Oreotoxin formulations

Abstract

The present invention discloses an aqueous pharmaceutical solution formulation comprising olotoxin.

Description

Technical Field

[0001] The present invention relates to pharmaceutical preparations comprising orellanine. Background Art

[0002] Orellanine (see Formula I below) is a selective nephrotoxin that occurs in relatively large amounts in several fungal species of the Cortinarius family.

[0003]

[0004] WO 2010 / 040750 discloses that orellanine can be used for the treatment of renal cell carcinoma. Orellanine is taken up in human renal cancer cells and is highly effective in killing cancer cells, whether they are derived from primary tumors or metastatic tumor tissue. After a brief exposure to orellanine, cell death occurs for many days, indicating that the toxin is actively taken up and retained by the cells.

[0005] Orellanine drugs are being developed for the treatment of renal cancer. It would therefore be desirable to provide pharmaceutical formulations of orellanine.

[0006] The present inventors have sought to provide a stable formulation of orellanine suitable for administration by intravenous injection. Summary of the invention

[0007] Therefore, the present invention provides a pharmaceutical aqueous solution formulation comprising:

[0008] 0.1 to 40 mg / ml of orellanine; and

[0009] Buffers;

[0010] The pH of the formulation is in the range of 7 to 8.

[0011] The inventors have found that such formulations are stable and suitable for administration by intravenous injection. DETAILED DESCRIPTION

[0012] The pharmaceutical formulation is an aqueous formulation. Water for injection (WFI) can be used in aqueous formulations to be administered by intravenous injection.

[0013] The amount of orellanine in the formulation is 0.1 to 40 mg / ml and can be 0.5 to 30 mg / ml, 1 to 20 mg / ml of orellanine, 5 to 15 mg / ml of orellanine, 8 to 12 mg / ml of orellanine, 9 to 11 mg / ml of orellanine, or about 10 mg / ml of orellanine. In one embodiment, the formulation comprises 10 mg / ml of orellanine. The amount of orellanine in the formulation can be determined by conventional methods, including high performance liquid chromatography (HPLC).

[0014] The inventors have found that the solubility of orellanine is pH dependent. Outside the preferred pH range, the solubility of orellanine is low and it is not possible to provide a concentration of orellanine preferably for use in a pharmaceutical formulation administered by intravenous injection. The pH of the formulation is in the range of 7 to 8, and may be in the range of 7.1 to 7.6, or in the range of 7.2 to 7.5, or in the range of 7.3 to 7.4. In one embodiment, the pH is about 7.4 (7.39 to 7.41). The pH can be determined by standard methods such as according to Ph. Eur. 2.2.3.

[0015] The inventors have found that the concentration of the buffer has an effect on the solubility of orellanine. The buffer concentration may be 10 to 200 mM, or 20 to 180 mM, or 30 to 120 mM, or 40 to 80 mM, or 40 to 60 mM.

[0016] The buffer may be a phosphate buffer such as NaH2PO4-Na2HPO4 or citrate-Na2HPO4.

[0017] The buffer ensures that the pH is within the selected range. Additional adjustment of the pH can be performed by adding a base or an acid, such as sodium hydroxide or hydrochloric acid, for example with 0.2 M sodium hydroxide or hydrochloric acid.

[0018] The inventors have found that the osmotic mass molar concentration of the preparation affects the solubility of orellanine. The osmotic mass molar concentration of the preparation can be greater than 200 mOsm, or can be in the range of 250 to 350 mOsm. The osmotic mass molar concentration can be determined by standard methods such as according to Ph. Eur. 2.2.35. In one embodiment of the invention, the osmotic mass molar concentration of the preparation is such that it is within the isotonic range, i.e. the preparation has an isotonicity similar to the isotonicity of blood.

[0019] The ionic strength of the formulation can be increased by adding sodium chloride. The formulation can include 0.05 to 2 wt % sodium chloride (wherein the weight is based on the weight of the formulation), or 0.1 to 1 wt % sodium chloride, or 0.5 to 0.9 wt % sodium chloride, or about 0.8 wt % sodium chloride.

[0020] The inventors have discovered that by controlling pH and osmolality, a stable formulation can be prepared in which the concentration of orellanine is suitable for intravenous administration.

[0021] In one embodiment, the pharmaceutical aqueous solution formulation comprises 0.1 to 40 mg / ml of orellanine; a phosphate buffer, wherein the concentration of the buffer is 10 to 200 mM; and 0.1 to 1 wt % sodium chloride; wherein the pH of the formulation is in the range of 7 to 8 and optionally wherein the osmolarity of the formulation is greater than 200 mOsm.

[0022] In another embodiment, the pharmaceutical aqueous solution formulation comprises 1 to 20 mg / ml of orellanine; a phosphate buffer, wherein the concentration of the buffer is 10 to 200 mM; and 0.1 to 1 wt % sodium chloride; wherein the pH of the formulation is in the range of 7 to 8 and optionally wherein the osmolarity of the formulation is greater than 200 mOsm.

[0023] In another embodiment, the pharmaceutical aqueous solution formulation comprises from 5 to 15 mg / ml of orellanine; a phosphate buffer, wherein the concentration of the buffer is 10 to 200 mM; and 0.1 to 1 wt % sodium chloride; wherein the pH of the formulation is in the range of 7 to 8 and optionally wherein the osmolarity of the formulation is greater than 200 mOsm.

[0024] In another embodiment, the pharmaceutical aqueous solution formulation comprises 0.1 to 40 mg / ml of orellanine; a phosphate buffer selected from NaH2PO4-Na2HPO4 or citrate-Na2HPO4, wherein the concentration of the buffer is 10 to 200 mM; and 0.1 to 1 wt % sodium chloride; wherein the pH of the formulation is in the range of 7 to 8 and optionally wherein the osmolarity of the formulation is greater than 200 mOsm.

[0025] In another embodiment, the pharmaceutical aqueous solution formulation comprises 1 to 20 mg / ml of orellanine; a phosphate buffer selected from NaH2PO4-Na2HPO4 or citrate-Na2HPO4, wherein the concentration of the buffer is 10 to 200 mM; and 0.1 to 1 wt % sodium chloride; wherein the pH of the formulation is in the range of 7 to 8 and optionally wherein the osmolarity of the formulation is greater than 200 mOsm.

[0026] In another embodiment, the pharmaceutical aqueous solution formulation comprises 5 to 15 mg / ml of orellanine; a phosphate buffer selected from NaH2PO4-Na2HPO4 or citrate-Na2HPO4, wherein the concentration of the buffer is 10 to 200 mM; and 0.1 to 1 wt % sodium chloride; wherein the pH of the formulation is in the range of 7 to 8 and optionally wherein the osmolarity of the formulation is greater than 200 mOsm.

[0027] In another embodiment, the pharmaceutical aqueous solution formulation comprises 0.1 to 40 mg / ml of orellanine; a phosphate buffer, wherein the concentration of the buffer is 40 to 60 mM; and 0.5 to 0.9 wt % sodium chloride; wherein the pH of the formulation is in the range of 7.2 to 7.5 and optionally wherein the osmolarity of the formulation is in the range of 250 to 350 mOsm.

[0028] In another embodiment, the pharmaceutical aqueous solution formulation comprises 1 to 20 mg / ml of orellanine; a phosphate buffer, wherein the concentration of the buffer is 40 to 60 mM; and 0.5 to 0.9 wt % sodium chloride; wherein the pH of the formulation is in the range of 7.2 to 7.5 and optionally wherein the osmolarity of the formulation is in the range of 250 to 350 mOsm.

[0029] In another embodiment, the pharmaceutical aqueous solution formulation comprises 5 to 15 mg / ml of orellanine; a phosphate buffer, wherein the concentration of the buffer is 40 to 60 mM; and 0.5 to 0.9 wt % sodium chloride; wherein the pH of the formulation is in the range of 7.2 to 7.5 and optionally wherein the osmolarity of the formulation is in the range of 250 to 350 mOsm.

[0030] In another embodiment, the pharmaceutical aqueous solution formulation comprises 0.1 to 40 mg / ml of orellanine; a phosphate buffer selected from NaH2PO4-Na2HPO4 or citrate-Na2HPO4, wherein the concentration of the buffer is 40 to 60 mM; and 0.5 to 0.9 wt % sodium chloride; wherein the pH of the formulation is in the range of 7.2 to 7.5 and optionally wherein the osmolarity of the formulation is in the range of 250 to 350 mOsm.

[0031] In another embodiment, the pharmaceutical aqueous solution formulation comprises 1 to 20 mg / ml of orellanine; a phosphate buffer selected from NaH2PO4-Na2HPO4 or citrate-Na2HPO4, wherein the concentration of the buffer is 40 to 60 mM; and 0.5 to 0.9 wt % sodium chloride; wherein the pH of the formulation is in the range of 7.2 to 7.5 and optionally wherein the osmolarity of the formulation is in the range of 250 to 350 mOsm.

[0032] In another embodiment, the pharmaceutical aqueous solution formulation comprises 5 to 15 mg / ml of orellanine; a phosphate buffer selected from NaH2PO4-Na2HPO4 or citrate-Na2HPO4, wherein the concentration of the buffer is 40 to 60 mM; and 0.5 to 0.9 wt % sodium chloride; wherein the pH of the formulation is in the range of 7.2 to 7.5 and optionally wherein the osmolarity of the formulation is in the range of 250 to 350 mOsm.

[0033] The formulation can be prepared by a method comprising the following steps:

[0034] a) adding a buffer to an aqueous solution of orellanine;

[0035] b) stirring; and

[0036] c) Adjusting the pH by adding further buffers, by adding a base (eg sodium hydroxide), and / or by adding an acid (eg hydrochloric acid).

[0037] Alternatively, the formulation may be prepared by a method comprising the following steps:

[0038] a) adding orellanine (optionally as an aqueous solution) to a buffer;

[0039] b) stirring; and

[0040] c) Adjusting the pH by adding further buffers, by adding a base (eg sodium hydroxide), and / or by adding an acid (eg hydrochloric acid).

[0041] The steps of stirring and adjusting pH may be repeated several times to ensure that the final pH is within the selected range. The inventors have observed that the pH of the formulation may change upon stirring, necessitating pH adjustment to provide a pH within the selected range and to provide a stable formulation.

[0042] The buffer can be prepared by combining the buffer components (eg, NaH2PO4 and Na2HPO4, or sodium citrate and Na2HPO4) with water for injection and with sodium chloride.

[0043] The formulation can be filtered (e.g., through a 0.22 μm PVDF filter) before packaging. The formulation can be packaged in conventional containers, such as glass injection vials. The vials can be stoppered and sealed with a flip off cap.

[0044] The formulation can be used in a method of treating cancer, such as renal cancer. The formulation can be used to treat a patient suffering from or susceptible to renal cell carcinoma. The treatment can be used to treat a tumor located in one or both kidneys of the patient. Alternatively, the renal cell carcinoma may have metastasized such that at least one renal cell carcinoma tumor is present in at least one non-renal tissue.

[0045] The formulation is suitable for administration by intravenous injection.

[0046] The formulation can be administered in a single dose, a series of daily doses, or in intermittent dosing (e.g., multiple doses or a sequence of doses administered between 1 and about 30 days apart, between 1 and about 14 days apart, or between 1 and about 7 days apart).

[0047] When administered as a single dose, an orellanine dosage of between about 1 mg / kg and about 100 mg / kg, or between about 2 mg / kg and about 25 mg / kg, or between about 5 mg / kg and about 15 mg / kg may be administered to the patient.

[0048] The formulation may be administered in two or more doses. The dose may be administered daily or intermittently (e.g., consecutive doses separated by at least one non-administration day). When administered in multiple doses, a dose of orellanine between about 0.5 mg / kg and about 10 mg / kg may be administered to the patient, or a dose between about 1 mg / kg and 5 mg / kg, or a dose of about 2 mg / kg.

[0049] If the consecutive doses are administered intermittently, the consecutive doses may be administered at intervals of between two and seven days. The formulation may be administered to the patient in three, four, five, or six or more doses, and wherein each dose is administered at intervals of between three and five days. The formulation may be administered to the patient in four, five, or six or more doses, administered at intervals of between three and four days, wherein each dose comprises between about 1 mg / kg to about 20 mg / kg of orellanine, or about 2 mg / kg to about 10 mg / kg of orellanine, or about 5 mg / kg of orellanine.

[0050] The daily dose of the preparation can be applied for at least two days. The typical daily dose applied to the patient is between 0.1 and 10 mg / kg, or between 1 and 5 mg / kg, or about 2 mg / kg. The treatment regimen can include daily application of the preparation between 5 days and about 30 days, or preferably between 10 days and about 20 days, or most preferably about 14 days.

[0051] In some cases, it may be desirable to perform multiple intermittent administration regimens, daily administration regimens, or combinations thereof, as described above, in combination with a rest and / or recovery period. Thus, in some cases, it may be desirable to administer the formulation according to a daily or intermittent administration method, measure the tumor's response to the therapy, and then perform subsequent daily or intermittent administration therapy as needed to eliminate or otherwise reduce the size of the renal cancer tumor. Such administration strategies are well known to those of ordinary skill in the field of oncology.

[0052] In one embodiment of the invention, a patient suffering from renal cell carcinoma may be treated with the formulation by daily injections of about 0.5-5 mg of orellanine / kg body weight, most preferably about 2 mg of orellanine / kg body weight, for 7-21 consecutive days, or for about 14 consecutive days. In order to clear any orellanine that is not absorbed into the tumor tissue, thereby minimizing any undesirable side effects that may occur in the extracellular space, the patient is subjected to hemodialysis for 1-5 hours, most preferably about 2 hours, 1 to 5 hours after each daily injection of the formulation, most preferably about 2 hours after such injection.

[0053] The preferred dosage and dosage regimen described above are based on a human being with a body weight of 70 kg and suffering from renal cell carcinoma with a tumor load of about 1 kg. However, as those of ordinary skill in the field of cancer medicine are readily aware, such preferred dosage and dosage regimen are greatly affected by patient characteristics, such as age, sex, weight, general condition, and especially the tumor load and response to treatment of individual patients. As always, the ultimate responsibility for selecting an appropriate dosage and treatment strategy falls on the physician in charge of the patient.

[0054] The present invention will now be described by reference to examples which are not intended to limit the invention: Example

[0055] Solubility studies using buffer solutions (pH-dependent solubility tests)

[0056] Phase solubility studies were performed in different buffers to analyze the effect of pH on the solubility of orellanine. Four buffers were tested:

[0057] ● pH 4.5, 50 mM acetate buffer - Prepare from 1.81 g sodium acetate and 14 ml acetic acid (2N) and adjust to 1 l with water for injection (WFI).

[0058] ● pH 5.8, 50 mM phosphate buffer - Prepare from 92 ml of NaH2PO4 and 8 ml of Na2HPO4 and adjust to 200 ml with WFI.

[0059] • pH 6.8, 50 mM phosphate buffer - Prepare from 51 ml of NaH2PO4 and 49 ml of Na2HPO4 and adjust to 200 ml with WFI.

[0060] ● pH 7.4, 50 mM phosphate buffer - Prepare from 19 ml of NaH2PO4 and 81 ml of Na2HPO4 and adjust to 200 ml with WFI.

[0061] The pH of each buffer was adjusted using a pH meter and 0.2 M sodium hydroxide or hydrochloric acid.

[0062] The solubility of orellanine in each buffer was tested by weighing 40 mg of orellanine into a vial, adding 2 ml of buffer, stirring the suspension for 1 hour, and then stirring at 400 rpm for 24 hours at room temperature. Each suspension was centrifuged at 5300 rpm for 15 minutes. The supernatant was filtered with a 0.45 μm nylon filter. Table 1 shows the results of the test:

[0063] Table 1:

[0064] Using 50 mM phosphate buffer, pH 7.4, the maximum concentration of orellanine achieved was 1.7 mg / ml.

[0065] Solubility studies at pH 7.4 using different buffer concentrations

[0066] The effect of buffer concentration was evaluated. Three buffers were used:

[0067] ● pH 7.4, 25 mM phosphate buffer - Prepare from 0.95 ml NaH2PO4 (0.1 M) and 4.05 ml Na2HPO4 (0.1 M) and adjust to 20 ml with WFI.

[0068] ● pH 7.4, 50 mM phosphate buffer - Prepare from 19 ml of NaH2PO4 (0.1 M) and 81 ml of Na2HPO4 (0.1 M), adjust to 200 ml with WFI.

[0069] ● pH 7.4, 100 mM phosphate buffer - Prepare from 38 ml NaH2PO4 (0.1 M) and 162 ml Na2HPO4 (0.1 M), adjust to 200 ml with WFI.

[0070] The pH of each buffer was adjusted using a pH meter and 0.2 M sodium hydroxide or hydrochloric acid.

[0071] The solubility of orellanine in each buffer was tested by weighing 20 mg of orellanine into a 5 ml vial, adding 2 ml of buffer, stirring the suspension for 3 hours, and then adjusting the pH to 7.4 with 0.2 M sodium hydroxide or hydrochloric acid. The suspension was then stirred at 400 rpm for 24 hours at room temperature. Each suspension was filtered through a 0.2 μm PVDF syringe filter. Table 2 shows the test results:

[0072] Table 2:

[0073] For these formulations, the solubility ranged from 3.7 to 4.9 mg / ml. 50 mM appears to be the optimal concentration of the buffer from a solubility and purity perspective.

[0074] Solubility studies using different buffer salts at pH 7.4

[0075] Four buffers at pH 7.4 and 50 mM were evaluated:

[0076] ● pH 7.4, 50 mM phosphate buffer - Prepare from 19 ml of NaH2PO4 (0.1 M) and 81 ml of Na2HPO4 (0.1 M), adjust to 200 ml with WFI.

[0077] • pH 7.4, 50 mM phosphate buffer - Prepare from 28.1 ml KH2PO4 (0.2 M) and 21.9 ml NaOH (0.2 M), adjust to 200 ml with WFI.

[0078] • pH 7.4, 50 mM Tris-HCl buffer - Prepare from 54.7 ml of tris(hydroxymethyl)aminomethane (0.1 M) and 45.3 ml of hydrochloric acid (0.1 M) and adjust to 200 ml with WFI.

[0079] • pH 7.4, 50 mM citrate phosphate buffer - Prepare from 2.4 ml citric acid monohydrate (0.2 M) and 47.6 ml Na2HPO4 (0.2 M) and adjust to 200 ml with WFI.

[0080] The pH of each buffer was adjusted using a pH meter and 0.2 M sodium hydroxide or hydrochloric acid.

[0081] The solubility of orellanine in each buffer was tested by weighing 20 mg of orellanine into a 5 ml vial, adding 2 ml of buffer, stirring the suspension for 3 hours, and then adjusting the pH to 7.4 with 0.2 M sodium hydroxide or hydrochloric acid. The suspension was then stirred at 400 rpm for 24 hours at room temperature. Each suspension was filtered through a 0.2 μm PVDF syringe filter. Table 3 shows the test results:

[0082] Table 3:

[0083] Optimal solubility was provided by NaH2PO4-Na2HPO4 and citrate-Na2HPO4 buffers.

[0084] Solubility studies at pH 7.4 using different ionic strengths

[0085] The ionic strength of the NaH2PO4-Na2HPO4 buffer was adjusted by adding sodium chloride. The ionic strength of the 50 mM NaH2PO4-Na2HPO4 buffer was estimated to be approximately 130 mM. The proportions of sodium chloride added to the buffer were 0.41%, 0.98%, and 2.15%.

[0086] The solubility of orellanine in each buffer was tested by weighing 20 mg of orellanine into a 5 ml vial, adding 2 ml of buffer, stirring the suspension for 3 hours, and then adjusting the pH to 7.4 with 0.2 M sodium hydroxide or hydrochloric acid. The suspension was then stirred at 400 rpm for 24 hours at room temperature. Each suspension was filtered through a 0.2 μm PVDF syringe filter. Table 4 shows the test results:

[0087] Table 4:

[0088] Increasing the ionic strength increases the osmolarity of the solution and enhances the solubility of orellanine.

[0089] Preparation adjustment

[0090] Two buffers were selected: 50 mM NaH2PO4-Na2HPO4 pH 7.4 and citrate-Na2HPO4 pH 7.4, each with 0.70% sodium chloride added. The addition of 0.70% sodium chloride provides a nearly isotonic solution.

[0091] The formulations were prepared by weighing 40 mg of orellanine into a 5 ml vial, adding 2 ml of buffer, stirring the suspension for 1 hour, adjusting the pH to 7.4 with sodium hydroxide (0.2 M), stirring the suspension at 400 rpm for 24 hours at room temperature, and adjusting the pH to 7.4 with sodium hydroxide (0.2 M). Each suspension was filtered through a 0.2 μm PVDF syringe filter. Table 5 shows the properties of the formulations:

[0092] Table 5:

[0093] A solubility of greater than 10 mg / ml was achieved by adjusting the pH to 7.4 twice: first after 1 hour of stirring and again after overnight stirring.

Claims

1. A pharmaceutical aqueous solution preparation, comprising: From 0.1 to 40 mg / ml of orellanine; and Buffers; The pH of the formulation is in the range of 7 to 8.

2. The formulation according to claim 1, comprising 1 to 20 mg / ml of orellanine.

3. The formulation of claim 2, comprising 5 to 15 mg / ml of orellanine.

4. A formulation according to any preceding claim, wherein the pH of the formulation is in the range of 7.1 to 7.

6.

5. The formulation according to claim 4, wherein the pH of the formulation is in the range of 7.2 to 7.

5.

6. The formulation of any preceding claim, wherein the concentration of the buffer is 10 to 200 mM.

7. The formulation of claim 6, wherein the concentration of the buffer is 40 to 60 mM.

8. A formulation according to any preceding claim, wherein the buffer is a phosphate buffer.

9. The formulation of claim 8, wherein the buffer is NaH2PO4 - Na2HPO4 or citrate-Na2HPO4.

10. The formulation of any preceding claim, wherein the formulation has an osmolality greater than 200 mOsm.

11. The formulation of any preceding claim, wherein the formulation has an osmolality in the range of 250 to 350 mOsm.

12. A formulation according to any preceding claim comprising sodium chloride.

13. The formulation of claim 12, comprising 0.1 to 1 wt% sodium chloride.

14. A formulation according to any preceding claim for use in a method of treating cancer.

15. The formulation for use according to claim 14, wherein the cancer is renal cancer.

16. A formulation for use according to claim 14 or claim 15, wherein the method comprises providing the formulation to a subject in need thereof for at least two days, wherein the daily dose is between 0.1 and 10 mg / kg body weight.

17. A method for preparing a formulation according to any one of claims 1 to 13, comprising the following steps: a) adding a buffer to an aqueous solution of orellanine, or adding orellanine, optionally as an aqueous solution, to a buffer; b) stirring; and c) Adjusting the pH by adding further buffers, by adding a base (eg sodium hydroxide) and / or by adding an acid (eg hydrochloric acid).

18. The method of claim 17, wherein steps (b) and (c) are performed at least twice.

19. A method for treating cancer, wherein the method comprises intravenously administering the formulation according to any one of claims 1 to 13 to a subject in need thereof.

20. The method of claim 19, comprising providing the formulation to a subject in need thereof for at least two days, wherein the daily dose is between 0.1 and 10 mg / kg body weight.

21. A kit for intravenous administration, comprising a formulation according to any one of claims 1 to 13 packaged in a container, and optionally further comprising information for providing the formulation to a subject in need thereof.

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