WO2023164092A1 - Treatment of psychiatric disorders, brain injuries, and autism spectrum disorder - Google Patents

Treatment of psychiatric disorders, brain injuries, and autism spectrum disorder Download PDF

Info

Publication number
WO2023164092A1
WO2023164092A1 PCT/US2023/013767 US2023013767W WO2023164092A1 WO 2023164092 A1 WO2023164092 A1 WO 2023164092A1 US 2023013767 W US2023013767 W US 2023013767W WO 2023164092 A1 WO2023164092 A1 WO 2023164092A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
entheogen
mushroom
psilocybin
aeruginascin
Prior art date
Application number
PCT/US2023/013767
Other languages
French (fr)
Inventor
Leonard LERER
Bernard Lerer
Karin Lerer BLAKOLMER
Original Assignee
Parow Entheobiosciences Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Parow Entheobiosciences Llc filed Critical Parow Entheobiosciences Llc
Publication of WO2023164092A1 publication Critical patent/WO2023164092A1/en

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • 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

Definitions

  • the present invention relates to the use of entheogens to treat psychiatric disorders, brain injuries, and autism, and in particular to the use of entheogens to treat obsessive compulsive disorder (OCD), schizophrenia, depression, post-traumatic stress disorder (PTSD), anxiety, autism spectrum disorder, traumatic brain injury (TBI), concussion, and chronic traumatic encephalopathy (CTE).
  • OCD obsessive compulsive disorder
  • PTSD depression
  • anxiety anxiety
  • autism spectrum disorder traumatic brain injury
  • TBI traumatic brain injury
  • concussion concussion
  • chronic traumatic encephalopathy CTE
  • Obsessive-compulsive disorder features a pattern of unwanted thoughts and fears (obsessions) that lead to repetitive behaviors (compulsions) (Stein DJ, Costa DLC et al: Obsessive-compulsive disorder. Nat Rev Dis Primers. 5(1): 52). These obsessions and compulsions interfere with daily activities and cause significant distress. Obsessive-compulsive disorder usually includes both obsessions and compulsions. But it is also possible to have only obsession symptoms or only compulsion symptoms. OCD obsessions are repeated, persistent and unwanted thoughts, urges or images that are intrusive and cause distress or anxiety. The sufferer might try to ignore them or get rid of them by performing a compulsive behavior or ritual.
  • Schizophrenia is a devastating neuropsychiatric disorder with lifetime impact, that is only partly addressed by available treatment with antipsychotic drugs (APDs) (Jauhar S, Johnstone M et al: Schizophrenia. The Lancet. Volume 399, Issue 10323, P473-486, January 29, 2022).
  • APDs antipsychotic drugs
  • the main target of APDs is the treatment of psychosis, and they do not attenuate the emergence of debilitating negative symptoms and cognitive deficits. Effective treatment of negative symptoms is crucial, as the persistence of these symptoms while psychosis is in remission is an impediment preventing schizophrenia patients from leading a fully functional life.
  • Major depressive disorder is the leading cause of disability worldwide in terms of total years lost due to disability and is associated with excess mortality (Abbafati, C.; Machado, D.B.; Cislaghi, B.; Salman, O.M.; Karanikolos, M.; McKee, M.; Abbas, K.M ; Brady, O.J.; Larson, H.J.; Trias-Llimos, S.; et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020, 396, 1204-1222).
  • Post-traumatic stress disorder is a mental health condition that is triggered by a cosmic event - either experiencing it or witnessing it (Bryant RA: Post-traumatic stress disorder: a state-of-the-art review of evidence and challenges. Psychiatry. 2019 Oct;18(3):259- 269). Symptoms may include flashbacks, nightmares and severe anxiety, as well as uncontrollable thoughts about the event. Post-traumatic stress disorder symptoms may start within one month of a traumatic event, but sometimes symptoms may not appear until years after the event. These symptoms cause significant problems in social or work situations and in relationships. They can also interfere with the ability to go about normal daily tasks. PTSD symptoms are generally grouped into four types: intrusive memories, avoidance, negative changes in thinking and mood, and changes in physical and emotional reactions. Symptoms can vary over time or vary from person to person.
  • Anxiety disorders are the most prevalent psychiatric disorders (with a current worldwide prevalence of 7.3%. Among them, specific phobias are the most common, with a prevalence of 10.3%, then panic disorder (with or without agoraphobia) is the next most common with a prevalence of 6.0%, followed by social phobia (2.7%) and generalized anxiety disorder (2.2%). There is a high comorbidity between anxiety (especially generalized anxiety disorders or panic disorders) and depressive disorders. Additionally, anxiety disorders are often associated, which renders treatment even more complex. As a result, anxiety disorders often remain underdiagnosed and undertreated in primary care (Thibaut F: Anxiety disorders: a review of current literature. Dialogues Clin Neurosci. 2017 Jun; 19(2): 87-88).
  • Entheogens are chemical substances, typically of plant origin, that is ingested to produce a nonordinary state of consciousness for religious or spiritual purposes (Nichols DE: Psychedelics. Pharmacol Rev. 2016 Apr; 68(2): 264-355).
  • entheogens such as the psychedelic compound, psilocybin
  • psilocybin the psychedelic compound
  • ACS Chem. Neurosci. 2 ACS Chem. Neurosci. 2
  • Autism spectrum disorder is a developmental and behavioral disorder, representing one of the most disabling chronic conditions in childhood. Picardi et al., Parental burden and its correlates in families of children with autism spectrum disorder: a multicentre study with two comparison groups. Clin Pract Epidemiol Ment Health. 2018;14: 143. It is characterized by abnormalities in social interactions, deficits in communication, restricted interests, and repetitive behavior. Frye RE. Social skills deficits in autism spectrum disorder: potential biological origins and progress in developing therapeutic agents. CNS Drugs. 2018;32:713-734. ASD children are often subjected to criticism and bullying and react by isolation, aggression, hostility, or self-harm.
  • ASD affects not only the persons diagnosed with this disorder but their entire families. Indeed, parents experience a significant level of stress due to the severity and permanency of ASD, development of co-morbidities, and the lack of health support for their children.
  • Vohra R Madhavan et al., Access to services, quality of care, and family impact for children with autism, other developmental disabilities, and other mental health conditions. Autism. 2014;18:815-826.
  • the daily challenges of caring for these children are troublesome, affect all aspects of the child's care and harm the parent's mental health. Bonis, Stress and parents of children with autism: a review of literature. Issues Ment Health Nurs. 2016;37: 153-163.
  • TBI traumatic brain injury
  • mimild-TBI concussion
  • CTE chronic traumatic encephalopathy
  • TBI traumatic brain injury
  • mimild-TBI concussion
  • CTE chronic traumatic encephalopathy
  • Psilocybin is a naturally occurring psychedelic compound produced by over 200 mushrooms, that is being increasingly researched as treatment for a range of different psychiatric disorders (Carhart-Harris RL and Goodwin GM. (2017) The therapeutic potential of psychedelic drugs: past, present and future. Neuropsychopharmacology). Five separate trials have reported improvements in depressive symptoms after psilocybin-assisted psychotherapy including one in which 'treatment-resistant depression' was the primary criterion for inclusion (Carhart-Harris R, Giribaldi B, Watts R et al Trial of Psilocybin versus Escital opram for Depression. N Engl J Med.
  • Treatment procedures typically involve psychological preparation prior to one or two therapist-supported drug sessions followed by psychological integration.
  • a consistent model i.e. involving appropriate psychological support
  • sustained improvements in well-being in healthy individuals were observed after a single dose of psilocybin in a double-blind design incorporating an active placebo (Griffiths R, Richards W, Johnson M et al (2008) Mystical-type experiences occasioned by psilocybin mediate the attribution of personal meaning and spiritual significance 14 months later.
  • psilocybin Like all serotonergic psychedelics, psilocybin initiates its characteristic effects via serotonin 2A receptor (5-HT2AR) agonism (Vollenweider FX, Vollenweider-Scherpenhuyzen MF, Babler A et al (1998); Vollenweider FX, Smallridge JW: Classic Psychedelic Drugs: Update on Biological Mechanisms. Pharmacopsychiatry. 2022 Jan 25. doi: 10.1055/a- 1721 -2914. Online ahead of print). Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action. Neuroreport 9:3897-3902).
  • 5-HT2AR signaling has been associated with better responses to conventional antidepressants (Qesseveur G, Petit AC, Nguyen HT et al. (2016) Genetic dysfunction of serotonin 2A receptor hampers response to antidepressant drugs: a translational approach. Neuropharmacology 105: 142-153; Petit AC, Quesseveur G, Gressier F et al (2014) Converging translational evidence for the involvement of the serotonin 2A receptor gene in major depressive disorder.
  • 5-HT2AR antagonists have been found to augment the antidepressant effects of SSRIs (Ostroff RB, Nelson JC (1999) Risperidone augmentation of selective serotonin reuptake inhibitors in major depression. J Clin Psychiatry 60:256-259) and many effective antidepressant augmentation medications have 5-HT2AR antagonist properties (Carpenter LL, Jocic Z, Hall JM et al (1999) Mirtazapine augmentation in the treatment of refractory depression. J Clin Psychiatry 60:45-49).
  • the present invention relates to the use of entheogens to treat psychiatric disorders, brain injuries, and autism, and in particular to the use of entheogens to treat obsessive compulsive disorder (OCD), schizophrenia, depression, post-traumatic stress disorder (PTSD), anxiety, autism spectrum disorder, traumatic brain injury (TBI), concussion, and chronic traumatic encephalopathy (CTE).
  • OCD obsessive compulsive disorder
  • PTSD depression
  • anxiety anxiety
  • autism spectrum disorder traumatic brain injury
  • TBI traumatic brain injury
  • concussion concussion
  • chronic traumatic encephalopathy CTE
  • the present invention provides methods of treating a psychiatric disorder in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin or a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mushroom cell culture composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the psychiatric disorder is selected from the group consisting of obsessive compulsive disorder (OCD), schizophrenia, post-traumatic stress disorder (PTSD), depressive and anxiety disorders, alcohol and substance use disorders and nicotine addiction.
  • OCD obsessive compulsive disorder
  • PTSD post-traumatic stress disorder
  • depressive and anxiety disorders alcohol and substance use disorders and nicotine addiction.
  • the effective amount of a mushroom cell culture extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder.
  • the psychiatric disorders are OCD and schizophrenia.
  • the psychiatric disorder is PTSD.
  • the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
  • PCB phycobiliprotein
  • the method further comprise co-administering a 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • the present invention provides methods of providing prophylaxis, and primary, secondary and tertiary prevention against PTSD and/or depression in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin or a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mushroom cell culture composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the effective amount of a mushroom cell culture composition or entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD and depression.
  • the mushroom cell culture composition or entheogen composition is administered to the subject within 4 weeks of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 week of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 3 days of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 day of a psychological trauma or other traumatic event.
  • the mushroom cell culture composition or entheogen composition is produced by a process comprising mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
  • PCB phycobiliprotein
  • the methods further comprise co-administering a 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • the present invention provides a mushroom cell culture extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin or entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin for use in treating a psychiatric disorder in a subject in need thereof.
  • the mushroom cell culture composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition or mushroom cell culture extract comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition or mushroom cell culture extract comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition or mushroom cell culture extract comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
  • the amount of mushroom cell culture composition or entheogen composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder.
  • the psychiatric disorder is PTSD.
  • the psychiatric disorders OCD and or schizophrenia.
  • the mushroom cell culture composition is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
  • PCB phycobiliprotein
  • the entheogen composition or mushroom cell culture extract is co-administered with a 5-HTIA receptor agonist.
  • the 5- HT IA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, Fl 3714 and F15599.
  • the present invention provides a mushroom cell culture extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin or entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin for use in prophylaxis, or primary, secondary and tertiary prevention of PTSD and/or depression.
  • the mushroom cell culture or entheogen composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition or mushroom cell culture extract comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen or mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen or mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the effective amount of a mushroom cell culture composition or entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD and depression.
  • the mushroom cell culture composition or entheogen composition is administered to the subject within 4 weeks of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 week of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 3 days of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 day of a psychological trauma or other traumatic event.
  • the mushroom cell culture composition or entheogen composition is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
  • PCB phycobiliprotein
  • the entheogen or mushroom cell culture composition is co-administered with a 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • the present invention provides a process for mushroom cell culture comprising: culturing mushrooms on substrate to provide a mushroom cell culture, and contacting the mushroom cell culture with a PC composition.
  • the mushrooms cells are psychoactive mushroom cells or parts of a psychoactive mushroom.
  • the mushroom cell culture produces one or more psychoactive compounds or other compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the process further comprises processing the mushroom cell culture to provide an aqueous fraction comprising a psychoactive compound. In some preferred embodiments, the process further comprises processing the mushroom cell culture to provide an extract comprising a psychoactive compound. In some preferred embodiments, the process further comprises processing the mushroom cell culture to provide a purified composition comprising a psychoactive compound. In some preferred embodiments, the process further comprises the step of formulating the aqueous fraction, extract or purified composition comprising a psychoactive compound with a pharmaceutically acceptable carrier. In some preferred embodiments, the pharmaceutically acceptable carrier does not naturally occur with the aqueous fraction, extract or purified composition comprising a psychoactive compound.
  • the present invention provides a process for production of psychoactive compounds comprising: culturing mushroom cells from a psychoactive mushroom on substrate to cell culture; contacting the mushroom cell with a PC composition; and processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds.
  • the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises producing an aqueous fraction from the mushroom cell culture that comprises one or more psychoactive compounds.
  • the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises extracting the mushroom cell culture with an aqueous solvent, an organic solvent of combinations thereof to provide an extract that comprises one or more psychoactive compounds.
  • the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises processing the mushroom cell culture to provide a purified composition comprising a psychoactive compound.
  • the process further comprises the step of formulating the aqueous fraction, extract or purified composition comprising a psychoactive compound with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier does not naturally occur with the aqueous fraction, extract or purified composition comprising a psychoactive compound.
  • the present invention provides a mushroom cell culture composition or formulation thereof produced by a process as described above.
  • the present invention provides a formulation comprising a mushroom cell culture composition and a pharmaceutically acceptable carrier that does not naturally occur with the mushroom cell culture.
  • the present invention provides a formulation comprising psilocybin or psilocin and a 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • the psilocybin or psilocin is provided in a mushroom cell culture extract.
  • the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
  • the formulations further comprise one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the psilocybin or psilocin is chemically synthesized.
  • the baeocystin, aeruginascin, norpsilocin, or norbaeocystin are chemically synthesized.
  • the formulations described in the eighth and ninth aspects are provides for use in treating a psychiatric disorder.
  • the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
  • the present invention provides a kit comprising: a) a first formulation or composition comprising psilocybin or psilocin; and b) a second formulation or composition comprising a 5-HTIA receptor agonist; wherein the first formulation or composition and second formulation or composition are provided in different containers or oral delivery vehicles.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • the psilocybin or psilocin is provided in a mushroom cell culture extract.
  • the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
  • the kit further comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the psilocybin or psilocin is chemically synthesized.
  • the baeocystin, aeruginascin, norpsilocin, or norbaeocystin are chemically synthesized.
  • the kit of the eleventh aspect is provided for use in treating a psychiatric disorder.
  • the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
  • the present invention provides methods of treating autism spectrum disorder in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition.
  • the entheogen composition comprises psilocybin or psilocin. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition is a mushroom cell culture composition.
  • the entheogens in the composition are chemically synthesized.
  • the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the effective amount of a mycelial cell culture extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of autism spectrum disorder.
  • the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
  • the present invention provides an entheogen composition for use in treating autism spectrum disorder in a subject in need thereof.
  • the entheogen composition comprises psilocybin or psilocin. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen is provided as a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mushroom cell culture composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the amount of mushroom cell culture composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder.
  • the mushroom cell culture composition is produced by a process comprising culturing mycelial cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
  • the present invention provides methods of treating a brain injury in a subject in need thereof comprising administering to the subject an effective amount of an entheogen composition.
  • the brain injury is selected from the group consisting of traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma of the brain.
  • the entheogen composition comprises psilocybin or psilocin. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition is a mushroom extract.
  • the entheogens are chemically synthesized.
  • the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
  • the effective amount of an entheogen or mushroom extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the brain injury.
  • the entheogen composition is a psilocybin-free entheogen composition.
  • the administration of the entheogen composition is immediately before, during of immediately after anesthesia or sedation of the subject.
  • the present invention provides an entheogen composition for use in treating a brain injury in a subject in need thereof.
  • the brain injury is selected from the group consisting of traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma of the brain.
  • the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition is a mushroom extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mushroom extract is a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the mycelial cell culture composition is produced by a process comprising culturing mycelial cells in a culture vessel. In some preferred embodiments, the process comprising culturing mycelial cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
  • the amount of entheogen or mycelial cell culture composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the brain injury.
  • the entheogen composition is a psilocybin-free entheogen composition.
  • the administration of the entheogen composition is immediately before, during of immediately after anesthesia or sedation of the subject. In some preferred embodiments, the entheogen composition is co-administered with an anesthesia or sedation agent.
  • the present invention provides an entheogen composition comprising a phycocyanin (PC) composition and a mushroom extract.
  • PC phycocyanin
  • the mushroom extract comprises psilocybin. In some preferred embodiments, the mushroom extract is psilocybin-free. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the phycocyanin composition is extracted from Spirulina.
  • the phycocyanin composition is characterized by one or more of the following characteristics: a) the protein fraction of the composition comprises greater than about 30% of a protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; b) the protein fraction of the composition comprises greater than about 5% of a protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6 14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; c) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 45,578 kDa and an isoelectric point of about 6.2 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; d) the protein fraction of the composition comprises greater than about
  • the PC composition has at least two of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least three of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least four of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least five of characteristics a, b, c, d, e, f, g, h, i, j and k.
  • the PC composition has at least six of characteristics a, b, c, d, e, f, g, h, i, j and k.
  • the purified PC composition of claim 1, wherein the composition has at least seven of characteristics a, b, c, d, e, f, g, h, i, j and k.
  • the PC composition has at least eight of characteristics a, b, c, d, e, f, g, h, i, j and k.
  • the PC composition has at least nine of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least ten of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has all eleven characteristics a, b, c, d, e, f, g, h, i, j and k.
  • the phycocyanin composition is produced by a process comprising: encapsulating Spirulina to provide capsules; and contacting the capsules with an aqueous medium under conditions such that the compound of interest passes from the capsule into the aqueous solution.
  • the PC composition comprises a dried powder comprising a purified PC, the powder having a residual moisture content of less than about 5% w/w of the powder.
  • the psilocybin-free mushroom extract is extracted from a mushroom or part thereof that produces psilocybin, a psilocybin analog or related typtamine.
  • the psilocybin-free mushroom extract is extracted from a mushroom or part thereof that comprises the psilocybin biosynthetic gene cluster (psiD, psiH, psiK, psiM).
  • the psilocybin-free mushroom extract is an aqueous extract.
  • the psilocybin-free mushroom extract is produced by a process comprising: encapsulating a raw material comprising a mushroom or part thereof that produces psilocybin, a psilocybin analog or related typtamine to provide capsules; and contacting the capsules with an aqueous medium under conditions such that compounds of interest passes from the capsule into the aqueous solution.
  • the methods further comprise the step of removing psilocybin from the extract by chromatography.
  • the psilocybin-free mushroom extract comprises a dried powder having a residual moisture content of less than about 5% w/w of the powder.
  • the psilocybin-free mushroom extract is a psilocybin- free mycelial extract.
  • the composition comprises from 1 to 99% w/w phycocyanin. In some preferred embodiments, the composition comprises from 10 to 90% w/w phycocyanin. In some preferred embodiments, the composition comprises from 1 to 99% w/w psilocybin-free mushroom extract. In some preferred embodiments, the composition comprises from 10 to 90% w/w psilocybin-free mushroom extract.
  • the composition is formulated as a topical formulation, an oral formulation, a mucosal formulation, an ophthalmological formulation, an aerosol formulation, and an intranasal formulation.
  • the composition further comprises one or more excipients or pharmaceutically acceptable carriers.
  • the oral formulation is an oral delivery vehicle.
  • the oral delivery vehicle is selected from the group consisting of a capsule, a tablet and a gummi gel.
  • the present invention provides methods of treating or preventing a psychiatric disease or disorder comprising administering an effective amount of the composition of the sixteenth aspect to a subject suffering from a psychiatric disease or disorder.
  • the psychiatric disease or disorder is selected from the group consisting of anxiety disorder and depression.
  • the present invention provides method of improving or altering cognitive function or sociability, or reducing aggressive behavior comprising administering an effective amount of the composition of the sixteenth aspect to a subject suffering from a psychiatric disease or disorder.
  • the present invention provides a composition of the sixteenth aspect for use in treating or preventing a psychiatric disease or disorder in a subject in need thereof.
  • the psychiatric disease or disorder is anxiety disorder and depression.
  • the present invention provides a composition of the sixteenth aspect for use in of improving or altering cognitive function or sociability, or reducing aggressive behavior in a subject in need thereof.
  • the present invention provides methods of improving neuroplasticity in a subject in need thereof comprising administering to the subject an entheogen composition comprising psilocybin or psilocin and one or more entourage compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the composition comprises psilocybin.
  • the subject in need thereof has a disease, condition or disorder that could benefit from the promotion of neuroplasticity.
  • the disease, condition or disorder associated with decreased neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD.
  • administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43, and Synaptophysin in one or more areas of the brain selected from the group consisting of the prefrontal cortex, amygdala, hippocampus, and striatum.
  • administration of the entheogen composition increases the level of two or more proteins selected from the group consisting of PSD-95, GAP43 and Synaptophysin.
  • administration of the entheogen composition increases the level of PSD-95, GAP43 and Synaptophysin.
  • the area of the brain is the prefrontal cortex. In some preferred embodiments, the area of the brain is the amygdala. In some preferred embodiments, the area of the brain is the hippocampus. In some preferred embodiments, the area of the brain is the striatum.
  • the entheogen composition comprises two or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition is a mushroom extract.
  • the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
  • the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with neural atrophy.
  • the entheogens in the composition are chemically synthesized.
  • the entheogen composition is co-administered with a 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F 13714 and F15599.
  • the present invention provides an entheogen composition for use in treating a disease, condition or disorder associated with neural atrophy.
  • the entheogen composition comprises psilocybin.
  • the disease, condition or disorder associated with decreased neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD.
  • administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43 and Synaptophysin in one or more areas of the brain selected from the group consisting of the prefrontal cortex, amygdala, hippocampus, and striatum.
  • administration of the entheogen composition increases the level of two or more proteins selected from the group consisting of PSD-95, GAP43 and Synaptophysin.
  • administration of the entheogen composition increases the level of PSD-95, GAP43 and Synaptophysin.
  • the area of the brain is the prefrontal cortex. In some preferred embodiments, the area of the brain is the amygdala. In some preferred embodiments, the area of the brain is the hippocampus. In some preferred embodiments, the area of the brain is the striatum.
  • the entheogen composition comprises two or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition is a mushroom extract.
  • the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel.
  • the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
  • the entheogens in the composition are chemically synthesized.
  • the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with reduced neuroplasticity.
  • the entheogen composition is co-administered with a 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • FIGs. 1 A to ID provide data on psilocybin dose response. These graphs depict (FIG. 1 A) full range dose response of HTR through the course of 18 min, (FIG. IB) taken from A the distinct shape of faster and higher onset following a rapid decrease in response (doses high then 3mg/kg), (FIG. 1C) taken from A the distinct shape of flatter response that carries over time (doses lower then 1.6 mg/kg), (FIG. lD)dose response curve taken from the data shown in A.
  • FIGs. 2A to 2D provide data on the effects of serotonin receptor modulators on psilocybin-induced HTR (Head Twitch Response).
  • the bar graphs show the cumulative HTR recorded by the mice during 20 min session immediately following injection of either psilocybin (vehicle) alone or combined with a receptor modulator.
  • FIG. 2A Shows the effect of a combined psilocybin + 5HT2A antagonist, which completely reduced HTR in both concentrations.
  • FIG. 2B Shows the effect of a combined psilocybin + 5HT1 A agonist, which reduced HTR in dose depended manner.
  • FIG. 2C Shows the effect of a combined psilocybin + TAAR1 antagonist, which did not alter HTR.
  • FIG. 2D Shows the effect of a combined psilocybin + 5HT2C antagonist, which increased HTR with 4 mg/kg and reduced the HTR-increasing effect at 8mg/kg.
  • FIGs. 3 A and 3B Effect of psilocybin 4.4 mg/kg and escitalopram 5 mg/kg on total marbles buried over 30 minutes.
  • 3B Effect of psilocybin 4.4 mg/kg, M100907 2 mg/kg and M100907 2 mg/kg + psilocybin 4.4 mg/kg on total marbles buried over 30 minutes.
  • ##p ⁇ 01 vs. psilocybin, n 6-16 (Tukey's multiple comparisons test).
  • FIGs 5A and 5B 5A:. Effect of psilocybin 4.4 mg/kg, buspirone 5 mg/kg and buspirone 5 mg/kg+ psilocybin 4.4 mg/kg on total marbles buried over 30 minutes.
  • FIG. 6A and 6B Effect of psilocybin 4.4 mg/kg, buspirone 5 mg/kg and psilocybin 4.4 mg/kg + buspirone 5 mg/kg on HTR over a 20-minute measurement period.
  • 6B Total HTR over 20 minutes.
  • FIGs. 7A-7C Effect of psilocybin 4.4 mg/kg and buspirone 5 mg/kg on distance travelled in the open field over 30 minutes.
  • 7B Effect of psilocybin 4.4 mg/kg and buspirone 5 mg/kg on time spent in the center of the open field over 30 minutes.
  • FIG. 8 Graphs comparing the effect of chemical psilocybin and full spectrum mushroom extract on synaptic protein levels across the prefrontal cortex, hippocampus, amygdala and striatum in a mouse model.
  • a “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats). "Administering” or "administration of a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art.
  • a PBP can be administered, intravenously, arterially, intradermally, intra-muscularly, intraperitoneally, intravenously, subcutaneously, sublingually, orally (by ingestion), intranasally (by inhalation), intrapulmonary (by nebulization or instillation) intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct).
  • a compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug.
  • direct administration including self-administration
  • indirect administration including the act of prescribing a drug.
  • a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.
  • a “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent, such as the phycocyanin and psilocybin-free mushroom extract compositions described herein, is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect.
  • the full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations.
  • the precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of symptoms of the condition being treated. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
  • a “prophylactically effective amount” or a “prophylactically effective dose” of a drug or agent, such as a phycocyanin and psilocybin-free mushroom extract composition is an amount of a drug or an agent that, when administered to a subject will have the intended prophylactic effect.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations.
  • the precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of symptoms of the condition being treated. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
  • Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results include, but are not limited to, alleviation, amelioration, or slowing the progression, of one or more symptoms associated with the disease or disorder being treated.
  • treatment may be prophylactic.
  • the present invention relates to the use of entheogens to treat psychiatric disorders, brain injuries, and autism, and in particular to the use of entheogens to treat obsessive compulsive disorder (OCD), schizophrenia, depression, post-traumatic stress disorder (PTSD), anxiety, autism spectrum disorder, traumatic brain injury (TBI), concussion, and chronic traumatic encephalopathy (CTE).
  • OCD obsessive compulsive disorder
  • PTSD depression
  • anxiety anxiety
  • autism spectrum disorder traumatic brain injury
  • TBI traumatic brain injury
  • concussion concussion
  • chronic traumatic encephalopathy CTE
  • the present invention provides solution to multiple problems.
  • the problem of treatment of the listed disorders is addressed via the use of fullspectrum entheogen compositions that include entourage compounds (e.g., baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof) in addition to psilocybin.
  • the problem of providing full-spectrum entheogen compositions is addressed by improved culture of mushroom parts or cells.
  • Other problems and solutions for those problems presented in the specification will be apparent to those of skill in the art.
  • Entheogens are naturally occurring compounds with psychedelic properties that have historically been used for religious and spiritual ceremonies and also for medicinal purposes.
  • OCD obsessive compulsive disorder
  • PTSD post-traumatic stress disorder
  • PTSD schizophrenia
  • depressive and anxiety disorders alcohol and substance use disorders and nicotine addiction
  • autism spectrum disorder traumatic brain injury (TBT)
  • concussion concussion
  • chronic traumatic encephalopathy CTE
  • the present invention is not limited to any particular entheogen composition or formulation.
  • the entheogen compositions may be prepared by a variety of methods including, but not limited to, chemical synthesis, extraction and/or purification from plants or fungi, and extraction and/or purification from mushroom cell cultures.
  • the entheogen compositions comprise psilocybin.
  • the entheogen compositions comprise psilocybin and additionally one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof.
  • the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof and is essentially free of psilocybin.
  • Exemplary entheogen compositions are provided below.
  • the compounds listed may, for example, be chemically synthesized (see, e.g., Sherwood et al., (2020) Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin; J. Nat. Prod. 83(2) 461-67, incorporated herein by reference in its entirety), may be provided in an extract from mushrooms or from mushroom cell cultures as described in detail below, or be isolated from mushrooms or mushroom cell cultures as described in detail below.
  • he listed composition comprises the listed compounds.
  • the listed compositions consist essentially of the listed compounds.
  • the listed compositions consist of the listed compounds.
  • composition Compounds in composition
  • Biotechnology-based production of entheogens offers a promising route to ensure a cost- effective, cGMP, robust supply for clinical development.
  • Naturally derived psychedelic compounds may offer significant therapeutic advantages over synthetic molecules because of entourage effects. These reflect the action of additional components that may act synergistically or additively with the principal molecule or have pharmacokinetic effects.
  • additional components may act synergistically or additively with the principal molecule or have pharmacokinetic effects.
  • psilocybin which is derived from multiple mushroom species that contain a wealth of additional active components.
  • Anecdotal and clinical reports as well as preclinical studies suggest a discernible difference between the effects of chemically synthesized psilocybin and those of psychedelic mushrooms and also differences among the effects of different mushroom strains.
  • the present invention is based on the systematic evaluation of the psychedelic properties and therapeutic potential of entheogen compounds derived from cell culture and in particular on mushroom cell cultured, psychoactive compound-containing mushroom compositions.
  • the term "mushroom culture compositions” refers to compositions comprising a mushroom cell culture and products derived therefrom specifically including 1) fractions, such as aqueous fractions, prepared from the mushroom cell culture that contain one or more psychoactive compounds, 2) extracts, such as aqueous solvent extracts or organic solvent extracts, prepared from the mushroom cell culture that comprise one or more psychoactive compounds, and 3) purified preparations of one or more psychoactive compounds prepared from a mushroom cell culture by methods including one or more of solvent extraction, concentration by solvent removal, chromatography and other methods of extraction, purification and fractionation.
  • the term "psychoactive compounds” refers to chemical substances that change a person's mental state by affecting the way the brain and nervous system work, and specifically, includes, but is not limited to psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof.
  • the biomass used to prepare a mushroom extract useful in the compositions of the present invention is any fungi (including hybrids) of part thereof (e g., mycelia, primordia, fruiting bodies, etc.) containing the psilocybin biosynthetic gene cluster (psiD, psiH, psiK, psiM) or other mushrooms, fungi, lichens, etc. producing psilocybin or psilocybin analogs or related tryptamines.
  • the biomass is mycelial biomass.
  • Exemplary mushroom species include, but are not limited to, Conocybe sp., Copelandia sp., Galerina sp., Gerronema sp., Gymnopilus sp., Hypholoma sp., Inocybe sp., Mycena sp., Panaeolina sp., Panaeolus sp., Pluteus sp., andPsilocybe sp.
  • Exemplary Psilocybes include, but are not limited to, P. acutipilea, P. angustipleurocystidiata, P. antioquensis, P. aquamarine, P. argentipes, P.
  • cordispora P. cubensis, P. cyanescens, P. cyanofibrillosa, P. dumontii, P. eucalypta, P. fagicola, P. fagicola mesocystidiata, P. farinacea, P. fimetaria, P. fuliginosa, P. furtadoana, P. galindoi, P. goniospora, P. graveolens, P. guatapensis, P. guilartsis, P. heimii, P. heliconiae, P. herrerae, P. hispanica, P. hoogshagenii , P.
  • pericystis P. pintonii, P. pleurocystidiosa, P. plutonia, P. portoricensis, P. pseudoaztecorum, P. puberula, P. quebecensis, P. ramulosa, P. rostrata, P. rzedowskii, P. samuiensis, P. sanctorum, P. schultesii, P. semilanceata, P. septentrionalis, P. serbica, P. sierrae, P. sylvatica, P. singerii, P. strictipes, P. stuntzii, P. subacutipilea, P.
  • the mushroom cell cultures of the present invention preferably utilize cells, parts or tissues of psilocybin-producing mushrooms.
  • the invention is not limited to any particular method of mushroom cell culture. Suitable methods include liquid culture, semi-solid culture, static culture, static-dynamic culture, biofilm cultures, hybrid culture and various methods of bioreactor and non-bioreactor culture of mushroom cells including mycelia and sclerotia.
  • the mushroom cell cultures are conducted on manufactured substrates and may preferably be grown in culture vessels.
  • the mushroom cell cultures are supplemented with a phycocyanin (PC) composition.
  • PC phycocyanin
  • PC compositions useful in the present invention are produced by processes based on a chemical reaction between a gelling agent and a multivalent cation. Exposing the gelling agent to the multivalent cation (or vice versa) facilitates the formation of a gel and/or a membrane, trapping the cellular debris and other large-size molecular assemblies, but allowing diffusion of water-soluble small molecules and proteins into the surrounding aqueous medium, usually water, thereby permitting the purification of the desired substance such as the PCB or PC.
  • the multivalent cation may also act on the cell wall of, for example Spirulina, thereby making it more fragile and porous and consequently allowing the extracellular diffusion of PCB and other water-soluble molecules.
  • the methods of the present invention result not only in the purification of, for example PC but also permit the extraction of PC from Spirulina in an aqueous solution in a single step.
  • Phycobiliproteins are water-soluble proteins that play a key role in photosynthesis. Phycobiliproteins take the form of a complex between proteins and covalently bound phycobilins that act as chromophores. They are the most important constituents of the phycobilisome.
  • the major phycobiliproteins are C-phycocyanin, R-phycoerythrin, B-phycoerythrin and allophycocyanin.
  • the blue phycobiliprotein (PBP) pigment complex contained in Spirulina that is generally called phycocyanin (PC) plays an important role in the photosynthetic chain acting as the link between light energy and chlorophyll.
  • PBP blue phycobiliprotein
  • PC phycocyanin
  • a number of different algae including Porphyridium cruentum Galdieria sulphuria and Aphanizomenon flos aquae (AF A), other cyanobacteria and other organisms contain varying amounts of PBP complexe
  • PC compositions used in the compositions described herein are described in co-pending application 16/685,363, filed Nov. 15, 2019, and which is incorporated herein by reference in its entirety.
  • PC is known to degrade rapidly at temperatures over 55°C and at low PH, with a loss of blue color and consequently utility as a food colorant.
  • the destruction of the Spirulina cell wall, an integral part of the extraction of PC, can damage the PC and if the extraction takes place over a long period cause the degradation of the PC.
  • the nature of the raw biomass for example whether it is fresh or dried, the type of drying process including temperature and time of exposure to heat, the preparation of the raw material, the addition of heat protecting substances, the length of time and temperature of storage, the final water content, the method of packaging, the level of oxidation of the protein and fats, exposure to bacteria and yeasts and fungi, residual chemicals and nutrients and range of other factors affect the quality and yield of PC.
  • PC Once the PC has been extracted, it can be further purified. Purification requires separation of blue- or other colored PBPs such as PC from the green-colored chlorophylls and is usually undertaken using filtration technologies including various types of centrifuges and filtration units. This can include disk centrifuges, decanting centrifuges, charcoal filtration, ultrafiltration, nanofiltration, reverse and forward osmosis filtration, high-pressure filtration, tangential filtration and a range of technologies that separate out the PC from the chlorophyll and cell debris and other undesirable substances.
  • filtration technologies including various types of centrifuges and filtration units. This can include disk centrifuges, decanting centrifuges, charcoal filtration, ultrafiltration, nanofiltration, reverse and forward osmosis filtration, high-pressure filtration, tangential filtration and a range of technologies that separate out the PC from the chlorophyll and cell debris and other undesirable substances.
  • Purification using filtration is an expensive process and contributes substantially to the high current cost of PBPs such as PC. Purification using filtration is also wasteful as it generally requires large quantities of disposable filters or filters that require cleaning with acid and alkaline solutions that are potential environmental pollutants if not managed and disposed of appropriately. Also, in order to reduce the cost of purification using filtration, additional prefiltration production steps may be used to precipitate out undesirable substances using chitosan and other precipitating or flocculating or purifying agents. These steps are time-consuming, expensive and potentially polluting and can damage the PC. Also, the use of certain precipitating and flocculating and purifying agents may result in the PC no longer qualifying as being of organic origin, if the PC was purified from organic biomass.
  • the liquid rich in PC is generally dried using spray drying processes.
  • sugars such as trehalose or maltodextrin and salts such as sodium citrate may be added in order to facilitate the spray drying process, protect the PC from the heat of the spray drying process and enhance the color and solubility of the powder emanating from the spray drying process.
  • salts such as sodium citrate
  • the form of delivery of the extracted and sometimes purified algae extract depends on its applications.
  • PC as a food colorant is delivered as a dry powder.
  • the document CN103285375 teaches the production of PC microspheres with an external oily layer, produced by the action of a calcium solution on an emulsion containing PC and sodium alginate in the water phase, paraffin and emulsifiers.
  • the document CN101322568 describes capsules containing Spirulina, sodium alginate, chitosan, additives and calcium chloride. PC diffusion during storage is not mentioned and does not occur because the PC is degraded by heat during the pasteurization process.
  • PC compositions useful in the present invention are produced by processes based on a chemical reaction between a gelling agent and a multivalent cation. Exposing the gelling agent to the multivalent cation (or vice versa) facilitates the formation of a gel and/or a membrane, trapping the cellular debris and other large-size molecular assemblies, but allowing diffusion of water-soluble small molecules and proteins into the surrounding aqueous medium, usually water, thereby permitting the purification of the desired substance such as PC.
  • the multivalent cation may also act on the cell wall of, for example Spirulina, thereby making it more fragile and porous and consequently allowing the extracellular diffusion of PC and other water-soluble molecules.
  • the methods of the present invention result not only in the purification of, for example PBP but also permit the extraction of PBP from Spirulina in an aqueous solution in a single step.
  • the methods described may be utilized with other species of microalgae, including, but not limited to Chlorella, Porphyndium, Aphanizomenon flos aquae (AFA) and Galdieria.
  • the processes to prepare purified PC from Spirulina comprises the following steps:
  • the processes to prepare purified PC from Spirulina comprises the following steps:
  • the processes to prepare purified PC from Spirulina comprises the following steps:
  • the different methods previously described to produce the purified PC compositions can be used simultaneously, sequentially or repeatedly.
  • the PC containing organism biomass is specially prepared through heat, cold, chemical and biological processes and physical mechanisms and techniques to facilitate the purification process of this invention.
  • the PC composition comprises a dried powder comprising a purified PBP composition as described above, the powder having a residual moisture content of less than about 10% w/w of the powder.
  • the PC composition comprises fresh or freshly harvested PBP containing organism biomass.
  • the microcapsule or other form contains chemicals or biological agents that enhance or retard or selectively control the purification of the PC.
  • the microcapsule or other form contains a natural substance that is purified or concentrated in conjunction with or ‘chaperoned’ into the aqueous solution by the PC or some other substance that exists in the organism biomass.
  • the aqueous solution into which the PC diffuses is modified by changing its temperature, through agitation or mixing or the addition of chemical or biological agents such as acids, alkalis, salts and antimicrobial agents to enhance or retard diffusion of PC and to prevent contamination.
  • the microcapsule or other form is frozen, dried or treated in some other way to enhance or retard diffusion of the PC into the aqueous solution.
  • the biomass used to prepare a PC composition useful in the present invention is the genus Arthrospira, and more preferably to the species Arthrospira platensis (commonly known as Spirulina).
  • the gelling agent is sodium alginate which reacts with most multivalent cations and especially well with the calcium ions.
  • Preferred sources of calcium ions are, for example, calcium chloride and calcium gluconate. The present invention is not limited to the use of calcium ions.
  • the multivalent cations may be provided by salts of manganese, magnesium, zinc, or barium.
  • the first solution is prepared with 10% to 60% Spirulina (wet weight), 0.1% to 5% sodium alginate (dry weight) and water.
  • Spirulina dry weight
  • sodium alginate dry weight
  • additives can be added to the solution including flavorings, color agents, preservatives, moisteners, natural antibiotics, thickeners, sugars, anti-foaming agents, salts, acids and alkalis.
  • the second solution is an aqueous solution containing multivalent cations at a concentration between 0.005 and 0.5 mole per liter, preferably between 0.05 and 0.2 mole per liter (corresponding for example to the range 5.5 - 22 g/L of calcium chloride).
  • This solution can also contain flavorings, color additives, preservatives and acidifying agents.
  • the present invention is not limited to any particular mechanism of action. Indeed, an understanding of the mechanism of action is not necessary to practice the invention. Nevertheless, it is contemplated that the microcapsules generated by dropping the first solution into the second solution are irregular, roughly egg-shaped with a maximum dimension of less than 6 mm and have a solid texture. Other forms can include tubular or spaghetti-like shapes, sausages, disks and irregular shapes.
  • the setting time, during which the microcapsules or other forms are immersed in the solution B, is preferably less than 1 hour.
  • the purification of the preferably takes place as the last step of the process. Specifically, the microcapsules are immersed in a large volume of water and the medium containing the microcapsules is kept at a low temperature (between 0 and 6°C) for from about 1 to 60 hours. The diffusion of the purified PC can demonstrated when the water become progressively bluer and shows a purple fluorescence when exposed to light. These observed phenomena as characteristic of the PC complex of Spirulina.
  • the microcapsules or other forms may be manufactured in a workshop or a factory and the extraction is undertaken at a consumer's home or at an industrial site or on a farm or in a laboratory.
  • microcapsules are packaged into a porous container such as a bag, a sachet, a pod or the like, with a pore size much lower than the microcapsule size to ensure that PC can diffuse while the microcapsules remain in the container.
  • the process steps are conducted at low temperatures (e g., between 0 and 6°C) to prevent PC degradation or natural or chemical preservatives are added.
  • the process of purification described in this invention has a number of advantages over existing algal biomass purification methods.
  • the main advantage is that it is possible to limit or even avoid the use of membrane-based filtration in the production of purified algal extracts such as PC, thereby substantially reducing the cost of production and improving the quality of such extracts.
  • the process described also does not require the use of salts and other substances to precipitate out the PC and the consequent expense of dialysis, osmosis, gels and exchange columns to remove such salts.
  • This invention also can increase the yield of PBP extraction and the concentration of proteins of interest in the purified extract. This invention permits the combination of extraction and purification in a single step, if desired.
  • compositions containing a high quality protein fraction that is enriched for PC are useful for producing compositions containing a high quality protein fraction that is enriched for PC.
  • the protein content and quality of the PC purified extracts obtained by these methods differ substantially from other described PC purified extracts, generally purified using membrane filtration methods.
  • the present invention provides the purified PC protein compositions are characterized by one or more of the following characteristics: a) the protein fraction of the composition comprises greater than about 30% of a protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; b) the protein fraction of the composition comprises greater than about 5% of a protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; c) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 45,578 kDa and an isoelectric point of about 6.2 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; d) the protein fraction of the composition comprises
  • the composition has at least two of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least three of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least four of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least five of characteristics a, b, c, d, e, f, g, h, i, j and k.
  • the composition has at least six of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least seven of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least eight of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least nine of characteristics a, b, c, d, e, f, g, h, i, j and k.
  • the composition has at least ten of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has all eleven characteristics a, b, c, d, e, f, g, h, i, j and k. It will be understood by those of skill in the art that in preferred embodiments, the compositions of the present invention may be identified by any subcombination of one or more of the characteristics identified above.
  • the purified phycobiliprotein composition is produced by a process comprising: mixing PCB containing organism biomass with water and gelling agent, forming a droplet, introducing a droplet of the first solution into a second solution containing a salt of divalent cations under conditions such that microcapsules form, and obtaining an extract enriched for PCB by mixing the microcapsules with a volume of an aqueous solution under conditions such that the phycobiliprotein diffuses from the microcapsules into the aqueous solution.
  • the purified PC composition is produced by a process comprising: mixing PC containing organism biomass with water and a salt of divalent cations, forming a droplet, introducing a droplet of the first solution into a second solution containing a gelling agent under conditions that microcapsules form, and obtaining an extract enriched for PC by mixing the microcapsules with a volume of an aqueous solution under conditions such that the PCB diffuses from the microcapsules into the aqueous solution.
  • the purified PC composition is produced by a process comprising: mixing PCB containing organism biomass with water, coating a droplet, extruded tube, sausage or some other form of this first solution with a second solution of gelling agent and introducing a droplet, extruded tube, sausage or some other form of the first solution coated with the second solution into a third solution containing a salt of divalent cations under conditions such that a coated droplet, extruded tube sausage or another form is obtained, and thereafter obtaining purified extract rich in PC by mixing the droplet, extruded tube, sausage or other form with a volume of an aqueous solution under conditions such that the PC diffuses from the droplet, extruded tube, sausage or other form into the aqueous solution.
  • the different methods previously described to produce the purified PC compositions can be used simultaneously, sequentially or repeatedly.
  • the PC compositions are provided as a dried powder.
  • the residual moisture in the powder is less than 5%, more preferably less than 4%, and most preferably less than 3% or 1%.
  • the powder may be produced by spray-drying, spray -freeze drying, refractance window drying, microwave drying, air drying, fluidized bed drying, vacuum drying, natural drying, microwave drying or foam drying the solutions of purified PCB.
  • the present invention provides methods for mushroom cell culture comprising culturing mushroom cells on substrate to provide a mushroom cell culture and contacting (or supplementing) the mushroom cell with a PC composition as described above.
  • the mushroom cells are psychoactive mushroom cells.
  • the mushroom cell culture produces one or more psychoactive compounds or other compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the methods further comprise processing the mushroom cell culture to provide or remove an aqueous fraction or extract from the culture comprising one or more psychoactive compounds, preferably one or more of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the extract may be made by separating an aqueous fraction from the mushroom cell culture.
  • the mushroom cell culture may be extracted with an aqueous or organic solvent or combinations thereof.
  • the fractions or extracts thus obtained may be further concentrated, for example by removal or of the aqueous or organic solvent by evaporation, lyophilization or spray drying, or by concentration via column chromatography.
  • the extract is prepared from the mushroom biomass by the encapsulation method described for PC purification, followed by a chromatography step, such as one or more of FLASH chromatography, ion-exchange chromatography, Centrifugal Partition chromatography or size-exclusion chromatography.
  • a chromatography step such as one or more of FLASH chromatography, ion-exchange chromatography, Centrifugal Partition chromatography or size-exclusion chromatography.
  • the chromatography steps may be selected so that psilocybin is removed from the extract.
  • the gelling agent used for encapsulation of the biomass is sodium alginate which reacts with most multivalent cations and especially well with the calcium ions.
  • Preferred sources of calcium ions are, for example, calcium chloride and calcium gluconate. The present invention is not limited to the use of calcium ions.
  • the multivalent cations may be provided by salts of manganese, magnesium, zinc, or barium.
  • the first solution is prepared with 10% to 60% of the mushroom biomass (wet weight), 0.1% to 5% sodium alginate (dry weight) and water.
  • additives can be added to the solution including flavorings, color agents, preservatives, moisteners, natural antibiotics, thickeners, sugars, anti-foaming agents, salts, acids and alkalis.
  • the second solution is an aqueous solution containing multivalent cations at a concentration between 0.005 and 0.5 mole per liter, preferably between 0.05 and 0.2 mole per liter (corresponding for example to the range 5.5 - 22 g/L of calcium chloride).
  • This solution can also contain flavorings, color additives, preservatives and acidifying agents.
  • the present invention is not limited to any particular mechanism of action. Indeed, an understanding of the mechanism of action is not necessary to practice the invention. Nevertheless, it is contemplated that the microcapsules generated by dropping the first solution into the second solution are irregular, roughly egg-shaped with a maximum dimension of less than 6 mm and have a solid texture. Other forms can include tubular or spaghetti-like shapes, sausages, disks and irregular shapes.
  • the setting time, during which the microcapsules or other forms are immersed in the solution B, is preferably less than 1 hour.
  • the purification of the preferably takes place as the last step of the process. Specifically, the microcapsules are immersed in a large volume of water and the medium containing the microcapsules is kept at a low temperature (between 0 and 6°C) for from about 1 to 60 hours.
  • the microcapsules or other forms may be manufactured in a workshop or a factory and the extraction is undertaken at a consumer's home or at an industrial site or on a farm or in a laboratory.
  • microcapsules are packaged into a porous container such as a bag, a sachet, a pod or the like, with a pore size much lower than the microcapsule size to ensure that water soluble compounds can diffuse while the microcapsules remain in the container.
  • the process steps are conducted at low temperatures (e.g., between 0 and 6°C).
  • the mushroom extracts are provided as a dried powder.
  • the residual moisture in the powder is less than 5%, more preferably less than 4%, and most preferably less than 3% or 1%.
  • the powder may be produced by spray-drying, spray -freeze drying, refractance window drying, microwave drying, air drying, fluidized bed drying, vacuum drying, natural drying, microwave drying or foam drying the solutions of purified PBP.
  • psilocybin or psilocin, or a composition comprising psilocybin such as the mushroom cell culture extracts described herein are co-administered with a 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • the present invention also provides formulations comprising psilocybin or psilocin, or a composition comprising psilocybin such as the mushroom cell culture extracts described herein, and a 5-HTIA receptor agonist, as well as kits comprising a psilocybin or psilocin formulation and a 5-HTIA receptor agonist formulation.
  • the mushroom cell culture extracts and fractions may be further formulated with one or more pharmaceutically acceptable carriers or delivery vehicles.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • the mushroom cell extracts of the present invention may be formulated with different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the mushroom cell extracts of the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation and nebulization (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
  • mushroom cell extracts of the present invention will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • the mushroom cell extracts the present invention suitable for administration is provided in a physiologically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the mushroom cell extract is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the actual dosage amount of a mushroom cell extract of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration: Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • the present invention provides entheogen compositions as described above for the treatment of psychiatric disorders.
  • the present invention provides mushroom cell culture compositions comprising psychoactive compounds for the treatment of psychiatric disorders.
  • the present invention is not limited to the treatment of any particular psychiatric disorders. Indeed, the treatment of a variety of psychiatric disorders is contemplated, including, but not limited to, post-traumatic stress disorder (PTSD), depressive and anxiety disorders, schizophrenia, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
  • PTSD post-traumatic stress disorder
  • OCD obsessive compulsive disorder
  • the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of depression, schizophrenia, anxiety, PTSD or OCD.
  • the entheogen compositions comprise one or more of the following compounds: psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin.
  • the entheogen compositions comprise psilocybin and one or more of the following compounds: baeocystin, aeruginascin, norpsilocin, norbaeocystin.
  • the effective dosage of the entheogen composition is a subpsychedelic dose.
  • a "subpsychedelic dose” as used herein is a sub-hallucinogenic doses of a psychedelic substance such as a psilocybin.
  • Sub-psychedelic dosing can be achieved via several preferred schedules: 1) Sub-chronic, sub-psychedelic doses of psilocybin given over several days with or without one or more the entourage compounds baeocystin, aeruginascin, norpsilocin, and norbaeocystin ; 2) A sustained release preparation of psilocybin with or without one or more the entourage compounds baeocystin, aeruginascin, norpsilocin, and norbaeocystin which will allow the full psychedelic dose to be administered over 24 hours and thus avoid acute psychotogenic effects; 3) A psychedelic dose of psilocybin with or without one or more the entourage compounds baeocystin,
  • the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of OCD.
  • Obsessive symptoms to be treated include: fear of contamination or dirt; doubting and having difficulty tolerating uncertainty, needing things orderly and symmetrical, aggressive or undesirable thoughts about losing control and self-harming or harming others; and unwanted thoughts, including aggression, or sexual or religious subjects.
  • Compulsive symptoms to be treated include: excessive hand-washing; checking doors repeatedly to make sure they are locked, checking the stove repeatedly to make sure it is off; counting in certain patterns; silently repeating a prayer, word or phrase; arranging items in a certain order.
  • administration of the entheogen composition comprising one or more psychoactive compounds alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof.
  • the dosage is a sub-perceptual or sub-psychedelic dose.
  • the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of schizophrenia.
  • the symptoms to be treated include: hallucinations, delusions and thought disorder and negative symptoms which include impairments of volition, social functioning and affect as well as deficits in cognition, specifically executive functions.
  • the dosage is sub-chronic, sub-psychedelic or sustained release preparation of psilocybin which will allow the full psychedelic dose to be administered over 24 hours or a psychedelic dose of psilocybin in conjunction with 5-HT2A receptor antagonist, that blocks psilocybin-induced psychedelic effects or a psychedelic dose of psilocybin in conjunction with a 5-HT1A agonist such as buspirone which will prevent the possible psychosis enhancing effects while at the same time exerting a possible dopaminergically mediated beneficial effect via dopamine D2 receptor antagonist effects of buspirone.
  • a 5-HT1A agonist such as buspirone
  • the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of PTSD.
  • the symptoms to be treated include: Symptoms of intrusive memories, including, but not limited to recurrent, unwanted distressing memories of the traumatic event, reliving the traumatic event as if it were happening again (i.e., flashbacks), upsetting dreams or nightmares about the traumatic event, and severe emotional distress or physical reactions to something that reminds the subject of the traumatic event; Symptoms of avoidance including, but not limited to trying to avoid thinking or talking about the traumatic event; avoiding places, activities or people that remind the subject of the traumatic event, and negative changes in thinking and mood; Symptoms of negative changes in thinking and mood including, but not limited to, negative thoughts about the subject, other people or the world; hopelessness about the future; memory problems, including not remembering important aspects of the traumatic event, difficulty maintaining close relationships, feeling detached from family and friends, lack of interest in activities the subject once enjoyed, difficulty experiencing positive emotions,
  • administering alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof.
  • the dosage is a sub-perceptual or sub-psychedelic dose.
  • the present invention provides methods of providing prophylaxis for PTSD in a subject in need thereof comprising administering to the subject an effective amount of an entheogen composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the method comprises administering to the subject an effect amount of an entheogen composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the effective amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD.
  • the entheogen composition is preferably administered to the subject when the subject presents with a traumatic event or injury or soon thereafter.
  • the entheogen composition is administered to the subject within 4 weeks, 1 week, 3 days or 1 day of a psychological trauma or other traumatic event.
  • the dosage is a sub-perceptual or sub-psychedelic dose.
  • the present invention provides entheogen compositions as described above for the treatment of autism spectrum disorder.
  • the present invention provide mushroom cell culture compositions comprising psychoactive compounds, including entheogens, for the treatment of autism spectrum disorder.
  • the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of ASD.
  • the entheogen compositions comprise one or more of the following compounds: psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin.
  • the entheogen compositions comprise psilocybin and one or more of the following compounds: baeocystin, aeruginascin, norpsilocin, norbaeocystin.
  • the effective dosage of the mycelial culture compositions is a microdose or subpsychedelic dose as described above.
  • the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of ASD, including but not limited to symptoms related to social communication and interaction and behavioral symptoms.
  • Symptoms related to social communication and interaction include one or more of failure to respond to name, resistance to holding or cuddling, poor eye contact, lack of facial expression, delayed speech or failure to speak, inability to maintain conversation, speaking with an abnormal tone, repetitive use of words, failure to understand or comply with directions, failure to express emotions, and inability to recognize nonverbal cues.
  • Behavioral symptoms include one or more of performance of repetitive movements, performance of activities that can lead to self-harm such as head-banging, performance of specific routines, lack of coordination or performance of odd movement patterns, sensitivity to light or touch, and abnormal fixation on object or activity.
  • administration of the entheogen composition comprising one or more psychoactive compounds alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof.
  • the dosage is a microdose or subpsychedelic dose.
  • the subject is less than 60 years old. In some preferred embodiments, the subject is less than 50 years old. In some preferred embodiments, the subject is less than 40 years old. In some preferred embodiments, the subject is less than 30 years old. In some preferred embodiments, the subject is less than 20 years old. In some preferred embodiments, the subject is less than 10 years old. In some preferred embodiments, the subject is less than 9 years old. In some preferred embodiments, the subject is less than 8 years old. In some preferred embodiments, the subject is less than 7 years old. In some preferred embodiments, the subject is less than 6 years old. In some preferred embodiments, the subject is less than 5 years old. In some preferred embodiments, the subject is less than 4 years old. In some preferred embodiments, the subject is less than 3 years old. In some preferred embodiments, the subject is less than 2 years old.
  • the present invention provides entheogen compositions as described above for the treatment of brain injuries including, but not limited to, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multisystem trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma).
  • TBI traumatic brain injury
  • CTE chronic traumatic encephalopathy
  • concussion multisystem trauma including brain injury
  • multisystem trauma multisystem trauma including brain injury
  • severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma).
  • the present invention provides mushroom cell culture compositions comprising psychoactive compounds, including entheogens, for the treatment of brain injuries including, but not limited to, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma).
  • TBI traumatic brain injury
  • CTE chronic traumatic encephalopathy
  • concussion multi-system trauma including brain injury
  • multi-system trauma multi-system trauma including brain injury
  • severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma).
  • the present invention provides full spectrum entheogen compositions or full spectrum mushroom extracts (FSMEs) comprising one or more psychoactive compounds for the treatment of brain injuries.
  • the FSME is an extract from the fruiting body of mushrooms.
  • the present invention provides mushroom cell culture compositions comprising one or more psychoactive compounds for the treatment of brain injuries.
  • the mushroom cell culture compositions comprise one or more, two or more, three or more, four or more, or all of the following compounds: psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin.
  • the mushroom cell culture compositions comprise psilocybin and one or more, two or more, three or more of all of the following compounds: baeocystin, aeruginascin, norpsilocin, norbaeocystin.
  • the effective dosage of the entheogen composition is a microdose or sub-hallucinogenic dose.
  • the entheogen composition is administered immediately before, during or immediately after anesthesia or sedation. In some embodiments, the entheogen composition is administered concurrently or serially to a subject in need thereof with an anesthesia or sedation agent. In some embodiments, administration of the entheogen composition not only treats the brain injury but also to prevent sequelae such as neurodegeneration, traumatic encephalopathy, PTSD, addiction disorders, cognitive decline and post-traumatic depression.
  • the present invention provides entheogen compositions as described above comprising one or more psychoactive compounds for the treatment of one or more symptoms of brain injuries.
  • Symptoms of mild TBI include, but are not limited to, headache, nausea or vomiting, fatigue or drowsiness, problems with speech, dizziness or loss of balance, blurred vision, ringing in the ears, a bad taste in the mouth or changes in the ability to smell, sensitivity to light or sound, memory or concentration problems, mood changes or mood swings, feeling depressed or anxious, difficulty sleeping, sleeping more than usual.
  • Symptoms of moderate to severe TBI include, but are not limited to, persistent headache or headache that worsens, repeated vomiting or nausea, convulsions or seizures, inability to awaken from sleep, weakness or numbness in fingers and toes, loss of coordination, profound confusion, agitation, combativeness or other unusual behavior, slurred speech, and coma and other disorders of consciousness.
  • Symptoms of CTE include, but are not limited to, difficulty thinking (cognitive impairment), memory loss, problems with planning, organization and carrying out tasks (executive function), impulsive behavior, aggression, mood disorders, depression or apathy, emotional instability, substance misuse, suicidal thoughts or behavior, Parkinsonism, and motor neuron disease.
  • administration of the entheogen composition comprising one or more psychoactive compounds alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof.
  • the present invention provides methods of promoting neuroplasticity in a subject in need thereof comprising administering to the subject an entheogen composition comprising psilocybin or psilocin and one or more entourage compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
  • the entheogen composition comprises psilocybin.
  • the entheogen composition is an entheogen composition as described supra. 223.
  • the entheogen composition is co-administered with a 5-HTIA receptor agonist.
  • the present invention is not limited to the use of any particular 5-HTIA receptor agonist.
  • the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
  • the subject in need thereof has a disease, condition or disorder associated with neural atrophy or the impairment of neuroplasticity .
  • the disease, condition or disorder associated with neural atrophy or the impairment of neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD.
  • the psychiatric disease or disorder includes, but is not limited to, depression, anxiety, social anxiety disorder, obsessive compulsive disorder and major depressive disorder.
  • the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or Huntington's disease.
  • the brain injury or disorder is traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma).
  • administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43 and Synaptophysin in one or more areas of the brain selected from the group consisting of the frontal cortex, amygdala, hippocampus, and striatum.
  • the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with neural atrophy or the impairment of neuroplasticity.
  • Drugs Chemically synthesized psilocybin and psilocin will be provided by Usona Institute, Madison, Wisconsin, U.S.A. Naturally-derived psilocybin, psilocin and mushroom extracts will be provided by Back of the Yards Algae Sciences (BYAS), Chicago, U.S.A. Other drugs and chemicals will be purchased from Sigma-Aldrich Israel Ltd. Drugs will be administered by intraperitoneal (i.p.) or subcutaneous (s.c.) injection or, for sub-chronic administration of 7 days or more, by Alzet osmotic minipumps implanted subcutaneously. Animals. Outbred C57BL/6 mice will be used. Mice will be aged 12 weeks.
  • mice Parallel groups of male and female mice will be used for all experiments. When mice are re-used for additional experiments, this will be after administration of a single drug dose and with an interval of at least 7 days. Animals will be housed under standard conditions with a 12-hour light dark cycle. Additional strains of mice will be used if they are more applicable for specific experiments e.g. ICR mice for marble burying. Rats (Sprague Dawley strain) will be used for pharmacokinetics experiments and where otherwise indicated. Behavioral assays will be performed at the same time each day. Animals from different experimental groups will be tested in counterbalanced order.
  • affinity of psilocin, the active metabolite of psilocybin, for 5-HT2A, 5-HTC and 5-HT1A receptors will be examined in HEK-293 cells that over-express these receptors.
  • radioligands and competitors will be used: [3H]8-hydroxy-2-(di-n-propylamine) tetralin and indatraline (5-HT1A receptor) [3H]ketanserin and spiperone(5-HT2A receptor) [3H] mesulgerine and mianserin(5-HT2C receptor).
  • HTR as a marker of psilocybin action by determining modification of HTRs following administration to psilocybin treated animals of the specific 5-HT2A receptor (5-HT2AR) antagonist, M100907; the 5-HT2CR antagonist, SB242084, the 5-HT1 AR agonist, 8OHDPAT, and the trace amine associated receptor 1 (TAAR1) antagonist, EPPTB.
  • 5-HT1 A agonist 8OH-DPAT inhibits HTR induced by psilocybin
  • 5-HT1A agonists and partial agonists including but not limited to flesinoxan, gepirone, ipsapirone and buspirone.
  • Psilocybin and most other naturally occurring psychedelic agents are tryptamine, phenethylamine or ergoline derivatives. It is important for future drug development to evaluate compounds that belong to a different chemical category but are nevertheless 5HT2AR agonists and inducers of HTR.
  • the antiviral agent efavirenz
  • efavirenz which has been reported to have psychedelic effects but is not a tryptamine, phenethylamine or ergoline.
  • 5-HTP (5-hydroxytryptophan) will be used as a control in these experiments since this agent stimulates 5-HT2AR but is not psychedelic at regular doses.
  • mice will be administered the maximally effective doses of all agonists and antagonists as determined in the dose-response experiments. After 45 min. the animals will be sacrificed and tissue from frontal cortex will be taken for real time PCR evaluation of treatment effects on the immediate early genes (IEG), c-fos, egr-1 and egr-2.
  • IEG immediate early genes
  • c-fos egr-1 and egr-2.
  • mice will be administered psilocybin, efavirenz or 5HTP at the dose shown to have maximum effect in the dose response studies, after having received sub-chronic escitalopram at a dose of lOmg/kg/day via osmotic minipumps for two weeks.
  • SSRI serotonin reuptake inhibitor
  • sub-chronic administration (7-14 days via osmotic minipumps implanted subcutaneously), the maximal dose that did not induce HTR will be administered daily (sub-psychedelic dose).
  • sub-psychedelic dose the maximal dose that did not induce HTR will be administered daily (sub-psychedelic dose).
  • sustained release psilocybin at full psychedelic dose over 24 hours, administered i.p. or orally.
  • psilocybin administered concurrently with a 5-HT1 A agonist such as buspirone in order to determine whether psychedelic effects (represented by HTR in the rodent) are required in order to achieve therapeutic effects.
  • mice The following mouse models will be used.
  • Activity and motor function Open field Test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84-99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3; b) Sensorimotor gating - Pre-pulse inhibition, a model for positive symptoms, will be used to test whether psilocybin has an exacerbating effect on psychosis.
  • the test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84-99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3; c) Cognitive function - Tests in this category will be used to examine the effect of psilocybin on cognitive and negative features, especially cognitive flexibility. These tests will include:
  • Neonatal injections of MK801 treatment will be used as a translational model of schizophrenia.
  • mice are injected with 0.5mg/kg MK80I s.c. daily, from PND6 to PND13.
  • Treatment with sub-chronic, sub-psychedelic dose of psilocybin- rich mushroom extract (PRME), sustained release psilocybin, psilocybin + ketanserin, psilocybin + buspirone, psilocybin-free mushroom extract (PFME) or phycocyanin-rich spirulina extract (PRSE) will commence 14 days before the beginning of the behavioral battery and will continue through the battery.
  • Acute injections of MK801 will be used as a model for schizophrenia to specifically determine the effect of treatments on positive symptoms.
  • a single injection of MK801 or amphetamine will be used prior to measurement of the behavioral and electrophysiological endpoints.
  • Mice will be pretreated with sub-chronic, sub-psychedelic doses of psilocybin-rich mushroom extract (PRME), sustained release psilocybin, psilocybin + ketanserin, psilocybin + buspirone, psilocybin-free mushroom extract (PFME) or phycocyanin-rich spirulina extract (PRSE) as in the neonatal MK801 model above.
  • PRME psilocybin-rich mushroom extract
  • PFME psilocybin + ketanserin
  • PFME psilocybin + buspirone
  • PFME psilocybin-free mushroom extract
  • PRSE phycocyanin-
  • Expt 1 Neonatal MK801/adult sub-chronic psilocybin experiment.
  • Expt 2 Neonatal MK801/ acute psychedelic psilocybin + ketanserin/buspirone or sustained release administration of psychedelic psilocybin dose experiment.
  • Expt 3 Acute MK801/ acute psychedelic psilocybin + ketanserin or buspirone or sustained release administration of psychedelic psilocybin.
  • mice will be needed for this experiment.
  • Method MK801 or vehicle administration daily from PND6 to PND13. Combination experiment in which the mushroom extract that gave the best results in Expt. 1 will be tested in combination with PRSE. Psychedelic drug treatment will start on PND70, 14 days before beginning of behavioral battery. Behavioral battery on PND84-99. Sacrifices on PND100. Sub-psychedelic dose of PRME will be as defined in prior dose response experiments.
  • a) Activity and motor function'. Open field Test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84-99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3.
  • Sensorimotor gating - Pre-pulse inhibition a model for positive symptoms, will be used to test whether psilocybin has an exacerbating effect on psychosis. The test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84- 99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3.
  • Cognitive function - Tests in this category will be used to examine the effect of psilocybin on cognitive and negative features, especially cognitive flexibility. These tests will include:
  • NMD NRI subunit, parvalbumin (PV), double staining of GAD67 and synaptophysin in the frontal cortex.
  • PV parvalbumin
  • FC Fear conditioning
  • Mice are exposed to a single 2-hour restraint episode in clean, ventilated, 50ml conical vials (such as Falcon tubes) one week prior to conducting a fear conditioning protocol, in which each mouse is entered into the conditioning chamber for two minutes of uninterrupted exploration followed by two blocks of 20-second 80Db 2000Hz tone (conditioned stimulus) paired with 2-second 0.5mA footshock (unconditioned stimulus), and a further 60 seconds of uninterrupted exploration of the chamber.
  • Plasma corticosterone level is measured 30 minutes after the end of FC protocol.
  • mice Four days after conditioning mice undergo 2-day intensive extinction protocol and the time mice spend freezing in each extinction trial is measured. The combination of pre-exposure to stress with FC protocol induces higher plasma corticosterone level and resistance to fear extinction. 24 days later a recall test is performed.
  • Psilocybin will be administered according to the following regimens: i) Single injection i.p. of the maximal HTR-inducing dose 30 min. after the completion of the FC protocol and withdrawal of blood for plasma corticosterone level. ii) Sub-chronic treatment with psilocybin via osmotic minipumps or i.p. injection at a sub-psychedelic daily dose, commencing 30 min. after the completion of the FC protocol and withdrawal of blood for plasma corticosterone level, and continuing until the recall test on day 30. iii) Single i.p.
  • OCD - Marble Burying and Nestlet Shredding are for evaluating levels of repetitive compulsive behavior characteristic of OCD and autism and the effects of treatments to reduce these behaviors.
  • each mouse is introduced into a cage with 25 marbles that are presented on the top of the sawdust (unburied). The number of marbles buried by the animal after 30 minutes' exposure is recorded.
  • the Nestlet Shredding test weighted cotton fibers are introduced into cage, and thereafter an animal is entered into that cage. After the animal spends 30 min in the cage, it is returned to its home cage and the unshredded cotton nestlets are weighed (the more compulsive the animal the less unshredded material will remain in the cage).
  • Sapap3 KO mice Mice with genetic deletion of the Sapap3 gene exhibit increased anxiety and compulsive grooming behavior leading to facial hair loss and skin lesions. Both behaviors are alleviated by SSRIs which are an effective treatment in some cases of OCD. We will examine whether treatment with psilocybin alleviates the increased anxiety and compulsive grooming behavior of Sapap3 KO mice.
  • CUS Depression - Chronic Unpredictable Stress
  • UMS Unpredictable Mild Stress
  • Inflammation-induced depression models On the background of the strong association between depression and inflammation, depression models in rodents can be implemented using inflammatory agents such as lipopolysaccharide (LP and the viral mimetic Poly EC. Treated animals lose weight, eat and drink less, and decrease their motor activity for several hours to days, depending on the nature of the inflammatory agent and dose. These sickness behaviors correspond with elevations in proinflammatory cytokines at the periphery and in the brain. In the LPS-induced model of depression, which we will implement, sickness behaviors will typically resolve within 24 hours. When sickness behaviors have resolved, the rodents display depressive- like behaviors.
  • LP lipopolysaccharide
  • the viral mimetic Poly EC the viral mimetic Poly EC.
  • Neuroplasticity gene expression.
  • the therapeutic action of psychedelic compounds in treatment resistant psychiatric disorders is related to their effects on neuroplasticity i.e., the ability of neural networks in the brain to change through growth, reorganization, and new connections.
  • synaptic plasticity is particularly evident in the prefrontal cortex of mice treated with psychedelics which is a brain region rich in 5-HT2A receptors to which classical psychedelics preferentially bind. This is consistent with prior reports that neuroplastic mechanisms play a key role in the rapid antidepressant action of the hallucinatory anesthetic agent, ketamine and also in the slower in onset effects of specific serotonin reuptake inhibitors.
  • mice receiving acute, psychedelic or sub-chronic, sub-psychedelic psilocybin treatment will be evaluated and also mice treated with psychedelic doses administered over 24 hours by sustained release.
  • Using western blotting we will assay key molecular markers of synaptic plasticity such as GAP-43, PSD-95 and synaptophysin.
  • psilocybin In common with other serotonergic psychedelic agents, psilocybin is thought to act via the 5-HT2A receptor to which its active metabolite, psilocin, binds with high affinity. In rodents, psilocybin induces a characteristic head twitch response (HTR), which is highly correlated with the psychedelic trip in humans in terms of intensity.
  • HTR head twitch response
  • Drugs were administered by intraperitoneal (i.p.) injection immediately before the assessment of HTR commenced.
  • Psilocybin was administered at doses of 0.1 mg/kg to 51.2 mg/kg i.p. alone, or at a dose of 4.4 mg/kg i.p. preceded by:
  • 5-HT2A receptor antagonist M107900 (0.5, and 2 mg/kg i.p.);
  • 5-HT2C receptor antagonist RS-102221 (2, 4, and 8 mg/kg i.p.);
  • 5-HT1A receptor agonist 8OH-DPAT (1, and 2 mg/kg i.p );
  • TAAR 1 antagonist EPPTB (1, and 10 mg/kg i.p.).
  • HTR was measured for 20 minutes in a custom-built magnetometer using mini magnets tagged onto the ears of the mice. Recording and data display employed proprietary software
  • FIG. 1A to ID provide data on psilocybin dose response. These graphs depict (FIG. 1A full range dose response of HTR through the course of 18 min, (FIG. IB) taken from (i) the distinct shape of faster and higher onset following a rapid decrease in response (doses high then 3mg/kg), (FIG. 1C) taken from A the distinct shape of flatter response that carries over time (doses lower then 1.6 mg/kg), (FIG. lD)dose response curve taken from the data shown in A.
  • FIGS. 2A to 2D provide data on the effects of serotonin receptor modulators on psilocybin-induced HTR (Head Twitch Response).
  • the bar graphs show the cumulative HTR recorded by the mice during 20 min session immediately following injection of either psilocybin (vehicle) alone or combined with a receptor modulator.
  • FIG. 2A Shows the effect of a combined psilocybin + 5HT2A antagonist, which completely reduced HTR in both concentrations.
  • FIG. 2B Shows the effect of a combined psilocybin + 5HT1 A agonist, which reduced HTR in dose depended manner.
  • FIG. 2C Shows the effect of a combined psilocybin + TAAR1 antagonist, which did not alter HTR.
  • FIG. 2D Shows the effect of a combined psilocybin + 5HT2C antagonist, which increased HTR with 4 mg/kg and reduced the HTR-increasing effect at 8mg/kg.
  • Psilocybin induced a dose dependent increase in the frequency of HTR over 20 minutes. With doses in excess of 25.6 mg/kg i.p. there was a reduction in the frequency of HTR. With higher doses of psilocybin, the increase in HTR frequency was more rapid as was the decline.
  • the 5-HT2A receptor antagonist, Ml 07900 completely blocked HTR.
  • the 5-HT2C receptor antagonist, RS-102221, enhanced HTR at lower doses but reduced it at higher doses.
  • Fig 6a shows the time course of the effect of psilocybin and buspirone on HTR.
  • Psilocybin PSIL was administered at a dose of 4.4 mg/kg i.p. alone, or preceded by the 5- HT2A receptor antagonist, M107900 (2 mg/kg i.p.), the 5-HT2C receptor antagonist, RS-102221 (4 mg/kg i.p ), the 5-HT1 A receptor agonist, 8OH-DPAT (2 mg/kg i.p ), or buspirone (Busp).
  • Escitalopram(ESC, 5mg/kg i.p. was administered as a positive control. Marble burying was measured.
  • MTT Marble-burying test
  • Twenty glass marbles were placed equidistant from each other in a 5 x 4 pattern. The experiment was done under dim light in a quiet room to reduce the influence of anxiety on behavior. The mice were left in the cage with the marbles for a 30-min period after which the test was terminated by removing the mice. Number of buried marbles was counted after 10, 20 and 30 minutes. All mice underwent a pretest without any injection and the number of marbles buried was counted. Only mice that buried at least 15 marbles were selected to perform the test after drug administration. 80 % of pretested mice fulfilled this criterion and were used in the definitive experiment which took place at least a week following the pretest.
  • the effect of psilocybin was not statistically different from the effect exerted by a positive control, the SSRI escitalopram (48.43% reduction in marble-burying relative to vehicle; p ⁇ 0.0001, Fig 3a).
  • mice were placed in an open field on completion of the MBT and were monitored for 30 minutes using the Ethovision Video Tracking System (Noldus Information Technology BV). As shown in Fig. 7a there was no significant difference in distance travelled between vehicle treated mice and those administered psilocybin or buspirone. Similarly, there was no difference in time spent by the mice in the center of the open field (center duration) (Fig 7b) or in the periphery of the open field (periphery duration) (Fig 7c) under treatment with psilocybin or buspirone compared to treatment with vehicle.
  • center duration center duration
  • Fig 7c periphery of the open field
  • test compound will be administered in the following formats: Psilocybin in Vehicle; Psilocybin in Mycelial Extract; Mycelial Extract alone; Vehicle without test compound.
  • CNTNAP2 The contactin-associated protein-like 2 (CNTNAP2)' gene encodes a neuronal transmembrane protein member of the neurexin superfamily involved in neuron-glia interactions and clustering of K+ channels in myelinated axons31. This gene also plays a role in synapse formation and stabilization.
  • the dendritic spine dynamics are affected in the Cntnap2 knockout (KO) mice causing reduced stability in newly formed spines.
  • Loss-of-function mutations in Cntnap2 have been implicated in ASD and cortical dysplasia-focal epilepsy syndrome. Feliciano, Cntnap2-/- autism model. Nat Genet. 2011 ;43 : 1053- 1053.
  • Studies on Cntnap2-/- mice have shown spontaneous seizures, stereotypic motor movements, and behavioral inflexibility. Penagarikano et al., supra.
  • Knockout pups emitted fewer isolation-induced ultrasonic vocalizations (distress calls) to their mothers and spent less time interacting with other mice in a juvenile play test. Id. These behavioral patterns are typical for ASD.
  • mice 1- Establishing two groups of mice: CASPR-KO and WT groups.
  • mice 3- In total, -200 male mice will be included in this study.
  • mice were allowed to explore the arena without objects for 5 min to habituate to their surroundings. During the first session for 5 min the mice were left to explore two identical objects that found at constant locations, 15 cm from the sidewalls, in the already familiar black plastic arena. 24 h later the mice were introduced to the arena for a test session in which one of the familiar objects was replaced with a novel object (Place recognition: same object is moved). The time spent by the mouse in exploring each object will recorded for 5 min. Elevated Plus Maze Test
  • the elevated plus-maze consisted of four arms (30*5 cm), two open and the other two closed.
  • the platform was made of white plexiglass.
  • the apparatus was elevated 45 cm above the floor.
  • the test (described in Waif and Frye, The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc. 2007;2:322-8), will be initiated by placing the mouse on the central platform of the maze, facing one of the open arms, and letting it move freely. Each session will be lasted for 10 min. The time spent in the close and open arms will be recorded. First day is habituation and second day is the test itself.
  • a three-chamber social test will be performed (as described in Silverman et al., Behavioural phenotyping assays for mouse models of autism. Nat Rev Neurosci. 2010; 11 :490- 502, with some modifications).
  • the social test apparatus consisted of a transparent acrylic box divided into three chambers. Two cylindrical wire cages were placed, one in chamber 1 and the other in chamber 2.
  • the test animal will be introduced to the middle chamber and allowed to adjust for 5 minutes, then in the next day an unfamiliar mouse will be introduced into a wire cage in one of the side-chambers and an empty wire cage on the other side-chamber. The time spent by the test mouse in exploring the wire cage with an unfamiliar mouse inside it will be recorded for 5 minutes.
  • DMT reduces infarct size and improves functional recovery following transient focal brain ischemia in rats
  • N N-dimethyltryptamine reduces infarct size and improves functional recovery following transient focal brain ischemia in rats.
  • Experimental neurology 327, 113245.
  • Preclinical research in rodents has shown that psychedelics enhance synaptic plasticity in key brain regions linked to psychological functioning (Ly et al., (2016) Psychedelics promote structural and functional neural plasticity. Cell reports, 23, 3170-3182).
  • psychedelics may be dissociated from their psychological effects (i.e., the 'psychedelic trip') and may be achieved at sub-psychedelic doses (i.e., lower doses than required to induce discernible perceptual effects in humans) and without 5-HT2A receptor-mediated behavioral stereotypies in rodents.
  • mushrooms have prebiotic functions based on their abundances in carbohydrates, like chitin, hemicellulose, y and P-glucans, mannans, xylans, and galactans (Cerletti et al., (2021) Edible Mushrooms and Beta-Glucans: Impact on Human Health. Nutrients, 13, 2195).
  • mycelial extract devoid of the main psychoactive tryptamines (psilocin and psilocybin), on affective, metabolic and cognitive functions is feasible and necessary.
  • Psychedelics exert significant modulatory effects on immune responses by altering signaling pathways involved in inflammation, cellular proliferation, and cell survival (Szabo, Psychedelics and immunomodulation: novel approaches and therapeutic opportunities. Frontiers in immunology, 6, 358). Kuypers, ((2019) Psychedelic medicine: The biology underlying the persisting psychedelic effects. Medical hypotheses, 125, 21-24) suggested that that low doses of psychedelics exert their effects on mental well-being through indirect modulation of the gut-brain axis.
  • Astaxanthin a vivid red pigment 'carotenoid' is a well-known nutraceutical and is considered to be one of the strongest antioxidants in nature, and of possible high potential as an anti-neuroinflammatory agent (Galasso et al., (2016) On the neuroprotective role of astaxanthin: new perspectives. Marine drugs, 16, 247). Due to its high free-radical scavenging activity and ability to easily pass through the blood-brain barrier, astaxanthin is a potential natural biomolecule for application in the protection against traumatic insults to the brain.
  • Phycocyanin is a fluorescent, water-soluble protein that is part of the photosynthetic system of blue-green algae such as Spirulina. Like astaxanthin, phycocyanin is a potent antioxidant with strong anti-microbial, neurotrophic, and anti-inflammatory properties (Min et al., (2015) Assessment of C-phy cocyanin effect on astrocyte s-mediated neuroprotection against oxidative brain injury using 2D and 3D astrocyte tissue model.
  • Outcome measures will include standard measures of cognition and behavior, brain histology, synaptic plasticity, biomarker, gene expression and -omics, inflammatory response, and microbiome.
  • mice Outbred C57BL/6 mice will be used. Mice will be aged 12 weeks. Parallel groups of male and female mice will be used for all experiments. Animals will be housed under standard conditions with a 12-hour light-dark cycle. Behavioral assays will be performed at the same time each day. Animals from different experimental groups will be tested in counterbalanced order.
  • Natural psilocybin (PSI), psilocybin rich full spectrum mushroom extract (PRME), psilocybin free mycelial extract (PFME), phycocyanin rich spirulina extract (PRSE), and astaxanthin (AST) will be provided by Back of the Yards Algae Sciences (BYAS) (IL, USA). Other chemicals will be purchased from Sigma-Aldrich (IL, USA).
  • Treatments will be administered to groups of mice that undergo head trauma. Treatments will be administered in the context of a series of experiments in which the treatments will first be examined alone and then in systematically designed combinations: a) Natural, mycelial derived psilocybin (PSI) b) Full spectrum mushroom extract containing psilocybin (FSME) c) Mycelial extract free of psilocybin (PFME) d) Phycocyanin rich spirulina extract (PRSE) e) Naturally derived astaxanthin (AST) f) Vehicle (VEH)
  • PSI Natural, mycelial derived psilocybin
  • FSME Full spectrum mushroom extract containing psilocybin
  • PFME Mycelial extract free of psilocybin
  • PRSE Phycocyanin rich spirulina extract
  • AST Naturally derived astaxanthin
  • VH Vehicle
  • HTR head twitch response
  • mice will be administered increasing doses (0 - 30 mg/kg) of PSI and FSME and psychedelic dosage will be established.
  • increasing doses of PFME will be administered to establish HTR is not induced.
  • HTR will be evaluated by the use of a magnetometer as previously described. Following on, mice will be administered a single administration of the highest doses of PSI, FSME, and PFME that do not induce HTR. Fifteen minutes after administration mice will be sacrificed and frontal cortex specimens will be obtained and frozen for analysis of IEG.
  • mice are lightly anesthetized with isoflurane (i.e., until unresponsive to paw or tail pinch) and placed immediately under a vertical PVC tube. Mice are suspended chest-down on a slit piece of aluminum foil 10 cm above a foam cushion.
  • the mouse is quickly positioned so that its head is directly in the path of the falling weight by first resting the weight on the scalp midline between bregma and lambda. Incisions in the scalp or emplacement of a protective skull helmet are not necessary. The weight is then pulled rapidly upward by an attached string to the desired drop distance and released. The downward traverse of the falling weight is restricted by the string such that upon contact, the weight travels no more than 1 cm beyond the original position of the dorsal surface of the head. Immediately upon impact, the mouse falls freely onto the foam cushion. In this arrangement, the impact-induced acceleration and fall always involve a 180° horizontal rotation of the mouse body. The mouse is moved immediately to a holding cage to recover. Mice will undergo five consecutive daily applications of the trauma which is analogous to repeated mild traumatic brain injury. Mice will be subjected to 5 consecutive, daily exposures to the head trauma model.
  • mice Because of the large number of cognitive and behavioral outcome measures, mice will be treated in two cohorts (A and B) and the tests will be performed on the two cohorts separately as indicated:
  • Cognitive function tests Novel object recognition (A); Radial arm water maze (B).
  • Affective/ Anxiety - like outcome measures Forced swim test (depression-like) (A); Elevated plus maze (anxiety-like) (B); Marble burying test (obsessional like) (A).
  • Microbiome and Biomarkers Fecal pellets will be collected for microbiome analysis on days 0, +10 and +19 and blood will be collected for biomarker analysis.
  • psychedelic mushrooms contain intermediate products of the psilocybin biosynthetic pathway such as baeocystin, norbaeocystin and aeruginascin that may influence the nature of the effect of psilocybin (“entourage effect”) along with other components such as harmines with monoamine oxidase inhibiting properties.
  • efficacy effect the nature of the effect of psilocybin
  • HTR mouse head twitch response
  • TST mouse tail suspension test
  • mice Male C57Bl/6j mice were used in all head twitch experiments.
  • PSIL (98.75% purity) was provided by Usona Institute.
  • FSME a methanol extract of Psilocybe cubensis with a psilocybin content of 1.5%, was provided by BYAS-PEB.
  • Drug doses were calculated so that equal injection volumes of PSIL and FSME contained equal concentrations of psilocybin on a mg per kg basis.
  • Control mice received vehicle (VEH) injections (0.9% NaCl solution). HTR was measured over 20 minutes in a magnetometer-based system using ear clip magnets. The TST was conducted 48 hours following administration of PSIL, FSME and vehicle.
  • Behavioral tests were conducted using a Noldus Ethovision system by observers blind to treatment status.
  • Individual male zebrafish (Danio rerio) were used in an open arena, behavioral phenotyping experiment.
  • the drug dose, 3mg/L of PSIL and FSME was administered in a beaker containing 200ml of water over 10 minutes.
  • Control fish were placed in a beaker containing 200ml of water for 10 minutes.
  • the fish were then placed in an 50x50x4cm arena and video-tracked using idTracker software, with trajectories recorded for 20 minutes immediately after treatment and for 20 minutes at 80 minutes after treatment.
  • mice Animals. Outbred C57BL/6 mice will be used. Mice will be aged 12 weeks. Parallel groups of male and female mice will be used for all experiments. Altogether, 344 mice will be used in this project (172 of each gender). Mice will be group-housed for the duration of the experiment, and maintained under standard conditions with a 12-hour light dark cycle. Behavioral assays will be performed at the same time each day. Mice from different experimental groups will be tested in counterbalanced order.
  • PRSE Physicalcyanin-rich Spirulina Extract
  • PFME Psilocybin-free mushroom extract
  • BYAS Yards Algae Sciences
  • Other drugs and chemicals will be purchased from Sigma- Aldrich Israel Ltd.
  • Drugs will be administered by intraperitoneal (i.p.) injection.
  • PFME based on the doses previously administered to examine HTR induced by psilocybin, to determine whether the psilocybin free extract induces HTR. In the event that HTR is observed at a particular dose, subsequent experiments will be performed at a dose lower than the observed HTR-inducing dose.
  • mice will be administered a single injection of the highest dose of PFME that does not induce HTR. 15 minutes after injection mice will be sacrificed and frontal cortex specimens will be obtained and frozen for analysis of IEG.
  • mice will be administered PRSE or PFME or both by IP injection for 18 days. Behavioral and cognitive testing will commence on day 11 for 7 days. Mice will be treated in two cohorts of 12 for each treatment category and each cohort will undergo a different module of behavioral and cognitive testing. Mice will be sacrificed on day 19, a cardiac blood sample will be taken, and brain areas will be dissected. female (N)
  • synaptic plasticity In organisms with complex nervous systems, memory storage is believed to be heavily based on changes in synapses, specialized sites of cell-cell contact that connect the nerve cells within the nervous system. Changes in synaptic connections, broadly referred to as synaptic plasticity, represent an important neuronal plasticity process, only one of multiple neuronal plasticity processes. Rosenberg et al. (2014) The roles of protein expression in synaptic plasticity and memory consolidation. Fron. Molecular Neurosci. 7(86): 1-14.
  • This example provides data on levels of the following synaptic plasticity markers in a mouse model after administration of purified psilocybin (PSIL) or a full spectrum mushroom extract (FSME).
  • PSIL purified psilocybin
  • FSME full spectrum mushroom extract
  • PSD-95 which regulates the trafficking and localization of glutamate receptors such as a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type or N-methyl-D-aspartate (NMD A) type-receptors.
  • AMPA a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
  • NMD A N-methyl-D-aspartate
  • GAP43 which is expressed at high levels in neuronal growth cones during development and axonal regeneration, and it is phosphorylated after long-term potentiation and after learning. GAP43 is a proven marker for developing and regenerating neurons, and to a lesser extent, reactive glial cells.
  • Synaptophysin which is a presynaptic vesicular protein that is often used as a marker for synaptic plasticity and integrity, decreases with increasing age in hippocampal and cortical regions.
  • mice C57B1/6J mice ( ⁇ 30g) were administered chemically produced psilocybin provided by USONA or full spectrum, psilocybin-containing mushroom extract (FSME) provided by PEB, by intraperitoneal injection, both at a psilocybin dose of 4.4 mg/kg. 10-12 days later the mice were sacrificed, and brain areas were dissected and frozen immediately at -180C until assay.
  • the synaptic proteins, GAP43, PSD95 and synaptophysin were assayed by Western Blot analysis as ratios to actin housekeeping gene.

Abstract

The present invention relates to the use of entheogens to treat psychiatric disorders, brain injuries, and autism, and in particular to the use of entheogens to treat obsessive compulsive disorder (OCD), schizophrenia, depression, post-traumatic stress disorder (PTSD), anxiety, autism spectrum disorder, traumatic brain injury (TBI), concussion, and chronic traumatic encephalopathy (CTE).

Description

TREATMENT OF PSYCHIATRIC DISORDERS, BRAIN INJURIES, AND AUTISM SPECTRUM DISORDER
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Prov. Appl. 63/313,816, filed February 25, 2022, U.S. Prov. Appl. 63/319,550, filed March 14, 2022, U.S. Prov. Appl. 63/359,282, filed July 8, 2022, U.S. Prov. Appl. 63/405,620, filed September 12, 2022, U.S. Prov. Appl. 63/405,617, filed September 12, 2022, and U.S. Prov. Appl. 63/405,615, filed September 12, 2022, the contents of which are incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
The present invention relates to the use of entheogens to treat psychiatric disorders, brain injuries, and autism, and in particular to the use of entheogens to treat obsessive compulsive disorder (OCD), schizophrenia, depression, post-traumatic stress disorder (PTSD), anxiety, autism spectrum disorder, traumatic brain injury (TBI), concussion, and chronic traumatic encephalopathy (CTE).
BACKGROUND OF THE INVENTION
Obsessive-compulsive disorder (OCD) features a pattern of unwanted thoughts and fears (obsessions) that lead to repetitive behaviors (compulsions) (Stein DJ, Costa DLC et al: Obsessive-compulsive disorder. Nat Rev Dis Primers. 5(1): 52). These obsessions and compulsions interfere with daily activities and cause significant distress. Obsessive-compulsive disorder usually includes both obsessions and compulsions. But it is also possible to have only obsession symptoms or only compulsion symptoms. OCD obsessions are repeated, persistent and unwanted thoughts, urges or images that are intrusive and cause distress or anxiety. The sufferer might try to ignore them or get rid of them by performing a compulsive behavior or ritual. These obsessions typically intrude when the sufferer trying to think of or do other things. Schizophrenia (SCZ) is a devastating neuropsychiatric disorder with lifetime impact, that is only partly addressed by available treatment with antipsychotic drugs (APDs) (Jauhar S, Johnstone M et al: Schizophrenia. The Lancet. Volume 399, Issue 10323, P473-486, January 29, 2022). The main target of APDs is the treatment of psychosis, and they do not attenuate the emergence of debilitating negative symptoms and cognitive deficits. Effective treatment of negative symptoms is crucial, as the persistence of these symptoms while psychosis is in remission is an impediment preventing schizophrenia patients from leading a fully functional life. There is considerable evidence that schizophrenia is associated with cortical atrophy and cell loss in the frontal cortex and other brain regions (Osimo EF, Beck K, Reis Marques T, Howes OD. Synaptic loss in schizophrenia: a meta-analysis and systematic review of synaptic protein and mRNA measures. Mol Psychiatry. 2019 Apr;24(4):549-61). Recently, serotonergic psychedelics were demonstrated to increase synapse number and function in the prefrontal cortex, along with increased neurogenesis, suggesting that psychedelics may offer a novel direction for treatment of schizophrenia patients whose acute psychotic symptoms have been stabilized by APDs.
Major depressive disorder is the leading cause of disability worldwide in terms of total years lost due to disability and is associated with excess mortality (Abbafati, C.; Machado, D.B.; Cislaghi, B.; Salman, O.M.; Karanikolos, M.; McKee, M.; Abbas, K.M ; Brady, O.J.; Larson, H.J.; Trias-Llimos, S.; et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: A systematic analysis for the Global Burden of Disease Study 2019. Lancet 2020, 396, 1204-1222). About 30% of patients with major depression fail to achieve remission despite treatment with multiple antidepressants and are considered to have treatmentresistant depression (Fava M: Diagnosis and definition of treatment-resistant depression. Biol Psychiatry 2003; 53:649-659). In patients who respond to antidepressants, the time to onset of effect is typically several weeks, during which time patients remain symptomatic and at risk of suicidal behavior and self-harm (Rush AJ, Trivedi MH, Wisniewski SR, et al: Acute and longer- term outcomes in depressed outpatients requiring one or several treatment steps: a STAR*D report. Am J Psychiatry 2006; 163:1905-1917). There is a need to develop novel treatments that provide rapid relief of depressive symptoms, especially in patients with treatment-resistant depression.
Post-traumatic stress disorder (PTSD) is a mental health condition that is triggered by a terrifying event - either experiencing it or witnessing it (Bryant RA: Post-traumatic stress disorder: a state-of-the-art review of evidence and challenges. Psychiatry. 2019 Oct;18(3):259- 269). Symptoms may include flashbacks, nightmares and severe anxiety, as well as uncontrollable thoughts about the event. Post-traumatic stress disorder symptoms may start within one month of a traumatic event, but sometimes symptoms may not appear until years after the event. These symptoms cause significant problems in social or work situations and in relationships. They can also interfere with the ability to go about normal daily tasks. PTSD symptoms are generally grouped into four types: intrusive memories, avoidance, negative changes in thinking and mood, and changes in physical and emotional reactions. Symptoms can vary over time or vary from person to person.
Anxiety disorders are the most prevalent psychiatric disorders (with a current worldwide prevalence of 7.3%. Among them, specific phobias are the most common, with a prevalence of 10.3%, then panic disorder (with or without agoraphobia) is the next most common with a prevalence of 6.0%, followed by social phobia (2.7%) and generalized anxiety disorder (2.2%). There is a high comorbidity between anxiety (especially generalized anxiety disorders or panic disorders) and depressive disorders. Additionally, anxiety disorders are often associated, which renders treatment even more complex. As a result, anxiety disorders often remain underdiagnosed and undertreated in primary care (Thibaut F: Anxiety disorders: a review of current literature. Dialogues Clin Neurosci. 2017 Jun; 19(2): 87-88).
Entheogens are chemical substances, typically of plant origin, that is ingested to produce a nonordinary state of consciousness for religious or spiritual purposes (Nichols DE: Psychedelics. Pharmacol Rev. 2016 Apr; 68(2): 264-355). There has been increasing interest in the use of entheogens, such as the psychedelic compound, psilocybin, for the treatment of mental disorders including depression (Geiger HA, Wurst MG et al: DARK Classics in chemical neuroscience. Psilocybin. ACS Chem. Neurosci. 2).
Autism spectrum disorder (ASD) is a developmental and behavioral disorder, representing one of the most disabling chronic conditions in childhood. Picardi et al., Parental burden and its correlates in families of children with autism spectrum disorder: a multicentre study with two comparison groups. Clin Pract Epidemiol Ment Health. 2018;14: 143. It is characterized by abnormalities in social interactions, deficits in communication, restricted interests, and repetitive behavior. Frye RE. Social skills deficits in autism spectrum disorder: potential biological origins and progress in developing therapeutic agents. CNS Drugs. 2018;32:713-734. ASD children are often subjected to criticism and bullying and react by isolation, aggression, hostility, or self-harm. Schroeder et al., Shedding light on a pervasive problem: A review of research on bullying experiences among children with autism spectrum disorders. J Autism Dev Disord. 2014;44: 1520-1534; Vernberg and Biggs, Preventing and treating bullying and victimization. 2010. These difficulties are exacerbated by an inflexible adherence to routines and overreaction to sensory stimuli. Once developed, this disorder descends to permanent lifelong abnormalities and disability. Thurm and Swedo, The importance of autism research. Dialogues Clin Neurosci. 2012;14:219. Globally, recent epidemiological studies referred to prevalence rates of ASD of 1 in 54 children. Control CfD and Prevention. Data & statistics on autism spectrum disorder. 2019. The estimated prevalence rate of ASD in Europe is 1 in 66 children (http://asdeu.eu/prevalence/). Worryingly, it grew 32-fold over the last 50 years. Elsabbagh et al., Global prevalence of autism and other pervasive developmental disorders. Autism Res. 2012;5: 160-79. Furthermore, the prevalence rate may be underdiagnosed for poorer populations who experience health inequality. Kerub et al., Autism spectrum disorder: Evolution of disorder definition, risk factors and demographic characteristics in Israel. Isr Med Assoc J. 2018;20:576-581.
It is noteworthy that ASD affects not only the persons diagnosed with this disorder but their entire families. Indeed, parents experience a significant level of stress due to the severity and permanency of ASD, development of co-morbidities, and the lack of health support for their children. Vohra R, Madhavan et al., Access to services, quality of care, and family impact for children with autism, other developmental disabilities, and other mental health conditions. Autism. 2014;18:815-826. The daily challenges of caring for these children are troublesome, affect all aspects of the child's care and harm the parent's mental health. Bonis, Stress and parents of children with autism: a review of literature. Issues Ment Health Nurs. 2016;37: 153-163.
The increase in the number of people with ASD has led to a very significant negative impact on the economy. Leigh and Du forecast that the family economic burden of ASD in the US would reach $461 billion (up to 3.6 % of the GDP) and would likely exceed the economic burden of diabetes by 2025. Leigh and Du, Brief report: forecasting the economic burden of autism in 2015 and 2025 in the united states. J Autism Dev Disord. 2015;45:4135-4139.; Investigators P, Control CfD and Prevention. Prevalence of autism spectrum disorder among children aged 8 years-autism and developmental disabilities monitoring network, 11 sites, United States, 2010. Morbidity and Mortality Weekly Report Surveillance Summaries (Washington, DC: 2002). 2014;63:1-21. Therefore, the successful development of diagnostics and treatment of ASD will have a significant impact on the global economy. Unfortunately, no reliable diagnostics from laboratory tests, effective treatment, or preventive measures have been found against this disorder to date Sharma et al., Autism spectrum disorder: classification, diagnosis and therapy. Pharmacol Ther. 2018;190:91-104.
The Center for Disease Control and Prevention (CDC) reports that traumatic brain injury (TBI) including concussion (mild-TBI) is a major cause of death and disability in the United States. This silent epidemic has been placed in the spotlight through epidemiological studies and popular press coverage of the suffering of athletes and veterans exposed to repetitive symptomatic and asymptomatic concussions. TBI manifests in cumulative, multiple, long-term neurological abnormalities, the prototype being chronic traumatic encephalopathy (CTE). The symptoms of CTE include confusion, impaired judgment, impulse control problems, aggression, depression, anxiety, suicidality, parkinsonism, and, eventually, progressive dementia. These symptoms may manifest years or even decades after the last brain trauma or the end of an active athletic career. Despite increased awareness of TBI and CTE over the past decade, much remains to be done to advance our understanding of these conditions.
Currently, there are no approved drugs for the prevention or treatment of TBI and CTE. Changes in brain function are apparent in football players after exposure to non-concussive head impacts. This provides clear evidence that impact-induced changes in brain function are occurring in the absence of currently visible structural damage and accumulate as the frequency of concussive incidents occurs. Therefore, prevention would need to act at cellular and molecular levels.
In this context, there is growing interest in the maladaptation and interconnection of (i) brain plasticity i.e., changes that occur at synapses, the communicational' junctions between brain neurons, (ii) the immune system, and (iii) the gastrointestinal system. The effect of repeated mild-TBI on brain synaptic maladaptation and dysfunction has been demonstrated in mouse models indicating that impacts below those required to generate visible neuropathological sequelae still cause substantial neuronal dysfunction when delivered with sufficient frequency (Jamjoom et al., (2021) The synapse in traumatic brain injury. Brain, 144, 18-31). There is evidence implicating a dysregulated immune response in the exacerbation of TBI-induced neurological dysfunction and CTE (McKee and Lukens, (2016) Emerging roles for the immune system in traumatic brain injury. Frontiers in immunology, 7, 556; Simon et al., (2017) The far- reaching scope of neuroinflammation after traumatic brain injury. Nature Reviews Neurology, 13, 171-191) TBI has been shown to induce an immune-mediated neuroinflammatory response that can last for many years post-injury. Neuronal cell apoptosis (cell suicide) also plays a crucial role in the pathogenesis of head injury (Lin et al., (2014) Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death. Cell death & disease, 5, el 147-el 147). Inhibition of apoptosis can potentially prevent or even reverse the deleterious effects of repeated concussion and oxidative stress, as the occurrence of oxidative free radicals also plays an important role in these biochemical cascades. A breakthrough study published in Nature Scientific Reports (Angoa-Perez et al., (2020) Repetitive, mild traumatic brain injury results in a progressive white matter pathology, cognitive deterioration, and a transient gut microbiota dysbiosis. Scientific reports, 10, 1-11) explains the systemic effect of repetitive mild-TBI on gastrointestinal function. These studies show the emerging role of gutmicrobiome dysbiosis (imbalance) in the development of chronic neurological diseases. Other studies corroborate these observations and highlight the influence of the gut-brain axis in a myriad of other neurological disorders (Morais et al., (2020) The gut microbiota-brain axis in behaviour and brain disorders. Nature Reviews Microbiology, 1-15).
The Center for Disease Control and Prevention (CDC) reports that traumatic brain injury (TBI) including concussion (mild-TBI) is a major cause of death and disability in the United States. This silent epidemic has been placed in the spotlight through epidemiological studies and popular press coverage of the suffering of athletes and veterans exposed to repetitive symptomatic and asymptomatic concussions. TBI manifests in cumulative, multiple, long-term neurological abnormalities, the prototype being chronic traumatic encephalopathy (CTE). The symptoms of CTE include confusion, impaired judgment, impulse control problems, aggression, depression, anxiety, suicidality, parkinsonism, and, eventually, progressive dementia. These symptoms may manifest years or even decades after the last brain trauma or the end of an active athletic career. Despite increased awareness of TBI and CTE over the past decade, much remains to be done to advance our understanding of these conditions.
Currently, there are no approved drugs for the prevention or treatment of TBI and CTE. Changes in brain function are apparent in football players after exposure to non-concussive head impacts. This provides clear evidence that impact-induced changes in brain function are occurring in the absence of currently visible structural damage and accumulate as the frequency of concussive incidents occurs. Therefore, prevention would need to act at cellular and molecular levels. Tn this context, there is growing interest in the maladaptation and interconnection of (i) brain plasticity i.e., changes that occur at synapses, the communicational' junctions between brain neurons, (ii) the immune system, and (iii) the gastrointestinal system. The effect of repeated mild-TBI on brain synaptic maladaptation and dysfunction has been demonstrated in mouse models indicating that impacts below those required to generate visible neuropathological sequelae still cause substantial neuronal dysfunction when delivered with sufficient frequency (Jamjoom et al., 2021). There is evidence implicating a dysregulated immune response in the exacerbation of TBI-induced neurological dysfunction and CTE (McKee and Lukens, 2016) (Simon et al., 2017). TBI has been shown to induce an immune-mediated neuroinflammatory response that can last for many years post-injury. Neuronal cell apoptosis (cell suicide) also plays a crucial role in the pathogenesis of head injury (Lin et al., 2014). Inhibition of apoptosis can potentially prevent or even reverse the deleterious effects of repeated concussion and oxidative stress, as the occurrence of oxidative free radicals also plays an important role in these biochemical cascades. A breakthrough study published in Nature Scientific Reports (Angoa- Perez et al., 2020) explains the systemic effect of repetitive mild-TBI on gastrointestinal function. These studies show the emerging role of gut-microbiome dysbiosis (imbalance) in the development of chronic neurological diseases. Other studies corroborate these observations and highlight the influence of the gut-brain axis in a myriad of other neurological disorders (Morais et al., 2020).
Psilocybin is a naturally occurring psychedelic compound produced by over 200 mushrooms, that is being increasingly researched as treatment for a range of different psychiatric disorders (Carhart-Harris RL and Goodwin GM. (2017) The therapeutic potential of psychedelic drugs: past, present and future. Neuropsychopharmacology). Five separate trials have reported improvements in depressive symptoms after psilocybin-assisted psychotherapy including one in which 'treatment-resistant depression' was the primary criterion for inclusion (Carhart-Harris R, Giribaldi B, Watts R et al Trial of Psilocybin versus Escital opram for Depression. N Engl J Med. 2021 04 15; 384(15): 1402-1411, Carhart-Harris RL, Nutt DJ (2016) Question-based drug development for psilocybin - authors' reply. Lancet Psychiatry 3:807; Ross S, Bossis A, Guss J et al (2016) Rapid and sustained symptom reduction following psilocybin treatment for anxiety and depression in patients with life-threatening cancer: a randomized controlled trial J Psychopharmacol 30: 1165-1180; Griffiths RR, Johnson MW, Carducci MA et al (2016) Psilocybin produces substantial and sustained decreases in depression and anxiety in patients with life-threatening cancer: a randomized double-blind trial. J Psychopharmacol 30: 1181-1197; Grob CS, Danforth AL, Chopra GS et al (2011) Pilot study of psilocybin treatment for anxiety in patients with advanced-stage cancer. Arch Gen Psychiatry 68:71-78. Psilocybin has shown promise in the treatment of obsessive compulsive disorder (Moreno FA, Wiegand CB, Taitano EK et al (2006) Safety, tolerability, and efficacy of psilocybin in 9 patients with obsessive-compulsive disorder. J Clin Psychiatry 67: 1735-1740), alcohol (Bogenschutz MP, Forcehimes AA, Pommy JA et al (2015) Psilocybin-assisted treatment for alcohol dependence: a proof-of- concept study. J Psychopharmacol 29:289-299) and tobacco addiction (Johnson MW, Garcia- Romeu A, Cosimano MP et al (2014) Pilot study of the 5-HT2AR agonist psilocybin in the treatment of tobacco addiction. J Psychopharmacol 28:983-992) and anxiety related to terminal diagnoses (Griffiths et al. 2016; Ross et al. 2016; Grob et al. 2011). Treatment procedures typically involve psychological preparation prior to one or two therapist-supported drug sessions followed by psychological integration. Using a consistent model (i.e. involving appropriate psychological support), sustained improvements in well-being in healthy individuals were observed after a single dose of psilocybin in a double-blind design incorporating an active placebo (Griffiths R, Richards W, Johnson M et al (2008) Mystical-type experiences occasioned by psilocybin mediate the attribution of personal meaning and spiritual significance 14 months later. J Psychopharmacol 22:621-632).
Studies involving other serotonergic psychedelics combined with psychological support have found similarly promising outcomes: Sustained reductions in end-of-life anxiety were observed after LSD-assisted psychotherapy (Gasser P, Holstein D, Michel Y et al (2014) Safety and efficacy of lysergic acid diethylamide-assisted psychotherapy for anxiety associated with life-threatening diseases. J Nerv Ment Dis 202:513-520), and reduced depressive symptoms were seen after ayahuasca in patients with 'recurrent depression' (Osorio Fde L, Sanches RF, Macedo LR et al (2015) Antidepressant effects of a single dose of ayahuasca in patients with recurrent depression: a preliminary report. Rev Bras Psiquiatr 37: 13-20; Sanches RF, de Lima Osorio F, Dos Santos RG et al (2016) Antidepressant effects of a single dose of ayahuasca in patients with recurrent depression: a SPECT study. J Clin Psychopharmacol 36:77-81). Naturalistic, observational studies of ayahuasca support its long-term well-being promoting and anti -addiction properties (Thomas G, Lucas P, Capler NR et al (2013) Ayahuasca-assisted therapy for addiction: results from a preliminary observational study in Canada. Curr Drug Abuse Rev 6:30- 42; Bouso JC, Gonzalez D, Fondevila S et al (2012) Personality, psychopathology, life attitudes and neuropsychological performance among ritual users of Ayahuasca: a longitudinal study. PLoS One 7:e42421) and a recent population survey found lower rates of suicidality and psychological distress in association with psychedelic drug use (Hendricks PS, Thome CB, Clark CB, et al (2015) Classic psychedelic use is associated with reduced psychological distress and suicidality in the United States adult population. J Psychopharmacol 29(3) :280-8). Further evidence favoring the therapeutic potential of psychedelics can be found in literature documenting the extensive research carried out with these compounds in the mid-twentieth century, e.g. two relevant meta-analyses have found positive safety and efficacy data for LSD for alcohol dependence (Krebs TS, Johansen PO (2012) Lysergic acid diethylamide (LSD) for alcoholism: meta-analysis of randomized controlled trials. J Psychopharmacol 26:994-1002) and mood disorders (Rucker JJ, Jelen LA, Flynn S et al (2016) Psychedelics in the treatment of unipolar mood disorders: a systematic review. J Psychopharmacol 30: 1220-1229). See Sarris J et al. Psychedelic medicines for mood disorders: current evidence and clinical considerations Curr Opin Psychiatry 2021, 33:000 - 000.
Like all serotonergic psychedelics, psilocybin initiates its characteristic effects via serotonin 2A receptor (5-HT2AR) agonism (Vollenweider FX, Vollenweider-Scherpenhuyzen MF, Babler A et al (1998); Vollenweider FX, Smallridge JW: Classic Psychedelic Drugs: Update on Biological Mechanisms. Pharmacopsychiatry. 2022 Jan 25. doi: 10.1055/a- 1721 -2914. Online ahead of print). Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action. Neuroreport 9:3897-3902). 5-HT2AR signaling has been associated with better responses to conventional antidepressants (Qesseveur G, Petit AC, Nguyen HT et al. (2016) Genetic dysfunction of serotonin 2A receptor hampers response to antidepressant drugs: a translational approach. Neuropharmacology 105: 142-153; Petit AC, Quesseveur G, Gressier F et al (2014) Converging translational evidence for the involvement of the serotonin 2A receptor gene in major depressive disorder. Prog Neuro-Psychopharmacol Biol Psychiatry 54:76-82), and preclinical work indicates that 5-HT2AR signalling may mediate (at least some of) the therapeutic effects of SSRIs (Nic Dhonnchadha BA, Ripoil N, Clenet F et al (2005) Implication of 5-HT2 receptor subtypes in the mechanism of action of antidepressants in the four plates test. Psychopharmacology 179:418-429; Buchbom T, Schroder H, Hollt V et al (2014) Repeated lysergic acid diethylamide in an animal model of depression: normalisation of learning behaviour and hippocampal serotonin 5-HT2 signalling. J Psychopharmacol 28:545-552). 5-HT2AR antagonists have been found to augment the antidepressant effects of SSRIs (Ostroff RB, Nelson JC (1999) Risperidone augmentation of selective serotonin reuptake inhibitors in major depression. J Clin Psychiatry 60:256-259) and many effective antidepressant augmentation medications have 5-HT2AR antagonist properties (Carpenter LL, Jocic Z, Hall JM et al (1999) Mirtazapine augmentation in the treatment of refractory depression. J Clin Psychiatry 60:45-49).
What is needed in the art is identification of effective entheogen compositions for the treatment of disorders such as OCD, schizophrenia and other psychiatric disorders.
SUMMARY OF THE INVENTION
The present invention relates to the use of entheogens to treat psychiatric disorders, brain injuries, and autism, and in particular to the use of entheogens to treat obsessive compulsive disorder (OCD), schizophrenia, depression, post-traumatic stress disorder (PTSD), anxiety, autism spectrum disorder, traumatic brain injury (TBI), concussion, and chronic traumatic encephalopathy (CTE).
In a first aspect, the present invention provides methods of treating a psychiatric disorder in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin or a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the psychiatric disorder is selected from the group consisting of obsessive compulsive disorder (OCD), schizophrenia, post-traumatic stress disorder (PTSD), depressive and anxiety disorders, alcohol and substance use disorders and nicotine addiction. In some preferred embodiments, the effective amount of a mushroom cell culture extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder. In some preferred embodiments, the psychiatric disorders are OCD and schizophrenia. In some preferred embodiments, the psychiatric disorder is PTSD.
In some preferred embodiments, the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
In some preferred embodiments, the method further comprise co-administering a 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
In a second aspect, the present invention provides methods of providing prophylaxis, and primary, secondary and tertiary prevention against PTSD and/or depression in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin or a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the effective amount of a mushroom cell culture composition or entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD and depression.
In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 4 weeks of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 week of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 3 days of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 day of a psychological trauma or other traumatic event.
In some preferred embodiments, the mushroom cell culture composition or entheogen composition is produced by a process comprising mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
In some preferred embodiments, the methods further comprise co-administering a 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599. Tn a third aspect, the present invention provides a mushroom cell culture extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin or entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin for use in treating a psychiatric disorder in a subject in need thereof.
In some preferred embodiments, the mushroom cell culture composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition or mushroom cell culture extract comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition or mushroom cell culture extract comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition or mushroom cell culture extract comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction. In some preferred embodiments, the amount of mushroom cell culture composition or entheogen composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder. In some preferred embodiments, the psychiatric disorder is PTSD. In some preferred embodiments, the psychiatric disorders OCD and or schizophrenia.
In some preferred embodiments, the mushroom cell culture composition is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
In some preferred embodiments, the entheogen composition or mushroom cell culture extract is co-administered with a 5-HTIA receptor agonist. In some preferred embodiments, the 5- HT IA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, Fl 3714 and F15599.
In a fourth aspect, the present invention provides a mushroom cell culture extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin or entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin for use in prophylaxis, or primary, secondary and tertiary prevention of PTSD and/or depression.
In some preferred embodiments, the mushroom cell culture or entheogen composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition or mushroom cell culture extract comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen or mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen or mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the effective amount of a mushroom cell culture composition or entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD and depression.
In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 4 weeks of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 week of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 3 days of a psychological trauma or other traumatic event. In some preferred embodiments, the mushroom cell culture composition or entheogen composition is administered to the subject within 1 day of a psychological trauma or other traumatic event.
In some preferred embodiments, the mushroom cell culture composition or entheogen composition is produced by a process comprising culturing mushroom cells in a culture vessel. Tn some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
In some preferred embodiments, the entheogen or mushroom cell culture composition is co-administered with a 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
In a fifth aspect, the present invention provides a process for mushroom cell culture comprising: culturing mushrooms on substrate to provide a mushroom cell culture, and contacting the mushroom cell culture with a PC composition. In some preferred embodiments, the mushrooms cells are psychoactive mushroom cells or parts of a psychoactive mushroom. In some preferred embodiments, the mushroom cell culture produces one or more psychoactive compounds or other compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the process further comprises processing the mushroom cell culture to provide an aqueous fraction comprising a psychoactive compound. In some preferred embodiments, the process further comprises processing the mushroom cell culture to provide an extract comprising a psychoactive compound. In some preferred embodiments, the process further comprises processing the mushroom cell culture to provide a purified composition comprising a psychoactive compound. In some preferred embodiments, the process further comprises the step of formulating the aqueous fraction, extract or purified composition comprising a psychoactive compound with a pharmaceutically acceptable carrier. In some preferred embodiments, the pharmaceutically acceptable carrier does not naturally occur with the aqueous fraction, extract or purified composition comprising a psychoactive compound.
In a sixth aspect, the present invention provides a process for production of psychoactive compounds comprising: culturing mushroom cells from a psychoactive mushroom on substrate to cell culture; contacting the mushroom cell with a PC composition; and processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds.
In some preferred embodiments, the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises producing an aqueous fraction from the mushroom cell culture that comprises one or more psychoactive compounds. Tn some preferred embodiments, the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises extracting the mushroom cell culture with an aqueous solvent, an organic solvent of combinations thereof to provide an extract that comprises one or more psychoactive compounds. In some preferred embodiments, the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises processing the mushroom cell culture to provide a purified composition comprising a psychoactive compound.
In some preferred embodiments, the process further comprises the step of formulating the aqueous fraction, extract or purified composition comprising a psychoactive compound with a pharmaceutically acceptable carrier. In some preferred embodiments, the pharmaceutically acceptable carrier does not naturally occur with the aqueous fraction, extract or purified composition comprising a psychoactive compound.
In a seventh aspect, the present invention provides a mushroom cell culture composition or formulation thereof produced by a process as described above.
In an eighth aspect, the present invention provides a formulation comprising a mushroom cell culture composition and a pharmaceutically acceptable carrier that does not naturally occur with the mushroom cell culture.
In a ninth aspect, the present invention provides a formulation comprising psilocybin or psilocin and a 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
In some preferred embodiments according to the eighth or ninth aspects, the psilocybin or psilocin is provided in a mushroom cell culture extract. In some preferred embodiments, the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition. In some preferred embodiments, the formulations further comprise one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the psilocybin or psilocin is chemically synthesized. Tn some preferred embodiments, the baeocystin, aeruginascin, norpsilocin, or norbaeocystin are chemically synthesized.
In a tenth aspect, the formulations described in the eighth and ninth aspects are provides for use in treating a psychiatric disorder. In some preferred embodiments, the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
In an eleventh aspect, the present invention provides a kit comprising: a) a first formulation or composition comprising psilocybin or psilocin; and b) a second formulation or composition comprising a 5-HTIA receptor agonist; wherein the first formulation or composition and second formulation or composition are provided in different containers or oral delivery vehicles. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599. In some preferred embodiments, the psilocybin or psilocin is provided in a mushroom cell culture extract. In some preferred embodiments, the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition. In some preferred embodiments, the kit further comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the psilocybin or psilocin is chemically synthesized. In some preferred embodiments, the baeocystin, aeruginascin, norpsilocin, or norbaeocystin are chemically synthesized. In some preferred embodiments, the kit of the eleventh aspect is provided for use in treating a psychiatric disorder. In some preferred embodiments, the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
In a twelfth aspect, the present invention provides methods of treating autism spectrum disorder in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition.
In some preferred embodiments, the entheogen composition comprises psilocybin or psilocin. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition is a mushroom cell culture composition. In some preferred embodiments, the entheogens in the composition are chemically synthesized.
In some preferred embodiments, the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the effective amount of a mycelial cell culture extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of autism spectrum disorder.
In some preferred embodiments, the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
In a thirteenth aspect, the present invention provides an entheogen composition for use in treating autism spectrum disorder in a subject in need thereof.
In some preferred embodiments, the entheogen composition comprises psilocybin or psilocin. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen is provided as a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mushroom cell culture composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the amount of mushroom cell culture composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder.
In some preferred embodiments, the mushroom cell culture composition is produced by a process comprising culturing mycelial cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
In a fourteenth aspect, the present invention provides methods of treating a brain injury in a subject in need thereof comprising administering to the subject an effective amount of an entheogen composition. In some preferred embodiments, the brain injury is selected from the group consisting of traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma of the brain.
In some preferred embodiments, the entheogen composition comprises psilocybin or psilocin. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition is a mushroom extract. In some preferred embodiments, the entheogens are chemically synthesized.
In some preferred embodiments, the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
In some preferred embodiments, the effective amount of an entheogen or mushroom extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the brain injury.
In some preferred embodiments, the entheogen composition is a psilocybin-free entheogen composition.
In some preferred embodiments, the administration of the entheogen composition is immediately before, during of immediately after anesthesia or sedation of the subject.
In a fifteenth aspect, the present invention provides an entheogen composition for use in treating a brain injury in a subject in need thereof. In some preferred embodiments, the brain injury is selected from the group consisting of traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma of the brain.
In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition is a mushroom extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the mushroom extract is a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the mycelial cell culture composition is produced by a process comprising culturing mycelial cells in a culture vessel. In some preferred embodiments, the process comprising culturing mycelial cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
In some preferred embodiments, the amount of entheogen or mycelial cell culture composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the brain injury.
In some preferred embodiments, the entheogen composition is a psilocybin-free entheogen composition.
In some preferred embodiments, the administration of the entheogen composition is immediately before, during of immediately after anesthesia or sedation of the subject. In some preferred embodiments, the entheogen composition is co-administered with an anesthesia or sedation agent.
In a sixteenth aspect, the present invention provides an entheogen composition comprising a phycocyanin (PC) composition and a mushroom extract.
In some preferred embodiments, the mushroom extract comprises psilocybin. In some preferred embodiments, the mushroom extract is psilocybin-free. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the phycocyanin composition is extracted from Spirulina.
In some preferred embodiments, the phycocyanin composition is characterized by one or more of the following characteristics: a) the protein fraction of the composition comprises greater than about 30% of a protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; b) the protein fraction of the composition comprises greater than about 5% of a protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6 14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; c) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 45,578 kDa and an isoelectric point of about 6.2 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; d) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 35,014 kDa and an isoelectric point of about 5.9 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; e) the protein fraction of the composition comprises greater than about 0.30% of a protein having a molecular weight of about 24,688 kDa and an isoelectric point of about 5.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; f) the protein fraction of the composition comprises greater than about 2% of a protein having a molecular weight of about 22,522 kDa and an isoelectric point of about 5.9 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; g) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 21,023 kDa and an isoelectric point of about 7.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; h) the protein fraction of the composition comprises greater than about 0.50% of a protein having a molecular weight of about 13,417 kDa and an isoelectric point of about 7.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; i) the protein fraction of the composition comprises a ratio of major protein constituents to minor protein constituents of less than 3.5: 1 based on the aggregate mass of the proteins, wherein major protein constituents are the protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the minor protein constituents are the remainder of the proteins; j) the protein fraction of the composition comprises less than about 75% on a mass basis of the combined amounts of the protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; k) the composition produces a solution having a color value of greater than 200 E 10%/l cm when 250 mg of a dry powder of the composition are dissolved in one liter of water and absorbance is measured at 618 nm. In some preferred embodiments, the PC composition has at least two of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least three of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least four of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least five of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least six of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the purified PC composition of claim 1, wherein the composition has at least seven of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least eight of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least nine of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has at least ten of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the PC composition has all eleven characteristics a, b, c, d, e, f, g, h, i, j and k.
In some preferred embodiments, the phycocyanin composition is produced by a process comprising: encapsulating Spirulina to provide capsules; and contacting the capsules with an aqueous medium under conditions such that the compound of interest passes from the capsule into the aqueous solution.
In some preferred embodiments, the PC composition comprises a dried powder comprising a purified PC, the powder having a residual moisture content of less than about 5% w/w of the powder.
In some preferred embodiments, the psilocybin-free mushroom extract is extracted from a mushroom or part thereof that produces psilocybin, a psilocybin analog or related typtamine. In some preferred embodiments, the psilocybin-free mushroom extract is extracted from a mushroom or part thereof that comprises the psilocybin biosynthetic gene cluster (psiD, psiH, psiK, psiM). In some preferred embodiments, the psilocybin-free mushroom extract is an aqueous extract.
In some preferred embodiments, the psilocybin-free mushroom extract is produced by a process comprising: encapsulating a raw material comprising a mushroom or part thereof that produces psilocybin, a psilocybin analog or related typtamine to provide capsules; and contacting the capsules with an aqueous medium under conditions such that compounds of interest passes from the capsule into the aqueous solution. In some preferred embodiments, the methods further comprise the step of removing psilocybin from the extract by chromatography.
In some preferred embodiments, the psilocybin-free mushroom extract comprises a dried powder having a residual moisture content of less than about 5% w/w of the powder.
In some preferred embodiments, the psilocybin-free mushroom extract is a psilocybin- free mycelial extract.
In some preferred embodiments, the composition comprises from 1 to 99% w/w phycocyanin. In some preferred embodiments, the composition comprises from 10 to 90% w/w phycocyanin. In some preferred embodiments, the composition comprises from 1 to 99% w/w psilocybin-free mushroom extract. In some preferred embodiments, the composition comprises from 10 to 90% w/w psilocybin-free mushroom extract.
In some preferred embodiments, the composition is formulated as a topical formulation, an oral formulation, a mucosal formulation, an ophthalmological formulation, an aerosol formulation, and an intranasal formulation.
In some preferred embodiments, the composition further comprises one or more excipients or pharmaceutically acceptable carriers.
In some preferred embodiments, the oral formulation is an oral delivery vehicle. In some preferred embodiments, the oral delivery vehicle is selected from the group consisting of a capsule, a tablet and a gummi gel.
In a seventeenth aspect, the present invention provides methods of treating or preventing a psychiatric disease or disorder comprising administering an effective amount of the composition of the sixteenth aspect to a subject suffering from a psychiatric disease or disorder. In some preferred embodiments, the psychiatric disease or disorder is selected from the group consisting of anxiety disorder and depression.
In an eighteenth aspect, the present invention provides method of improving or altering cognitive function or sociability, or reducing aggressive behavior comprising administering an effective amount of the composition of the sixteenth aspect to a subject suffering from a psychiatric disease or disorder.
In a nineteenth aspect, the present invention provides a composition of the sixteenth aspect for use in treating or preventing a psychiatric disease or disorder in a subject in need thereof. Tn some preferred embodiments, the psychiatric disease or disorder is anxiety disorder and depression.
In a twentieth aspect, the present invention provides a composition of the sixteenth aspect for use in of improving or altering cognitive function or sociability, or reducing aggressive behavior in a subject in need thereof.
In a twenty-first aspect, the present invention provides methods of improving neuroplasticity in a subject in need thereof comprising administering to the subject an entheogen composition comprising psilocybin or psilocin and one or more entourage compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the composition comprises psilocybin.
In some preferred embodiments, the subject in need thereof has a disease, condition or disorder that could benefit from the promotion of neuroplasticity. In some preferred embodiments, the disease, condition or disorder associated with decreased neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD.
In some preferred embodiments, administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43, and Synaptophysin in one or more areas of the brain selected from the group consisting of the prefrontal cortex, amygdala, hippocampus, and striatum. In some preferred embodiments, administration of the entheogen composition increases the level of two or more proteins selected from the group consisting of PSD-95, GAP43 and Synaptophysin. In some preferred embodiments, administration of the entheogen composition increases the level of PSD-95, GAP43 and Synaptophysin.
In some preferred embodiments, the area of the brain is the prefrontal cortex. In some preferred embodiments, the area of the brain is the amygdala. In some preferred embodiments, the area of the brain is the hippocampus. In some preferred embodiments, the area of the brain is the striatum.
In some preferred embodiments, the entheogen composition comprises two or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. Tn some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the entheogen composition is a mushroom extract. In some preferred embodiments, the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
In some preferred embodiments, the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with neural atrophy.
In some preferred embodiments, the entheogens in the composition are chemically synthesized.
In some preferred embodiments, wherein the entheogen composition is co-administered with a 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F 13714 and F15599.
In a twenty-second aspect, the present invention provides an entheogen composition for use in treating a disease, condition or disorder associated with neural atrophy. In some preferred embodiments, the entheogen composition comprises psilocybin. In some preferred embodiments, the disease, condition or disorder associated with decreased neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD.
In some preferred embodiments, administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43 and Synaptophysin in one or more areas of the brain selected from the group consisting of the prefrontal cortex, amygdala, hippocampus, and striatum. In some preferred embodiments, administration of the entheogen composition increases the level of two or more proteins selected from the group consisting of PSD-95, GAP43 and Synaptophysin. In some preferred embodiments, administration of the entheogen composition increases the level of PSD-95, GAP43 and Synaptophysin.
In some preferred embodiments, the area of the brain is the prefrontal cortex. In some preferred embodiments, the area of the brain is the amygdala. In some preferred embodiments, the area of the brain is the hippocampus. In some preferred embodiments, the area of the brain is the striatum.
In some preferred embodiments, the entheogen composition comprises two or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
In some preferred embodiments, the entheogen composition is a mushroom extract. In some preferred embodiments, the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel. In some preferred embodiments, the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
In some preferred embodiments, the entheogens in the composition are chemically synthesized.
In some preferred embodiments, the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with reduced neuroplasticity.
In some preferred embodiments, wherein the entheogen composition is co-administered with a 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGs. 1 A to ID provide data on psilocybin dose response. These graphs depict (FIG. 1 A) full range dose response of HTR through the course of 18 min, (FIG. IB) taken from A the distinct shape of faster and higher onset following a rapid decrease in response (doses high then 3mg/kg), (FIG. 1C) taken from A the distinct shape of flatter response that carries over time (doses lower then 1.6 mg/kg), (FIG. lD)dose response curve taken from the data shown in A.
FIGs. 2A to 2D provide data on the effects of serotonin receptor modulators on psilocybin-induced HTR (Head Twitch Response). The bar graphs show the cumulative HTR recorded by the mice during 20 min session immediately following injection of either psilocybin (vehicle) alone or combined with a receptor modulator. (Compared to vehicle *=p<0.05, **=p<0.005, ***=p<0.0005, ****=p<0.0001). FIG. 2A Shows the effect of a combined psilocybin + 5HT2A antagonist, which completely reduced HTR in both concentrations. FIG. 2B Shows the effect of a combined psilocybin + 5HT1 A agonist, which reduced HTR in dose depended manner. FIG. 2C Shows the effect of a combined psilocybin + TAAR1 antagonist, which did not alter HTR. FIG. 2D Shows the effect of a combined psilocybin + 5HT2C antagonist, which increased HTR with 4 mg/kg and reduced the HTR-increasing effect at 8mg/kg.
FIGs. 3 A and 3B. 3 A: Effect of psilocybin 4.4 mg/kg and escitalopram 5 mg/kg on total marbles buried over 30 minutes. One way ANOVA: F2.35 = 13.32 p<0.0001. **p<0.01 vs. VEH, n=8-16 (Tukey's multiple comparisons test). 3B. Effect of psilocybin 4.4 mg/kg, M100907 2 mg/kg and M100907 2 mg/kg + psilocybin 4.4 mg/kg on total marbles buried over 30 minutes. Two-way ANOVA: M100907 FI,40 = 7.74, p=0.008, psilocybin FI,40 = 24.80, p<0.0001, Interaction FI,4O = 0.169, p=0.68. **p<0.01 vs. vehicle, n=7-16 (Tukey's multiple comparisons test).
FIGs. 4A and 4B. 4A:. Effect of psilocybin 4.4 mg/kg, 8-OH-DPAT 2mg/kg and 8-OH- DPAT 2 mg/kg + psilocybin 4.4 mg/kg on total marbles buried over 30 minutes Two way ANOVA: psilocybin Fi,37 = 10.43, p=0.0026, 8-OHDPAT Fl, 37 = 74.25, p<0.0001 . Interaction Fi, 37 = 0.9324, p = 0.3405) **p<0.01 vs. VEH. ##p< 01 vs. psilocybin, n = 6-16 (Tukey's multiple comparisons test). 4B: Effect of psilocybin 4.4 mg/kg, WAY100635 2 mg/kg and WAY100635 2 mg/kg + psilocybin 4.4 mg/kg on total marbles buried over 30 minutes .Two- way ANOVA: WAY100635 Fi,6i = 0.4162, p=0.5212, psilocybin Fi,6i = 42.47, pO.OOOl, Interaction Fi,6i = 0.0003 , p=0.9845.
**p<0.001 vs. vehicle, n=16-17 (Tukey's multiple comparisons test). FIGs 5A and 5B. 5A:. Effect of psilocybin 4.4 mg/kg, buspirone 5 mg/kg and buspirone 5 mg/kg+ psilocybin 4.4 mg/kg on total marbles buried over 30 minutes. Two way ANOVA: buspirone FI,75 = 8.532, p=0.0046; psilocybin Fi, 75 = 6.530, p= 0.0126; Interaction Fi, 75 = 5.805, p = 0.0184 **p<0.01 vs. VEH., n=19-20(Tukey's multiple comparisons test). 5B: Effect of buspirone 5 mg/kg, WAY100635 2 mg/kg and WAY100635 2 mg/kg + buspirone 5 mg/kg on total marbles buried over 30 minutes. Two-way ANOVA: WAY100635 FI,59 = 3.078, p=0.084, buspirone FI,59 = 19.45, p<0.0001, Interaction Fi, 59 = 1.219, p =0.274. **p<0.001 vs. VEH, n=15- 16 (Tukey's multiple comparisons test).
FIG. 6A and 6B. 6A: Effect of psilocybin 4.4 mg/kg, buspirone 5 mg/kg and psilocybin 4.4 mg/kg + buspirone 5 mg/kg on HTR over a 20-minute measurement period. Two-way ANOVA: Time F 2.915, 93.2s = 5.001, p = 0.0032; Treatment F 32, 288 = 4.874, p <0.0001; Time x Treatment F27, 288 = 2.852, p<0.0001. 6B: Total HTR over 20 minutes. F 3, 32 = 12.87, p <0.0001; ***p<0.001 vs. vehicle, ##p= 0.0002 BUSP vs. PSIL and p=0.0009 BUSP + PSIL vs. PSIL, n=6-12 (Tukey's multiple comparisons test).
FIGs. 7A-7C. 7A: Effect of psilocybin 4.4 mg/kg and buspirone 5 mg/kg on distance travelled in the open field over 30 minutes. One way ANOVA: F2,3o = 1.044; p=0.3645 . p N.S. vs. VEH. 7B: Effect of psilocybin 4.4 mg/kg and buspirone 5 mg/kg on time spent in the center of the open field over 30 minutes. One way ANOVA: F 2, 30 = 4.934; p=0.0140. p N.S. vs. VEH. 7C: Effect of psilocybin 4.4 mg/kg and buspirone 5 mg/kg on time spent in the periphery of the open field over 10 minutes. One way ANOVA: F 2, 30 = 5.003; p = 0.0133. p N.S vs VEH, n= 9- 15 (Tukey's multiple comparisons test).
FIG. 8: Graphs comparing the effect of chemical psilocybin and full spectrum mushroom extract on synaptic protein levels across the prefrontal cortex, hippocampus, amygdala and striatum in a mouse model.
DEFINITIONS
A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats). "Administering" or "administration of a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a PBP can be administered, intravenously, arterially, intradermally, intra-muscularly, intraperitoneally, intravenously, subcutaneously, sublingually, orally (by ingestion), intranasally (by inhalation), intrapulmonary (by nebulization or instillation) intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some aspects, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.
A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent, such as the phycocyanin and psilocybin-free mushroom extract compositions described herein, is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of symptoms of the condition being treated. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
A “prophylactically effective amount” or a “prophylactically effective dose” of a drug or agent, such as a phycocyanin and psilocybin-free mushroom extract composition, is an amount of a drug or an agent that, when administered to a subject will have the intended prophylactic effect. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of symptoms of the condition being treated. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.
“Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation, amelioration, or slowing the progression, of one or more symptoms associated with the disease or disorder being treated. In certain embodiments, treatment may be prophylactic.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the use of entheogens to treat psychiatric disorders, brain injuries, and autism, and in particular to the use of entheogens to treat obsessive compulsive disorder (OCD), schizophrenia, depression, post-traumatic stress disorder (PTSD), anxiety, autism spectrum disorder, traumatic brain injury (TBI), concussion, and chronic traumatic encephalopathy (CTE).
The present invention provides solution to multiple problems. In some preferred embodiments, the problem of treatment of the listed disorders is addressed via the use of fullspectrum entheogen compositions that include entourage compounds (e.g., baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof) in addition to psilocybin. In other preferred embodiments, the problem of providing full-spectrum entheogen compositions is addressed by improved culture of mushroom parts or cells. Other problems and solutions for those problems presented in the specification will be apparent to those of skill in the art.
1. ENTHEOGENS
Entheogens are naturally occurring compounds with psychedelic properties that have historically been used for religious and spiritual ceremonies and also for medicinal purposes. In spite of legal barriers, there is rapidly increasing interest among clinicians and researchers in the use of natural or synthetic psychedelics for the treatment of psychiatric disorders. The focus of the present invention is on disorders that are characterized by a high level of therapeutic resistance to standard pharmacological and psychological treatments. Central among these are obsessive compulsive disorder (OCD) schizophrenia, post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, alcohol and substance use disorders and nicotine addiction, autism spectrum disorder, traumatic brain injury (TBT), concussion, and chronic traumatic encephalopathy (CTE).
The present invention is not limited to any particular entheogen composition or formulation. The entheogen compositions may be prepared by a variety of methods including, but not limited to, chemical synthesis, extraction and/or purification from plants or fungi, and extraction and/or purification from mushroom cell cultures. In some preferred embodiments, the entheogen compositions comprise psilocybin. In some particularly preferred embodiments, the entheogen compositions comprise psilocybin and additionally one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof. In some preferred embodiments, the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof and is essentially free of psilocybin.
Exemplary entheogen compositions are provided below. The compounds listed may, for example, be chemically synthesized (see, e.g., Sherwood et al., (2020) Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin; J. Nat. Prod. 83(2) 461-67, incorporated herein by reference in its entirety), may be provided in an extract from mushrooms or from mushroom cell cultures as described in detail below, or be isolated from mushrooms or mushroom cell cultures as described in detail below. In some preferred embodiments, he listed composition comprises the listed compounds. In some preferred embodiments, the listed compositions consist essentially of the listed compounds. In some preferred embodiments, the listed compositions consist of the listed compounds.
Composition Compounds in composition
1. psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin
2. psilocybin, baeocystin
3. psilocybin, baeocystin, aeruginascin
4. psilocybin, baeocystin, aeruginascin, norpsilocin
5. psilocybin, baeocystin, norpsilocin, norbaeocystin
6. psilocybin, baeocystin, norbaeocystin
7. psilocybin, baeocystin, norpsilocin 8. psilocybin, aeruginascin
9. psilocybin, aeruginascin, norpsilocin
10. psilocybin, aeruginascin, norbaeocystin
11. psilocybin, aeruginascin, norpsilocin, norbaeocystin
12. psilocybin, norpsilocin
13. psilocybin, norpsilocin, norbaeocystin
14. psilocybin, norbaeocystin
15. baeocystin, aeruginascin, norpsilocin, norbaeocystin
16. baeocystin
17. baeocystin, aeruginascin
18. baeocystin, aeruginascin, norpsilocin
19. baeocystin, norpsilocin, norbaeocystin
20. baeocystin, norbaeocystin
21. baeocystin, norpsilocin
22. aeruginascin
23. aeruginascin, norpsilocin
24. aeruginascin, norbaeocystin
25. aeruginascin, norpsilocin, norbaeocystin
26. norpsilocin
27. norpsilocin, norbaeocystin
28. norbaeocystin
29. psilocin, baeocystin, aeruginascin, norpsilocin, norbaeocystin
30. psilocin, baeocystin
31. psilocin, baeocystin, aeruginascin
32. psilocin, baeocystin, aeruginascin, norpsilocin
33. psilocin, baeocystin, norpsilocin, norbaeocystin
34. psilocin, baeocystin, norbaeocystin
35. psilocin, baeocystin, norpsilocin
36. psilocin, aeruginascin
37. psilocin, aeruginascin, norpsilocin
38. psilocin, aeruginascin, norbaeocystin 39. psilocin, aeruginascin, norpsilocin, norbaeocystin
40. psilocin, norpsilocin
41. psilocin, norpsilocin, norbaeocystin
42. psilocin, norbaeocystin
Biotechnology-based production of entheogens offers a promising route to ensure a cost- effective, cGMP, robust supply for clinical development. Naturally derived psychedelic compounds may offer significant therapeutic advantages over synthetic molecules because of entourage effects. These reflect the action of additional components that may act synergistically or additively with the principal molecule or have pharmacokinetic effects. A case in point is psilocybin which is derived from multiple mushroom species that contain a wealth of additional active components. Anecdotal and clinical reports as well as preclinical studies suggest a discernible difference between the effects of chemically synthesized psilocybin and those of psychedelic mushrooms and also differences among the effects of different mushroom strains.
The present invention is based on the systematic evaluation of the psychedelic properties and therapeutic potential of entheogen compounds derived from cell culture and in particular on mushroom cell cultured, psychoactive compound-containing mushroom compositions. As used herein, the term "mushroom culture compositions" refers to compositions comprising a mushroom cell culture and products derived therefrom specifically including 1) fractions, such as aqueous fractions, prepared from the mushroom cell culture that contain one or more psychoactive compounds, 2) extracts, such as aqueous solvent extracts or organic solvent extracts, prepared from the mushroom cell culture that comprise one or more psychoactive compounds, and 3) purified preparations of one or more psychoactive compounds prepared from a mushroom cell culture by methods including one or more of solvent extraction, concentration by solvent removal, chromatography and other methods of extraction, purification and fractionation. As used herein, the term "psychoactive compounds" refers to chemical substances that change a person's mental state by affecting the way the brain and nervous system work, and specifically, includes, but is not limited to psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin, and combinations thereof.
In some preferred embodiments, the biomass used to prepare a mushroom extract useful in the compositions of the present invention is any fungi (including hybrids) of part thereof (e g., mycelia, primordia, fruiting bodies, etc.) containing the psilocybin biosynthetic gene cluster (psiD, psiH, psiK, psiM) or other mushrooms, fungi, lichens, etc. producing psilocybin or psilocybin analogs or related tryptamines. In some preferred embodiments, the biomass is mycelial biomass. Exemplary mushroom species include, but are not limited to, Conocybe sp., Copelandia sp., Galerina sp., Gerronema sp., Gymnopilus sp., Hypholoma sp., Inocybe sp., Mycena sp., Panaeolina sp., Panaeolus sp., Pluteus sp., andPsilocybe sp. Exemplary Psilocybes include, but are not limited to, P. acutipilea, P. angustipleurocystidiata, P. antioquensis, P. aquamarine, P. argentipes, P. armandii, P. aucklandii, P. Australiana, P. aztecorum, P. aztecorum bonetii, P. azurescens, P. baeocystis, P. bander iliensis, P. barrerae, P. bohemica, P. brasiliensis, P. brunneocystidiata, P. caeruleoannulata, P. caerulescens, P. caerulescens ombrophila, P. caerulipes, P. carbonaria, P. chiapanensis, P. collybioides, P. Columbiana, P. coprinifacies, P. cordispora, P. cubensis, P. cyanescens, P. cyanofibrillosa, P. dumontii, P. eucalypta, P. fagicola, P. fagicola mesocystidiata, P. farinacea, P. fimetaria, P. fuliginosa, P. furtadoana, P. galindoi, P. goniospora, P. graveolens, P. guatapensis, P. guilartensis, P. heimii, P. heliconiae, P. herrerae, P. hispanica, P. hoogshagenii , P. hoogshagenii convexa, P. inconspicua, P. indica, P. isabelae, P. jacobsii, P. jaliscana, P. kumaenorum, P. laurae, P. lazoi, P. liniformans, P. liniformans americana, P. mairei, P. makarorae, P. mammillata, P. meridensis, P. Mexicana, P. moseri, P. muliercula, P. natalensis, P. natarajanii, P. ochreata, P. papuana, P. paulensis, P. pelliculosa, P. pericystis, P. pintonii, P. pleurocystidiosa, P. plutonia, P. portoricensis, P. pseudoaztecorum, P. puberula, P. quebecensis, P. ramulosa, P. rostrata, P. rzedowskii, P. samuiensis, P. sanctorum, P. schultesii, P. semilanceata, P. septentrionalis, P. serbica, P. sierrae, P. sylvatica, P. singerii, P. strictipes, P. stuntzii, P. subacutipilea, P. subaeruginascens, P. subaeruginosa, P. subcaerulipes, P. subcubensis, P. sub tropi calls, P. subyungensis, P. subzapotecorum, P. tampanensis, P. tasmaniana, P. uruguayensis, P. uxpanapensis, P. venenata, P. veraecrucis, P. villarrealii, P. wassoniorum, P. weilii, P. weldenii, P. wrightii, P. xalapensis, P. yungensis, and P. zapotecorum.
The mushroom cell cultures of the present invention preferably utilize cells, parts or tissues of psilocybin-producing mushrooms. The invention is not limited to any particular method of mushroom cell culture. Suitable methods include liquid culture, semi-solid culture, static culture, static-dynamic culture, biofilm cultures, hybrid culture and various methods of bioreactor and non-bioreactor culture of mushroom cells including mycelia and sclerotia. Tn preferred embodiments, the mushroom cell cultures are conducted on manufactured substrates and may preferably be grown in culture vessels. In some particularly preferred embodiments, the mushroom cell cultures are supplemented with a phycocyanin (PC) composition. Suitable PCB compositions are described in US Pat. Publ. 2020/0155642, which is incorporated herein by reference in its entirety.
In some preferred embodiments, PC compositions useful in the present invention are produced by processes based on a chemical reaction between a gelling agent and a multivalent cation. Exposing the gelling agent to the multivalent cation (or vice versa) facilitates the formation of a gel and/or a membrane, trapping the cellular debris and other large-size molecular assemblies, but allowing diffusion of water-soluble small molecules and proteins into the surrounding aqueous medium, usually water, thereby permitting the purification of the desired substance such as the PCB or PC. The multivalent cation may also act on the cell wall of, for example Spirulina, thereby making it more fragile and porous and consequently allowing the extracellular diffusion of PCB and other water-soluble molecules. Thus, the methods of the present invention result not only in the purification of, for example PC but also permit the extraction of PC from Spirulina in an aqueous solution in a single step.
Phycobiliproteins are water-soluble proteins that play a key role in photosynthesis. Phycobiliproteins take the form of a complex between proteins and covalently bound phycobilins that act as chromophores. They are the most important constituents of the phycobilisome. The major phycobiliproteins are C-phycocyanin, R-phycoerythrin, B-phycoerythrin and allophycocyanin. The blue phycobiliprotein (PBP) pigment complex contained in Spirulina that is generally called phycocyanin (PC) plays an important role in the photosynthetic chain acting as the link between light energy and chlorophyll. A number of different algae including Porphyridium cruentum Galdieria sulphuria and Aphanizomenon flos aquae (AF A), other cyanobacteria and other organisms contain varying amounts of PBP complexes.
In some embodiments, the PC compositions used in the compositions described herein are described in co-pending application 16/685,363, filed Nov. 15, 2019, and which is incorporated herein by reference in its entirety.
The purification of PC from organisms such as Spirulina is a delicate and potentially complex process, especially because these natural proteins are fragile and sensitive to heat and acids. For example, PC is known to degrade rapidly at temperatures over 55°C and at low PH, with a loss of blue color and consequently utility as a food colorant.
The destruction of the Spirulina cell wall, an integral part of the extraction of PC, can damage the PC and if the extraction takes place over a long period cause the degradation of the PC. The nature of the raw biomass, for example whether it is fresh or dried, the type of drying process including temperature and time of exposure to heat, the preparation of the raw material, the addition of heat protecting substances, the length of time and temperature of storage, the final water content, the method of packaging, the level of oxidation of the protein and fats, exposure to bacteria and yeasts and fungi, residual chemicals and nutrients and range of other factors affect the quality and yield of PC.
Once the PC has been extracted, it can be further purified. Purification requires separation of blue- or other colored PBPs such as PC from the green-colored chlorophylls and is usually undertaken using filtration technologies including various types of centrifuges and filtration units. This can include disk centrifuges, decanting centrifuges, charcoal filtration, ultrafiltration, nanofiltration, reverse and forward osmosis filtration, high-pressure filtration, tangential filtration and a range of technologies that separate out the PC from the chlorophyll and cell debris and other undesirable substances.
Purification using filtration is an expensive process and contributes substantially to the high current cost of PBPs such as PC. Purification using filtration is also wasteful as it generally requires large quantities of disposable filters or filters that require cleaning with acid and alkaline solutions that are potential environmental pollutants if not managed and disposed of appropriately. Also, in order to reduce the cost of purification using filtration, additional prefiltration production steps may be used to precipitate out undesirable substances using chitosan and other precipitating or flocculating or purifying agents. These steps are time-consuming, expensive and potentially polluting and can damage the PC. Also, the use of certain precipitating and flocculating and purifying agents may result in the PC no longer qualifying as being of organic origin, if the PC was purified from organic biomass.
Following purification, the liquid rich in PC is generally dried using spray drying processes. Prior to spray drying sugars such as trehalose or maltodextrin and salts such as sodium citrate may be added in order to facilitate the spray drying process, protect the PC from the heat of the spray drying process and enhance the color and solubility of the powder emanating from the spray drying process. As a result of the purification and drying steps, there is an inevitable loss in the yield and quality of the PC. This impacts on the quality, color value, pricing and profitability of the final product. .
The form of delivery of the extracted and sometimes purified algae extract depends on its applications. For example PC as a food colorant is delivered as a dry powder. For human consumption as a nutraceutical, functional food, health food or pharmaceutical, there are processes to manufacture capsules containing Spirulina or PC preparations, to be swallowed. The document CN103285375 teaches the production of PC microspheres with an external oily layer, produced by the action of a calcium solution on an emulsion containing PC and sodium alginate in the water phase, paraffin and emulsifiers. The document CN101322568 describes capsules containing Spirulina, sodium alginate, chitosan, additives and calcium chloride. PC diffusion during storage is not mentioned and does not occur because the PC is degraded by heat during the pasteurization process.
In some preferred embodiments, PC compositions useful in the present invention are produced by processes based on a chemical reaction between a gelling agent and a multivalent cation. Exposing the gelling agent to the multivalent cation (or vice versa) facilitates the formation of a gel and/or a membrane, trapping the cellular debris and other large-size molecular assemblies, but allowing diffusion of water-soluble small molecules and proteins into the surrounding aqueous medium, usually water, thereby permitting the purification of the desired substance such as PC. The multivalent cation may also act on the cell wall of, for example Spirulina, thereby making it more fragile and porous and consequently allowing the extracellular diffusion of PC and other water-soluble molecules. Thus, the methods of the present invention result not only in the purification of, for example PBP but also permit the extraction of PBP from Spirulina in an aqueous solution in a single step. The methods described may be utilized with other species of microalgae, including, but not limited to Chlorella, Porphyndium, Aphanizomenon flos aquae (AFA) and Galdieria.
In some preferred embodiments, the processes to prepare purified PC from Spirulina comprises the following steps:
• Mixing dried or fresh Spirulina, water and a gelling agent, for example sodium alginate to provide a first solution (Solution A); • Forming microcapsules by dropping small drops of Solution A using a dropper, pipette or some other industrial device or machine into a second solution of a salt of divalent cations;
• Diluting the microcapsules into a predetermined volume of water or other liquid to provide an aqueous solution to permit purification through diffusion of the PC from the microcapsules; and
• Removal of the microcapsules and any debris from the liquid.
In some preferred embodiments, the processes to prepare purified PC from Spirulina comprises the following steps:
• Mixing dried or fresh Spirulina, water and divalent cation, for example calcium chloride to provide a first solution (Solution A);
• Forming microcapsules by dropping small drops of Solution A using a dropper, pipette or some other industrial device or machine into a second solution of a gelling agent, such as sodium alginate;
• Diluting the microcapsules into a predetermined volume of water or other liquid to provide an aqueous solution to permit purification through diffusion of the PC from the microcapsules; and
• Removal of the microcapsules and any debris from the liquid.
In some preferred embodiments, the processes to prepare purified PC from Spirulina comprises the following steps:
• Mixing dried or fresh Spirulina with water;
• Transforming the Spirulina and water mixture into a droplet, extruded tube or sausage or some other form;
• Coating this droplet, extruded tube or sausage or some other form with a layer of a solution of gelling agent such as sodium alginate;
• Coating the droplet, extruded tube or sausage or some other form that has been coated with the gelling agent, with a salt of divalent cations;
• Placing the droplets, extruded tubes or sausages or some other forms that have been coated with the gelling agent and thereafter the divalent cation solution into a predetermined volume of water or other liquid to provide an aqueous solution to permit purification through diffusion of the PC; and
• Removal of the droplets, extruded tubes or sausages or some other form and any debris from the liquid.
In some preferred embodiments, the different methods previously described to produce the purified PC compositions can be used simultaneously, sequentially or repeatedly.
In some preferred embodiments, the PC containing organism biomass is specially prepared through heat, cold, chemical and biological processes and physical mechanisms and techniques to facilitate the purification process of this invention.
In some preferred embodiments, the PC composition comprises a dried powder comprising a purified PBP composition as described above, the powder having a residual moisture content of less than about 10% w/w of the powder.
In some preferred embodiments, the PC composition comprises fresh or freshly harvested PBP containing organism biomass.
In some preferred embodiments, the microcapsule or other form contains chemicals or biological agents that enhance or retard or selectively control the purification of the PC.
In some preferred embodiments, the microcapsule or other form contains a natural substance that is purified or concentrated in conjunction with or ‘chaperoned’ into the aqueous solution by the PC or some other substance that exists in the organism biomass.
In some preferred embodiments the aqueous solution into which the PC diffuses is modified by changing its temperature, through agitation or mixing or the addition of chemical or biological agents such as acids, alkalis, salts and antimicrobial agents to enhance or retard diffusion of PC and to prevent contamination.
In some preferred embodiments, the microcapsule or other form is frozen, dried or treated in some other way to enhance or retard diffusion of the PC into the aqueous solution.
In some preferred embodiments, the biomass used to prepare a PC composition useful in the present invention is the genus Arthrospira, and more preferably to the species Arthrospira platensis (commonly known as Spirulina). In some preferred embodiments, the gelling agent is sodium alginate which reacts with most multivalent cations and especially well with the calcium ions. Preferred sources of calcium ions are, for example, calcium chloride and calcium gluconate. The present invention is not limited to the use of calcium ions. Tn other embodiments, the multivalent cations may be provided by salts of manganese, magnesium, zinc, or barium.
In some preferred embodiments, the first solution is prepared with 10% to 60% Spirulina (wet weight), 0.1% to 5% sodium alginate (dry weight) and water. Several additives can be added to the solution including flavorings, color agents, preservatives, moisteners, natural antibiotics, thickeners, sugars, anti-foaming agents, salts, acids and alkalis.
In some preferred embodiments, the second solution is an aqueous solution containing multivalent cations at a concentration between 0.005 and 0.5 mole per liter, preferably between 0.05 and 0.2 mole per liter (corresponding for example to the range 5.5 - 22 g/L of calcium chloride). This solution can also contain flavorings, color additives, preservatives and acidifying agents.
The present invention is not limited to any particular mechanism of action. Indeed, an understanding of the mechanism of action is not necessary to practice the invention. Nevertheless, it is contemplated that the microcapsules generated by dropping the first solution into the second solution are irregular, roughly egg-shaped with a maximum dimension of less than 6 mm and have a solid texture. Other forms can include tubular or spaghetti-like shapes, sausages, disks and irregular shapes. The setting time, during which the microcapsules or other forms are immersed in the solution B, is preferably less than 1 hour.
The purification of the preferably takes place as the last step of the process. Specifically, the microcapsules are immersed in a large volume of water and the medium containing the microcapsules is kept at a low temperature (between 0 and 6°C) for from about 1 to 60 hours. The diffusion of the purified PC can demonstrated when the water become progressively bluer and shows a purple fluorescence when exposed to light. These observed phenomena as characteristic of the PC complex of Spirulina.
In preferred embodiments, the microcapsules or other forms may be manufactured in a workshop or a factory and the extraction is undertaken at a consumer's home or at an industrial site or on a farm or in a laboratory.
In some preferred embodiments, microcapsules are packaged into a porous container such as a bag, a sachet, a pod or the like, with a pore size much lower than the microcapsule size to ensure that PC can diffuse while the microcapsules remain in the container. In some preferred embodiments, the process steps are conducted at low temperatures (e g., between 0 and 6°C) to prevent PC degradation or natural or chemical preservatives are added.
The process of purification described in this invention has a number of advantages over existing algal biomass purification methods. The main advantage is that it is possible to limit or even avoid the use of membrane-based filtration in the production of purified algal extracts such as PC, thereby substantially reducing the cost of production and improving the quality of such extracts. The process described also does not require the use of salts and other substances to precipitate out the PC and the consequent expense of dialysis, osmosis, gels and exchange columns to remove such salts. This invention also can increase the yield of PBP extraction and the concentration of proteins of interest in the purified extract. This invention permits the combination of extraction and purification in a single step, if desired. As a result, it is possible to make purified extract of Spirulina with very high PC concentrations. This permits purification of PC without using chemical solvents or any product from animal origin, allowing the making of an PC-rich extract compatible with vegan, halal and kosher food requirements. This invention is not damaging to delicate and sensitive proteins and other molecules including PC thereby permitting the purification of highly active natural extracts. This invention permits low-waste, low water consumption purification with positive impacts in terms of sustainability and compatibility with circular economy principles. The processes can be applied to a wide range of algae and other natural substances. This invention is easily scalable with fairly low capital expenditure costs on machinery and technologies.
The methods described herein are useful for producing compositions containing a high quality protein fraction that is enriched for PC. The protein content and quality of the PC purified extracts obtained by these methods differ substantially from other described PC purified extracts, generally purified using membrane filtration methods.
In some preferred embodiments, the present invention provides the purified PC protein compositions are characterized by one or more of the following characteristics: a) the protein fraction of the composition comprises greater than about 30% of a protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; b) the protein fraction of the composition comprises greater than about 5% of a protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; c) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 45,578 kDa and an isoelectric point of about 6.2 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; d) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 35,014 kDa and an isoelectric point of about 5.9 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; e) the protein fraction of the composition comprises greater than about 0.30% of a protein having a molecular weight of about 24,688 kDa and an isoelectric point of about 5.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; f) the protein fraction of the composition comprises greater than about 2% of a protein having a molecular weight of about 22,522 kDa and an isoelectric point of about 5.9 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; g) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 21,023 kDa and an isoelectric point of about 7.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; h) the protein fraction of the composition comprises greater than about 0.50% of a protein having a molecular weight of about 13,417 kDa and an isoelectric point of about 7.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; i) the protein fraction of the composition comprises a ratio of major protein constituents to minor protein constituents of less than 3.5: 1 based on the aggregate mass of the proteins, wherein major protein constituents are the protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the minor protein constituents are the remainder of the proteins; j) the protein fraction of the composition comprises less than about 75% on a mass basis of the combined amounts of the protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; and k) the composition produces a solution having a color value of greater than 180 E 10%/lcm when 250 mg of a dry powder of the composition are dissolved in one liter of water and absorbance is measured at 618 nm.
In some preferred embodiments, the composition has at least two of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least three of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least four of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least five of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least six of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least seven of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least eight of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least nine of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has at least ten of characteristics a, b, c, d, e, f, g, h, i, j and k. In some preferred embodiments, the composition has all eleven characteristics a, b, c, d, e, f, g, h, i, j and k. It will be understood by those of skill in the art that in preferred embodiments, the compositions of the present invention may be identified by any subcombination of one or more of the characteristics identified above.
In some preferred embodiments, the purified phycobiliprotein composition is produced by a process comprising: mixing PCB containing organism biomass with water and gelling agent, forming a droplet, introducing a droplet of the first solution into a second solution containing a salt of divalent cations under conditions such that microcapsules form, and obtaining an extract enriched for PCB by mixing the microcapsules with a volume of an aqueous solution under conditions such that the phycobiliprotein diffuses from the microcapsules into the aqueous solution.
In some preferred embodiments, the purified PC composition is produced by a process comprising: mixing PC containing organism biomass with water and a salt of divalent cations, forming a droplet, introducing a droplet of the first solution into a second solution containing a gelling agent under conditions that microcapsules form, and obtaining an extract enriched for PC by mixing the microcapsules with a volume of an aqueous solution under conditions such that the PCB diffuses from the microcapsules into the aqueous solution.
In some preferred embodiments, the purified PC composition is produced by a process comprising: mixing PCB containing organism biomass with water, coating a droplet, extruded tube, sausage or some other form of this first solution with a second solution of gelling agent and introducing a droplet, extruded tube, sausage or some other form of the first solution coated with the second solution into a third solution containing a salt of divalent cations under conditions such that a coated droplet, extruded tube sausage or another form is obtained, and thereafter obtaining purified extract rich in PC by mixing the droplet, extruded tube, sausage or other form with a volume of an aqueous solution under conditions such that the PC diffuses from the droplet, extruded tube, sausage or other form into the aqueous solution.
In some preferred embodiments, the different methods previously described to produce the purified PC compositions can be used simultaneously, sequentially or repeatedly. In some preferred embodiments, the PC compositions are provided as a dried powder.
In some preferred embodiments, the residual moisture in the powder is less than 5%, more preferably less than 4%, and most preferably less than 3% or 1%. In other preferred embodiments, the powder may be produced by spray-drying, spray -freeze drying, refractance window drying, microwave drying, air drying, fluidized bed drying, vacuum drying, natural drying, microwave drying or foam drying the solutions of purified PCB.
Accordingly, in some further preferred embodiments, the present invention provides methods for mushroom cell culture comprising culturing mushroom cells on substrate to provide a mushroom cell culture and contacting (or supplementing) the mushroom cell with a PC composition as described above. In some preferred embodiments, the mushroom cells are psychoactive mushroom cells. In some preferred embodiments, the mushroom cell culture produces one or more psychoactive compounds or other compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the methods further comprise processing the mushroom cell culture to provide or remove an aqueous fraction or extract from the culture comprising one or more psychoactive compounds, preferably one or more of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the extract may be made by separating an aqueous fraction from the mushroom cell culture. Tn other preferred embodiments, the mushroom cell culture may be extracted with an aqueous or organic solvent or combinations thereof. In some preferred embodiments, the fractions or extracts thus obtained may be further concentrated, for example by removal or of the aqueous or organic solvent by evaporation, lyophilization or spray drying, or by concentration via column chromatography.
In some preferred embodiments the extract is prepared from the mushroom biomass by the encapsulation method described for PC purification, followed by a chromatography step, such as one or more of FLASH chromatography, ion-exchange chromatography, Centrifugal Partition chromatography or size-exclusion chromatography. In some embodiments, where a psilocybin-free extract is desired, the chromatography steps may be selected so that psilocybin is removed from the extract. In some preferred embodiments, the gelling agent used for encapsulation of the biomass is sodium alginate which reacts with most multivalent cations and especially well with the calcium ions. Preferred sources of calcium ions are, for example, calcium chloride and calcium gluconate. The present invention is not limited to the use of calcium ions. In other embodiments, the multivalent cations may be provided by salts of manganese, magnesium, zinc, or barium.
In some preferred embodiments, the first solution is prepared with 10% to 60% of the mushroom biomass (wet weight), 0.1% to 5% sodium alginate (dry weight) and water. Several additives can be added to the solution including flavorings, color agents, preservatives, moisteners, natural antibiotics, thickeners, sugars, anti-foaming agents, salts, acids and alkalis.
In some preferred embodiments, the second solution is an aqueous solution containing multivalent cations at a concentration between 0.005 and 0.5 mole per liter, preferably between 0.05 and 0.2 mole per liter (corresponding for example to the range 5.5 - 22 g/L of calcium chloride). This solution can also contain flavorings, color additives, preservatives and acidifying agents.
The present invention is not limited to any particular mechanism of action. Indeed, an understanding of the mechanism of action is not necessary to practice the invention. Nevertheless, it is contemplated that the microcapsules generated by dropping the first solution into the second solution are irregular, roughly egg-shaped with a maximum dimension of less than 6 mm and have a solid texture. Other forms can include tubular or spaghetti-like shapes, sausages, disks and irregular shapes. The setting time, during which the microcapsules or other forms are immersed in the solution B, is preferably less than 1 hour.
The purification of the preferably takes place as the last step of the process. Specifically, the microcapsules are immersed in a large volume of water and the medium containing the microcapsules is kept at a low temperature (between 0 and 6°C) for from about 1 to 60 hours.
In preferred embodiments, the microcapsules or other forms may be manufactured in a workshop or a factory and the extraction is undertaken at a consumer's home or at an industrial site or on a farm or in a laboratory.
In some preferred embodiments, microcapsules are packaged into a porous container such as a bag, a sachet, a pod or the like, with a pore size much lower than the microcapsule size to ensure that water soluble compounds can diffuse while the microcapsules remain in the container. In some preferred embodiments, the process steps are conducted at low temperatures (e.g., between 0 and 6°C).
In some preferred embodiments, the mushroom extracts are provided as a dried powder. In some preferred embodiments, the residual moisture in the powder is less than 5%, more preferably less than 4%, and most preferably less than 3% or 1%. In other preferred embodiments, the powder may be produced by spray-drying, spray -freeze drying, refractance window drying, microwave drying, air drying, fluidized bed drying, vacuum drying, natural drying, microwave drying or foam drying the solutions of purified PBP.
In some preferred embodiments, psilocybin or psilocin, or a composition comprising psilocybin such as the mushroom cell culture extracts described herein, are co-administered with a 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599. The present invention also provides formulations comprising psilocybin or psilocin, or a composition comprising psilocybin such as the mushroom cell culture extracts described herein, and a 5-HTIA receptor agonist, as well as kits comprising a psilocybin or psilocin formulation and a 5-HTIA receptor agonist formulation.
In some preferred embodiments, the mushroom cell culture extracts and fractions may be further formulated with one or more pharmaceutically acceptable carriers or delivery vehicles. As used herein, "pharmaceutically of physiologically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
The mushroom cell extracts of the present invention may be formulated with different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The mushroom cell extracts of the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation and nebulization (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Upon formulation, mushroom cell extracts of the present invention will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
Further in accordance with the present invention, the mushroom cell extracts the present invention suitable for administration is provided in a physiologically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
In accordance with the present invention, the mushroom cell extract is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
The actual dosage amount of a mushroom cell extract of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration: Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
2. TREATMENT OF PSYCHIATRIC DISORDERS
In some preferred embodiments, the present invention provides entheogen compositions as described above for the treatment of psychiatric disorders. In some further preferred embodiments, the present invention provides mushroom cell culture compositions comprising psychoactive compounds for the treatment of psychiatric disorders. The present invention is not limited to the treatment of any particular psychiatric disorders. Indeed, the treatment of a variety of psychiatric disorders is contemplated, including, but not limited to, post-traumatic stress disorder (PTSD), depressive and anxiety disorders, schizophrenia, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
In some particularly preferred embodiments, the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of depression, schizophrenia, anxiety, PTSD or OCD. In some particularly preferred embodiments, the entheogen compositions comprise one or more of the following compounds: psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin. In some particularly preferred embodiments, the entheogen compositions comprise psilocybin and one or more of the following compounds: baeocystin, aeruginascin, norpsilocin, norbaeocystin. In some preferred embodiments, and specifically for treatment of the disorders described herein, the effective dosage of the entheogen composition is a subpsychedelic dose.
A "subpsychedelic dose" as used herein is a sub-hallucinogenic doses of a psychedelic substance such as a psilocybin. Sub-psychedelic dosing can be achieved via several preferred schedules: 1) Sub-chronic, sub-psychedelic doses of psilocybin given over several days with or without one or more the entourage compounds baeocystin, aeruginascin, norpsilocin, and norbaeocystin ; 2) A sustained release preparation of psilocybin with or without one or more the entourage compounds baeocystin, aeruginascin, norpsilocin, and norbaeocystin which will allow the full psychedelic dose to be administered over 24 hours and thus avoid acute psychotogenic effects; 3) A psychedelic dose of psilocybin with or without one or more the entourage compounds baeocystin, aeruginascin, norpsilocin, and norbaeocystin in conjunction with a 5- HT2A receptor antagonist, that blocks psilocybin-induced psychedelic effects; 4) Use of a psychedelic dose of psilocybin with or without one or more the entourage compounds baeocystin, aeruginascin, norpsilocin, and norbaeocystin in conjunction with a 5-HT1A agonist such as buspirone which will prevent the possible psychosis enhancing effects while at the same time exerting a possible dopaminergically mediated beneficial effect via dopamine D2 receptor antagonist effects of buspirone. This approach is supported by our recent findings. We have demonstrated that the 5-HT1A agonist, 80H-DPAT, blocks the psilocybin induced head twitch response (HTR) in mice.
In some particularly preferred embodiments, the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of OCD. Obsessive symptoms to be treated include: fear of contamination or dirt; doubting and having difficulty tolerating uncertainty, needing things orderly and symmetrical, aggressive or horrific thoughts about losing control and self-harming or harming others; and unwanted thoughts, including aggression, or sexual or religious subjects.
Compulsive symptoms to be treated include: excessive hand-washing; checking doors repeatedly to make sure they are locked, checking the stove repeatedly to make sure it is off; counting in certain patterns; silently repeating a prayer, word or phrase; arranging items in a certain order. In some particularly preferred embodiments, administration of the entheogen composition comprising one or more psychoactive compounds alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof. In some preferred embodiments, the dosage is a sub-perceptual or sub-psychedelic dose.
In some particularly preferred embodiments, the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of schizophrenia. The symptoms to be treated include: hallucinations, delusions and thought disorder and negative symptoms which include impairments of volition, social functioning and affect as well as deficits in cognition, specifically executive functions. In some preferred embodiments, the dosage is sub-chronic, sub-psychedelic or sustained release preparation of psilocybin which will allow the full psychedelic dose to be administered over 24 hours or a psychedelic dose of psilocybin in conjunction with 5-HT2A receptor antagonist, that blocks psilocybin-induced psychedelic effects or a psychedelic dose of psilocybin in conjunction with a 5-HT1A agonist such as buspirone which will prevent the possible psychosis enhancing effects while at the same time exerting a possible dopaminergically mediated beneficial effect via dopamine D2 receptor antagonist effects of buspirone.
In some particularly preferred embodiments, the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of PTSD. The symptoms to be treated include: Symptoms of intrusive memories, including, but not limited to recurrent, unwanted distressing memories of the traumatic event, reliving the traumatic event as if it were happening again (i.e., flashbacks), upsetting dreams or nightmares about the traumatic event, and severe emotional distress or physical reactions to something that reminds the subject of the traumatic event; Symptoms of avoidance including, but not limited to trying to avoid thinking or talking about the traumatic event; avoiding places, activities or people that remind the subject of the traumatic event, and negative changes in thinking and mood; Symptoms of negative changes in thinking and mood including, but not limited to, negative thoughts about the subject, other people or the world; hopelessness about the future; memory problems, including not remembering important aspects of the traumatic event, difficulty maintaining close relationships, feeling detached from family and friends, lack of interest in activities the subject once enjoyed, difficulty experiencing positive emotions, feeling emotionally numb, and changes in physical and emotional reactions; and Symptoms of changes in physical and emotional reactions (also called arousal symptoms) including, but not limited to being easily startled or frightened, always being on guard for danger, self-destructive behavior, such as drinking too much or driving too fast, trouble sleeping, trouble concentrating, irritability, angry outbursts or aggressive behavior, and overwhelming guilt or shame. For children 6 years old and younger, signs and symptoms may also include: re- enacting the traumatic event or aspects of the traumatic event through play and frightening dreams that may or may not include aspects of the traumatic event.
In some particularly preferred embodiments, administration of an entheogen composition comprising one or more psychoactive compounds alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof. In some preferred embodiments, the dosage is a sub-perceptual or sub-psychedelic dose. In some preferred embodiments, the present invention provides methods of providing prophylaxis for PTSD in a subject in need thereof comprising administering to the subject an effective amount of an entheogen composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the method comprises administering to the subject an effect amount of an entheogen composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the effective amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD. In some preferred embodiments, the entheogen composition is preferably administered to the subject when the subject presents with a traumatic event or injury or soon thereafter. For example, in some embodiments, the entheogen composition is administered to the subject within 4 weeks, 1 week, 3 days or 1 day of a psychological trauma or other traumatic event. In some preferred embodiments, the dosage is a sub-perceptual or sub-psychedelic dose. 3. TREATMENT OF AUTISM SPECTRUM DISORDER
The US Centers for Disease Control and Prevention lists the following possible treatments for ASD (on the world wide web at cdc.gov/ncbddd/autism/treatment.html): 1) Behavior and communication approaches; 2) Dietary approaches; 3) Complementary and alternative treatments; 4) Drug treatment. It was demonstrated that with intensive applied behavioral analysis, some children at an early age may reach a degree of improvement. Lovaas 01. Behavioral treatment and normal educational and intellectual functioning in young autistic children. J Consult Clin Psychol. 1987;55 :3. For example, such children may show some gains in cognitive and adaptive functioning and education as compared to those who did not receive treatment. Warren et al., A Systematic Review of Early Intensive Intervention For Autism Spectrum Disorders. Pediatrics. 201 !;127:el303-el311. Some dietary treatments have been developed to address ASD symptoms. However, a systematic review of 19 randomized control trials could not find any evidence of a positive effect. Sathe et al., Nutritional and Dietary Interventions For Autism Spectrum Disorder: A Systematic Review. Pediatrics. 2017;139. Studies have been conducted to find drug treatments against ASD such as oxytocin and propranolol. Bernaerts et al., Behavioral effects of multiple-dose oxytocin treatment in autism: a randomized, placebo-controlled trial with long-term follow-up. Mol Autism. 2020;11 :1-14; Sagar-Ouriaghli et al., Propranolol for treating emotional, behavioural, autonomic dysregulation in children and adolescents with autism spectrum disorders. J Psychopharmacol. 2018;32:641- 653.Balovaptan, a selective antagonist of the vasopressin VIA receptor is being tested for the treatment of some behavioral deficits of adult ASD patients but there is only cautious optimism regarding this drug. Initial clinical trials on a small number of ASD patients showed that serotonin reuptake inhibitors, venlafaxine and fluoxetine, may attenuate anxiety and repetitive behaviors in adults suffering from neurodevelopmental disorders. Schnider et al., Discovery of balovaptan, a vasopressin la receptor antagonist for the treatment of autism spectrum disorder. J Med Chem. 2020;63: 1511-1525; Hollander et al., Venlafaxine in children, adolescents, and young adults with autism spectrum disorders: an open retrospective clinical report. J Child Neurol. 2000;15:132-135; Mouti et al., Fluoxetine for Autistic Behaviors (FAB trial): study protocol for a randomized controlled trial in children and adolescents with autism. Trials. 2014;15: 1 -8; LeClerc and Easley, Pharmacological therapies for autism spectrum disorder: A review. P T. 2015;40:389.
Manipulations of the enteric microbiome using microbiota transfer therapy look promising for the treatment of ASD. However, despite some progress, such approaches are still far from regular clinical use. Further studies are needed to find the right doses, administration routes, and protocols of treatment to assess their therapeutic efficacy and the extent of possible side effects. Furthermore, there is no established pharmacological treatment for the core symptoms of ASD available today. The only FDA-approved medications for the associated irritability are risperidone and aripiprazole, the drugs with substantial metabolic adverse events. Conley, Risperidone side effects. J Clin Psychiatry. 2000;61 :20-25; Valicenti-McDermott and Demb. Clinical effects and adverse reactions of off-label use of aripiprazole in children and adolescents with developmental disabilities. J Child Adolesc Psychopharmacol. 2006;16:549- 560. Importantly, studies tend to be aimed at the symptoms rather than the etiology of this disorder.
Accordingly, in some preferred embodiments, the present invention provides entheogen compositions as described above for the treatment of autism spectrum disorder. In some further preferred embodiments, the present invention provide mushroom cell culture compositions comprising psychoactive compounds, including entheogens, for the treatment of autism spectrum disorder. In some particularly preferred embodiments, the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of ASD. In some particularly preferred embodiments, the entheogen compositions comprise one or more of the following compounds: psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin. In some particularly preferred embodiments, the entheogen compositions comprise psilocybin and one or more of the following compounds: baeocystin, aeruginascin, norpsilocin, norbaeocystin. In some preferred embodiments, and specifically for treatment of the disorders described herein, the effective dosage of the mycelial culture compositions is a microdose or subpsychedelic dose as described above.
In some particularly preferred embodiments, the present invention provides entheogen compositions comprising one or more psychoactive compounds for the treatment of one or more symptoms of ASD, including but not limited to symptoms related to social communication and interaction and behavioral symptoms. Symptoms related to social communication and interaction include one or more of failure to respond to name, resistance to holding or cuddling, poor eye contact, lack of facial expression, delayed speech or failure to speak, inability to maintain conversation, speaking with an abnormal tone, repetitive use of words, failure to understand or comply with directions, failure to express emotions, and inability to recognize nonverbal cues. Behavioral symptoms include one or more of performance of repetitive movements, performance of activities that can lead to self-harm such as head-banging, performance of specific routines, lack of coordination or performance of odd movement patterns, sensitivity to light or touch, and abnormal fixation on object or activity. In some particularly preferred embodiments, administration of the entheogen composition comprising one or more psychoactive compounds alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof. In some preferred embodiments, the dosage is a microdose or subpsychedelic dose.
In some preferred embodiments, the subject is less than 60 years old. In some preferred embodiments, the subject is less than 50 years old. In some preferred embodiments, the subject is less than 40 years old. In some preferred embodiments, the subject is less than 30 years old. In some preferred embodiments, the subject is less than 20 years old. In some preferred embodiments, the subject is less than 10 years old. In some preferred embodiments, the subject is less than 9 years old. In some preferred embodiments, the subject is less than 8 years old. In some preferred embodiments, the subject is less than 7 years old. In some preferred embodiments, the subject is less than 6 years old. In some preferred embodiments, the subject is less than 5 years old. In some preferred embodiments, the subject is less than 4 years old. In some preferred embodiments, the subject is less than 3 years old. In some preferred embodiments, the subject is less than 2 years old.
4. TREATMENT OF BRAIN IN JURIE S
In some embodiments, the present invention provides entheogen compositions as described above for the treatment of brain injuries including, but not limited to, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multisystem trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma). In some embodiments, the present invention provides mushroom cell culture compositions comprising psychoactive compounds, including entheogens, for the treatment of brain injuries including, but not limited to, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma). In some preferred embodiments, the present invention provides full spectrum entheogen compositions or full spectrum mushroom extracts (FSMEs) comprising one or more psychoactive compounds for the treatment of brain injuries. In some particularly preferred embodiments, the FSME is an extract from the fruiting body of mushrooms. In some particularly preferred embodiments, the present invention provides mushroom cell culture compositions comprising one or more psychoactive compounds for the treatment of brain injuries. In some particularly preferred embodiments, the mushroom cell culture compositions comprise one or more, two or more, three or more, four or more, or all of the following compounds: psilocybin, baeocystin, aeruginascin, norpsilocin, norbaeocystin. In some particularly preferred embodiments, the mushroom cell culture compositions comprise psilocybin and one or more, two or more, three or more of all of the following compounds: baeocystin, aeruginascin, norpsilocin, norbaeocystin. In some preferred embodiments, and specifically for treatment of the disorders described herein, the effective dosage of the entheogen composition is a microdose or sub-hallucinogenic dose.
In some preferred embodiments, the entheogen composition is administered immediately before, during or immediately after anesthesia or sedation. In some embodiments, the entheogen composition is administered concurrently or serially to a subject in need thereof with an anesthesia or sedation agent. In some embodiments, administration of the entheogen composition not only treats the brain injury but also to prevent sequelae such as neurodegeneration, traumatic encephalopathy, PTSD, addiction disorders, cognitive decline and post-traumatic depression.
In some particularly preferred embodiments, the present invention provides entheogen compositions as described above comprising one or more psychoactive compounds for the treatment of one or more symptoms of brain injuries. Symptoms of mild TBI include, but are not limited to, headache, nausea or vomiting, fatigue or drowsiness, problems with speech, dizziness or loss of balance, blurred vision, ringing in the ears, a bad taste in the mouth or changes in the ability to smell, sensitivity to light or sound, memory or concentration problems, mood changes or mood swings, feeling depressed or anxious, difficulty sleeping, sleeping more than usual. Symptoms of moderate to severe TBI include, but are not limited to, persistent headache or headache that worsens, repeated vomiting or nausea, convulsions or seizures, inability to awaken from sleep, weakness or numbness in fingers and toes, loss of coordination, profound confusion, agitation, combativeness or other unusual behavior, slurred speech, and coma and other disorders of consciousness. Symptoms of CTE include, but are not limited to, difficulty thinking (cognitive impairment), memory loss, problems with planning, organization and carrying out tasks (executive function), impulsive behavior, aggression, mood disorders, depression or apathy, emotional instability, substance misuse, suicidal thoughts or behavior, Parkinsonism, and motor neuron disease.
In some particularly preferred embodiments, administration of the entheogen composition comprising one or more psychoactive compounds alleviates, prevents, reduces the frequency of, or provide improvement of one or more of the foregoing symptoms in a subject in need thereof.
5. PROMOTION OF NEUROPLASTICITY
As described in Example 9, the entheogen composition of the present invention have been shown to increase markers of neuroplasticity in the brain. Accordingly, in some preferred embodiments, the present invention provides methods of promoting neuroplasticity in a subject in need thereof comprising administering to the subject an entheogen composition comprising psilocybin or psilocin and one or more entourage compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin. In some preferred embodiments, the entheogen composition comprises psilocybin. In some preferred embodiments, the entheogen composition is an entheogen composition as described supra. 223. In some preferred embodiments, the entheogen composition is co-administered with a 5-HTIA receptor agonist. The present invention is not limited to the use of any particular 5-HTIA receptor agonist. In some preferred embodiments, the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
In some preferred embodiments, the subject in need thereof has a disease, condition or disorder associated with neural atrophy or the impairment of neuroplasticity . In some preferred embodiments, the disease, condition or disorder associated with neural atrophy or the impairment of neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD. In some preferred embodiments, the psychiatric disease or disorder includes, but is not limited to, depression, anxiety, social anxiety disorder, obsessive compulsive disorder and major depressive disorder. In some preferred embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, or Huntington's disease. In some preferred embodiments, the brain injury or disorder is traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma or other conditions (i.e., where the brain injury is not result of direct physical brain trauma).
In some preferred embodiments, administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43 and Synaptophysin in one or more areas of the brain selected from the group consisting of the frontal cortex, amygdala, hippocampus, and striatum.
In some preferred embodiments, the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with neural atrophy or the impairment of neuroplasticity.
EXPERIMENTAL
Example 1 (Prophetic)
Drugs. Chemically synthesized psilocybin and psilocin will be provided by Usona Institute, Madison, Wisconsin, U.S.A. Naturally-derived psilocybin, psilocin and mushroom extracts will be provided by Back of the Yards Algae Sciences (BYAS), Chicago, U.S.A. Other drugs and chemicals will be purchased from Sigma-Aldrich Israel Ltd. Drugs will be administered by intraperitoneal (i.p.) or subcutaneous (s.c.) injection or, for sub-chronic administration of 7 days or more, by Alzet osmotic minipumps implanted subcutaneously. Animals. Outbred C57BL/6 mice will be used. Mice will be aged 12 weeks. Parallel groups of male and female mice will be used for all experiments. When mice are re-used for additional experiments, this will be after administration of a single drug dose and with an interval of at least 7 days. Animals will be housed under standard conditions with a 12-hour light dark cycle. Additional strains of mice will be used if they are more applicable for specific experiments e.g. ICR mice for marble burying. Rats (Sprague Dawley strain) will be used for pharmacokinetics experiments and where otherwise indicated. Behavioral assays will be performed at the same time each day. Animals from different experimental groups will be tested in counterbalanced order.
1. Validation of Specific Assays and Protocols
1.1 Screening Assays for 5-HT2A Receptor Affinity. Receptor Binding and Functional Assays Classical psychedelic substances from diverse chemical groups (such as tryptamines, ergolines and phenylethylamines) bind with high (micromolar or lower) affinity to serotonergic receptors of the 5-HT2A subtype. These compounds also bind with moderate to high affinity to other serotonergic receptors such as 5-HT2C and 5-HT1 A and their activity at these receptors may influence psychedelic activity. In the current validation assays, affinity of psilocin, the active metabolite of psilocybin, for 5-HT2A, 5-HTC and 5-HT1A receptors will be examined in HEK-293 cells that over-express these receptors.
The following radioligands and competitors will be used: [3H]8-hydroxy-2-(di-n-propylamine) tetralin and indatraline (5-HT1A receptor) [3H]ketanserin and spiperone(5-HT2A receptor) [3H] mesulgerine and mianserin(5-HT2C receptor).
In addition to radioligand binding, the following functional assays will be conducted in order to determine and compare functional activation of these receptors by psilocin:
5-HT2A Human Serotonin GPCR Cell Based Agonist Calcium Flux Assay. 5-HT2C Human Serotonin GPCR Cell Based Agonist Calcium Flux Assay. 5-HT1A Human Serotonin GPCR Cell Based Agonist Calcium Flux Assay
1.2. Screening Assays for Psychedelic Activity. Implementing dose response studies, we will validate in our laboratory psilocybin effects to induce the well-established, 5-HT2A serotonergic receptor mediated behavioral effect, the head twitch response (HTR). This behavior has been shown to represent a reliable analogue of psychedelic effects in humans. Dose response studies will be designed to establish the psilocybin dose that induces maximal HTR as well as the maximal dose that does not induce HTR, for use in subsequent experiments. Psilocybin will be administered by i.p. injection 10 minutes before measurement of HTR. HTR will be measured by head-mounted magnets and magnetometer detection coils.
There is evidence that 5-HT1A, 5-HT2C and possibly TAAR1 receptors modulate psychedelic effects. It is important to elucidate the degree to which these receptors modulate psilocybin action in inducing HTR. Therefore, we will further characterize HTR as a marker of psilocybin action by determining modification of HTRs following administration to psilocybin treated animals of the specific 5-HT2A receptor (5-HT2AR) antagonist, M100907; the 5-HT2CR antagonist, SB242084, the 5-HT1 AR agonist, 8OHDPAT, and the trace amine associated receptor 1 (TAAR1) antagonist, EPPTB.
In addition, based on preliminary evidence we have obtained that the 5-HT1 A agonist, 8OH-DPAT inhibits HTR induced by psilocybin, we will check the effect of other 5-HT1A agonists and partial agonists including but not limited to flesinoxan, gepirone, ipsapirone and buspirone.
Psilocybin and most other naturally occurring psychedelic agents are tryptamine, phenethylamine or ergoline derivatives. It is important for future drug development to evaluate compounds that belong to a different chemical category but are nevertheless 5HT2AR agonists and inducers of HTR. In this context we will administer animals the antiviral agent, efavirenz, which has been reported to have psychedelic effects but is not a tryptamine, phenethylamine or ergoline. We will evaluate this drug's effects on HTR and the degree to which this effect is influenced by 5-HT2AR, 5-HT2CR, 5-HT1AR and TAAR1. 5-HTP (5-hydroxytryptophan) will be used as a control in these experiments since this agent stimulates 5-HT2AR but is not psychedelic at regular doses.
After the behavioral experiments have been completed, a further experiment will be performed to examine the effects of psilocybin, efavirenz and 5HTP on immediate early gene (TEG) expression. In this experiment mice will be administered the maximally effective doses of all agonists and antagonists as determined in the dose-response experiments. After 45 min. the animals will be sacrificed and tissue from frontal cortex will be taken for real time PCR evaluation of treatment effects on the immediate early genes (IEG), c-fos, egr-1 and egr-2. It has been shown that enhanced expression of all three genes is a characteristic feature of 5HT2A agonists that are psychedelic whereas 5HT2A agonists that lack psychedelic properties enhance expression of c-fos only and do not enhance egr-1 and egr-2. The information from this experiment will provide a molecular complement to the behavioral information already gathered and support our planned use of IEG expression in the screening of compounds with psychedelic potential.
Finally, we will conduct an experiment to determine the effect of prior treatment with the specific serotonin reuptake inhibitor (SSRI) antidepressant (escitalopram) on psilocybin, efavirenz and 5-HTP induced HTR. It has been reported that individuals under treatment with SSRIs manifest reduced responses to psychedelics. It is important to clarify this observation on the preclinical level. In this experiment, mice will be administered psilocybin, efavirenz or 5HTP at the dose shown to have maximum effect in the dose response studies, after having received sub-chronic escitalopram at a dose of lOmg/kg/day via osmotic minipumps for two weeks. We anticipate that HTR response to psilocybin, efavirenz and 5-HTP will be attenuated. Such a finding would have important implications for clinical studies with the BYAS entheogen compounds. In a parallel experiment we will determine whether observed effects on HTR are also reflected in effects on IEG expression.
1.3 Outcome Measure Assays: Depression-like, Anxiety-like, Obsessional-like, Cognitive, Social, Schizophrenia. As part of our screening protocol for novel entheogen molecules, we will examine their effect on a series of outcome measures that reflect phenotypic proxies for the clinical manifestations of target psychiatric disorders. Validation of these outcome measures is a key requirement, and this will be done by examining the effect on them of chemically synthesized psilocybin. Dosing for these validation studies will be based on the results of the prior HTR dose response studies. To examine the effects of a single dose of psilocybin, the i.p. injected dose that induced maximal HTR will be used (psychedelic dose). For sub-chronic administration (7-14 days via osmotic minipumps implanted subcutaneously), the maximal dose that did not induce HTR will be administered daily (sub-psychedelic dose). In addition we will examine the effects of sustained release psilocybin at full psychedelic dose over 24 hours, administered i.p. or orally. We will also examine the effect of psilocybin administered concurrently with a 5-HT1 A agonist such as buspirone in order to determine whether psychedelic effects (represented by HTR in the rodent) are required in order to achieve therapeutic effects.
The following phenotypic outcome measures will be examined after a single psychedelic dose of psilocybin and after 14 days of psilocybin administration at the sub-psychedelic dose: a. Motor: Open field test (OFT), Rotarod (RTR); b. Depression-like: Forced swim test (FST), Tail suspension test (TST), Sucrose preference test (SPT); c. Anxiety-like: Elevated plus maze (EPM), Fear extinction (FE), OFT, Light dark box (LDB); d. OCD-like: Marble Burying (MB) and Nestlet Shredding (NS); e. Cognitive: Novel object recognition (NOR), T-Maze (TM), Y-Maze (YM), Open field habituation (OFH); f. Social: Three chambers sociability (TCS); Social interaction in pairs (SIP), Tube test (TT)
1.4. Disease Model Assays. We will evaluate the therapeutic potential of entheogen compounds for the treatment of PTSD, OCD and depression using phenotypic mouse models of these disorders. In the current assay development phase, these disease models will be validated by the use of psilocybin - single administration at the dose that induced maximal HTR (psychedelic dose) or sub-chronic administration via osmotic minipumps or i.p injections at the maximal sub-HTR inducing dose (sub-psychedelic dose). In addition we will examine the effect of single i.p. injection of a sustained release psilocybin preparation at full psychedelic dose or oral administration of a sustained release preparation at full psychedelic dose and single administration of a psychedelic dose of psilocybin administered i.p. concomitantly with a dose of 5-HT1A agonist such as buspirone to block HTR.
The following mouse models will be used.
Schizophrenia. In the current project, we will seek preclinical proof of concept for the use of a mushroom-derived form of the serotonergic psychedelic, psilocybin, to treat schizophrenia. We will seek this evidence in two translational mouse models, testing the effect on rodent analogues of positive, negative, and cognitive symptoms. The therapeutic effect of mushroom-derived psilocybin will be tested in the neonatal MK801 model of schizophrenia which models negative and cognitive as well as positive symptoms and in the acute MK801 model of schizophrenia which primarily model positive symptoms and will indicate the extent to which the treatment is likely to exacerbate psychotic symptoms. The findings of this project will provide preclinical support for the use of mushroom cell derived psilocybin with or without spirulina extract, to treat schizophrenia and ameliorate negative symptoms or prevent their onset.
The following key outcome measures will be examined: a) Activity and motor function: Open field Test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84-99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3; b) Sensorimotor gating - Pre-pulse inhibition, a model for positive symptoms, will be used to test whether psilocybin has an exacerbating effect on psychosis. The test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84-99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3; c) Cognitive function - Tests in this category will be used to examine the effect of psilocybin on cognitive and negative features, especially cognitive flexibility. These tests will include:
- Intellicage sucrose preference and cognitive flexibility starting before psilocybin treatment and running throughout the entire protocol
- Y-maze: During behavioral and cognitive battery on PND92. d) Tests performed post-sacrifice:
- Immunohistochemistry staining ofNMDA NRl subunit, parvalbumin (PV), double staining of GAD67 and synaptophysin in the frontal cortex.
- Western blot of SK3, PV, synaptophysin, BDNF and BDNF receptor in the frontal cortex.
Methods. Neonatal injections of MK801 treatment will be used as a translational model of schizophrenia. In the neonatal MK801 model mice are injected with 0.5mg/kg MK80I s.c. daily, from PND6 to PND13. Treatment with sub-chronic, sub-psychedelic dose of psilocybin- rich mushroom extract (PRME), sustained release psilocybin, psilocybin + ketanserin, psilocybin + buspirone, psilocybin-free mushroom extract (PFME) or phycocyanin-rich spirulina extract (PRSE) will commence 14 days before the beginning of the behavioral battery and will continue through the battery. The sub-psychedelic dose of PRME and lack of psychedelic effects of PFME will have been established previously by head twitch response (HTR) experiments Psychedelic dose will be administered 24 hours before the commencement of the behavioral battery and will be repeated 24 hours before the second week of the battery
Acute injections of MK801 will be used as a model for schizophrenia to specifically determine the effect of treatments on positive symptoms. A single injection of MK801 or amphetamine will be used prior to measurement of the behavioral and electrophysiological endpoints. Mice will be pretreated with sub-chronic, sub-psychedelic doses of psilocybin-rich mushroom extract (PRME), sustained release psilocybin, psilocybin + ketanserin, psilocybin + buspirone, psilocybin-free mushroom extract (PFME) or phycocyanin-rich spirulina extract (PRSE) as in the neonatal MK801 model above.
Experimental Design
Expt 1: Neonatal MK801/adult sub-chronic psilocybin experiment.
Aims:
1) To test the hypothesis that sub-psychedelic doses of psilocybin (PRME) will have a beneficial effect on positive, negative and cognitive symptoms
2) To determine the effect of PFME which does not contain psilocybin but does contain entourage molecules.
3) To determine the effect of phycocyanin rich Spirulina extract (PRSE)
Method: MK801 or vehicle administration daily from PND6 to PND13; adult sub-chronic psilocybin treatment at sub-psychedelic dose starting 14 days before starting behavioral battery, on PND70. Behavioral battery on PND84-99. Sacrifice on PND100. Sub-psychedelic dose of PRME will be as defined in prior dose response experiments. 240 mice will be needed for this experiment.
Figure imgf000066_0001
Figure imgf000067_0001
Expt 2: Neonatal MK801/ acute psychedelic psilocybin + ketanserin/buspirone or sustained release administration of psychedelic psilocybin dose experiment.
Aim: To test the following hypotheses 1) That psychedelic doses of psilocybin given in conjunction with 5-HT2A antagonist ketan serin will have a robust beneficial effect on negative symptoms without exacerbating positive symptoms in the neonatal MK801 model of schizophrenia used in this study.
2) That psychedelic doses of psilocybin given in conjunction with 5-HT1A agonist, buspirone, will have a robust beneficial effect on negative symptoms without exacerbating positive symptoms in the neonatal MK801 model of schizophrenia used in this study.
3) That psychedelic doses of psilocybin given as a slow release preparation (over 24 hours) will have a robust beneficial effect on negative symptoms without exacerbating positive symptoms in the neonatal MK801 model of schizophrenia used in this study.
Method: MK801 or vehicle administration daily from PND6 to PND13; Acute psilocybin (PRME) treatment at psychedelic dose, alone or in conjunction with ketanserin (5-HT2A antagonist) or buspirone (5-HT1A agonist) or slow release form of PRME on PND84 and PND91 Behavioral battery on PND84-99. Sacrifice on day PND100. Psychedelic dose of PRME will be as defined in prior dose response experiments. 210 mice will be needed for this experiment
Figure imgf000068_0001
Expt 3: Acute MK801/ acute psychedelic psilocybin + ketanserin or buspirone or sustained release administration of psychedelic psilocybin.
Aim: To test the hypothesis that psychedelic doses of psilocybin (PRME) given in conjunction with the 5-HT2A antagonist, ketanserin or the 5-HT1 A agonist, buspirone, or as a sustained release preparation, will not exacerbate positive symptoms in the acute MK801 model of schizophrenia Method: MK801 or vehicle administration on PND 90 preceded 24 hours before by administration of a psychedelic dose of psilocybin (PRME) by in conjunction with the 5-HT2A antagonist, ketanserin or the 5-HT1A agonist, buspirone, or i.p. injection of a sustained release format of PRME at a psychedelic dose. Open Field activity measurement and PPT to follow immediately after MK8012 administration.
210 mice will be needed for this experiment.
Figure imgf000069_0001
Expt. 4: Combination Treatment
Aim: To test whether combined treatment of PRME/PFME with PRSE will have greater beneficial effect on negative symptoms compared with the individual treatments.
Method: MK801 or vehicle administration daily from PND6 to PND13. Combination experiment in which the mushroom extract that gave the best results in Expt. 1 will be tested in combination with PRSE. Psychedelic drug treatment will start on PND70, 14 days before beginning of behavioral battery. Behavioral battery on PND84-99. Sacrifices on PND100. Sub-psychedelic dose of PRME will be as defined in prior dose response experiments.
60 mice will be needed for this experiment.
Figure imgf000070_0001
The following key outcome measures will be examined: a) Activity and motor function'. Open field Test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84-99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3. b) Sensorimotor gating - Pre-pulse inhibition, a model for positive symptoms, will be used to test whether psilocybin has an exacerbating effect on psychosis. The test will be performed (i) at baseline before starting treatment; (ii) during the behavioral battery on PND 84- 99 in Experiments 1, 2 and 4; (iii) on PND 90 in Experiment 3. c) Cognitive function - Tests in this category will be used to examine the effect of psilocybin on cognitive and negative features, especially cognitive flexibility. These tests will include:
Intellicage sucrose preference and cognitive flexibility starting before psilocybin treatment and running throughout the entire protocol.
Y-maze: During behavioral and cognitive battery on PND92. d) Tests performed post-sacrifice:
Immunohistochemistry staining of NMD A NRI subunit, parvalbumin (PV), double staining of GAD67 and synaptophysin in the frontal cortex.
Western blot of SK3, PV, synaptophysin, BDNF and BDNF receptor in the frontal cortex. PTSD. Fear conditioning (FC) with pre-exposure: Mice are exposed to a single 2-hour restraint episode in clean, ventilated, 50ml conical vials (such as Falcon tubes) one week prior to conducting a fear conditioning protocol, in which each mouse is entered into the conditioning chamber for two minutes of uninterrupted exploration followed by two blocks of 20-second 80Db 2000Hz tone (conditioned stimulus) paired with 2-second 0.5mA footshock (unconditioned stimulus), and a further 60 seconds of uninterrupted exploration of the chamber. Plasma corticosterone level is measured 30 minutes after the end of FC protocol. Four days after conditioning mice undergo 2-day intensive extinction protocol and the time mice spend freezing in each extinction trial is measured. The combination of pre-exposure to stress with FC protocol induces higher plasma corticosterone level and resistance to fear extinction. 24 days later a recall test is performed.
Psilocybin will be administered according to the following regimens: i) Single injection i.p. of the maximal HTR-inducing dose 30 min. after the completion of the FC protocol and withdrawal of blood for plasma corticosterone level. ii) Sub-chronic treatment with psilocybin via osmotic minipumps or i.p. injection at a sub-psychedelic daily dose, commencing 30 min. after the completion of the FC protocol and withdrawal of blood for plasma corticosterone level, and continuing until the recall test on day 30. iii) Single i.p. injection of a sustained release psilocybin preparation at full psychedelic dose or oral administration of a sustained release preparation at full psychedelic dose, iv) Single psychedelic dose of psilocybin administered i.p. concomitantly with a dose of 5- HT1A agonist such as buspirone to block HTR. i.) In experiments comparing chemical psilocybin and PRME, the same treatment schedule will be used for both compounds. In all case PRME administered to the mice will contain exactly the same psilocybin dose in mg/kg as the chemical psilocybin.
OCD - Marble Burying and Nestlet Shredding. These two tests are for evaluating levels of repetitive compulsive behavior characteristic of OCD and autism and the effects of treatments to reduce these behaviors. In the marble burying, each mouse is introduced into a cage with 25 marbles that are presented on the top of the sawdust (unburied). The number of marbles buried by the animal after 30 minutes' exposure is recorded. In the Nestlet Shredding test, weighted cotton fibers are introduced into cage, and thereafter an animal is entered into that cage. After the animal spends 30 min in the cage, it is returned to its home cage and the unshredded cotton nestlets are weighed (the more compulsive the animal the less unshredded material will remain in the cage). These behaviors are reduced by SSRIs which alleviate OCD features.
These tests will be performed in the context of the outcome measure validation described above. To examine the effects of a single dose of psilocybin on these outcome measures, the i.p. injected dose that induced maximal HTR will be administered 30 minutes prior to testing for marble burying and nestlet shredding. For sub-chronic administration (14 days via osmotic minipumps implanted subcutaneously), the dose below that which induced HTR will be administered daily (sub-psychedelic dose).
Dosage of drugs and administration schedules will be as for the PTSD - Fear Conditioning experiment above.
The SSRI, escital opram, will be used as a positive control in these experiments.
Sapap3 KO mice. Mice with genetic deletion of the Sapap3 gene exhibit increased anxiety and compulsive grooming behavior leading to facial hair loss and skin lesions. Both behaviors are alleviated by SSRIs which are an effective treatment in some cases of OCD. We will examine whether treatment with psilocybin alleviates the increased anxiety and compulsive grooming behavior of Sapap3 KO mice.
Dosage of drugs and administration schedules will be as for the PTSD - Fear Conditioning experiment above.
Outcome measures to be tested: EPM, OFT, LDB for anxiety-like features; grooming behavior, MB, NS for OCD-like features.
The SSRI, escital opram, will be used as a positive control in these experiments.
Depression - Chronic Unpredictable Stress (CUS). A depressive like behavioral state is induced in rodents by exposure to chronic CUS. This model demonstrates face validity in that exposed animals display anhedonic features, construct validity in that biochemical and cellular changes following CUS resemble similar changes in depressed patients, and predictive validity because the neurobiological and behavioral post CMS changes are effectively reversed by antidepressant drugs. We will use the Unpredictable Mild Stress (UMS) version of the CUS protocol described by Dunlap et al (2020) in which stress exposure lasts for 7 days. At the end of the UMS period, psilocybin treatment will be administered, and outcome measures will be taken.
Dosage of drugs and administration schedules will be as for the PTSD - Fear Conditioning experiment above.
Outcome measures to be tested: OFT, FST, TST, SPT, EPM, LDB, NOR, TMAZE, TCS The SSRI, escitalopram, will be used as a positive control in these experiments.
Inflammation-induced depression models. On the background of the strong association between depression and inflammation, depression models in rodents can be implemented using inflammatory agents such as lipopolysaccharide (LP and the viral mimetic Poly EC. Treated animals lose weight, eat and drink less, and decrease their motor activity for several hours to days, depending on the nature of the inflammatory agent and dose. These sickness behaviors correspond with elevations in proinflammatory cytokines at the periphery and in the brain. In the LPS-induced model of depression, which we will implement, sickness behaviors will typically resolve within 24 hours. When sickness behaviors have resolved, the rodents display depressive- like behaviors. Thus, 24 hours after injection of LPS, when motor activity is back to normal and 24 hours after injection of LPS, when motor activity is back to normal and appetite present, treated animals show increased immobility in the forced swim test and tail suspension test as well as decreased sucrose preference. We will employ the LPS model and will determine whether the depression-like features induced by LPS in mice are reversed by pretreatment with psilocybin and psilocybin-rich mushroom extract.
Dosage of drugs and administration schedules will be as for the PTSD - Fear Conditioning experiment above
Outcome measures to be tested: OFT, FST, TST, SPT, EPM, LDB, NOR, TMAZE, TCS The SSRI, escitalopram, will be used as a positive control in these experiments.
Neuroplasticity, gene expression. There is increasing evidence that the therapeutic action of psychedelic compounds in treatment resistant psychiatric disorders is related to their effects on neuroplasticity i.e., the ability of neural networks in the brain to change through growth, reorganization, and new connections. Particular emphasis has been given to synaptic plasticity in which there is an increase in key synaptic elements such as dendrites and dendritic spines (termed structural plasticity) and also in the functionality of synapses (termed functional plasticity). Synaptic plasticity is particularly evident in the prefrontal cortex of mice treated with psychedelics which is a brain region rich in 5-HT2A receptors to which classical psychedelics preferentially bind. This is consistent with prior reports that neuroplastic mechanisms play a key role in the rapid antidepressant action of the hallucinatory anesthetic agent, ketamine and also in the slower in onset effects of specific serotonin reuptake inhibitors.
We will implement measures of synaptic plasticity as key endpoints for assessing the therapeutic potential of novel entheogen compounds in treatment refractory psychiatric disorders. Accordingly, in this preliminary phase of the program we will validate the effect of psilocybin on synaptic plasticity measures and examine the relationship of neuroplastic effects to the phenotypic outcome measures we will be examining.
In the experiments we will perform to validate phenotypic outcome measures and disease models, we will obtain brain tissue for studies of synaptic plasticity in the frontal cortex, amygdala and hippocampus. Mice receiving acute, psychedelic or sub-chronic, sub-psychedelic psilocybin treatment will be evaluated and also mice treated with psychedelic doses administered over 24 hours by sustained release. Using western blotting we will assay key molecular markers of synaptic plasticity such as GAP-43, PSD-95 and synaptophysin. We will also use immunohistochemical techniques to examine dendritogenesis, spinogenesis and synaptogenesis in the frontal cortex of mice from these experiments. Key issues to be evaluated are the relationship between neuroplastic effects and functional phenotypes and whether "psychedelic" (HTR inducing) doses of psilocybin are required to induce neuroplasticity or sub-chronic, subpsychedelic doses (non-HTR inducing) are sufficient.
Example 2. Pharmacological Characterization of the Head Twitch Response:
A Rodent Analogue of the Psychedelic Trip in Humans
In common with other serotonergic psychedelic agents, psilocybin is thought to act via the 5-HT2A receptor to which its active metabolite, psilocin, binds with high affinity. In rodents, psilocybin induces a characteristic head twitch response (HTR), which is highly correlated with the psychedelic trip in humans in terms of intensity. Using psilocybin, we sought to determine the role of other serotonergic receptors and the trace amine associate receptor 1 in mediating HTR induced by psilocybin. Methods. Male C57BL/6J mice (11 weeks old, ~30g) were group housed under a regular 12 hr. light dark cycle.
Drugs were administered by intraperitoneal (i.p.) injection immediately before the assessment of HTR commenced.
Psilocybin was administered at doses of 0.1 mg/kg to 51.2 mg/kg i.p. alone, or at a dose of 4.4 mg/kg i.p. preceded by:
5-HT2A receptor antagonist, M107900 (0.5, and 2 mg/kg i.p.);
5-HT2C receptor antagonist, RS-102221 (2, 4, and 8 mg/kg i.p.);
5-HT1A receptor agonist, 8OH-DPAT (1, and 2 mg/kg i.p );
TAAR 1 antagonist EPPTB (1, and 10 mg/kg i.p.).
HTR was measured for 20 minutes in a custom-built magnetometer using mini magnets tagged onto the ears of the mice. Recording and data display employed proprietary software
Results. Figures 1 A to ID provide data on psilocybin dose response. These graphs depict (FIG. 1A full range dose response of HTR through the course of 18 min, (FIG. IB) taken from (i) the distinct shape of faster and higher onset following a rapid decrease in response (doses high then 3mg/kg), (FIG. 1C) taken from A the distinct shape of flatter response that carries over time (doses lower then 1.6 mg/kg), (FIG. lD)dose response curve taken from the data shown in A.
Figures 2A to 2D provide data on the effects of serotonin receptor modulators on psilocybin-induced HTR (Head Twitch Response). The bar graphs show the cumulative HTR recorded by the mice during 20 min session immediately following injection of either psilocybin (vehicle) alone or combined with a receptor modulator. (Compared to vehicle *=p<0.05, **=p<0.005, ***=p<0.0005, ****=p<0.0001). FIG. 2A Shows the effect of a combined psilocybin + 5HT2A antagonist, which completely reduced HTR in both concentrations. FIG. 2B Shows the effect of a combined psilocybin + 5HT1 A agonist, which reduced HTR in dose depended manner. FIG. 2C Shows the effect of a combined psilocybin + TAAR1 antagonist, which did not alter HTR. FIG. 2D Shows the effect of a combined psilocybin + 5HT2C antagonist, which increased HTR with 4 mg/kg and reduced the HTR-increasing effect at 8mg/kg.
Psilocybin induced a dose dependent increase in the frequency of HTR over 20 minutes. With doses in excess of 25.6 mg/kg i.p. there was a reduction in the frequency of HTR. With higher doses of psilocybin, the increase in HTR frequency was more rapid as was the decline. The 5-HT2A receptor antagonist, Ml 07900, completely blocked HTR. The 5-HT2C receptor antagonist, RS-102221, enhanced HTR at lower doses but reduced it at higher doses.
Surprisingly, 5-HT1A receptor agonist, 8OH-DPAT, significantly attenuated HTR. There was no effect of the TAAR1 antagonist, EPPTB on HTR.
We further examined the effect of co-administration of buspirone 5 mg/kg with psilocybin 4.4 mg/kg to determine whether co-administration of buspirone would attenuate the HTR-enhancing effect of psilocybin. Fig 6a shows the time course of the effect of psilocybin and buspirone on HTR. Two way ANOVA showed a significant main effect of treatment (F 32, 2ss = 4.874, p <0.0001) and time (F 2.915, 93.28 = 5.001, p = 0.0032) and a significant treatment x time interaction treatment (F 27, 288 = 2.852, p<0.0001). When evaluating the total number of HTRs during the 20 minute measurement period (Fig 6b) there were significant effects of psilocybin (Fi, 32 = 19.22, p=0.0001) and buspirone (FI,32 = 7.483, p=0.0001) and a significant psilocybin x buspirone interaction (Fi, 32 = 5.237 ,p=0.0289).
Discussion. Using magnetometer-based automated evaluation of HTR in C57BL/6J mice, we have shown a clear dose response relationship for psilocybin. Our findings have important implications for the use of this technology to evaluate the psychedelic potency of other natural and synthetic compounds. We have confirmed the key role of 5-HT2A receptors in HTR and also the significant contribution of 5-HT2C receptors to the response. Our finding that 5-HT1A receptors play a key role in HTR is novel and has intriguing therapeutic implications. We did not find a modulatory role for TAAR1 receptors in psilocybin induced HTR although we have found such an effect for HTR induced by 5-hydroxytryptophan (5-HTP) in our lab.
Example 3. Pre-clinical studies on the anti-obsessional properties of psilocybin
Notwithstanding the relative efficacy of serotonin uptake blockers and cognitive- behavioral therapy (CBT), a third or more patients with OCD do not respond to standard treatments. Reports from one clinical trial and several preclinical studies suggest that 5-HT2A receptor agonists with psychedelic properties may have unique efficacy in the treatment of OCD. We are examining the effect the psychedelic agent, psilocybin, on marble burying (MB), a rodent proxy of obsessional behavior, using psychedelic and sub-psychedelic doses as determined by the head twitch response in mice. In addition, we are conducting a series of experiments to determine the role of 5-HT2A, 5-HT2C and 5-HT1 A receptors in the anti-obsessional effect of psilocybin.
Methods. Male ICR mice (30±2 gm) were group housed under a regular 12 hr. light dark cycle. Drugs were administered by intraperitoneal(i .p ) injection 30 minutes before assessment of MB. Psilocybin (PSIL) was administered at a dose of 4.4 mg/kg i.p. alone, or preceded by the 5- HT2A receptor antagonist, M107900 (2 mg/kg i.p.), the 5-HT2C receptor antagonist, RS-102221 (4 mg/kg i.p ), the 5-HT1 A receptor agonist, 8OH-DPAT (2 mg/kg i.p ), or buspirone (Busp).
Escitalopram(ESC, 5mg/kg i.p. was administered as a positive control. Marble burying was measured.
Marble-burying test (MBT) was performed in transparent cages containing ~4.5 cm sawdust. Twenty glass marbles were placed equidistant from each other in a 5 x 4 pattern. The experiment was done under dim light in a quiet room to reduce the influence of anxiety on behavior. The mice were left in the cage with the marbles for a 30-min period after which the test was terminated by removing the mice. Number of buried marbles was counted after 10, 20 and 30 minutes. All mice underwent a pretest without any injection and the number of marbles buried was counted. Only mice that buried at least 15 marbles were selected to perform the test after drug administration. 80 % of pretested mice fulfilled this criterion and were used in the definitive experiment which took place at least a week following the pretest.
Statistical analysis compared cumulative MB over a 30-minute period among the different treatment groups by ANOVA and post-hoc tests.
Results and Discussion. Mice administered psilocybin buried 32.84% fewer marbles over 30 minutes than vehicle treated mice (p=0.001, Fig 3a). The effect of psilocybin was not statistically different from the effect exerted by a positive control, the SSRI escitalopram (48.43% reduction in marble-burying relative to vehicle; p<0.0001, Fig 3a). Using a two-way- ANOVA design, we examined the effects of acute treatment with psilocybin and pretreatment with the 5-HT2A antagonist, Ml 00907 (volinanserin) (Fig 3b). A strong main effect of psilocybin was noted (Fi, 40=24.8, p<0.0001). We also observed a main effect of M100907 (Fi, 40=7.7, p=0.008, Fig. lb). However, there was no interaction between psilocybin and M100907 and the post-hoc comparison of M100907 and vehicle was not significant (p>0.10) while the post-hoc comparison of M100907+psilocybin and vehicle was (p<0.0001). These findings indicate that the mechanism whereby psilocybin reduces marble-burying behavior is likely independent of 5-HT2A signaling.
Could stimulation of 5-HT1A receptors underlie psilocybin’s effect on marble-burying? Psilocybin and the 5-HT1A agonist, 8-OH-DPAT, both exerted significant main effects to reduce marble-burying (Fi, 37=10.4, p=0.0026 and Fi, 37=74.3, p<0.0001, respectively, Fig. 4a). However, psilocybin did not interact with 8-OH-DPAT (FI,37=0.9, p=0.34 for psilocybin-DPAT interaction term) indicating that 5-HT1A stimulation was unlikely to account for PSI’s effects in the marbleburying paradigm. Moreover, the combined effect of psilocybin and 8-OH-DPAT was significantly greater than that of vehicle or psilocybin alone (p<0.0001 and p<0.0001 respectively). To consolidate this observation, we tested the effects of treatment with psilocybin preceded by the 5-HT1A receptor antagonist WAY100635. Using a two-way-ANOVA design, we noted a strong main effect of psilocybin (Fi, 61=42.47, p<0.0001, Fig. 4b), while the main effect of WAY100635 and the psilocybin-WAY100635 interaction were both not significant (Fi, 61=0.4, p=0.521 and Fi, 6i=0. 0003, p=0.985, respectively, Fig. 4b). Post-hoc testing confirmed that pretreatment with WAY100635 does not block the effect of psilocybin to reduce marble-burying (p>0.10, Fig 2b).
Buspirone is a 5-HT1A receptor partial agonist and a weak dopamine D2 receptor antagonist (Di Ciano et al., 2017; Loane and Politis, 2012). Unlike 8-OH-DPAT, we found an interaction ofbuspirone with psilocybin (Fi, 75=5.805, p=0.018, Fig. 5a). Thus, although psilocybin and buspirone alone both significantly reduced marble-burying compared to vehicle (Fi, 75=6.53, p=0.015; F 1,75=68.53, p=0.005; respectively, Fig.5a), their co-admini strati on did not yield a further reduction in marble-burying. We also tested the effect of pretreatment with WAY100635 on the reduction in marble-burying induced by buspirone (buspirone Fi, 59=19.45, p<0.0001, WAY100635 Fi, 59=3.07, p=0.08, WAY100635 X buspirone Fi, 59=1.2, p=0.274, Fig. 5b). Contrasting with the lack of effect of WAY100635 on the psilocybin-induced reduction in marbleburying (Fig 4b), we found a significant effect of WAY100635 to attenuate the effect ofbuspirone on marble-burying (buspirone vs. vehicle, pO.OOl; WAY100635 +buspirone vs. vehicle, p>0.10).
A key question not addressed in studies thus far is whether the effect of psilocybin and other psychedelic compounds on marble-burying is transient or persistent. We examined marbleburying in a subset of mice treated with vehicle or psilocybin 7 days following the initial MBT. No significant effect of psilocybin was observed (vehicle 18.6 ± 1.6, n=5; psilocybin 17.75 ± 2.21 n=4; p =0.53). We further examined whether the effect of psilocybin on marble-burying requires a bolus injection of the full dose of the drug (at 4.4 mg/kg) or whether the same effect can be achieved by administering the same quantity of drug in staggered fashion over a period of 3.5 hours i.e. i.p. injections of 1.1 mg. kg every 60 minutes with the MBT performed 30 minutes after the last injection. When administered in this fashion, no significant effect of psilocybin on marbleburying was observed (vehicle 19±0.89 n=6; psilocybin 19±1.32 n=9; p>0.10).
The results of our study support a significant effect of psilocybin to reduce the number of marbles buried in the MBT by male ICR mice when the drug is administered 30 minutes before the test, as previously reported by Matsushima et al (2009) and by Odland et al (2021a). Matsushima et al (2009) studied male ICR mice and observed a significant effect of psilocybin at a dose of 1.5 mg/kg. Odland et al (2021a) studied female NMRI mice and observed a significant effect of psilocybin at 1.0 mg/kg. In our study, 1.5 mg/kg was not sufficient to obtain a significant effect on male ICR mice and we used dose of 4.4 mg/kg psilocybin for our experiments. The reason for this difference between our study and that of Matsushima et al (2009) is not clear.
Our findings regarding the role of 5-HT1 A receptors in the effect of psilocybin on marble-burying are intriguing. As reported by others (Bruins et al., 2008; Egashira et al., 2008), the prototypical 5-HT1A agonist, 8-OH-DPAT, significantly reduced marble-burying in our study. Combined administration of 8-OH-DPAT and psilocybin exerted an additive anti-marble- burying effect. There was no interaction between 8-OH-DPAT and psilocybin, consistent with a differential mode of action exerted by these two compounds. Moreover, pretreatment with the 5- HT1A antagonist, WAY100635, which has been shown to block the effect of 8-OH-DPAT on marble-burying (Egashira et al., 2008) and was shown to have this effect for the 5-HT1 A partial agonist, buspirone, in our study, did not attenuate the effect of psilocybin. Taken together, these two findings suggest that the effect of psilocybin to reduce marble-burying is not mediated by the 5-HT1 A receptor but by a different, as yet unelucidated mechanism.
While the 5-HT1A partial agonist, buspirone, significantly reduced marble-burying in our study, its effect was not additive to the effect of psilocybin as was the case for 8-OH-DPAT and psilocybin. In this context it is noteworthy that the psychedelic effects induced by psilocybin are attenuated by buspirone as shown by Pokorny et al. (2016) in a study of healthy volunteers. Consistent with this observation we showed that co-administration of buspirone and psilocybin blocked the effect of psilocybin to induce HTR in mice while not affecting anti-marble-burying. The clinical implication of this finding is that co-treatment with psilocybin and buspirone could potentially permit the anti-obsessional effects of psilocybin while blocking its psychedelic effects. Further exploration of this interaction is an important topic for further study that has high relevance for the treatment of OCD.
As previously demonstrated by Odland et al (2021 ), the effect of psilocybin and buspirone to reduce marble-burying in our study was not a consequence of reduced motor activity. In our measurements of open field activity there was no difference in the effect of psilocybin and buspirone compared to vehicle. Our activity measurements were not performed during the MBT, 30-60 minutes after the psilocybin injection but after the MBT, 60-90 minutes after psilocybin administration. Nevertheless, it is noteworthy that the activity measurements performed by Odland et al (2021a) were during the MBT and no difference in the effect of psilocybin compared to vehicle was observed.
We did not find an effect of psilocybin on marble-burying that extended beyond that observed after 30 minutes. No effect was observed when a subset of mice were retested after 7 days. Also, we found that a bolus injection of psilocybin was needed to achieve an effect on marble-burying and there was no effect of psilocybin on the MBT when the same dose was spaced over 4 hours rather than being administered at once. Implications for the anti-obsessional effect of psilocybin in humans await studies in which the longer-term effects of psilocybin administration on OCD are examined and the dose required is clarified. In the study by Moreno et al (2006) clinical observations were not performed beyond 24 hours following oral administration of psilocybin. However, doses of psilocybin that did not induce prominent psychedelic effects were found to have anti-obsessional effects while in our study a bolus of drug sufficient to induce prominent HTR in mice was required, as demonstrated by our HTR data.
In conclusion, the results of our study confirm the previously reported effect of psilocybin to reduce marble-burying in mice, confirm that this effect is not blocked by a 5-HT2A receptor antagonist and suggest that the effect is not mediated by 5-HT1A receptors since the effects of psilocybin and 8-OH-DPAT are additive and the effect of psilocybin is not blocked by the 5-HT1 A antagonist, WAY 100635. The results further show that co-treatment with the 5- HT1A partial agonist, buspirone, blocks effects of psilocybin on HTR, a rodent correlate of psychedelic effects while not impeding its effect on marble-burying. Further studies are indicated to identify the receptor mechanisms of psilocybin in reducing marble-burying in mice with implications for the mechanism of the putative anti-obsessional effect of psilocybin in humans.
Example 4. Open Field Test
Mice were placed in an open field on completion of the MBT and were monitored for 30 minutes using the Ethovision Video Tracking System (Noldus Information Technology BV). As shown in Fig. 7a there was no significant difference in distance travelled between vehicle treated mice and those administered psilocybin or buspirone. Similarly, there was no difference in time spent by the mice in the center of the open field (center duration) (Fig 7b) or in the periphery of the open field (periphery duration) (Fig 7c) under treatment with psilocybin or buspirone compared to treatment with vehicle.
Example 5 (Prophetic) - ASD Therapy
After four decades in which legal barriers precluded extensive research with psychedelic drugs, there is currently growing interest in the potential therapeutic role of these agents in psychiatric disorders. Nutt, Psychedelic drugs — a new era in psychiatry? Dialogues in clinical neuroscience. 2019;21: 139. There were early uncontrolled reports of positive therapeutic effects of LSD in autistic patients but until very recently no controlled studies. A recent randomized controlled trial showed a significant reduction in social anxiety after MDMA-assisted psychotherapy in autistic adults. Mogar and Aldrich, The use of psychedelic agents with autistic schizophrenic children. Behav Neuropsychiatry. 1969; 1 :44-50; Danforth et al., Reduction in social anxiety after MDMA-assisted psychotherapy with autistic adults: a randomized, doubleblind, placebo-controlled pilot study. Psychopharmacology. 2018;235:3137-3148. There are no studies on the use psilocybin or other classical tryptaminergic psychedelics for the treatment of autism. In the present project we will employ psilocybin derived from mycelial culture in order to seek preclinical proof of concept for the use of this psychedelic agent to treat autism. Furthermore, we will explore possible entourage effects of additional components of the mycelial extract. We will also examine the possible utility of sub-psychedelic doses which would considerably simplify treatment administration.
Methods
This study will focus on naturally sourced psilocybin produced from mycelial culture by Back of the Yards Algae Sciences (BYAS). The test compound will be administered in the following formats: Psilocybin in Vehicle; Psilocybin in Mycelial Extract; Mycelial Extract alone; Vehicle without test compound. There will be two treatment regimens: Acute high dose (two consecutive daily i.p. injections); Subchronic low dose (10 consecutive i.p. injections).
For mouse models of ASD, we will use one commonly recognized models of ASD based on the synapse-related human mutation, Cntnap2. Penagarikano et al., Absence of CNTNAP2 Leads to Epilepsy, Neuronal Migration Abnormalities, and Core Autism-Related Deficits. Cell. 2011;147:235-246. It has been shown that the pathogenesis of ASD, at least in part, can be associated with synaptic dysfunction, which can lead to functional and cognitive impairments. Guang et al., Synaptopathology involved in autism spectrum disorder. Front Cell Neurosci. 2018;12:470. Previous studies have shown that mutations in genes, such as SHANK3, CNTNAP2, NLGN3, and others converge on common synapse-related cellular pathways, which are strongly linked to ASD. Stessman et al., Targeted sequencing identifies 91 neurodevelopmental-disorder risk genes with autism and developmental-disability biases. Nat Genet. 2017;49:515-526. The contactin-associated protein-like 2 (CNTNAP2)' gene encodes a neuronal transmembrane protein member of the neurexin superfamily involved in neuron-glia interactions and clustering of K+ channels in myelinated axons31. This gene also plays a role in synapse formation and stabilization. The dendritic spine dynamics are affected in the Cntnap2 knockout (KO) mice causing reduced stability in newly formed spines. Loss-of-function mutations in Cntnap2 have been implicated in ASD and cortical dysplasia-focal epilepsy syndrome. Feliciano, Cntnap2-/- autism model. Nat Genet. 2011 ;43 : 1053- 1053. Studies on Cntnap2-/- mice have shown spontaneous seizures, stereotypic motor movements, and behavioral inflexibility. Penagarikano et al., supra. Knockout pups emitted fewer isolation-induced ultrasonic vocalizations (distress calls) to their mothers and spent less time interacting with other mice in a juvenile play test. Id. These behavioral patterns are typical for ASD.
The following steps will be done:
1- Establishing two groups of mice: CASPR-KO and WT groups.
2- The groups will receive 4 types of treatments using two ways of drug delivery = total of 16 groups, n=12.
3- In total, -200 male mice will be included in this study.
4- Large scale of behavioral studies will be done before and after treatments (See details below). 5- Statistical analysis of the behavioral experiments
6- Sacrifice of animals and preparation and storage of brain regions for subsequent analysis.
Subsequent analyses
7- Biochemical validation: synaptogenesis, NO signaling, mTOR pathway, ROS production.
8- Global Proteomics followed by large scale of bioinfmatics.
9- Phospho-Proteomics followed by large scale of bioinformatics.
Detailed information on the behavioral tests:
Open Field Test The motor activity of the mice will tested in an open field consisting of a white plastic arena (60 cm x 60 cm) with a floor divided into 10 cm x 10 cm squares. In the first session (habituation) the mouse will be introduced to the field for 5 min. On the next day the mouse will introduced to the same field, and different parameters will be calculated using Ethovision system. Amal et al., Long-term consequences of a single treatment of mice with an ultra-low dose of Delta9-tetrahydrocannabinol (THC). Behav Brain Res. 2010;206:245-53. Object and Place Recognition Tests
These tests, as described in Amal et al., utilizes the tendency of mice to explore novel stimuli. The test consisted of two parts: a familiarization session and a test session. 24 h before these sessions the mice were allowed to explore the arena without objects for 5 min to habituate to their surroundings. During the first session for 5 min the mice were left to explore two identical objects that found at constant locations, 15 cm from the sidewalls, in the already familiar black plastic arena. 24 h later the mice were introduced to the arena for a test session in which one of the familiar objects was replaced with a novel object (Place recognition: same object is moved). The time spent by the mouse in exploring each object will recorded for 5 min. Elevated Plus Maze Test
The elevated plus-maze consisted of four arms (30*5 cm), two open and the other two closed. The platform was made of white plexiglass. The apparatus was elevated 45 cm above the floor. The test, (described in Waif and Frye, The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc. 2007;2:322-8), will be initiated by placing the mouse on the central platform of the maze, facing one of the open arms, and letting it move freely. Each session will be lasted for 10 min. The time spent in the close and open arms will be recorded. First day is habituation and second day is the test itself.
Three-Chambered Social Test
A three-chamber social test will be performed (as described in Silverman et al., Behavioural phenotyping assays for mouse models of autism. Nat Rev Neurosci. 2010; 11 :490- 502, with some modifications). The social test apparatus consisted of a transparent acrylic box divided into three chambers. Two cylindrical wire cages were placed, one in chamber 1 and the other in chamber 2. For the sociability test the test animal will be introduced to the middle chamber and allowed to adjust for 5 minutes, then in the next day an unfamiliar mouse will be introduced into a wire cage in one of the side-chambers and an empty wire cage on the other side-chamber. The time spent by the test mouse in exploring the wire cage with an unfamiliar mouse inside it will be recorded for 5 minutes.
Example 6 (prophetic) Therapeutic potential psychedelics in TBI
1.1.1. Synaptogenic effects: There is growing evidence that psychedelics such as psilocybin, DMT, and LSD have significant therapeutic potential in the treatment of various conditions including depression, addiction, and PTSD. Indeed, pioneering research on psychedelics has moved them from 'agents of rebellion towards breakthrough therapeutics' (McCall, (2020) Psychedelics move from agents of rebellion towards therapeutics. Nature Medicine). There is some early evidence of the potential utility of psychedelics in the treatment of brain injury. For example, DMT reduces infarct size and improves functional recovery following transient focal brain ischemia in rats (Nardai et al., (2020) N, N-dimethyltryptamine reduces infarct size and improves functional recovery following transient focal brain ischemia in rats. Experimental neurology, 327, 113245). Preclinical research in rodents has shown that psychedelics enhance synaptic plasticity in key brain regions linked to psychological functioning (Ly et al., (2018) Psychedelics promote structural and functional neural plasticity. Cell reports, 23, 3170-3182). Enhancement of synaptic density by psychedelics has recently been demonstrated by PET studies in pigs (Raval et al., (2021) A Single Dose of Psilocybin Increases Synaptic Density and Decreases 5-HT2A Receptor Density in the Pig Brain. International Journal of Molecular Sciences, 22, 835) and is supported by the measurement of plasma BDNF in humans (Hutten et al., (2020) Low doses of LSD acutely increase BDNF blood plasma levels in healthy volunteers. ACS Pharmacology & Translational Science, 4, 461 -466). These effects are thought to underlie the clinical action of psychedelics in neuropsychiatric disorders and are currently the focus of intense research.
1.1.2. Sub-psychedelic effects: Observations in rodents (Higgins et al., (2021) Low doses of psilocybin and ketamine enhance motivation and attention in poor performing rats: Evidence for an antidepressant property. Frontiers in Pharmacology, 12, 299) and humans (Hutten et al., (2020) Low doses of LSD acutely increase BDNF blood plasma levels in healthy volunteers. ACS Pharmacology & Translational Science, 4, 461-466) suggest that the therapeutic effects of psychedelics may be dissociated from their psychological effects (i.e., the 'psychedelic trip') and may be achieved at sub-psychedelic doses (i.e., lower doses than required to induce discernible perceptual effects in humans) and without 5-HT2A receptor-mediated behavioral stereotypies in rodents. In line with this observation, a psychedelic analog devoid of effects on the head twitch response, a rodent behavior analogous to the psychedelic trip in humans, was found to induce antidepressant and anti-addiction like effects as well enhancement of synaptic plasticity in mice (Cameron et al., (2021) A non-hallucinogenic psychedelic analogue with therapeutic potential. Nature, 589, 474-479). Clinical evidence for a therapeutic effect of subpsychedelic doses was shown in a clinical trial of OCD treatment (Moreno et al., (2006) Safety, tolerability, and efficacy of psilocybin in 9 patients with obsessive-compulsive disorder. The Journal of clinical psychiatry, 67, 0-10). These findings support the potential efficacy of subpsychedelic doses of psychedelic compounds in the prevention and treatment of TBI.
1.1.3. Potential therapeutic effects of psilocybin- free, "full spectrum" mycelial extract: It is well established that mushroom fruiting bodies, mycelia, and spores accumulate a variety of bioactive metabolites with immunomodulatory, cardiovascular, hepatoprotective, antifibrotic, anti-inflammatory, antidiabetic, antiviral, antioxidant, antitumor, and antimicrobial properties (Al-Obaidi et al., (2021) Mycopharmaceuticals and Nutraceuticals: Promising Agents to Improve Human Well-Being and Life Quality. Journal of Fungi, 7, 503), in addition to a variety of tryptamines and beta-carbolines with monoamine oxidase inhibiting properties (Blei et al., (2020) Simultaneous production of psilocybin and a cocktail of P-carboline monoamine oxidase inhibitors in “magic” mushrooms. Chemistry (Weinheim an der Bergstrasse, Germany), 26, 729). Moreover, mushrooms have prebiotic functions based on their abundances in carbohydrates, like chitin, hemicellulose, y and P-glucans, mannans, xylans, and galactans (Cerletti et al., (2021) Edible Mushrooms and Beta-Glucans: Impact on Human Health. Nutrients, 13, 2195). Thus, exploring the effect of mycelial extract, devoid of the main psychoactive tryptamines (psilocin and psilocybin), on affective, metabolic and cognitive functions is feasible and necessary.
1.1.4. Entourage effects: As noted, psychedelic mushrooms contain a variety of psychoactive compounds besides psilocybin. Thus, it is not surprising that anecdotal reports suggest a discernible difference between the effects of chemically synthesized psilocybin and those of psychedelic mushrooms and differences among the effects of different mushroom strains. These reports are consistent with the observations of Gartz regarding the effects of the mushroom, Inocybe aeruginascens (high in aeruginascin content) as compared to the effects of mushrooms with a high psilocybin and psilocin content. Gartz (1989) Analysis of aeruginascin in fruit bodies of the mushroom Inocybe aeruginascens. International Journal of Crude Drug Research, 27, 141-144. Gartz observed an increased mood-enhancing effect of the mushrooms high in aeruginascin content as compared to those high in psilocybin. These observations led Gartz to propose an "entourage effect" of psychedelic mushrooms whereby additional components of the mushroom extract enhance the effect of psilocybin. A preclinical study that is relevant in this context compared the effects of an extract from Psilocybe argentipes to pure psilocybin on marble-burying behavior in mice (an animal model used to study OCD) (Matsushima et al., (2009) Effects of Psilocybe argentipes on marble-burying behavior in mice. Bioscience, biotechnology, and biochemistry, 73, 1866-1868). The results of the study showed that the psilocybin-containing mushroom extract was more effective (at the same psilocybin dose) in reducing marble-burying than pure psilocybin, supporting the hypothesis of an entourage effect from the mushroom derived preparation. Further evidence for a psychedelic mushroom entourage effect is a study showing that psychedelic mushroom extracts were significantly more potent on in-vivo serotonin 5-HT2A receptor behavioral assays (as demonstrated by the head-twitch response in mice) than the same dose of pure psilocin, the active metabolite of psilocybin (Zhuk et al., (2015) Research on acute toxicity and the behavioral effects of methanolic extract from psilocybin mushrooms and psilocin in mice. Toxins, 7, 1018- 1029). These observations provide support for a "full-spectrum" therapeutic approach that utilizes all the components of the mushroom extract and not only psilocybin. 1.1.5. Immunological and microbiome effects: Along with the reported effects of psychedelics on synaptic plasticity, there are also several studies demonstrating the potential of low doses of classical psychedelics as modulators of immune response and in reducing microbiome dysbiosis. Psychedelic mushrooms such as Psilocybe natalensis have been shown to have antioxidant and anti-inflammatory activity (Nkadimeng et al., (2020) Phytochemical, Cytotoxicity, Antioxidant and Anti-Inflammatory Effects of Psilocybe Natalensis Magic Mushroom. Plants, 9, 1127). Psychedelics exert significant modulatory effects on immune responses by altering signaling pathways involved in inflammation, cellular proliferation, and cell survival (Szabo, Psychedelics and immunomodulation: novel approaches and therapeutic opportunities. Frontiers in immunology, 6, 358). Kuypers, ((2019) Psychedelic medicine: The biology underlying the persisting psychedelic effects. Medical hypotheses, 125, 21-24) suggested that that low doses of psychedelics exert their effects on mental well-being through indirect modulation of the gut-brain axis.
2.2. Therapeutic potential of algae in TBI. Algae have a long history as food and medicine. The Aztecs reserved Spirulina, a blue-green algae, for nobles and warriors, and considered it a divine food. In modern times, algae are regarded as a 'superfood' for athletes and the compounds derived from algae provide a panoply of potent, natural, therapeutic, and wellness-enhancing biomolecules. Two archetypal algae extracts, astaxanthin and phycocyanin, are ideal candidates as neuroprotective and neuro-regenerative agents. Astaxanthin, a vivid red pigment 'carotenoid' is a well-known nutraceutical and is considered to be one of the strongest antioxidants in nature, and of possible high potential as an anti-neuroinflammatory agent (Galasso et al., (2018) On the neuroprotective role of astaxanthin: new perspectives. Marine drugs, 16, 247). Due to its high free-radical scavenging activity and ability to easily pass through the blood-brain barrier, astaxanthin is a potential natural biomolecule for application in the protection against traumatic insults to the brain. Published animal model research provides further evidence of a potential role for astaxanthin in neuroprotection (Wu et al., (2020) Astaxanthin-shifted gut microbiota is associated with inflammation and metabolic homeostasis in mice. The Journal of Nutrition, 150, 2687-2698). Phycocyanin is a fluorescent, water-soluble protein that is part of the photosynthetic system of blue-green algae such as Spirulina. Like astaxanthin, phycocyanin is a potent antioxidant with strong anti-microbial, neurotrophic, and anti-inflammatory properties (Min et al., (2015) Assessment of C-phy cocyanin effect on astrocyte s-mediated neuroprotection against oxidative brain injury using 2D and 3D astrocyte tissue model. Scientific reports, 5, 1-11). Both these biomolecules can modulate gut microbiota in mouse models acting as prebiotics, promoting the growth and diversity of gut-friendly bacteria and reducing gut-microbiome dysbiosis (Li et al., (2020) Effects of phycocyanin on pulmonary and gut microbiota in a radiation-induced pulmonary fibrosis model. Biomedicine & Pharmacotherapy, 132, 110826).
Based on these promising data, we propose a synergistic approach, leveraging the potential of "full spectrum" mushroom and algal extracts, for the prevention and treatment of TBI and CTE. Our core hypothesis is that the combination of Psilocybe mushroom (in subpsychedelic formulations) and nutraceutical algae extracts will have neuroprotective, antiinflammatory, immune modulation, neurogenesis, neuroplasticity, neurobiome, cognitive, behavioral, and other effects relevant to the prevention and treatment of TBI and CTE.
We aim to test whether:
• The combination of sub-psychedelic mushroom and nutraceutical algal extracts (phycocyanin and astaxanthin) delivered in a daily dosing schedule has neuroprotective functionality;
• The combination of sub-psychedelic mushroom and nutraceutical algal extracts (phycocyanin and astaxanthin) delivered immediately post-injury can reduce the severity of TBI and demonstrate potential in ameliorating CTE symptoms and,
• The combination treatment delivered pre- and post-injury will have maximal efficacy.
Outcome measures will include standard measures of cognition and behavior, brain histology, synaptic plasticity, biomarker, gene expression and -omics, inflammatory response, and microbiome.
3. METHODS
3.1 Animals. Outbred C57BL/6 mice will be used. Mice will be aged 12 weeks. Parallel groups of male and female mice will be used for all experiments. Animals will be housed under standard conditions with a 12-hour light-dark cycle. Behavioral assays will be performed at the same time each day. Animals from different experimental groups will be tested in counterbalanced order.
3.2 Therapeutic Agents. Natural psilocybin (PSI), psilocybin rich full spectrum mushroom extract (PRME), psilocybin free mycelial extract (PFME), phycocyanin rich spirulina extract (PRSE), and astaxanthin (AST) will be provided by Back of the Yards Algae Sciences (BYAS) (IL, USA). Other chemicals will be purchased from Sigma-Aldrich (IL, USA).
3.3 Treatments The following treatments will be administered to groups of mice that undergo head trauma. Treatments will be administered in the context of a series of experiments in which the treatments will first be examined alone and then in systematically designed combinations: a) Natural, mycelial derived psilocybin (PSI) b) Full spectrum mushroom extract containing psilocybin (FSME) c) Mycelial extract free of psilocybin (PFME) d) Phycocyanin rich spirulina extract (PRSE) e) Naturally derived astaxanthin (AST) f) Vehicle (VEH)
- PSI will be administered at a sub-psychedelic dose established by dose-response studies using the head twitch response (HTR).
- FSME will contain identical PSI concentration to that of PSI administered alone.
- Absence of HTR induced by PFME dose will be established by dose-response studies.
These treatments will be administered in systematically studied combinations to identify possible summation or potentiation of therapeutic effects. Choice of components of the combination therapies will be based on the outcome of studies with the individual therapies. All treatment groups will receive the same number of injections. Examples of possible combination therapies are: a) FSME + PRSE + VEH b) FSME +AST + VEH c) PRSE + AST + VEH d) FSME + PRSE + AST e) PFME + PRSE + VEH f) PFME +AST + VEH g) PFME + PRSE + AST h) VEH only (triple inj ection)
Possible combination treatment groups are summarized in the following Table. Combination treatments to be administered to mice that undergo head trauma model.
Figure imgf000090_0001
Treatment Schedules
Following the aims of the project, treatments will be administered according to the following schedules:
1. Pre-trauma (PrT) - to determine prevention of the effects of the trauma.
2. Post-trauma (PoT) - to determine amelioration of the effects of the trauma.
3. PrT + PoT - to determine prevention and amelioration of the effects of the trauma.
3.4 Experimental Models
3.4.1. Mouse model to establish psychedelic potency of therapeutic agents. It is well established that psychedelic drugs induce a characteristic head twitch response (HTR) in mice which reflects the psychedelic potency of the drug in humans. Furthermore, psychedelic drugs which are 5-HT2A receptor agonists characteristically enhance the levels of the immediate-early response genes (TEG) egrl and egr2. To determine sub-psychedelic dose, calibration experiments will be conducted. Mice will be administered increasing doses (0 - 30 mg/kg) of PSI and FSME and psychedelic dosage will be established. Similarly, increasing doses of PFME will be administered to establish HTR is not induced. HTR will be evaluated by the use of a magnetometer as previously described. Following on, mice will be administered a single administration of the highest doses of PSI, FSME, and PFME that do not induce HTR. Fifteen minutes after administration mice will be sacrificed and frontal cortex specimens will be obtained and frozen for analysis of IEG.
3.4.2. Mouse model of mild chronic traumatic brain injury. Because the focus of this project is on repeated, mild TBI exposure, a modification of Marmarou's weight-drop method will be used, as described by (Kane et al., (2012) A mouse model of human repetitive mild traumatic brain injury. Journal of neuroscience methods, 203, 41-49). Briefly, mice are lightly anesthetized with isoflurane (i.e., until unresponsive to paw or tail pinch) and placed immediately under a vertical PVC tube. Mice are suspended chest-down on a slit piece of aluminum foil 10 cm above a foam cushion. The mouse is quickly positioned so that its head is directly in the path of the falling weight by first resting the weight on the scalp midline between bregma and lambda. Incisions in the scalp or emplacement of a protective skull helmet are not necessary. The weight is then pulled rapidly upward by an attached string to the desired drop distance and released. The downward traverse of the falling weight is restricted by the string such that upon contact, the weight travels no more than 1 cm beyond the original position of the dorsal surface of the head. Immediately upon impact, the mouse falls freely onto the foam cushion. In this arrangement, the impact-induced acceleration and fall always involve a 180° horizontal rotation of the mouse body. The mouse is moved immediately to a holding cage to recover. Mice will undergo five consecutive daily applications of the trauma which is analogous to repeated mild traumatic brain injury. Mice will be subjected to 5 consecutive, daily exposures to the head trauma model.
3.5 Outcome Measures. Because of the large number of cognitive and behavioral outcome measures, mice will be treated in two cohorts (A and B) and the tests will be performed on the two cohorts separately as indicated:
3.5.1. Cognitive and behavioral tests:
(i) Cognitive function tests: Novel object recognition (A); Radial arm water maze (B).
(ii) Activity and motor function tests: Rotarod (A); Open field (B).
(iii) Affective/ Anxiety - like outcome measures: Forced swim test (depression-like) (A); Elevated plus maze (anxiety-like) (B); Marble burying test (obsessional like) (A).
(iv) Sociability and Aggression Measures: Social interaction in pairs (A); Tube dominance test (B).
3.5.2. Microbiome and Biomarkers: Fecal pellets will be collected for microbiome analysis on days 0, +10 and +19 and blood will be collected for biomarker analysis.
3.6. Follow-Up Measures: For treatment interventions showing the most striking effect immediately following the trauma, additional experiments will be implemented to allow for longer-term follow-up extending to 3 months following the exposure to head trauma and treatment. Cognitive and behavioral outcome measures will be obtained as well as microbiome and post-sacrifice outcome measures including histology, inflammatory markers, and synaptic plasticity measures.
Example 7
Anecdotal reports suggest that the behavioral and pharmacological effects of psilocybin- containing, “full spectrum” psychedelic mushroom extract (FSME) differ from those of chemical psilocybin (PSIL) in their nature and intensity. A limited number of rodent studies have compared synthetic psilocybin (or psilocin) with crude psychedelic mushroom extract. Furthermore, psychedelic mushrooms contain intermediate products of the psilocybin biosynthetic pathway such as baeocystin, norbaeocystin and aeruginascin that may influence the nature of the effect of psilocybin (“entourage effect”) along with other components such as harmines with monoamine oxidase inhibiting properties. In the current study, we compared the effect of PSIL to that of FSME on the mouse head twitch response (HTR), which is correlated with psychedelic effects in humans, the mouse tail suspension test (TST) and in a behavioral phenotypic zebrafish model.
Methods: Male C57Bl/6j mice were used in all head twitch experiments. PSIL (98.75% purity) was provided by Usona Institute. FSME, a methanol extract of Psilocybe cubensis with a psilocybin content of 1.5%, was provided by BYAS-PEB. Drug doses were calculated so that equal injection volumes of PSIL and FSME contained equal concentrations of psilocybin on a mg per kg basis. Control mice received vehicle (VEH) injections (0.9% NaCl solution). HTR was measured over 20 minutes in a magnetometer-based system using ear clip magnets. The TST was conducted 48 hours following administration of PSIL, FSME and vehicle. Behavioral tests were conducted using a Noldus Ethovision system by observers blind to treatment status. Individual male zebrafish (Danio rerio) were used in an open arena, behavioral phenotyping experiment. The drug dose, 3mg/L of PSIL and FSME was administered in a beaker containing 200ml of water over 10 minutes. Control fish were placed in a beaker containing 200ml of water for 10 minutes. The fish were then placed in an 50x50x4cm arena and video-tracked using idTracker software, with trajectories recorded for 20 minutes immediately after treatment and for 20 minutes at 80 minutes after treatment.
Results: FSME induced a significantly greater number of head twitches over 20 minutes at a psilocybin dose of 4.4 mg/kg than PSIL at the same dose (F=4.41, df 1,21, p=0.04; FSME n=l 1 , PSTL n=l 2). The difference was evident over the entire time course. On the tail suspension test (TST) conducted 48 hours after i.p. administration of PSIL (4.4 mg/kg), FSME (psilocybin content 4.4. mg/kg) or VEH, both PSIL (209.3±93.6 sec, n=l l, p=0.01) and FSME (198.14± 61.3 sec, n=10, p=0.0006) showed significantly less inactivity than VEH (296.0±33.8 sec, n=l 1). On the highly active measure of the TST, FSME (11.38± 10.13 sec, n=10) induced significantly more activity than both VEH (0.04 ± 0.12, n=l l, p=0.0007) and PSIL (0.53±1.04 sec, n=l 1, p=0.001) In the zebrafish experiment, during the first 20 minute recording period, 2D spatiotemporal reconstructions of the zebrafish swim paths demonstrated clearly visible differences in swimming patterns including velocity, distance swum, average distance to perimeter and middle and changes in direction, between the control and the FSME/PSIL groups. There were also a number of clear differences in swimming patterns between the FSME and PSIL groups, especially related to the mean time spent in the comers of the arena (P<0.05; FSME/PSIL n=9). At 80 minutes, the swimming pattern of the PSIL group closely resembled that of the control group, whereas the FSME group continued to show a similar, slightly attenuated swimming pattern to that observed in the initial 20-minute recording period.
Conclusions: A prior study by Zhuk et al. ((2015) Research on acute toxicity and the behavioral effects of methanolic extract from psilocybin mushrooms and psilocin in mice. Toxins, 7, 1018-1029) suggested that mushroom extract has greater potency in inducing HTR than psilocin (the active metabolite of psilocybin). Our findings in mice are in accordance with this observation. We have further shown that on the TST, a screening test for antidepressant potential, FSME induces a stronger effect than PSIL when the same dose of psilocybin is administered with both preparations. Furthermore, this work provides evidence of a robust and measurable zebrafish response to PSIL and FSME. The more sustained effect of FSME may be indicative of an “entourage effect”.
Example 8 (Prophetic)
1. Animals. Outbred C57BL/6 mice will be used. Mice will be aged 12 weeks. Parallel groups of male and female mice will be used for all experiments. Altogether, 344 mice will be used in this project (172 of each gender). Mice will be group-housed for the duration of the experiment, and maintained under standard conditions with a 12-hour light dark cycle. Behavioral assays will be performed at the same time each day. Mice from different experimental groups will be tested in counterbalanced order.
2. Drugs. PRSE (Phycocyanin-rich Spirulina Extract) and PFME (Psilocybin-free mushroom extract) will be provided by Back of the Yards Algae Sciences (BYAS), Chicago, U.S.A. Other drugs and chemicals will be purchased from Sigma- Aldrich Israel Ltd. Drugs will be administered by intraperitoneal (i.p.) injection.
3. Experimental Design
3.1. Effect of PFME on the head twitch response (HER) and immediate early response genes (IEG) in mice. It is well established that psychedelic drugs induce a characteristic head twitch response (HTR) in mice which reflects the psychedelic potency of the drug in humans. Furthermore, psychedelic drugs which are 5-HT2A receptor agonists characteristically enhance the levels of the immediate early response genes (IEG) egrl and egr2 whereas 5-HT2A agonists that are not psychedelic do not have this effect. To determine whether PFME has any psychedelic effects we will therefore establish its effect on these two variables. Expt 1. Effect of PFME on HTR in mice. Mice will be administered increasing doses of
PFME, based on the doses previously administered to examine HTR induced by psilocybin, to determine whether the psilocybin free extract induces HTR. In the event that HTR is observed at a particular dose, subsequent experiments will be performed at a dose lower than the observed HTR-inducing dose.
Figure imgf000094_0001
Figure imgf000095_0002
Expt 2. Effect of PFME on IEG in mice. Mice will be administered a single injection of the highest dose of PFME that does not induce HTR. 15 minutes after injection mice will be sacrificed and frontal cortex specimens will be obtained and frozen for analysis of IEG.
Figure imgf000095_0003
3.2. Separate and combined effects of PRSE and PFME on anxiety and depressionlike measures cognitive function, sociability and aggression in mice.
Expt 3. Mice will be administered PRSE or PFME or both by IP injection for 18 days. Behavioral and cognitive testing will commence on day 11 for 7 days. Mice will be treated in two cohorts of 12 for each treatment category and each cohort will undergo a different module of behavioral and cognitive testing. Mice will be sacrificed on day 19, a cardiac blood sample will be taken, and brain areas will be dissected. female (N)
Figure imgf000095_0001
Figure imgf000096_0001
*Dose to be determined
4. Outcome Measures a. Behavioral , cognitive and social outcome measures: Mice will be tested in two separate cohorts, A and B. which will undergo the following tests: • Activity and motor function tests: Rotarod (A); Open field (B)
• Affective/ Anxiety-like outcome measures: Forced swim test: Depression like (A); Elevated plus maze: Anxiety like (B); Marble burying test (A)
• Cognitive function tests: Novel object recognition (A); Radial arm water maze (B)
• Sociability and Aggression Measures: Social interaction in pairs (A); Three chambers sociability and social novelty test (B); Tube dominance test (B) b. Microbiome: Fecal pellets will be collected for microbiome analysis on days 0, +10 and +19
The following table summarizes the experimental protocol:
Figure imgf000096_0002
Figure imgf000097_0001
Post-Sacrifice Outcome Measures
To be measured on samples collected at sacrifice:
• Cardiac blood collected for inflammation related assays;
• Brain areas dissected for synaptic plasticity markers.
Example 9
In organisms with complex nervous systems, memory storage is believed to be heavily based on changes in synapses, specialized sites of cell-cell contact that connect the nerve cells within the nervous system. Changes in synaptic connections, broadly referred to as synaptic plasticity, represent an important neuronal plasticity process, only one of multiple neuronal plasticity processes. Rosenberg et al. (2014) The roles of protein expression in synaptic plasticity and memory consolidation. Fron. Molecular Neurosci. 7(86): 1-14.
This example provides data on levels of the following synaptic plasticity markers in a mouse model after administration of purified psilocybin (PSIL) or a full spectrum mushroom extract (FSME).
• PSD-95, which regulates the trafficking and localization of glutamate receptors such as a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type or N-methyl-D-aspartate (NMD A) type-receptors. Overexpression of PSD-95 enhances the amplitude of the AMPA receptor-mediated synaptic current.
• GAP43, which is expressed at high levels in neuronal growth cones during development and axonal regeneration, and it is phosphorylated after long-term potentiation and after learning. GAP43 is a proven marker for developing and regenerating neurons, and to a lesser extent, reactive glial cells. • Synaptophysin, which is a presynaptic vesicular protein that is often used as a marker for synaptic plasticity and integrity, decreases with increasing age in hippocampal and cortical regions.
C57B1/6J mice (~30g) were administered chemically produced psilocybin provided by USONA or full spectrum, psilocybin-containing mushroom extract (FSME) provided by PEB, by intraperitoneal injection, both at a psilocybin dose of 4.4 mg/kg. 10-12 days later the mice were sacrificed, and brain areas were dissected and frozen immediately at -180C until assay. The synaptic proteins, GAP43, PSD95 and synaptophysin, were assayed by Western Blot analysis as ratios to actin housekeeping gene. Effect of psilocybin and FSME on levels of synaptic proteins across 4 brain regions - prefrontal cortex, amygdala, hippocampus and striatum, was tested by nested one-way analysis of variance (ANOVA) using Graphpad-Prism. The figure shows the effect of psilocybin and FSME on GAP43, PSD95 and synaptophysin compared to saline vehicle across the 4 brain regions as means and 95% confidence intervals.
Over both treatments a significant enhancement of the levels of all three synaptic proteins is seen in FIG. 8 - GAP43, nested one-way ANOVA, p=0.017; PSD95, nested one-way ANOVA, p=0.037; synaptophysin, nested one-way ANOVA, p=0.0054. Dunnetf s post hoc test is significant for the effect of FSME compared to vehicle for GAP43 (p=0.0053), PSD95 (p=0.023) and synaptophysin (p-0.0026) but not for psilocybin.

Claims

What is claimed is:
1. A method of treating a psychiatric disorder in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin or a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
2. The method of claim 1, wherein the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
3. The method of claim 1, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
4. The method of claim 1, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
5. The method of claim 1, wherein the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
6. The method of claim 5, wherein the mushroom cell culture composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
7. The method of claim 5, wherein the mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
8. The method of claim 5, wherein the mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
9. The method of any one of claims 1 to 8, wherein the psychiatric disorder is selected from the group consisting of obsessive compulsive disorder (OCD), schizophrenia, post-traumatic stress disorder (PTSD), depressive and anxiety disorders, alcohol and substance use disorders and nicotine addiction.
10. The method of any one of claims 1 to 9, wherein the effective amount of a mushroom cell culture extract or entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder.
11. The method of any one of claims 1 to 10, wherein the psychiatric disorders are OCD and/or schizophrenia.
12. The method of any one of claims 1 to 10, wherein the psychiatric disorder is PTSD.
13. The method of any one of claims 1 to 12, wherein the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
14. The method of claim 13, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
15. The method of any one of claims 1 to 14, further comprising co-administering a 5-HTIA receptor agonist.
16. The method of claim 15, wherein the 5-HT IA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
17. A method of providing prophylaxis, and/or primary, secondary and tertiary prevention against PTSD and/or depression in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin or a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
18. The method of claim 17, wherein the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
19. The method of claim 17, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
20. The method of claim 17, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
21. The method of claim 17, wherein the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
22. The method of claim 21, wherein the mushroom cell culture composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
23. The method of claim 21, wherein the mushroom cell culture composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
24. The method of claim 21, wherein the mushroom cell culture composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
25. The method of any one of claims 17 to 24, wherein the effective amount of a mushroom cell culture composition or entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD and depression.
26. The method of any one of claims 17 to 25, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 4 weeks of a psychological trauma or other traumatic event.
27. The method of any one of claims 17 to 25, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 1 week of a psychological trauma or other traumatic event.
28. The method of any one of claims 17 to 25, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 3 days of a psychological trauma or other traumatic event.
29. The method of any one of claims 17 to 25, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 1 day of a psychological trauma or other traumatic event.
30. The method of any one of claims 17 to 29, wherein the mushroom cell culture composition or entheogen composition is produced by a process comprising mushroom cells in a culture vessel.
31. The method of claim 30, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
32. The method of any one of claims 17 to 19, further comprising co-administering a 5-HTIA receptor agonist.
33. The method of claim 32, wherein the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
34. Mushroom cell culture extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin or entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin for use in treating a psychiatric disorder in a subject in need thereof.
35. Use of claim 34, wherein the mushroom cell culture composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
36. Use of any one of claims 34 to 35, wherein the entheogen composition or mushroom cell culture extract comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
37. Use of any one of claims 34 to 35, wherein the entheogen composition or mushroom cell culture extract comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
38. Use of any one of claims 34 to 35, wherein the entheogen composition or mushroom cell culture extract comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
39. Use of any one of claims 34 to 38, wherein the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
40. Use of any one of claims 34 to 39, wherein the amount of mushroom cell culture composition or entheogen composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder.
41. Use of any one of claims 34 to 40, wherein the psychiatric disorder is PTSD.
42. Use of any one of claims 34 to 40, wherein the psychiatric disorder is OCD and/or schizophrenia.
43. Use of any one of claims 34 to 42, wherein the mushroom cell culture composition is produced by a process comprising culturing mushroom cells in a culture vessel.
44. Use of claim 43, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
45. Use of any one of claims 34 to 44, further comprising co-administering a 5-HTIA receptor agonist.
46. Use of claim 45, wherein the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
47. Mushroom cell culture extract comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin or entheogen composition comprising psilocybin or psilocin and at least one compound selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin for use in prophylaxis, and/or primary, secondary and tertiary prevention of PTSD and/or depression.
48. Use of claim 47, wherein the mushroom cell culture composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
49. Use of any one of claims 47 to 48, wherein the entheogen composition or mushroom cell culture extract comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
50. Use of any one of claims 47 to 48, wherein the entheogen composition or mushroom cell culture extract comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
51. Use of any one of claims 47 to 48, wherein the entheogen composition or mushroom cell culture extract comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
52. Use of any one of claims 47 to 51, wherein the effective amount of a mushroom cell culture composition or entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with PTSD and depression.
53. Use of any one of claims 47 to 52, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 4 weeks of a psychological trauma or other traumatic event.
54. Use of any one of claims 47 to 52, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 1 week of a psychological trauma or other traumatic event.
55. Use of any one of claims 47 to 52, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 3 days of a psychological trauma or other traumatic event.
56. Use of any one of claims 47 to 52, wherein the mushroom cell culture composition or entheogen composition is administered to the subject within 1 day of a psychological trauma or other traumatic event.
57. Use of any one of claims 47 to 56, wherein the mushroom cell culture composition or entheogen composition is produced by a process comprising culturing mushroom cells in a culture vessel.
58. Use of claim 57, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
59. Use of any one of claims 47 to 58, further comprising co-administering a 5-HTIA receptor agonist.
60. Use of claim 59, wherein the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
61. A process for mushroom cell culture comprising: culturing mushrooms on substrate to provide a mushroom cell culture, and contacting the mushroom cell culture with a PC composition.
62. The process of claim 61, wherein the mushrooms cells are psychoactive mushroom cells.
63. The process of claim 62, wherein the mushroom cell culture produces one or more psychoactive compounds or other compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
64. The process of any one of claims 61 to 63, further comprising processing the mushroom cell culture to provide an aqueous fraction comprising a psychoactive compound.
65. The process of any one of claims 61 to 63, further comprising processing the mushroom cell culture to provide an extract comprising a psychoactive compound.
66. The process of any one of claims 61 to 63, further comprising processing the mushroom cell culture to provide a purified composition comprising a psychoactive compound.
67. The process of any one of claims 61 to 63, further comprising the step of formulating the aqueous fraction, extract or purified composition comprising a psychoactive compound with a pharmaceutically acceptable carrier.
68. The process of claim 67, wherein the pharmaceutically acceptable carrier does not naturally occur with the aqueous fraction, extract or purified composition comprising a psychoactive compound.
69. A process for production of psychoactive compounds comprising: culturing mushroom cells from a psychoactive mushroom on substrate to cell culture; contacting the mushroom cell with a PC composition; and processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds.
70. The process of claim 69, wherein the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises producing an aqueous fraction from the mushroom cell culture that comprises one or more psychoactive compounds.
71. The process of claim 69, wherein the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises extracting the mushroom cell culture with an aqueous solvent, an organic solvent of combinations thereof to provide an extract that comprises one or more psychoactive compounds.
72. The process of claim 69, wherein the step of processing the mushroom cell culture to provide a mushroom cell culture composition comprising one or more psychoactive compounds comprises processing the mushroom cell culture to provide a purified composition comprising a psychoactive compound.
73. The process of any one of claims 69 to 72, further comprising the step of formulating the aqueous fraction, extract or purified composition comprising a psychoactive compound with a pharmaceutically acceptable carrier.
74. The process of claim 73, wherein the pharmaceutically acceptable carrier does not naturally occur with the aqueous fraction, extract or purified composition comprising a psychoactive compound.
75. A mushroom cell culture composition or formulation thereof produced by a process of any one of claims 69 to 74.
76. A formulation comprising a mushroom cell culture composition and a pharmaceutically acceptable carrier that does not naturally occur with the mushroom cell culture.
77. A formulation comprising psilocybin or psilocin and a 5-HTIA receptor agonist.
78. Formulation of claim 77, wherein the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
79. Formulation of any one of claims 77 to 78, wherein the psilocybin or psilocin is provided in a mushroom cell culture extract.
80. Formulation of claim 79, wherein the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
81. Formulation of claim 80, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
82. Formulation of any one of claims 77 to 81, further comprising one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
83. Formulation of any one of claims 77 to 82, wherein the psilocybin or psilocin is chemically synthesized.
84. Formulation of any one of claims 77 to 83 for use in treating a psychiatric disorder.
85. Use of claim 84, wherein the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
86. A kit comprising: a) a first formulation or composition comprising psilocybin or psilocin; and b) a second formulation or composition comprising a 5-HTIA receptor agonist; wherein the first formulation or composition and second formulation or composition are provided in different containers or oral delivery vehicles.
87. Kit of claim 86, wherein the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
88. Kit of any one of claims 86 to 87, wherein the psilocybin or psilocin is provided in a mushroom cell culture extract.
89. Kit of claim 88, wherein the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
90. Kit of claim 89, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycobiliprotein (PCB) composition.
91. Kit of any one of claims 86 to 90, further comprising one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
92. Kit of any one of claims 86 to 91, wherein the psilocybin or psilocin is chemically synthesized.
93. Kit of any one of claims 86 to 92 for use in treating a psychiatric disorder.
94. Use of claim 93, wherein the psychiatric disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), schizophrenia, depressive and anxiety disorders, obsessive compulsive disorder (OCD), alcohol and substance use disorders and nicotine addiction.
95. A method of treating autism spectrum disorder in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition. 6. The method of claim 95, wherein the entheogen composition comprises psilocybin or psilocin. 7. The method of any one of claims 95 to 96, wherein the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
98. The method of any one of claims 95 to 96, wherein the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
99. The method of any one of claims 95 to 96, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
100. The method of any one of claims 95 to 96, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
101. The method of any one of claims 95 to 100, wherein the entheogen composition is a mushroom cell culture composition.
102. The method of any one of claims 95 to 101, wherein the method comprises administering to the subject an effective amount of a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
103. The method of any one of claims 95 to 102, wherein the effective amount of a mushroom cell culture extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of autism spectrum disorder.
104. The method of any one of claims 95 to 103, wherein the mushroom cell culture extract is produced by a process comprising culturing mushroom cells in a culture vessel.
105. The method of claim 104, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
106. Entheogen composition for use in treating autism spectrum disorder in a subject in need thereof.
107. Use of claim 106, wherein the entheogen composition comprises psilocybin or psilocin.
108. Use of any one of claims 106 to 107, wherein the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
109. Use of any one of claims 106 to 107, wherein the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
110. Use of any one of claims 106 to 107, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
111. Use of any one of claims 106 to 107, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
112. Use of any one of claims 106 to 111, wherein the entheogen is provided as a mushroom cell culture composition comprising one or more compounds selected from the group consisting of psilocybin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
113. Use of any one of claims 106 to 112, wherein the mushroom cell culture composition comprises psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
114. Use of any one of claims 106 to 113, wherein the amount of mushroom cell culture composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the psychiatric disorder.
1 15. Use of any one of claims 106 to 1 14, wherein the mushroom cell culture composition is produced by a process comprising culturing mycelial cells in a culture vessel.
116. Use of claim 115, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
117. A method of treating a brain injury in a subject in need thereof comprising: administering to the subject an effective amount of an entheogen composition.
118. The method of claim 117, wherein the brain injury is selected from the group consisting of traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma of the brain.
119. The method of any one of claims 117 to 118, wherein the entheogen composition comprises psilocybin or psilocin.
120. The method of any one of claims 117 to 119, wherein the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
121. The method of any one of claims 117 to 119, wherein the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
122. The method of any one of claims 117 to 119, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
123. The method of any one of claims 117 to 119, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
124. The method of any one of claims 117 to 123, wherein the entheogen composition is a mushroom extract.
125. The method of claim 124, wherein the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel.
126. The method of claim 125, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
127. The method of any one of claims 117 to 126, wherein the effective amount of an entheogen or mushroom extract is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the brain injury.
128. The method of any one of claims 117 to 127, wherein the entheogen composition is a psilocybin-free entheogen composition.
129. The method of any one of claims 117 to 128, wherein the administration of the entheogen composition is immediately before, during, or immediately after anesthesia or sedation of the subject.
130. Entheogen composition for use in treating a brain injury in a subject in need thereof.
131. Use of claim 130, wherein the brain injury is selected from the group consisting of traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), concussion, polytraumatic injury (multi-system trauma including brain injury), and severe brain injury due to metabolic, inflammatory and other conditions related to severe trauma of the brain.
132. Use of any one of claims 130 to 131, wherein the entheogen composition comprises one or more compounds selected from the group consisting of psilocybin or psilocin, baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
133. Use of any one of claims 130 to 131, wherein the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
134. Use of any one of claims 130 to 131, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
135. Use of any one of claims 130 to 131, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
136. Use of any one of claims 130 to 135, wherein the entheogen composition is a mushroom extract comprising one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
137. Use of any one of claims 130 to 136, wherein the mushroom extract is a mushroom cell culture composition comprising psilocybin and one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
138. Use of claim 137, wherein the mycelial cell culture composition is produced by a process comprising culturing mycelial cells in a culture vessel.
139. Use of claim 138, wherein the process comprising culturing mycelial cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
140. Use of any one of claims 130 to 139, wherein the amount of entheogen or mycelial cell culture composition administered is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms of the brain injury.
141 . Use of any one of claims 130 to 140, wherein the entheogen composition is a psilocybin- free entheogen composition.
142. Use of any one of claims 130 to 141, wherein the administration of the entheogen composition is immediately before, during, or immediately after anesthesia or sedation of the subject.
143. Use of any one of claims 130 to 142, wherein the entheogen composition is coadministered with an anesthesia or sedation agent.
144. A composition comprising a phycocyanin (PC) composition and a mushroom extract.
145. The composition of claim 144, wherein the mushroom extract comprises psilocybin.
146. The composition of claim 144, wherein the mushroom extract is psilocybin-free. 47. The composition of any one of claims 144 to 146, wherein the entheogen composition comprises one or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
148. The composition of any one of claims 144 to 146, wherein the entheogen composition comprises at least two compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
149. The composition of any one of claims 144 to 146, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
150. The composition of any one of claims 144 to 146, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
151. The composition of claim any one of claims 144 to 150, wherein the phycocyanin composition is extracted from Spirulina.
152. The composition of any one of claims 144 to 151, wherein the phycocyanin composition is characterized by one or more of the following characteristics: a) the protein fraction of the composition comprises greater than about 30% of a protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; b) the protein fraction of the composition comprises greater than about 5% of a protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; c) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 45,578 kDa and an isoelectric point of about 6.2 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; d) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 35,014 kDa and an isoelectric point of about 5.9 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; e) the protein fraction of the composition comprises greater than about 0.30% of a protein having a molecular weight of about 24,688 kDa and an isoelectric point of about 5.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; f) the protein fraction of the composition comprises greater than about 2% of a protein having a molecular weight of about 22,522 kDa and an isoelectric point of about 5.9 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; g) the protein fraction of the composition comprises greater than about 1% of a protein having a molecular weight of about 21,023 kDa and an isoelectric point of about 7.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; h) the protein fraction of the composition comprises greater than about 0.50% of a protein having a molecular weight of about 13,417 kDa and an isoelectric point of about 7.3 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; i) the protein fraction of the composition comprises a ratio of major protein constituents to minor protein constituents of less than 3.5: 1 based on the aggregate mass of the proteins, wherein major protein constituents are the protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the minor protein constituents are the remainder of the proteins; j) the protein fraction of the composition comprises less than about 75% on a mass basis of the combined amounts of the protein having a molecular weight of about 17,695 kDa and an isoelectric point of about 6.29 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry and the protein having a molecular weight of about 19,833 kDa and an isoelectric point of about 6.14 as assayed by two dimensional gel electrophoresis followed by quantitation by densitometry; k) the composition produces a solution having a color value of greater than 200 E 10%/ 1cm when 250 mg of a dry powder of the composition are dissolved in one liter of water and absorbance is measured at 618 nm.
153. The composition of claim 152, wherein the PC composition has at least two of characteristics a, b, c, d, e, f, g, h, i, j and k.
154. The composition of claim 152, wherein the PC composition has at least three of characteristics a, b, c, d, e, f, g, h, i, j and k.
155. The composition of claim 152, wherein the PC composition has at least four of characteristics a, b, c, d, e, f, g, h, i, j and k.
156. The composition of claim 152, wherein the PC composition has at least five of characteristics a, b, c, d, e, f, g, h, i, j and k.
157. The composition of claim 152, wherein the PC composition has at least six of characteristics a, b, c, d, e, f, g, h, i, j and k.
158. The composition of claim 152, wherein the purified PC composition of claim 1 , wherein the composition has at least seven of characteristics a, b, c, d, e, f, g, h, i, j and k.
159. The composition of claim 152, wherein the PC composition has at least eight of characteristics a, b, c, d, e, f, g, h, i, j and k.
160. The composition of claim 152, wherein the PC composition has at least nine of characteristics a, b, c, d, e, f, g, h, i, j and k.
161. The composition of claim 152, wherein the PC composition has at least ten of characteristics a, b, c, d, e, f, g, h, i, j and k.
162. The composition of claim 152, wherein the PC composition has all eleven characteristics a, b, c, d, e, f, g, h, i, j and k.
163. The composition of any one of claims 144 to 162, wherein the phycocyanin composition is produced by a process comprising: encapsulating Spirulina to provide capsules; and contacting the capsules with an aqueous medium under conditions such that the compound of interest passes from the capsule into the aqueous solution.
164. The composition of any one of claims 144 to 163, wherein the PC composition comprises a dried powder comprising a purified PC, the powder having a residual moisture content of less than about 5% w/w of the powder.
165. The composition of any one of claims 144 to 164, wherein the psilocybin-free mushroom extract is extracted from a mushroom or part thereof that produces psilocybin, a psilocybin analog or related typtamine.
166. The composition of any one of claims 144 to 165, wherein the psilocybin-free mushroom extract is extracted from a mushroom or part thereof that comprises the psilocybin biosynthetic gene cluster (psiD, psiH, psiK, psiM).
167. The composition of any one of claims 144 to 166, wherein the psilocybin-free mushroom extract is an aqueous extract.
168. The composition of any one of claims 144 to 167, wherein the psilocybin-free mushroom extract is produced by a process comprising: encapsulating a raw material comprising a mushroom or part thereof that produces psilocybin, a psilocybin analog or related typtamine to provide capsules; and contacting the capsules with an aqueous medium under conditions such that compounds of interest passes from the capsule into the aqueous solution.
169. The composition of claim 68, further comprising the step of removing psilocybin from the extract by chromatography.
170. The composition of any one of claims 144 to 169, wherein the psilocybin-free mushroom extract comprises a dried powder having a residual moisture content of less than about 5% w/w of the powder.
171. The composition of any one of claims 144 to 170, wherein the psilocybin-free mushroom extract is a psilocybin-free mycelial extract.
172. The composition of any one of claims 144 to 171, wherein the composition comprises from 1 to 99% w/w phycocyanin.
173. The composition of any one of claims 144 to 172, wherein the composition comprises from 10 to 90% w/w phycocyanin.
174. The composition of any one of claims 144 to 173, wherein the composition comprises from 1 to 99% w/w psilocybin-free mushroom extract.
175. The composition of any one of claims 144 to 174, wherein the composition comprises from 10 to 90% w/w psilocybin-free mushroom extract.
176. The composition of any one of claims 144 to 175, wherein the composition is formulated as a topical formulation, an oral formulation, a mucosal formulation, an ophthalmological formulation, an aerosol formulation, and an intranasal formulation.
177. The composition of claim 176, further comprising one or more excipients or pharmaceutically acceptable carriers.
178. The composition of any one of claims 176 or 177, wherein the oral formulation is an oral delivery vehicle.
179. The composition of claim 178, wherein the oral delivery vehicle is selected from the group consisting of a capsule, a tablet and a gummi gel.
180. A method of treating or preventing a psychiatric disease or disorder comprising administering an effective amount of the composition of any one of claims 144 to 179 to a subject suffering from a psychiatric disease or disorder.
181. The method of claim 180, wherein the psychiatric disease or disorder is selected from the group consisting of anxiety disorder and depression.
182. A method of improving or altering cognitive function or sociability, or reducing aggressive behavior comprising administering an effective amount of the composition of any one of claims 144 to 179 to a subject suffering from a psychiatric disease or disorder.
183. Composition of any one of claims 144 to 179 for use in treating or preventing a psychiatric disease or disorder in a subject in need thereof.
184. Composition of claim 183, wherein the psychiatric disease or disorder is anxiety disorder and depression.
185. Composition of any one of claims 144 to 179 for use in of improving or altering cognitive function or sociability, or reducing aggressive behavior in a subject in need thereof.
186. A method of improving neuroplasticity in a subj ect in need thereof comprising administering to the subject an entheogen composition comprising psilocybin or psilocin and one or more entourage compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
187. The method of claim 186, wherein the composition comprises psilocybin.
188. The method of any one of claims 186 to 187, wherein the subject in need thereof has a disease, condition or disorder that could benefit from the promotion of neuroplasticity.
189. The method of claim 188, wherein the disease, condition or disorder associated with decreased neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD.
190. The method of any one of claims 186 to 189, wherein administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43, and Synaptophysin in one or more areas of the brain selected from the group consisting of the prefrontal cortex, amygdala, hippocampus, and striatum.
191. The method of claim 190, wherein administration of the entheogen composition increases the level of two or more proteins selected from the group consisting of PSD-95, GAP43 and Synaptophysin.
192. The method of claim 190, wherein administration of the entheogen composition increases the level of PSD-95, GAP43 and Synaptophysin.
193. The method of any one of claims 190 to 192, wherein the area of the brain is the prefrontal cortex.
194. The method of any one of claims 190 to 192, wherein the area of the brain is the amygdala.
195. The method of any one of claims 190 to 192, wherein the area of the brain is the hippocampus.
196. The method of any one of claims 190 to 192, wherein the area of the brain is the striatum.
197. The method of any one of claims 186 to 196, wherein the entheogen composition comprises two or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
198. The method of any one of claims 186 to 196, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
199. The method of any one of claims 186 to 196, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
200. The method of any one of claims 186 to 199, wherein the entheogen composition is a mushroom extract.
201. The method of claim 200, wherein the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel.
202. The method of claim 201, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
203. The method of any one of claims 186 to 202, wherein the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with neural atrophy.
204. Entheogen composition for use in treating a disease, condition or disorder associated with neural atrophy.
205. Use of claim 204, wherein the entheogen composition comprises psilocybin.
206. Use of any one of claims 204 to 205, wherein the disease, condition or disorder associated with decreased neuroplasticity is selected from the group consisting of a psychiatric disease or disorder, substance abuse disorder, a brain injury or disorder, autism spectrum disorder, brain surgery, stroke, epilepsy, neurodegenerative disease, long term memory loss, short term memory loss, aphasia, age associated cognitive decline, cognitive impairment, dementia, PTSD, and ADHD.
207. Use any one of claims 205 to 206, wherein administration of the entheogen composition increases the level of one or more protein selected from the group consisting of PSD-95, GAP43 and Synaptophysin in one or more areas of the brain selected from the group consisting of the prefrontal cortex, amygdala, hippocampus, and striatum.
208. Use of claim 207, wherein administration of the entheogen composition increases the level of two or more proteins selected from the group consisting of PSD-95, GAP43 and Synaptophysin.
209. Use of claim 207, wherein administration of the entheogen composition increases the level of PSD-95, GAP43 and Synaptophysin.
210. Use of any one of claims 207 to 209, wherein the area of the brain is the prefrontal cortex.
211. Use of any one of claims 207 to 209, wherein the area of the brain is the amygdala.
212. Use of any one of claims 207 to 209, wherein the area of the brain is the hippocampus.
213. Use of any one of claims 207 to 209, wherein the area of the brain is the striatum.
214. Use of any one of claims 204 to 213, wherein the entheogen composition comprises two or more compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
215. Use of any one of claims 204 to 213, wherein the entheogen composition comprises at least three compounds selected from the group consisting of baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
216. Use of any one of claims 204 to 213, wherein the entheogen composition comprises baeocystin, aeruginascin, norpsilocin, and norbaeocystin.
217. Use of any one of claims 204 to 216, wherein the entheogen composition is a mushroom extract.
218. Use of claim 217, wherein the mushroom extract is produced by a process comprising culturing mushroom cells in a culture vessel.
219. Use of claim 218, wherein the process comprising culturing mushroom cells in a culture vessel further comprises treating the culture with a phycocyanin composition.
220. Use of any one of claims 204 to 219, wherein the administered amount of the entheogen composition is sufficient to alleviate, prevent, reduce the frequency of, or provide improvement of one or more symptoms associated with reduced neuroplasticity.
221. Method or use of any one of claims 186 to 220, wherein the entheogen composition is coadministered with a 5-HTIA receptor agonist.
222. Method or use of claim 221, wherein the 5-HTIA receptor agonist is selected from the group consisting of buspirone, 8-OH-DPAT, F13714 and F15599.
PCT/US2023/013767 2022-02-25 2023-02-24 Treatment of psychiatric disorders, brain injuries, and autism spectrum disorder WO2023164092A1 (en)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US202263313816P 2022-02-25 2022-02-25
US63/313,816 2022-02-25
US202263319550P 2022-03-14 2022-03-14
US63/319,550 2022-03-14
US202263359282P 2022-07-08 2022-07-08
US63/359,282 2022-07-08
US202263405620P 2022-09-12 2022-09-12
US202263405615P 2022-09-12 2022-09-12
US202263405617P 2022-09-12 2022-09-12
US63/405,617 2022-09-12
US63/405,615 2022-09-12
US63/405,620 2022-09-12

Publications (1)

Publication Number Publication Date
WO2023164092A1 true WO2023164092A1 (en) 2023-08-31

Family

ID=87766641

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/013767 WO2023164092A1 (en) 2022-02-25 2023-02-24 Treatment of psychiatric disorders, brain injuries, and autism spectrum disorder

Country Status (1)

Country Link
WO (1) WO2023164092A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4542608A (en) * 1982-09-28 1985-09-24 Everbloom Mushrooms Mushroom cultivation
WO2020212952A1 (en) * 2019-04-17 2020-10-22 Compass Pathfinder Limited Treatment of depression and other various disorders with psilocybin
US20200375967A1 (en) * 2016-07-23 2020-12-03 Paul Edward Stamets Compositions of psilocybin and analogs
US20210010015A1 (en) * 2018-03-19 2021-01-14 Teknologian Tutkimuskeskus Vtt Oy Heterologous production of psilocybin
WO2021067626A2 (en) * 2019-10-01 2021-04-08 Intima Bioscience, Inc. Genetic engineering of fungi to modulate tryptamine expression
US20210251976A1 (en) * 2016-07-23 2021-08-19 Turtle Bear Holdings, Llc Tryptamine compositions for enhancing neurite outgrowth
WO2021207824A1 (en) * 2020-04-17 2021-10-21 Revive Therapeutics Ltd. Use of psilocybin in the treatment of neurological brain injury and migraines

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4542608A (en) * 1982-09-28 1985-09-24 Everbloom Mushrooms Mushroom cultivation
US20200375967A1 (en) * 2016-07-23 2020-12-03 Paul Edward Stamets Compositions of psilocybin and analogs
US20210251976A1 (en) * 2016-07-23 2021-08-19 Turtle Bear Holdings, Llc Tryptamine compositions for enhancing neurite outgrowth
US20210010015A1 (en) * 2018-03-19 2021-01-14 Teknologian Tutkimuskeskus Vtt Oy Heterologous production of psilocybin
WO2020212952A1 (en) * 2019-04-17 2020-10-22 Compass Pathfinder Limited Treatment of depression and other various disorders with psilocybin
WO2021067626A2 (en) * 2019-10-01 2021-04-08 Intima Bioscience, Inc. Genetic engineering of fungi to modulate tryptamine expression
WO2021207824A1 (en) * 2020-04-17 2021-10-21 Revive Therapeutics Ltd. Use of psilocybin in the treatment of neurological brain injury and migraines

Similar Documents

Publication Publication Date Title
KR102476644B1 (en) Compositions containing tannic acid and uses thereof
KR20090080105A (en) Treatment of pervasive developmental disorders
KR102403047B1 (en) Composition containing benzoate compound and tannic acid for treating central nervous system disorder
WO2011119227A2 (en) Compositions for the treatment of central nervous system disorders including depression employing novel drug combination therapy to reduce suicidality in patients
ES2955870T3 (en) Compositions comprising bacterial strains
JP2020520928A (en) Composition comprising a bacterial strain
WO2015100091A1 (en) Methods for treating, preventing, or reducing neuroinflammation or cognitive impairment
TW202116333A (en) Compositions comprising bacterial strains
WO2022115796A1 (en) Compositions and methods for treating neurological conditions
WO2023164092A1 (en) Treatment of psychiatric disorders, brain injuries, and autism spectrum disorder
US9851354B2 (en) Methods of treating fragile X syndrome and related disorders
TW202140773A (en) Compositions comprising bacterial strains
Liu et al. The potent analgesia of intrathecal 2R, 6R-HNK via TRPA1 inhibition in LF-PENS-induced chronic primary pain model
TWI686192B (en) Compositions containing tannic acids and uses thereof
JP5634062B2 (en) Composition for improving brain function and method for improving brain function
JP2011136932A (en) Composition for improving cerebral function and method for improving cerebral function
WO2012035871A1 (en) Brain function enhancement composition and method for enhancing brain function
ITMI20110143A1 (en) NEW ASSOCIATION FOR PAIN TREATMENT
KR102527378B1 (en) Composition for improvement, prevention and treatment of diseases caused by a decrease in dopamine with 2&#39;-fucosyllactose
WO2012036191A1 (en) Composition for improving brain function and method for improving brain function
Abdel-Maksoud et al. NMDA/glutamate mechanism of anxiolytic-like action of zinc in vogel conflict test in mice
WO2011041920A2 (en) Nutraceutical composition that comprises extract of shilajit, folic acid, vitamin b12 and vitamin b6 and the use thereof for preventing and/or treating neurodegenerative diseases and/or the cognitive deterioration associated with cerebral ageing
Filpa Adaptive changes of the gastrointestinal neuromuscolar function in a mouse model of Catechol-O-Methyl trasferase genetic reduction: implication in the pathogenesis of Irritable Bowel syndrome.
Zhang Opioid System's involvement in Ketamine's Antidepressant-like Effects
JP6401028B2 (en) Medium / long-term memory disorder prevention / improvement agent consisting of whale muscle extract

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23760669

Country of ref document: EP

Kind code of ref document: A1