Resiniferatoxin preparations

Abstract

The present invention discloses a safer resiniferatoxin (RTX) formulation for intrathecal, intraganglionic, intraarticular and pericardial administration. More specifically, an ethanol-free formulation of RTX is disclosed, which comprises a solubilizing component, a monosaccharide or sugar alcohol, a salt buffer and RTX, and has a narrower pH range and specific gravity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 556,824, filed on September 11, 2017, the entire contents of which are hereby incorporated by reference in their entirety. Field of the Invention

[0003] The present disclosure provides a resiniferatoxin (RTX) formulation with lower toxicity for administration. Since RTX is a very water-insoluble compound, the formulation of the present disclosure provides a high concentration of the RTX active ingredient in the formulation, wherein a very small amount of liquid is injected into the sheath, into the ganglion, around the ganglion, into the pericardium, or into the joint cavity (intra-articular). More specifically, the present disclosure provides an ethanol-free formulation of RTX, the ethanol-free formulation of RTX comprising a solubilizing component, a monosaccharide or a sugar alcohol, a salt buffer, and RTX. Background Art

[0004] Transient receptor potential cation channel subfamily V member 1 (TrpV1) or vanilloid receptor-1 (VR1) is a polycation channel that is prominently expressed in nociceptive primary afferent neurons (Caterina et al., (1997) Nature 389:816-824; Tominaga et al., (1998) Neuron 531-543). Activation of TrpV1 usually occurs in nerve endings by application of painful heat and is upregulated under certain types of inflammatory stimuli. Activation of TrpV1 in peripheral tissues by chemical agonists results in the opening of calcium ion channels and the transmission of pain sensation (Szallasi et al., (1999) Mol. Pharmacol. 56:581-587). However, direct administration of certain TrpV1 agonists to the cell bodies of neurons (ganglia) expressing TrpV1 causes calcium ion channels to open and triggers a cascade of events leading to programmed cell death ("apoptosis") (Karai et al., (2004) Journal of Clinical Investigation. 113: 1344-1352).

[0005] RTX is known to be a TrpV1 agonist and acts as a very potent analog of capsaicin, the main component of the spicy taste of red peppers. RTX is a tricyclic diterpenoid isolated from certain plants of the genus Eurphorbia. The homovanillin group is an important structural feature of capsaicinoids and is the most prominent feature that distinguishes resiniferatoxins from typical phorbol-related compounds. Naturally occurring or native RTX has the following structure:

[0006]

[0007] U.S. Patents 4,939,194; 5,021,450 and 5,232,684 describe RTX and similar compounds such as tintoxin and other compounds (20-homovanillate diterpenoid esters such as 12-deoxyphorbol 13-phenylacetate 20-homovanillate and mezerein 20-homovanillate). Other resiniferatoxin-type phorboid vanilloids have also been identified (Szallasi et al., (1999) Brit. J. Phrmacol. 128:428-434).

[0008] In U.S. Pat. No. 8,338,457 (the disclosure of which is incorporated herein by reference), RTX in a stock formulation containing 1 mg / mL RTX, 10% ethanol, 10% Tween 80, and 80% saline was diluted with 0.9% saline. The vehicle for injection was a 1:10 dilution of the RTX stock formulation, which used 0.9% saline as a diluent. Therefore, previous injections were to dissolve the hydrophobic RTX molecules in ethanol and inject the formulation containing about 1-2% (v / v) ethanol directly into the ganglion. However, it is not advisable to inject ethanol (or other organic solvents) directly into the brain, spinal cord (subdural), or ganglia because these compounds can non-specifically kill any cells they contact, and nerves are particularly sensitive. Therefore, there is a need in the art to develop RTX formulations for administration that do not contain any organic solvents (e.g., ethanol) and can still keep the RTX molecules in solution. The present disclosure is dedicated to achieving such non-ethanol formulations. SUMMARY OF THE INVENTION

[0010] The present disclosure provides an ethanol-free formulation of RTX that can be administered by injection in a relatively small volume, comprising about 10 μg / mL to about 200 μg / mL of RTX in the formulation, and having enough monosaccharides or sugar alcohols to maintain a specific gravity between 1.0 and 1.3. RTX is soluble in a saline buffered aqueous solution of at least one or a mixture of PEG (0-40%), polysorbate (0-5%) and cyclodextrin (0-5%), with a pH of about 6.5 to about 7.5, and contains an antioxidant.

[0011] Preferably, the formulation comprises about 25-50 μg / mL of RTX. Preferably, the monosaccharide or sugar alcohol is selected from the group consisting of dextrose, mannitol, and combinations thereof. Preferably, the solubilizer is selected from the group consisting of polysorbate (20, 60 or 80), polyethylene glycol (PEG100, 200, 300, 400 or 600), cyclodextrin, and combinations thereof. Preferably, the buffer is selected from the group consisting of phosphate buffer, acetate buffer, citrate buffer, and combinations thereof. Preferably, the formulation further comprises an antioxidant. More preferably, the antioxidant is selected from the group consisting of ascorbic acid, citric acid, potassium bisulfate, sodium bisulfate acetone, sodium bisulfate, thioglycerol, potassium pyrosulfite, sodium pyrosulfite, and combinations thereof. DETAILED DESCRIPTION OF THE INVENTION

[0013] definition

[0014] "Intraganglionic administration" is administration within a ganglion. Intraganglionic administration can be achieved by direct injection into the ganglion, and also includes selective nerve root injection or periganglionic administration, in which the compound enters the ganglion through the connective tissue sleeve around the nerve and from the nerve root outside the spine. Typically, intraganglionic administration is used in conjunction with imaging techniques, such as using MRI or X-ray contrast dyes or agents to visualize the target ganglion and the area of ​​administration. The amount of administration varies from about 50 μl administered directly into the ganglion to 2 ml for periganglionic administration around the ganglion.

[0015] The term "subarachnoid space" or cerebrospinal fluid (CSF) space encompasses common usage and refers to the anatomical space between the pia mater and the arachnoid mater that contains CSF.

[0016] "Intrathecal administration" means administering the composition directly into the subarachnoid space of the spinal cord. The amount of intrathecal administration for adults is 2 to 50 μg.

[0017] "Intra-articular administration" is the injection of a compound in an aqueous solution into a joint cavity, such as the knee or elbow. The amount of intra-articular administration for an adult human knee joint is 3 to 10 ml volume and 5 to 50 μg of RTX. The amount for a young human or livestock (dog or cat) knee joint is smaller and proportional in volume to the relative size of the knee joint of each species.

[0018] The present disclosure provides an ethanol-free formulation of RTX for intrathecal, intraarticular, intraganglionic or peripheral ganglion administration, comprising about 10 μg / mL to about 200 μg / mL of RTX in the formulation, and having sufficient monosaccharides to maintain a specific gravity between 1.0 and 1.3. RTX is soluble in a saline buffered aqueous solution of at least one or a mixture of PEG (0-40%), polysorbate (0-5%) and cyclodextrin (0-5%), pH value is about 6.5 to about 7.5, and contains an antioxidant.

[0019] RTX can be injected directly into the ganglion or nerve root (intrathecal or intraganglionic) using standard neurosurgical techniques to create a temporary environment in the dorsal root or autonomic ganglion. RTX can also be injected directly into the joint cavity to treat arthritis pain in this particular joint. The duration of RTX taking effect may be longer than the time the temporary environment maintains. Any dose can be used as needed and as tolerated by the patient. It can be administered with the aid of image analysis using MRI or X-ray contrast dyes to provide direct delivery to the perikarya. For example, the process can be performed in conjunction with procedures such as CAT scans, fluoroscopy or open MRI.

[0020] For intraganglionic administration, the usual injection volume is 50 to 300 μl, and the total amount of RTX delivered ranges from about 50 ng to about 50 μg. For intraarticular administration, the usual amount injected into the knee of an adult is 3 ml to 10 ml, and the total amount of RTX delivered is 5 ng to 50 μg. Usually, the amount administered is 200 ng to 10 μg. RTX can be administered as a single dose or infused over a period of time (usually 1 to 10 minutes).

[0021] For intrathecal administration, an amount of about 0.5cc to 5cc (usually 3cc) is injected into the subarachnoid space. The total amount of RTX in the injection volume is usually about 500ng to about 200μg. Typically, the amount administered is 20μg to 50μg. RTX can be administered as a single dose or infused over a period of time (usually 1 to 10 minutes).

[0022] Table 1. RTX solution formulation

[0023]

[0024]

[0025] Example 1: Preparation of formulation

[0026] The formulations in Table 1 are prepared as follows (using formulations 3 and 5 as examples). Formulation 3 is prepared by preparing a 30mM phosphate buffer of pH 7.2. 1.43% w / v of polysorbate 80 and 0.86% w / v of NaCl are then mixed to form an aqueous component. 20 mg of RTX is added to 100 mL of aqueous components in a volumetric flask. 30 mL of PEG 300 is then added, and the solution is sonicated to dissolve the solids. The aqueous component is added to about 80% volume and then sonicated to mix. It should be noted that sometimes RTX is initially precipitated at the interface of the aqueous solution and PEG, but returns to the solution after sonication. The entire mixture in the flask is diluted to a certain volume with the aqueous component and mixed by a version process. The entire formulation is filtered through a 0.2 μm polytetrafluoroethylene (PTFE) filter.

[0027] Preparation 5 was prepared by preparing a 30mM pH 7.2 phosphate buffer. 3.0% w / v polysorbate 80, 0.8% w / v dextrose and 0.54% w / v NaCl were then mixed together to form an aqueous component. 20 mg of RTX was added to 100 mL of the aqueous component in a volumetric flask. The aqueous component was added to about 80% volume and then ultrasonicated to dissolve all solids. The entire mixture in the flask was diluted to a certain volume with the aqueous component and mixed by conversion. The entire preparation was filtered through a 0.2 μm PTFE filter.

[0028] A formulation according to Formulation 11 was prepared using 200 μg of RTX, 20 mg of polysorbate 80 (commercially available Tween(C) 80 was used), 5.4 mg of sodium chloride, 50 mg of dextrose, and 30 mM phosphate buffer, water (WFI) to 1 mL.

[0029] Example 2: Solubility Comparison

[0030] Independent of the formulation described in Example 1, 12 surfactants were tested to compare the recovery of RTX based on HPLC analysis of samples (after ambient and cold (5°C) storage). Table 2 shows the percentage recovery for the different solvents tested:

[0031] Table 2 Solubility of RTX in various solutions

[0032]

[0033] This study demonstrates that RTX is insoluble in water. Furthermore, none of the aqueous surfactant solutions showed recoveries close to ethanol, which reported an ambient temperature recovery of 98.4% and a low temperature recovery of 99.8%. The closest percentage recoveries were those of sodium dodecyl sulfate solution, at only 24.0%, and 0.5% Tween 80, at 20.2%. Example 2 demonstrates that aqueous solubility of RTX is difficult to achieve in non-ethanol solvents. Many common solvents fail to provide usable solutions. Example 2 further demonstrates that RTX is insoluble in unmodified aqueous solutions.

[0034] Example 3: Purity and potency of RTX solution

[0035] Formulations 1-10 of Table 1 were also tested to measure the purity and potency of RTX. These measurements demonstrate the stability of RTX in solution, demonstrating that RTX remained in solution when aliquots were withdrawn for testing. Testing was performed at the initial moment of solution preparation, and at set time periods after solution preparation. Formulations 1 to 10 (described above) were studied in Example 3.

[0036] For purity, potency, and related substances testing, approximately 2 mL of each formulation was filtered through a 0.2 μm, 13 mm PTFE filter and approximately the first 1 mL of the filtrate was discarded. Unfiltered samples were also analyzed as shown below. All samples were analyzed by HPLC with an injection volume of 50 μL. Table 3.1 shows the results for filtered and unfiltered purity and potency.

[0037] Table 3.1 Overview of RTX preparation analysis and testing (t=0)

[0038]

[0039] In further analysis, 100 μL of each formulation was diluted 1:10 in cerebrospinal fluid (CSF) and tested for appearance, potency, purity, and related substances. After dilution, all solutions remained clear visually. The samples were filtered through a 0.2 μm, 13 mm PTFE filter and the first 800 μL of filtrate was discarded. All samples were analyzed with an injection volume of 50 μL. The results are shown in Table 3.2:

[0040] Table 3.2 RTX solution detection in CSF

[0041] preparation purity(%) Efficacy (%) 1 99.44 134.48 2 99.32 93.65 3 99.07 109.51 4 98.98 62.68 5 98.95 130.19 6 99.20 131.16 7 99.40 133.71 8 99.66 96.23 9 99.14 94.37 10 98.82 77.40

[0042] This study demonstrated high purity and potency.It is generally believed that high potency values ​​(eg, values ​​exceeding 100%) reflect filter compatibility issues with low concentration CSF filtered samples.

[0043] Example 4: RTX stability over time

[0044] In further studies, samples as described above were stored and analyzed after 0.5 and 1 month of storage. Tables 4.1 and 4.2 list the potency results at 0.5 and 1 month.

[0045] Table 4.1 Efficacy profile of RTX formulations t = 0.5 months

[0046]

[0047] Table 4.2 Efficacy profile of RTX prototype formulations t = 1 month

[0048]

[0049] The data in Table 4.1 show that the formulations with mannitol maintained pH consistency better than the formulations with dextrose (as can be seen by comparing Formulation 1 to Formulation 7, Formulation 2 to Formulation 8, Formulation 5 to Formulation 6, and Formulation 9 to Formulation 10).

[0050] In addition, the results in Table 4.1 show that at -20°C, Formulations 1 and 3 achieve optimal storage. At 5°C, all formulations except Formulation 4 have more than 90% of the potency of Formulation 3, which provides the highest potency. For 25°C / 60%RH, Formulations 3 and 5 have the best potency. For 40°C / 75%RH, Formulation 5 has the best potency. For 60°C, Formulations 1 and 5 have the best potency.

[0051] Purity was also checked after 0.5 and 1 month. These results are shown in Tables 4.3 and 4.4.

[0052] Table 4.3 Purity profile of RTX preparations t = 0.5 months

[0053]

[0054]

[0055] Table 4.4 Purity profile of RTX prototype formulations t = 1 month

[0056]

[0057] The results in Table 4.3 show that at -20°C, all formulations showed comparable purity to the data at t=0. At 5°C, formulations 2, 3, 8, and 9 showed the best purity results, while the purity of the other formulations dropped by 0.2-0.9%. For 25°C / 60%RH, formulations 3 and 5 showed the best response, with a drop in purity of about 4%. Table 4.4 shows the corresponding measurement results for some formulations after 1 month.

[0058] Example 5: pH stability

[0059] Formulations 1-10 were also studied to determine their pH at the time of preparation (t=0) and after 0.5 and 1 month. These results are shown in Tables 5.1 and 5.2.

[0060] Table 5.1 pH profile of RTX formulations t = 0.5 months

[0061]

[0062] Table 5.2 pH profile of RTX formulations t = 1 month

[0063]

[0064] As shown in the above Tables 5.1 and 5.2, the formulations exhibited good pH stability over time. In particular, for Table 5.2, the pH values ​​of samples stored at a temperature of less than or equal to 40°C did not change significantly. For the formulations stored at 60°C, the pH value of each formulation was further reduced compared to the results at t = 0.5 months.

Claims

1. An ethanol-free formulation of RTX comprising 200 μg / mL of RTX dissolved in a solution comprising 3% or 7% w / v polysorbate 80, 0.8% w / v dextrose and a saline buffer, wherein the pH of the formulation is 6.5 to 7.

5.

2. The ethanol-free formulation of RTX according to claim 1, wherein the saline buffer is selected from the group consisting of a phosphate buffer, an acetate buffer, and a citrate buffer, or a combination thereof.

3. The ethanol-free formulation of RTX according to claim 1, further comprising an antioxidant.

4. The ethanol-free formulation of RTX according to claim 3, wherein the antioxidant is selected from the group consisting of ascorbic acid, citric acid, potassium bisulfate, sodium bisulfate acetone, sodium bisulfate, thioglycerol, potassium metabisulfite, and sodium metabisulfite, or a combination thereof. The ethanol-free formulation of RTX according to claim 1 , comprising a phosphate buffer.

6. The ethanol-free formulation of RTX according to claim 1, wherein the polysorbate 80 is present at 3% w / v and the solution comprises 30 mM phosphate buffer.

7. The ethanol-free formulation of RTX according to claim 6, further comprising 0.54% NaCl.

8. The ethanol-free formulation of RTX according to claim 6 or 7, having a pH of 7.

2.

9. The ethanol-free formulation of RTX according to claim 1, wherein the polysorbate 80 is present at 7% w / v and the solution comprises 30 mM phosphate buffer.

10. The ethanol-free formulation of RTX according to claim 9, further comprising 0.44% NaCl.

11. The ethanol-free formulation of RTX according to claim 9 or 10, having a pH of 7.

2.

12. An ethanol-free formulation of RTX comprising 200 μg / mL of RTX dissolved in a solution comprising 7% w / v polysorbate 80, 0.8% w / v dextrose, and phosphate buffer, wherein the pH of the formulation is 7.

2.

13. An ethanol-free formulation of RTX comprising 200 μg / mL of RTX dissolved in a solution comprising 3% w / v polysorbate 80, 0.8% w / v dextrose, and phosphate buffer, wherein the pH of the formulation is 7.

2.

14. The ethanol-free formulation of RTX according to claim 12, wherein the formulation retains 80.2% potency after 0.5 month at 60°C.

15. The ethanol-free formulation of RTX according to claim 13, wherein the formulation retains 78.0% potency after 0.5 month at 60°C.

16. The ethanol-free formulation of RTX according to claim 12 or 14, wherein the formulation retains 85.2% potency after 1 month at 40°C and 75% relative humidity.

17. The ethanol-free formulation of RTX according to claim 13 or 15, wherein the formulation retains 90.0% potency after 1 month at 40°C and 75% relative humidity.

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