Preparation of Stable Erythrosine Salts, Esters and Conjugates and Their Use

Abstract

Pharmaceutically acceptable salts, esters and conjugates of silibinin, as well as compositions, and methods for their manufacture, and their use in the treatment of diseases or conditions treatable with silibinin or silybin are provided.

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 388,414, filed on July 12, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] Field Disclosed are silibinin salts, esters and conjugates with improved stability, physical properties and / or handling properties compared to silibinin, pharmaceutical compositions containing these silibinin salts, esters and conjugates, and methods for using them in conditions treatable with silibinin.

Background Art

[0003] Background Silibinin, the structure of which is illustrated in Formula I

Chemical Formula

Chemical Formula

[0004] When administered to a subject, silibinin is metabolized to form silicic acid. Silibinin undergoes a dephosphorylation reaction by an acid catalyst and / or an enzyme, losing a phosphate group and exposing the hydroxy group of silicic acid. Silibinin exists as a zwitterion in which the phosphate and amine moieties are ionized with respect to each other. The presence of the zwitterion limits the solubility of silibinin and also reduces the ability to form salts with alternative acids that may exist under physiologically acceptable conditions. Removal of the phosphate group can form intermolecular salts of silicic acid that cannot be prepared with silibinin by other methods. As the non-ionized form, silicic acid is much more lipophilic than silibinin and can thus more effectively cross the blood-brain barrier to elicit a response. Silicic acid has a high affinity for and can activate the 5-HT2A receptor, which plays an important role in regulating behaviors such as mood, sexual behavior, aggression, impulsivity, cognitive function, appetite, pain, sleep, and memory.

[0005] Evidence of the therapeutic effects of silicic acid in a wide range of clinical applications, such as mental disorders, pain disorders, and neurological disorders, has generated great interest in this compound. However, the poor physical properties of silicic acid in the solid state, for example, poor crystallinity with limited improvement in bulk purity upon crystallization, sensitivity to autcatalytic oxidation during handling and long-term storage, and low water solubility, have hindered the development of silicic acid-based pharmaceuticals.

[0006] Multiple studies have shown that classical psychedelics, such as silicic acid, which are serotoninergic hallucinogens, can induce therapeutic changes in people with various mental disorders. However, since natural silicic acid is present in relatively small amounts in actual mushrooms (Tyls et al. European Neuropsychopharmacology 2014 24(3): 342-356) and unmodified silicic acid is relatively unstable in solution, most studies have focused on the prodrug, silibinin, which is metabolized to silicic acid after ingestion and is involved in the demonstrated therapeutic and behavioral effects.

[0007] The results of completed and published studies show that exposure to psilocin (by psilocybin administration) leads to a significant improvement in symptoms of anxiety, depression, and substance use disorders.

[0008] Furthermore, the clinical safety of psilocin has been widely studied as a single agent and as an adjunctive therapy in adult populations. Psilocin is most commonly administered orally as psilocybin capsules and has been evaluated in non-blinded and double-blinded controlled trials. The dosing regimens of psilocybin range from 0.014 mg / kg to 0.6 mg / kg (which approximately corresponds to a psilocin dose range of 7 μg / kg to 0.32 mg / kg based on an estimated dose-normalized bioavailability of 52.7% (F. Hasler et al. Pharmaceutica Acta Helvetiae 1997 72(3), 175-184)) and are administered either as a single dose or as multiple escalating doses at intervals of several weeks.

[0009] Among adverse psychological experiences, those frequently reported include anxiety, induction of negative emotional states, and delusional or paranoid thinking. In relation to physical adverse events, they are cardiovascular (i.e., increased blood pressure and heart rate), as well as nausea and headache. Furthermore, hallucinogenic effects accompanied by increased anxiety have been reported at a low dose of 5 mg of psilocybin corresponding to approximately 2 mg of psilocin. See, for example, Griffith et al. Pharmaceutica Acta Helvetiae 2011 72(3) 175-184.

[0010] U.S. Patent No. 11,312,684 discloses that psilocin benzoate and psilocin succinate salts are preferred salt forms for manufacturing pharmaceutical compositions having excellent storage life stability and resistance to oxidative degradation. For some salts, a stability of up to 3 weeks has been disclosed.

[0011] Psilocin salts, esters, conjugates, and their formulations need to have improved stability, physical properties, and / or handling properties compared to psilocin.

Summary of the Invention

[0012] Summary One aspect of the present disclosure relates to the preparation of silocin salts, esters, and conjugates. Another aspect of the present disclosure relates to pharmaceutical compositions containing these silocin salts, esters, and conjugates. Another aspect of the present disclosure relates to methods for manufacturing these silocin salts, esters, and conjugates.

[0013] Yet another aspect of the present disclosure relates to methods of using these silocin salts, esters, and conjugates and their pharmaceutical compositions for the treatment of diseases and disorders treatable with silocin or siloviscin.

[0014] Definitions For ease of understanding of the present invention, several terms are defined below and throughout the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms herein are used to describe particular aspects of the invention, but their use is not intended to limit the invention, except as outlined in the claims.

[0015] Terms such as "a," "an," "the," etc. are not intended to refer to only a single entity, but include general classes that can be used to exemplify specific instances. As used herein, the term "about" refers to a value that is within 10% above or below the stated value. The term "administer" or "administering" refers to the method of giving a dosage of a compound or pharmaceutical composition to a subject.

[0016] As used herein, terms such as "pharmacologically effective amount" and "therapeutically effective amount" refer to an amount sufficient to relieve one or more symptoms of a disease or condition when an ester or conjugate or a composition thereof comprising a silosine salt, ester and conjugate, and salts, esters and conjugates thereof disclosed herein is administered to a subject including mammals such as humans.

[0017] The amount of a given composition described herein corresponding to such amount can vary depending on various factors such as a given drug, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, sex, weight) or host being treated.

[0018] As used herein, the terms "treating", "treat" or "treatment" refer to the administration of a compound or pharmaceutical composition for therapeutic purposes. Using for "treating a disorder" or "therapeutic treatment" refers to administering a treatment to a subject already suffering from a disease to improve the disease or one or more of its symptoms and improve the condition of the subject (e.g., by reducing one or more symptoms of inflammation). The compositions of the present disclosure can also be used as a primary preventive means, i.e., to prevent a condition or reduce the risk of developing a condition. Prevention refers to the prophylactic treatment of a subject who may not have fully developed a condition or disorder but is susceptible to or at risk of that condition. Thus, in the claims and aspects, the disclosed compositions and methods can be used for either therapeutic or prophylactic purposes.

[0019] The terms "salt", "salts", "salt form", "conjugate", "conjugates" and "conjugate form" as used herein are interchangeable and mean to include any compound formed when silosine is mixed with 1-hydroxy-2-naphthoic acid, 2-hydroxy-3-naphthoic acid, galactaric acid (mucic acid), glutamic acid, mandelic acid, naphthalene-2-sulfonic acid, camphorsulfonic acid, pantothenic acid or isethionic acid.

[0020] Without being bound by a particular theory, the salt, ester, and conjugate forms of the compounds disclosed herein exhibit binding properties, in addition to or instead of the ionic bonds of conventional salts, and are thought to enhance their stability.

[0021] Other features and advantages of the present disclosure will become apparent from the following detailed description, examples, and claims.

[0022] Detailed Description The present disclosure provides salt forms, ester, and conjugate forms of silosine, pharmaceutical compositions containing these salts, esters, and conjugates, and methods for their manufacture useful in any treatment in which silosine can be used.

[0023] The salts, esters, and conjugates of this disclosure have the following general formula A:

Chemical formula

[0024] Here, s / e / c of formula A represents a salt, ester, or conjugate.

[0025] In one non-limiting embodiment, the salt, ester, or conjugate is prepared from a mixture of silosine with 1-hydroxy-2-naphthoic acid, 2-hydroxy-3-naphthoic acid, galactaric acid (mucic acid), glutamic acid, mandelic acid, naphthalene-2-sulfonic acid, camphorsulfonic acid, pantothenic acid, or isethionic acid, or reagents such as, but not limited to, chlorosulfonic acid, sulfur trioxide pyridine complex, sulfur trioxide N,N-dimethylformamide complex, sulfur trioxide triethylamine complex, or sulfur trioxide trimethylamine complex.

[0026] A non-limiting example of a silosine salt, ester, or conjugate of the present disclosure is silosine isethionate. Another non-limiting example of a silosine salt, ester, or conjugate of the present disclosure is silosine mucart. In one non-limiting embodiment, the prepared salt, ester or conjugate is zwitterionic.

[0027] A non-limiting example of the zwitterion of the present disclosure is silosine-O-sulfate illustrated in Formula A1. [Chemical formula]

[0028] The salts, esters and conjugates of the present disclosure exhibit improved stability, physical properties, and / or handling properties compared to silosine. For example, the silosine mucic acid conjugate has been shown to be stable for at least 7 months.

[0029] Furthermore, studies in human subjects have shown that the salts and conjugates of the present disclosure exhibit a strong calming effect in patients without the hallucinogenic effects and / or anxiolytic effects often seen with silosine administration at similar doses.

[0030] Surprisingly, unlike the results of silibinin administration reported by Griffith et al. (Pharmaceutica Acta Helvetiae 2011 72(3) 175-184), 9 out of 10 subjects administered the silosine mucic acid conjugate at an equivalent effective amount of 2 mg of silosine did not have hallucinogenic effects but strongly suggested anxiolytic effects.

[0031] These anxiolytic effects at such low doses without hallucinogenic effects were completely unexpected, especially in patient populations that were frequently blood-sampled during the same period.

[0032] Also disclosed herein are pharmaceutical compositions comprising the silosine salt form, ester or conjugate form of the present disclosure and a pharmaceutically acceptable excipient. The pharmaceutical compositions of the present disclosure exhibit improved stability, physical properties, and / or handling properties compared to silosine-containing pharmaceutical compositions.

[0033] Furthermore, studies in patients have shown that the salts and conjugates of the present disclosure exhibit a strong calming effect in patients without the hallucinogenic effects and / or anxiolytic effects often seen with psilocybin administration.

[0034] Generally, the dosing regimens found to be useful for psilocin can also be used for the salts, esters and conjugates of the present disclosure.

[0035] In addition, microdosing, i.e., the regular intake of the psilocin salts or conjugates disclosed herein, at very low doses (ranging from 0.5 to 5 mg depending on the condition to be treated) can also be an effective dosing approach with treatment potential.

[0036] Examples of pharmaceutically acceptable excipients include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other excipients include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

[0037] The pharmaceutical compositions of the present disclosure may contain one or more solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants suitable for the particular dosage form desired. Remington's Pharmaceutical Sciences, Eighteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1990) discloses various excipients used in the formulation of pharmaceutical compositions and known techniques for their preparation. The use of any conventional excipient medium is considered to be within the scope of the present disclosure, except when it produces undesirable biological effects or otherwise interacts in a detrimental manner with other component(s) of the pharmaceutical composition and is incompatible with the compounds of the invention.

[0038] Some examples of materials that can serve as pharmaceutically acceptable excipients include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; natural and synthetic phospholipids such as soybean and egg yolk phosphatides, lecithin, hydrogenated soybean lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, dioleoyl lecithin, hydroxylated lecithin, lysophosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, distearoyl phosphatidylethanolamine (DSPE) and its pegylated esters such as DSPE-PEG750 and DSPE-PEG2000, phosphatidic acid, phosphatidylglycerol and phosphatidylserine; hydroxypropyl-beta-cyclodextrin and sulfonic acid substituted cyclodextrins (e.g., CAPTISOL™). Preferred commercial grades of lecithin include those available under the trade names Phosal® or Phospholipon®.Also includes: Phosal 53 MCT, Phosal 50 PG, Phosal 75 SA, Phospholipon 90H, Phospholipon 90G and Phospholipon 90 NG; soy - phosphatidylcholine (SoyPC) and DSPE - PEG2000 are particularly preferred; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen - free water; isotonic saline; Ringer's solution; 5% dextrose solution and combinations with the aforementioned aqueous solutions; ethyl alcohol, and phosphate buffer, and other non - toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, release agents, coating agents, sweetening agents, flavors and fragrances, preservatives and antioxidants may also be present in the composition, at the discretion of the formulator.

[0039] The pharmaceutical compositions of the present disclosure can be administered parenterally, orally, nasally, rectally, topically, or buccally. As used herein, the term "parenteral" refers to subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intra - articular injection, arterial injection, intra - synovial injection, sternal injection, intramedullary injection, lesion injection, or intracranial injection, and any suitable infusion technique.

[0040] When used in the methods and compositions of the present disclosure, a pharmaceutically acceptable silosine salt, ester, or conjugate can be contained in any suitable amount in any suitable carrier substance formulated for intravenous injection and generally is present in an amount of from 0.01 to 95% by weight of the total weight of the composition. In certain embodiments, the pharmaceutically acceptable silosine salt, ester, or conjugate is present in an amount of from 0.01 to 5% by weight of the total weight of the composition. In some embodiments, an aqueous solution suitable for intravenous injection containing a pharmaceutically acceptable silosine salt, ester, or conjugate may be formulated in physiological saline. The formulation of infusions is well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy (23rd ed), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988 - 1999, Marcel Dekker, New York). Compositions for infusion use may be provided in unit dosage form (e.g., single-dose ampules) or in vials containing several doses and may have suitable preservatives added. Solutions of pharmaceutically acceptable silosine salts, esters, or conjugates suitable for intravenous injection may have a pH of from about 3 and about 9. Further, solutions of pharmaceutically acceptable silosine salts, esters, or conjugates suitable for intravenous injection may encompass concentrations of pharmaceutically acceptable silosine salts, esters, or conjugates between about 0.1 mg / mL and about 50 mg / mL. In some embodiments, the aqueous solution has any one of the pharmaceutically acceptable salts, esters, or conjugates of silosine described herein between about 1 mg / mL and about 15 mg / mL. In certain embodiments, the aqueous solution has any one of the pharmaceutically acceptable salts, esters, or conjugates of silosine described herein between about 0.1 mg / mL and about 1 mg / mL.

[0041] A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Such solutions include, but are not limited to, 1,3 - butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils have conventionally been used as solvents or suspending media (by way of example, synthetic monoglycerides or diglycerides). Fatty acids, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, or their polyoxyethylated versions. These oil solutions or suspensions can also contain, but are not limited to, long-chain alcohol diluents or dispersants such as carboxymethylcellulose, or similar dispersants. Other commonly used surfactants, such as, but not limited to, Tweens or Spans or other similar emulsifiers or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0042] Compositions for oral administration can be in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets, commonly used excipients include, but are not limited to, lactose and corn starch. Lubricants such as, but not limited to, magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dried corn starch. When administering an aqueous suspension or emulsion orally, the active ingredient can be suspended or dissolved in an oily phase combined with an emulsifier or suspending agent. If desired, certain sweetening, flavoring, or coloring agents can be added.

[0043] Preferably, it is an oral preparation such as a capsule, which is packaged in an amber glass bottle, fixed with a polypropylene cap with a seal liner, and maintained at 15-25 °C in a sealed container in a dry place until administration.

[0044] In a non-limiting aspect, the silocin salt, ester or conjugate is administered in a nasal spray formulation. In a non-limiting aspect, the silocin salt, ester or conjugate is administered by time-release administration programmed by a nasal spray transducer.

[0045] Non-limiting devices for such administration are described in PCT / US2021 / 028068, filed Apr. 20, 2021, the teachings of which are hereby incorporated by reference in their entirety.

[0046] In a non-limiting aspect, the silocin salt, ester or conjugate is administered in a nasal spray, where the therapeutically active amount of the silocin salt, ester or conjugate is dissolved or suspended in a solution or mixture of excipients (e.g., preservatives, viscosity modifiers, emulsifiers, buffers) in a non-pressurized dispenser that delivers a spray containing a quantified dose of the active ingredient.

[0047] The silocin salts, esters and conjugates of the present disclosure and their pharmaceutical compositions are useful in methods of treating or alleviating the symptoms of any disease or condition treatable with silocin or siloviscin.

[0048] Such a method involves administering a silodosin salt, ester, or conjugate of the present disclosure to a subject in need thereof in an amount sufficient to treat or alleviate the symptoms of a disease or condition. Non-limiting examples of diseases or conditions include neurological injury, neurodegenerative diseases, inflammatory conditions, chronic pain, or psychological conditions. In certain embodiments, the disease or condition is an inflammatory condition (e.g., pneumonia, neuroinflammation, rheumatoid arthritis, atherosclerosis, psoriasis, type II diabetes, inflammatory bowel disease, Crohn's disease, multiple sclerosis, and / or sepsis). In specific embodiments, the inflammatory condition is chronic obstructive pulmonary disease (COPD), or Alzheimer's disease. In certain embodiments, the disease or condition is a neurological injury (e.g., stroke, traumatic brain injury, or spinal cord injury). In some embodiments, the disease or condition is chronic pain (e.g., pain resulting from postoperative pain, tension headache, chronic low back pain, fibromyalgia, nephropathy, multiple sclerosis, herpes zoster, complex regional pain syndrome, migraine, or sciatica). In specific embodiments, the chronic pain condition results from trigeminal autonomic cephalgia (e.g., paroxysmal and chronic cluster headache (CH), paroxysmal and chronic episodic migraine (PH), and short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (SUNCT)). In some embodiments, the trigeminal autonomic cephalgia is a transient or chronic CH. In certain embodiments, the condition is a psychological condition (such as depression, anxiety disorder, addiction, post-traumatic stress disorder, eating disorder, selective mutism, or obsessive-compulsive disorder). In specific embodiments, the psychological condition is depression or anxiety.

[0049] The features and advantages of the silodosin salts, esters, and conjugates and compositions prepared in the invention of the present disclosure are more fully shown by the following examples provided for illustrative purposes and should not be construed as limiting the present disclosure in any way.

Examples

[0050] Example Preparation of Silodosin Salts, Esters, and Conjugates Materials: The following acids were used in the preparation of new silodosin salts and conjugates. 1-Hydroxy-2-naphthoic acid 3-Hydroxy-2-naphthoic acid Galactaric acid (mucic acid) L-Glutamic acid Mandelic acid (R-2-hydroxy-2-phenylacetic acid) Pantothenic acid Isethionic acid [2-hydroxyethylsulfonic acid] Naphthalene-2-sulfonic acid (1S)-(+)-10-Camphorsulfonic acid

[0051] In addition, shirosine esters and shirosine-O-sulfate were prepared, which, like shiroshibin, are zwitterions. Shirosine-O-sulfate can be prepared by reacting shirosine with a number of reagents including, but not limited to, chlorosulfonic acid, sulfur trioxide pyridine complex, sulfur trioxide N,N-dimethylformamide complex, sulfur trioxide triethylamine complex or sulfur trioxide trimethylamine complex.

[0052] General procedure: To prepare salts, esters and adducts, equimolar or greater amounts of shirosine and the corresponding acid or other reagent were used. Various solvents were used to prepare shirosine salts, esters and adducts, and then the salt, ester, or adduct product was recrystallized; typical solvents used are, for example, methanol, ethanol, tetrahydrofuran, acetone, ethyl acetate and ether, etc.

[0053] Example 1: Siroxine, 204.2 mg (1.0 mmol), and the corresponding acid (1.0 mmol) selected from [2-hydroxy-3-naphthoic acid, galactaric acid (mucic acid), glutamic acid, mandelic acid, naphthalene-2-sulfonic acid, camphorsulfonic acid] were weighed into an 8 ml vial, and 2.0 ml of methanol was added with stirring using a magnetic bar. Precipitates were immediately formed with mucic acid and 1-hydroxy-2-naphthoic acid. All other salts or conjugates formed a clear solution, although glutamic acid formed a viscous mass.

[0054] The precipitate was diluted with approximately 4 mL of ethyl acetate and then collected on a frit. The collected product was washed successively with ethyl acetate and ether and then dried under high vacuum.

[0055] In reactions where no precipitate formed, the clear solution was transferred to a small round-bottom flask. The solvent was removed using a rotavapor; bubbles formed when high vacuum was applied. The bubbles were then treated with a combination of solvents including, but not limited to, ethyl acetate, acetone, tetrahydrofuran, and ether to precipitate the product.

[0056] Example 2: As described below, the pantothenate of siroxine was prepared. Calcium pantothenate 476.6 mg (1.0 MMOL) was dissolved in 2.0 ml of deionized water and treated with 2.0 mL of 1NHCL; a clear solution was obtained. To this solution, siroxine, 408.4 mg (2.0 mmol), a stirred solution in 2.0 mL of methanol was added. After stirring at room temperature for 2 hours, the solvent was removed and dried under high vacuum to obtain a white solid containing the pantothenate of siroxine and 1 mmol of calcium chloride.

[0057] Example 3: As described herein, the isethionate of siromycin was prepared. Sodium isethionate, 145.4 mg (1.0 mmol) was dissolved in 2.0 mL of deionized water and treated with 1.0 ml of 1N HCl; a clear solution was obtained. This solution was added to a stirred solution of siromycin 204.2 mg (1.0 mmol) in 3.0 ml of methanol. After stirring at room temperature for 2 hours, the solvent was removed and dried under high vacuum to obtain a white solid containing the isethionate of siromycin and 1 millimole of sodium chloride.

[0058] Example 4: Siromycin - O - sulfate was prepared, for example, by treating a solution of siromycin 204.0 mg (1.0 mmol) in 5.0 mL of pyridine with sulfur trioxide pyridine complex 318.3 mg (2.0 mmol). The reaction was continued overnight, after which the solvent was removed under high vacuum. Cold water was added to the residue and insoluble product collected on a frit. The product was washed with cold water and methanol and dried under high vacuum. Recrystallization from methanol gave the pure product.

[0059] Example 5: Purity evaluation Purity evaluation was performed by high - performance liquid chromatography (HPLC) for the following siromycin salts, esters and conjugates.

Table A

[0060] The purity, analysis, and identification by retention time of the synthesized psilocin salts, esters, and conjugates were determined using reverse-phase HPLC. Chromatographic separation was performed on a 4.6×250 mm, 5 μm column Zorbax SB-Phenyl using gradient elution and UV detection at 220 nm. Additionally, the psilocin salts, esters, and conjugates were analyzed for identification from 200 nm to 400 nm using diode array detection. The chromatographic conditions used are shown in Table 1. A stock solution of the sample was prepared with approximately 0.5 mg / mL of psilocin in methanol. The sample was diluted 5-fold with dilute acetic acid (0.2% v / v) to an approximate concentration of 0.1 mg / mL Psilocin in dilute acetic acid:methanol (80:20). The purity values were determined using peak area percent. The assay values of the psilocin salts, esters, and conjugates were calculated using psilocin base as an external reference standard. The assay values are "as is" and do not take into account water, residual solvents, or inorganic impurities. The retention time and UV spectrum of psilocin O-sulfate did not allow for a positive identification of psilocin. The results are shown in Table 2.

[0061]

Table 1-1

Table 1-2

[0062]

Table 2

[0063] Example 6: Stability Study of Psilocin Mucoadhesive Additional stability studies were performed on psilocin mucoadhesive (also known as psilocin mucate or conjugate or psilocin galactarate or conjugate) capsules. Appearance was evaluated by presenting the sample on a watch glass against black and white backgrounds. The assay conditions for Test / Method TM-022-0392 for evaluating stability and impurities are as follows:

[0064] Mobile phase: MPA: 945 mL water, 5 mL H3PO4, pH adjusted to 5.7 with NaOH; MPB: acetonitrile Diluent: 80:20 water:MeOH Standard: 0.4 mg / mL Psilocin Mucic RS in diluent Sample: 0.4 mg / mL Psilocin Mucic API in diluent The HPLC conditions are shown in Table 3.

[0065]

Table 3

[0066] System suitability: RSD NMT 2.0% (5 injections), tailing NMT 2.0, plates NLT 10000 Calculation was performed against the external standard in brackets. The identification of psilocin mucate was verified by ultraviolet (UV) evaluation, where the UV spectrum of the main peak of the standard sample was consistent with the UV spectrum of the main peak in the sample, and the retention time ratio was 0.98 - 1.02. The results are shown in Table 4 below.

[0067]

Table 4

[0068] Example 7: A Phase 1, open-label, single-treatment, single-dose, single-period, pharmacokinetic study of a hard gelatin capsule containing psilocin in the form of a mucic acid conjugate In this study, silocin mucart, a tryptamine derivative presented as a white to light brown solid crystalline powder, was administered. The molecular weight of this compound is 414.41 g / mol. In terms of equivalence, 4.05 mg of silocin mucart corresponds to 2 mg of silocin. In humans, mucic acid is enzymatically cleaved in the body during metabolism to produce silocin, which functions as an agonist for various serotonin receptors, including the 5-HT2A receptor, which underlies the hallucinogenic and therapeutic effects of silocin (Cao et al. Science (New York, N.Y.) 2022 375(6579):403-411; Lowe et al. Molecules 2021 26(10), 2948).

[0069] In this clinical trial, silocin was provided as white / opaque hard gelatin capsules containing 4.05 mg of silocin mucic acid conjugate corresponding to 2 mg of silocin. Ten healthy subjects between the ages of 21 and 50, body mass index 18.5 - 30.0 kg / m 2 (including males weighing 50 kg or more and females weighing 45 kg or more), non-smokers, or those who quit smoking 24 hours before administration, were orally administered 1 capsule with water. The purpose of this study was to evaluate the bioavailability and pharmacokinetic parameters of the mucic acid conjugate by measuring the plasma concentration of silocin and calculating from those measurements.

[0070] Blood samples (8 mL) were taken in sodium light, using K3 EDTA blood tubes, at -1.00 (before administration) and 0.25, 0.50, 0.75, 1.00, 1.50, 2.00, 3.00, 5.00, 8.00, 12.00, 16.00, and 24.00 hours after administration. The total number of blood samples taken during the study period was 13.

[0071] Furthermore, a psychiatrist independent of the principal investigator of the study administered the Mini-Mental State Examination (MMSE) test to each subject to evaluate the "mood" of each subject and to determine whether the subject's score had decreased. The MMSE test is a scale of 11 questions that tests five areas of cognitive function (orientation, registration, attention and calculation, recall, language). When a hallucinogenic substance is administered, the MMSE score is expected to be below 25. The results are shown in Table 5 below.

[0072]

Table 5

[0073] Surprisingly, unlike the results of psilocybin administration as reported by Griffith et al. (Pharmaceutica Acta Helvetiae 2011 72(3) 175-184), nine out of ten subjects administered the silosine mucic acid conjugate did not have hallucinogenic effects but strongly suggested anxiolytic effects. These anxiolytic effects at such low doses without hallucinogenic effects were completely unexpected, especially in the patient population that was frequently blood sampled during the same period.

Images