentage of lyzed RBC was determined by the following equation:

% Hemolysis = 100 x (A-Ao)/(A₁₀o-Ao) where A, A_Q and A₁₀₀ (0.80) were the absorbances for the sample, control with no Nys and control in the presence of 20 μg/ml of AmB as Fungizone, respectively.

At a level of about 50μg/ml, Nys alone causes 50% hemolysis. Nys prepared as a

solid dispersion with HSA causes 50% hemolysis at about 270μg/ml.

Example 5

Antifungal Activity of a Solid Dispersion of Nvstatin and Human Serum Albumin The solid dispersion of Nys and HSA was dissolved in Bacto YPD broth media at a

Nys level of 30 μg/ml. The aqueous solution was filtered through 0.22 μm syringe filter for

sterilization. For Nys alone, drug was dissolved in dimethylsulfoxide (DMSO) and used without filtration. An aliquot of filtrate (1.6 ml) was placed into a microcentπfuge tube and two-fold dilution was made until a Nys level of 0.2 μg/ml was obtained. To each tube, inoculum (50μl) containing 5x10³ colony forming unit ml of Saccharomyces cerevisiae (ATCC 4921 deposited November 19, 1975 with the American Type Culture Collection at 12301 Parklawn Drive, Rockville Maryland) in YPD broth medium was added. Incubation was done at 30 °C for 24 hours. A solvent control and medium control were simultaneously studied to check the growth inhibiting activities of DMSO and sterility of broth medium, respectively. The minimal inhibitory concentration (MIC) was defined as the minimum concentration of Nys that showed a full inhibition of Saccharomyces cerevisiae in the microcentrifuge tube, examined by naked eyes and confirmed by the measurement of absorbance by UV/VIS spectrophotometry at 600 nm.

The MIC of Nys alone for Saccharomyces cerevisiae was 3μg/ml. Similarly, the MIC of Nys of the solid dispersion with HSA was 3 μg/ml. Therefore, the solid dispersion of nystatin with human serum albumin showed no decrease in antifungal activity.

The above-described embodiments of the present invention are meant to be illustrative of preferred embodiments and are not intended to limit the scope of the present invention. Variations of the invention will be readily apparent to persons skilled in the art and may be made without departing from the spirit or scope of the invention. These variations are intended to be within the scope of the present invention. The only limitations to the scope of the present invention are set out in the following appended claims.

Claims

CLAIMS:

1. A method of preparing a solid dispersion of polyene macrolides for administration to a patient, the polyene macrolide dispersion having improved solubility, the method comprising the steps of:

(a) dissolving the polyene macrolide in a cosolvent;

(b) mixing a serum albumin solution (SA) with the solution in (a); and

(c) freeze-drying the polyene macrolide-SA solution in (b) to form a solid dispersion.

2. A method of preparing a polyene macrolide composition for administration to a patient comprising reconstituting the solid dispersion of step (c) in claim 1 with a suitable sterile aqueous vehicle.

3. The method of claim 1 further comprising the step of incubating the solution of step (b) at elevated temperatures and under suitable conditions prior to the step of freeze drying.

4. The method of claim 2, wherein the polyene macrolide is nystatin.

5. The method of claim 2, wherein the polyene macrolide is amphotericin B.

6. The method of treating a patient for a fungal infection comprising the step of administering the polyene macrolide composition produced according to the method of claim 1 to the patient.

7. The method of treating a patient for a fungal infection comprising the step of administering the polyene macrolide composition produced according to the method of claim 2 to the patient.

8. The method of treating a patient for a fungal infection comprising the step of administering the polyene macrolide composition produced according to the method of claim 3 to the patient.

9. Polyene macrolide solid dispersions produced according to the method of claim 1.

10. Polyene macrolide solid dispersions produced according to the method of claim 2.

11. Polyene macrolide solid dispersions produced according to the method of claim 3.

12. Polyene macrolide solid dispersions produced according to the method of claim 4.

13. Polyene macrolide solid dispersions produced according to the method of claim 5.

14. A method of preparing a polyene macrolides and human serum albumin (HSA) composition for administration to a patient, the polyene macrolide having improved solubility, the method comprising the steps of:

(a) dissolving a polyene macrolide in a cosolvent;

(b) mixing a HSA solution to the solution in (a);

(c) incubating the solution in (b) at a temperature of above 25 °C;

(d) freeze-drying the polyene macrolide-SA solution in (c) to form a solid dispersion;

and

(e) reconstituting the solid dispersion with a suitable sterile aqueous vehicle prior to

administration.