Crotoxin stabilized preparation

Abstract

A highly stable acidified formulation of crotoxin is disclosed, comprising physiological saline and benzalkonium chloride, optionally thiamine, to facilitate the therapeutic use of crotoxin. A method for preparing a highly stable formulation of crotoxin is also disclosed.

Description

Technical Field

[0001] The present invention relates to a stable formulation of crotoxin for clinical administration that facilitates handling and enables waste reduction. A method of formulating a solution of crotoxin that provides the aforementioned properties is also disclosed.

Background Art

[0002] Crotoxin was first isolated in 1934 and has been extensively studied because it corresponds to the main toxic component of Crotalus durissus snake venom. Its neurotoxicity is due to its high affinity for nicotinic acetylcholine receptors located in the diaphragm of animals injected with the toxin. The A subunit (crotoxin) of the protein not only targets the receptor but also inhibits the enzymatic activity of its B subunit (crotactin). The two subunits are bound by a covalent bond and can be separated under specific conditions. The two subunits bind spontaneously when recombined either in vitro or in vivo. Crotoxin has been found to be highly toxic to cancer cells both in vitro and in vivo, leading to human clinical trials in subjects with cancer. Subsequently, crotoxin has been found to exhibit further pharmacological properties, including anti-inflammatory and antiviral activities. The storage and administration of protein-based tumor drugs are often difficult and can lead to significant losses of drug material due to inappropriate packaging and handling. It is reported that these costs are in the range of $1.8 billion to $3 billion per year. This application describes a formulation of crotoxin that enables simple storage and administration of the drug and avoids unnecessary waste.

[0003] A known method for purifying crotoxin involves suspending crude snake venom in water and adjusting the pH to 2.0 with dilute HCl. The preparation is then heated to 90°C for 10 minutes and centrifuged. The supernatant is removed, and its pH is adjusted to 4.7 (with diluted ammonia) to produce a precipitate. The pH-adjusted supernatant and ammonia solution are centrifuged, retaining a precipitate mainly containing crotoxin. This method has several drawbacks: 1. Not all crotoxin is recovered from the crude snake venom; 2. The recovered crotoxin is not of high purity; and 3. The activity of crotoxin is reduced by the heat process. The advantages are: 1. Precipitation of exogenous proteins; 2. Killing of viral or exogenous contaminants; and 3. It is rapid.

[0004] U.S. Patent No. 5,164,196 and European Patent No. 0246,861 disclose stable compositions of substances based on the cytotoxic activity of crotoxin B. Snake venom is dissolved in ammonium formate at pH 4.0, and the crotoxin peak is isolated by size exclusion chromatography (one flowing on G75 and the other on S200 resin). The eluent fractions corresponding to the main cuts are combined (approximately 25 mL containing 23-25 ​​mg of protein), filtered (pore size 0.22 μm), concentrated to 5-7 mL by ultrafiltration in a 50 mL Amicon cell equipped with a Filtron membrane (Omega, exclusion limit molecular weight 3000 (mol. wt. limit 3K)), filtered again (pore size 0.22 μm), and lyophilized. The lyophilized crotoxin is dissolved in phosphate buffer (35 mM KHPO4, pH 7.0), and the solution is loaded onto a DEAE column. Crotoxin elution is achieved with an NaCl gradient up to 1.0 M. Crotoxin heterodimers elute from the anion exchange resin as a relatively broad peak due to the presence of isoforms. The resulting DEAE crotoxin solution is diafiltration with volatile 50 mM ammonium formate (pH 4.5) and freeze-dried. Typically, 250 mg of snake venom is purified in this manner. The inventors concluded that ion exchange chromatography is unsuitable as a typical first step for purification because it is a time-consuming and not a large-scale process. However, this process does produce pure protein.

[0005] In other known methods, organic acids are used to aid in the dissociation of proteins into their constituent subunits, which helps in the separation process during purification. Organic acids such as citric acid, acetic acid, and formic acid can facilitate this process, and any residual acidic buffer is removed by dialysis or lyophilization, and the proteins are suspended in neutral pH 6.9 phosphate buffer.

[0006] Reverse-phase chromatography can also be used for purification, but this technique also causes the dissociation of subunits in organic solutions that are not readily compatible with the drugs for injection. Further considerations for these processes include maintaining the cleanliness of the manufacturing process and preventing microbial or endotoxin contamination that could impair the final product.

[0007] The use of animal-derived products also requires consideration of accidental contamination with viral material, usually viruses or mycoplasmas, which must be removed or inactivated during the manufacturing process. Since monoclonal antibodies are produced using animal-derived cell lines, the current standard practice for monoclonal antibody production involves lowering the pH to 4.0 for several hours, followed by filtration through a membrane designed to retain the virus. The World Health Organization's 2004 "Annex 4 Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products" recognizes that most proteins are damaged by exposure to the acidic conditions necessary to kill viruses. For example, at pH 5.0–5.5, known conditions for inactivating factor VIII, very few viruses are killed. Immunoglobulin solutions are an exception. Various studies have shown that low pH treatments, such as pH 4 used in immunoglobulin production, inactivate some enveloped viruses. Based on these and other results, WHO guidelines suggest that one manufacturer may incubate its globulin preparation at pH 4.0 for at least 6 hours at 37°C, while another manufacturer may follow a solvent / cleaning treatment by incubating at pH 4.25 in a container at 20°C for at least 21 days. In either process, the pH never falls below 4.0 and never exceeds 30 days.

[0008] Previous reports indicated that the antibody is fairly stable and should maintain its functional activity for up to 12 months under refrigerated conditions at 2–8°C. For longer-term storage, it is recommended to store the antibody frozen in smaller aliquots and to avoid repeated freeze-thaw cycles, which can negatively affect the antibody's functional activity. Generally, antibodies can be left at room temperature for up to one week without losing activity. Therefore, antibodies can be transported at ambient temperature. However, longer-term storage at room temperature is not recommended due to the lack of guarantee of antibody performance under such conditions. Current monoclonal antibody preparations often use sucrose to stabilize concentrated proteins for refrigerated or cryopreserved storage, giving them a shelf life of up to 3 years. However, manufacturer recommendations for storage of the monoclonal antibody, pembrolizumab, suggest storage at room temperature for up to 6 hours from the time of dilution. This includes the time for storing the dilution solution at room temperature and for infusion. Storage under refrigeration at 2–8°C (36–46°F) for up to 96 hours from the time of dilution is recommended. Similar guidance is provided by manufacturers of monoclonal antibodies, atezolizumab, demonstrating the chemical and physical stability of diluted products in use from the time of dilution for up to 24 hours at 30°C or below, and for up to 30 days at 2–8°C.

[0009] Previously, oral administration of crotoxin had been reported to be ineffective, likely because the protein could not withstand the acidic conditions of the stomach, which had become the accepted theory. HCl is the primary acid in the stomach. [Overview of the Initiative] [Means for solving the problem]

[0010] A stable crotoxin formulation containing approximately 0.9% physiological saline and a pH of approximately 1.5 to 4.5 is disclosed, and the formulation is acidified using HCl. In one embodiment, the stable crotoxin formulation contains a pH of approximately 3.5 to 4.5, exhibits stability under refrigerated conditions for up to 10⁶ months at a temperature of approximately 1 to 10°C, and has a crotoxin concentration of approximately 350 to 403 μg / mL as measured by A280nm absorbance. In one embodiment, the stable crotoxin formulation further contains approximately 0.009% by weight of benzalkonium chloride. In one embodiment, the stable crotoxin formulation further contains approximately 0.01 to 0.1% by weight of thiamine. [Modes for carrying out the invention]

[0011] A formulation of crotoxin is disclosed that is stable for extended periods under normal refrigerated drug storage conditions and also exhibits exceptional room-temperature stability. This stable formulation of crotoxin for clinical administration facilitates handling and reduces waste, allows the protein to be maintained in solution, preserves its biological activity without dissociation, is compatible with preservatives, and allows for a multi-use vial format.

[0012] According to one embodiment of the present invention, when the pH was lowered using hydrochloric acid (HCl), crotoxin was found to be stable for extended periods in physiological saline, even at acidic pH levels below 2.0. According to another embodiment of the present invention, when HCl was used to lower the pH and the crotoxin was stored for extended periods at pH 1.5 in a simple solution of 0.9% physiological saline, it was found that crotoxin could maintain its active state. Furthermore, when UV absorption was used to help estimate the protein concentration in the solution, it was confirmed that a decrease in the absorbance level of the protein at 280 nm occurred when crotoxin was in a low pH solution. It was considered that the change in absorbance resulting from the low pH may be the result of a structural change that makes the protein size more compact, thereby hiding the amino acids that contribute to the absorbance level. These absorbance changes were most evident in physiological saline at pH levels below 4.0. Recognizing the pH-induced structural effect, and despite concerns about its success, it was considered that this structural change may play a role in protecting amino acids within the protein from oxidation, thereby protecting the safety of the critical phospholipase enzyme. As a result, while storage under acidic conditions is harmful to the majority of proteins, solution formulations at pH 2.5–4.0 appear to be stable for extended periods, allowing for easy storage and use of crotoxin.

[0013] Crotoxin is not soluble in water unless it is acidic (<3.0). Crotoxin subunits dissociate at pH less than 2.5, although this has been reported to vary depending on the storage solution. At higher pH levels, generally above 4.0, the presence of at least 35 mM salt is necessary to maintain chlorotoxin solubility. Even then, chlorotoxin may precipitate from the solution (at pH 4 or approximately pH 4, and above pH 4), and the pH must be adjusted to less than 2.5 or above 9.5 to redissolve it. Changing pH is a delicate operation unless high concentrations of salt (>0.1M) are present. Certain buffers, such as citrate solution, can cause dissociation of chlorotoxin subunits at pH less than 4.0, thereby allowing any free cortactin to lyse cells at the injection site, or red blood cells if administered intravenously, making the product undesirable as a drug.

[0014] Using physiological saline (pH 5.0) and phosphate-buffered saline (pH 6.9-7.2), accelerated stability studies of crotoxin were completed at 49°C, starting with a range of formulations. This temperature is higher than the usual accelerated temperature used in standard stability tests, which is typically 42°C, because crotoxin exhibits inherent stability at high temperatures and can withstand boiling above 95°C for 10 minutes. The presence of protein precipitation and a decrease in the A280 value determined that crotoxin is more stable at a lower pH of 5.0. Two preservatives, methylparaben and benzalkonium chloride, were evaluated under accelerated conditions. The use of propylene glycol as a preservative was excluded in previous tests because it appeared to interfere with the efficacy of crotoxin. Solutions containing methylparaben accelerated protein precipitation, and benzalkonium chloride was preferred over the methylparaben solution.

[0015] Similar observations were made when the solution was stored at room temperature, and degradation was observed with increasing protein precipitation. While the lower pH solution maintained its potency for 24 months compared to 12-18 months when the protein was stored at a more neutral pH, the drug's potency decreased in processes that involved visual changes in the solution.

[0016] When stored under refrigeration (1-10°C), the potency of crotoxin was maintained for 10 years in a ready-to-use formulation in 0.9% physiological saline at pH 3.0-4.0, using HCl for acidification and benzalkonium chloride (approximately 0.009%) as a system preservative. During this period, intermittent exposure to room temperature had no detectable effect on the drug's activity. During formulation, the addition of benzalkonium chloride causes turbidity in the solution, but the solution becomes clear after standing at room temperature for 24 hours. It is also thought that the cationic charge of the preservative interacts with proteins, and by blocking protein-protein interactions via charges associated with amino acid components, it prevents protein aggregation, thereby contributing to the stability of the protein solution.

[0017] The accumulation of protein charge can occur from oxidative reactions with hydroxyl ions in water. The vitamin thiamine is an excellent antioxidant neutralizer of these radicals and can be incorporated into this formulation in concentrations ranging from 0.01 to 0.1%, thereby further enhancing the formulation's stability. When exposed to high temperatures, thiamine was found to protect peptides, particularly the readily reduced sulfur amino acids, methionine, and cysteine, from oxidation.

[0018] In one embodiment, the formulation contains about 0.9% by weight of physiological saline. In one embodiment, the formulation contains about 0.9% by volume of physiological saline. In one embodiment, the formulation contains about 0.009% by weight of benzalkonium chloride. In one embodiment, the formulation contains about 0.009% by volume of benzalkonium chloride. In one embodiment, the stable formulation of crotoxin further contains about 0.01-0.1% by weight of thiamine. In one embodiment, the stable formulation of crotoxin further contains about 0.01-0.1% by volume of thiamine.

[0019] The resulting formulation has several advantages. The low pH, in addition to inactivating exogenous substances, helps prevent the protein solution from being contaminated by microorganisms as a whole. The preservative, as a cleaning agent, is known to inactivate enveloped viruses and further protect patients from such substances, while also protecting the solution from contamination by repeated needle insertions. Finally, the simple storage conditions should instill confidence in its handling and transport, contributing to minimizing drug loss.

[0020] Table 1 shows the test results of crotoxin along with benzalkonium chloride in 0.9% physiological saline stored under refrigerated conditions of 2–8°C over a period of 106 months.

[0021] [Table 1] ND: Not implemented

Claims

1. A crotoxin preparation containing physiological saline and benzalkonium chloride, exhibiting stability and a pH of approximately 1.5 to 4.5, and further acidified using HCl.

2. A preparation of crotoxin according to claim 1, comprising approximately 0.9% by weight of physiological saline.

3. A preparation of crotoxin according to claim 2, comprising about 0.009% by weight of benzalkonium chloride.

4. A preparation of crotoxin according to claim 3, containing a pH of approximately 3.5 to approximately 4.5, and exhibiting stability under refrigerated conditions for up to 106 months at a temperature of approximately 1 to 10°C.

5. The crotoxin formulation according to claim 3, wherein the concentration of crotoxin measured at 280 nm absorbance is approximately 350 to approximately 403 μg / mL.

6. The crotoxin formulation according to claim 3, wherein the concentration measured at 280 nm absorbance contains approximately 350 to approximately 375 μg / mL after 106 months of storage at approximately 2 to 10°C.

7. A preparation of crotoxin according to claim 2, further comprising about 0.009% by volume of benzalkonium chloride.

8. A preparation of crotoxin according to claim 7, containing a pH of approximately 3.5 to approximately 4.5, and exhibiting stability under refrigerated conditions for up to 106 months at a temperature of approximately 1 to 10°C.

9. The crotoxin formulation according to claim 7, wherein the concentration of crotoxin measured by absorbance at 280 nm is approximately 350 to approximately 403 μg / mL.

10. The crotoxin formulation according to claim 7, wherein the concentration measured at 280 nm absorbance contains approximately 350 to approximately 375 μg / mL after 106 months of storage at approximately 2 to 10°C.

11. A preparation of crotoxin according to claim 1, comprising approximately 0.9% by volume of physiological saline.

12. A preparation of crotoxin according to claim 8, further comprising about 0.009% by weight of benzalkonium chloride.

13. A preparation of crotoxin according to claim 12, containing a pH of approximately 3.5 to approximately 4.5, and exhibiting stability under refrigerated conditions for up to 106 months at a temperature of approximately 1 to 10°C.

14. The crotoxin preparation according to claim 12, wherein the concentration of crotoxin measured by absorbance at 280 nm is approximately 350 to approximately 403 μg / mL.

15. The crotoxin formulation according to claim 12, wherein the concentration measured at 280 nm absorbance contains approximately 350 to approximately 375 μg / mL after storage at approximately 2 to 10°C for 106 months.

16. A preparation of crotoxin according to claim 8, further comprising about 0.009% by volume of benzalkonium chloride.

17. A preparation of crotoxin according to claim 16, containing a pH of approximately 3.5 to approximately 4.5, and exhibiting stability under refrigerated conditions for up to 106 months at a temperature of approximately 1 to 10°C.

18. The crotoxin preparation according to claim 16, wherein the concentration of crotoxin measured by absorbance at 280 nm is approximately 350 to approximately 403 μg / mL.

19. The crotoxin formulation according to claim 16, wherein the concentration measured at 280 nm absorbance contains approximately 350 to approximately 375 μg / mL after storage at approximately 2 to 10°C for 106 months.

20. A preparation of crotoxin according to claim 1, further comprising about 0.01 to 0.1% by weight of thiamine.

21. A preparation of crotoxin according to claim 1, further comprising about 0.01 to 0.1 volume percent of thiamine.

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