WO2022261761A1

WO2022261761A1 - Cultured mycelia from psychoactive psilocybe species and methods of production and use thereof

Info

Publication number: WO2022261761A1
Application number: PCT/CA2022/050950
Authority: WO
Inventors: Sharan SIDHU; Bernd Keller; Sarah NEUMANN
Original assignee: Numinus Bioscience
Current assignee: Numinus Bioscience
Priority date: 2021-06-14
Filing date: 2022-06-14
Publication date: 2022-12-22
Anticipated expiration: 2023-12-14

Abstract

An composition or extract obtained from mycelium of psychoactive species of mushrooms, including species of Psilocybe and, methods of obtaining said composition or extract and methods of use thereof.

Description

PSYCHOACTIVE SPECIES CULTURED MYCELIUM

TECHNICAL FIELD

[0001] This disclosure generally relates to the field of psychoactives from fungi. BACKGROUND OF THE ART

[0002] The use of Psilocybe mushroom species in medicinal, religious, and spiritual practices date back 3000 years in traditional cultural paradigms. Psilocybe spp. produce psilocybin, a tryptamine that when ingested is dephosphorylated to psilocin, the bioactive metabolite. Psilocin exerts a neurological effect by binding to serotonin 2A receptors with high affinity and produces a range of effects collectively coined the “psychedelic experience”. Recent studies have shown that psilocybin can be used as an effective therapeutic tool for several mental health conditions. Psilocybin is currently being delivered in two ways i) as a synthetic isolate, and ii) as whole fruiting body/mushroom extracts. Synthetic psilocybin allows for accurate formulary and production consistency, but lacks the synergistic compounds inherent in the species. Whole fruiting bodies present additional challenges on batch consistency, reproducibility and sustainability of a finished product that consistently meets label claims.

SUMMARY

[0003] In accordance with one aspect, there is provided a composition and/or an extract obtained from a biomass comprising mycelium of one or more psychoactive species cultured under controlled conditions. In some embodiments, the biomass consists essentially of mycelium of one or more psychoactive species. In some embodiments, the biomass comprises, consists essentially or consists of mycelium of a species of the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni. In one embodiment, the compositions are dried, preferably freeze dried.

[0004] In one embodiment, the mycelium are cultured in a liquid medium, optionally in a bioreactor, wherein the mycelium may be cultured and harvested in a continuous process or a batch process. In an embodiment, the bioreactor is an airlift reactor, preferably comprising an agitator, e.g. an impeller.

[0005] In one embodiment, the mycelium are cultured on a dry medium in a controlled environment, where the mycelium may be cultured and harvested in a continuous batch process or a batch process. [0006] In one embodiment, the extract is obtained by solvent extraction. The solvent may be ethanol and/or water. In one embodiment, the material is sonicated in ethanol. In another embodiment, the extract is obtained by percolation extraction.

[0007] In one embodiment, the extract comprises at least one of an alkaloid, indoleamine, indoleamine derivative, amino acid, modified amino acid, derivative of amino acid, tryptamine, lecithin, vitamin, metabolite, hormone, essential nutrient, antioxidant, pyridine-nucleoside, phenolic glycoside, glycoside, monoamine alkaloid, carbohydrate, coenzyme, monosaccharide, ribonucleic acid, sugar, sugar alcohol, purine nucleoside, acyl carnitines, ketone, fatty acid, neurotransmitter, and/or lactams.

[0008] In accordance with another aspect, there is provided a composition comprising, consisting or consisting essentially of dried mycelium of one or more psychoactive species cultured in a liquid medium or a dry media under controlled conditions. Preferably the mycelium is dried and ground into granular particles. In some embodiments, mycelium is of a species of the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni. Suitably, the mycelium are cultured in a bioreactor and are harvested in a continuous process or a batch process.

[0009] In another aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an extract or a composition as provided herein. The pharmaceutical composition may be in a microdosage or macrodosage form.

[0010] In an embodiment, a pharmaceutical composition comprises at least one of an alkaloid, indoleamine, indoleamine derivative, amino acid, modified amino acid, derivative of amino acid, tryptamine, lecithin, vitamin, metabolite, hormone, essential nutrient, antioxidant, pyridine-nucleoside, phenolic glycoside, glycoside, monoamine alkaloid, carbohydrate, coenzyme, monosaccharide, ribonucleic acid, sugar, sugar alcohol, purine nucleoside, acyl carnitines, ketone, fatty acid, neurotransmitter, and/or lactams.

[0011] Pharmaceutical compositions as provided herein may be used in treating a mental health condition, which may be selected from depression, including type II bipolar depression, postpartum depression and situational depression, anxiety, and mood disorders. The pharmaceutical compositions may also be used in treating a disease or condition selected from Obsessive Compulsive Disorders, Substance Use Disorders, Adjustment Reactions, Post- Traumatic Stress Disorder (PTSD), Sleep Dysregulation, headaches, including migraine, concussion and cluster headaches, chronic pain, fibromyalgia, Alzheimer’s disease, Parkinson's disease, and behavioural disorders, including anorexia, bulimia, and binge eating disorder.

[0012] In another aspect, there is provided a process for producing an extract comprising: culturing mycelium of a psychoactive species in a liquid medium under controlled conditions; drying the mycelium, preferably by flash freezing; and obtaining an extract from the cultured mycelium by a solvent extraction. In some embodiment, the solvent may be ethanol and/or water. In some embodiments, the psychoactive species is from the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni. The solvent extraction can include ultrasonication, and/or percolation extraction. In an embodiment, the mycelium may be dried to a moisture content of less than 30%, in a range of 10-20%, in a range of 5-10%, or less than 5%.

[0013] In another aspect, a process for culturing mycelium of a psychoactive species is provided, the process comprises: inoculating a media with parental mycelium of a psychoactive species; culturing a first biomass comprising cultured mycelium from the parental mycelium, the cultured mycelium and parental mycelium each comprising a same strain, or substantially the same strain, of mycelium; controlling an environmental condition while the media is inoculated and/or the first biomass is cultured; removing at least a portion of the first biomass from the media; and culturing a second biomass comprising cultured mycelium having the strain of mycelium of the parental mycelium from the portion of the first biomass or the parental mycelium, the second biomass having the same strain, or substantially the same strain, of mycelium as the parental mycelium. The second biomass may be cultured under about the same controlled environment condition(s) as the first biomass such that the mycelium of the first and second biomass may have about the same metabolomic profile. This process may be repeated to continuously culture additional new biomass comprising cultured mycelium.

[0014] In one embodiment, the first media is a wet media, preferably malt extract broth, Modified Melin-Norkrans medium, potato dextrose broth, and/or yeast malt extract broth. The wet media may have a single macro and micro nutritional profile.

[0015] In one embodiment, the first media is a dry media, preferably rye, bird seed, rice, whole oat, whole millet, and/or combinations thereof. The process may comprise expanding the first biomass by subdividing the first biomass onto additional media. The expanded first biomass may be subdivided between vessels for culturing mycelium of a psychoactive species . The first biomass and second biomass may be formed in a continuous batch process from the parental mycelium.

[0016] In one embodiment, the psychoactive species is from the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni.

[0017] In one embodiment, the process comprises inoculating the media, and/or culturing the first biomass at pressure comprising one of a hypobaric pressure, hyperbaric pressure, or atmospheric pressure. The process may also comprises inoculating the media, and/or culturing the first biomass at temperature of 1-20°C, 20-25°C, 25-30°C, 30-35°C, or 35°C-50°C. The process may also comprise inoculating the media, and/or culturing the first biomass at a relative humidity of greater than 50%, 85% to 95%, or greater than 90%. The process may also comprise inoculating the media, and/or culturing the first biomass at an oxygen concentration of about 21 %, greater than 21%, or in a range of 21-80%. The process may also comprise inoculating the media, and/or culturing the first biomass at a carbon dioxide concentration of less than 5%, less than 3%, or in a range of 0-1%.

[0018] In an embodiment, a moisture content of the first media is greater than 50%, less than 50%, 50-70%, greater than 70%. In another embodiment, a pH of the first media is about 7, in a range of 4.5-6, in a range of 5-7, or in a range of 7-9.

[0019] In another aspect, a process for producing an extract from a biomass comprising cultured mycelium of a psychoactive species is provided. The process comprises: contacting the biomass with a solvent to form a mixture of the solvent and an extract; optionally cooling the mixture; and separating the extract from the solvent.

[0020] In one embodiment, cooling the mixture comprises flash cooling the mixture. Flash cooling the mixture may comprise decreasing the temperature of the mixture from about a boiling point of the solvent to about a freezing point of the solvent.

[0021] In one embodiment, separating the extract from the solvent comprises freeze drying the mixture to sublimate the solvent. The process may also comprises forming droplets from the mixture before freeze drying the mixture to sublimate the solvent.

[0022] In one embodiment, the solvent is water or ethanol. The extract may be separated from the solvent by ultrasonication. [0023] In one embodiment, the psychoactive species is from the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni.

[0024] In another aspect, a process for producing an extract from a biomass is provided. The process comprises: inoculating a media with parental mycelium of a psychoactive species; culturing a first biomass comprising cultured mycelium from the parental mycelium, the cultured mycelium and parental mycelium each comprising a same strain, or substantially the same strain, of mycelium; controlling an environmental condition while the media is inoculated and/or the first biomass is cultured; removing at least a portion of the first biomass from the media; and culturing a second biomass comprising cultured mycelium from the portion of the first biomass of the parental mycelium, the second biomass comprising the same strain, or substantially the same strain, of mycelium as the parental mycelium, the second biomass cultured under the controlled environment condition; contacting at least the portion of the first biomass with a solvent to form a mixture of the solvent and an extract; optionally cooling the mixture; and separating the extract from the solvent.

[0025] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.

DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows the chemical structures of example compounds found in Psilocybe cultured mycelium: (A) Psilocybin, (B) Psilocin, (C) Baeocystin, (D) Norbaeocystin, (E) Tryptophan, (F) Aeruginascin, (G) Norpsilocin, (H) Serotonin, (I) Melatonin, (J) Betaine, (K) Ergothioneine, (L) Choline Alfoscerate, (M) Pantothenic acid, (N) D-Ribosylnicotinate, (O) Phosphocholine, (P) L-Histidine Trimethylbetane, and (Q) Glutathione.

[0027] FIG. 2 shows psilocybin, psilocin and tryptophan concentration assessment over 28 days assayed in an example cultured Psilocybe cubensis mycelium.

[0028] FIG. 3 shows the ratio of psilocybin:psilocin concentration over 28 days assayed in an example Psilocybe cubensis cultured mycelium. The figure evidences a viable concentration of psilocybin and a limited level of conversion to psilocin.

[0029] FIG. 4 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Psilocin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of psilocin at 0.90 minutes using a gradient of 22min. This Retention time has been confirmed using a commercially available standard.

[0030] FIG. 5 shows the Mass Chromatogram of Psilocin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of psilocin at a mass to charge ratio of 205.1337. The theoretical monoisotopic protonated ion [M+H]⁺ is 205.1335, therefore the mass error for this analysis is 1ppm. This

Fragmentation spectra has been confirmed by comparison with a commercially available standard.

[0031] FIG. 6 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Psilocybin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Psilocybin at 10.82 minutes using a gradient of 22min. This Retention time has been confirmed using a commercially available standard.

[0032] FIG. 7 shows the Mass Chromatogram of Psilocybin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Psilocybin at a mass to charge ratio of 285.0999. The theoretical monoisotopic protonated ion [M+H]⁺ is 285.0999, therefore the mass error for this analysis is Oppm. This

[0033] FIG. 8 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of 4-OH-TMT, dephosphorylated Aeruginascin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of 4-OH-TMT, dephosphorylated Aeruginascin at 0.89 minutes using a gradient of 22min.

[0034] FIG. 9 shows the Mass Chromatogram of 4-OH-TMT, dephosphorylated Aeruginascin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of 4-OH-TMT, dephosphorylated Aeruginascin at a mass to charge ratio of 219.1496. The theoretical monoisotopic protonated ion [M+H]⁺ is 219.1492, therefore the mass error for this analysis is 1.8ppm.

[0035] FIG. 10 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Aeruginascin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Aeruginascin at 9.98 minutes using a gradient of 22min.

[0036] FIG. 11 shows the Mass Chromatogram of Aeruginascin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Aeruginascin at a mass to charge ratio of 299.1157. The theoretical monoisotopic protonated ion [M+H]⁺ is 299.1155, therefore the mass error for this analysis is 0.7ppm.

[0037] FIG. 12 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Tryptophan generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Tryptophan at 2.15 minutes using a gradient of 22min. This Retention time has been confirmed using a commercially available standard.

[0038] FIG. 13 shows the Mass Chromatogram of Tryptophan generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Tryptophan at a mass to charge ratio of 205.0974. The theoretical monoisotopic protonated ion [M+H]⁺ is 205.0972, therefore the mass error for this analysis is 1ppm.

[0039] FIG. 14 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Betaine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Betaine at 1.67 minutes using a gradient of 25min, as well as a total ion chromatogram above. This Retention time has been confirmed using a commercially available standard.

[0040] FIG. 15 shows the Mass Chromatogram of Betaine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Betaine at a mass to charge ratio of 118.0861. The theoretical monoisotopic protonated ion [M+H]⁺ is 118.0863, therefore the mass error for this analysis is -1.7ppm.

[0041] FIG. 16 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Ergothioneine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Ergothioneine at 3.49 minutes using a gradient of 22min. This Retention time has been confirmed using a commercially available standard.

[0042] FIG. 17 shows the Mass Chromatogram of Ergothioneine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Ergothioneine Psilocybin at a mass to charge ratio of 230.0957. The theoretical monoisotopic protonated ion [M+H]⁺ is 230.0958, therefore the mass error for this analysis is - 0.4ppm.

[0043] FIG. 18 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of alpha- Glycerophosphocholine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of alpha- Glycerophosphocholine at 1.62 minutes using a gradient of 25min. This Retention time has been confirmed using a commercially available standard.

[0044] FIG. 19 shows the Mass Chromatogram of alpha-Glycerophosphocholine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of alpha-Glycerophosphocholine at a mass to charge ratio of 258.1097. The theoretical monoisotopic protonated ion [M+H]⁺ is 258.1096, therefore the mass error for this analysis is 0.4ppm. This Fragmentation spectra has been confirmed by comparison with a commercially available standard.

[0045] FIG. 20 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Choline generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Choline at 0.97 minutes using a gradient of 22min. This Retention time has been confirmed using a commercially available standard.

[0046] FIG. 21 shows the Mass Chromatogram of Choline generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Choline at a mass to charge ratio of 104.1069. The theoretical monoisotopic protonated ion [M+H]⁺ is 104.1070, therefore the mass error for this analysis is -1ppm.

[0047] FIG. 22 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Pantothenic Acid generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Pantothenic Acid at 2.60 minutes using a gradient of 22min.

[0048] FIG. 23 shows the Mass Chromatogram of Pantothenic Acid generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Pantothenic Acid at a mass to charge ratio of 220.179. The theoretical monoisotopic protonated ion [M+H]⁺ is 220.1180, therefore the mass error for this analysis is - 0.5ppm.

[0049] FIG. 24 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Nicotinic acid ribonucleoside generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Nicotinic acid ribonucleoside at 3.28 minutes using a gradient of 22min.

[0050] FIG. 25 shows the Mass Chromatogram of Nicotinic acid ribonucleoside generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Nicotinic acid ribonucleoside at a mass to charge ratio of 256.0816. The theoretical monoisotopic protonated ion [M+H]⁺ is 256.0816, therefore the mass error for this analysis is Oppm.

[0051] FIG. 26 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of

Trimethylhistidine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Trimethylhistidine at 1.47 minutes using a gradient of 22min.

[0052] FIG. 27 shows the Mass Chromatogram of Trimethylhistidine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Trimethylhistidine at a mass to charge ratio of 198.1238. The theoretical monoisotopic protonated ion [M+H]⁺ is 198.1237, therefore the mass error for this analysis is 0.5ppm.

[0053] FIG. 28 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of

Trimethyllysine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Trimethyllysine at 1.57 minutes using a gradient of 22min.

[0054] FIG. 29 shows the Mass Chromatogram of Trimethyllysine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Trimethyllysine at a mass to charge ratio of 189.1599. The theoretical monoisotopic protonated ion [M+H]⁺ is 189.1598, therefore the mass error for this analysis is 0.5ppm. [0055] FIG. 30 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Glucosamine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Glucosamine at 1.68 minutes using a gradient of 25min.

[0056] FIG. 31 shows the Mass Chromatogram of Glucosamine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Glucosamine at a mass to charge ratio of 180.0864. The theoretical monoisotopic protonated ion [M+H]⁺ is 180.0866, therefore the mass error for this analysis is -1.1 ppm.

[0057] FIG. 32 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of L- Carnitine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Psilocybin at 1.61 minutes using a gradient of 22min.

[0058] FIG. 33 shows the Mass Chromatogram of L-Carnitine generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Psilocybin at a mass to charge ratio of 162.1125. The theoretical monoisotopic protonated ion [M+H]⁺ is 162.1125, therefore the mass error for this analysis is Oppm.

[0059] FIG. 34 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Melatonin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Melatonin at 6.93 minutes using a gradient of 25min.

[0060] FIG. 35 shows the Mass Chromatogram of Melatonin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Melatonin at a mass to charge ratio of 233.1286. The theoretical monoisotopic protonated ion [M+H]⁺ is 233.1285, therefore the mass error for this analysis is 0.4ppm.

[0061] FIG. 36 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Baeocystin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Baeocystin at 6.27 minutes using a gradient of 7min.

[0062] FIG. 37 shows the Mass Chromatogram of Baeocystin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Baeocystin at a mass to charge ratio of 271.0846. The theoretical monoisotopic protonated ion [M+H]⁺ is 271.0842, therefore the mass error for this analysis is 1.5ppm.

[0063] FIG. 38 shows the Single Ion Monitoring (SIM) Chromatographic Spectra of Norpsilocin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of total ion current signal of Norpsilocin at 6.79 minutes using a gradient of 8min.

[0064] FIG. 39 shows the Mass Chromatogram of Norpsilocin generated from the analysis of example Psilocybe species cultured mycelium. Shows the relative abundance of the protonated ion [M+H]⁺ of Norpsilocin at a mass to charge ratio of 191.184. The theoretical monoisotopic protonated ion [M+H]⁺ is 191.1179, therefore the mass error for this analysis is 2.6ppm.

[0065] FIG. 40 shows the psilocybin, psilocin and total psilocybin and psilocin content of ethanol extracted Psilocybe cubensis cultured mycelium per EXAMPLE 2.

[0066] FIG. 41 shows the percentage increase in psilocybin extract from example Psilocybe cubensis cultured mycelium relative to the unextracted cultured mycelium material.

[0067] FIG. 42 shows an example bioreactor for culturing mycelium.

DETAILED DESCRIPTION

[0068] Several factors contribute to the challenges of developing natural product therapeutics that are utilized for the treatment of conditions that require practitioner supervision. This includes meeting label claims for natural products that have inherent variability due to environmental conditions of culturing, genetic variability of starting materials as well as epigenetic factors that result in fluctuations in the metabolites of interest that initiate the therapeutic effect. The fluctuations may be addressed by using clonal parental strains, tightly controlling process environments (temperature, humidity, light intensity), standardizing processes such as inoculation, spawning, propagating, cultivating, harvesting and extraction as well as standardizing substrates used.

[0069] Psilocybin is produced by mushrooms from the genus Psilocybe and it is the fruiting body of the species that has historically and more recently been utilized to achieve the “psychedelic experience” in medicinal, religious and spiritual practice as well as more recently in compassionate access use and clinical trial applications. [0070] In view of the factors identified above, the process of obtaining a natural psilocybin extract at a commercialized scale may become cost prohibitive and require an extensive amount of critical checkpoints to ensure standardized process control at several levels in the process flow. Further, although the culturing may be able to achieve this standardization of processes, the substrates used for spawning and culturing are also natural and may contribute to variability in the phytochemical profile and metabolomic output of the Psilocybe mushrooms. These are considerations for the development of Psilocybe mushroom derived products for use in psychedelic assisted psychotherapy for mental health conditions as well as other relevant conditions.

[0071] For confidence of regulatory approval, the presentation of a product with a narrow margin of variability increases. Additionally, reproducibility, sustainability, and scalability to consistently produce input materials and extracts are key considerations for the commercial viability of using a naturally derived Psilocybe species product, extract, and/or derivative for use in dosage formats for therapeutic applications. These considerations are not limited to the Psilocybe species, but apply to other species of mushrooms that contain psychoactive compounds that are therapeutically affective for mental health and other relevant conditions. This includes genres of Inocybe, Panaeolus, Gymnopilus, Copelandia, Hyboloma, Piuteus, Conocybe, and Panaeolina that produce psychoactive tryptamines such as psilocybin and psilocin, aeruginascin, baeocystin ibotenic acid and muscimol [1]

[0072] As used herein, “psychoactive species” includes any species of mushrooms that contains psychoactive compounds that are therapeutically affective for mental health and other relevant conditions, and can include Conocybe species, including Conocybe cyanopus, Conocybe siligineoides R. Heim, Conocybe kuehneriana Singer (Conocybe velutipes accepted name); Copelandia species, including Copelandia affinis Horak (Panaeolus cyanescens), Copelandia anomala (Panaeolus cyanescens), Copelandia bispora (Panaeolus bisporus), Copelandia cambodginiensis (Panaeolus cambodginiensis), Copelandia chlorocystis (Panaeolus chlorocystis), Copelandia cyanescens (Panaeolus cyanescens), Copelandia lentisporus (Panaeolus lentisporus), Copelandia tirunelveliensis (Panaeolus tirunelveliensis), Copelandia tropica, Copelandia tropicalis (Panaeolus tropicalis), Copelandia westii (Panaeolus cyanescens); Galerina species; Galerina steglichii Besl; Gymnopilus species, including Gymnopilus aeruginosus, Gymnopilus braendlei, Gymnopilus cyanopalmicola, Gymnopilus dilepis, Gymnopilus dunensis, Gymnopilus intermedius, Gymnopilus lateritius, Gymnopilus luteofolius, Gymnopilus luteoviridis, Gymnopilus luteus, Gymnopilus palmicola, Gymnopilus purpuratus, Gymnopilus subpurpuratus, Gymnopilus subspectabilis, Gymnopilus validipes, Gymnopilus viridans; Inocybe species, including Inocybe aeruginascens, Inocybe coelestium, Inocybe corydalina, Inocybe corydalina var. corydalina, Inocybe corydalina var. erinaceomorpha, Inocybe haemacta, Inocybe tricolor; Panaeolus species, including Panaeolus affinis, Panaeolus africanus, Panaeolus axfordii, Panaeolus bisporus, Panaeolus cambodginiensis, Panaeolus castaneifolius, Panaeolus chlorocystis, Panaeolus cinctulu, Panaeolus cyanescens, Panaeolus fimicola, Panaeolus lentisporus, Panaeolus microspores, Panaeolus moellerianus, Panaeolus olivaceus, Panaeolus rubricaulis, Panaeolus tirunelveliensis, Panaeolus tropicalis, Panaeolus venezolanus; Pholiotina species, including Pholiotina cyanopus, Pholiotina smithii; Pluteus species, including Pluteus albostipitatus, Pluteus americanus, Pluteus cyanopus, Pluteus glaucus, Pluteus glaucotinctus, Pluteus nigroviridis, Pluteus phaeocyanopus, Pluteus salicinus, Pluteus saupei, Pluteus villosus; Psilocybe species, including Psilocybe acutipilea, Psilocybe allenii Borov, Psilocybe alutacea, Psilocybe angulospora, Psilocybe antioquiensis, Psilocybe araucariicola, Psilocybe atlantis, Psilocybe aquamarina, Psilocybe armandii, Psilocybe aucklandi, Psilocybe aztecorum, Psilocybe aztecorum var. aztecorum, Psilocybe aztecorum var. bonetii, Psilocybe azurescen, Psilocybe baeocystis, Psilocybe banderillensis, Psilocybe bispora, Psilocybe brasiliensis, Psilocybe brunneocystidiata, Psilocybe cubensis, Psilocybe caeruleoannulata, Psilocybe caerulescens, Psilocybe caerulescens var. caerulescens, Psilocybe caerulescens var. ombrophila, Psilocybe caerulipes, Psilocybe callosa, Psilocybe carbonaria, Psilocybe caribaea, Psilocybe chuxiongensis, Psilocybe collybioides, Psilocybe Columbiana, Psilocybe congolensis, Psilocybe cordispora, Psilocybe cubensis, Psilocybe cyanescens, Psilocybe cyanofibrillosa, Psilocybe dumontii, Psilocybe egonii, Psilocybe eximia, Psilocybe fagicola, Psilocybe fagicola var. fagicola, Psilocybe fagicola var. mesocystidiata Guzman, Psilocybe farinacea, Psilocybe fimetaria, Psilocybe fuliginosa, Psilocybe furtadoana, Psilocybe tampanensis, Psilocybe galindii, Psilocybe gallaeciae, Psilocybe graveolens, Psilocybe guatapensis, Psilocybe guilartensis, Psilocybe heimii, Psilocybe herrerae, Psilocybe hispanica, Psilocybe hoogshagenii, Psilocybe hoogshagenii var. hoogshagenii (Psilocybe caerulipes var. gastonii, Psilocybe zapotecorum), Psilocybe hoogshagenii var. convexa (Psilocybe semperviva), Psilocybe hopii, Psilocybe inconspicua, Psilocybe indica, Psilocybe isabelae , Psilocybe jacobsii, Psilocybe jaliscana, Psilocybe kumaenor, Psilocybe laurae, Psilocybe lazoi, Psilocybe liniformans, Psilocybe liniformans var. liniformans, Psilocybe liniformans var. Americana, Psilocybe Mexicana, Psilocybe mairei, Psilocybe makarorae, Psilocybe mammillata, Psilocybe medullosa, Psilocybe meridensis, Psilocybe meridionalis, Psilocybe mescaleroensis, Psilocybe Mexicana, Psilocybe moseri, Psilocybe muliercula, Psilocybe naematoliformis, Psilocybe natalensis Gartz, Psilocybe natarajanii (Psilocybe aztecorum var. bonetii), Psilocybe neorhombispora, Psilocybe neoxalapensis, Psilocybe ovoideocystidiata, Psilocybe ovoideocystidiata, Psilocybe papuana, Psilocybe paulensis (Psilocybe banderiliensis var. paulensis), Psilocybe pelliculosa, Psilocybe pintonii, Psilocybe pleurocystidiosa, Psilocybe plutonia, Psilocybe portoricensis, Psilocybe pseudoaztecorum , Psilocybe puberula, Psilocybe quebecensis, Psilocybe rickii, Psilocybe rostrate, Psilocybe rzedowskii, Psilocybe semilanceata, Psilocybe samuiensis, Psilocybe schultesii, Psilocybe semilanceata, Psilocybe septentrionalis (Psilocybe subaeriginascens Hohn. var. septentrionalis), Psilocybe serbica, Psilocybe sierrae (Psilocybe subfimetaria), Psilocybe silvatica, Psilocybe singer, Psilocybe strictipes, Psilocybe stuntzii, Psilocybe subacutipilea, Psilocybe subaeruginascens, Psilocybe subaeruginosa, Psilocybe subbrunneocystidiata, Psilocybe subcaerulipes, Psilocybe subcubensis, Psilocybe subpsilocybioides, Psilocybe subtropicalis, Psilocybe tampanensis, Psilocybe tampanensis, Psilocybe thaicordispora, Psilocybe thaiaerugineomaculans, Psilocybe thaiduplicatocystidiata, Psilocybe uruguayensis, Psilocybe uxpanapensis, Psilocybe venenata (Psilocybe fasciata Hongo; Stropharia caerulescens), Psilocybe villarrealiae, Psilocybe weraroa, Psilocybe wassoniorum, Psilocybe weilii, Psilocybe weldenii, Psilocybe weraroa, Psilocybe wrightii, Psilocybe xalapensis, Psilocybe yungensis, Psilocybe zapotecorum, Psilocybe zapotecoantillarum, Psilocybe zapotecocaribaea, Psilocybe zapotecorum.

[0073] In some embodiments, the psychoactive species is a species of the genus Psilocybe. In some embodiments, Psilocybe cubensis, Psilocybe cyanescens, or Psilocybe alleni.

[0074] As used herein, “psychoactive species cultured mycelium” (abbreviated as PSCM) refers to cultured mycelium from psychoactive mushroom species.

[0075] As used herein, “culturing mycelium” or “cultured mycelium” of a psychoactive species refers to growing mycelium, or mycelium grown, on a media in a controlled environment respectively, to limit contaminants and provide about the same metabolomic profile for the mycelium as its parent mycelium when grown and tested under the same environmental conditions. The term “about” as used in this definition is defined below. In an example, having about the same metabolomic profile refers to a cultured mycelium of a psychoactive species having about the same desired psychoactive compound concentration, e.g. psilocybin, as its parent mycelium. In another example, having the about the same metabolomics profile refers to a cultured mycelium of a psychoactive species having about the same concentration of at least one, in some embodiments, more than one or all of, indole alkaloids, derivatives of indoleamines, amino acids and modified amino acids, ketones, nucleosides, carbohydrates, essential nutrients, neurotransmitters, vitamins, purine nucleosides, antioxidant compounds, oligosaccharides, coenzymes, lecithins, and glycerides (or specific compounds falling within these classes) as its parent mycelium; in some embodiments, the same concentration of the psychoactive species and at least one, some or all of derivatives of indoleamines, amino acids and modified amino acids, ketones, nucleosides, carbohydrates, essential nutrients, neurotransmitters, vitamins, purine nucleosides, antioxidant compounds, oligosaccharides, coenzymes, lecithins, and glycerides as its parent mycelium (or specific compounds falling within these classes).

[0076] Although terms such as “maximize”, “minimize” and “optimize” may be used in the present disclosure, it should be understood that such term may be used to refer to improvements, tuning and refinements which may not be strictly limited to maximal, minimal or optimal.

[0077] The terms "preferably," "preferred," "prefer," "optionally," "may," and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention.

[0078] The term “substantially” as used herein may be applied to modify any quantitative representation which could permissibly vary without resulting in a change in the basic function to which it is related.

[0079] Terms such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges. In the same manner, all ratios recited herein also include all sub ratios falling within the broader ratio.

[0080] The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0081] The term "about" can refer to a variation of± 5%, ± 10%, ± 20%, or± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values and ranges proximate to the recited range that are equivalent in terms of the functionality of the composition, or the embodiment. [0082] In one embodiment, the invention provides a novel process for producing biomass and downstream materials, such as compositions, extracts, and derivatives from mycelium of psychoactive species of mushrooms, and pharmaceutical compositions comprising the compositions, extracts, and derivatives. In another embodiment, there is provided novel compositions and/or extracts produced by processes provided herein. The term “composition” used herein may refer to a composition produced from a biomass comprising PSCM.

[0083] In one embodiment, there is provided a process for producing a composition comprising, consisting or consisting essentially of dried PSCM of one or more psychoactive species cultured in a liquid medium or a dry medium under controlled conditions. Preferably, the dried mycelium is granulated into particles. In an example, the PSCM may be freeze dried.

[0084] In one embodiment, there is provided a process of obtaining an extract from psychoactive mushrooms comprising culturing mycelium from one or more species of psychoactive mushrooms and obtaining the extract therefrom.

[0085] The mycelium are suitably cultured under controlled conditions. As used herein “controlled conditions” or “controlled environment” refer to condition(s) in which at least one of the conditions such as nutrients provided, temperature, pressure, and gas levels (one or more of oxygen, CO2 and nitrogen) are controlled. In some embodiments, at least two, at least three, or all of nutrients provided, temperature, pressure, and gas levels are controlled. In some embodiments, further conditions are controlled, including one or more of pH and duration, humidity, moisture content of media, spectrum and/or intensity of light exposure, and agitation.

[0086] Producing compositions, extracts, and starting materials for drug substances, from PSCM may resolve several challenges of producing a natural extract that contains several secondary metabolites and additional compounds. In an aspect, psychoactive mushroom mycelium may be cultured in a vessel that allows for respiration, media change, control of environmental conditions within the vessel such as temperature, moisture, air flow, etc., removal of mycelium for downstream processing, concentration of metabolites for ultimate use in formulations for several mental health conditions as well as other conditions that may benefit from the therapeutic use of naturally derived materials, such as materials and extracts, containing psilocybin. The viability of the invention has been assessed using Psilocybe species and Psilocybe PSCM. In an example, Psilocybe species and Psilocybe PSCM include Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe alleni which were cultured and extracted using the method described in this disclosure. The use of PSCM reduces the process workflow required to produce the desired Psilocybe species-derived materials and extracts by eliminating spawning, cultivation and harvesting processes and key factors that can increase variability. Use of PSCM may also remove the impact of environmental conditions, the introduction of different phytochemical compositions of the substrates used for different stages of cultivation, as well as time of harvesting and inter cultivation batch variability within a single flush and from flush to flush of the same batch.

[0087] In an aspect, methods for culturing mycelium of a psychoactive species described in this disclosure may reduce the workflow to a process of 1) inoculating media in a vessel by a pre determined parental mycelium stock, 2) culturing a biomass comprising cultured mycelium in the vessel, and 3) removing the cultured mycelium for downstream processing to obtain Psilocybe species biomass, extracts and derivatives that contains psilocybin, additional tryptamines, amino acids, and additional compounds of interest. Biomass may be cultured on media such that the biomass has the same strain, or substantially the same strain, of mycelium as the parental strain, which may in turn be removed for further processing and/or extraction. This process may be repeated indefinitely to allow consistent production of mycelium having the same genetic strain, i.e. the same genetic strain from generation-to-generation of PSCM produced according to the processes described in this disclosure. The media may be standardized and have a single macro and micro nutritional profile, thereby reducing the influence of nutrition on the metabolomic output. Environmental conditions can be kept consistent so that PSCM, and extracts thereof, are produced with consistent yield and with substantially repeatable chemical compounds. The culture may be grown in a continuous batch from a parental mycelium to reduce genetic variability as well as batch to batch variability which would occur with mushroom flushes. The extract may be reproducible because it is continuous, and provide a constant dosage starting material that is sustainable in any scale of vessel, rendering the process commercially viable. Additionally, growing PSCM in a vessel may allow controlled environment conditions which can provide a consistent and repeatable product having the same, or similar, genetic profile as the parental mycelium of a psychoactive species. Environment conditions within the reactor may also be optimized to increase PSCM yield and output of desired compounds in the PSCM and extracts thereof.

[0088] In an embodiment, the method for culturing mycelium of a psychoactive species may be cultured using a wet method. The wet method may comprise 1) inoculating wet media in a vessel by a pre-determined parental mycelium stock, 2) culturing a biomass comprising cultured mycelium in the vessel and 3) removing the biomass for downstream processing. The parental mycelium stock may be any of the Psilocybe PSCM. The wet media may be standardized and have a single macro and micro nutritional profile, thereby reducing the influence of nutrition on the metabolomic output. In an embodiment, the wet media may be malt extract broth, Modified Melin-Norkrans medium which may preferably be used for ectomycorrhizal and saprotrophic fungi, potato dextrose broth, and/or yeast malt extract broth. The wet media may be selected based on the Psilocybe species being cultured to maximize mycelium growth and/or yield of desired compounds such as psilocybin. The wet method may utilize a vessel that allows for respiration and media change. Environmental conditions within the vessel may also be adjusted to optimize mycelium yield and selectivity of desired psychoactive compound of the mycelium, e.g. psilocybin. Example environmental conditions include temperature, pressure, humidity, moisture content of media (i.e. the substrate on which mycelium is cultured), pH of the media, oxygen concentration, carbon dioxide concentration (CO2), and/or air flow rate. Removal of mycelium from the vessel may occur after a time period selected to maximize growth and/or yield of the mycelium. In an embodiment, a time period to maximize growth and/or yield of the mycelium post inoculation, e.g. the time period for culturing (post-inoculation) may be greater than one day depending on the Psilocybe species being cultured. In an embodiment, a time period to maximize growth and/or yield of the mycelium post inoculation is greater than 5 days, greater than 10 days, or in a range of 14-30 days depending on the Psilocybe species being cultured.

[0089] Potency of mycelium produced using the wet method may be in a range of greater than 0.1 milligram psilocybin per gram mycelium. In an embodiment, the potency may be in a range of 0.13-0.4 milligram psilocybin per gram mycelium. In an example, yield of mycelium using the wet method may be about 100 g to 2000 g dried biomass comprising mycelium per Liter of wet media. In another example, the yield of mycelium using the wet method may be 500-1500 g dried biomass comprising mycelium per Liter wet media. In another example, the yield of mycelium using the wet method may be 740-1150 g dried biomass comprising mycelium per Liter wet media.

[0090] FIG. 42 illustrates an example bioreactor which may be used as a vessel for the wet method. In an example, the vessel is an airlift bioreactor 1. In an embodiment, the airlift bioreactor 1 comprising an internal loop gas draft tube 7 inside a jacketed column 2, and a sparger 6 and/or impeller agitator system 12. Wet media (liquid) 13 may be provided by bioreactor 1 by a pump 14. The gas draft tube 7 may be T-shaped, cylindrical, or other desired shape. A cylindrical gas draft tube 7 is illustrated in FIG. 42. Probes 11 may be provided to measure pH, temperature, oxygen concentration, and other environmental conditions within the reactor. Compressed gas, e.g. air and/or oxygen, may be used for aeration and agitation. Gas sparger 6 may induce liquid upflow with the suspended particles of mycelium 9 in the draft tube 7. Gas source 10 may provide gas to the bioreactor 1, such that sparger 6 may introduce gas, e.g. air, into the reactor. Subsequently, the gas escapes from the top through a gas collector 4 of the bioreactor 1 and the wet media liquid with the suspended particles of mycelium may be led through the gas-free downcomer (illustrated by downward arrows) to outlet 15 where biomass of suspended particles 9 may be collected and dried for further processing. Pump 16 may be provided to withdraw the biomass comprising wet media and suspended particle 9 through outlet 15. An agitator system 12, e.g. an impeller, may be provided to circulate the liquid and suspended particle 9 throughtout the reactor to promote oxygen saturation in the liquid. Controller 17 may be in communication with pumps 14, 16, agitator system 12, gas source 10, and probes 11 to control and maintain the environment conditions as desired setpoint(s) to promote consistent, repeatable growth of mycelium in bioreactor 1. In an example, a tip speed of an impeller may be in a range from 0.1- 1 m/s. In another example the tip speed may be about 0.51 m/s. In an example a gas discharge velocity from the sparger may be 0.001 m/s to 0.1 m/s. In another example the gas discharge velocity from the sparger may be about 0.055 m/s. In an example a Volumetric Mass Transfer Coefficient (K_LA) may be 10-100/h, preferably about 78/h. In an example, a volume of air sparged (in aerobic cultures) per unit volume of liquid growth medium per minute (vvm) may be about 1 vvm. In an example, a dissolved oxygen (DO) content may be about 20%, or in a range of about 16-26%. Calcium ion (Ca²⁺) and/or non-ionic detergents may be added to the wet media to induce pelleted mycelium. Increasing DO above 20% may also induce pelleted mycelium. Filamentous mycelium may be induced by decreasing DO below 20% and increasing shear stress by increase tip speed above 0.51 m/s and/or gas discharge velocity above 0.055 m/s. In an example, a preferred pH in the vessel may be in a range of 4.5-6, a range of 5-7, or about 7.

[0091] In another embodiment, the method for culturing mycelium of a psychoactive species may be cultured using a dry method. The dry method may comprise 1) inoculation of dry media in a vessel by a pre-determined parental mycelium stock, 2) expansion into further dry media and 3) the removal of the biomass for downstream processing. The parental mycelium stock may be any Psilocybe PSCM described in this disclosure. The dry media may be standardized and have minor fluctuations macro and micro nutritional profile, but does not contribute to variability in metabolomic profile of the biomass comprising mycelium. In an embodiment, the dry media may be rye, bird seed, rice, whole oat, whole millet, and/or combinations thereof. The dry method may allow the mycelium to fully colonize a dry media, which is then expanded to introduce fresh nutrients allowing for biomass production. In an example, the vessel may be an incubator in which inoculated dry media, e.g. rye, is placed to allow mycelium cultures to grow in a controlled environment. The vessel may comprise a recirculator and microbial controls to minimize contaminants entering the vessel. Continuing the example, the inoculated dry media may be placed in one more spawn bags configured to allow respiration of mycelium within the bag which are in turn placed into the vessel. This may approach may reduce contamination of the mycelium as filtration of the air entering the vessel and/or spawn bag(s) may reduce influx of contamination in comparison to other methods of culturing mycelium. Dry media cultured with biomass comprising mycelium may be extracted from the vessel after a time period for harvest. Dry media cultured with biomass comprising mycelium, and/or the biomass comprising mycelium, may be added to new dry media to inoculate the new dry media which can be placed in the vessel for further culturing of mycelium on the new dry media. This cycle may be repeated to continuously inoculate new dry media to produce mycelium having the same, or substantially the same, genetic strain as the original parental mycelium. Environmental conditions within the vessel may be adjusted to optimize mycelium yield and selectivity of desired psychoactive compound of the mycelium, e.g. psilocybin. Example environmental conditions include temperature, pressure, humidity, moisture content of media (i.e. the substrate on which mycelium is cultured), pH of the media, oxygen concentration, carbon dioxide concentration (CO2), and/or air flow rate. In an embodiment, a time period to maximize growth and/or yield of the mycelium post inoculation, e.g. the time period for culturing (post-inoculation) may be greater than one day depending on the Psilocybe species being cultured. In an embodiment, the time period is greater than 5 days, greater than 10 days, or in an range of 14-45 days.

[0092] Potency of mycelium produced using the dry method may be in a range of greater than 0.1 milligram psilocybin per gram mycelium. In an embodiment, the potency of mycelium may be 0.1-0.43 milligram psilocybin per gram mycelium. In another example, the yield of mycelium using the dry method may be 1000-1500 gram dried biomass per 760 inch³ (12454 cm³) grain spawn bag, or about 1 gram/inch³ to 2 gram/inch³ of dried biomass of mycelium per volume of dry media. In another example the yield of mycelium using the wet method may be 1241.3 gram per 760 inch³ (12454 cm³) grain spawn bag, i.e. 1.63 g/inch³of dried biomass of mycelium per volume of dry media. An example grain spawn bag may be 8" X5" X19" about 760 inch³ (12454 cm³) and include a 0.2 micron filter patch to allow for air exchange while excluding potential contaminants. The filter may have an area of 1.5 in x 1.5 in (2.25 inch²). Various sized spawn bags may be used and the example spawn bags used in the examples are non-limiting.

[0093] Method for culturing mycelium of a psychoactive species described in this disclosure may allow for biomass production in a continuous batch, or a batch process, from a parental mycelium to reduce genetic variability as well as batch to batch variability. The biomass material may be reproducible because it may be produced continuously from the same parental mycelium, and the ability to produce a constant dosage starting material is sustainable making the vessels scalable. In contrast, traditional method for culturing mushroom may require mushroom flushes which increases genetic variability, and batch to batch variability, reducing the ability to produce psychoactive species of mushrooms having consistent chemical compounds.

[0094] Example environmental conditions for a vessel used to culture PSCM according to this disclosure may include a pressure of at least one of a hypobaric pressure, hyperbaric pressure, or atmospheric pressure. Temperature may be controlled in the vessel and held constant, for example in a range of 15-45°C. In an embodiment, the temperature may be in a range of 1-20°C, 20-25°C, 25-30°C, 30-35°C, and/or greater than 35°C. Relative humidity may be controlled, for example at a relative humidity of greater than 50%. In an embodiment, the relative humidity is 85% to 95%, and/or greater than 90%. Moisture content of media may be greater than 50%, less than 50%, 50-70%, greater than 70%. pH of the media may be a pH of at least one of about 7, in a range of 4.5-6, in a range of 5-7, in a range of 7-9. Oxygen concentration in the vessel may be controlled for example at about 21%, greater than 21%, and/or in a range of 21-80%. CO2 concentration in the vessel may also be controlled, for example less than 5%, less than 3%, and/or in a range of 0-1%. Gas flow through the vessel may be modulated to maintain desired oxygen and/or CO2 concentrations in the vessel. In some embodiments, biomass cultured according to the processes described in this disclosure may be cultured in about the same environmental conditions as a parental mycelium.

[0095] As detailed in the Examples, the present inventors have surprisingly and unexpectedly found that a PSCM contains the same key actives as an extract from the fruiting body of psychoactive species of mushrooms. In addition to indole alkaloids, a variety off other classes of compounds have been detected in Psilocybe PSCM including, but not limited to, derivatives of indoleamines, amino acids and modified amino acids, ketones, nucleosides, carbohydrates, essential nutrients, neurotransmitters, vitamins, purine nucleosides, antioxidant compounds, oligosaccharides, coenzymes, lecithins, and glycerides. Some detected compounds may contribute beneficially to downstream pharmaceutical formulations and/or act synergistically/or modulate the effects of psilocin. Present inventors have also developed processing and extraction steps that yield compositions and/or extracts comprising the compounds found in Psilocybe PSCM, including the classes of compound mentioned above, such as psilocybin.

[0096] In exemplary embodiments, the PSCM of Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe alleni, produced according to a wet method of culturing PSCM according to this disclosure, contains a combination of, in some embodiments all of, the compound identified in Table 1:

Table 1 Example compounds identified in cultured Psilocybe mycelium according to wet method of culturing PSCM.

V indicates compound present in PSCM sample; x indicates not present in sample [0097] In another exemplary embodiment, the PSCM produced according to a dry method of culturing PSCM according to this disclosure contains a combination of, in some embodiments all of, the compound identified in Table 2:

Table 2 Example compounds identified in cultured Psilocybe mycelium according to dry method of culturing PSCM.

V indicates compound present in PSCM sample; x indicates not present in sample

[0098] As detailed in the Examples, the present inventors have surprisingly and unexpectedly found that a PSCM extract (i.e. an extract from mycelium) contains the same key actives as an extract from the fruiting body, and the PSCM.

[0099] In an exemplary embodiment, the PSCM extract contains a combination of, in some embodiments all of, the compounds identified in Table 3:

[0100] By utilizing PSCM and, in preferred embodiments, Psilocybe PSCM the downstream processing is simpler as it may not require removal of large amounts of fibrous materials. In an example, PSCM comprises less than 10% of fibrous material in comparison to the fruiting body of a mushroom from which the PSCM is derived. The vessel containing the media is inoculated with a pre-determined parental mycelium stock chosen for its metabolomic profile and allowed to grow. The vessel may allow for respiration and scheduled media changes to ensure nutrient availability. For example, for the wet method of culturing mycelium of a psychoactive species described in this disclosure, the vessel may provide an opening and/or feeding mechanism to exchange media to ensure nutrient availability. In another example, for the dry method of culturing mycelium of a psychoactive species, additional new dry media may be introduced to expand the mycelium onto the additional dry media. The vessel, e.g. an incubator, may provide a controlled environment that may not need to be changed depending on the phase of growth and is therefore consistent. Example conditions of the environment that may be controlled include temperature, pressure, humidity, moisture content of media (i.e. the substrate on which mycelium is cultured), and/or air flow rate. As the biomass comprising PSCM grows and expands it may be collected and dried then may undergo further processing depending on what the PSCM is designated for. Composition(s) and extract(s) from the PSCM may be used as a drug substance; accordingly, a composition comprising PSCM and/or an extract of PSCM may be refined and then used in the formulation of a drug product. For example, a dried PSCM, or an extract or PSCM, may be designated as a drug substance and used in drug formulations. Composition(s) and extract(s) may also go through several additional steps of post processing before being used in the formulation of a drug product, or to produce derivatives and isolates for use in drug formulations. All derived materials from PSCM may contain psilocybin and other beneficial compounds. Compositions and extracts derived from PSCM produced by the methods describe in this disclosure may comprise the same genetic strain of PSCM and consistent of amounts of component compounds in the mycelium batch cycle after batch cycle of harvesting and extracting Psilocybe PSCM. Non-destructive extraction method described in this disclosure may also provide consistent constituent compounds between Psilocybe PSCM, compositions, and extracts thereof.

[0101] As detailed in the Examples and Figures 4-39, these example compounds, among others, have been detected using quantitative high-resolution, accurate-mass (HRAM) liquid chromatography mass spectrometry (LC-MS), specifically the OrbiTrap Exploris 120. Discovery and detection workflow included the comparison of measured mass of protonated ions with the theoretical monoisotopic protonated ion mass, confirmation of retention time of available standards and a confirmed comparison of fragmentation spectra of the compound with commercially available standard compound as well as reviewing extracted chromatograms. Table 3 shows a list of compounds that have been detected in the Psilocybe PSCM. These compounds, which include tryptamines, amino acids, vitamins and pre-cursors and additional compounds of interest, may be optimized by selection of mycelium parental stock, and/or by selection of specific environmental conditions in the vessel used to culture PSCM.

Indole alkaloids

[0102] The compounds assayed in the mycelium (table 3) are important for the therapeutic potential of the extract and associated therapeutic formulations. Psilocybe species contain bioactive indole alkaloids [2], which are important for receptor binding and interactions with other proteins and enzymes making them important fungal metabolites from a pharmaceutical and industrial prospective. Indole alkaloids are also referred to as tryptamines, indoleamine, hallucinogenic alkaloids, tryptamine alkaloids, indoleamine hallucinogens [2,3], and are derived from the amino acid tryptophan (figure 1e) [4] by way of tryptamine [4,5] Psilocybin, psilocin (figure 1b), baeocystin (figure 1c), norbaeocystin (figure 1d), aeruginascin (figure 1f), norpsilocin (figure 1g) are indole alkaloids found in Psilocybe mushrooms [2, 6, 7] and have been detected in the Psilocybe PSCM per the Examples. These alkaloids have hallucinogenic, anxiolytic, and psychoactive activities and are potential tools for psychotherapy as well as novel treatments for additional conditions. Natural Psilocybe extracts can have the same clinical efficacy as pure psilocybin, see reference [50]: in this study, psilocybin mushroom extract was more effective at the same dose, pointing to a synergistic interplay of chemistries within the extract and that there are additional contributing compounds beyond psilocybin and psilocin.

[0103] Psilocybin and psilocin are the primary hallucinogenic alkaloids found in psychedelic mushrooms [2] (also referred to as hallucinogenic, entheogenic, magic, medicinal, neurotropic, psychoactive, sacred and saint mushrooms). The fungal metabolite psilocin is the active serotonergic agonist and thereby the bioactive component found in trace amounts in Psilocybe mushrooms comparative to psilocybin and, in vivo, psilocybin is dephosphorylated to the bioactive compound psilocin. The need to implement new tools to combat the growing mental health crisis has resulted in clinical investigation of the use of psychedelic assisted psychotherapy to treat a variety of mental health conditions as well as other relevant disorders. Psilocybin and psilocin are structurally like serotonin (figure 1h) [3, 7, 8] and considered serotonergic hallucinogens. The effects of serotonergic hallucinogens are mediated by interactions with the 5-HT2A receptors [9,10] Serotonin is an indoleamine hormone neurotransmitter, synthesized mainly by intestinal cells in humans and plays an important role in sleep, cognition, memory, temperature regulation and behavior [5] Dysregulation in the serotonin system has been associated with alterations in stress hormones, such as cortisol, and mood disorders. Psilocybin administration show increases in cortisol levels and activation of networks that lead to the increased control over emotional processes, and relief of negative thinking and persistent negative emotions [8]

[0104] Data derived from psilocybin psychedelic assisted psychotherapy studies support the therapeutic potential of psilocybin as treatment for several different conditions. These include OCD [11], substance use disorder [8,12], mood/depression [12-15], anxiety [12, 15] and migraine headaches [16] Further clinical trials have been approved for investigations in additional conditions including concussion headaches and anorexia [17]

[0105] As mentioned, evidence suggests that additional compounds inherent in Psilocybe species contribute to its beneficial effects. This includes other tryptamine alkaloids like Baeocystin and Norbaeocystin found in Psilocybe mushroom fruiting bodies and that have been characterized in Psilocybe PSCM per the Examples. Baeocystin is a tryptamine alkaloid that is N- methyltryptamine carrying an additional phosphoryloxy substituent at position 4. It is an organic phosphate, a tryptamine alkaloid and a secondary amino compound derived from tryptamine [2, 7] . It was first isolated from Psilocybe baeocystis by Leung and Paul in 1968. Later, other researchers isolated it from other species such as Psilocybe semilanceata, Panaeolus renenosus, Panaeolus subbaiteatus and Piuteus salicinus [2, 6, 50] Although there are mixed reports of psychoactive capacity it has been hypothesized that baeocystin may work synergistically with psilocin by “competing” for monoamine oxidases which rapidly degrade psilocin playing an essential role in the modulation of the psychedelic effects. Norbaeocystin a derivative analogue of psilocybin and a N-demethylated derivative of baeocystin, is an intermediate in the biosynthesis of psilocybin. Again, considered nonpsychedelic, Norbaeocystin aswell may play an essential role in generating and modulating specific psychedelic effects.

[0106] Aeruginascin is the N-trimethyl analogue of psilocybin [51] Both aeruginasin and its hydrolysis product 4-hydroxyN,N,N-trimethyltryptamine, 4-OH-TMT have been detected in the Psilocybe PSCM per the Examples. Evidence shows binding at 5-HTI_A, 5-HT_2A, and 5-HT_2B receptors [52], although the binding affinity is less so than psilocin.

Serotonin

[0107] As serotonin and associated receptors are involved in several systems [18, 19], it therefore stands to reason that dysfunction in the serotoninergic system would be associated with several diseases and disorders. This has been shown for several conditions including anxiety and depression [20, 21], migraine headaches [22-25], schizophrenia [26-29], vomiting [30, 31], obsessive compulsive disorder [32-34], addiction [35-37], and neurodegenerative disorders [38- 41] Due to the high affinity of psilocybin and psilocin for 5-HT2A receptors [9, 10], coupled with the fact that serotonin is involved in several systems such as the nervous, gastrointestinal, and cardiovascular systems merits the assessment of efficacy in additional disorders/conditions.

Amino acids

[0108] In addition to indole alkaloids, a variety off additional compounds have been detected in the Psilocybe PSCM including amino acids, saccharides, lecithins and glycerides. Essential amino acids are amino acids that can not be synthesised by humans and vertebrates from metabolic intermediates, they are the basic building blocks of amino acids and proteins. All amino acids are composed of an amino (-NH2) and carboxylic acid (-COOH) functional group. Amino acids are the nitrogen backbone of important compounds for the human body to function, such as neurotransmitters and hormones. There are nine essential amino acids including: phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histidine, leucine, and lysine. All nine of these essential amino acids have been identified in PSCM extract per the Examples. These nine amino acids are important to create the compounds that humans need to live and grow, and are all necessary for optimal health and bodily functions [81]

[0109] Tryptophan is an essential amino acid found in most proteins and is precursor of serotonin by conversion to 5-hydroxy-tryptophan (5-HTP) which in turn is converted to serotonin [53] It is a natural sedative and present in many foods as well as Psilocybe mushrooms and has been detected in Psilocybe PSCM per the Examples. About 2-10% of the tryptophan consumed through diet is destined for the serotonin pathway in humans where it is converted to 5-HTP [53] Studies have shown that low levels of tryptophan result in low levels of serotonin [54]

[0110] Non-essential Amino Acids are created through metabolic pathways from the essential 9 amino acids. Non-essential amino acids are not necessary in the human diet as they can be synthesised using essential amino acids, however they are still used in the creation of proteins. Per Table 3, in addition to the 9 essential amino acids, a number of other amino acids were identified in Psilocybe PSCM.

Other compounds

[0111] Betaine (figure 1j) is a trimethyl glycine consisting of glycine with three methyl groups that serves as a methyl group donor in several metabolic pathways primarily in methionine cycle- primarily in the human liver and kidneys, and as an osmoprotectant [61] As an osmoprotectant, betaine protects cells, proteins, and enzymes from environmental stress inclusive of low water, high salinity, or extreme temperature [61, 62] Methylation is an important biochemical process involved in many critical pathways. Insufficient dietary intake of methyl groups results in a reduction in methylation which results in disruption kidney protein and fat metabolism leading to elevated plasma fat and homocysteine [62] and betaine is approved as a supplement to help support liver function. Betaine also has an immune modulatory role and is involved in oxidative stress, inhibiting transcription protein factors in innate responses as well as inflammasome protein [61] Betaine has been detected in the Psilocybe PSCM per the Examples .

[0112] Ergothioneine (figure 1k) is a thiourea derivative of histidine that is

N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine in which the hydrogen at position 2 on the imdazole ring is replaced by a mercapto group. Ergothioneine has antioxidant and anti inflammatory properties [63] It is synthesized by few bacteria fungi and where mushrooms appear to be the highest source [63] Ergothioneine has been detected in Psilocybe PSCM per the Examples. Ergothioneine does accumulate through diet, which appears to decrease through age [63, 64] It is an important chemoprotective agent and functions as a disease preventing molecule [63, 64] and is also associated with aging [63]

[0113] Choline Alfoscerate (alpha-GPC) (figure 11) is a member of the class of phosphocholines that is the choline ester of sn-glycero-3-phosphate found in the brain [65] It is one of the major osmolytes in the renal medullary cells. It has a role as a parasympatholytic, a neuroprotective agent, a human metabolite, a Saccharomyces cerevisiae metabolite, an Escherichia coli metabolite, and a mouse metabolite [65] Alpha-GPC quickly delivers choline to the brain and is important in the biosynthetic pathway of acetylcholine, a neurotransmitter and neuromodulator involved in arousal, attention, memory, and motivation and has been found to support cognitive health and/or brain function. It has been shown to increase the neurotransmitter acetylcholine which aids in leaning and memory and is used as supplement in North America and in European countries as a prescription medication for a variety of conditions. Due to its increase in the projection of acetylcholine, Alpha-GPC has been used for treatment of Alzheimer’s, Dementia, and stroke. Alpha-GPC can also be taken as a supplement to increase overall brain health and memory [66] Alpha-GPC’s health benefit are not limited to the brain, it is also used by athletes to reduce reduction of choline levels. Athletes use alpha-GPC to increase both endurance performance and growth hormone secretions, thereby increasing overall athletic performance [67] Alpha-gpc has also been detected the Psilocybe PSCM per the Examples. [0114] Pantothenic acid (figure 1m) also known as Vitamin B5 is a water-soluble vitamin widely distributed in both plant and animal tissues and is readily available as a retail supplement [68] Pantothenic acid has been detected in Psilocybe PSCM per the Examples. It is a key component in the generation of coenzyme A, essential for the breakdown of fatty acids and plays an important role in the metabolic function of all cells [69] Vitamin B5 is a key building block in compounds in the Vitamin B complex, and as such contributes to the overall function and health of the human body [68]

[0115] D-Ribosylnicotinate (figure 1n) more commonly known as nicotinic acid riboside belongs to a group of organic compounds known as glycosylamines. Nicotinic Acid Riboside exists in all living species from bacteria to humans, and is involved in a variety of metabolic pathways catalyzed by the enzyme nicotinamide riboside kinase [70] D-Ribosylnicotinate is detected in Psilocybe PSCM per the Examples.

[0116] Composed of cysteine, glycine, and glutamic acid, glutathione (figure 1q) [71] is a tripeptide found in most human cells. Glutathione has been detected in the Psilocybe PSCM per the Examples. Glutathione is incredibly important in detoxification of the body and in reducing oxidative stress, regulates cellular growth and apoptosis, is vital to mitochondrial function, and additionally, plays a crucial role in the regeneration of vitamins C and E. When glutathione is in its reduced state, it exists as GSH. Several diseases are associated with depleted GSH, including neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. Thus, increasing intracellular and intramitochondrial levels of glutathione can play a critical role in overall health and well-being [72]

[0117] Phosphocholine (figure 1o) [73] is a precursor to choline, as it is an intermediate in the synthesis of phosphatidylcholine, which is a choline phospholipid (Phosphatidylcholine and the CDP-choline cycle). Phosphocholine has been detected in the Psilocybe PSCM per the Examples. Choline is an essential nutrient necessary for proper lipid metabolism, liver, muscle, and brain function, as well as for cell membrane signaling, repair, and lipid transport [74] Produced endogenously in small amounts via the hepatic phosphatidylethanolamine N- methyltransferase pathway, but not in amounts sufficient to meet metabolic demands, this nutrient must be consumed through the diet. Consequences of choline deficiency include fatty liver disease, muscle damage, and an increased risk for cardiovascular disease, cancer and cognitive decline [75] Choline is also needed to produce acetylcholine, which is a neurotransmitter needed for proper brain and nervous system functioning, and additionally, dietary supplementation of choline may prevent Alzheimer’s disease progression [76]

[0118] L-Histidine Trimethylbetaine (figure 1p) [77], also known as hercynine, is a metabolite belonging to the class of compounds referred to as histidine and derivatives [78] and has been detected in the Psilocybe PSCM per the Examples. It is an intermediate in the synthesis of ergothioneine, an amino acid that that can modulate inflammation, mitigate oxidative damage, protect against acute respiratory diseases and neuronal damage, and prevent damage to the lungs, kidneys, liver, and gastrointestinal tract. Ergothioneine is not produced endogenously and is strictly acquired through diet [79, 80]

[0119] Monosaccharides are a type of carbohydrate known as simple sugars with the most common among them glucose, galactose, and fructose. Monosaccharides have the general formula of (ChhOj_n with N being three to seven carbons [82] Monosaccharides are the simplest form of carbohydrate and cannot be farther hydrolyzed into smaller components [83] They exist in either linear chains or ring structures, and may contain a functional group such as aldehyde and ketones [82] Glucose is the major cell fuel in the body and, is present unbound in both body fluids and tissue. Monosaccharides are essential for the function of the body at a cellular level, and are required to create both disaccharides and polysaccharides [83]

[0120] Disaccharides are formed when two monosaccharides undergo a condensation reaction, thereby losing a water molecule and creating a glycosidic bond [82] . The most common of the disaccharides include lactose, maltose, and sucrose, and are all key carbohydrates in many foods [82] Lactose is found in milk and dairy products; maltose is component in starch, and sucrose is a constituent in fruits, vegetables, and sweeteners [82]

[0121] Phosphatidiylcholines also known as Lecithins, are a group of phospholipids that are important to the structure and metabolism of cells. Lecithins are made up of phosphoric acid, cholines, esters of glycerol, and two fatty acids, the differences in combination and type lead to high degree of variability in lecithin’s and therefore a wide variety in biological function. The most common source of lecithin’s is from soy bean oil, and is widely used as an emulsifying agent. Lecithin’s can be found in baked goods, chocolates, cosmetics, and as a dietary supplement [84]

[0122] Diglycerides also known as Diacylglycerols or DAG’s, are made up of two fatty acids esterified to a glycerol backbone. They can be produced naturally through the hydrolytic activity of lipase enzymes during the maturation of oil in fruits and seeds or can be produced chemically or enzymatically in an industrial setting. Diglycerides are common food ingredients, primarily used as emulsifying agents. Diglycerides can be frequently found in a variety of products including baked goods, frozen deserts and sauces. DAG’s have been praised for being a healthy cooking oil alternative to the traditional triglycerides. The anti-obesity effects of DAG’s are contributed to difference in polymorphic behaviour and melting points from triglycerides [85]

Pharmaceutical formulations and uses thereof

[0123] Pharmaceutical formulations may comprise compositions and/or extracts according to this disclosure. Composition described in this disclosure may comprise, consist of, or consist essentially of mycelium, preferably dried mycelium, of one or more psychoactive species which may for example be cultured in a liquid medium or a dry medium under controlled conditions according to a process described in this disclosure.

[0124] In one embodiment, there is provided a method treating for mental health conditions as well as other relevant conditions using a PSCM or extract, whether alone or formulated into a dosage form. In one embodiment, the extract is obtained from the mycelium of Psilocybe species, which contain psilocybin as well as other tryptamines and other compounds of interest (as per table 3). In some embodiments, the treatment comprises psychedelic assisted psychotherapy.

[0125] As used herein, “therapeutically effective amount” refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.

[0126] As used herein “subject” refers to an animal being administered a therapeutic, in one embodiment a mammal, in one embodiment a human patient. As used herein “treatment”, and grammatical variations thereof, refers to administering a compound or composition of the present invention. The treatment may require administration of multiple doses, which may be at regular intervals. A treatment may be preventative.

[0127] Because the PSCM contains the same key actives as the fruiting body, in some embodiments, PSCM, preferably in a dried form without extraction, may be used in microdosing applications. In an embodiment, the PSCM may be dried to a moisture content of less than 30%, in a range of 10-20%, in a range of 5-10%, or less than 5%

[0128] Because the PSCM extract contains the same key actives as an extract from the fruiting body, the extract is suitable for use in treatments of conditions amenable to treatment with psilocybin or extract from the psilocybe fruiting body.

[0129] Such conditions can include: mental health conditions such as depression, including type II bipolar depression, postpartum and situational depression (such as may be associated with cancer, palliative care, multiple sclerosis (MS) or cognitive impairment), anxiety, and mood disorders; Obsessive Compulsive Disorders; Substance Use Disorders; Adjustment Reactions; Post-Traumatic Stress Disorder (PTSD); Sleep Dysregulation; headaches, including migraine, concussion and cluster headaches; chronic pain; fibromyalgia; behavioural disorders, including anorexia, bulimia, and binge eating disorder; neurodegenerative disorders, including Alzheimer’s; and neuroinflammatory conditions.

[0130] All documents referenced herein are incorporated by reference, however, it should be appreciated that any patent, publication, or other disclosure material, in whole or in part, that is incorporated by reference herein is incorporated only to the extent that the incorporated material does not conflict with definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

[0131] It will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense. It will further be understood that it is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims. EXAMPLES

EXAMPLE 1 : CULTURING OF PSILOCYBE MYLELIUM AND EXTRACTION METHOD

1.1 Wet method of culturing PSCM

[0132] Psilocybe cubensis were cultured and tested according to the following steps:

1.1.1 Inoculation of plates with parental mycelium stock.

[0133] Parental mycelium aliquots were removed from storage and allowed to thaw. Using aseptic techniques, the mycelium was plated on to prepared agar plates. The plates were incubated until the mycelium had fully propagated over the plate.

1.1.2 Inoculation of liquid media vessel

[0134] A section of the plate was taken and transferred to the liquid media vessel using aseptic technique. The mycelium was assayed periodically for Psilocybin, Psilocin and Tryptophan using HPLC. The OrbiTrap was used to complete screen of alkaloids and additional compounds.

[0135] Mycelium removed from the vessel may be dried and tested, described below, and/or further processed, e.g. by extraction methods according to this disclosure.

1.1.3 Drying

[0136] Approximately 300 mL of liquid nitrogen was poured into a mortar to precool the mortar for each sample preparation. More liquid nitrogen was poured as needed. The tissue culture mycelium sample was added portion wise to the liquid nitrogen and allowed to freeze. The frozen tissue was crushed using the pestle until it turned into a granular powder. The shock frozen tissue culture powder was transferred into a pear-shaped round flask and the nitrogen was allowed to evaporate. The flask was immediately attached to the freeze-dryer for 12 to 24 h depending on the sample amount.

[0137] Alternatively, tissue culture mycelium sample may be dried by a freeze drying device, e.g. Harvest Right™ freeze dryer (Model #: HRFDL). The freeze drying device may flash freeze the sample with liquid nitrogen. The duration for freeze drying will be based on the quantity of material and loading. For example, 6kg wet grains may take 34 hours to dry whereas 100g wet grains may take 13 hours. Freeze drying may occur at e.g. -10F or -23.33°C to evaporate the frozen water crystals through sublimation.

[0138] In another alternative, freeze drying may occur according to an optimized freeze drying process. In some instances, previous methods of freeze drying were found to contain moisture even after 5-10 days of drying which may cause degradation of psilocybin upon storage. This method may reduce moisture to less than 5%, and reduce drying time, to reduce degradation of psilocybin in dry sample. A full loading (6kg) of grain mycelium were compared by drying the samples with “old” method at -22°C for 6 days and “new” settings of drying at a primary setting of -21 °C and secondary setting of 12°C for 30 hours. Moisture in the mycelium was 2.23% for the new method and 25.26% for the old method indicating superior performance for the new method reducing potential for degradation of psilocybin.

[0139] Particle size after freeze drying is preferably less than or equal to 500mn.

1.1.4 T esting - example 1 Preparation for Analytical Testing

[0140] Samples were tested in triplicates. 0.02 to 0.025 g (20 to 25 mg) of the dried tissue culture sample was weighed into a 2.0 mL-micro centrifugation tube. 1.5 ml_ HPLC-grade methanol was added and the samples were vortexed for 10 sec, making sure that the tissue culture powder was fully covered in methanol. The samples were subject to sonication (sonar bath) for 25 min, followed by vortexing for 10 sec and centrifugation at 2500 rpm for 20 min until the tissue culture formed a pellet. 1.0 ml_ of the supernatant was filtered through a 0.2 pm Nylon 66 syringe filter into a 2.0 mL-amber vial.

Preparation for OrbiT rap

[0141] Acetyl nitrile may be added to the filtered sample prepared per step 1.1.4 and centrifuged. The sample was then transferred to a analysis vial and loaded on to the OrbiTrap.

[0142] 1.1.5 Testing Example 2

[0143] In another example, samples may be tested with a TSQ Altis Plus Triple Quadrupole Mass Spectrometer. Samples were weighed in 25 ± 5 mg into a 15 ml_ Falcon tube (3 x per sample), and 5.0 ml_ Methanol was added followed by the internal standard consisting of 50 pl_ Tryptophan-d5 [1000 ppm], 50 mI_ Choline-d9 [1000 ppm] and 50 mI_ Carnitine-d3 [1000 ppm]. The solution was vortexed for 5 minutes, sonicated for 5 minutes, vortexed for 25 minutes, and then finally centrifuge at 4000rpm for 15min at at -8 ° C. The methanolic supernatant was decanted into an empty 15 ml_ Falcon tube, and another 5.0 ml_ Methanol was added. The solution was vortexed for 5 minutes, sonicated for 5 minutes, vortexed for 25 minutes, and then finally centrifuged at 4000rpm for 15min at at -8 ° C. The methanolic supernatant was combined into the new 15 mL Falcon tube with the first portion of methanolic supernatant. The combined methanolic extracts may be pipetted (1 mL) into a 1.5 mL amber sample vial, and submitted for ORBITRAP analysis. Pipette 100 pL of the combined methanolic extracts into a 1.5 mL amber sample vial, add exactly 900 pL Acetonitrile: dd. H20 (1:1) to the sample vial, label sample vial and submit for analysis by TSQ ALTIS.

[0144] Example compounds identified are illustrated in Tables 1 and 2.

[0145] Figure 2 shows psilocybin, psilocin and tryptophan concentration assessment over 28 days assayed Psilocybe cubensis mycelium cultured according to this Example. FIG. 3 shows the ratio of psilocybin:psilocin concentration over 28 days assayed in Psilocybe cubensis cultured mycelium. The figure evidences a viable concentration of psilocybin and a limited level of conversion to psilocin.

[0146] Figures 4-39 show the Single Ion Monitoring (SIM) Chromatographic Spectra and Mass Chromatogram of compounds Psilocybe cubensis mycelium cultured according to this Example. All the results were generated using Orbitrap Exploris 120 Mass Spectrometer in combination with a Vanquish Flex Liquid Chromatographer.

1.2 Dry Method of Culturing PSCM

1.2.1 Inoculation of plates with parental mycelium stock.

[0147] Parental mycelium aliquots were removed from storage and allowed to thaw. Using aseptic techniques, the mycelium was plated on to prepared [NTD: dry media] agar plates. The plates were incubated until the mycelium had fully propagated over the plate.

1.2.2 Inoculation of dry media [0148] A section of the plate was taken and transferred to dry media (rye) in a grain spawn bag using aseptic technique. The grain spawn bag was placed in an incubator vessel to culture the mycelium in a controlled environment. The mycelium was allowed to fully colonize the dry media and then expanded to new dry media (rye) in additional grain spawn bags to introduce fresh nutrients allowing for continuous biomass production.

[0149] The mycelium was assayed periodically for Psilocybin, Psilocin and Tryptophan using TSQ ALTIS and OrbiT rap. The OrbiT rap was used to complete screen of alkaloids and additional compounds. Example compounds identified are illustrated in Table 3. The parental strain of mycelium remained consistent through each expansion of biomass of mycelium to new grain spawn bags.

[0150] Mycelium removed from the vessel may be dried and tested, described below, and/or further processed, e.g. by extraction methods according to this disclosure.

1.2.3 Drying and Testing

[0151] Drying and testing of mycelium removed from the vessel were conducted according to the steps described in 1.1.3 to 1.1.5.

EXAMPLE 2: EXTRACTION METHODS FOR TISSUE CULTURE EXTRACTION

[0152] Several methods of extraction were tested to develop a viable extract from Psychoactive Species Cultured Mycelium, PSCM. Methods of extraction optimized for extraction of fruiting body were found not to be optimal for extracts derived from PSCM. Methods of extraction optimized for PSCM are ethanol based i) ultrasonication extraction, and ii) Soxhlet extraction.

2.1 Ultrasonication Extraction

2.1.1 First Example of Ultrasonication Extraction

[0153] 5.0 g of freeze-dried tissue culture material (Psilocybe cubensis mycelium cultured in a liquid media vessel) were weighed into a 300 mL-beaker and 200 mL of ethanol 96% was added. The material was subject to sonication for 60 min, then allowed to settle, followed by sonication for another 60 min. The mixture was then centrifuged at 4000 rpm at -8°C. The supernatant was dried by rotary evaporation (122 mbar, 45°C bath temperature). The pellet was suspended in another 100 ml_ of ethanol 96% and sonicated for 60 minutes. The mixture was then centrifuged at 4000 rpm at -8°C. The supernatant was then unified with the supernatant from the former extraction step. The supernatants were dried by rotary evaporation (122 mbar, 45°C bath temperature).

[0154] If required, complete drying may be performed in a dehydrator, suitably at about 35°C.

2.1.2 Second Example of Ultrasonication Extraction

[0155] An ultrasonic processor featuring ultrasonic cycle and amplitude control was employed. A 20mm probe was employed for extraction. A total of 5g dried and ground culture material (Psilocybe cubensis mycelium) was placed inside a beaker and 100ml ethanol (reagent alcohol 90.5%) was added. The beaker was placed inside the acoustic chamber with a temperature probe and the contents were extracted for an hour. Once the extraction was completed, the extract was transferred to 50 ml falcon tubes and centrifuged for 15 min at 4000rpm, 15°C to collect the supernatant. The precipitate was subjected to second round of extraction with another aliquot of 100ml ethanol. The extract was again centrifuged, and the supernatant was collected and combined with the one collected in previous step. Final combined extract was evaporated using a rotary evaporator temp 45°C and 120mBar pressure to recover the dry extract. The final extract was dried further in dehydrator at 35C for 1 hour and stored in an airtight container at room temperature and until further analysis. A dry yield of 14.6% and an average potency of 39.4mg/g was shown by analytical testing (average).

2.2 Soxhlet Extraction

[0156] 5.0 g of freeze-dried tissue culture material (mycelium cultured in a liquid medium) was placed into an extraction thimble. After closing the thimble well, the recirculation chiller was switched on (set to 4°C). The thimble was placed into Soxhlet apparatus [Soxhlet Setup: 1000 mL-round flask in 1000 mL-magnetic stirrer- heating mantle, small magnetic stirrer, Soxhlet apparatus 250 ml_ capacity with ball condenser, chiller operated with water.] 500 mL of ethanol 96% in total was poured into the Soxhlet apparatus (250 mL siphoned into the round flask immediately). The stirrer (low rpm) and heating unit (medium) were switched on. The ethanol was allowed to boil and the extraction was allowed to proceed for 3 hours. After being allowed to cool off, the extract was centrifuged at 4000 rpm at -8°C and the supernatant was dried by rotary evaporation (122 mbar, 45°C bath temperature). [0157] If required, complete drying may be performed in a dehydrator, suitably at about 35°C.

Discussion

[0158] Psilocybin and psilocin content of ethanol extracted psilocybe cubensis PSCM per 2.1 and 2.2 are shown in FIG. 40. FIG. 41 shows the percentage increase in psilocybin extract from Psilocybe cubensis cultured mycelium relative to the unextracted cultured mycelium material.

[0159] The Examples evidence that Psilocybe mycelium cultured in a liquid medium can with suitable extraction processes yield an extract containing psilocybin and other tryptamines specially indole alkaloids as well as additional compounds of therapeutic benefits for use in psychedelic assisted psychotherapy for mental health and other related conditions.

2.2 Percolation Extraction

[0160] Percolation extraction was conducted with a condenser, Mini-Chiller, attached to a power source, Toption Freeze dryer, Funnel, Separation funnel, and Liquid Nitrogen dewar. The condenser was connected to the mini chiller using the quick-connect tubes and set to 2°C allowing circulating water to come to temperature. 100 ml of distilled water was heated to 100°C and a glass funnel was placed in a beaker and lined with a round filter paper folded into a cone shape. 5 g of PSCM material was weighed into the filter paper.

[0161] Liquid nitrogen was poured into an open dewar and a sieve placed into the liquid nitrogen. A separation funnel was placed in a clamp stand and placed over the sieve.

[0162] Once the mini chiller reached 2 °C and the water 100 °C, the hot water was poured over the PSCM to be extracted in the filter paper and allowed to drip through the condenser and into a clean beaker. All the PSCM powdered material was wet in the glass funnel. The percolated droplets were cooled by passing through the glass condenser such that the percolate temperature reduces from 100 °C to about 15 °C within seconds, avoiding heat degradation of the extracted compounds.

[0163] Liquid that had passed through the condenser was transferred from the beaker into the separation funnel for flash cooling. The separation funnel was opened to allow the extract to drip dropwise into the liquid nitrogen forming iced spheres. The sieve was periodically used to remove frozen drops of liquid nitrogen and to place them in an uncovered glass vessel to be used to freeze dry the PSCM material. Liquid nitrogen was provided to the glass vessel to keep drops of the PSCM material completely frozen.

[0164] When all of the percolation extract was frozen, the glass vessel (petri dishes) were placed in a freeze dryer to start the freeze dryer process of the drops of the PSCM material allowing ice crystals to sublimate. The freeze dryer was run until droplets are completely dry, at least 72 hours. After drying, the dried extract was collected, weighted, tested, and stored in amber glass vials to avoid light exposure.

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Claims

WHAT IS CLAIMED IS:

1. An extract obtained from a biomass comprising mycelium of one or more psychoactive species cultured under controlled conditions.

2. The extract of claim 1, wherein the biomass consists or consists essentially of mycelium of one or more psychoactive species.

3. The extract of claim 2, wherein the biomass comprises, consists essentially or consists of mycelium of a species of the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni.

4. The extract of any one of claims 1 to 3, wherein the mycelium are cultured in a liquid medium.

5. The extract of any one of claims 1 to 4, wherein the mycelium are culturedin a bioreactor.

6. The extract of any one of claims 1 to 5, wherein the mycelium are cultured and harvested in a continuous process.

7. The extract of any one of claims 1 to 6, wherein the extract is obtained by an ethanol extraction.

8. The extract of claim 7, wherein the material is sonicated in ethanol.

9. The extract of any one of claims 1-6, wherein the extract is obtained by percolation extraction.

10. The extract of any one of claims 1-9, wherein the extract comprises at least one of alkaloids, indoleamines, indoleamine derivatives, amino acids, modified amino acids, derivative of amino acids, ketones, nucleosides, carbohydrates, essential nutrients, neurotransmitters, vitamins, purine nucleosides, antioxidant compounds, oligosaccharides, coenzymes, lecithins, and glycerides.

11. A composition comprising, consisting or consisting essentially of dried mycelium of one or more psychoactive species cultured in a liquid medium or a dry medium under controlled conditions.

12. The composition of claim 11 , wherein the composition is freeze dried.

13. The composition of any one of claims 11-12, wherein mycelium is of a species of the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni.

14. The composition of claim 11 or 13, wherein the mycelium are cultured in a bioreactor and are harvested in a continuous process or a batch process.

15. A pharmaceutical composition comprising a therapeutically effective amount of the extract of any one of claims 1 to 10 or the composition of any one of claims 11 to 14.

16. A pharmaceutical composition comprising the composition of any one of claims 11 to 14, in a microdosage form.

17. The pharmaceutical composition of claim 15 in a macrodosage form.

18. The pharmaceutical composition of any one of claims 15 to 17 for use in a method of treating a mental health condition.

19. The pharmaceutical composition of claim 18, wherein the mental health condition is selected from depression, including type II bipolar depression, postpartum depression and situational depression, anxiety, and mood disorders.

20. The pharmaceutical composition of any one of claims 15 to 17 for use in treating a disease or condition including any one of Obsessive Compulsive Disorders, Substance Use Disorders, Adjustment Reactions, Post-Traumatic Stress Disorder (PTSD), Sleep Dysregulation, headaches, including migraine, concussion and cluster headaches, chronic pain, fibromyalgia, Alzheimer’s disease, Parkinson's disease, and behavioural disorders, including anorexia, bulimia, and binge eating disorder.

21. A method of treating a mental health condition comprising administering a therapeutically effective amount of an extract according to any one of claims 1 to 10, a composition according to any one of claims 11 to 14 or a pharmaceutical composition of any one of claims 15 to 17 to a subject in need thereof.

22. The method of claim 21, wherein the mental health condition is selected from depression, including type II bipolar depression, postpartum and situational depression, anxiety, and mood disorders.

23. A method of treating a condition selected from Obsessive Compulsive Disorders, Substance Use Disorders, Adjustment Reactions, Post-Traumatic Stress Disorder (PTSD), Sleep Dysregulation, headaches, including migraine, concussion and cluster headaches, chronic pain, fibromyalgia and behavioural disorders, including anorexia, bulimia, and binge eating disorder, comprising administering a therapeutically effective amount of an extract according to any one of claims 1 to 10, a composition according to any one of claims 11 to 14 or a pharmaceutical composition of any one of claims 15 to 17 to a subject in need thereof.

24. A process for culturing mycelium of a psychoactive species, the process comprising: inoculating a media with parental mycelium of a psychoactive species; culturing a first biomass comprising cultured mycelium from the parental mycelium, the cultured mycelium and parental mycelium each comprising a same strain, or substantially the same strain, of mycelium; controlling an environmental condition while the media is inoculated and/or the first biomass is cultured; removing at least a portion of the first biomass from the media; and culturing a second biomass comprising cultured mycelium from the portion of the first biomass or the parental mycelium, the second biomass comprising the same strain, or substantially the same strain, of mycelium as the parental mycelium, the second biomass cultured under the controlled environment condition.

25. The process of claim 24, wherein the media is a wet media, preferably malt extract broth, modified melin-norkrans medium, potato dextrose broth, and/or yeast malt extract broth.

26. The process of claim 25, wherein the wet media has a single macro and micro nutritional profile.

27. The process of claim 24, wherein the media is a dry media, preferably rye, bird seed, rice, whole oat, whole millet, and/or combinations thereof.

28. The process of claim 27, comprising expanding the first biomass by subdividing the first biomass onto additional media.

29. The process of claim 27 or 28, wherein the biomass is subdivided between vessels for culturing mycelium of a psychoactive species.

30. The process of any one of claims 24-29, wherein the first biomass and second biomass are cultured in a continuous batch process from the parental mycelium.

31. The process of any one of claims 24-30, wherein the psychoactive species is from the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni.

32. The process of any one of claims 24-31 , wherein the environmental condition is a pressure comprising one of a hypobaric pressure, hyperbaric pressure, or atmospheric pressure.

33. The process of any one of claims 24-32, wherein the environmental condition is a temperature of 1-20°C, 20-25°C, 25-30°C, 30-35°C, or 35°C-50°C.

34. The process of any one of claims 24-33, wherein the environmental condition is a relative humidity of greater than 50%, 85% to 95%, or greater than 90%.

35. The process of any one of claims 24-34, wherein the environmental condition is an oxygen concentration of about 21%, greater than 21%, or in a range of 21-80%.

36. The process of any one of claims 24-35, wherein the environmental condition is a carbon dioxide concentration of less than 5%, less than 3%, or in a range of 0-1%.

37. The process of any one of claims 24-36, wherein a moisture content of the first media is greater than 50%, less than 50%, 50-70%, greater than 70%.

38. The process of any one of claims 24-37, wherein a pH of the first media is about 7, in a range of 4.5-6, in a range of 5-7, or in a range of 7-9.

39. The process of any one of claims 24-38 comprising drying the mycelium, preferably by flash freezing.

40. A process for producing an extract from a biomass comprising cultured mycelium of a psychoactive species, the process comprising: contacting the biomass with a solvent to form a mixture of the solvent and an extract; optionally cooling the mixture; and separating the extract from the solvent.

41. The process of claim 40, wherein cooling the mixture comprises flash cooling the mixture.

42. The process of claim 41, wherein flash cooling the mixture comprises decreasing the temperature of the mixture from about a boiling point of the solvent to about a freezing point of the solvent.

43. The process of any one of claims 40-42, wherein separating the extract from the solvent comprising freeze drying the mixture to sublimate the solvent.

44. The process of claim 43, comprising forming droplets from the mixture before freeze drying the mixture to sublimate the solvent.

45. The process of any one of claims 40-44, wherein the solvent is water or ethanol.

46. The process of claim 45, wherein the extract is separated from the solvent by ultrasonication.

47. The process of any one of claims 40-46, wherein the psychoactive species is from the genus Psilocybe, preferably Psilocybe cubensis, Psilocybe cyanescens, and/or Psilocybe alleni.

48. A process for producing an extract from a biomass comprising: inoculating a media with parental mycelium of a psychoactive species; culturing a first biomass comprising cultured mycelium from the parental mycelium, the cultured mycelium and parental mycelium each comprising a same strain, or substantially the same strain, of mycelium; controlling an environmental condition while the media is inoculated and/or the first biomass is cultured; removing at least a portion of the first biomass from the media; culturing a second biomass comprising cultured mycelium from the portion of the first biomass or the parental mycelium, the second biomass having the same strain, or substantially the same strain, of mycelium as the parental mycelium, the second biomass cultured under the controlled environment condition; contacting at least the portion of the first biomass with a solvent to form a mixture of the solvent and an extract; optionally cooling the mixture; and separating the extract from the solvent.