WO2022150840A1 - Psilocybin and norbaeocystin compositions and methods of treatment - Google Patents

Psilocybin and norbaeocystin compositions and methods of treatment Download PDF

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Publication number
WO2022150840A1
WO2022150840A1 PCT/US2022/070094 US2022070094W WO2022150840A1 WO 2022150840 A1 WO2022150840 A1 WO 2022150840A1 US 2022070094 W US2022070094 W US 2022070094W WO 2022150840 A1 WO2022150840 A1 WO 2022150840A1
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psilocybin
norbaeocystin
produced
molar ratio
pharmaceutical composition
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PCT/US2022/070094
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French (fr)
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Matthew Stephen MCMURRAY
John Andrew Jones
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Miami University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the general inventive concepts relate to the field of pharmaceutical compositions and medical therapeutics comprising the administration of two or more tryptamines, for example psilocybin and norbaeocystin.
  • Psilocybin (4-phosphoryloxy-/V,./V-dimethyltryptamine) has gained attention in pharmaceutical markets as a result of recent clinical studies.
  • the efficacy of psilocybin has been demonstrated for the treatment of anxiety in terminal cancer patients and alleviating the symptoms of post-traumatic stress disorder (PTSD).
  • PTSD post-traumatic stress disorder
  • the FDA has approved the first Phase lib clinical trial for the use of psilocybin as a treatment for depression that is not well controlled with currently available interventions such as antidepressants and cognitive behavioral therapies.
  • Norbaeocystin (3-(2-aminoethyl)-lH-indol-4-yl dihydrogen phosphate) is a tryptamine intermediate product in the psilocybin biosynthesis pathway that has been hypothesized to have its own neurological activity due to its structural similarity to psilocybin. This similarity suggests that it may compete with psilocybin for the same binding sites in the brain or metabolic process, thus enhancing psilocybin’s activity.
  • No natural sources are currently available that specifically over-produce norbaeocystin, a key precursor to psilocybin, and only recently has a synthetic route been published for its production (Sherwood, A.
  • a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms.
  • the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe.
  • the major tryptamine is psilocybin or a structurally similar analog.
  • the two or more tryptamines comprise one or more minor tryptamines present in Psilocybe.
  • the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin.
  • the one or more minor tryptamines is norbaeocystin.
  • the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
  • two tryptamines are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 25:1 to 1:25. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, two tryptamines are present in the composition in a molar ratio of 1:1.
  • a pharmaceutical composition comprising psilocybin and norbaeocystin.
  • the psilocybin is heterologously produced.
  • the norbaeocystin is heterologously produced.
  • the psilocybin is synthetically produced.
  • the norbaeocystin is synthetically produced.
  • composition comprising heterologously produced psilocybin and heterologously produced norbaeocystin.
  • the heterologously produced psilocybin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced norbaeocystin is purified.
  • the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of 1 : 1.
  • the pharmaceutical composition comprises cell media.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms.
  • the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe.
  • the major tryptamine is psilocybin or a structurally similar analog.
  • the two or more tryptamines comprise one or more minor tryptamines present in Psilocybe.
  • the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin.
  • the one or more minor tryptamines is norbaeocystin.
  • the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
  • two tryptamines are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 25:1 to 1:25. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, two tryptamines are present in the composition in a molar ratio of 1:1.
  • a pharmaceutical composition comprising psilocybin and norbaeocystin.
  • the psilocybin is heterologously produced.
  • the norbaeocystin is heterologously produced.
  • the administration of the pharmaceutical composition comprising psilocybin and norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
  • the heterologously produced psilocybin was produced in a prokaryotic cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced norbaeocystin is purified.
  • the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of 1 : 1.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient an effective amount of a first tryptamine and an effective amount of a second tryptamine that are present in Psilocybe magic mushrooms.
  • the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 100:1 to 1:100. In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 75:1 to 1:75.
  • the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 50:1 to 1:50. In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 25:1 to 1:25. In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 10:1 to 1:10. In some embodiments, the first tryptamine and the second tryptamine are are administered to the patient in a molar ratio of 1 : 1.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient an effective amount of a pharmaceutical composition comprising psilocybin and an effective amount of a pharmaceutical composition comprising norbaeocystin.
  • the psilocybin is heterologously produced.
  • the norbaeocystin is heterologously produced.
  • the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at substantially the same time. In further embodiments, the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at different times. In some embodiments, the pharmaceutical composition comprising psilocybin is administered before the pharmaceutical composition comprising norbaeocystin. In some embodiments the pharmaceutical composition comprising norbaeocystin is administered before the pharmaceutical composition comprising psilocybin.
  • the administration of the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
  • the heterologously produced psilocybin was produced in a prokaryotic cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced norbaeocystin is purified.
  • the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100. In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75. In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50. In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
  • the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10. In some embodiments, the psilocybin and the norbaeocystin are are administered to the patient in a molar ratio of 1 : 1.
  • the neurological disorder is selected from the group consisting of functional neurological disorder (FND), Guillain-Barre syndrome, Alzheimer’s disease, autism, migraine, headache, Traumatic Brain Injury, and Chronic Traumatic Encephalopathy.
  • FND functional neurological disorder
  • Guillain-Barre syndrome Alzheimer’s disease
  • autism migraine, headache, Traumatic Brain Injury, and Chronic Traumatic Encephalopathy.
  • the psychological disorder is selected from the group consisting of depression, anxiety, PTSD, obsessive compulsive disorder, bipolar disorder, eating disorder, substance abuse disorder, attention deficit hyperactivity disorder (ADHD), and schizophrenia.
  • the pharmaceutical composition comprises cell media.
  • one or more tryptamine is heterologously produced, for example in a prokaryotic cell.
  • one or more tryptamine is synthetically produced.
  • FIG. 1A shows an overview of study methods.
  • Recombinant E. coli were developed capable of high-level norbaeocystin production.
  • Norbaeocystin production was optimized and scaled up in a benchtop bioreactor.
  • Norbaeocystin concentration in cell broth was quantified using HPLC.
  • a rat with magnet affixed to its head was gavaged with filtered cell media containing psilocybin, norbaeocystin, or both, A magnetometer coil was used in order to record head twitches. Waveforms were then analyzed to determine the head twitch count.
  • FIG. IB shows a norbaeocystin biosynthesis pathway.
  • the E. coli strain contains three genes, one native ( trpB ) and two heterologous (psiD , psiK) that enable norbaeocystin biosynthesis from external supplementation of 4-hydroxyindole.
  • Tryptophan synthase (TrpB) condenses 4-hydroxyindole and serine to form 4-hydroxytryptophan.
  • P. cubensis tryptophan decarboxylase (PsiD) converts 4-hydroxytryptophan into 4-hydroxytryptamine while releasing a carbon dioxide and water.
  • P. cubensis kinase (PsiK) converts 4-hydroxytryptamine into norbaeocystin using a phosphate donated by ATP.
  • FIGs. 2A-2D show a summary of genetic optimization and scale-up results.
  • FIG. 2A Promoter library screening. Individual colonies from the operon (red bars) and pseudooperon (gray bars) libraries were selected and evaluated to discover elite production strains. Data for samples producing 0 mg/L of norbaeocystin (11.5 % of total colonies screened) are not shown.
  • FIG. 2B Normalized production of operon library members for norbaeocystin and psilocybin pathways organized in order of increasing promoter strength: G6 (low) - T7 (high).
  • FIG. 2C Effect of varying supplemental serine concentration in the initial fermentation media on strain performance in the bioreactor.
  • FIG. 2D Metabolite and growth curve profiles for a representative norbaeocystin bioreactor fed-batch fermentation. Data shown for one replicate of the 0 g/L serine condition.
  • FIGs. 3A-3C show dose-dependent effects of tryptamines alone and in combination.
  • FIG. 3A Psilocybin caused significant increases in the number of head twitches at IX, 2X, and 10X dosages.
  • FIG. 3B Norbaeocystin reduced the number of head twitches exhibited by rats at IX dosage, but not at other dosages.
  • FIG. 3C Animals receiving both norbaeocystin and psilocybin demonstrated increased head twitches at all dosages investigated, but most significantly at 2X and 10X dosages. Note: * p ⁇ 0.05; ** p ⁇ 0.01.
  • FIG. 4 shows that similar dosages of tryptamines and their combinations cause different effects on head twitch responses.
  • 2X dose combination only psilocybin+norbaeocystin increased head twitch responses (HTRs) compared to norbaeocystin alone.
  • psilocybin alone increased head twitch responses relative to norbaeocystin alone
  • psilocybin+norbaeocystin increased head twitches relative to both norbaeocystin and psilocybin alone.
  • * p ⁇ 0.05 there were no differences between the drugs in the magnitude of their effects.
  • FIGs. 5A-5F show HPLC standard curves used for metabolite profiles.
  • FIG. 5A 4- hydroxyindole
  • FIG. 5B 5-hydroxytryptamine
  • FIG. 5C norbaeocystin
  • FIG. 5D 5- hydroxytryptophan
  • FIG. 5E psilocybin
  • FIG. 5F glucose
  • FIG. 6 is a graph illustrating a media optimization study. Shake flasks with Norl were used to test varying media compositions. AMM is the control. No MOPS is AMM without the MOPS/tricine buffer added, and No Mix is AMM without any MOPS mix. Although not significantly different (p>Q.05), the observed increase in No MOPS motivated the use of AMM- no MOPS in all future experiments. Error bars represent ⁇ standard error of the mean for duplicate samples.
  • FIG. 7 shows an induction point sensitivity study. Norl was studied at 37 °C from 2 to 8 hours in 125 mL shake flasks. Error bars represent ⁇ standard error of the mean for duplicate samples.
  • FIGs. 8A-8H show bioreactor runs supplemented with 5 g/L of serine.
  • FIGs. 8E-8H Second trial.
  • FIGs. 8A and 8E Metabolite profiles.
  • FIGs. 8B and 8F Total cumulative glucose (gray) and ammonium phosphate dibasic (dashed line) fed compared to O ⁇ ⁇ oo (dotted line).
  • FIGS. 8C and 8G Total cumulative 4-hydroxyindole fed (gray) and 4- hydroxyindole (40 mg/mL in EtOH) feed rate (dotted line).
  • FIGs. 9A-9H show bioreactor runs supplemented with 1 g/L of serine.
  • FIGs. 9E-9H Second trial.
  • FIGs. 9A and 9E Metabolite profiles.
  • FIGs. 9B and 9F Total cumulative glucose (gray) and ammonium phosphate dibasic (dashed line) fed compared to O ⁇ ⁇ oo (dotted line).
  • FIGS. 9C and 9G Total cumulative 4-hydroxyindole fed (gray) and 4- hydroxyindole (40 mg/mL in EtOH) feed rate (dotted line).
  • FIGs. 10A-10H show bioreactor runs without supplemental serine.
  • FIGs. 10E-10H First trial.
  • FIGs. 10E-10H Second trial.
  • FIGs. 10A and 10E Metabolite profiles.
  • FIGs. 10B and 10F Total cumulative glucose (gray) and ammonium phosphate dibasic (dashed line) fed compared to O ⁇ ⁇ oo (dotted line).
  • FIGs. IOC and 10G Total cumulative 4-hydroxyindole fed (gray) and 4-hydroxyindole (40 mg/mL in EtOH) feed rate (dotted line).
  • FIGs. 10D and 10H Total cumulative 4-hydroxyindole fed (dotted line) compared to norbaeocystin production (dashed line) and transient molar yield on substrate (bold) shows a maximum molar yield of 58.2% and 77.5% with final molar yields at 57.8% and 54.3% for trials 1 and 2, respectively.
  • FIGs. 11A-11C show representative measurement of dissolved oxygen (DO) (bold), pH (dashed line), temperature (dotted line), and agitation rate (black line) in bioreactors.
  • FIG. 11 A Fed batch bioreactor run supplemented with 5 g/L serine.
  • FIG. 1 IB Run supplemented with 1 g/L serine.
  • FIG. 11C Run without supplemental serine.
  • FIGs. 12A-12C show locomotor effects of tryptamines.
  • FIG. 12A shows: norbaeocystin failed to alter distance traveled at any dose.
  • FIG. 12B shows: psilocybin alone also did not significantly affect distance traveled.
  • FIG. 12C shows: the combination of norbaeocystin and psilocybin increased locomotion at 0.2 mg/kg each.
  • * denotes significant (p ⁇ 0.05) increase in HTR compared to control; ⁇ denotes significant increase compared to 2 mg/kg each. .
  • FIG. 14 shows 1 H NMR spectra (500 MHz) for norbaeocystin in D2O with H2O suppression. Expanded figure inserts show splitting and peak assignments consistent with previous literature (Sherwood at ah, 2020). Protons marked with (*) are exchangeable with D2O and were not observed. Assigned peaks represent roughly 94% of total peak area observed.
  • FIG. 15 shows 13 C NMR spectra (125 MHz) for norbaeocystin in D2O.
  • FIG. 16 shows HPLC (A280) chromatograms of psilocybin-containing (top), norbaeocystin-containing (middle), and control (bottom) E. coli broth used in animal studies. Norbaeocystin, baeocystin, and psilocybin retention times marked with N, B, and P, respectively.
  • FIGs. 17A-17C shows the effects of tryptamines on stereotypical behavior during the locomotor test.
  • FIG. 17A shows norbaeocystin had no effect on rearing duration at any dosage tested.
  • FIG. 17B shows: psilocybin alone also did not affect rearing.
  • FIG. 17C shows: the combination of norbaeocystin and psilocybin reduced rearing behavior when administered at 2 mg/kg each compared to 0.2 mg/kg each, but not control. Note: ⁇ indicates significant decrease compared to 0.2 mg/kg each.
  • FIG. 18 shows the effect of psilocybin alone and combined with norbaeocystin on the change in sucrose preference after treatment (compared to pre-treatment). Effects of psilocybin alone and combined with norbaeocystin are compared to vehicle control and fluoxetine (a selective serotonin reuptake inhibitor). The greatest magnitude of effects was observed in the combined group. Note: significant difference from vehicle group denoted by asterisk (* p ⁇ 0.05,
  • FIG. 19 shows the effect of psilocybin alone and combined with norbaeocystin on immobility in the forced swim test. Effects are compared to vehicle control and fluoxetine (a selective serotonin reuptake inhibitor). The greatest magnitude of effects was observed in the combined group. Note: significant difference from vehicle group denoted by asterisk (* p ⁇ 0.05,
  • prokaryotic host cell means a prokaryotic cell that is susceptible to transformation, transfection, transduction, or the like, with a nucleic acid construct or expression vector comprising a polynucleotide.
  • prokaryotic host cell encompasses any progeny that is not identical due to mutations that occur during replication.
  • the term “recombinant cell” or “recombinant host” means a cell or host cell that has been genetically modified or altered to comprise a nucleic acid sequence that is not native to the cell or host cell.
  • the genetic modification comprises integrating the polynucleotide in the genome of the host cell.
  • the polynucleotide is exogenous in the host cell.
  • Psilocybe refers to Psilocybe ‘magic’ mushrooms as well as related psilocybin containing mushroom species.
  • intermediate of psilocybin means an intermediate in the production or biosynthesis of psilocybin, e.g., norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine.
  • side product of psilocybin means a side product in the production or biosynthesis of psilocybin, e.g., aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
  • the term “patient” or “user” means a member of the animal kingdom, including, but not limited to, a human.
  • the term “therapeutically effective amount” refers to that amount of any of the present compounds or compositions required to bring about a desired effect or behavior in a patient. The desired effect or behavior will vary dependent on the desired therapeutic, e.g., psychotherapeutic, response.
  • a therapeutically effective amount of said compounds or compositions can be administered by any means known in the art, including but not limited to, injection, parenterally, orally, bucally, transdermally, nasally, or where appropriate, topically.
  • heterologously produced in the context of heterologously produced psilocybin or norbaeocystin, means produced by heterologous expression of at least one psilocybin production gene selected from: psiD, psiK, psiM, and combinations thereof, in a host cell.
  • the host cell is a prokaryotic cell.
  • psilocybin can be enhanced by combining it with its metabolic precursor norbaeocystin, despite norbaeocystin having no observable effect on its own.
  • Norbaeocystin s potential for synergistic activity when combined with psilocybin was determined by measuring rodent behavioral responses to both drugs, alone and in combination. Results described herein demonstrate the pharmacological efficacy of E. coli- derived psilocybin and suggest that other tryptamines can augment its effectiveness, thus providing strong evidence for the existence of an entourage effect in psilocybin mushrooms.
  • a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms.
  • the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe.
  • the major tryptamine is psilocybin or a structurally similar analog.
  • the two or more tryptamines comprise one or more minor tryptamines present in Psilocybe.
  • the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin.
  • the one or more minor tryptamines is norbaeocystin.
  • the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
  • a pharmaceutical composition comprising psilocybin and norbaeocystin.
  • the psilocybin is heterologously produced.
  • the norbaeocystin is heterologously produced.
  • the psilocybin is synthetically produced.
  • the norbaeocystin is synthetically produced.
  • composition comprising heterologously produced psilocybin and heterologously produced norbaeocystin.
  • the heterologously produced psilocybin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced norbaeocystin is purified.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1.
  • a pharmaceutical composition comprising heterologously produced psilocybin and synthetically produced norbaeocystin.
  • the heterologously produced psilocybin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
  • the pharmaceutical composition comprises cell media.
  • composition comprising synthetically produced psilocybin and heterologously produced norbaeocystin.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1.
  • the pharmaceutical composition comprises cell media.
  • composition comprising synthetically produced psilocybin and synthetically produced norbaeocystin.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms.
  • the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe.
  • the major tryptamine is psilocybin or a structurally similar analog.
  • the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin.
  • the one or more minor tryptamines is norbaeocystin.
  • the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
  • administering to the patient a pharmaceutical composition comprising the two or more tryptamines produces a synergistic effect.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient a pharmaceutical composition according to any one of the embodiments described herein.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient a pharmaceutical composition comprising psilocybin and norbaeocystin.
  • the psilocybin is heterologously produced.
  • the norbaeocystin is heterologously produced.
  • the psilocybin is synthetically produced.
  • the norbaeocystin is synthetically produced.
  • the administration of the pharmaceutical composition comprising psilocybin and norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
  • administering to the patient the pharmaceutical composition comprising psilocybin and norbaeocystin produces a synergistic effect.
  • the heterologously produced psilocybin was produced in a prokaryotic cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced norbaeocystin is purified.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1. For example, for every X mole of heterologously produced psilocybin in the composition, there is X mole of heterologously produced norbaeocystin.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient heterologously produced psilocybin and synthetically produced norbaeocystin.
  • the heterologously produced psilocybin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
  • the pharmaceutical composition comprises cell media.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1.
  • the pharmaceutical composition comprises cell media.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient synthetically produced psilocybin and synthetically produced norbaeocystin.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
  • a method of treating a neurological or psychological disorder or condition in a patient comprising administering to the patient an effective amount of a pharmaceutical composition comprising psilocybin and an effective amount of a pharmaceutical composition comprising norbaeocystin.
  • the psilocybin is heterologously produced.
  • the norbaeocystin is heterologously produced.
  • the psilocybin is synthetically produced.
  • the norbaeocystin is synthetically produced.
  • the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at substantially the same time. In further embodiments, the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at different times. In some embodiments, the pharmaceutical composition comprising psilocybin is administered before the pharmaceutical composition comprising norbaeocystin. In some embodiments the pharmaceutical composition comprising norbaeocystin is administered before the pharmaceutical composition comprising psilocybin.
  • the administration of the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
  • administering to the patient the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin produces a synergistic effect.
  • the heterologously produced psilocybin was produced in a prokaryotic cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the heterologously produced psilocybin is purified.
  • the heterologously produced norbaeocystin is purified.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2.
  • the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of heterologously produced psilocybin administered to the patient, X mole of heterologously produced norbaeocystin is administered to the patient.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin administered to the patient in a molar ratio of from 4:1 to 1:4.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2.
  • the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of heterologously produced psilocybin administered to the patient, X mole of synthetically produced norbaeocystin is administered to the patient.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2.
  • the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of synthetically produced psilocybin administered to the patient, X mole of heterologously produced norbaeocystin is administered to the patient.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2.
  • the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of synthetically produced psilocybin administered to the patient, X mole of synthetically produced norbaeocystin is administered to the patient.
  • the neurological disorder is selected from the group consisting of functional neurological disorder (FND), Guillain-Barre syndrome, Alzheimer’s disease, autism, migraine, headache, Traumatic Brain Injury, and Chronic Traumatic Encephalopathy.
  • FND functional neurological disorder
  • Guillain-Barre syndrome Alzheimer’s disease
  • autism migraine, headache, Traumatic Brain Injury, and Chronic Traumatic Encephalopathy.
  • the psychological disorder is selected from the group consisting of depression, anxiety, PTSD, obsessive compulsive disorder, bipolar disorder, eating disorder, substance abuse disorder, attention deficit hyperactivity disorder (ADHD), and schizophrenia.
  • the pharmaceutical composition comprises cell media.
  • one or more tryptamine is heterologously produced, for example in a prokaryotic cell.
  • one or more tryptamine is synthetically produced.
  • one or more tryptamine is purified.
  • a range is intended to comprise every integer or fraction or value within the range.
  • the pharmaceutical composition is a parenteral dosage form. In some embodiments, the pharmaceutical composition is an oral dosage form. In some embodiments, the pharmaceutical composition comprises a tablet, capsule, dry powder, gel, film, solution or combination.
  • Pharmaceutically acceptable carriers are provided.
  • compositions described herein may comprise a pharmaceutical carrier.
  • the pharmaceutical carrier is a solvent (e.g. an alcohol), a polymer, a nanoparticle, a liposome, a lipoprotein, a gel, a sugar or sugars, protein or other matrix, or carriage device.
  • the route of delivery is by injection, oral, sublingual, buccal, transdermal, or nasal.
  • a method for the production of psilocybin or an intermediate or a side product thereof comprises contacting a host cell with at least one psilocybin production gene selected from: psiD, psiK, psiM, and combinations thereof to form a recombinant cell; culturing the recombinant cell; and obtaining the psilocybin or an intermediate or side product thereof.
  • the host cell is a prokaryotic cell.
  • the host cell is an E. coli cell.
  • a method for the production of psilocybin or an intermediate or a side product thereof comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell.
  • the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • any intermediate or side product of psilocybin may be produced by any of the methods described herein.
  • the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aemginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT).
  • the intermediate of psilocybin is norbaeocystin, baeocystin, 4- hydroxytryptophan, or 4-hydroxytryptamine.
  • the side product of psilocybin is aemginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4- OH-TMT).
  • the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4-hydroxytryptophan, 4-hydroxytryptamine, and combinations thereof.
  • the supplement is fed continuously to the host cell.
  • the host cell is grown in an actively growing culture. Continuous feeding is accomplished by using a series of syringe and/or peristaltic pumps whose outlet flow is directly connected to the bioreactor. The set point of these supplement addition pumps is adjusted in response to real-time measurement of cell biomass and specific metabolic levels using UV-vis absorption and HPLC analysis, respectively.
  • the fed-batch fermentation process is focused on maximizing production of target metabolites through harnessing the ability of an actively growing and replicating cell culture to regenerate key co-factors and precursors which are critical to the biosynthesis of target metabolites.
  • This process notably does not involve the centrifugal concentration and reconstitution of cell biomass to artificially higher cell density and/or into production media that was not used to build the initial biomass.
  • the production process involves the inoculation of the reactor from an overnight preculture at low optical density, followed by exponential phase growth entering into a fed-batch phase of production, culminating in a high cell density culture.
  • the psilocybin and intermediate or side products are found extracellularly in the fermentation broth.
  • the psilocybin and intermediate or side products are isolated. These target products can be collected through drying the fermentation broth after centrifugation to remove the cell biomass. The resulting dry product can be extracted to further purify the target compounds.
  • the products can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions psilocybin or any of the intermediate or side products into the organic phase.
  • contaminants from the fermentation broth can be removed through extraction leaving the psilocybin and/or intermediate or side products in the aqueous phase for collection after drying or crystallization procedures.
  • the methods described herein result in a titer of psilocybin of about 0.5 to about 50 g/L. In some embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 10 g/L. In yet further embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 2 g/L. In certain embodiments, the methods described herein result in a titer of psilocybin of about 1.0 to about 1.2 g/L. In further embodiments, the methods described herein result in a titer of psilocybin of about 1.16 g/L.
  • the methods described herein result in a molar yield of psilocybin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of psilocybin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of psilocybin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of psilocybin of about 40% to about 60%. In further embodiments, the methods described herein result in a molar yield of psilocybin of about 50%. Recombinant prokaryotic cells for the production of psilocybin or an intermediate or a side product thereof
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.
  • the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • a vector for introducing at least one gene associated with psilocybin production the gene may be selected from: psiD , psiK , and psiM and combinations thereof.
  • the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the expression vector comprises a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration.
  • the expression vector comprises a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
  • the expression vector comprises the nucleic acid sequence of SEQ ID NO: 18 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the expression vector is pPsilol6 or a vector having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • kits comprising an expression vector as described herein.
  • a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes.
  • container means such as, e.g., vials or test tubes.
  • Each of such container means comprises components or a mixture of components needed to perform a transfection.
  • kits may include, for example, one or more components selected from vectors, cells, reagents, lipid- aggregate forming compounds, transfection enhancers, or biologically active molecules.
  • a method for the production of norbaeocystin comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof; and culturing the host cell.
  • each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof; and culturing the host cell.
  • none of the expression vectors comprises psiM.
  • the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the prokaryotic cell is contacted with an expression vector comprising a psilocybin production gene selected from the group consisting of a psiD gene, a psiK gene, and combinations thereof, all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
  • the expression vectors comprises a psiM gene.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4-hydroxytryptophan, 4-hydroxytryptamine, and combinations thereof.
  • the supplement is fed continuously to the host cell.
  • the host cell is grown in an actively growing culture. Continuous feeding is accomplished by using a series of syringe and/or peristaltic pumps whose outlet flow is directly connected to the bioreactor. The set point of these supplement addition pumps is adjusted in response to real-time measurement of cell biomass and specific metabolic levels using UV-vis absorption and HPLC analysis, respectively.
  • the fed-batch fermentation process is focused on maximizing production of target metabolites through harnessing the ability of an actively growing and replicating cell culture to regenerate key co-factors and precursors which are critical to the biosynthesis of target metabolites.
  • This process notably does not involve the centrifugal concentration and reconstitution of cell biomass to artificially higher cell density and/or into production media that was not used to build the initial biomass.
  • the production process involves the inoculation of the reactor from an overnight preculture at low optical density, followed by exponential phase growth entering into a fed-batch phase of production, culminating in a high cell density culture.
  • the norbaeocystin is found extracellularly in the fermentation broth.
  • the norbaeocystin is isolated.
  • Norbaeocystin can be collected through drying the fermentation broth after centrifugation to remove the cell biomass.
  • the resulting dry product can be extracted to further purify the norbaeocystin.
  • the norbaeocystin can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions norbaeocystin into the organic phase.
  • contaminants from the fermentation broth can be removed through extraction leaving the norbaeocystin in the aqueous phase for collection after drying or crystallization procedures.
  • the methods described herein result in a titer of norbaeocystin of about 0.1 to about 50 g/L. In some embodiments, the methods described herein result in a titer of norbaeocystin of about 0.1 to about 10 g/L. In yet further embodiments, the methods described herein result in a titer of norbaeocystin of about 0.1 to about 3 g/L. In certain embodiments, the methods described herein result in a titer of norbaeocystin of about 0.5 to about 2.5 g/L.
  • the methods described herein result in a titer of norbaeocystin of about 0.5 to about 2.0 g/L. In further embodiments, the methods described herein result in a titer of norbaeocystin of about 1.5 g/L.
  • the methods described herein result in a molar yield of norbaeocystin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of norbaeocystin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of norbaeocystin of about 40% to about 60%. In further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 50%.
  • a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof. In certain embodiments, none of the expression vectors comprises psiM.
  • the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
  • the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
  • none of the expression vectors comprises a psiM gene.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the gene may be selected from: psiD , psiK , and combinations thereof.
  • the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene all under control of a single promoter in operon configuration.
  • the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration.
  • each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
  • none of the expression vectors comprises a psiM gene.
  • the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
  • the expression vector comprises the nucleic acid sequence of SEQ ID NO: 19 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • the expression vector is pETM6-C4-psiDK or a vector having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
  • kits comprising an expression vector as described herein.
  • a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes.
  • container means such as, e.g., vials or test tubes.
  • Each of such container means comprises components or a mixture of components needed to perform a transfection.
  • kits may include, for example, one or more components selected from vectors, cells, reagents, lipid- aggregate forming compounds, transfection enhancers, or biologically active molecules.
  • Example 1 Norbaeocystin production in E. coli.
  • Plasmid and pooled library construction and screening Plasmids containing the norbaeocystin production pathway were constructed using the previously reported pETM6- SDM2x plasmid backbone (Adams, A. M. et al. In vivo production of psilocybin in E. coli. Metab. Eng. 56, 111-119 (2019)). Single gene constructs were assembled using traditional restriction ligation cloning using Ndel and Xho I .
  • the transcriptional library promoters included T7 mutant promoters G6, H9, H10, and C4, along with the consensus (T7) promoter and were screened in 48-well plate format after transformation in the commercially available production host strain BL21 StarTM (DE3). Upon identification of the top seven mutants from the promoter library, the strains were rescreened to confirm high production levels prior to plasmid isolation and transformation into E. coli DH5oc for permanent storage. The plasmid DNA was then isolated from the DH5oc strain for promoter sequencing and retransformed into BL21 StarTM (DE3) for a final round of screening.
  • Standard screening conditions Standard screening was performed using 48-well plates with a rectangular cross section and a 2 mL working volume at 37 °C. Serine (1 g/L), 4- hydroxyindole (350 mg/L), and ampicillin (80 pg/mL) were supplemented in AMM-No MOPS for all experiments, unless otherwise noted. Overnight cultures were grown for 12-16 h at 37 °C in a shaker incubator (225 rpm) in the same media that was used for final production. Induction with 1 mM IPTG occurred 4 hours after inoculation. Samples were taken at 24 and 48 hours, unless otherwise noted, and subjected to HPLC and LC-MS analysis.
  • E. coli Broth Preparation for HTR Studies For use in the animal studies, control, psilocybin containing, and norbaeocystin containing broths were produced from fed batch bioreactor fermentations of Psilol6 (no 4-hydroxyindole supplement), Psilol6 (4-hydroxyindole supplement), and Norl (4-hydroxyindole supplement), respectively, using the conditions specified previously for Psilol6 and here for norbaeocystin. After the fermentation was concluded, the broth was centrifuged (5000 x g, 30 min) and filtered using a 0.2 pm bottle top filter, prior to administration to animals (described below). Filtered broth samples were stored at room temperature up to two months between production and use with negligible degradation observed. Metabolite concentrations in the broth were periodically quantified using HPFC analysis.
  • FIG. 16 Representative HPFC chromatograms for the negative control, norbaeocystin, and psilocybin containing broth are shown in FIG. 16.
  • the psilocybin containing broths also contained trace levels of norbaeocystin ( ⁇ 20 mg/F) and aeruginascin ( ⁇ 1 mg/F), low levels of baeocystin (approx. 150 mg/F), and high levels of psilocybin (approx. 1 g/F).
  • the norbaeocystin containing broths had high levels of norbaeocystin (approx.
  • the sequencing results revealed an interesting trend with the top producing strain containing the strongest mutant promoter, C4, controlling the transcription of both psiD and psiK in operon configuration (FIG. 2B).
  • the data also shows a mixed association between promoter strength and pathway performance, with two of the reduced strength promoters (G6, H10), as well as the strong T7 consensus promoter, leading to low levels of norbaeocystin production (FIG. 2B). This is in contrast with the similarly constructed psilocybin promoter library studies, which resulted in the best performance from the medium strength, H10, mutant promoter (FIG. 2B).
  • the norbaeocystin producing libraries showed a preference for the operon promoter configuration (FIG. 2A - black bars), resulting in the highest production strains with the lowest build-up of intermediate products when compared to their respective pseudo-operon libraries (FIG. 2A - gray bars).
  • the top norbaeocystin producer, Norl shows a 7-fold improvement in norbaeocystin production over the original T7 consensus construct (FIG. 2B).
  • Temperature, pH, and dissolved oxygen (DO) were held constant at 37°C, 6.5, and 30%, respectively. pH, DO, and foam were maintained automatically by the addition of 10 M NaOH, an agitation cascade (300-1000 rpm), and addition of Antifoam 204. Full oxygen saturation was defined under the conditions of 37 °C, 300 rpm agitation, pH 7.0, and 2 v/v per minute of standard air. The zero-oxygen set point was calibrated using a nitrogen gas flush. Samples were collected periodically for measurement of O ⁇ ⁇ oo and metabolite concentrations.
  • LC-MS Liquid Chromatography Mass Spectrometry
  • EIC extracted ion chromatogram
  • the source and detector conditions were as follows: sheath gas pressure of 80.0 psig, auxiliary gas pressure of 9.7 psig, sweep gas pressure of 0.5 psig, foreline vacuum pump pressure of 1.55 Torr, vaporizer temperature of 500 °C, ion transfer tube temperature of 300 °C, source voltage of 3049 V, and source current of 15.90 mA.
  • the broth was then dried under a vacuum in a round bottom flask.
  • the dried broth was continually mixed with 160 mL of hot (50 °C) ethanol for 30 minutes.
  • the mixture was then filtered through grade 615 filter paper in a Buchner funnel.
  • the filtrate was collected and analyzed by HPLC to confirm a lack of norbaeocystin.
  • the filter cake was then collected and resuspended in 50 mL of water, resulting in some insoluble product that was discarded.
  • the resuspension was quantified by HPLC, as described above, and a concentration of approximately 11.5 g/L was found, resulting in a -70% yield.
  • This norbaeocystin concentrate was then purified by preparative HPLC using an Agilent Polaris C18-A column (250 mm x 21.2 mm, 5 pm) with mobile phases of water (A) and acetonitrile (B), both containing 0.1% formic acid at a flow rate of 10 mL/min: 0 min, 5% B; 0.9 min, 5% B; 10 min, 40% B; 11.25 min, 100% B; 14.5 min, 100% B; 14.5 min, 5% B; 17 min,
  • Serine plays an important role in metabolism as it is directly utilized for protein synthesis, but it also serves as the precursor molecule for several other amino acids including tryptophan, cystine, and glycine. Serine is one of the major substrates for the norbaeocystin biosynthesis pathway, condensing with 4-hydroxyindole to form 4-hydroxytryptophan through the promiscuous activity of the native tryptophan synthase. This serves as the first concerted step towards norbaeocystin biosynthesis.
  • HTRs voltage
  • Offline Sorter v4.5, Plexon Inc, Dallas, TX
  • head twitches were identified from voltage recordings by a single observer, blinded to subject condition, by the presence of 1) amplitude exceeds background noise; 2) fundamental frequency of 20-40 Hz; 3) more than 2 bipolar peaks; and 4) duration ⁇ 120 ms.
  • the total number of HTRs was compared across doses within each drug condition using 2-way Poisson regression (proc glimmix, SAS v9.4), followed by post hoc tests when appropriate, corrected for multiple comparisons using the FDR method (Benjamini, Y.
  • HTRs head twitch responses
  • HTRs head twitch responses
  • coli-synthesized norbaeocystin is effective at eliciting robust behavioral responses in rats when directly gavaged in its filtered broth vehicle, but norbaeocystin alone does not increase HTRs.
  • norbaeocystin alone does not increase HTRs.
  • the individual dose response curves for these compounds do not address the existence of an entourage effect between psilocybin and its minor metabolites.
  • Psilocybin and norbaeocystin were produced using a genetically optimized recombinant strain of Escherichia coli capable of conversion of 4-hydroxyindole into each target product at high titers, productivities, and yields in a scalable bioprocess (Adams et al, 2019, process also reported in patent application PCT/US2020/051543). After production, the broth was centrifuged (5000 x g, 30 min) and filtered using a 0.2 um bottle top filter, prior to administration to animals (described below). Filtered broth samples were stored at room temperature up to two months between production and use with negligible degradation observed. Metabolite concentrations in the broth were quantified using high performance liquid chromatography as previously reported (Adams et al, 2019). Drugs and Drug Administration
  • Head movements were recorded using a magnetometer-based approach. After surgical implantation of a skull-mounted magnet and recovery (see above), rats were administered drug or vehicle and placed in a large polycarbonate tube ( ⁇ 56 cm diameter, ⁇ 30 cm height) surrounded by -150 turns of #30 enameled magnet wire. Changes in the position of the animal caused changes in the voltage across the wire, which was recorded at 1000 S/s and analyzed as detailed below.
  • This magnetometer-based approach is considerably more sensitive and easier to quantify than hand-coding of high-speed video recordings (Halberstadt & Geyer, 2013). HTRs were observed for 60 minutes, beginning immediately after drug delivery.
  • Locomotor behaviors were assessed using Accuscan Open Field chambers. Each chamber consists of 16 infrared beams arranged in two horizontal rows. Beams broken by the animal are interpreted by the software to determine the location of the rat, including rearing behavior. Animals were habituated to the chamber for 15 minutes, then gavaged and immediately placed back into the chamber for an additional 60 minutes. Beam breaks were recorded for the entire duration (including habituation). Results
  • HTRs Continuous recordings of HTRs (voltage) were exported to Offline Sorter (v4.5, Plexon Inc, Dallas, TX) for determination of the time of each HTR based on waveform characteristics. Specifically, head twitches were identified from voltage recordings by a single observer, blinded to subject condition, by the presence of 1) amplitude exceeds background noise; 2) fundamental frequency of 20-40 Hz; 3) more than 2 bipolar peaks; and 4) duration ⁇ 120 ms. Because the nonnormal nature of the data (count with low means) violated the assumptions of ANOVA, the total number of HTRs was compared across doses within each drug condition using 1-way poisson mixed models (proc glimmix, SAS v9.4), followed by post hoc tests when appropriate.
  • 1-way poisson mixed models proc glimmix, SAS v9.4
  • Locomotor data were analyzed using 1-way ANOVA, including analyses for the total distance travelled and total number of rearing events, with separate analyses for habituation and after gavage. Group means ⁇ standard error of the mean are presented in all figures.
  • Rodent Subjects A total of 24 adult (PND 90-120) male Long Evans rats (Charles River Laboratories, Raleigh, NC) were used in these experiments. Except where noted in the chronic variable stress paradigm, all animals were dual-housed in standard rat cages on a 12 hr: 12 hr light/dark cycle, and food and water were available ad libitum. Animals were divided into one of 4 treatment groups (vehicle, fluoxetine, psilocybin, and psilocybin+norbaeocystin). All procedures and protocols were conducted in accordance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals and the Animal Welfare Act, and were approved by Miami University’s Institutional Animal Care and Use Committee.
  • the chronic stress regimen is one of the most reliable methods for establishing a rodent model of depression / HPA dysfunction.
  • This regimen involves exposure of rodents to a series of randomly-alternating stressors (administered twice daily) over a period of 15 days. In addition, animals receive an extra overnight stressor on two out of every three days.
  • This random-stress procedure has been used previously and has been shown to produce reliable changes in basal stress-axis function (Herman et ah, 1995; Ostrander et ah, 2006; Ziegler et ah, 1999).
  • the stressors employed in our studies included the following:
  • Restraint tubes are Plexiglas restrainers, approximately 21 cm in length with an approximately 5 cm opening, equipped with ventilation holes.
  • Cage tilt with wet bedding Animals’ home cage was elevated on one end to produce up to 30° incline. Additionally, the bedding of an animal's home cage was moistened with water without producing standing water. Animals were housed in this condition for 2 hours.
  • Strobe Light Animals were placed in a darkened room with a stroboscopic light (up to 300 flashes/min) for up to 2 hours.
  • the stressors were given using a set-schedule model. Individual timing of the stressors was selected based on the stressor duration required to provoke an adrenocortical response, without endangering the animals’ well-being. For example, 1 hr exposure to cold in a cold room is required for an optimal pituitary- adrenal response, whereas only 30 min are needed for restraint stress. At least 2 hours to recover between the AM and PM stressor were provided each day. Drug administration occurred 1-week after the final stressor. Drugs and Drug Administration
  • Drugs were synthesized as described in the Head Twitch Response methods, but extracted, purified, and suspended in a water vehicle (for delivery to animals via gavage).
  • Cell broth containing psilocybin was centrifuged and filtered to remove cell biomass, followed by drying under reduced pressure to form a wet sticky solid.
  • This solid was then extracted with methanol, and the liquid fraction is dried under reduced pressure.
  • the resulting solid is dissolved in water and purified using reverse phase preparative HPLC. The collected fractions are concentrated under reduced pressure until spontaneous crystallization, resulting in a white solid.
  • Cell broth containing norbaeocystin was centrifuged and filtered to remove cell biomass, followed by drying under reduced pressure to form a wet sticky solid.
  • HTRs Head Twitch Responses
  • sucrose preference test is a commonly used metric of antidepressant efficacy. Animals in a depressive-like state demonstrate reduced preference for sweetened solutions, indicative of the anhedonia commonly experienced by individuals suffering from depression. After completing our chronic variable stress paradigm, animals were given overnight access (12 hr) to a sweetened water solution (2% sucrose), and preference for the sweetened solution assessed. All animals demonstrated suppressed preference, indicating anhedonia. Following treatment, overnight sucrose preference was again assessed (12 hr) and compared to pretreatment. We found that animals in the vehicle control group and fluoxetine groups showed no change in sucrose preference, but that animals in the psilocybin group demonstrated a significant increase in preference after treatment. Additionally, animals in the combined group (psilocybin+norbaeocystin) demonstrated the greatest increase in sucrose preference (FIG. 18).
  • the forced swim test is one of the most commonly used metrics of antidepressant efficacy in rodents (Slattery & Cryan, 2012). After completing our chronic variable stress paradigm, control animals demonstrated high levels of immobility, indicating their “despair-like” state. This state was alleviated in animals who had been treated with either psilocybin or fluoxetine (a selective serotonin reuptake inhibitor). Animals receiving both psilocybin and norbaeocystin showed the lowest levels of immobility, demonstrating the greater effectiveness of the combination of these drugs (FIG. 19).
  • norbaeocystin may allow for a wider dose range of psilocybin to be used, enabling lower doses of psilocybin to have greater effects in vivo , while simultaneously broadening the dose response curve to allow animals to tolerate even higher doses of psilocybin, increasing the drug’s effectiveness.
  • Our data support the idea that the addition of norbaeocystin allows for a wider range of psilocybin doses to be used clinically, facilitating more specific dosing (e.g. individualized medicine) and better therapeutic outcomes.
  • a third significant outcome of our results is that they demonstrate the relative safety of directly gavaging filtered E. coli broth as a drug vehicle.
  • the use of filtered cell culture media as a drug vehicle has never before been published.
  • the use of cell culture media as a drug vehicle could dramatically reduce the cost of drug production by cutting costly downstream purification steps. It would also reduce the time to market for such compounds and presents a low barrier to entry for screening of novel compounds in animal models.
  • Bioactive alkaloids from fungi Psilocybin.

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Abstract

Provided is a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms. Provided is a pharmaceutical composition comprising heterologously produced psilocybin and heterologously produced norbaeocystin. Also provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition comprising psilocybin and an effective amount of a pharmaceutical composition comprising norbaeocystin. Also provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition comprising psilocybin and an effective amount of a pharmaceutical composition comprising norbaeocystin. In some embodiments, the psilocybin is heterologously produced. In some embodiments, the norbaeocystin is heterologously produced.

Description

PSILOCYBIN AND NORBAEOCYSTIN COMPOSITIONS AND METHODS OF TREATMENT
FIELD
[0001] The general inventive concepts relate to the field of pharmaceutical compositions and medical therapeutics comprising the administration of two or more tryptamines, for example psilocybin and norbaeocystin.
CROSS-REFERENCE TO RELATED APPLICATIONS [0002] The instant application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/135,054, filed January 8, 2021, U.S. Provisional Application No. 63/146,044, filed February 5, 2021, and U.S. Provisional Application No. 63/219,430, filed July 8, 2021, each of which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which is submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on January 2, 2022, is named 315691-00019_Sequence_Listing.txt and is 39,095 bytes in size.
BACKGROUND
[0004] Because of its potential for treatment for a number of anxiety and mental-health related conditions, interest in psilocybin is significant. However, due to roadblocks in routing methods of obtaining drug targets (synthesis and/or extraction from a known biological source), large amounts are not currently available.
[0005] Psilocybin (4-phosphoryloxy-/V,./V-dimethyltryptamine) has gained attention in pharmaceutical markets as a result of recent clinical studies. The efficacy of psilocybin has been demonstrated for the treatment of anxiety in terminal cancer patients and alleviating the symptoms of post-traumatic stress disorder (PTSD). Most recently, the FDA has approved the first Phase lib clinical trial for the use of psilocybin as a treatment for depression that is not well controlled with currently available interventions such as antidepressants and cognitive behavioral therapies.
[0006] Clinical trials with psilocybin as a medication for individuals struggling with treatment- resistant depression are ongoing.
[0007] Norbaeocystin (3-(2-aminoethyl)-lH-indol-4-yl dihydrogen phosphate) is a tryptamine intermediate product in the psilocybin biosynthesis pathway that has been hypothesized to have its own neurological activity due to its structural similarity to psilocybin. This similarity suggests that it may compete with psilocybin for the same binding sites in the brain or metabolic process, thus enhancing psilocybin’s activity. No natural sources are currently available that specifically over-produce norbaeocystin, a key precursor to psilocybin, and only recently has a synthetic route been published for its production (Sherwood, A. M. et al. Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin. J. Nat. Prod. 83, 461-467 (2020)).
[0008] There remains a need for improved methods of producing psilocybin and norbaeocystin, for compositions comprising psilocybin and norbaeocystin, and for methods of treatment comprising administration of psilocybin and norbaeocystin.
SUMMARY
[0009] Provided is a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms. In some embodiments, the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe. In further embodiments, the major tryptamine is psilocybin or a structurally similar analog. In some embodiments, the two or more tryptamines comprise one or more minor tryptamines present in Psilocybe.
[0010] In some embodiments, the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin. In further embodiments, the one or more minor tryptamines is norbaeocystin. In further embodiments, the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
[0011] In some embodiments, two tryptamines are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 25:1 to 1:25. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, two tryptamines are present in the composition in a molar ratio of 1:1.
[0012] Provided is a pharmaceutical composition comprising psilocybin and norbaeocystin. In some embodiments, the psilocybin is heterologously produced. In some embodiments, the norbaeocystin is heterologously produced.
[0013] In some embodiments the psilocybin is synthetically produced. In some embodiments, the norbaeocystin is synthetically produced.
[0014] Provided is a pharmaceutical composition comprising heterologously produced psilocybin and heterologously produced norbaeocystin.
[0015] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced psilocybin is purified.
[0016] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced norbaeocystin is purified.
[0017] In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of 1 : 1.
[0018] In some embodiments of any of the pharmaceutical compositions described herein, the pharmaceutical composition comprises cell media.
[0019] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms. In some embodiments, the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe. In further embodiments, the major tryptamine is psilocybin or a structurally similar analog. In some embodiments, the two or more tryptamines comprise one or more minor tryptamines present in Psilocybe.
[0020] In some embodiments, the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin. In further embodiments, the one or more minor tryptamines is norbaeocystin. In further embodiments, the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
[0021] In some embodiments, two tryptamines are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 25:1 to 1:25. In some embodiments, two tryptamines are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, two tryptamines are present in the composition in a molar ratio of 1:1.
[0022] Also provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient a pharmaceutical composition comprising psilocybin and norbaeocystin. In some embodiments, the psilocybin is heterologously produced. In some embodiments, the norbaeocystin is heterologously produced.
[0023] In some embodiments, the administration of the pharmaceutical composition comprising psilocybin and norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
[0024] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0025] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0026] In some embodiments, the heterologously produced psilocybin is purified. In further embodiments, the heterologously produced norbaeocystin is purified.
[0027] In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10. In some embodiments, the psilocybin and the norbaeocystin are present in the composition in a molar ratio of 1 : 1.
[0028] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient an effective amount of a first tryptamine and an effective amount of a second tryptamine that are present in Psilocybe magic mushrooms.
[0029] In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 100:1 to 1:100. In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 75:1 to 1:75.
In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 50:1 to 1:50. In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 25:1 to 1:25. In some embodiments, the first tryptamine and the second tryptamine are administered to the patient in a molar ratio of from 10:1 to 1:10. In some embodiments, the first tryptamine and the second tryptamine are are administered to the patient in a molar ratio of 1 : 1.
[0030] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition comprising psilocybin and an effective amount of a pharmaceutical composition comprising norbaeocystin. In some embodiments, the psilocybin is heterologously produced. In some embodiments, the norbaeocystin is heterologously produced.
[0031] In some embodiments, the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at substantially the same time. In further embodiments, the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at different times. In some embodiments, the pharmaceutical composition comprising psilocybin is administered before the pharmaceutical composition comprising norbaeocystin. In some embodiments the pharmaceutical composition comprising norbaeocystin is administered before the pharmaceutical composition comprising psilocybin.
[0032] In some embodiments, the administration of the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
[0033] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0034] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0035] In some embodiments, the heterologously produced psilocybin is purified. In further embodiments, the heterologously produced norbaeocystin is purified.
[0036] In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100. In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75. In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50. In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25. In some embodiments, the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10. In some embodiments, the psilocybin and the norbaeocystin are are administered to the patient in a molar ratio of 1 : 1.
[0037] In some embodiments, the neurological disorder is selected from the group consisting of functional neurological disorder (FND), Guillain-Barre syndrome, Alzheimer’s disease, autism, migraine, headache, Traumatic Brain Injury, and Chronic Traumatic Encephalopathy.
[0038] In some embodiments, the psychological disorder is selected from the group consisting of depression, anxiety, PTSD, obsessive compulsive disorder, bipolar disorder, eating disorder, substance abuse disorder, attention deficit hyperactivity disorder (ADHD), and schizophrenia.
[0039] In some embodiments of any of the methods described herein, the pharmaceutical composition comprises cell media.
[0040] In some embodiments of any of the compositions or methods described herein, one or more tryptamine is heterologously produced, for example in a prokaryotic cell.
[0041] In some embodiments of any of the compositions or methods described herein, one or more tryptamine is synthetically produced.
DESCRIPTION OF THE FIGURES
[0042] FIG. 1A shows an overview of study methods. Recombinant E. coli were developed capable of high-level norbaeocystin production. Norbaeocystin production was optimized and scaled up in a benchtop bioreactor. Norbaeocystin concentration in cell broth was quantified using HPLC. A rat with magnet affixed to its head was gavaged with filtered cell media containing psilocybin, norbaeocystin, or both, A magnetometer coil was used in order to record head twitches. Waveforms were then analyzed to determine the head twitch count.
[0043] FIG. IB shows a norbaeocystin biosynthesis pathway. The E. coli strain contains three genes, one native ( trpB ) and two heterologous (psiD , psiK) that enable norbaeocystin biosynthesis from external supplementation of 4-hydroxyindole. Tryptophan synthase (TrpB) condenses 4-hydroxyindole and serine to form 4-hydroxytryptophan. P. cubensis tryptophan decarboxylase (PsiD) converts 4-hydroxytryptophan into 4-hydroxytryptamine while releasing a carbon dioxide and water. Finally, P. cubensis kinase (PsiK) converts 4-hydroxytryptamine into norbaeocystin using a phosphate donated by ATP.
[0044] FIGs. 2A-2D show a summary of genetic optimization and scale-up results. FIG. 2A: Promoter library screening. Individual colonies from the operon (red bars) and pseudooperon (gray bars) libraries were selected and evaluated to discover elite production strains. Data for samples producing 0 mg/L of norbaeocystin (11.5 % of total colonies screened) are not shown. FIG. 2B: Normalized production of operon library members for norbaeocystin and psilocybin pathways organized in order of increasing promoter strength: G6 (low) - T7 (high). Constructs for each operon promoter configuration were identified and screened providing evidence that the transcriptional optimization solution for the norbaeocystin pathway differed from that of the psilocybin pathway. FIG. 2C: Effect of varying supplemental serine concentration in the initial fermentation media on strain performance in the bioreactor. FIG. 2D: Metabolite and growth curve profiles for a representative norbaeocystin bioreactor fed-batch fermentation. Data shown for one replicate of the 0 g/L serine condition.
[0045] FIGs. 3A-3C show dose-dependent effects of tryptamines alone and in combination.
FIG. 3A: Psilocybin caused significant increases in the number of head twitches at IX, 2X, and 10X dosages. FIG. 3B: Norbaeocystin reduced the number of head twitches exhibited by rats at IX dosage, but not at other dosages. FIG. 3C: Animals receiving both norbaeocystin and psilocybin demonstrated increased head twitches at all dosages investigated, but most significantly at 2X and 10X dosages. Note: * p < 0.05; ** p < 0.01.
[0046] FIG. 4 shows that similar dosages of tryptamines and their combinations cause different effects on head twitch responses. At the 2X dose combination, only psilocybin+norbaeocystin increased head twitch responses (HTRs) compared to norbaeocystin alone. At the 10X dose combination, psilocybin alone increased head twitch responses relative to norbaeocystin alone, while psilocybin+norbaeocystin increased head twitches relative to both norbaeocystin and psilocybin alone. At the 20X dose combination, there were no differences between the drugs in the magnitude of their effects. Note: * p<0.05.
-Si- [0047] FIGs. 5A-5F show HPLC standard curves used for metabolite profiles. (FIG. 5A) 4- hydroxyindole, (FIG. 5B) 5-hydroxytryptamine, (FIG. 5C) norbaeocystin, (FIG. 5D) 5- hydroxytryptophan, (FIG. 5E) psilocybin, and (FIG. 5F) glucose.
[0048] FIG. 6 is a graph illustrating a media optimization study. Shake flasks with Norl were used to test varying media compositions. AMM is the control. No MOPS is AMM without the MOPS/tricine buffer added, and No Mix is AMM without any MOPS mix. Although not significantly different (p>Q.05), the observed increase in No MOPS motivated the use of AMM- no MOPS in all future experiments. Error bars represent ± standard error of the mean for duplicate samples.
[0049] FIG. 7 shows an induction point sensitivity study. Norl was studied at 37 °C from 2 to 8 hours in 125 mL shake flasks. Error bars represent ± standard error of the mean for duplicate samples.
[0050] FIGs. 8A-8H show bioreactor runs supplemented with 5 g/L of serine. (FIGs.8A-8D)
First trial. (FIGs. 8E-8H) Second trial. (FIGs. 8A and 8E) Metabolite profiles. (FIGs. 8B and 8F) Total cumulative glucose (gray) and ammonium phosphate dibasic (dashed line) fed compared to Oϋόoo (dotted line). (FIGs. 8C and 8G) Total cumulative 4-hydroxyindole fed (gray) and 4- hydroxyindole (40 mg/mL in EtOH) feed rate (dotted line). (FIGs. 8D and 8H) Total cumulative 4-hydroxyindole fed (dotted line) compared to norbaeocystin production (dashed line) and transient molar yield on substrate (bold) shows a maximum molar yield of 72.2% and 58.6% with final molar yields at 63.7% and 51.7% for trials 1 and 2, respectively.
[0051] FIGs. 9A-9H show bioreactor runs supplemented with 1 g/L of serine. (FIGs.9A-9D)
First trial. (FIGs. 9E-9H) Second trial. (FIGs. 9A and 9E) Metabolite profiles. (FIGs. 9B and 9F) Total cumulative glucose (gray) and ammonium phosphate dibasic (dashed line) fed compared to Oϋόoo (dotted line). (FIGs. 9C and 9G) Total cumulative 4-hydroxyindole fed (gray) and 4- hydroxyindole (40 mg/mL in EtOH) feed rate (dotted line). (FIGs. 9D and 9H) Total cumulative 4-hydroxyindole fed (dotted line) compared to norbaeocystin production (dashed line) and transient molar yield on substrate (bold) shows a maximum molar yield of 59.0% and 71.7% with final molar yields at 58.6% and 61.2% for trials 1 and 2, respectively. [0052] FIGs. 10A-10H show bioreactor runs without supplemental serine. (FIGs.lOA-lOD) First trial. (FIGs. 10E-10H) Second trial. (FIGs. 10A and 10E) Metabolite profiles. (FIGs. 10B and 10F) Total cumulative glucose (gray) and ammonium phosphate dibasic (dashed line) fed compared to Oϋόoo (dotted line). (FIGs. IOC and 10G) Total cumulative 4-hydroxyindole fed (gray) and 4-hydroxyindole (40 mg/mL in EtOH) feed rate (dotted line). (FIGs. 10D and 10H) Total cumulative 4-hydroxyindole fed (dotted line) compared to norbaeocystin production (dashed line) and transient molar yield on substrate (bold) shows a maximum molar yield of 58.2% and 77.5% with final molar yields at 57.8% and 54.3% for trials 1 and 2, respectively.
[0053] FIGs. 11A-11C show representative measurement of dissolved oxygen (DO) (bold), pH (dashed line), temperature (dotted line), and agitation rate (black line) in bioreactors. FIG. 11 A: Fed batch bioreactor run supplemented with 5 g/L serine. FIG. 1 IB: Run supplemented with 1 g/L serine. FIG. 11C: Run without supplemental serine.
[0054] FIGs. 12A-12C show locomotor effects of tryptamines. FIG. 12A shows: norbaeocystin failed to alter distance traveled at any dose. FIG. 12B shows: psilocybin alone also did not significantly affect distance traveled. FIG. 12C shows: the combination of norbaeocystin and psilocybin increased locomotion at 0.2 mg/kg each. Note: * denotes significant (p<0.05) increase in HTR compared to control; § denotes significant increase compared to 2 mg/kg each. .
[0055] FIGs. 13A-13C show effects of tryptamines, alone and in combination, on body weight. Neither psilocybin (FIG. 13 A) nor norbaeocystin alone (FIG. 13B) had any statistically significant effect on weight gain, up to 6 days after gavage. Although the highest dose of the combination of these drugs caused a short-term reduction in weight gain (FIG. 13C), the effect of dose did not reach statistical significance in our 2-way repeated measures mixed model (p = 0.10).
[0056] FIG. 14 shows 1 H NMR spectra (500 MHz) for norbaeocystin in D2O with H2O suppression. Expanded figure inserts show splitting and peak assignments consistent with previous literature (Sherwood at ah, 2020). Protons marked with (*) are exchangeable with D2O and were not observed. Assigned peaks represent roughly 94% of total peak area observed.
[0057] FIG. 15 shows 13C NMR spectra (125 MHz) for norbaeocystin in D2O. [0058] FIG. 16 shows HPLC (A280) chromatograms of psilocybin-containing (top), norbaeocystin-containing (middle), and control (bottom) E. coli broth used in animal studies. Norbaeocystin, baeocystin, and psilocybin retention times marked with N, B, and P, respectively.
[0059] FIGs. 17A-17C shows the effects of tryptamines on stereotypical behavior during the locomotor test. FIG. 17A: shows norbaeocystin had no effect on rearing duration at any dosage tested. FIG. 17B shows: psilocybin alone also did not affect rearing. FIG. 17C shows: the combination of norbaeocystin and psilocybin reduced rearing behavior when administered at 2 mg/kg each compared to 0.2 mg/kg each, but not control. Note: † indicates significant decrease compared to 0.2 mg/kg each.
[0060] FIG. 18 shows the effect of psilocybin alone and combined with norbaeocystin on the change in sucrose preference after treatment (compared to pre-treatment). Effects of psilocybin alone and combined with norbaeocystin are compared to vehicle control and fluoxetine (a selective serotonin reuptake inhibitor). The greatest magnitude of effects was observed in the combined group. Note: significant difference from vehicle group denoted by asterisk (* p<0.05,
**p<0.01).
[0061] FIG. 19 shows the effect of psilocybin alone and combined with norbaeocystin on immobility in the forced swim test. Effects are compared to vehicle control and fluoxetine (a selective serotonin reuptake inhibitor). The greatest magnitude of effects was observed in the combined group. Note: significant difference from vehicle group denoted by asterisk (* p<0.05,
**p<0.01).
DETAILED DESCRIPTION
[0062] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.
[0063] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. [0064] The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a cell” means one cell or more than one cell.
[0065] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0066] As used herein, the term “prokaryotic host cell” means a prokaryotic cell that is susceptible to transformation, transfection, transduction, or the like, with a nucleic acid construct or expression vector comprising a polynucleotide. The term “prokaryotic host cell” encompasses any progeny that is not identical due to mutations that occur during replication.
[0067] As used herein, the term “recombinant cell” or “recombinant host” means a cell or host cell that has been genetically modified or altered to comprise a nucleic acid sequence that is not native to the cell or host cell. In some embodiments the genetic modification comprises integrating the polynucleotide in the genome of the host cell. In further embodiments the polynucleotide is exogenous in the host cell.
[0068] As used herein the term “ Psilocybe ” refers to Psilocybe ‘magic’ mushrooms as well as related psilocybin containing mushroom species.
[0069] As used herein, the term “intermediate” of psilocybin means an intermediate in the production or biosynthesis of psilocybin, e.g., norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine.
[0070] As used herein, the term “side product” of psilocybin means a side product in the production or biosynthesis of psilocybin, e.g., aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
[0071] As used herein the term “patient” or “user” means a member of the animal kingdom, including, but not limited to, a human. [0072] As used herein, the term “therapeutically effective amount” refers to that amount of any of the present compounds or compositions required to bring about a desired effect or behavior in a patient. The desired effect or behavior will vary dependent on the desired therapeutic, e.g., psychotherapeutic, response. As will be understood by one skilled in the art, a therapeutically effective amount of said compounds or compositions can be administered by any means known in the art, including but not limited to, injection, parenterally, orally, bucally, transdermally, nasally, or where appropriate, topically.
[0073] As used herein, the term “heterologously produced” in the context of heterologously produced psilocybin or norbaeocystin, means produced by heterologous expression of at least one psilocybin production gene selected from: psiD, psiK, psiM, and combinations thereof, in a host cell. In certain embodiments, the host cell is a prokaryotic cell.
[0074] The materials, compositions, and methods described herein are intended to be used to provide novel routes for the production of psilocybin and intermediates or side products, and methods for the production of norbaeocystin, as well as methods of treatment comprising the administration of psilocybin and norbaeocystin.
[0075] Applicants discovered that the efficacy of psilocybin can be enhanced by combining it with its metabolic precursor norbaeocystin, despite norbaeocystin having no observable effect on its own. Norbaeocystin’ s potential for synergistic activity when combined with psilocybin was determined by measuring rodent behavioral responses to both drugs, alone and in combination. Results described herein demonstrate the pharmacological efficacy of E. coli- derived psilocybin and suggest that other tryptamines can augment its effectiveness, thus providing strong evidence for the existence of an entourage effect in psilocybin mushrooms.
I. Pharmaceutical Compositions
[0076] Provided is a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms. In some embodiments, the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe. In further embodiments, the major tryptamine is psilocybin or a structurally similar analog. In some embodiments, the two or more tryptamines comprise one or more minor tryptamines present in Psilocybe.
[0077] In some embodiments, the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin. In further embodiments, the one or more minor tryptamines is norbaeocystin. In further embodiments, the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
[0078] Provided is a pharmaceutical composition comprising psilocybin and norbaeocystin. In some embodiments, the psilocybin is heterologously produced. In some embodiments, the norbaeocystin is heterologously produced.
[0079] In some embodiments the psilocybin is synthetically produced. In some embodiments, the norbaeocystin is synthetically produced.
[0080] Provided is a pharmaceutical composition comprising heterologously produced psilocybin and heterologously produced norbaeocystin.
[0081] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced psilocybin is purified.
[0082] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced norbaeocystin is purified.
[0083] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0084] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
[0085] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0086] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0087] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0088] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
[0089] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0090] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
[0091] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
[0092] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1.
[0093] Provided is a pharmaceutical composition comprising heterologously produced psilocybin and synthetically produced norbaeocystin. [0094] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced psilocybin is purified.
[0095] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0096] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
[0097] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0098] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0099] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0100] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
[0101] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0102] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
[0103] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2. [0104] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
[0105] In some embodiments of any of the pharmaceutical compositions described herein, the pharmaceutical composition comprises cell media.
[0106] Provided is a pharmaceutical composition comprising synthetically produced psilocybin and heterologously produced norbaeocystin.
[0107] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced psilocybin is purified.
[0108] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0109] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
[0110] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0111] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0112] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0113] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5. [0114] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0115] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
[0116] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
[0117] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1.
[0118] In some embodiments of any of the pharmaceutical compositions described herein, the pharmaceutical composition comprises cell media.
[0119] Provided is a pharmaceutical composition comprising synthetically produced psilocybin and synthetically produced norbaeocystin.
[0120] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0121] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
[0122] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0123] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0124] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0125] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5. [0126] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0127] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
[0128] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
[0129] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
[0130] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms. In some embodiments, the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe. In further embodiments, the major tryptamine is psilocybin or a structurally similar analog.
[0131] In some embodiments, the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin. In further embodiments, the one or more minor tryptamines is norbaeocystin. In further embodiments, the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
[0132] In some embodiments, administering to the patient a pharmaceutical composition comprising the two or more tryptamines produces a synergistic effect.
[0133] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient a pharmaceutical composition according to any one of the embodiments described herein.
[0134] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient a pharmaceutical composition comprising psilocybin and norbaeocystin. In some embodiments, the psilocybin is heterologously produced. In some embodiments, the norbaeocystin is heterologously produced.
[0135] In some embodiments the psilocybin is synthetically produced. In some embodiments, the norbaeocystin is synthetically produced.
[0136] In some embodiments, the administration of the pharmaceutical composition comprising psilocybin and norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
[0137] In some embodiments, administering to the patient the pharmaceutical composition comprising psilocybin and norbaeocystin produces a synergistic effect.
[0138] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0139] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0140] In some embodiments, the heterologously produced psilocybin is purified. In further embodiments, the heterologously produced norbaeocystin is purified.
[0141] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0142] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75. [0143] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0144] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0145] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0146] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
[0147] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0148] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
[0149] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
[0150] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1. For example, for every X mole of heterologously produced psilocybin in the composition, there is X mole of heterologously produced norbaeocystin.
[0151] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient heterologously produced psilocybin and synthetically produced norbaeocystin.
[0152] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced psilocybin is purified.
[0153] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0154] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
[0155] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0156] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0157] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0158] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
[0159] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0160] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3.
[0161] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
[0162] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
[0163] In some embodiments of any of the pharmaceutical compositions described herein, the pharmaceutical composition comprises cell media. [0164] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient synthetically produced psilocybin and heterologously produced norbaeocystin.
[0165] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. In yet further embodiments, the heterologously produced psilocybin is purified.
[0166] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0167] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
[0168] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0169] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0170] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0171] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
[0172] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0173] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3. [0174] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
[0175] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are present in the composition in a molar ratio of 1:1.
[0176] In some embodiments of any of the pharmaceutical compositions described herein, the pharmaceutical composition comprises cell media.
[0177] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient synthetically produced psilocybin and synthetically produced norbaeocystin.
[0178] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
[0179] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
[0180] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
[0181] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
[0182] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
[0183] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 5:1 to 1:5.
[0184] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 4:1 to 1:4.
[0185] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 3:1 to 1:3. [0186] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of from 2:1 to 1:2.
[0187] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are present in the composition in a molar ratio of 1:1.
[0188] Provided is a method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition comprising psilocybin and an effective amount of a pharmaceutical composition comprising norbaeocystin. In some embodiments, the psilocybin is heterologously produced. In some embodiments, the norbaeocystin is heterologously produced.
[0189] In some embodiments the psilocybin is synthetically produced. In some embodiments, the norbaeocystin is synthetically produced.
[0190] In some embodiments, the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at substantially the same time. In further embodiments, the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin are administered at different times. In some embodiments, the pharmaceutical composition comprising psilocybin is administered before the pharmaceutical composition comprising norbaeocystin. In some embodiments the pharmaceutical composition comprising norbaeocystin is administered before the pharmaceutical composition comprising psilocybin.
[0191] In some embodiments, the administration of the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
[0192] In some embodiments, administering to the patient the pharmaceutical composition comprising psilocybin and the pharmaceutical composition comprising norbaeocystin produces a synergistic effect. [0193] In some embodiments, the heterologously produced psilocybin was produced in a prokaryotic cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0194] In some embodiments, the heterologously produced norbaeocystin was produced in a prokaryotic host cell. In further embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0195] In some embodiments, the heterologously produced psilocybin is purified. In further embodiments, the heterologously produced norbaeocystin is purified.
[0196] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
[0197] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
[0198] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
[0199] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
[0200] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
[0201] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5. [0202] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin administered to the patient in a molar ratio of from 4:1 to 1:4.
[0203] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3.
[0204] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2.
[0205] In some embodiments, the heterologously produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of heterologously produced psilocybin administered to the patient, X mole of heterologously produced norbaeocystin is administered to the patient.
[0206] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
[0207] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
[0208] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
[0209] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
[0210] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
[0211] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5.
[0212] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin administered to the patient in a molar ratio of from 4:1 to 1:4. [0213] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3.
[0214] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2.
[0215] In some embodiments, the heterologously produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of heterologously produced psilocybin administered to the patient, X mole of synthetically produced norbaeocystin is administered to the patient.
[0216] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
[0217] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
[0218] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
[0219] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
[0220] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
[0221] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5.
[0222] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin administered to the patient in a molar ratio of from 4:1 to 1:4.
[0223] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3. [0224] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2.
[0225] In some embodiments, the synthetically produced psilocybin and the heterologously produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of synthetically produced psilocybin administered to the patient, X mole of heterologously produced norbaeocystin is administered to the patient.
[0226] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
[0227] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
[0228] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
[0229] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
[0230] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
[0231] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 5:1 to 1:5.
[0232] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin administered to the patient in a molar ratio of from 4:1 to 1:4.
[0233] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 3:1 to 1:3.
[0234] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of from 2:1 to 1:2. [0235] In some embodiments, the synthetically produced psilocybin and the synthetically produced norbaeocystin are administered to the patient in a molar ratio of 1:1. For example, for every X mole of synthetically produced psilocybin administered to the patient, X mole of synthetically produced norbaeocystin is administered to the patient.
[0236] In some embodiments, the neurological disorder is selected from the group consisting of functional neurological disorder (FND), Guillain-Barre syndrome, Alzheimer’s disease, autism, migraine, headache, Traumatic Brain Injury, and Chronic Traumatic Encephalopathy.
[0237] In some embodiments, the psychological disorder is selected from the group consisting of depression, anxiety, PTSD, obsessive compulsive disorder, bipolar disorder, eating disorder, substance abuse disorder, attention deficit hyperactivity disorder (ADHD), and schizophrenia.
[0238] In some embodiments of any of the compositions or methods described herein, the pharmaceutical composition comprises cell media.
[0239] In some embodiments of any of the compositions or methods described herein, one or more tryptamine is heterologously produced, for example in a prokaryotic cell.
[0240] In some embodiments of any of the compositions or methods described herein, one or more tryptamine is synthetically produced.
[0241] In some embodiments of any of the compositions or methods described herein, one or more tryptamine is purified.
[0242] In some embodiments of any of the compositions or methods described herein, a range is intended to comprise every integer or fraction or value within the range.
Pharmaceutical composition
[0243] In some embodiments, the pharmaceutical composition is a parenteral dosage form. In some embodiments, the pharmaceutical composition is an oral dosage form. In some embodiments, the pharmaceutical composition comprises a tablet, capsule, dry powder, gel, film, solution or combination. Pharmaceutically acceptable carriers
[0244] The pharmaceutical compositions described herein may comprise a pharmaceutical carrier. In some embodiments, the pharmaceutical carrier is a solvent (e.g. an alcohol), a polymer, a nanoparticle, a liposome, a lipoprotein, a gel, a sugar or sugars, protein or other matrix, or carriage device.
Route of Delivery
[0245] In some embodiments, the route of delivery is by injection, oral, sublingual, buccal, transdermal, or nasal.
II. Methods, vectors, host cells and kits for the production of psilocybin or norbaeocystin
[0246] Provided herein are methods of in vivo psilocybin production using a prokaryotic host. (See WO2021/086513, which is hereby incorporated by reference in its entirety and for all purposes) Provided is a method for the production of psilocybin or an intermediate or a side product thereof. The method comprises contacting a host cell with at least one psilocybin production gene selected from: psiD, psiK, psiM, and combinations thereof to form a recombinant cell; culturing the recombinant cell; and obtaining the psilocybin or an intermediate or side product thereof. In certain embodiments, the host cell is a prokaryotic cell. In certain exemplary embodiments, the host cell is an E. coli cell.
[0247] Provided is a method for the production of psilocybin or an intermediate or a side product thereof comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof; and culturing the host cell. In certain embodiments, the prokaryotic host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae. [0248] In certain embodiments, the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0249] In certain embodiments, the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0250] In certain embodiments, the psiM comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0251] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
[0252] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0253] It is envisaged that any intermediate or side product of psilocybin may be produced by any of the methods described herein. In some embodiments, the intermediate or side product of psilocybin is norbaeocystin, baeocystin, 4-hydroxytryptophan, 4-hydroxytryptamine, aemginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT). In some embodiments the intermediate of psilocybin is norbaeocystin, baeocystin, 4- hydroxytryptophan, or 4-hydroxytryptamine. In some embodiments, the side product of psilocybin is aemginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4- OH-TMT).
[0254] In certain embodiments, the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4-hydroxytryptophan, 4-hydroxytryptamine, and combinations thereof. In certain exemplary embodiments, the supplement is fed continuously to the host cell. In further embodiments, the host cell is grown in an actively growing culture. Continuous feeding is accomplished by using a series of syringe and/or peristaltic pumps whose outlet flow is directly connected to the bioreactor. The set point of these supplement addition pumps is adjusted in response to real-time measurement of cell biomass and specific metabolic levels using UV-vis absorption and HPLC analysis, respectively. The fed-batch fermentation process is focused on maximizing production of target metabolites through harnessing the ability of an actively growing and replicating cell culture to regenerate key co-factors and precursors which are critical to the biosynthesis of target metabolites. This process notably does not involve the centrifugal concentration and reconstitution of cell biomass to artificially higher cell density and/or into production media that was not used to build the initial biomass. The production process involves the inoculation of the reactor from an overnight preculture at low optical density, followed by exponential phase growth entering into a fed-batch phase of production, culminating in a high cell density culture.
[0255] The psilocybin and intermediate or side products are found extracellularly in the fermentation broth. In certain embodiments, the psilocybin and intermediate or side products are isolated. These target products can be collected through drying the fermentation broth after centrifugation to remove the cell biomass. The resulting dry product can be extracted to further purify the target compounds. Alternatively, the products can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions psilocybin or any of the intermediate or side products into the organic phase. Furthermore, contaminants from the fermentation broth can be removed through extraction leaving the psilocybin and/or intermediate or side products in the aqueous phase for collection after drying or crystallization procedures.
[0256] In certain embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 50 g/L. In some embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 10 g/L. In yet further embodiments, the methods described herein result in a titer of psilocybin of about 0.5 to about 2 g/L. In certain embodiments, the methods described herein result in a titer of psilocybin of about 1.0 to about 1.2 g/L. In further embodiments, the methods described herein result in a titer of psilocybin of about 1.16 g/L.
[0257] In certain embodiments, the methods described herein result in a molar yield of psilocybin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of psilocybin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of psilocybin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of psilocybin of about 40% to about 60%. In further embodiments, the methods described herein result in a molar yield of psilocybin of about 50%. Recombinant prokaryotic cells for the production of psilocybin or an intermediate or a side product thereof
[0258] Provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and psiM and combinations thereof.
[0259] In certain embodiments, the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0260] In certain embodiments, the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0261] In certain embodiments, the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. [0262] In certain embodiments, the psiM comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0263] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged.
[0264] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
Expression vectors
[0265] Provided is a vector for introducing at least one gene associated with psilocybin production; the gene may be selected from: psiD , psiK , and psiM and combinations thereof.
[0266] In certain embodiments, the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0267] In certain embodiments, the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0268] In certain embodiments, the psiM comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM comprises the amino acid sequence of Genbank accession number KY984100.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiM is encoded by a nucleotide sequence comprising SEQ ID NO: 3 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0269] In certain embodiments, the expression vector comprises a psiD gene, a psiK gene and a psiM gene all under control of a single promoter in operon configuration. In certain embodiments, the expression vector comprises a psiD gene, a psiK gene and a psiM gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged. [0270] In certain embodiments, the expression vector comprises the nucleic acid sequence of SEQ ID NO: 18 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the expression vector is pPsilol6 or a vector having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0271] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
Kits
[0272] Provided is a transfection kit comprising an expression vector as described herein. Such a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes. Each of such container means comprises components or a mixture of components needed to perform a transfection. Such kits may include, for example, one or more components selected from vectors, cells, reagents, lipid- aggregate forming compounds, transfection enhancers, or biologically active molecules.
III. Methods, vectors, host cells and kits for the production of norbaeocystin
Methods
[0273] Provided is a method for the production of norbaeocystin comprising contacting a prokaryotic host cell with one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK and combinations thereof; and culturing the host cell. In certain embodiments, none of the expression vectors comprises psiM.
[0274] In certain embodiments, the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0275] In certain embodiments, the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0276] In certain embodiments, the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0277] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psilocybin production gene selected from the group consisting of a psiD gene, a psiK gene, and combinations thereof, all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged. In certain embodiments, none of the expression vectors comprises a psiM gene. [0278] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
[0279] In certain embodiments, the host cell is cultured with a supplement independently selected from the group consisting of 4-hydroxyindole, serine, methionine, 4-hydroxytryptophan, 4-hydroxytryptamine, and combinations thereof. In certain exemplary embodiments, the supplement is fed continuously to the host cell. In further embodiments, the host cell is grown in an actively growing culture. Continuous feeding is accomplished by using a series of syringe and/or peristaltic pumps whose outlet flow is directly connected to the bioreactor. The set point of these supplement addition pumps is adjusted in response to real-time measurement of cell biomass and specific metabolic levels using UV-vis absorption and HPLC analysis, respectively. The fed-batch fermentation process is focused on maximizing production of target metabolites through harnessing the ability of an actively growing and replicating cell culture to regenerate key co-factors and precursors which are critical to the biosynthesis of target metabolites. This process notably does not involve the centrifugal concentration and reconstitution of cell biomass to artificially higher cell density and/or into production media that was not used to build the initial biomass. The production process involves the inoculation of the reactor from an overnight preculture at low optical density, followed by exponential phase growth entering into a fed-batch phase of production, culminating in a high cell density culture.
[0280] The norbaeocystin is found extracellularly in the fermentation broth. In certain embodiments, the norbaeocystin is isolated. Norbaeocystin can be collected through drying the fermentation broth after centrifugation to remove the cell biomass. The resulting dry product can be extracted to further purify the norbaeocystin. Alternatively, the norbaeocystin can be extracted from the liquid cell culture broth using a solvent which is immiscible with water and partitions norbaeocystin into the organic phase. Furthermore, contaminants from the fermentation broth can be removed through extraction leaving the norbaeocystin in the aqueous phase for collection after drying or crystallization procedures.
[0281] In certain embodiments, the methods described herein result in a titer of norbaeocystin of about 0.1 to about 50 g/L. In some embodiments, the methods described herein result in a titer of norbaeocystin of about 0.1 to about 10 g/L. In yet further embodiments, the methods described herein result in a titer of norbaeocystin of about 0.1 to about 3 g/L. In certain embodiments, the methods described herein result in a titer of norbaeocystin of about 0.5 to about 2.5 g/L. In further embodiments, the methods described herein result in a titer of norbaeocystin of about 0.5 to about 2.0 g/L. In further embodiments, the methods described herein result in a titer of norbaeocystin of about 1.5 g/L.
[0282] In certain embodiments, the methods described herein result in a molar yield of norbaeocystin of about 10% to about 100%. In some embodiments, the methods described herein result in a molar yield of norbaeocystin of about 20% to about 80%. In yet further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 30% to about 70%. In certain embodiments, the methods described herein result in a molar yield of norbaeocystin of about 40% to about 60%. In further embodiments, the methods described herein result in a molar yield of norbaeocystin of about 50%.
Recombinant prokaryotic cells for the production of norbaeocystin
[0283] Provided is a recombinant prokaryotic cell comprising one or more expression vectors, wherein each expression vector comprises a psilocybin production gene selected from the group consisting of psiD, psiK, and combinations thereof. In certain embodiments, none of the expression vectors comprises psiM.
[0284] In certain embodiments, the recombinant prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
[0285] In certain embodiments, the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0286] In certain embodiments, the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0287] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged. In certain embodiments, none of the expression vectors comprises a psiM gene.
[0288] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter.
Expression vectors
[0289] Provided is a vector for introducing at least one gene associated with psilocybin production; the gene may be selected from: psiD , psiK , and combinations thereof. [0290] In certain embodiments, the psiD comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD comprises the amino acid sequence of Genbank accession number KY984101.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiD is encoded by a nucleotide sequence comprising SEQ ID NO: 1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0291] In certain embodiments, the psiK comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK comprises the amino acid sequence of Genbank accession number KY984099.1 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the psiK is encoded by a nucleotide sequence comprising SEQ ID NO: 2 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
[0292] In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene all under control of a single promoter in operon configuration. In certain embodiments, the prokaryotic cell is contacted with an expression vector comprising a psiD gene and a psiK gene, wherein each gene is under control of a separate promoter in pseudooperon configuration. In certain embodiments, each gene is in monocistronic configuration, wherein each gene has a promoter and a terminator. Any configuration or arrangement of promoters and terminators is envisaged. In certain embodiments, none of the expression vectors comprises a psiM gene.
[0293] In some embodiments, the promoter is selected from the group consisting of G6 mutant T7, H9 mutant T7, H10 mutant T7, C4 mutant T7, consensus T7, Lac, Lac UV5, tac, trc, GAP, and xylA promoter. [0294] In certain embodiments, the expression vector comprises the nucleic acid sequence of SEQ ID NO: 19 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments, the expression vector is pETM6-C4-psiDK or a vector having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.
Kits
[0295] Provided is a transfection kit comprising an expression vector as described herein. Such a kit may comprise a carrying means being compartmentalized to receive in close confinement one or more container means such as, e.g., vials or test tubes. Each of such container means comprises components or a mixture of components needed to perform a transfection. Such kits may include, for example, one or more components selected from vectors, cells, reagents, lipid- aggregate forming compounds, transfection enhancers, or biologically active molecules.
EXAMPLES
[0296] The following examples describe various compositions and methods for genetic modification of cells to aid in the production of psilocybin, according to the general inventive concepts.
Example 1: Norbaeocystin production in E. coli.
Materials and Methods
Bacterial strains, vectors, and media
[0297] All plasmids were propagated with E. coli DH5α , while all chemical production experiments used E. coli BL21 Star™ (DE3) as the production host. Unless otherwise noted, Andrew’s Magic Media (AMM) (He, W. et al. Production of chondroitin in metabolically engineered E. coli. Metab. Eng. 27, 92-100 (2015)) was used for both overnight growth and production media, while Luria Broth (LB) was used for plasmid propagation during cloning. The antibiotic ampicillin (80 pg/mL), was used to select for all pET -based vectors. A description of all plasmids and strains used in this study can be found in Table 1.
Table 1. Plasmid and Strain List
Figure imgf000048_0001
Figure imgf000049_0001
[0298] Plasmid and pooled library construction and screening: Plasmids containing the norbaeocystin production pathway were constructed using the previously reported pETM6- SDM2x plasmid backbone (Adams, A. M. et al. In vivo production of psilocybin in E. coli. Metab. Eng. 56, 111-119 (2019)). Single gene constructs were assembled using traditional restriction ligation cloning using Ndel and Xho I . All multigene plasmids were constructed in either operon or psuedooperon configuration using a modified version of the previously published ePathBrick (isocaudomer-based) methods while all pooled promoter libraries were constructed using standard ePathOptimize methods (Jones, J. A. et al. ePathOptimize: a combinatorial approach for transcriptional balancing of metabolic pathways. Nat. Publ. Gr. (2015) doi:10.1038/srepll301; Xu, P., Vansiri, A., Bhan, N. & Koffas, M. A. G. ePathBrick: A synthetic biology platform for engineering metabolic pathways in E. coli. Biol 1, 256-266 (2012)).
[0299] The transcriptional library promoters included T7 mutant promoters G6, H9, H10, and C4, along with the consensus (T7) promoter and were screened in 48-well plate format after transformation in the commercially available production host strain BL21 Star™ (DE3). Upon identification of the top seven mutants from the promoter library, the strains were rescreened to confirm high production levels prior to plasmid isolation and transformation into E. coli DH5oc for permanent storage. The plasmid DNA was then isolated from the DH5oc strain for promoter sequencing and retransformed into BL21 Star™ (DE3) for a final round of screening.
[0300] Standard screening conditions : Standard screening was performed using 48-well plates with a rectangular cross section and a 2 mL working volume at 37 °C. Serine (1 g/L), 4- hydroxyindole (350 mg/L), and ampicillin (80 pg/mL) were supplemented in AMM-No MOPS for all experiments, unless otherwise noted. Overnight cultures were grown for 12-16 h at 37 °C in a shaker incubator (225 rpm) in the same media that was used for final production. Induction with 1 mM IPTG occurred 4 hours after inoculation. Samples were taken at 24 and 48 hours, unless otherwise noted, and subjected to HPLC and LC-MS analysis.
[0301] E. coli Broth Preparation for HTR Studies: For use in the animal studies, control, psilocybin containing, and norbaeocystin containing broths were produced from fed batch bioreactor fermentations of Psilol6 (no 4-hydroxyindole supplement), Psilol6 (4-hydroxyindole supplement), and Norl (4-hydroxyindole supplement), respectively, using the conditions specified previously for Psilol6 and here for norbaeocystin. After the fermentation was concluded, the broth was centrifuged (5000 x g, 30 min) and filtered using a 0.2 pm bottle top filter, prior to administration to animals (described below). Filtered broth samples were stored at room temperature up to two months between production and use with negligible degradation observed. Metabolite concentrations in the broth were periodically quantified using HPFC analysis.
[0302] Representative HPFC chromatograms for the negative control, norbaeocystin, and psilocybin containing broth are shown in FIG. 16. The psilocybin containing broths also contained trace levels of norbaeocystin (<20 mg/F) and aeruginascin (<1 mg/F), low levels of baeocystin (approx. 150 mg/F), and high levels of psilocybin (approx. 1 g/F). The norbaeocystin containing broths had high levels of norbaeocystin (approx. 1.5 g/F) with no baeocystin, psilocybin, or aeruginascin due to the lack of the methyltransferase responsible for the synthesis of the latter metabolites. The control broth contained none of the aforementioned metabolites. Results
[0303] Prior to determining if an entourage effect may be present, we first developed a novel process for synthesizing pharmacologically relevant amounts of norbaeocystin, as existing synthetic methods were insufficient and resource intensive. Norbaeocystin production in E. coli was originally reported as a low-level accumulation of an undesired intermediate product in the psilocybin production pathway using the optimized psilocybin production strain, Psilol6 (Adams, A. M. et al. In vivo production of psilocybin in E. coli. Metab. Eng. 56, 111-119 (2019)). After reconstructing a norbaeocystin-specific production strain, containing PsiD and PsiK and controlled by the consensus T7 promoter, low-level accumulation of norbaeocystin was observed (29.6 ± 5.2 mg/L) under standard fermentation conditions (FIG. 2B).
[0304] Once a viable biosynthesis pathway in E. coli was identified, norbaeocystin production was optimized to generate sufficient product for animal behavioral studies. To accomplish this, a transcriptional library comprised of five isopropyl B-D-l-thogalactopyranoside (IPTG) -inducible T7 -lac promoter mutants of varied strength were used to construct two independent pooled libraries capable of norbaeocystin production: pETM6-xx5-psiDK (operon configuration, 5 possible variants) and pETM6-xx5-psiD-xx5-psiK (pseudooperon configuration, 25 possible variants). Nearly 50 operon library colonies and 150 pseudooperon library colonies were screened and their norbaeocystin production was quantified (FIG. 2A).
[0305] The sequencing results revealed an interesting trend with the top producing strain containing the strongest mutant promoter, C4, controlling the transcription of both psiD and psiK in operon configuration (FIG. 2B). The data also shows a mixed association between promoter strength and pathway performance, with two of the reduced strength promoters (G6, H10), as well as the strong T7 consensus promoter, leading to low levels of norbaeocystin production (FIG. 2B). This is in contrast with the similarly constructed psilocybin promoter library studies, which resulted in the best performance from the medium strength, H10, mutant promoter (FIG. 2B).
[0306] Similar to the development of a psilocybin overproducing strain, the norbaeocystin producing libraries showed a preference for the operon promoter configuration (FIG. 2A - black bars), resulting in the highest production strains with the lowest build-up of intermediate products when compared to their respective pseudo-operon libraries (FIG. 2A - gray bars). Upon final rescreening, the top norbaeocystin producer, Norl, shows a 7-fold improvement in norbaeocystin production over the original T7 consensus construct (FIG. 2B).
Example 2: Fermentation Optimization.
Materials and Methods
[0307] Scale-up Studies: All bioreactor studies were performed in an Eppendorf BioFlol20 bioreactor with 1.5 L working volume. The vessel was mixed using a direct drive shaft containing two Rushton-type impellers positioned equidistance under the liquid surface. The overnight cultures were grown for 9-12 h in a 250 mL baffled shake flask at 37 °C in a modified AMM supplemented with serine (0-5 g/L), thiamine (15 mg/L), and ampicillin (80 pg/mL), as noted. The bioreactor was then inoculated at 2% v/v to an initial ODeoo of approximately 0.05- 0.10 and induced 4 hours post-inoculation using 1 mM IPTG. Temperature, pH, and dissolved oxygen (DO) were held constant at 37°C, 6.5, and 30%, respectively. pH, DO, and foam were maintained automatically by the addition of 10 M NaOH, an agitation cascade (300-1000 rpm), and addition of Antifoam 204. Full oxygen saturation was defined under the conditions of 37 °C, 300 rpm agitation, pH 7.0, and 2 v/v per minute of standard air. The zero-oxygen set point was calibrated using a nitrogen gas flush. Samples were collected periodically for measurement of Oϋόoo and metabolite concentrations. Once the initial 20 g/L of glucose was exhausted, as determined by HPLC, a separate feed stream of 500 g/L glucose and 90 g/L (NH4)2HP04 was set to a flow rate ranging from 2.0 to 4.0 mL/L/hr. Once the initial 150 mg/L of 4-hydroxyindole was consumed and build-up of the key intermediate, 4-hydroxytryptophan, was low (approx. 12 hours), 4-hydroxyindole (40 mg/mL in EtOH) was periodically adjusted from 0 up to 47 mg/L/hr using an external syringe pump. The concentration of norbaeocystin, all pathway intermediates, and glucose were quickly analyzed via HPLC after each sample was taken with an approximate 45-minute delay. This allowed for modifications to be made to the reaction vessel in near realtime to feed rates for glucose and 4-hydroxyindole.
[0308] Analytical Methods: Metabolite analysis was performed on a Thermo Scientific Ultimate 3000 High-Performance Liquid Chromatography (HPLC) system equipped with Diode Array Detector (DAD), Refractive Index Detector (RID), and Thermo Scientific ISQ™ EC single quadrupole mass spectrometer (MS). Samples were prepared for HPLC and LC-MS analysis by using a 3: 1 ratio of MilliQ water to sample broth, vortexed briefly, and then centrifuged at 15,000 x g for 5 min. A volume of 2 pL of the resulting supernatant was then injected for HPLC and LC-MS analysis. Authentic Standards were purchased for psilocybin (Cerilliant). Norbaeocystin was quantified using a standard purified and characterized as described below.
[0309] Quantification of aromatic metabolites was performed using absorbance at 280 nm from the DAD and the metabolites were separated using an Agilent Zorbax Eclipse XDB-C18 analytical column (3.0 mm x 250 mm, 5 pm) with mobile phases of water (A) and acetonitrile (B) both containing 0.1% formic acid at a rate of 1 mL/min: 0 min, 5% B; 0.43 min, 5% B; 5.15 min, 19% B; 6.44 min, 100% B; 7.73 min, 100% B; 7.73 min, 5% B; 9.87 min, 5% B. This method resulted in the following observed retention times as verified by analytical standards (when commercially available) and MS analysis (as described below): 4-hydroxyindole (6.6 min), 4-hydroxytryptophan (3.4 min), 4-hydroxytryptamine (3.2 min), norbaeocystin (1.6 min), baeocystin (1.9 min) and psilocybin (2.2 min).
[0310] Liquid Chromatography Mass Spectrometry (LC-MS) data was collected where the full MS scan was used to provide an extracted ion chromatogram (EIC) of our compounds of interest. Analytes were measured in positive ion mode at the flow rate, solvent gradient, and column conditions described above. The instrument was equipped with a heated electrospray ionization (HESI) source and supplied > 99% purity nitrogen from a Peak Scientific Genius XE 35 laboratory nitrogen generator. The source and detector conditions were as follows: sheath gas pressure of 80.0 psig, auxiliary gas pressure of 9.7 psig, sweep gas pressure of 0.5 psig, foreline vacuum pump pressure of 1.55 Torr, vaporizer temperature of 500 °C, ion transfer tube temperature of 300 °C, source voltage of 3049 V, and source current of 15.90 mA.
[0311] A Bio-Rad Aminex HPX-87H column coupled with a RI detector was used for quantification of sugars and organic acids as described in Adams, A. M. et al. In vivo production of psilocybin in E. coli. Metab. Eng. 56, 111-119 (2019). All error is reported as the group mean ± standard error of the mean. [0312] Norbaeocystin Purification: Approximately 1.6 g/L norbaeocystin in 500 mL of E. coli broth was centrifuged (5,000 x g, 30 minutes), and filtered through a 0.2 pm bottle top filter to remove cells and extracellular particulates. The broth was then dried under a vacuum in a round bottom flask. The dried broth was continually mixed with 160 mL of hot (50 °C) ethanol for 30 minutes. The mixture was then filtered through grade 615 filter paper in a Buchner funnel. The filtrate was collected and analyzed by HPLC to confirm a lack of norbaeocystin. The filter cake was then collected and resuspended in 50 mL of water, resulting in some insoluble product that was discarded. The resuspension was quantified by HPLC, as described above, and a concentration of approximately 11.5 g/L was found, resulting in a -70% yield.
[0313] This norbaeocystin concentrate was then purified by preparative HPLC using an Agilent Polaris C18-A column (250 mm x 21.2 mm, 5 pm) with mobile phases of water (A) and acetonitrile (B), both containing 0.1% formic acid at a flow rate of 10 mL/min: 0 min, 5% B; 0.9 min, 5% B; 10 min, 40% B; 11.25 min, 100% B; 14.5 min, 100% B; 14.5 min, 5% B; 17 min,
5% B. Injections of 500 μL were used, resulting in a retention time of approximately 9.2 minutes for norbaeocystin. The fraction collection was triggered by peak height in the UV absorbance spectrum at 280 nm. The collected fractions were then pooled and dried under vacuum prior to analysis by LC-MS, 1 H NMR (FIG. 14), and 13C NMR (FIG. 15). The sample accounted for -94% of the 1 H NMR peak area and -98% of the HPLC A280 peak area observed in the respective spectrums. 1H NMR (400 MHz, D2O) d (ppm) 7.18 (1H, d, J = 8.2 Hz), 7.13 (IH, s), 7.08 (1H, m), 6.94 (IH, d, J = 8.2 Hz), 3.26 (2H, t, J = 7.2 Hz), 3.18 (2H, t, J = 7.1 Hz); 13C NMR (125 MHz, D20) d (ppm) 145.74, 138.65, 124.25, 122.51, 118.55, 108.86, 108.63, 107.84, 40.85, 23.89; LC-MS (ESI) calculated for C10H14N2O4P+ 257.069 [M + H]+, found 257.1.
Results
[0314] After selection and identification of Norl was complete, variations in media composition were tested in a shake flask study to maximize norbaeocystin production (FIG. 6). The results from the media study were used to inform the selection of AMM-no MOPS (AMM without the MOPS/Tricine buffer component) for 1.5 L bioreactor studies using Norl. These scaled up studies incorporated automated control of pH, foam, dissolved oxygen, and temperature, mimicking industrial conditions, while also enhancing cell growth and norbaeocystin production using a fed-batch process for glucose, ammonium phosphate dibasic, and the substrate 4- hydroxyindole.
[0315] In an attempt to further maximize norbaeocystin production in the bioreactor, the amino acid serine was identified as a necessary and potentially limiting substrate. Serine plays an important role in metabolism as it is directly utilized for protein synthesis, but it also serves as the precursor molecule for several other amino acids including tryptophan, cystine, and glycine. Serine is one of the major substrates for the norbaeocystin biosynthesis pathway, condensing with 4-hydroxyindole to form 4-hydroxytryptophan through the promiscuous activity of the native tryptophan synthase. This serves as the first concerted step towards norbaeocystin biosynthesis.
[0316] Due to the high demands on serine for both native metabolism and norbaeocystin biosynthesis, it was unclear if wild-type metabolism could support the necessary serine biosynthesis demand. To this end, various levels of serine were supplemented in the media and investigated at scale as a means to further evaluate this potential pathway bottleneck. Interestingly, no statistically significant difference (p > 0.17) was observed between differing serine supplementation levels, with an average of over 1.58 ± 0.08 g/L of norbaeocystin being produced across all serine supplementation conditions (FIG. 2C). Furthermore, 4- hydroxytryptophan, the pathway intermediate resulting from the promiscuous activity of tryptophan synthase, was observed to build up throughout the fermentation when 4- hydroxyindole was provided in excess (FIGs. 8-10). This indicates that the norbaeocystin pathway serves as a sufficient serine sink to trigger natural serine biosynthesis and the resulting native flux towards serine exceeds the needs of both metabolism and the optimized exogenous pathway.
[0317] Comparing the metabolite profiles under differing serine supplementation levels, fermentation with enhanced serine supplementation tended to consume the initial 4- hydroxyindole substrate more quickly, building up a temporary excess of the 4- hydroxytryptophan intermediate (FIGs. 8-10). A representative temporal metabolite profile for a fed batch bioreactor trial is shown (FIG. 2D). Across all 6 bioreactor trials, an average productivity of 19.3 ± 2.1 mg/L/hr over the course of the fermentation run at a final substrate yield of 57.9 ± 1.8% mol norbaeocystin/mol 4-hydroxyindole was observed. These results represent the highest reported production metrics to date for norbaeocystin from any recombinant organism.
Example 3: Rodent Behavior-Head Twitch Response Study.
Materials and Methods
[0318] Rodent Head Twitch Response Studies: A total of 36 adult (PND 90-120) male Long Evans rats (Charles River Laboratories, Raleigh, NC) were used in these experiments. All animals were individually housed in standard rat cages throughout testing to protect cranial implants and kept on a 12 hr: 12 hr light/dark cycle. Food and water were available ad libitum throughout all experiments. Animals were divided into one of 3 treatment groups (norbaeocystin, psilocybin, and norbaeocystin+psilocybin) and received only vehicle and the drug(s) assigned to their group. All procedures and protocols were conducted in accordance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals and the Animal Welfare Act and were approved by Miami University’s Institutional Animal Care and Use Committee.
Surgery
[0319] Prior to beginning surgery, animals were given 48 hr access to acetaminophen via drinking water (37 mg/ml, Children’s Dye Free Pain and Fever Reliever, Walgreen Co.) for preventive pain relief. All surgeries were then conducted on isoflurane anesthetized animals (5% for induction and 3% for maintenance). Once deeply sedated, animals were secured in a stereotaxic frame and the surgical site sterilized. The skull was then exposed, and four holes were drilled into the periphery of the skull in a rectangular formation and 4 stainless steel screws (1/8” 1-72) were inserted into each of the holes. A single neodymium magnet (MGLN10-5-5, MiSUMi Corp.) was then placed on top of the screws. Both the screws and the magnet were then covered in dental acrylic to secure them to the skull. Once complete, animals were returned to their home cage and closely monitored for the following 3 days. During this period, they were provided acetaminophen (37 mg/ml), as described above. Animals were given a one-week recovery following surgery before beginning behavioral testing. Drugs and Drug Administration
[0320] Filtered E. coli broth containing each compound (or control broth) was administered to rats via intraoral gavage. Animals received either vehicle or doses of their associated drug condition as follows: 0, 0.1, 0.2, 1, 2 mg/kg psilocybin; 0, 0.13, 0.25, 1.26, 2.52 mg/kg norbaeocystin; 0+0, 0.1+0.13, 0.2+0.25, 1+1.26, 2+2.52 mg/kg psilocybin+norbaeocystin. Animals received no more than 3 treatments, and the order of drug presentation was randomized across subjects, with one week separating each drug exposure to prevent the development of tolerance or sensitization. Total volume of each gavage ranged from 0.03-3.5 mL.
Head Twitch Response Testing
[0321] Head movements were recorded using a magnetometer-based approach described previously (Halberstadt, A. L. & Geyer, M. A. Characterization of the head-twitch response induced by hallucinogens in mice: detection of the behavior based on the dynamics of head movement. Psychopharmacology (Berl). 227, 727-39 (2013)). This magnetometer-based approach is considerably more sensitive and easier to quantify than hand-coding high-speed video recordings. After surgical implantation of a skull-mounted magnet and recovery (see above), rats were administered drug or vehicle and placed in a large polycarbonate tube (~56 cm diameter, ~30 cm height) surrounded by -2000 turns of #30 enameled magnet wire. Changes in the position of the animal caused changes in the voltage across the wire, which was minimally amplified with a gain of 2, bandpass filtered with a cutoff frequency of 10 kHz to exclude noise, recorded at 1000 S/s, and analyzed as detailed below. HTRs were observed for 60 minutes, beginning immediately after drug delivery.
Data Analysis
[0322] Continuous recordings of HTRs (voltage) were exported to Offline Sorter (v4.5, Plexon Inc, Dallas, TX) for determination of the time of each HTR based on waveform characteristics. Specifically, head twitches were identified from voltage recordings by a single observer, blinded to subject condition, by the presence of 1) amplitude exceeds background noise; 2) fundamental frequency of 20-40 Hz; 3) more than 2 bipolar peaks; and 4) duration <120 ms. The total number of HTRs was compared across doses within each drug condition using 2-way Poisson regression (proc glimmix, SAS v9.4), followed by post hoc tests when appropriate, corrected for multiple comparisons using the FDR method (Benjamini, Y. & Hochberg, Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B 57, 289-300 (1995)). Additionally, Poisson regression was also used to compare the change in HTR number compared to the average of our control animals, with post hoc Tukey tests conducted as appropriate. Body weight was analyzed independently for each drug condition using 2-way repeated measures mixed models, including factors for group and time since gavage (days). Locomotor data were analyzed using 1-way mixed models, including analyses for the total distance travelled and total number of rearing events, with separate analyses for habituation and post-gavage periods, as well as the percent change from the habituation period. Group means ± standard error of the mean are presented in all figures.
Results
[0323] Having developed a novel method to produce gram-scale quantities of norbaeocystin, we set out to test the effectiveness of psilocybin and norbaeocystin, individually and in combination, at eliciting head twitch responses (HTRs), also referred to as wet dogs shakes. HTRs are high- frequency paroxysmal head and body rotations that occur in rodents after 5-HT2A receptor activation. These behaviors are widely used as an indicator of hallucinogenic effects and they are one of the only behaviors that can reliably distinguish hallucinogenic from non-hallucinogenic 5- HT2A agonists. HTRs were assessed for 60 minutes, beginning immediately following gavage of filtered cell culture media containing each substance (or media from control E. coli strains).
[0324] Across this study, we found a significant interaction between drug and dose [2-way Poisson regression F(8,75)=5.33, p<0.001]. When administered alone, psilocybin significantly increased HTRs in a dose-dependent fashion (FIG. 3B), with significant increases in HTRs evident at intermediate dosages (0.2 and 1 mg/kg; p < 0.05 or p < 0.01, depending on the comparison). Additionally, although administration of norbaeocystin alone did significantly affect HTRs (FIG. 3A), instead of increasing HTRs as expected, it decreased HTRs at the lowest dosage tested (0.13 mg/kg) compared to control and other doses (all p < 0.05). Lastly, combining psilocybin with norbaeocystin, at roughly equal dosages, also resulted in increased HTRs in a dose-dependent fashion (FIG. 3C), especially at the intermediate doses (0.2 and 1 mg/kg psilocybin). The dramatic decrease in HTR at the highest dose of psilocybin (alone and combined with norbaeocystin) suggest that locomotor impairments may have resulted; however, a separate assessment found no major effects on locomotion for any dose of any drug or combination (FIG. 12). Together, these data indicate that E. coli-synthesized psilocybin alone, and in combination with E. coli-synthesized norbaeocystin, is effective at eliciting robust behavioral responses in rats when directly gavaged in its filtered broth vehicle, but norbaeocystin alone does not increase HTRs. However, the individual dose response curves for these compounds do not address the existence of an entourage effect between psilocybin and its minor metabolites.
[0325] To address this question, we calculated the change in the number of HTRs elicited by each drug/dose from the average of control animals (dose - control), and again found a significant interaction between drug and dose [2-way Poisson regression, F(6,38) = 2.71, p = 0.027]. Comparisons within each dosage allowed us to determine that combining these two drugs caused even greater behavioral responses than psilocybin alone (FIG. 4). This was most apparent at the moderately high dosage tested (1.0 mg/kg psilocybin + 1.26 mg/kg norbaeocystin), where the combination of norbaeocystin and psilocybin elicited significantly more head twitches than psilocybin alone at the same dosage (p = 0.05). Additionally, this same trend existed in all other dosages tested, although these effect sizes were proportionately smaller and did not reach significance. These data suggested that a synergistic interaction between psilocybin and norbaeocystin likely exists, providing strong support for the existence of an entourage effect between these two compounds in Psilocybe and other psilocybin-producing mushrooms.
Example 4: Rodent Behavior-Head Twitch Response Study.
Materials and Methods
Rodent Head Twitch Response Studies'. A total of 40 adult (PND 90-120) male Long Evans rats (Charles River Laboratories, Raleigh, NC) were used in these experiments. All animals were individually housed in standard rat cages throughout testing to protect cranial implants, and kept on a 12 hr: 12 hr light/dark cycle. Food and water were available ad libitum throughout all experiments. Animals were divided into one of 3 treatment groups (norbaeocystin, psilocybin, and norbaeocystin+psilocybin) and received only vehicle and the drug(s) assigned to their group. All procedures and protocols were conducted in accordance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals and the Animal Welfare Act, and were approved by Miami University’s Institutional Animal Care and Use Committee.
Surgery
[0326] Prior to beginning surgery, animals were given 48 hr access to acetaminophen via drinking water (37 mg/ml, Children’s Dye Free Pain and Fever Reliever, Walgreen Co.) for preventive pain relief. All surgeries were then conducted on isoflurane anesthetized animals (5% for induction and 3% for maintenance). Once deeply sedated, animals were secured in a stereotaxic frame and the surgical site sterilized. The skull was then exposed, and four holes were drilled into the periphery of the skull in a rectangular formation and 4 stainless steel screws (1/8” 1-72) were inserted into each of the holes. A single neodymium magnet (MGLN10-5-5, MiSUMi Corp.) was then placed on top of the screws. Both the screws and the magnet were then covered in dental acrylic to secure them to the skull. Once complete, animals were returned to their home cage and closely monitored for the following 3 days. During this period, they were provided acetaminophen (37 mg/ml), as described above. Animals were given one week following surgery before beginning behavioral testing.
Psilocybin and Norbaeocystin Production
[0327] Psilocybin and norbaeocystin were produced using a genetically optimized recombinant strain of Escherichia coli capable of conversion of 4-hydroxyindole into each target product at high titers, productivities, and yields in a scalable bioprocess (Adams et al, 2019, process also reported in patent application PCT/US2020/051543). After production, the broth was centrifuged (5000 x g, 30 min) and filtered using a 0.2 um bottle top filter, prior to administration to animals (described below). Filtered broth samples were stored at room temperature up to two months between production and use with negligible degradation observed. Metabolite concentrations in the broth were quantified using high performance liquid chromatography as previously reported (Adams et al, 2019). Drugs and Drug Administration
[0328] Filtered E. coli broth containing each compound (or control broth) was administered to rats via intraoral gavage. Animals received either vehicle or doses of their associated drug condition as follows: 0, 0.1, 0.2, 1, 2 mg/kg psilocybin; 0, 0.13, 0.25, 1.26, 2.52 mg/kg norbaeocystin; 0+0, 0.1+0.13, 0.2+0.25, 1+1.26, 2+2.52 mg/kg psilocybin+norbaeocystin. Animals received no more than 3 treatments, and the order of drug presentation was randomized across subjects, with one week separating each drug exposure to prevent the development of tolerance or sensitization (Gewirtz & Marek, 2000). Total volume of each gavage ranged from 0.03-3.5 ml.
Head Twitch Response Testing
[0329] Head movements were recorded using a magnetometer-based approach. After surgical implantation of a skull-mounted magnet and recovery (see above), rats were administered drug or vehicle and placed in a large polycarbonate tube (~56 cm diameter, ~30 cm height) surrounded by -150 turns of #30 enameled magnet wire. Changes in the position of the animal caused changes in the voltage across the wire, which was recorded at 1000 S/s and analyzed as detailed below. This magnetometer-based approach is considerably more sensitive and easier to quantify than hand-coding of high-speed video recordings (Halberstadt & Geyer, 2013). HTRs were observed for 60 minutes, beginning immediately after drug delivery.
Locomotor Testing
[0330] Locomotor behaviors were assessed using Accuscan Open Field chambers. Each chamber consists of 16 infrared beams arranged in two horizontal rows. Beams broken by the animal are interpreted by the software to determine the location of the rat, including rearing behavior. Animals were habituated to the chamber for 15 minutes, then gavaged and immediately placed back into the chamber for an additional 60 minutes. Beam breaks were recorded for the entire duration (including habituation). Results
[0331] Continuous recordings of HTRs (voltage) were exported to Offline Sorter (v4.5, Plexon Inc, Dallas, TX) for determination of the time of each HTR based on waveform characteristics. Specifically, head twitches were identified from voltage recordings by a single observer, blinded to subject condition, by the presence of 1) amplitude exceeds background noise; 2) fundamental frequency of 20-40 Hz; 3) more than 2 bipolar peaks; and 4) duration <120 ms. Because the nonnormal nature of the data (count with low means) violated the assumptions of ANOVA, the total number of HTRs was compared across doses within each drug condition using 1-way poisson mixed models (proc glimmix, SAS v9.4), followed by post hoc tests when appropriate. Additionally, mixed models compared the effects of each drug within each dosage. Locomotor data were analyzed using 1-way ANOVA, including analyses for the total distance travelled and total number of rearing events, with separate analyses for habituation and after gavage. Group means ± standard error of the mean are presented in all figures.
Example 5: Antidepressant Efficacy Study.
Materials and Methods
[0332] Rodent Subjects: A total of 24 adult (PND 90-120) male Long Evans rats (Charles River Laboratories, Raleigh, NC) were used in these experiments. Except where noted in the chronic variable stress paradigm, all animals were dual-housed in standard rat cages on a 12 hr: 12 hr light/dark cycle, and food and water were available ad libitum. Animals were divided into one of 4 treatment groups (vehicle, fluoxetine, psilocybin, and psilocybin+norbaeocystin). All procedures and protocols were conducted in accordance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals and the Animal Welfare Act, and were approved by Miami University’s Institutional Animal Care and Use Committee.
Chronic Variable Stress Paradigm
[0333] The chronic stress regimen is one of the most reliable methods for establishing a rodent model of depression / HPA dysfunction. This regimen involves exposure of rodents to a series of randomly-alternating stressors (administered twice daily) over a period of 15 days. In addition, animals receive an extra overnight stressor on two out of every three days. This random-stress procedure has been used previously and has been shown to produce reliable changes in basal stress-axis function (Herman et ah, 1995; Ostrander et ah, 2006; Ziegler et ah, 1999). The stressors employed in our studies included the following:
1. Physical Restraint: Animals were placed in plastic restraint tubes for up to 30 min. Restraint tubes are Plexiglas restrainers, approximately 21 cm in length with an approximately 5 cm opening, equipped with ventilation holes.
2. Cold Incubator: Animals in their home cage were placed in a cold-chamber incubator (4C) for 1 hour.
3. Crowding: Up to 6 animals were placed in a clean single standard rat cage overnight, taking weight into consideration. Density was such that they would be considered crowded in the cage, yet still had access to food and water.
4. Isolation: Animals were single-housed overnight in a clean cage.
5. Rotation: Animals in their home cages were placed on an orbital shaker and rotated for 1 hr. (Approx. 100 rpm in the shaker display).
6. Cage tilt with wet bedding: Animals’ home cage was elevated on one end to produce up to 30° incline. Additionally, the bedding of an animal's home cage was moistened with water without producing standing water. Animals were housed in this condition for 2 hours.
7. Strobe Light: Animals were placed in a darkened room with a stroboscopic light (up to 300 flashes/min) for up to 2 hours.
[0334] The stressors were given using a set-schedule model. Individual timing of the stressors was selected based on the stressor duration required to provoke an adrenocortical response, without endangering the animals’ well-being. For example, 1 hr exposure to cold in a cold room is required for an optimal pituitary- adrenal response, whereas only 30 min are needed for restraint stress. At least 2 hours to recover between the AM and PM stressor were provided each day. Drug administration occurred 1-week after the final stressor. Drugs and Drug Administration
[0335] Drugs were synthesized as described in the Head Twitch Response methods, but extracted, purified, and suspended in a water vehicle (for delivery to animals via gavage). Cell broth containing psilocybin was centrifuged and filtered to remove cell biomass, followed by drying under reduced pressure to form a wet sticky solid. This solid was then extracted with methanol, and the liquid fraction is dried under reduced pressure. The resulting solid is dissolved in water and purified using reverse phase preparative HPLC. The collected fractions are concentrated under reduced pressure until spontaneous crystallization, resulting in a white solid. Cell broth containing norbaeocystin was centrifuged and filtered to remove cell biomass, followed by drying under reduced pressure to form a wet sticky solid. This solid was then washed with hot ethanol, and the insoluble fraction was dissolved in water and purified using reverse phase preparative HPLC. The collected fractions were concentrated under reduced pressure, resulting in a light brown solid. All drugs were administered via gavage in a water vehicle (1 ml/kg). Psilocybin and norbaeocystin were administered at a dosage of lmg/kg, with the combined dosage of psilocybin and norbaeocystin including lmg/kg of each compound. Fluoxetine was provided via intragastric infusion in a water vehicle at a dose of 15 mg/kg, and was administered three times prior to behavioral testing: 23.5 hrs, 5hrs, and lhr. This schedule of administration has been previously shown to recapitulate the behavioral and neurobiologic al changes that occur with chronic exposure, but in a shorter timeframe (Slattery & Cryan, 2012).
Sucrose Preference Test
[0336] Beginning at the start of their dark cycle, animals were presented with two bottles side- by-side in their home cage, one containing 2% sucrose and one containing standard drinking water. Animal’s preference for sucrose (% of total fluid intake) was examined after 12 hours.
Forced Swim Test
[0337] Methods used here were previously published (Slattery & Cryan, 2012). Animals were brought into the room and allowed to habituate for 5 minutes. After habituation, the animal was placed into an 18 gallon plastic tube (55.6 cm diameter, 45.4 cm height), filled two thirds full with water (~24C). The animal was allowed to swim for 5 minutes, and behavior recorded for later analysis. After 5 minutes, the animal was dried in a fresh clean towel and placed into a warming cage (on a heating pad) for 10 minutes before returning to their homecage and the vivarium. Video recordings were hand-coded by two observers blind to the animal’s treatment group. The duration of the animal’s immobility (time spent floating without climbing, treading water, or diving) was coded by each observer, with final concurrence rates >95% across observers.
Data Analysis
[0338] All behavioral data were analyzed by one-way (group) Analysis of Variance, followed by post hoc Tukey Tests. Group means ± standard error of the mean are presented in all figures.
Results
Head Twitch Responses
[0339] Head Twitch Responses (HTRs) were assessed for 60 minutes, beginning immediately following gavage of filtered cell culture media containing each substance (or media from control E. coli strains). Across this study, we found a significant interaction between drug and dose [F(8,75)=5.33, p<0.001]. When administered alone, psilocybin significantly increased HTRs in a dose-dependent fashion (FIG. 3A), with significant increases in HTRs evident at intermediate dosages (0.2 and 1 mg/kg; p < 0.05 or p < 0.01, depending on the comparison). Additionally, administration of norbaeocystin alone did significantly affect HTRs (FIG. 3B). Fastly, combining psilocybin with norbaeocystin, at roughly equal dosages, also resulted in increased HTRs in a dose-dependent fashion (FIG. 3C), especially at the intermediate doses (0.2 and 1 mg/kg psilocybin+norbaeocystin). The dramatic decrease in HTR at the highest dose of psilocybin (alone and combined with norbaeocystin) is likely a result of 5-HT2C receptor activation, which occurs at higher dosages of psilocybin and inhibits neurons expressing 5-HT1A receptors, thereby blocking head twitch responses. Together, these data indicate that E. coli- synthesized psilocybin alone, and in combination with E. coli- synthesized norbaeocystin, is effective at eliciting robust behavioral responses in rats when directly gavaged in its filtered broth vehicle, but norbaeocystin alone does not increase HTRs. However, the individual dose response curves for these compounds do not address the existence of an entourage effect between psilocybin and its minor metabolites.
[0340] To address this question, we calculated the change in the number of HTRs elicited by each drug/dose from the average of control animals (dose - control), and again found a significant interaction between drug and dose [F(6,38) = 2.71, p = 0.027]. Comparisons within each dosage allowed us to determine that combining these two drugs caused even greater behavioral responses than psilocybin alone (FIG. 4). This was most apparent at the moderately high dosage tested (1.0 mg/kg psilocybin + 1.26 mg/kg norbaeocystin), where the combination of norbaeocystin and psilocybin elicited significantly more head twitches than psilocybin alone at the same dosage (p = 0.05). Additionally, this same trend existed in all other dosages tested, although these effect sizes were proportionately smaller and did not reach significance. These data suggested that a synergistic interaction between psilocybin and norbaeocystin exists.
Locomotor Behavior
[0341] Animals were habituated to the locomotor chamber for 15 minutes prior to gavage, and then locomotion assessed for 60 minutes after gavage. There were no significant differences in the distance traveled during habituation (prior to gavage). Thus, habituation data were excluded from our analyses. When administered alone, neither norbaeocystin (FIG. 12A) nor psilocybin (FIG. 12B) affected locomotion at any dosage studied. However, when combined, 0.2 mg/kg norbaeocystin + psilocybin did increase locomotion [F(2,17)=3.63,p=0.049] compared to control and 2 mg/kg (both p=0.03) (FIG. 12C).
[0342] In addition to distance traveled, we also investigated whether these treatments affect rearing, a common stereotypical and/or exploratory behavior exhibited by rodents. Similar to distance traveled, no dosage of norbaeocystin or psilocybin alone caused statistically significant effects on the duration of rearing. However, the combination of norbaeocystin and psilocybin did affect the duration of rearing [F(2,17)=3.87, p=0.041], with post hoc analyses showing that 2 mg/kg reduced rearing duration compared to 0.2 mg/kg (p=0.013) (FIGs. 17A-17C). Sucrose Preference Test
[0343] The sucrose preference test is a commonly used metric of antidepressant efficacy. Animals in a depressive-like state demonstrate reduced preference for sweetened solutions, indicative of the anhedonia commonly experienced by individuals suffering from depression. After completing our chronic variable stress paradigm, animals were given overnight access (12 hr) to a sweetened water solution (2% sucrose), and preference for the sweetened solution assessed. All animals demonstrated suppressed preference, indicating anhedonia. Following treatment, overnight sucrose preference was again assessed (12 hr) and compared to pretreatment. We found that animals in the vehicle control group and fluoxetine groups showed no change in sucrose preference, but that animals in the psilocybin group demonstrated a significant increase in preference after treatment. Additionally, animals in the combined group (psilocybin+norbaeocystin) demonstrated the greatest increase in sucrose preference (FIG. 18).
Forced Swim
[0344] The forced swim test is one of the most commonly used metrics of antidepressant efficacy in rodents (Slattery & Cryan, 2012). After completing our chronic variable stress paradigm, control animals demonstrated high levels of immobility, indicating their “despair-like” state. This state was alleviated in animals who had been treated with either psilocybin or fluoxetine (a selective serotonin reuptake inhibitor). Animals receiving both psilocybin and norbaeocystin showed the lowest levels of immobility, demonstrating the greater effectiveness of the combination of these drugs (FIG. 19).
Conclusions
[0345] Our results demonstrated that psilocybin alone dose-dependently affects head twitch responses, but not locomotion, and also that its effectiveness can be enhanced by the addition of norbaeocystin, even though norbaeocystin alone does not affect either behavior. Specifically, psilocybin treatment alone resulted in the expected inverted-U shaped dose-response curve, with the highest dose showing no difference from control. However, when combined with norbaeocystin, the highest dosage of psilocybin increased head twitches compared to control. These results suggest that psilocybin and norbaeocystin may interact pharmacologically to enhance the overall response of the animal. Further, the comparatively minimal effect of these compounds on locomotor behaviors suggests that this interaction may not cause general disturbances in behavior.
[0346] In addition to the effectiveness of these compounds at eliciting head twitch responses, we also assessed their therapeutic potential via commonly used rodent behavioral paradigms (forced swim and sucrose preference). We found that the already strong therapeutic effect of psilocybin in both tests could be enhanced by combining it with norbaeocystin. These data support the above claim that psilocybin and norbaeocystin may interact on a pharmacological level, and extend the behavioral context into treatment-relevant domains. These effects were observed in both behavioral tasks, indicating strong divergent validity, while the effects of fluoxetine were restricted to only the forced swim test. This is in line with prior studies of fluoxetine, which have reported inconsistent and small effects (for example, see Kirsch et al., 2008; Alboni et al., 2017).
[0347] Combined, our data have three important implications. First, they imply that other tryptamines may supplement or augment the effectiveness of psilocybin. Across the moderate and high dosages tested (0.2-2 mg/kg), the addition of an equal amount of norbaeocystin enhanced animals’ response to psilocybin (head twitch and antidepressant) by approximately 50%. Thus, the addition of norbaeocystin may allow for a wider dose range of psilocybin to be used, enabling lower doses of psilocybin to have greater effects in vivo , while simultaneously broadening the dose response curve to allow animals to tolerate even higher doses of psilocybin, increasing the drug’s effectiveness. Our data support the idea that the addition of norbaeocystin allows for a wider range of psilocybin doses to be used clinically, facilitating more specific dosing (e.g. individualized medicine) and better therapeutic outcomes.
[0348] A second major implication of our results is that they demonstrate the pharmacological efficacy of E. coli- derived psilocybin. Synthetically produced psilocybin is known to elicit responses similar to those presented here, but synthetic psilocybin is expensive and time- consuming to produce. The E. coli- derived psilocybin used by this study can be rapidly produced in large amounts at pharmacologically relevant concentrations. Having verified the efficacy of E. coli- derived psilocybin, our results confirm that this method of production is a viable means of producing these chemicals and likely other pharmaceutically relevant molecules. Use of these production methods will improve access to these compounds and lower barriers to their use in a variety of contexts, including pre-clinical, clinical, and religious.
[0349] A third significant outcome of our results is that they demonstrate the relative safety of directly gavaging filtered E. coli broth as a drug vehicle. As far as applicant is aware, the use of filtered cell culture media as a drug vehicle has never before been published. As the use of recombinant microorganisms for chemical production becomes more common, the use of cell culture media as a drug vehicle could dramatically reduce the cost of drug production by cutting costly downstream purification steps. It would also reduce the time to market for such compounds and presents a low barrier to entry for screening of novel compounds in animal models.
Bibliography
1. Johnson, J. R. et al. Multicenter, Double-Blind, Randomized, Placebo-Controlled, Parallel-Group Study of the Efficacy, Safety, and Tolerability of THC:CBD Extract and THC Extract in Patients with Intractable Cancer-Related Pain. J. Pain Symptom Manage. 39(2), 167-179 (2010).
2. Russo, E. B. Taming THC: Potential cannabis synergy and phytocannabinoid-terpenoid entourage effects. British Journal of Pharmacology vol. 163 1344-1364 (2011).
3. Weston-Green, K. The United Chemicals of Cannabis: Beneficial Effects of Cannabis Phytochemicals on the Brain and Cognition, in Recent Advances in Cannabinoid Research (IntechOpen, 2019). doi: 10.5772/intechopen.79266.
4. Cogan, P. S. The ‘entourage effect’ or ‘hodge-podge hashish’: the questionable rebranding, marketing, and expectations of cannabis polypharmacy. Expert Rev. Clin. Pharmacol. 13, 835-845 (2020).
5. Santiago, M., Sachdev, S., Arnold, J. C., Mcgregor, I. S. & Connor, M. Absence of Entourage: Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Functional Activity of A9-THC at Human CB1 and CB2 Receptors. Cannabis Cannabinoid Res. 4, 165-176 (2019).
6. Sherwood, A. M. et al. Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin. /. Nat. Prod. 83(2), 461-467 (2020). Zhuk, O. et al. Research on Acute Toxicity and the Behavioral Effects of Methanolic Extract from Psilocybin Mushrooms and Psilocin in Mice. Toxins (Basel). 7, 1018-1029 (2015). Matsushima, Y., Shirota, O., Kikura-Hanajiri, R., Goda, Y. & Eguchi, F. Effects of Psilocybe argentipes on marble-burying behavior in mice. Biosci. Biotechnol. Biochem. 73, 1866-8 (2009). Adams, A. M. et al. In vivo production of psilocybin in E. coli. Metab. Eng. 56, 111-119 (2019). Milne, N. et al. Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives. Metab. Eng. 60, 25-36 (2020). Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic synthesis of psilocybin. Angew. Chemie Int. Ed. 56, 12352-12355 (2017). Leung, A. Y. & Paul, A. G. Baeocystin and norbaeocystin: New analogs of psilocybin from Psilocybe baeocystis. J. Pharm. Sci. 57, 1667-1671 (1968). Wurst, M., Kysilka, R. & Flieger, M. Psychoactive tryptamines from basidiomycetes. Folia Microbiologica vol. 47 3-27 (2002). Gartz, J. Extraction and analysis of indole derivatives from fungal biomass. J. Basic Microbiol. 34, 17-22 (1994). Halberstadt, A. L. & Geyer, M. A. Multiple receptors contribute to the behavioral effects of indoleamine hallucinogens. Neuropharmacology 61, 364-81 (2011). Halberstadt, A. L. & Geyer, M. A. Characterization of the head-twitch response induced by hallucinogens in mice: detection of the behavior based on the dynamics of head movement. Psychopharmacology (Berl). 227 , 727-39 (2013). Canal, C. E. & Morgan, D. Head-twitch response in rodents induced by the hallucinogen 2,5-dimethoxy-4-iodoamphetamine: a comprehensive history, a re-evaluation of mechanisms, and its utility as a model. Drug testing and analysis vol. 4 556-576 (2012). Gonzalez-Maeso, J. et al. Hallucinogens Recruit Specific Cortical 5-HT2A Receptor- Mediated Signaling Pathways to Affect Behavior. Neuron 53, 439-452 (2007). de la Fuente Revenga, M. et al. Fully automated head-twitch detection system for the study of 5-HT2A receptor pharmacology in vivo. Sci. Rep. 9, 1-14 (2019). Jones, J. A. et al. ePathOptimize: a combinatorial approach for transcriptional balancing of metabolic pathways. Nat. Publ. Gr. (2015) doi:10.1038/srepll301. Rossen, J. W. A., Tedim, A. P. & Murray, A. K. The Novel Coronavirus COVID-19 Outbreak: Global Implications for Antimicrobial Resistance. Front. Microbiol. \ www.frontiersin.org 1, 1020 (2020). Fitzpatrick, K. M., Drawve, G. & Harris, C. Facing new fears during the COVID-19 pandemic: The State of America’s mental health. J. Anxiety Disord. 75, 102291 (2020). Rudenstine, S. et al. Depression and Anxiety During the COVID-19 Pandemic in an Urban, Low-Income Public University Sample. J. Trauma. Stress 2020, jts.22600 (2020). Chen, Y., Zhou, H., Zhou, Y. & Zhou, F. Prevalence of self-reported depression and anxiety among pediatric medical staff members during the COVID-19 outbreak in Guiyang, China. Psychiatry Research vol. 288 113005 (2020). He, W. et al. Production of chondroitin in metabolically engineered E. coli. Metab. Eng. 27, 92-100 (2015). Xu, P., Vansiri, A., Bhan, N. & Koffas, M. A. G. ePathBrick: A synthetic biology platform for engineering metabolic pathways in E. coli. Biol 1, 256-266 (2012). Gewirtz, J. C. & Marek, G. J. Behavioral evidence for interactions between a hallucinogenic drug and group II metabotropic glutamate receptors. Neuropsychopharmacology 23, 569-576 (2000). Benjamini, Y. & Hochberg, Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. Ser. B 57, 289-300 (1995). Adams, A. M., Kaplan, N. A., Wei, Z., Brinton, J. D., Monnier, C. S., Enacopol, A. L., Ramelot, T. A., & Jones, J. A. (2019). In vivo production of psilocybin in E. coli. Metabolic Engineering, 56, 111-119. doi.org/10.1016/j.ymben.2019.09.009. Jones, J. A., Vernacchio, V. R., Lachance, D. M., Lebovich, M., Fu, L., Shirke, A. N., Schultz, V. L., Cress, B., Linhardt, R. J., & Koffas, M. A. G. (2015). ePathOptimize: a combinatorial approach for transcriptional balancing of metabolic pathways. Scientific Reports, 5(1), 11301. doi.org/10.1038/srepll301. Ben-Shabat, S., Fride, E., Sheskin, T., Tamiri, T., Rhee, M. H., Vogel, Z., Bisogno, T., De Petrocellis, L., Di Marzo, V., & Mechoulam, R. (1998). An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity. European Journal of Pharmacology , 353(1), 23-31. doi.org/10.1016/S0014- 2999(98)00392-6. Mahmood, Z. A. (2013). Bioactive alkaloids from fungi: Psilocybin. In K. G. Ramawat & J. M. Merillon (Eds.), Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes (pp. 523-552). Springer, doi.org/10.1007/978-3-642- 22144-6_19. Slattery, D. A., & Cryan, J. F. (2012). Using the rat forced swim test to assess antidepressant- like activity in rodents. Nature Protocols , 7(6), 1009-1014. doi . org / 10.1038/nprot .2012.044. Ostrander, M.M., Ulrich-Lai, Y., Choi, D.C., Richtand, N.M., Herman, J.P., 2006. Hypoactivity of the hypothalamo-pituitary-adrenocortical axis during recovery from chronic variable stress. Endocrinology 147, 2008-17. Herman, J.P., Adams, D., Prewitt, C., 1995. Regulatory Changes in Neuroendocrine Stress-Integrative Circuitry Produced by a Variable Stress Paradigm. Neuroendocrinology 61, 180-190. doi: 10.1159/000126839. Ziegler, D.R., Cass, W.A., Herman, J.P., 1999. Excitatory influence of the locus coemleus in hypothalamic -pituitary-adrenocortical axis responses to stress. J. Neuroendocrinol. 11, 361-369. Kirsch, L, Deacon, B.J., Huedo-Medina, T.B., Scoboria, A., Moore, T.J., Johnson, B.T., 2008. Initial severity and antidepressant benefits: a meta-analysis of data submitted to the Food and Drug Administration. PLoS Medicine, 5(2), e45. http s ://doi . org / 10.1371 /j oumal .pmed .0050045. Alboni, S., van Dijk, R.M., Poggini, S., Milior, G., Perrotta, M., Drenth, T., Brunello, N., Wolfer, D.P., Limatola, C., Amrein, L, Cirulli, F., Maggi, L., Branchi, L, 2017. Fluoxetine effects on molecular, cellular and behavioral endophenotypes of depression are driven by the living environment. Molecular psychiatry, 22(4), 552-561. doi.org/10.1038/mp.2015.142. Table 2: Sequences
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Figure imgf000084_0001
Figure imgf000085_0001
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Figure imgf000089_0001
Figure imgf000090_0001
[0350] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.

Claims

CLAIMS What is claimed is:
1. A pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms.
2. The pharmaceutical composition of claim 1, wherein the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe.
3. The pharmaceutical composition of claim 2, wherein the major tryptamine is psilocybin or a structurally similar analog.
4. The pharmaceutical composition of claim 2, wherein the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin.
5. The pharmaceutical composition of claim 4, wherein the one or more minor tryptamines is norbaeocystin.
6. The pharmaceutical composition of claim 4, wherein the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4- hydroxytryptophan, 4-hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy- N,N,N-trimethyltryptamine (4-OH-TMT).
7. A pharmaceutical composition comprising psilocybin and norbaeocystin.
8. The pharmaceutical composition of claim 7, wherein the psilocybin is heterologously produced.
9. The pharmaceutical composition of claim 7 or 8, wherein the norbaeocystin is heterologously produced.
10. The pharmaceutical composition of claim 7, wherein the psilocybin is synthetically produced.
11. The pharmaceutical composition of claim 7 or 10, wherein the norbaeocystin is synthetically produced.
12. The pharmaceutical composition of claim 8, wherein the heterologously produced psilocybin was produced in a prokaryotic host cell.
13. The pharmaceutical composition of claim 12, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
14. The pharmaceutical composition of claim 8, wherein the heterologously produced psilocybin is purified.
15. The pharmaceutical composition of claim 9, wherein the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
16. The pharmaceutical composition of claim 15, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
17. The pharmaceutical composition of claim 7, wherein the heterologously produced norbaeocystin is purified.
18. The pharmaceutical composition of any one of claims 7-17, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
19. The pharmaceutical composition of claim 18, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
20. The pharmaceutical composition of claim 19, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
21. The pharmaceutical composition of claim 20, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
22. The pharmaceutical composition of claim 21, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
23. The pharmaceutical composition of claim 22, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 1:1 to 1:1.
24. A method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient a pharmaceutical composition comprising two or more tryptamines that are present in Psilocybe magic mushrooms.
25. The method of claim 24, wherein the two or more tryptamines comprise a major tryptamine present in Psilocybe and one or more minor tryptamines present in Psilocybe.
26. The method of claim 24, wherein the major tryptamine is psilocybin or a structurally similar analog.
27. The method of claim 24, wherein the one or more minor tryptamines is selected from an intermediate and/or a side product of psilocybin.
28. The method of claim 27, wherein the one or more minor tryptamines is norbaeocystin.
29. The method of claim 27, wherein the intermediate or side product of psilocybin is selected from the group consisting of norbaeocystin, baeocystin, 4-hydroxytryptophan, 4- hydroxytryptamine, aeruginascin, psilocin, norpsilocin, or 4-hydroxy-N,N,N-trimethyltryptamine (4-OH-TMT).
30. A method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient a pharmaceutical composition comprising psilocybin and norbaeocystin.
31. The method of claim 30, wherein the psilocybin is heterologously produced.
32. The method of claim 30 or 31, wherein the norbaeocystin is heterologously produced.
33. The method of claim 30, wherein the psilocybin is synthetically produced.
34. The method of claim 30 or 33, wherein the norbaeocystin is synthetically produced.
35. The method of claim 30, wherein the administration of the pharmaceutical composition comprising psilocybin and norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
36. The method of claim 31, wherein the heterologously produced psilocybin was produced in a prokaryotic cell.
37. The method of claim 36, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
38. The method of claim 32, wherein the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
39. The method of claim 38, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
40. The method of claim 31, wherein the heterologously produced psilocybin is purified.
41. The method of claim 32, wherein the heterologously produced norbaeocystin is purified.
42. The method of any one of claims 30-41, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 100:1 to 1:100.
43. The method of claim 42, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 75:1 to 1:75.
44. The method of claim 43, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 50:1 to 1:50.
45. The method of claim 44, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 25:1 to 1:25.
46. The method of claim 45, wherein the psilocybin and the norbaeocystin are present in the composition in a molar ratio of from 10:1 to 1:10.
47. The method of claim 46, wherein the and the norbaeocystin are present in the composition in a molar ratio of 1 : 1.
48. A method of treating a neurological or psychological disorder or condition in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition comprising psilocybin and an effective amount of a pharmaceutical composition comprising norbaeocystin.
49. The method of claim 48, wherein the psilocybin is heterologously produced.
50. The method of claim 48 or 49, wherein the norbaeocystin is heterologously produced.
51. The method of claim 48, wherein the psilocybin is synthetically produced.
52. The method of claim 48 or 51, wherein the norbaeocystin is synthetically produced.
53. The method of any one of claims 48-52, wherein the administration of the pharmaceutical composition comprising psilocybin and norbaeocystin results in a greater improvement of symptoms in the patient than administration of an equivalent amount of psilocybin alone.
54. The method of claim 49, wherein the heterologously produced psilocybin was produced in a prokaryotic cell.
55. The method of claim 54, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
56. The method of claim 40, wherein the heterologously produced norbaeocystin was produced in a prokaryotic host cell.
57. The method of claim 56, wherein the prokaryotic cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Vibrio natriegens, Bacillus subtilis, Bacillus megaterium, Escherichia coli Nissle 1917, Clostridium acetobutlyicum, Streptomyces coelicolor, Lactococcus lactis, Pseudomonas putida, Streptomyces clavuligerus, and Streptomyces venezuelae.
58. The method of claim 49, wherein the heterologously produced psilocybin is purified.
59. The method of claim 40, wherein the heterologously produced norbaeocystin is purified.
60. The method of any one of claims 48-59, wherein the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 100:1 to 1:100.
61. The method of claim 60, wherein the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 75:1 to 1:75.
62. The method of claim 61, wherein the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 50:1 to 1:50.
63. The method of claim 62, wherein the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 25:1 to 1:25.
64. The method of claim 63, wherein the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of from 10:1 to 1:10.
65. The method of claim 64, wherein the psilocybin and the norbaeocystin are administered to the patient in a molar ratio of 1 : 1.
66. The method of any one of claim 40-65, wherein the neurological disorder is selected from the group consisting of functional neurological disorder (FND), Guillain-Barre syndrome, Alzheimer’s disease, autism, migraine, headache, Traumatic Brain Injury, and Chronic Traumatic Encephalopathy.
67. The method of any one of claims 40-65, wherein the psychological disorder is selected from the group consisting of depression, anxiety, PTSD, obsessive compulsive disorder, bipolar disorder, eating disorder, substance abuse disorder, attention deficit hyperactivity disorder (ADHD), and schizophrenia.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11905535B2 (en) 2019-10-01 2024-02-20 Empyrean Nueroscience, Inc. Genetic engineering of fungi to modulate tryptamine expression

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190105313A1 (en) * 2016-07-23 2019-04-11 Paul Edward Stamets Psilocybin compositions
US20190142851A1 (en) * 2017-11-16 2019-05-16 CaaMTech, LLC Compositions comprising a psilocybin derivative and a cannabinoid
WO2021086513A1 (en) * 2019-10-28 2021-05-06 Miami University Methods for the production of psilocybin and intermediates or side products

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190105313A1 (en) * 2016-07-23 2019-04-11 Paul Edward Stamets Psilocybin compositions
US20190142851A1 (en) * 2017-11-16 2019-05-16 CaaMTech, LLC Compositions comprising a psilocybin derivative and a cannabinoid
WO2021086513A1 (en) * 2019-10-28 2021-05-06 Miami University Methods for the production of psilocybin and intermediates or side products

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. KY984100.1
ADAMS ALEXANDRA M ET AL: "In vivo production of psilocybin in E. coli", METABOLIC ENGINEERING, ACADEMIC PRESS, AMSTERDAM, NL, vol. 56, 21 September 2019 (2019-09-21), pages 111 - 119, XP085876197, ISSN: 1096-7176, [retrieved on 20190921], DOI: 10.1016/J.YMBEN.2019.09.009 *
ADAMS ALEXANDRA M. ET AL: "Development of an E. coli-based norbaeocystin production platform and evaluation of behavioral effects in rats", METABOLIC ENGINEERING COMMUNICATIONS, vol. 14, 12 March 2022 (2022-03-12), NL, pages e00196, XP055912131, ISSN: 2214-0301, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S2214030122000050/pdfft?md5=9e357069270222eb1ebc39ece357e30f&pid=1-s2.0-S2214030122000050-main.pdf> DOI: 10.1016/j.mec.2022.e00196 *
ADAMS, A. M. ET AL.: "In vivo production of psilocybin in E. coli", METAB. ENG., vol. 56, 2019, pages 111 - 119, XP085876197, DOI: 10.1016/j.ymben.2019.09.009
ADAMS, A. M., KAPLAN, N. A., WEI, Z., BRINTON, J. D., MONNIER, C. S., ENACOPOL, A. L.,RAMELOT, T. A., JONES, J. A.: "In vivo production of psilocybin in E. coli.", METABOLIC ENGINEERING, vol. 56, 2019, pages 111 - 119, XP085876197, DOI: 10.1016/j.ymben.2019.09.009
ALBONI, S.VAN DIJK, R.M.POGGINI, S.MILIOR, G.PERROTTA, M.DRENTH, T.BRUNELLO, N.WOLFER, D.P.LIMATOLA, C.AMREIN, I.: "Fluoxetine effects on molecular, cellular and behavioral endophenotypes of depression are driven by the living environment", MOLECULAR PSYCHIATRY, vol. 22, no. 4, 2017, pages 552 - 561
ALLEN J W ET AL: "Psychoactive mushroom use in Koh Samui and Koh Pha-Ngan, Thailand", JOURNAL OF ETHNOPHARMACOLOGY, ELSEVIER IRELAND LTD, IE, vol. 35, no. 3, 1 January 1992 (1992-01-01), pages 205 - 228, XP025813291, ISSN: 0378-8741, [retrieved on 19920101], DOI: 10.1016/0378-8741(92)90020-R *
BENJAMINI, Y., HOCHBERG, Y.: "Powerful Approach to Multiple Testing", J. R. STAT. SOC. SER. B, vol. 57, 1995, pages 289 - 300
BENJAMINI, Y.HOCHBERG, Y: "Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing", J. R. STAT. SOC. SER. B, vol. 57, 1995, pages 289 - 300
BEN-SHABAT, S.FRIDE, E.SHESKIN, T.TAMIRI, T.RHEE, M. H.VOGEL, Z.BISOGNO, T.DE PETROCELLIS, L.DI MARZO, V.MECHOULAM, R.: "An entourage effect: inactive endogenous fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 353, no. 1, 1998, pages 23 - 31
CANAL, C. E.MORGAN, D: "Head-twitch response in rodents induced by the hallucinogen 2,5-dimethoxy-4-iodoamphetamine: a comprehensive history, a re-evaluation of mechanisms, and its utility as a model", DRUG TESTING AND ANALYSIS, vol. 4, 2012, pages 556 - 576
CHEN, Y.ZHOU, H.ZHOU, Y.ZHOU, F: "Prevalence of self-reported depression and anxiety among pediatric medical staff members during the COVID-19 outbreak in Guiyang, China", PSYCHIATRY RESEARCH, vol. 288, 2020, pages 113005
COGAN, P. S: "The 'entourage effect' or 'hodge-podge hashish': the questionable rebranding, marketing, and expectations of cannabis polypharmacy", EXPERT REV. CLIN., vol. 13, 2020, pages 835 - 845
DE LA FUENTE REVENGA, M. ET AL.: "Fully automated head-twitch detection system for the study of 5-HT2A receptor pharmacology in vivo", SCI. REP., vol. 9, 2019, pages 1 - 14
FITZPATRICK, K. M.DRAWVE, G.HARRIS, C: "Facing new fears during the COVID-19 pandemic: The State of America's mental health", J. ANXIETY DISORD., vol. 75, 2020, pages 102291
FRICKE, J.BLEI, F.HOFFMEISTER, D: "Enzymatic synthesis of psilocybin", ANGEW. CHEMIE INT. ED., vol. 56, 2017, pages 12352 - 12355, XP055583973, DOI: 10.1002/anie.201705489
GARTZ, J: "Extraction and analysis of indole derivatives from fungal biomass", J. BASIC MICROBIOL., vol. 34, 1994, pages 17 - 22, XP002484501, DOI: 10.1002/jobm.3620340104
GEWIRTZ, J. C., MAREK, G. J.: "Behavioral evidence for interactions between a hallucinogenic drug and group II metabotropic glutamate receptors", NEUROPSYCHOPHARMACOLOGY, vol. 23, 2000, pages 569 - 576
GONZALEZ-MAESO, J. ET AL.: "Hallucinogens Recruit Specific Cortical 5-HT2A Receptor-Mediated Signaling Pathways to Affect Behavior", NEURON, vol. 53, 2007, pages 439 - 452
HALBERSTADT, A. L.GEYER, M. A: "Characterization of the head-twitch response induced by hallucinogens in mice: detection of the behavior based on the dynamics of head movement", PSYCHOPHARMACOLOGY (BERL), vol. 227, 2013, pages 727 - 39
HALBERSTADT, A. L.GEYER, M. A: "Characterization of the head-twitch response induced by hallucinogens in mice: detection of the behavior based on the dynamics of head movement", PSYCHOPHARMACOLOGY (BERL)., vol. 227, 2013, pages 727 - 39
HALBERSTADT, A. L.GEYER, M. A: "Multiple receptors contribute to the behavioral effects of indoleamine hallucinogens", NEUROPHARMACOLOGY, vol. 61, 2011, pages 364 - 81, XP028227974, DOI: 10.1016/j.neuropharm.2011.01.017
HE, W. ET AL.: "Production of chondroitin in metabolically engineered E. coli", METAB. ENG., vol. 27, 2015, pages 92 - 100, XP055842558, DOI: 10.1016/j.ymben.2014.11.003
HERMAN, J.P.ADAMS, D.PREWITT, C.: "Regulatory Changes in Neuroendocrine Stress-Integrative Circuitry Produced by a Variable Stress Paradigm", NEUROENDOCRINOLOGY, vol. 61, 1995, pages 180 - 190
JANIS FRICKE ET AL: "Enzymatic Synthesis of Psilocybin", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 56, no. 40, 25 August 2017 (2017-08-25), pages 12352 - 12355, XP055583973, ISSN: 1433-7851, DOI: 10.1002/anie.201705489 *
JANIS FRICKE ET AL: "Production Options for Psilocybin: Making of the Magic", CHEMISTRY - A EUROPEAN JOURNAL, vol. 25, no. 4, 18 January 2019 (2019-01-18), DE, pages 897 - 903, XP055766088, ISSN: 0947-6539, DOI: 10.1002/chem.201802758 *
JOHNSON, J. R. ET AL.: "Multicenter, Double-Blind, Randomized, Placebo-Controlled, Parallel-Group Study of the Efficacy, Safety, and Tolerability of THC:CBD Extract and THC Extract in Patients with Intractable Cancer-Related Pain", J. PAIN SYMPTOM MANAGE., vol. 39, no. 2, 2010, pages 167 - 179, XP026990959, DOI: 10.1016/j.jpainsymman.2009.06.008
JONES, J. A. ET AL.: "ePathOptimize: a combinatorial approach for transcriptional balancing of metabolic pathways", NAT. PUBL. GR., 2015
JONES, J. A.VERNACCHIO, V. R.LACHANCE, D. M.LEBOVICH, M.FU, L.SHIRKE, A. N.SCHULTZ, V. L.CRESS, B.LINHARDT, R. J.KOFFAS, M. A. G.: "ePathOptimize: a combinatorial approach for transcriptional balancing of metabolic pathways", SCIENTIFIC REPORTS, vol. 5, no. 1, 2015, pages 11301
KIRSCH, I.DEACON, B.J.HUEDO-MEDINA, T.B.SCOBORIA, A.MOORE, T.J.JOHNSON, B.T.: "Initial severity and antidepressant benefits: a meta-analysis of data submitted to the Food and Drug Administration", PLOS MEDICINE, vol. 5, no. 2, 2008, pages e45, Retrieved from the Internet <URL:https://doi.orgI10.1371/journal.pmed.0050045>
LEUNG ET AL.: "Baeocystin and norbaeocystin: New analogs of psilocybin from Psilocybe baeocystis", JOURNAL OF PHARMACEUTICAL SCIENCE, vol. 57, no. 10, 1 October 1968 (1968-10-01), pages 1667 - 1671, XP055537692, DOI: 10.1002/jps.2600571007 *
LEUNG, A. Y.PAUL, A. G: "Baeocystin and norbaeocystin: New analogs of psilocybin from Psilocybe baeocystis", J. PHARM. SCI., vol. 57, 1968, pages 1667 - 1671, XP055537692, DOI: 10.1002/jps.2600571007
MAHMOOD, Z. A.: "Natural Products: Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes", 2013, SPRINGER, article "Bioactive alkaloids from fungi: Psilocybin", pages: 523 - 552
MATSUSHIMA, Y.SHIROTA, O.KIKURA-HANAJIRI, R.GODA, Y.EGUCHI, F: "Effects of Psilocybe argentipes on marble-burying behavior in mice", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 73, 2009, pages 1866 - 8, XP055705802, DOI: 10.1271/bbb.90095
MERLIN M D ET AL: "Species identification and chemical analysis of psychoactive fungi in the Hawaiian islands", JOURNAL OF ETHNOPHARMACOLOGY, ELSEVIER IRELAND LTD, IE, vol. 40, no. 1, 1 September 1993 (1993-09-01), pages 21 - 40, XP025813287, ISSN: 0378-8741, [retrieved on 19930901], DOI: 10.1016/0378-8741(93)90086-K *
MILNE N. ET AL: "Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives", METABOLIC ENGINEERING, vol. 60, 1 July 2020 (2020-07-01), AMSTERDAM, NL, pages 25 - 36, XP055834851, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2019.12.007 *
MILNE, N. ET AL.: "Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives", METAB. ENG., vol. 60, 2020, pages 25 - 36, XP055834851, DOI: 10.1016/j.ymben.2019.12.007
OSTRANDER, M.M.ULRICH-LAI, Y.CHOI, D.C.RICHTAND, N.M.HERMAN, J.P.: "Hypoactivity of the hypothalamo-pituitary-adrenocortical axis during recovery from chronic variable stress", ENDOCRINOLOGY, vol. 147, 2006, pages 2008 - 17
ROSSEN, J. W. A.TEDIM, A. P.MURRAY, A. K: "The Novel Coronavirus COVID-19 Outbreak: Global Implications for Antimicrobial Resistance", FRONT. MICROBIOL., 2020, pages 1020
RUDENSTINE, S.: "Depression and Anxiety During the COVID-19 Pandemic in an Urban, Low-Income Public University Sample ", J. TRAUMA. STRESS, 2020, pages 22600
RUSSO, E. B.: "Taming THC: Potential cannabis synergy and phytocannabinoid-terpenoid entourage effects", BRITISH JOURNAL OF PHARMACOLOGY, vol. 163, 2011, pages 1344 - 1364, XP055420723, DOI: 10.1111/j.1476-5381.2011.01238.x
SANTIAGO, M.SACHDEV, S.ARNOLD, J. C.MCGREGOR, I. S.CONNOR, M: "Absence of Entourage: Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Functional Activity of A9-THC at Human CB 1 and CB2 Receptors", CANNABIS CANNABINOID RES., vol. 4, 2019, pages 165 - 176
SHERWOOD, A. M. ET AL.: "Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin", J. NAT. PROD., vol. 83, no. 2, 2020, pages 461 - 467
SLATTERY, D. A.CRYAN, J. F.: "Using the rat forced swim test to assess antidepressant-like activity in rodents", NATURE PROTOCOLS, vol. 7, no. 6, 2012, pages 1009 - 1014, XP037546589, DOI: 10.1038/nprot.2012.044
WESTON-GREEN, K: "Recent Advances in Cannabinoid Research", 2019, INTECHOPEN, article "The United Chemicals of Cannabis: Beneficial Effects of Cannabis Phytochemicals on the Brain and Cognition"
WURST, M., KYSILKA, R. & FLIEGER, M.: "Psychoactive tryptamines from basidiomycetes", FOLIA MICROBIOLOGICA, vol. 47, 2002, pages 3 - 27
XU, P.VANSIRI, A.BHAN, N.KOFFAS, M. A. G: "ePathBrick: A synthetic biology platform for engineering metabolic pathways in E. coli", BIOL, vol. 1, 2012, pages 256 - 266, XP007923085, DOI: 10.1021/sb300016b
ZHUK, O. ET AL.: "Research on Acute Toxicity and the Behavioral Effects of Methanolic Extract from Psilocybin Mushrooms and Psilocin in Mice", TOXINS (BASEL)., vol. 7, 2015, pages 1018 - 1029
ZIEGLER, D.R.CASS, W.A.HERMAN, J.P.: "Excitatory influence of the locus coeruleus in hypothalamic-pituitary-adrenocortical axis responses to stress", J. NEUROENDOCRINOL., vol. 11, 1999, pages 361 - 369

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* Cited by examiner, † Cited by third party
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US11905535B2 (en) 2019-10-01 2024-02-20 Empyrean Nueroscience, Inc. Genetic engineering of fungi to modulate tryptamine expression

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