sign in sign up
<<
Home , Amanita muscaria, Amanita pantherina, Bicuculline, Conocybe, Glutamic acid, Ibotenic acid

REVIEW ARTICLE

Fungal , Ibotenic , and : Analytical Methods and Biologic Activities

Katarzyna Stebelska, PhD

idea to use them for religious ceremonies or Abstract: Psychoactive of fungal origin, psilocin, ibotenic support. As an example, it was experimentally established acid, and muscimol among them have been proposed for recreational that , psilocin of fungal origin, is able to use and popularized since the 1960s, XX century. Despite their well- produce mystical states under favorable circumstances documented , they reached reputation of being safe and (involving acquired cultural frame of references).10–16 Some nonaddictive. Scientific efforts to find any medical application for details of the study design, particularly the usage of as these hallucinogens in psychiatry, psychotherapy, and even for an active placebo, may suggest that the authors of the first religious rituals support are highly controversial. Even if they show of these experiments known as Good Friday Experiment any healing potential, their usage in psychotherapy is in some cases were aware that religious rituals alone are not neutral for inadequate and may additionally harm seriously suffering patients. mental health.10–28 The states of altered induced Hallucinogens are thought to reduce cognitive functions. However, by religious settings and facilitated by psychedelic drugs fi in case of indolealkylamines, such as psilocin, some recent ndings increase susceptibility to suggested messages and are not free suggest their ability to improve perception and mental skills, what from negative emotions such as fear, terror, anxiety, shame, would motivate the consumption of “magic .” The pres- fi and sense of guilt. Therefore, any religious cult, particularly ent article offers an opportunity to nd out what are the main symp- if combined with psychoactive compounds intake, should toms of intoxication with mushrooms containing psilocybin/psilocin, be considered as being able to alter psychosocial identity. muscimol, and . The progress in analytical methods Psychotic syndromes evoked in religious purposes are not for detection of them in fungal material, , and body fluids is fully safe and may induce development of really serious psy- reviewed. Findings on the mechanisms of their biologic activity are chopathologies including confusional syndrome and schizo- summarized. Additionally, therapeutic potential of these fungal psy- phrenia, a disease of at least partly psychosocial etiology.29–35 choactive compounds and health risk associated with their abuse are discussed. It should be pointed out that psilocybin/psilocin, although still scientifically explored as potentially helpful and safe psycho- Key Words: hallucinogens, psilocin, ibotenic acid, muscimol therapy adjunctive,4,10–16,36–40 is presently used to model and to investigate psychosis similar to positive (Ther Monit 2013;35:420–442) symptoms of the disease.41–50 The psychoactive principles of mushrooms, namely psilocybin and psilocin, were isolated and identified INTRODUCTION 51 Psychoactive or fungi accompanied religious rituals by Hofmann in 1958. Both these substances, similarly as 1–4 LSD, were originally proposed as incapacitating agents and in many ancient civilizations worldwide. Hallucinogenic com- 7 pounds contained in fungi of Psilocybe and genera used for intelligence purposes. Ibotenic acid and muscimol seemed to be the most active substances of fly respon- have been used as recreational drugs of abuse since the begin- 52–54 ning of the 1960s, XX century.5,6 Although hallucinogens are sible for its hallucinogenic properties. Exposure to these thought to be safe because of relatively low physiologic tox- substances, especially chronic, not only may constitute a sig- fi icity and dependence potential7,8 nowadays the context of ni cant risk to human mental health but may also induce hallucinogenic compounds consumption, mainly by young other serious disorders including cardiovascular complica- people seeking for unusual experiences, is usually nega- tions and neurodegenerative diseases. Although chemically tive5,6,9 but not exclusively. One exception seems to be the unstable, ibotenic acid should be considered as highly toxic agent if one is aware of activity and Received for publication June 19, 2012; accepted January 9, 2013. of its endogenous analogue glutamate. Simultaneously, the From the Department of Analytical and Ecological Chemistry, Opole University, fungal psychedelics of high biologic activity display signifi- Opole, Poland. cant therapeutic potential. Supported by Wroclaw Research Centre EIT+ under the Project “Biotechnologies and advanced medical Technologies”—BioMed (POIG.01.01.02-02-003/08) Hallucinogenic fungi and psychedelics isolated from financed from the European Regional Development Fund (Operational Pro- them are illegal in many countries or their processing and gramme Innovative Economy, 1.1.2). distribution stay under strict regulation.12 However, because The author declares no conflicts of interest. they are easily available from natural , they have Correspondence: Katarzyna Stebelska, PhD, Department of Analytical and Ecological Chemistry, Opole University, Pl Kopernika 11, PL-45-040 become quite popular among growing population of drug Opole, Poland (e-mail: [email protected]). users and are frequent cause of serious poisoning or even – – Copyright © 2013 by Lippincott Williams & Wilkins accidental deaths resulted from their biologic activity.5 9,55 59

420 Ther Drug Monit  Volume 35, Number 4, August 2013 Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

Therefore, analytical methods elaborated for detection of these when is present in the medium from the stage of substances in fungal material and body fluids are also reviewed culture inoculation.75 Replacement of nutrient medium does in this article. not affect psilocybin content.74 Conditions of potassium depletion do not influence considerably mycelium growth but lower psilocybin production.75 Lack of succinate in the Psilocybin/Psilocin as a medium decreases of psilocybin.74 Psilocybin is a secondary metabolite of some of Psilocybin seems to be present at higher amounts in Agrocybe, , , , , caps than in the stems.76,77 , , Panaeolina, , , and P. serbica var. bohemica (synonymous with P. bohemica) , and Psilocybe genera.60–63 The major part of the were found to contain high amounts of psilocybin up to 1% of known psychoactive species belongs to the Psilocybe. dry weight. seemed to contain minor They are saprotrophic on wood, dung, , , and litter. amounts of psilocybin and low amounts of psilocin (0.63% , P. serbica var. bohemica, P. serbica and 0.11%, respectively). The contents of psilocybin and psi- var. arcana, and P. semilanceata are probably the only spe- locin in the sample of P. cyanescens were 0.32% and 0.51%, cies containing sufficient for intoxication amounts of psilocin/ respectively.78 var. bohemica collected in psilocybin that can grow in natural or near natural habitats in Europe were found to contain minor amounts of psilocybin the Middle and North Europe.60,64–66 Psilocybe cubensis is the (content between 0.33% and 0.62%) and almost no psilocin. most popular natural source of psilocybin in the warmer Psilocybe cyanescens from the Pacific Northwest, United regions of the Americas. In the United States, it can be found States, contained both psilocybin (0.41%–0.98%) and psilocin in the wild throughout the southeast to Texas and the Pacific (0.23%–0.93%).79 Northwest. The is common in Mexico and other Cen- During mycelium development, some processes are tral American countries, and it is also known from South controlled by light or are light responsive,80 and psilocybin American, Asian, Australian and Oceanian localities.62,67,68 biosynthesis is also somehow under control of ultraviolet occurs commonly in Australia and (UV) radiation. Psilocybe genus mushrooms produce fruit is widespread in and .68,69 bodies after UV or blue light irradiation.81 The caps of fruit Season-dependent availability of psilocybin-containing bodies formed from poorly enlightened cultures develop blu- mushrooms has stimulated the search for methods to cultivate ish phototropic zone originating from oxidation products of these fungi. Culturing of the mushrooms became common compounds. It is also thought that the highest level of after reports on the methodology seemed in both scientific psilocybin coincides with sporulation time. literature and specialist books. It seems that most recreation- ally used mushrooms are cultivated rather than picked wild at Analytical Methods Used for present. For illicit home growers, P. cubensis and its varieties Psilocybin/Psilocin Detection and Isolation are generally the mushrooms of choice because they are easy to culture and fruit readily in vitro, producing a significant Methods of Extraction amount of biomass with each flush.60,67 Psilocybin due to the presence of highly polar phos- isolated from hallucinogenic mushrooms phate group is well soluble in but poorly soluble in within Psilocybe genus such as psilocybin, , nor- and ethanol. Psilocybin is practically insoluble in baeocystin are tryptophan derivatives formed through decar- nonpolar solvents such as and . Psilocin is boxylation, indole ring at position 4, and slightly more soluble in some nonpolar solvents (1-chlorobutane N- through the activity of S-adenosylmethionine or dichloromethane).82–84 and O-. Biosynthetic pathway of psilocybin Water recrystallized psilocybin ( of in P. cubensis involves conversion of tryptophan to trypt- 220–2808C) forms white needles and contains crystal water. , N-methyltryptamine, N,N-dimethyltryptamine, and Psilocybin recrystallized from methanol contains crystal meth- psilocin or alternatively conversion of 4-hydroxytryptamine anol and melts at 185–1958C.85,86 to psilocybin.70 Psilocybe cubensis cultures have the ability Preparation of fungal material to extraction usually to hydroxylate derivatives at position 4.71 consists in its air drying at room temperature or slightly elevated Psilocybe and monocaryotic mycelium do not temperature (but not above 408C) and/or freeze-drying.87,88 contain psychoactive compounds. Mycelium starts to produce Comparable analysis reveals that freeze-dried material contains psilocybin/psilocin at further stages of mushroom develop- the highest amounts of psilocybin/psilocin. Pulverization of ment.72 Quantitative analysis of P. cubensis fruit bodies col- dried material is obtained by the use of a mortar or in laboratory lected from natural sources or from artificial cultures grown mills and homogenizers.55,74,89,90 Macerated homogenates pre- under strictly controlled conditions reveals that the content of pared in a mortar demand separation of solid components.82,91 psilocin varies greatly (eg, from a 3.2–13.3 mg/g for artificial Extraction of dried and powdered material is usually culture).73 The first flush collected from culture cultivated on assisted by mechanical agitation.74,87,92–94 Sonication in a water rice grain generally does not contain psilocin.73 bath was used to assist extraction process.55,95–97 To obtain the The amount of psilocybin depends on composition of extract of psilocybin/psilocin, a micropercolator was also used.98 nutrient medium. Media depleted in source of are less The time of extraction, depending on the amount of material, effective in the production.74,75 Tryptophan supple- solvent volume, and the exact mechanical agitation technique, mentation stimulates psilocybin biosynthesis. This happens ranges from a few minutes to several hours.78,88,94 Effectiveness

2013 Lippincott Williams & Wilkins 421 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013 of extraction improves and its time may be shortened if the The obtained supernatant after evaporation to dryness procedure is carried out at elevated temperature.89,96 However, and dissolving in appropriate solvent is usually directly heating to the temperature above 708C leads to psilocybin analyzed without further treatment. Sometimes the samples dephosphorylation, particularly if the extraction is carried out undergo solid-phase extraction for initial purification. For at acidic pH conditions.82,91 To improve effectiveness, extraction example, Chem Elut column was used to extract body fluid may be repeated several times and the obtained extracts com- samples.55 Initial purification of urine samples containing bined before analysis.89,90,97,98 Methanol is the most often used psilocin was performed by means of solid-phase extraction solvent for extraction of psilocybin/psilocin because it is able to using cation-exchange sorbent.105 To isolate indole alkaloids denature and to prevent enzymatic conversion of psilo- from fungal tissue, the crude methanolic extract was purified cybintopsilocin.55,75,78,87,88,90,92,97 According to Kysilka and by -exchange chromatography using cation-exchange Wurst,94 the optimal solvent for the extraction of psilocybin is sorbent.75 Purification and preconcentration could be per- a mixture of methanol and water (75:25) saturated with potas- formed using cartridges containing octadecyl sorbent.106 sium nitrate. The indole alkaloids could be extracted with 75% Before high-performance liquid chromatography (HPLC) ethanol; however, the obtained solution maintains phosphatase analysis, psilocin-containing samples after deproteinization activity.78,94 It was established that water solutions of and alkalization to pH 8.5 were extracted using liquid–liquid are more effective than water solutions of .78 Ethyl extraction technique or online solid-phase extraction with is also sometimes used for extraction.89 Aprocedure cation-exchange sorbent.84 of 1-step extraction directly to chloroform was also described.95 Sometimes a method suitable for alkaloids isolation is Methods of Qualitative Analysis and Isolation used, namely the extraction to acidified water solution followed Blue compound being a product of psilocin oxidation is by extraction to nonpolar solvent at basic pH.78,82,83,91,99,100 The easily formed in iodine vapors. Enzymatic oxidation of psilocin macerated material is heated during the procedure what leads to is catalyzed by p-diphenol oxidase.107 Similarly, oxidation with conversion of psilocybin to psilocin. ferric chloride, a component of the Keller reagent used for Thin Several examples of extraction methods used for Layer Chromatography (TLC) detection of indole compounds, psilocybin/psilocin analysis are briefly described in Table 1. makes possible visualization of psilocin spots on TLC plates.51,60 The obtained extract is separated from suspension by Under UV illumination, psilocin is visible as absorbent filtration79,87,92,95,97 or centrifugation.55,82,95 spot.108 Psilocybin shows blue fluorescence under UV light.108 To analyze body fluids (plasma or urine) for the presence Identification of indole compounds such as psilocybin and psi- of psilocin, it is necessary to hydrolyze conjugate of psilocin with locin in water solutions or after chromatographic separation on glucuronic acid. It is usually performed by the use of Escherichia TLC plates may be performed applying the Ehrlich reagent con- coli or Helix pomatia b-glucuronidase at slightly acidic condi- taining p-dimethylbenzaldehyde or para-Dimethylcinnamic acid, tions (pH 5–6).102–105 Acidic or alkaline was reported 2,4-dimethylcinnamic acid reagent containing 4-(dimethylamino)- to be ineffective.104 The addition of methanol to the sample cinnamaldehyde.71,75,108–110 Psilocin was visualized with diazo- denatures proteins.104 To deproteinize the ice cold sample, tized p-nitroaniline followed by alkali.105 Psilocin identification 20% of the solution of polyethylene glycol 6000 was also used.84 may be based on UV and infrared spectrometry.83 Centrifugation is an effective method to separate precipitated Thin-layer chromatography was reported as an appro- proteins from supernatant containing the exact analytes.104 priate method of psilocybin/psilocin isolation using cellulose

TABLE 1. Exemplary Methods of Extraction Used for Psilocybin/Psilocin Determination Dry Material/ Solvent Extraction Time Solvent Ratio Extraction Method References Methanol 12 h 250 mg/7 mL Shaking 87 Methanol 30 s 10 mg/2 mL Vortexing 92 Methanol 30 min — Sonication 55 10 mM of HCl in methanol 60 min 100 mg/100 mL Sonication 88 Methanol 15 min 100 mg/3 + 2 mL Sonication 97 10% of acetic acid 0.05 g—0.5 g Homogenization in a mortar 82 Methanol 8 h 25–100 mg/10 mL Shaking 74 Methanol 12 and 24 h 1 g Homogenization in a mortar. 69,101 The suspension was left in methanol for 12 or 24 h Methanol 3 · 24 h 10 g/3 · 100 mL The suspension was left in 90 methanol for 3 · 24 h 3 · 30 min 20 mg/3 · 1 mL Sonication 89 Chloroform 1 h 50 mg/1 mL Sonication 95 Water solution of acetic acid, 1h—maceration, 10 min 10–50 mg/10 mL Maceration, extraction, 99 pH 4, chloroform chloroform extraction

422 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol plates or silica gel plates.60,71,87,111–113 The methods of paper leads to the appearance of active form of the drug, namely chromatography108 or bidirectional paper chromatography74,75 psilocin. Blockage of alkaline phosphatase by means of were also used for separation. competitive substrates prevents the symptoms of intoxication.120 A method of psilocybin isolation using preparative In opposition to psilocybin, psilocin is easily absorbed from the thin-layer chromatography on silica gel plates followed by gastrointestinal tract to the circulating blood.121 Pharmacoki- reextraction was developed.90 netic animal studies on 14C-labeled psilocybin analogue reveal The method of isolation on cellulose sorbent was also that psilocybin is absorbed in 50% by the digestive tract after elaborated followed by step.90 oral dose application.122,123 Similarly, HPLC studies revealed about 50% (52.7% 6 20%) bioavailability of psilocin after oral 114 Analytical Methods of Quantitative Determination administration of psilocybin. Biodistribution of the drug after HPLC quantitative analysis of psychoactive substances intravenous injection is similar for all organs including 122 originating from fungi of Psilocybe genus is usually performed (animal study). After 8 hours, the tissue content of the drug is by reversed-phase chromatography.84,87,89,92,98,101,102,104,114 Ion- very low except for and adrenal tissue still expressing 122 pair chromatography applying reversed-phase column was also radioactivity after 48 hours. successfully used.87,98 As a method of determination, ion- After oral application of psilocybin, plasma concentra- exchange chromatography was also proposed using cation- tion of psilocin increases rapidly, reaches maximum after exchange sorbent.88 Electrochemical detection is preferred 50–100 minutes, and slowly decreases during the next 5– 84,114,124 due to low psilocin absorbance in UV light.84,114 Liquid chro- 6 hours. Psilocybin injected intravenously rapidly under- matography (LC) combined with (MS) is an goes dephosphorylation and disappears faster from circulatory 114 effective method for detection of psilocybin and psilocin.92,102,104 system (when compared with oral administration). Pharmaco- Thermolabile compounds including psilocybin should kinetic parameters of psilocin after oral or intravenous adminis- not be analyzed directly by chromatography (GC)–MS) tration of psilocybin determined in humans are shown in Table 2. technique. The GC-MS analysis may be performed after deriv- Animal studies reveal that psilocin is in 65% excreted atization with N-Methyl-N-(trimethylsilyl)trifluoroacetamide with urine and in 15%–20% with bile and within 8 hours carried out to protect psilocin against thermal decomposi- from administration.122 A small amount (about 10%–20%) tion.95,103,105 Alternatively, electrospray ionization (ESI)–MS remains in organism for a longer time (14C-labelled metabolites technique89,104,115,116 or chemical- ionization117 may be used. of psilocin have been identified in urine after 7 days from oral Fluorescent psilocin derivative was detected by this ESI-MS administration).122 About 25% of the whole dose was shown to technique.89 It is possible to detect psilocin O-glucuronide be excreted unaltered in mice.122 Psilocin renal elimination directly without earlier hydrolysis using LC-ESI-MS-MS profiles were studied also in humans.125 The psilocin technique.104 rate within 2–4 hours postdrug administration was equal to More sophisticated detection system based on chemilu- 55.5 6 33.8 mg/h.125 Within 24 hours, only a small amount minescence measurements was successfully used for psilocin of the whole dose (mean 3.4%) was shown to be excreted as determination.69,99 free psilocin.125 In the same study, it was demonstrated that Ion mobility spectrometry may be an alternative after the treatment of urine samples with glucuronidase to mea- method for rapid psilocin analysis.95 sure the total psilocin concentration, the extent of psilocin Capillary zone electrophoresis was also used for detection excretion after 24 hours from administration increases only of psilocybin/psilocin and other indole compounds.55,97,118,119 A 2-fold (compared with free psilocin measurements). To explain new method was developed for the screening of urine samples for this, it was proposed that hydroxyl groups of psilocin metab- the presence of 19 drugs of abuse including psilocin.106 The olites are also conjugated to O-glucuronide and in this form are addition of b-cyclodextrin and organic solvents to the background excreted by kidneys.125 The glucuronidation is catalyzed by glu- electrolyte made possible selective separation of analytes.106 curonosyltransferases: microsomal originating from liver or intestinal tissues. After oral administration, psilocybin Biologic Activity of Psilocybin/Psilocin in converted to psilocin is a substrate to UGT1A10 expressed Mammalian Organisms by small intestine. After absorption within circulatory system the conversion of psilocin to glucuronide is mediated by Psilocybin/Psilocin UGT1A9.126 Other identified psilocin metabolites, namely Psilocybin introduced to the digestive tract is very quickly 4-hydroxy-3-yl-acetic acid 4HIAA, 4-hydroxy-3-yl- hydrolyzed. Dephosphorylation under acidic environment of 4HIA, and 4-hydroxytryptophol 4I-IT, are the products of its stomach or under activity of alkaline phosphatase in intestine and oxidation.114,122

TABLE 2. Pharmacokinetic Parameters of Psilocin Determined After Psilocybin Dose Administration

Psilocybin Dose Way of Administration cmax (ng/mL) tmax (min) t1/2 (min) Fabs (%) References 0.224 mg/kg (10–20 mg) Orally 8.2 105 163.3 52.7 114 1 mg Intravenous 12.9 1.9 74.1 — 114 0.2 mg/kg Orally 6–21 70–90 135 — 84

2013 Lippincott Williams & Wilkins 423 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

Psilocin Pharmacodynamics Psilocin as an alkaloid of the structure resembling molecular structure is able to bind and activate serotonin receptors. Chemical structures of psilocin, its precursor psilocybin, and serotonin are shown in Figure 1. Both physiologic and psychotic effects of psilocybin/psilocin intoxication result from its agonistic activity toward 5-hydroxytryptamine(5-HT) receptors located within or beyond the central . Psilocybin should be considered as a prodrug. Product of its dephosphorylation, namely psilocin, as relatively lipophilic substance easily crosses blood–brain barrier. Psilocybin/psilocin evoked effects are thought to be 127 mediated mainly by activation of 5-HT2A . In vivo studies in human subjects reveal that , 5-HT2A , prevents most of the psychotic symptoms usually developing after psilocybin/psilocin administration.47,128 However, some of the symptoms are not fully diminished and even intensify under combined psilocybin/ketanserin action (particularly cognitive deficits measured as difficulties in atten- tion focusing and “reduction in vigilance”).41,129 The conclusion fi FIGURE 2. Psilocybin/psilocin derivatives tested in papers by of the ndings is that system mediates psychotic Sard et al131 and Bray et al132 effects, but they result from both stimulation of 5-HT2A recep- tors in cortical regions and activation of inhibitory 5-HT1A receptors located in dorsal raphe nucleus, leading to a decrease N-dimethyltryptamine (Ki = 82 and 84 nM), despite relatively in tonic release of serotonin into prefrontal cortex. poor agonistic activity against 5-HT2C (EC50 =90nM),seemed From binding studies, it is known that psilocin as to be able to inhibit serotonin-induced pruritus.131 serotonin binds with high affinity to 5-HT2A (Ki = However, psilocin psychoactivity is mostly dependent 6 nM) and 5-HT2C (Ki = 10 nM) and with lower affinity to on ligation of 5-HT2A receptors and inhibitory 5-HT1A receptors. 127,130 5-HT1A receptor (Ki = 190 nM). Psilocybin and psilocin Although 5-HT2A receptors are expressed by excitatory glu- are known to inhibit some serotonin biologic activities depen- tamatergic pyramidal , the overall effect on neuronal dent on 5-HT2C ligation, for example, serotonin-induced pru- activity is thought to be inhibitory due to the presence of 133 ritus (after its topical administration). Similarly, N-methylated 5-HT2A receptors also on GABAergic . derivatives of psilocin and psilocybin (see Fig. 2), namely Triggered signaling pathways are different for various 1-methyl-psilocin and 1-methyl-psilocybin, exhibit comparable ligands of 5-HT2A receptor depending on -induced con- inhibitory action on serotonin activity.131 The 1-methylopsilocin formational changes. Normally, serotonin stimulates phospho- selectively binds 2 isoforms of 5-HT2C receptor (Ki = 7.0 and lipase C releasing 2 important signaling molecules 33 nM) and functionally activates the receptor (EC50 = 12 nM). 1,4,5-trisphosphate and diacylglycerol and leads to expression The experimental data are in agreement with binding energies of transcription factor c-fos in a manner dependent on Gq/11 calculated from the data originating from computational pre- .134–136 Subsequent elevation of leads to diction of the binding site structure in interaction with the stud- potassium channels closure and depolarization. Similarly, psilo- 132 ied derivatives. The N-methylated psilocybin (Ki = 540 or cybin as serotonin agonist by binding to 5-HT2A receptor stim- .10,000 nM), similar to psilocybin, inhibits serotonin-induced ulates hydrolysis of phosphatidylinositol 4,5-bisphosphate.137 scratching. The 1-butyl psilocin (EC50 = 595 nM, Ki =4.4or Stimulation of 5-HT2A receptor activates also phospholipase 14 nM) was inactive in the behavioral test. The 4-fluoro-N, A2 and psilocin, but not its precursor psilocybin, and induces arachidonic acid release.138 More detailed investigations show that hallucinogenic serotonin activate additional signal- ing pathways and induce transcription factors typically after activation of 5-HT1 receptor, although the receptor is not involved.136 It is also known that designated mGluR2 forms functional complex with 5-HT2A receptor and modulates the serotonin receptor coupling to Gi protein, what implicates connection with psychosis induction and explains why ligation with hallucinogenic molecules results in the acti- vation of specific signaling pathway.139 Activation of mGluR2 receptor breaks the signaling cascade and corrects symptoms of psychosis.139 FIGURE 1. Chemical structures of psilocybin, psilocin, and its Ex vivo studies on rat brain showed that psilocin affects endogenous analogue serotonin. hippocampal pyramidal cells. Application of fungal extracts

424 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol containing psilocin and psilocybin decreases electrical activity of after administration of lower doses only increases sensitiveness these neurons including activity stimulated by .140 to light, what should not be interpreted as psychotic perceptual 156 The activation of 5-HT1A receptor induces release of Gi/0 changes. However, subjects suffering from are protein subunits that activate potassium channel and inactivate more sensitive to action of hallucinogens and undergo psy- calcium channel leading to hyperpolarization and inhibition of chotic syndrome after intake of lower psilocybin doses.157 neuronal activity. Neurons expressing 5-HT1A receptors are Administration of higher doses (the amount of about 10–20 highly responsive to psilocin action. Indeed, direct application mg/70 kg, orally) leads to the elevation of plasma concentra- of psilocin to neurons of dorsal raphe nucleus decreases fre- tion of the drug up to the value of 5–12 ng/mL.114 Psychotic quency of neuronal firing, what suggests the involvement of syndrome starts to develop when psilocin plasma concentration 114 5-HT1A autoreceptor (its activation leads to serotonin decrease reaches the value of about 4–6ng/ml. Psychotic syndrome is in synaptic cleft).141 sometimes called schizophrenic phase.146 The symptoms of the According to one of the existing hypotheses, the mech- psychosis intensify during the next 30–60 minutes and start to anism of waking dream is involved in visual hallucinations decline after 2–3hours.13,50,114,150,151 Psilocybe semilanceata occurrence in course of psilocybin intoxication and in fruit bodies ingestion results in similar symptoms of intoxica- psychopathology of acute schizophrenic psychosis.142 The state tion including psychotic episode.158 It is estimated that the of waking dream is associated with low level of serotonin consumption of hallucinogenic mushrooms in the amount of in brain.143–145 During rapid eye movement phase, serotonergic 2 g (about 10 fruit bodies of P. semilanceata) corresponding to neurons of dorsal raphe nucleus become inactive and their 10–20 mg of psilocybin results in mild psychotic effect usually normal inhibitory activity on other brain areas transiently dis- experienced as a pleasure (so-called “good trip”). The con- appears. Similar phenomenon takes place under action of psi- sumption of 12 g (about 60 specimens) corresponding to locin possibly due to stimulation of 5-HT1A autoreceptors 60–120 mg of psilocybin is negatively experienced (as present at higher density than other subtypes of 5-HT receptors so-called “bad trip”).159 The psychosis subsides after 5–6hours on serotonergic neurons of .146,147 from its appearance. The psychotic symptoms include disor- Psilocybin administration decreases binding of radio- ders of visual perception such as illusions or hypnagogic labeled ligand of D2 receptors in . The experiences, alterations of time, color and motion perception effect results from a rise of endogenic dopamine release and (eg, kaleidoscopic vision), , usually positively ligation.48 It should be mentioned here that despite psilocybin experienced sense of derealization and depersonalization, does not show any affinity to dopamine receptor of D2 type, sometimes converting to drowsiness, anxiety, modulating interactions between serotonergic and dopaminer- impaired mood, dysphoria, or emotional state described as gic neuronal systems are known to exist. extreme fear (mood swings).15,46,48,128,151,153,160–162 Body (MAO) inhibitors are taken by image perception is affected—intoxicated subjects reported psilocin users to intensify its hallucinogenic effects. MAO is impression of swelling and numbness.158 It seems that psi- the catalyzing serotonin decomposition in synaptic locybin impairs cognitive functions: decreases reaction time, cleft. Because psilocin may be MAO substrate, its presence impairs ability to focus and sustain attention mainly by pro- causes inhibition of the enzyme. Brain serotonin is elevated ducing strong unusual emotions and visions difficult to and simultaneously 5-HIAA level decreases as a result of ignore, and thinking processes are disturbed by ego disor- MAO blockage.148 ders and delusions.41–50,128,129 Similar to schizophrenics sub- Early studies revealed that psilocybin elevates total jects to whom psilocybin was administered have difficulties level of ATP in .149 More detailed to use contextual information, what may be interpreted as findings applying positron emission tomography determined cognitive deficit.45 However, by lexical decision tasks, it is elevated glucose metabolic rate under action of psilocybin. possible to show that psilocybin-induced psychosis, similar Increased of some cortical regions (particularly in to schizophrenia-associated psychosis, predisposes to anterior cingulate) and decreased metabolic activity in thalamus semantic network spreading.50 Religious or sensual people were observed after psilocybin administration in humans.43,46 may experience mystical feelings.10–16 Personality character- ized with specific curiosity (openness to cognitive or per- Somatic and Psychotic Effects of Psilocybin/ ceptual experimentation, also by means of psychoactive Psilocin Intoxication substances) and sensibility to a beauty of nature or works Dose dependent increases of blood pressure and heart of art predisposes to pleasurable reactions (including these rate were observed in course of psilocybin/psilocin intoxica- of mystical type).163 At high doses, the schizophrenic phase tion.13,15,150–152 The autonomic effects are maximal within is followed by the -like phase because pleasur- 1–2 hours after drug administration.150 Psilocybin elicits neu- able effects are replaced with dysphoria, paranoid thinking, roendocrine effects similar to serotonin-induced changes in and negatively experienced loss of ego control.13,146,158 Low humans150 (see Table 3). age and emotional excitability before the drug intake addi- Intensity of psilocybin intoxication depends on a dose of tionally predispose to such negative responses.163 Accidental the drug. Psilocybin at a dose of 8–10 mg (0.1–0.2 mg/kg, deaths resulted from jumping out of a window after the orally) evokes only physiologic symptoms such as mydriasis, consumption of magic mushrooms were reported,8,55 what rarely hypotonic reaction associated with and vomiting, suggests unbearable emotional state leading to suicidal slightly increased blood pressure and accelerated heart fre- attemptorastateofwakingdream(flying is a common quency, and paresthesias.13,15,124,150–152,156 Pupillary dilation dream motif). The state of psychotic episode obtained after

2013 Lippincott Williams & Wilkins 425 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

TABLE 3. Dose-Dependent Psychological and Somatic Effects of Psilocybin Treatment Acute Psychological Effects (at the point of maximal drug effect, – Psilocybin Dose 50 150 min postdrug administration) Approximations Sensory of cmax Based Plasma Altered States of Perception, Cognitive 114 on Data from Concentration/cmax Consciousness Hallucinations Mood, Affective States Functions 3–5 mg/70 kg Approximately 2 ng/mL No increases in ASC scores No increase in VR Drowsiness, increased No alternations (0.045–0.071 mg/kg) score sensitivity 8–10 mg/70 kg Approxmiately 5 ng/mL OB,DED,VR,AA,RV, global Illusions, elementary and (0.115–0.143 mg/kg) ASC scores [, linearity complex between a dose and a mean hallucinations, score or number of synesthesias, sensory subjects, derealization, alternations, alterations depersonalization in perception of time phenomena and space, ASC VR [ 15 mg/70 kg Approxmiately 9 ng/mL AMRS scale (0.215 mg/kg) “general inactivation” [, “dreaminess” [, introversion [ 20 mg/70 kg Approximately 14 ng/mL Rating (0.315 mg/kg) Scale scores “somatesthesia,” “affect,” “perception,” “cognition,” “volition,” “intensity”[ 30 mg/70 kg Approxmiately 19 ng/mL Measurements done based on Mood lability: Not measured 90% of participants (0.429 mg/kg) questionnaire considering: harmony, reported sense of unity, happiness, grief, headaches; mean transcendence of time and joy, anxiety, duration space, sense of sacredness, dysphoria, fear, 16 h sense of objective reality, paranoid thinking, positive mood, ineffability, subscale DED[ paradoxicality, transiency; all ratings [ Approximately Femoral venous blood 220 ng/mL Possibly a state of waking Death case 55Accidental death case, 2 mg/kg, (free psilocin), 4600 ng/mL (total dream (flying is a common death resulted from 90 mg/45–50 kg psilocin); heart blood 60 ng/mL dream motif), euphoria? jumping out of (free psilocin), 170 ng/mL (total a window after flying psilocin); postmortem analysis attempt Psilocybin Dose Subacute Acute Somatic Effects, References, Study Approximations of cmax Psychological Long-Term Measured Usually 1–2h Participants, Based on Data from114 Effects Effects Postdrug Administration Exclusion Criteria 3–5 mg/70 kg Headaches No No changes in cardiac Arterial blood pressure 15,47,50,125,150–154 healthy, no personal or (0.045–0.071 mg/kg) electrophysiology, blood [, heart rate [; rarely family history of psychiatric pressure, and heart rate tend drop of blood disorders, no history of drug abuse, to increase pressure with usually had previous experiences dizziness, fainting, with hallucinogens or vomiting 8–10 mg/70 kg Working Fatigue Retrospective ratings: positive Arterial blood pressure (0.115–0.143 mg/kg) impairment headaches change to ASC—56%, in [, heart rate [; the relationship to the environment 38%, in aesthetic experiencing 37%, in the relationship to one’s body 30%, in awareness of personal problems 29%, in relationships to other 25%; concentration or memory problems were reported, complaints that required psychotherapy (rarely)

426 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

TABLE 3. (Continued) Dose-Dependent Psychological and Somatic Effects of Psilocybin Treatment Psilocybin Dose Subacute Acute Somatic Effects, References, Study Approximations of cmax Psychological Long-Term Measured Usually 1–2h Participants, Based on Data from114 Effects Effects Postdrug Administration Exclusion Criteria 15 mg/70 kg Emotional excitation [, Attention impairment Fatigue, headaches Arterial blood pressure [, heart rate [; (0.215 mg/kg) dreaminess [, ability Y prolactin [ heightened mood [, dazed state [, sensitivity [, concentration Y, tiredness [, introversion [ 20 mg/70 kg Emotional excitability [, Impairment of thinking AMRS scale Arterial blood pressure [, heart rate[; (0.315 mg/kg) anxiety, fear of loss of process, reaction time Y, “dreaminess” [, prolactin [ and out of the normal self control, paranoid semantic priming promoted melancholia, physiologic range, thyroid- thinking, labile mood, efficiency-activation stimulating hormone [, primitive Y, inactivation [, adrenocorticotropic hormone [, emotionality, introversion [, [, g-glutamyltransferase [, emotional blunting extroversion Y, aspartate aminotransferase [ fatigue, headaches 30 mg/70 kg Positive changes in Arterial blood pressure [; 10,11, 13,14, 15, 154 (0.429 mg/kg) mood, personal and, heart rate [ (by about 10 b/ participant were male social behavior min) Protestant theology students, hallucinogen naïve, double-blind study (only in theory) Approximately 2 mg/kg, 90 mg/45–50 kg

ASC, the Altered States of Consciousness Rating Scale 5D consists of the following subscales: oceanic boundlessness (OB; measures: positively experienced phenomena of derealization, depersonalization, and altered sense of time, elevated mood), dread of ego dissolution (DED; measures negatively sensed depersonalization and derealization, anxiety and cognitive deficits connected to ego disorders), visionary restructuralization (VR; measures phenomena of elementary and visual (pseudo-)hallucinations, synesthesias, factors influencing imagination), auditory alterations (AA; measures disorders in auditory perception and hallucinations), reduction of vigilance (RV; measures impairment of alertness and associated disorders of cognitive functions).155 AMRS, Altman Mania Rating Scale. even single psilocybin treatment may return particularly vasoconstrictor activity of serotonin agonists, the decreased under action of other psychedelic substances such as blood flow through small arteries or blood vessels supplying or marihuana (so-called “flashback” effect).8,151,164 to skin may lead to disturbances of circulation in lower Psychological and dose-dependent somatic effects of and upper endings, formation of venous ulcers, and difficulties psilocybin intoxication are shown in Table 3. Table 4 sum- in wound healing. The risk of abortion resulted from vaso- marizes pharmacodynamic findings regarding psychotic prop- constrictor activity of psilocin as serotonin agonist also exists. erties of psilocybin. Postdrug administration transient headaches were also reported.151,154,158 Subchronic intoxication observed during Psilocybin/Psilocin Side Effects and Toxicity 8 weeks of psilocin daily administration in rats leads to elevation The toxicity of psilocybin is low.165,166 For rodents, of level in plasma, what likely composes protective 166 (LD )equalsto285mg/kginmiceand mechanism against vasoconstrictor activity of psilocin. 50 – to 280 mg/kg in rats.165 Psilocybin administration results in A destruction of blood brain barrier under action of psi- 169 transient elevation of blood concentration of some biochemical locin is possible. Similarly, serotonin released in high – 170 markers such as aspartate aminotransferase aspartate transami- amounts from platelets may damage blood brain barrier. nase and g-glutamyltransferase gamma-glutamyltransferase.150 The literature of the subject describes 1 case of 33-year-old In humans, even high doses able to induce psychosis patient who developed a monoparesis of the left leg and bilat- (about 30 mg/70 kg, orally) only slightly and transiently eral anopia 2 weeks after the consumption of magic mushrooms affect heart rate and blood pressure without noticeable due to multifocal cerebral demyelination within the corpus cal- 169 influence on heart function.13,15,150,152 Arterial pressure increase losum of the right cerebral hemisphere and both optic nerves. partly results from the mechanism controlling circulatory sys- tem involving central activation of 5-HT2 and 5-HT1A recep- Potential Therapeutic Psilocybin/ tors. In addition, psilocin may express ligand activity toward Psilocin Applications 5-HT2A receptors placed on the surface of smooth muscle Since the beginning of 1960s of XX century, the cells leading to vasoconstriction. Disturbances of blood clotting therapeutic potential of psilocybin has been a subject of are possible.167 Rare cardiomyopathies have been reported: scientific interest, particularly in psychiatry and psychotherapy. usually single cases of supraventricular , myocar- Harmfulness of the habit of psilocybin-containing mushrooms dial infarction,56 and Takotsubo cardiomyopathy.168 Due to consumption, including all aspects of human health and risk for

2013 Lippincott Williams & Wilkins 427 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

TABLE 4. Pharmacodynamic Findings Versus Dose-Dependent Psychological Effects of Psilocybin Treatment Acute Psychological Effects (at the point of maximal drug effect, 50–150 min postdrug Psilocybin Dose cmax administration) Pharmacodynamic Effects Approximations of cmax Based on Data From114 Altered States of Consciousness Sensory Perception, Hallucinations Mood, Affective States 0.25 mg/kg + placebo Approxmiately 11 ng/mL Increase in OB, VR and DED scores (about Increase in reaction time [working memory 45% placebo conditions) deficits measured with visual manual delayed response task (DRT)] 0.25 mg/kg+ 20 mg Increases in OB, VR, and DED Reduction of increase in reaction time ketanserin (5-HT2A scores (about 20% vs placebo antagonist) conditions) 0.25 mg/kg+ 40 mg No increases in OB, VR, and No increase in reaction time ketanserin (5-HT2A DED scores (vs placebo antagonist) conditions) 0.25 mg/kg+ 0.021 mg Increase in OB score (30%), Increase in reaction time (D2 increase in VR scores (40%); antagonist) increase in DED score (65% vs placebo conditions) 0.25 mg/kg+ 0.5 mg Increases in OB, VR, and DED Reduction of increase in reaction time (mixed 5- scores (about 10%–20% vs HT2 and D2 placebo conditions) antagonist) 0.25 mg/kg+ 1 mg No increases in OB, VR, and No increase in reaction time risperidone (mixed 5- DED scores (vs placebo HT2 and D2 conditions) antagonist) 0.25 mg/kg Approxmiately 11 ng/mL Derealization, loss of ego boundaries; OB [, Sensory perception alternations, illusions, VR [, DED [ complex scenery hallucinations, VR score [ 0.215 mg/kg Appr. 9 ng/mL Increases in 5D-ASC scores: OB [ (40%); RV [ (30%); attentional tracking reduced, DED [ (20%); VR [ (50%); AA [ (15%); spatial working memory not affected RV [ (30%) 0.215 mg/kg+ 50 mg Increases in 5D-ASC scores: VR [ (15%); RV RV [ (50%); attentional tracking reduced, ketanserin (5-HT2A [ (50%) spatial working memory not affected antagonist) 0.115 mg/kg Approxmiately 5 ng/mL Increases in 5D-ASC scores: OB [ (10%); VR Increase in VR score [ (15%). Binocular [ (15%) rivalry switch rate Y, phase duration [ (from 2 s to about 2.7 s comparing to placebo conditions, measurement done 90 min after drug administration) 0.25 mg/kg Approxmiately 11 ng/mL Increases in 5D-ASC scores: OB [ (30%); Increase in VR score [ (40%). Binocular DED [ (20%); VR [ (40%); AA [ (15%); rivalry switch rate Y, phase duration [ (from RV [ (25%) 2 s to about 3 s comparing to placebo conditions, measurement done 90 min after drug administration) 0.25 mg/kg Approxmiately 11 ng/mL Increases in 5D-ASC scores: OB [(40%); Binocular rivalry switch rate Y, phase duration DED (20%) [; VR (50%)[; RV(30%) [ [ 0.25 mg/kg+ 50 mg Increase in 5D-ASC score RV [ (50%) Binocular rivalry switch rate Y, phase duration ketanserin (5-HT2A [ antagonist) 0.28 mg/kg Approxmiately 12 ng/mL Brief Psychiatric Rating Scale total score [. Deficits in the AX-Continuous Performance psychosis factor (conceptual disorganization, Test (measure of visual context-dependent suspiciousness, hallucinatory behavior, information processing), correct detection of unusual thought content) [. activation AX sequences Y, incorrect detection of BX factor (tension, mannerism and posturing, sequences Y excitement) [. anxiety/depression factor (somatic concern, anxiety, guilt feelings, depressive mood) [. anergia factor (emotional withdrawal, motor retardation, blunted affect)[. hostility factor (hostility, suspiciousness, uncooperativeness) [ 0.2 mg/kg + activation Approxmiately 9 ng/mL Not measured Difficulties in attention focusing, subjects found task (word association) the activation task very difficult; number of associated and spoken words Y

428 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

TABLE 4. (Continued) Pharmacodynamic Findings Versus Dose-Dependent Psychological Effects of Psilocybin Treatment Acute Psychological Effects (at the point of maximal drug effect, 50–150 min postdrug Psilocybin Dose cmax administration) Pharmacodynamic Effects Approximations of cmax Based on Data From114 Altered States of Consciousness Sensory Perception, Hallucinations Mood, Affective States 15 mg for subjects ,50 Approxmiately 19 ng/mL Derealization, depersonalization phenomena, Alternations in the perception of time and kg, 20 mg for subjects los of ego boundaries 3D-ASC scores [ space, elementary and complex .50 kg + + hallucinations, synesthesias D- Psilocybin Dose References Approximations of cmax Based on Data From114 Cognitive Functions 0.25 mg/kg + placebo Psilocybin-induced cognitive deficits and 47 psychotic symptoms are dependent on activation 5-HT2A receptors 0.25 mg/kg+ 20 mg ketanserin (5-HT2A antagonist) 0.25 mg/kg+ 40 mg ketanserin (5-HT2A antagonist) 0.25 mg/kg+ 0.021 mg haloperidol (D2 antagonist) 0.25 mg/kg+ 0.5 mg risperidone (mixed 5- HT2 and D2 antagonist) 0.25 mg/kg+ 1 mg risperidone (mixed 5- HT2 and D2 antagonist) 0.25 mg/kg Emotional disturbances, euphoria, anxiety, 4 Difficulties in thinking, attention Decreased [11C] raclopride receptor binding 48 subjects had high ratings in OB score, 3 focusing potential bilaterally in the striatum subjects had high ratings in DED score interpreted as an increase in endogenic dopamine release and binding 0.215 mg/kg Psilocybin-induced activation of 5-HT2A 128 receptors (leading to increased cortical stimulation) and activation of 5-HT1A receptors in raphe nucleus (leading to a decrease in tonic inhibitory serotonin release into prefrontal cortex), both effects impaired attention ability; activation of 5- HT1A receptors was responsible for the effect of reduced vigilance 0.215 mg/kg+ 50 mg ketanserin (5-HT2A antagonist) 0.115 mg/kg Psilocybin-induced activation of 5-HT1A 41 receptors affects brainstem oscillator functioning 0.25 mg/kg 0.25 mg/kg 129 0.25 mg/kg+ 50 mg ketanserin (5-HT2A antagonist)

(continued on next page)

2013 Lippincott Williams & Wilkins 429 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

TABLE 4. (Continued) Pharmacodynamic Findings Versus Dose-Dependent Psychological Effects of Psilocybin Treatment Psilocybin Dose References Approximations of cmax Based on Data From114 Cognitive Functions 0.28 mg/kg Psilocybin psychotic action as 5-HT2A 45 agonist is associated with context- dependent information processing, similar effect was observed in schizophrenics and in ketamine (NMDA antagonist) treated subjects, support for the hypothesis of disruption as a mechanism underlying psilocybin effects 0.2 mg/kg + activation Glucose metabolic rates: in right 43 task (word frontotemporal cortical regions [, association) particularly in the anterior cingulate [,in the thalamus Y 15 mg for subjects ,50 Emotional lability, inability to control Difficulties in attention focusing, Glucose metabolic rates: global [ and in 46 kg, 20 mg for subjects emotions, euphoria, dysphoria, paranoid thinking process disturbed cortical regions [, subcortical regions [ .50 kg + ketamine + thinking with derealization and (although less stimulated); The increases D-amphetamine depersonalization phenomena noted in prefrontal cortex, anterior cingulate, temporomedial cortex and correlated with psychotic syndromes, ego pathologies in particular public health and safety, was recently estimated as very of personal satisfaction resulted from strong engagement in low.8 However, anyone interested in the usage of hallucino- absorbing scientificstudyconfirming positive significance gens for psychotherapy support should remember that seri- of religion and/or mystical experiences toward those they ously suffering patients would respond poorly to psilocybin were well disposed. and the drug could further negatively affect their mental Moreover, the study was designed in such a way that health because it is able to produce schizophrenia mimicking the participants were constantly monitored during drug session psychosis.41–50 Psilocybin at high doses (20–30 mg/70 kg) and also had an opportunity to talk with the observer. was shown to induce intense positive mystical-type Therefore, when answering mystical scale questionnaire, they experiences in healthy volunteers.10–16 Participants of the could be prompted by a suggestion, dishonest, or expressed mentioned studies on psilocybin-assisted psychotherapy metaphoric ideas (eg, having in memory and after the self-reported improvements of quality of life after the drug conceptual opposition death/resurrection). Communities con- session.10–16 The results of the psychological experiments glomerated around religious cult are usually oppressive and indicate positive influence of psilocybin-induced experien- their members are trained not only in conformity but also in ces on quality of social life including mood improvement cruelty by means of more or less sophisticated dangerous and inclination toward more altruistic social behaviors. psychosocial techniques. Therefore, religiously inclined However, the acquired data mostly originating from ques- individuals, particularly if well educated, may mistake, tionnaire measurements (mystical scale questionnaire, eg) intentionally or unknowingly, even basic emotions such as can be questioned taking into account the known, usually fear and pleasure. Moreover, religiously inclined people unpleasant physiologically and emotionally, symptoms of belonging to religious community are instructed to see any high-dose psilocybin intoxication. It is surprising that most part of their emotional life as something positive and precious of the participants confirmed mood improvement (regarded depending on intensity. It may lead to a kind rivalry, as an essential component of mystical experience) under excessive emotional expression or exaltation, progressive high-dose psilocybin action. One explanation could be that incuriosity (emotional devaluation similar to drug ) they completed questionnaires 7 hours after drug ingestion and finally result in the discovery of being manipulated or (the whole session lasted 8 hours). After that time, all forced for certain activities by cruel psychosocial or, like in unpleasant effects of intoxication had already subsided. this case, pharmacologic methods. Such a discovery is never The study by Griffiths et al13 was carefully designed to avoid satisfying (except for conformistic attitude) and may ruin expectancy effects as much as possible; however, the par- system of values making impossible social adaptation, what ticipants were recruited through announcements mentioning takes place, for example, in course of schizophrenia. Indeed, that the planned study aims to assess activity of “psychoac- about 40% of participants of the mentioned study experienced tive substance used sacramentally in some cultures.” All fear or paranoid thinking during drug session.13 Paranoid study participants declared positive attitude toward religion thinking included an impression of being manipulated or trea- and most of them were members of religious communities. ted malevolently—some of the participants decided it is an Postdrug session mood improvements reported by partici- occasion to inform about details on their private life.13 Look- pants of the experiment may be, at least partly, the effect ing for intense, extraordinary emotions, preferably lacking

430 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol any connection with reality (escapism) is common to drug IBOTENIC ACID AND MUSCIMOL users and alcoholics. Their psychological profile is often characterized by emotional blunting—in both cases antiso- Ibotenic Acid and Muscimol as cial personality and aggression are more likely to develop in Secondary Metabolites course of addiction than altruistic attitude. Similarly, as Major active substances of and theatralization of speech and behavior or myth-like confab- A. pantherina are ibotenic acid and muscimol—the products ulations (directly under action of alcohol or due to memory of the fungus secondary metabolism, thought to repel insects disorders in course of progressing the Korsakoff syndrome) in and slay their larvae. These 2 species are obligatory ectomy- alcoholics, behavioral and mental changes developing under corrhizal and grow under and coniferous trees, espe- action of fungal hallucinogens may be directed against others, cially , , and , in various types of woods. The particularly if domination becomes the main purpose of the drug mushrooms are cosmopolitan and occur wild in many conti- – user. Religious context promotes reproduction of the behavioral nents (Europe, Africa, Asia, Australia, and America).62,178 180 pattern. Therefore, expecting positive influence of psilocybin Muscimol is a product of ibotenic acid . treatment on quality of social life is simply ingenuousness. Both substances are polar and well soluble in water. Bio- The authors of the mentioned studies remark that fear and para- synthetic pathway of ibotenic acid and muscimol includes noia developing under psilocybin action could be dangerous for conversions of their precursor molecule b-hydroxyglutamic intoxicated subjects.13 Moreover, they take into account possi- acid (ring closure and decarboxylation).180 bility of long-term psychological effects of such experiences According to some authors, the custom of the cuticule and try to measure them. Indeed, personality changes in adults peeling off before mushrooms consumption aims to avoid after psilocybin single (!) treatment were demonstrated: partic- the highest content of these psychoactive substances in the ularly examined subjects became more open-minded.16 Such red cuticle and a yellowish flesh just under it.180 One of the personality change should not be univocally interpreted as pos- pigmentary compounds is muscaaurin I (chemical structure itive—it may reflect serious and dangerous ego disorders. shown in Fig. 3), being an aldimine derivative of ibotenic and The therapeutic potential of psilocin and psilocybin was betalamic .181,182 Apart from the poisonous properties, to also tested in therapy of habitual criminals. One study (although both and , the pigment shows antiox- critically reevaluated) demonstrated a short-term therapeutic idative potential and possibly protects against UV radiation. effect observed as a decreased frequency of relapse into Quantitative analysis does not support the statement crime.171,172 There is also an increasing interest in therapy of about higher content of ibotenic acid in the cuticle than in the anxiety neurosis, depression, and physical pain in patients with flesh. Tsujikawa et al183 examined samples of A. muscaria advanced-stage cancer by the use of psilocybin.36–38 Both these and A. pantherina and found that compounds tend examples of application are highly controversial and possibly to be more concentrated in the flesh than in the red cuticle. nonethical. Ibotenic acid contents ranged from 10 to 2845 ppm in the Psilocybin seemed to be effective in therapy of obses- cap of A. muscaria and from 188 to 269 ppm in the cap of sive-compulsive disorder. In standard therapy, selective sero- A. pantherina.183 Muscimol was found in concentrations of tonin inhibitors are applied, but the results of such 46–1052 ppm in the cap of A. muscaria and 1554–1880 ppm treatment are often unsatisfactory. Single cases supporting the in the cap of A. pantherina.183 Another analysis of A. musca- usefulness of the hallucinogen in correction of obsessive- ria fruit bodies184 revealed similar ranges of concentration compulsive disorder (OCD) symptoms can be found in the (for ibotenic acid, 182–1839 ppm in the cap and 627–1998 literature.131,173 Moreno and Delgado174 tested effectiveness of ppm in the stem and for muscimol, 46–1203 ppm in the cap psilocybin in OCD therapy. The study participants have been and 82–292 ppm in the stem). Tsunoda et al185 found the delivered 4 different psilocybin doses (the highest dose of ibotenic content ranging from 258 to 471 ppm and the mus- psychedelic action). Therapy was effective for all examined cimol content ranging from 18 to 27 ppm in A. muscaria patients. Similarly, marked effectiveness of psilocybin was samples. Størmer et al186 analyzed spores and fresh caps of observed in animal models of OCD.131,175,176 fly for the presence of ibotenic acid and they found the Psilocybin, psilocin, or its derivatives may be helpful in contents to be equal to 0.0054% and 0.0017%, respectively.186 therapy of neurologic or psychiatric diseases associated with pruritus. Serotonin and 5-HT2C agonists after topical cutaneous Insecticidal Activity of Ibotenic Acid application induce pruritus. In animal studies, the scratching The extract of A. muscaria fruit body is thought to be reaction in response to serotonin subcutaneous injection is poisonous or overwhelming for flies. Glutamate and ibotenic sometimes treated as an animal model of obsessive-compulsive acid are excitatory and inhibitory in insects disorder. The behavioral change is not observed when 5-HT2C due to the exclusive presence of glutamate-gated chloride receptors have been blocked. It was established that adminis- channels in invertebrates.187 Chloride channel activated by tration of psilocybin or to less extent psilocin diminishes the glutamate and ibotenic acid has been also identified in itching effect in mice.131 melanogaster.188 By the measurement of hyper- Cluster headache, condition of unknown cause occur- polarizing response, the presence of an inhibitory glutamate ring periodically, is often resistant to therapy. Interestingly, receptor was demonstrated on the cell body membrane of psilocybin treatment results are promising. Examined patients a cockroach motor .189 Inhibitory activity of ibotenic treated with the drug-reported pain attack interruption and acid and other structurally similar compounds were shown on remission time extension.177 snail periodically oscillating neuron.190 Also insect muscle

2013 Lippincott Williams & Wilkins 431 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

decarboxylation of ibotenic acid is significantly accelerated in 3 197 a mixture of dimethyl sulfoxide and H2OorD2O. Ibotenic acid and muscimol were determined in fresh, frozen, or freeze-dried mushrooms.183–185 The following sol- vents are usually applied for extraction of ibotenic acid and muscimol:75% methanol in water or 50%–70% ethanol in water.183–185,198,199 For briefly described methods of extraction used for ibotenic acid/muscimol analysis, see also Table 5. Sometimes extraction to water acidified with formic acid is applied.200,201 It is thought that decarboxylation of ibotenic acid to muscimol takes place under these conditions.200 How- ever, ibotenic acid was detected in water extracts containing formic acid.186 Methanol or ethanol extracts contain numerous impurities that are possible to eliminate by several step extraction with nonpolar solvents. Parapharmaceuticals, by producer declared to contain dried A. muscaria fruit bodies, were analyzed for the presence of ibotenic acid and muscimol applying modified the Stas- Otto extraction method commonly used for detection of psy- 183 FIGURE 3. Active substances of Amanita muscaria ibotenic, choactive alkaloids. However, it seemed that the method muscimol, their endogenous analogues glutamic acid and was not sufficiently sensitive to detect A. muscaria g-aminobutyric acid (GABA) and a pigmentary component in the examined material. muscaaurin I. Isoxazole derivatives ( in case of muscimol and carbamate and ethyl ester in case of ibotenic acid) were extracted to dichloromethane.200 Dansylated and ethyl ester fibers are responsible for ibotenic acid action. Ibotenic acid derivative of ibotenic acid were separated from other compo- stimulates chloride channels distributed on the nonsynaptic nents of the water–methanol extract by extraction of evapo- membrane of the coxal adductor muscle fibers but has no rated residue with ethyl acetate.184 affinity for glutamate receptors located on the excitatory post- synaptic membrane.191,192 Inhibitory glutamate-gated chloride Methods of Qualitative Analysis and Isolation channels have been detected within plasma membranes of Qualitative identification of ibotenic acid in water or cockroach corpus allatum—densely innervated endocrine water–ethanol extracts of pulverized A. muscaria fruit bodies gland synthesizing juvenile hormone. Juvenile hormone con- is difficult due to the presence of interfering con- trols processes of insect metamorphosis, vitellogenesis, and taminations. Fluorescamine or more often ninhydrin were reproduction. Ibotenic acid was shown to be able to activate used for TLC detection.185,198,202 Detection is easier when the channels what resulted in plasma membrane hyperpolar- bidirectional TLC is carried out in 2 different solvent sys- ization. Stimulation of these inhibitory receptors is followed tems.198 Preparative paper chromatography of extracts previ- by reduction of juvenile hormone synthesis.193 Stimulation of ously partly purified using ion-exchange chromatography was excitatory glutamate receptors, which are also present on the proven to be an effective method of ibotenic acid purification.199 surface of plasma membranes of corpus allatum, results in Amino acid contaminants may be eliminated by ion- antagonistic effect on juvenile hormone production.194,195 exchange chromatography using cation- and anion-exchange Flies of Drosophila genus that normally feed on fly sorbents.185,198–200 Crystallization step is necessary for satis- agaric fruit bodies seem to be resistant to fungal . factory purification of ibotenic acid.185 Ibotenic acid and to lower extent muscimol are toxic to larvae of carpophagous species (D. melanogaster and D. immigrans).196 Analytical Methods of Quantitative Determination Reversed-phase chromatography is the most often used HPLC technique to detect ibotenic acid and/or musci- Analytical Methods Used for Ibotenic 185,186,203 Acid/Muscimol Detection and Isolation mol. Reversed-phase HPLC may be combined with ion-pair chromatography.186 Dansylated analytes were also Methods of Extraction separated using HPLC method on octadecyl column.184 Separa- Muscimol is a product of ibotenic acid decarboxylation. tion applying LiChrosorb-NH2 and Nucleosil 5 CN columns was Pure ibotenic acid is a crystal colorless substance (melting also effective for muscimol and ibotenic acid determination.204 point of 150–1528C) readily soluble in water. Similarly, mus- UV absorption–based detection system was used for cimol with the melting point of 1758C, although more hydro- dansylared derivatives quantification.184 For more detailed phobic, is well soluble in water.180 Curiously, pure ibotenic identification, eluent from diode detector was analyzed with acid is extremely unstable in solution and rapidly decomposes mass spectrometer.184 GC–MS technique was used for detec- to muscimol without involvement of enzymes.197 The addition tion of carbamate and ethyl ester derivatives of both isoxa- of dimethyl sulfoxide to the water solution containing ibotenic soles in urine samples.200 Trimethylsilylation at 808C acid reduces its stability. It was established that spontaneous for 30 minutes with N,O-Bis(trimethylsilyl)trifluoroacetamide

432 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

TABLE 5. Exemplary Methods of Extraction Used for Ibotenic Acid/Muscimol Determination Solvent Extraction Time Dry Material/Solvent Ratio mg/mL Extraction Method References 75% methanol ? 2.5 g/70 mL Homogenization 185 Mixture of methanol: water 7:3 Shaking for 1 min, 50 mg/2 · 2 mL Shaking, sonication 184 sonication for 5 min 50% ethanol 1 h 500–2000 mg/100 mL Grinding and shaking 198 0.1% formic acid in water 10 min Spores: 5 mg/mL, fresh fruit bodies Freezing/thawing, sonication, 186 were suspended with acidified homogenization in liquid N2 water (in amount of twice their weight) results in conversion of isoxazoles to IBO-tri-TMS and MUS- muscimol was often thought to be mostly unmetabolized.3 di-TMS—both are possible to detect applying GC–MS.183 Indeed, both active compounds pass through organism LC/MS/MS technique was used for detection of dansylated quickly and partly unmetabolized. Ibotenic acid is in vivo muscimol and dansylated ibotenic acid ethyl ester.184 LC-MS metabolized to muscimol.213 The isoxazoles are excreted by was also used for direct (without derivatization) detection of kidneys. Both ibotenic acid and muscimol were detected in ibotenic acid in water extracts originating from fungal mate- urine collected 3–8 hours after ingestion of poisonous mush- rial186 and in urine samples.205 rooms in humans (similar concentrations were determined despite different symptoms and stages of intoxication).200 Biologic Activity of Ibotenic Acid and Muscimol in Mammalian Organisms Ibotenic Acid/Muscimol Pharmacodynamics Chemical structure of both isoxasoles closely resambles Ibotenic Acid/Muscimol Pharmacokinetics glutamic acid and a product of its enzymatic decarboxylation, A minimal dose to obtain symptoms of central nervous namely g-aminobutyric acid GABA (see Fig. 3). Structural system intoxication estimated for muscimol is 6 mg. In case similarity of ibotenic acid and muscimol to glutamic acid and of much less active but more dangerous ibotenic acid, the g-aminobutyric acid determinates their ability to bind and amount of 30–60 mg is sufficient for the psychedelic effect. activate receptors of the endogenous neurotransmitters. In contrast to g-aminobutyric acid, muscimol is able to Muscimol is an agonist of GABAA and cross blood–brain barrier possibly by the mechanism of active of GABAC receptors but evokes its activity mainly through 206–208 transport or due to muscimol or its metabolite/derivative/ GABAA receptors ligation. From radioligand binding study 209 adduct sufficiently high lipid membrane . Early using bovine brain GABAA receptors reconstituted in lipid studies resulted in conflicting data regarding muscimol distri- vesicles, it is known that muscimol binds to both high-affinity bution within brain tissue and questioned the a priori accepted (Kd = 10 nM) and low-affinity (Kd =0.27mM) sites and func- 214 theory of gamma-aminobutyric acid (GABA) receptor popula- tionally activates the receptor (EC50 =0.2mM). Wild-type rat 209 tion as the main binding site of muscimol. Muscimol was GABAA receptor subtype a1b2g2 expressed in cell culture binds 3 215 shown to be taken up by slices of cortex rat brain and the [ H]muscimol with Kd =21–24 nM. Kinetic parameters of uptake was likely dependent on amino acid transport system muscimol as GABAA agonist were calculated also from func- 206 3 localized in nerve terminals. The [ H]muscimol binding tional assays (apparent affinity KH = 10.9 mM, unbinding rate 216 studies carried out using crude synaptic membrane preparations koff = 40 /s; microscopic affinity constant Kn = 4.3 nM). of the rat brain revealed that GABA receptor is the main mus- Behavioral changes induced by muscimol in mice (, cimol binding site and that the binding was in vitro modulated ) depend on its high-affinity binding in 210 with . Muscimol high-affinity binding in forebrain and to a distinct subtype of GABAA receptors regions such as caudate–putamen, thalamus, and hippocampus containing a6 subunit and lacking a1 subunit.211 seemed to be dependent on distinct population of GABAA Additionally, muscimol inhibits GABA uptake by neu- receptors possibly containing a6subunitandlackinga1sub- rons and and, as already mentioned, is a substrate to unit as shown in knockout study in mice.211 the GABA-metabolizing enzyme, namely GABA transami- The fast conversion of radiolabeled muscimol to other nase.201,212,217,218 Chemical structure of muscimol was used as metabolites could be inhibited by pretreatment with an a basis for the design of GABA uptake inhibitors and GABA inhibitor of GABA-a-oxoglutarate .212 It has agonists.218 been also established with enzyme kinetics studies that mus- Ibotenic acid exhibits agonistic activity against N-Methyl- cimol is indeed an amine donor substrate to GABA transam- D- (NMDA) receptors and trans-1-Amino-1,3- inase.201 Another already identified metabolite of the fly dicarboxycyclopentane receptors (metabotropic quisqualate agaric isoxazoles is tricholomic acid shown to be active in Qm receptors). However, neurotoxicity of ibotenic acid invertebrates as agent able to inhibit neurons.180,190 Tricholo- results only from activation of NMDA receptors.219 Activa- mic acid and other unknown ibotenic acid/muscimol metab- tion of metabotropic Qm receptors followed by cellular signal olites may contribute to their activity.209 cascade initiated by phosphoinositide hydrolysis is not Because in some cultures drinking urine originating involved in ibotenic acid–induced excitotoxicity.219 In vitro from intoxicated subjects was practiced for ritual purposes, experiments revealed that ibotenic acid in contrast to kainic

2013 Lippincott Williams & Wilkins 433 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013 acid does not suppress glutamate ligation and exhibits low diminished with (potent GABA antagonist).233 220 affinity for sites binding radiolabeled . Therefore, In vitro experiments revealed that activation of GABAA it is concluded that ibotenic acid is not a ligand of kainate receptors after ligation of muscimol induces presynaptic receptors. and postsynaptic suppression of synaptic transmission It is postulated that ibotenic acid and muscimol are the between muscle afferents and spinal cord motor neurons.234 main compounds responsible for hallucinogenic properties of Muscimol applied microiontophoretically decreased firing fly agaric and the mechanism of their toxicity results from rate of cortical neurons.235 ligation of glutamate and GABA receptors, respectively. Ibotenic acid is able to excite spinal interneurons and High-dose muscimol treatment potentiates psychosis in Renshaw cells as demonstrated in anaesthetized cats.236 The schizophrenics. The subjects to whom muscimol was admin- study reveled that excitatory activity of ibotenic acid is istered reported the experience of vivid dreams.221 However, approximately 8 times higher than glutamate activity. How- the studies on the involvement of pedunculopontine tegmen- ever, after the recovery of excitation, the neurons became tal nucleus in regulation of paradoxical sleep seem to negate insensitive to the excitatory agents. The effect was abolished hallucinogenic properties of GABA and glutamate agonists. by bicuculline suggesting involvement of GABAA receptors. Pedunculopontine tegmental nucleus is one of the brain areas Therefore, it was concluded that ibotenic acid is in vivo involved in the process of paradoxical sleep appearance and decarboxylated to muscimol.236 Ibotenic acid (16 mg/kg, various paradoxical sleep–associated phenomena. Injections intraperitoneally) increases brain monoamines in rodents.225 of glutamic acid and muscimol into pedunculopontine teg- mental nucleus induce suppression of one of the paradoxical Somatic and Psychotic Effects Resulted From sleep hallmarks, namely hipoccampal theta rhythm in rats.222 Intoxication With A. muscaria and A.a pantherina However, it was also shown that muscimol locally adminis- First symptoms of fly agaric intoxication apparent within tered to dorsal raphe nucleus increases REM sleep.223 The 15–30 minutes after ingestion are signs of muscarinic poisoning influence of both on sleep architecture is likely the such as nausea, vomiting, diarrhea, vasodilatation, sweating, and result of complex GABA and glutamate-dependent mecha- salivation.3,58,207 Then, after about 30 minutes from oral intake, nisms modulating activity of cholinergic neurons within -like symptoms seem such as mydriasis, xerostomia, pedunculopontine tegmental nucleus and serotonergic neu- body temperature elevation, slightly increased blood pressure, rons of dorsal raphe nucleus.145,201,224 drowsiness, amentia, dizziness, hypersensitivity to light, eupho- Animal studies demonstrate that due to its inhibitory ria, motor hyperactivity, hallucinations, and .3,58,59,207 properties, muscimol reveals complex neuromodulatory activ- Mystical-type experiences were reported.59,207 The atropine-like ity. For example, muscimol (3 mg/kg, intraperitoneally) symptoms intensify during the next 2–3 hours. evokes serotonin rise and decreases catecholamine levels in turns into deep sleep—the state is accompanied by skeletal brain.225 Muscimol applied locally was shown to decrease muscles atony and tendon reflexes debility—the last symptoms stratial GABA release in rats.226 The study by Naik et al227 may suggest stroke.207 Similar spectrum of intoxication (hallu- revealed slight increase of the main catecholamine metabolite, cinations, muscle twitching, delirium, sleep) has been observed homovanilic acid, in striatum after muscimol administration. in human volunteers after pure ibotenic acid and muscimol Because muscimol alone decreases motor activity and musci- administration.237 Poisoning signs decline after about mol coadministered with drugs such as prevents 8hours.3,58,59,207,237 Somatic and psychotic effects observed in drug-induced motor hyperactivity dependent on dopamine course of intoxication with poisonous mushrooms or pure mus- neurotransmission, it is assumed that the isoxazole modulates cimol and ibotenic acid are summarized in Table 6. dopamine release.209 Within 30 minutes after intravenous injection of crude Muscimol, dose dependently, affects encephalogram in extract of A. muscaria changes in biochemical parameters have experimental animals distinctly from other typical hallucino- been observed in rats including decrease of ester- gens such as lysergic acid diethylamide (LSD) and mesca- ase activity, decline of liver glycogen, decline of blood line.228 Particularly, electroencephalogram pattern elicited by , and increase of blood glucose. No changes of serum muscimol consists of spikes, what characterizes substances of transaminase activity have been observed. After 6 hours, the convulsant activity.228 parameters come back to the line.242 The intravenous administration of muscimol reduces glucose utilization throughout the central nervous system.229,230 Side Effects and Toxicity of Ibotenic Acid/Muscimol However, the pattern of the effect was shown to be different Muscle twitching, spasms, cramps, and even from the known brain distribution of GABAergic neurons are frequent symptoms of fly agaric intoxication, particularly and GABA receptors.229 Another study revealed correlation in children.221,237,243 Systemic or local administration of pure between a decrease of cerebral glucose utilization and a bound ibotenic acid results in convulsions as established also in 244,245 fraction of muscimol (binding dependent on GABAA recep- animal studies. tors).231 Decreased brain metabolism was associated with Headaches and fatigue the next day after ingestion were decreased cerebral blood flow.230 Muscimol lowers cyclic gua- reported. In case of ibotenic acid intoxication, headache may nosine monophosphate level in also in case of its be prolonged up to a few weeks.237 232 elevation due to promoting convulsions activity of . Lethal dose LD50 of muscimol determined for rats Muscimol administered topically inhibits activity of is equal to 4.5 mg/kg after intravenous injection and spinal neurons in anaesthetized cats and the effect is 45 mg/kg after oral administration. The LD50 established

434 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

TABLE 6. Somatic and Psychotic Symptoms of Intoxication With Amanita muscaria or —Mushrooms Containing Ibotenic Acid and Muscimol. Therapeutic and Side Effects of Muscimol Somatic Symptoms of Intoxication, Therapeutic, and Long-Term Side Dose Side Effects Psychotic Symptoms of Intoxication Effects References Minimal effective dose of muscimol: 6 mg; minimal effective dose of ibotenic acid: 30–60 mg; one fruit body of A. muscaria (50–70 g) may contain up to 70 mg of ibotenic acid 6 fruit bodies of A. muscaria were Abdominal pain, nausea Visual and auditory hallucinations; 18- Fatigue, stomach 58 Case report eaten by 5 adults year-old girl lost consciousness complaints the next day 5 caps of A. pantherina were eaten by 2 h after ingestion: nausea, 2.5 h after ingestion: mania-like Disordered body 59 Case report 2 adults stomach-ache, diarrhea, stimulation, agitation, mystical perception, vomiting; transiently experiences, 4–5 h after ingestion difficulties to speak increased blood pressure the patient became more anxious, properly had difficulties to speak properly, reported , paresthesias of left arm A 72-year-old patient ate a few fruit Diarrhea, vomiting Neurologic disorders; hallucinations, Within 10 h after 207 Case report bodies of A. pantherina , skeletal muscle flassidity, ingestion neurologic hyporeflexia, facial assymetry symptoms subsided A. pantherina poisoning case Vomiting, urine analysis (4 h Hallucinations, the patient had No long-term effects 202 Case report postinjection): ibotenic acid difficulties in breathing 47.4 mg/mL, muscimol 9.9 mg/mL A. pantherina poisoning case Normal blood pressure, regular Dizziness, the patient was conscious No side effects the next heartbeat, urine analysis (8 h day postinjection): ibotenic acid 32.2 mg/mL, muscimol 6.0 mg/mL A. pantherina poisoning case Nausea, vomiting, normal blood Hallucinations No side effect the next pressure, regular heartbeat, day urine analysis (3 h postinjection): ibotenic acid 37.3 mg/mL, muscimol 7.6 mg/mL A. pantherina poisoning case Slightly increased blood Agitation, talkativeness, the patient No side effects the pressure, diarrhea, urine described the psychotic effects as next day analysis (6 h postinjection): similar to alcoholic intoxication ibotenic acid 55.2 mg/mL, muscimol 7.4 mg/mL A. pantherina fruit bodies, No muscle spasms, no vomiting 90 min after ingestion, alternations of No side effects 3 Self portionconsisting of about one half visual perception, wavy motions experimentation cup of static objects, altered perception of size and space, auditory hallucinations, impaired motor coordination, pleasurable emotionally altered state of consciousness lasting 7 h A. pantherina fruit bodies, Nausea Drowsiness, sleep No side effects Case report portionconsisting of about one half cup A. pantherina fruit bodies, portion — state (lasting 5 h), with no No side effects Case report consisting of about a cup of fried logical verbal contact with others, mushrooms the subject experienced vivid waking dreams somehow related to the reality around him A. muscaria, 30 g of dried caps Muscarinic effects such as Symptoms developed 1 h after No side effects Self increased salivation and ingestion, pleasant sedation, experimentation sweating, slight nausea alternations of visual perception, impaired motor coordination

(continued on next page)

2013 Lippincott Williams & Wilkins 435 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

TABLE 6. (Continued) Somatic and Psychotic Symptoms of Intoxication With Amanita muscaria or Amanita pantherina— Mushrooms Containing Ibotenic Acid and Muscimol. Therapeutic and Side Effects of Muscimol Somatic Symptoms of Intoxication, Therapeutic, and Long-Term Side Dose Side Effects Psychotic Symptoms of Intoxication Effects References Minimal effective dose of muscimol: 6 mg; minimal effective dose of ibotenic acid: 30–60 mg; one fruit body of A. muscaria (50–70 g) may contain up to 70 mg of ibotenic acid Muscimol 5 mg orally No stimulation, inactivation No side effects 237 Self- experimentation Muscimol 10 mg orally Myoclonic muscle twitching 90 min postingestion: dizziness, ataxia, No side effects mood improvement followed by sleep rich in dreams Muscimol 15 mg orally Myoclonic muscle cramps, 40 min postingestion: dizziness, Fatigue, inactivation slightly increased blood reflexes not changed, difficulties in the next day pressure speaking and attention focusing, loss of appetite, stimulation, echo- like pseudohallucinations (visual and auditory), sleep Ibotenic acid 20 mg orally Face skin flushing No stimulation, sleep Migraine attack the 237 Self- (paresthesias), no changes in next day experimentation blood pressure and heart rate postingestion; headache (occipitally localized) for the next 2 weeks Muscimol 5 mg orally Improvement in concentration 237 Tests in human performance subjects Muscimol 10–15 mg orally Impairment in concentration performance and ability Muscimol 5–9 mg orally Improvement in tardive No sedation 238 Schizophrenic dyskinesia patients Muscimol 7 mg orally Improvement in tardive Impairment in attention performance 239 Schizophrenic dykinesia and saccadic patients distractibility Muscimol orally. Within 120 min from ingestion: - 240,241 Patients prolactin [, growth hormone with the [, dose-dependent increases Huntington disease and chronic schizophrenia Muscimol 5 mg orally. - No relief of psychosis resulted from 221 Schizophrenic a disease, sedation, relaxation patients Muscimol 7–10 mg orally. Myoclonic muscle twitching Increases in psychosis symptoms measurements: confusion, affect, thought disorder, maximal effect 1–2 h after administration, sleep, vivid dreams in mice are 2.5 mg/kg (intraperitoneally), 3.8 mg/kg (sub- losses via hippocampal perforant pathway resembling defects 250 cutaneously), and 5.62 mg/kg (i.v.). The LD50 determined observed in the Alzheimer disease. in rabbits is 10 mg/kg (orally). Lethal doses of ibotenic acid Less toxic muscimol applied in similar experiments established in rats are 128 mg/kg (orally) and 42 mg/kg pharmacologically mimics cytotoxic agents by transient (intravenously). Ibotenic acid LD50 determined in mice inactivation of neuronal activity. The mechanism of its action equals to 15 mg/kg (intravenoulsy) and 38 mg/kg involves activation of GABA receptors followed by neuronal 246 (orally). depolarization caused by chloride ions influx. GABAA and To investigate an involvement of a particular brain area GABAC receptors are GABA-gated ion channels specific in the studied brain activity, ibotenic acid as a cytotoxic agent for chloride ions. After ligation, they trigger inward chlo- is directly injected into the brain tissue in vivo (animal ride current, what inhibits neuronal activity.251–253 For experiments).219,247–249 Due to its cytotoxic properties, ibo- example, it was established that microinjected muscimol tenic acid is used to model the Alzheimer disease in rats. This inhibits the dorsomedial and the caudal region compound injected into the entorhinal cortex causes neuronal of the lateral/dorsolateral periaqueductal gray. The inactivation

436 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol was able to diminish autonomic responses (such as increases in CONCLUDING REMARKS heart rate and blood pressure, adrenocorticotropic hormone Psilocybin/psilocin referring scientific literature, including elevation) to stress conditions in rats.254 analytical chemistry studies, is quite rich. Therapeutic strategies based on psilocybin biologic activity are being developed. Potential Therapeutic Muscimol Applications Agonists of serotonin receptors may find application in Due to its inhibitory activity, muscimol possesses anal- therapeutic areas such as psychiatry and neurology.4 The drugs gesic properties. When injected intravenously, muscimol poten- of activity against serotonergic system such as and 227 tiates analgesic effect of pilocarpine in rats and in are already used to treat migraine headaches.270,271 rats and mice.255,256 All these effects are mediated by GABAer- The results of search for potent and selective agonists of 5-HT2C gic system. Intrathecally injected muscimol, by GABAA ago- receptors among psilocybin derivatives seem to be promising. nistic activity, blocks nociception mediated by excitatory – 257 On the other hand, psilocybin/psilocin induced psychosis is aminoacids. Muscimol was shown to potentiate anaesthetic commonly used to model schizophrenia, what allows for better activity of , what allowed to establish GABAergic understanding of its complex etiology and psychopathology. transmission involvement in the mechanism of anaesthesia fl 258 Hallucinogens of y agaric seem to be far less frequently induced by propofol. a subject of chemical analysis and studies on the mechanisms of Intrathalamic microinjections of muscimol were able to their biologic activity in humans. It is not surprising if one is reduce tremor in patients suffering from essential tremor, what aware of the use of A. muscaria isoxazole neurotoxins for a long would implicate therapeutic use of the isoxazole compound to 258 time in neurobiology where ibotenic acid commonly serves as treat the neurologic disorder. brain-lesioning agent. Less poisonous muscimol and its deriva- Muscimol applied systemically or topically to cortex tives possess high therapeutic potential in neurology and psy- was shown to block or delay drug-induced convulsions in fi 232,260,261 chiatry, although the potential is still insuf ciently put to good animals. Pretreatment with muscimol (transmenin- account. geal administration) at 2.5 mM concentration prevents acetylcholine-induced seizures in rats and monkeys without 261 serious side effects. Therefore, it could be potentially used REFERENCES for treatment. 1. Crundwell E. The unnatural history of the fly agaric. Mycologist. 1987; Moreover, despite psychoactive properties, muscimol 21:178–181. applied in relatively low doses improves schizophrenia 2. Furst PT. Visionary plants and ecstatic . Expedition. 2004; symptoms working as an likely by the mechanisms 46:26–29. 3. Ott J. Psycho-mycological studies of Amanita—from ancient sacrament of neuronal activity suppression and/or paradoxical sleep – 221 to modern phobia. J Psychoactive Drugs. 1976;8:27 35. suppression. In agreement with the finding of muscimol 4. Sessa BSJ. Are treatments seeing a comeback in psy- anxiolytic activity are results originating from later studies chiatry? Prog Neurol Psychiatry. 2008;12:5–10. on the activation of GABA receptors localized in the lateral 5. Kuttner RE, Hickey RE. Culture and perception: a note on hallucino- – nucleus of the amygdale. Muscimol injections depending on genic drugs. J Natl Med Assoc. 1970;62:25 26. 6. Pavarin RM. Substance use and related problems: a study on the abuse concentration and the presence of selective antagonists affect of recreational and not recreational drugs in Northern Italy. Ann Ist fear learning and memory in rats. Particularly, activation of Super Sanita. 2006;42:477–484. fi GABAA receptors by relatively low doses of muscimol in 7. Johnson MW, Richards WA, Grif ths RR. Human hallucinogen research: guidelines for safety. J Psychopharmacol. 2008;22:603–620. the presence of GABAC antagonist leads to fear learning blockade.262 effect of muscimol was also shown in 8. van Amsterdam J, Opperhuizen A, van den Brink W. Harm potential of 212,237 magic mushroom use: a review. Regul Toxicol Pharmacol. 2011;59: mice. 423–429. Muscimol was proven to improve tardive dyskinesia in 9. Riley SC, James C, Gregory D, et al. Patterns of at schizophrenic patients.238,239 dance events in Edinburgh, Scotland. Addiction. 2001;96:1035–1047. Muscimol is able to prevent cellular death in ischaemia– 10. Pahnke W. Drugs and Mysticism: An Analysis of the Relationship Between Psychedelic Drugs and the Mystical Consciousness [PhD dis- reperfusion conditions when released in high amounts gluta- sertation]. Cambridge, MA: Harvard University; 1963. mate exhibits cytotoxic activity. The preventive activity of 11. Doblin R. Pahnke’s "Good Friday Experiment": a long-term follow-up muscimol was demonstrated in animal model of ischaemia– and methodological critique. J Transpersonal Psychol. 1991;23:1–28. reperfusion.263,264 Muscimol stimulation of modulating hetero- 12. Wark C., Galliher JF. , Richard Alpert (Ram Dass) and the changing definition of psilocybin. Int J . 2010;21:234–239. receptor GABAA on glutamatergic neurons reduces activation of 13. Griffiths RR, Richards WA, McCann U, et al. Psilocybin can occasion glutamate receptor NMDA, what protects pyramidal neurons mystical-type experiences having substantial and sustained personal against excessive excitability.265 The precise mechanisms of meaning and spiritual significance. . 2006;187: muscimol protective action is not known—it possibly involves 268–283. fi inhibition of NMDA-dependent activation of neuronal nitric 14. Grif ths RR, Richards WA, Johnson MW, et al. Mystical-type experi- 266 ences occasioned by psilocybin mediate the attribution of personal oxidesynthasenNOS. In vitro studies revealed that GABA meaning and spiritual significance 14 months later. J Psychopharmacol. and muscimol inhibit NO synthesis in rat ileal nerve terminals 2008;22:621–632. 267 fi through activation of GABAA and GABAC receptors. How- 15. Grif ths RR, Johnson MW, Richards WA, et al. Psilocybin occasioned mystical-type experiences: immediate and persisting dose-related ever, muscimol independently on its GABAA agonistic activity – 268 fl effects. Psychopharmacology. 2011;218:649 665. was also shown to stimulate NO release. Antiin ammatory 16. MacLean KA, Johnson MW, Griffiths RR. Mystical experiences occa- activity of muscimol in endotoxemia was recently demonstrated sioned by the hallucinogen psilocybin lead to increases in the personality in mice.268,269 domain of openness. J Psychopharmacol. 2011;25:1453–1461.

2013 Lippincott Williams & Wilkins 437 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

17. Murrell T, Taylor W, The cutaneous reaction to nicotinic acid (niacin)- and d- in healthy volunteers a double-blind, placebo- furfuryl. Arch Dermatol. 1959;79:545–552. controlled PET study with [18F]FDG. .1999; 18. Kobza Black A, Greaves MW, Hensby CN. The effect of systemic pred- 20:565–581. nisolone on arachidonic acid, and prostaglandin E2 and F2 levels in 44. Gouzoulis-Mayfrank E, Thelen B, Maier S, et al. Effects of the hallu- human cutaneous inflammation. Br J Clin Pharmacol. 1982;14:391–394. cinogen psilocybin on covert orienting of visual attention in humans. 19. Morrow JD, Awad JA, Oates JA, et al. Identification of skin as a major Neuropsychobiology. 2002;45:205–212. site of prostaglandin D2 release following oral administration of niacin 45. Umbricht D, Vollenweider FX, Schmid L, et al. Effects of the 5-HT2A in humans. J Invest Dermatol. 1992;98:812–815. agonist psilocybin on mismatch negativity generation and AX-continuous 20. Papaliodis D, Boucher W, Kempuraj D, et al. Niacin-induced skin performance task: implications for the neuropharmacology of cognitive “flush” involves release of prostaglandins D2 from mast cells and sero- deficits in schizophrenia. Neuropsychopharmacology.2003;28:170–181. tonin from platelets: evidence from human cells in vitro and on animal 46. Vollenweider FX, Leenders KL, Scharfetter C, et al. Positron emission model. J Pharmacol Exp Ther. 2008;327:665–672. tomography and fluorodeoxyglucose studies of metabolic hyperfrontality 21. Plummer NA, Hensby CN, Black AK, et al. Prostaglandin activity in and psychopathology in the psilocybin model of psychosis. Neuropsycho- sustained inflammation of human skin before and after aspirin. Clin Sci . 1997;16:357–372. Mol Med. 1977;52:615–620. 47. Vollenweider FX, Vollenweider-Scherpenhuyzen MFI, Bäbler A, et al. Psi- 22. Hudson C, Gotowiec A, Seeman M, et al. Clinical subtyping reveals locybin induces schizophrenia-like psychosis in humans via a serotonin-2 significant differences in calcium-dependent activity agonist action. Neuroreport. 1998;9:3897–3902. in schizophrenia. Biol Psychiatry. 1999;46:401–405. 48. Vollenweider FX, Vontobel P, Hell D, et al. 5-HT Modulation of dopa- 23. Messamore E. Relationship between the niacin skin flush response and mine release in ganglia in psilocybin-induced psychosis in man— essential fatty acids in schizophrenia. J Lipid Res. 1988;29:679–689. a PET study with [11C]raclopride. Neuropsychopharmacology. 1999; 24. Tavares H, Yacubian J, Talib LL, et al. Increased phospholipase A2 20:424–433. activity in schizophrenia with absent response to niacin. Schizophr Res. 49. Vollenweider FX, Csomor PA, Knappe B, et al. The effects of the 2003;61:1–6. preferential 5-HT2A agonist psilocybin on prepulse inhibition of startle 25. Ishii N, Nishihara Y. Pellagra encephalopathy among tuberculous in healthy human volunteers depend on interstimulus interval. Neuro- patients: its relation to isoniazid therapy. J Neurol Neurosurg Psychia- psychopharmacology. 2007;32:1876–1887. try. 1985;48:628–634. 50. Spitzer M, Thimm M, Hermle L, et al. Increased activation of indirect 26. Nagalski A, Bry1a J. Niacin in therapy. Postepy Hig Med Dosw. 2007; semantic associations under psilocybin. Biol Psychiatry. 1996;39:1055–1057. 61:288–302. 51. Hofmann A, Heim R, Brack A, et al. Psilocybin, ein psychotroper 27. Gibson GE, Blass JP. Nutrition and brain function. In: Siegel GJ, Wirkstoff aus dem Mexikanischen Rauschpilz Agranoff BW, Albers RW, et al, eds. Basic Neurochemistry: Molecular, Heim (Psilocybin, a psychotropic substance from the Mexican mush- Cellular and Medical Aspects. Philadelphia, PA: Lippincott-Raven, room Psilicybe mexicana Heim). Experientia. 1958;14:107–109. 2009;691–710. 52. Bowden K, Drysdale AC. A novel constituent of Amanita muscaria. 28. Prochwicz K. Paradygmat poprzedzania semantycznego w badaniach Tetrahedron Lett. 1965;6:727–728. nad schizofrenią. Neuropsychiatry Neuropsychol. 2008;3:21–28. 53. Müller GFR, Eugster CH. Muscimol, ein pharmakodynamisch wirk- 29. Jablensky A. The diagnostic concept of schizophrenia: its history, evolu- samer Stoff aus Amanita muscaria. Helv Chim Acta. 48;1965:910–922. tion, and future prospects. Dialogues Clin Neurosci. 2010;12:271–287. 54. Takemoto T, Nakajima T, Yokobe T. Structrure of ibotenic acid. 30. Araszkiewicz A, P1ocka-Lewandowska M. Schizofrenia u kobiet Yakugaku Zasshi. 1964;84:1232–1233. ime¸zczyzn._ Kosmos Problemy Nauk Biologicznych. 2003;52:97–103. 55. Asselborn G, Wennih R, Yegles M. Tragic flying attempt under the 31. Buchanan RW. Persistent negative symptoms in schizophrenia: an over- influence of “magic mushrooms”. Probl Forensic Sci. 2000;42:41–46. view. Schizophr Bull. 2007;33:1013–1022. 56. Borowiak KS, Ciechanowski K, Waloszczyk P. 32. Trémeau F. A review of emotion deficits in schizophrenia. Dialogues (Psilocybe semilanceata) intoxication with myocardial infarction. Clin Neurosci. 2006;8:59–70. J Toxicol Clin Toxicol. 1998;36:47–49. 33. Mees L, Zdanowicz N, Reynaert C, et al. Adolescents and young adults 57. Satora L, Goszcz H, Ciszowski K. Poisonings resulting from the inges- at ultrahigh risk of psychosis: detection, prediction and treatment. tion of magic mushrooms in Kraków. Przegl Lek. 2005;62:394–396. A review of current knowledge. Psychiatr Danub. 2011;23:S118–S122. 58. Satora L, Pach D, Butryn B, et al. Fly agaric (Amanita muscaria) 34. Dein S, Littlewood R. Religion and psychosis: a common evolutionary poisoning, case report and review. Toxicon. 2005;45:941–943. trajectory? Transcult Psychiatry. 2011;48:318–335. 59. Satora L, Pach D, Ciszowski K, et al. Panther cap Amanita pantherina 35. Myers NL. Update: schizophrenia across cultures. Curr Psychiatry Rep. poisoning case report and review. Toxicon. 2006;47:605–607. 2011;13:305–311. 60. Andersson C, Kristinsson J, Gry J. Occurrence and use of hallucinogenic 36. Grob CS. Psilocybin research with advanced-stage cancer patients. mushrooms containing psilocybin alkaloids. TemaNord. 2008;606:1–121. MAPS Bull. 2005;15:8. 61. Courtecuisse R, Deveaux M. Champignons hallucinogènes d’Europe et 37. Grob CS. The use of psilocybin in patients with advanced cancer and des Amériques: mise au point mycologique et toxicologique. Ann Toxicol existential anxiety. In: Winkelman M, Roberts T, eds. Psychedelic Med- Anal. 2004;16:36–64. icine: New Evidence for Hallucinogens as Treatments. Westport, CT: 62. Guzmán G, Allen JW, Jochen G. A worldwide geographical distribution Praeger/Greenwood; 2007:205–216. of the neurotropic fungi. An analysis and discussion. Ann Mus Rov. 38. Grob CS, Danforth AL, Chopra GS. Pilot study of psilocybin treatment 2000;14:189–280. for anxiety in patients with advanced-stage cancer. Arch Gen Psychiatry. 63. Stijve T, Kuyper TW. Absence of psilocybin in species of fungi previ- 2011;68:71–78. ously reported to contain psilocybin and related tryptamine derivatives. 39. Fisher G. Treatment of childhood schizophrenia utilizing LSD and psi- Persoonia.1988;13:463–465. locybin. Multidisciplinary Association for Psychedelic Studies Bulletin. 64. Borovicka J. The wood-rotting bluing Psilocybe species in Central 1997;7:18–25. Europe—an identification key. Czech Mycol. 2008;60:173–192. 40. Carhart-Harris RL, Leech R, Williams TM, et al. Implications for 65. Noordeloos ME. s.l. Fungi Europaei. Vol. 13. Alassio, psychedelic-assisted psychotherapy: functional magnetic resonance Italy: Edizioni Candusso; 2011. imaging study with psilocybin. Br J Psychiatry. 2012;200:238–244. 66. Stríbrný J, Borovicka J, Sokol M. Obsah psilocybinu a psilocinu v 41. Carter OL, Pettigrew JD, Hasler F, et al. Modulating the rate and rhythmic- nekterých druzích hub [Levels of psilocybin and psilocin in various ity of perceptual rivalry alternations with the mixed 5-HT2A and 5-HT1A types of mushrooms]. Soudní Lékarství. 2003;48:45–49. agonist psilocybin. Neuropsychopharmacology. 2005;30:1154–1162. 67. Beug MW. The genus Psilocybe in North America. Fungi. 2011;4:6–17. 42. Gouzoulis-Mayfrank E, Heekeren K, Thelen B, et al. Effects of the 68. Guzmán G, The genus Psilocybe. A systematic revision of the known hallucinogen psilocybin on habituation and prepulse inhibition of the species including the history, distribution and chemistry of the halluci- startle reflex in humans. Behav Pharmacol. 1998;9:561–566. nogenic species. Beihefte Nova Hedwigia. 1983;74:1–438. 43. Gouzoulis-Mayfrank E, Schreckenberger M, Sabri O, et al. Neurometa- 69. Anastos N, Lewis SW, Barnett NW, et al. The determination of psilocin bolic effects of psilocybin, 3,4-methylenedioxyethylamphetamine (MDE) and psilocybin in hallucinogenic mushrooms by HPLC utilizing a dual

438 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

reagent acidic potassium permanganate and Tris(2,20-bipyridyl)ruthe- 94. Kysilka R, Wurst M. A novel extraction procedure for psilocybin and nium(II) chemiluminescence detection system. J Forensic Sci. 2006; psilocin determination in mushroom samples. Planta Med. 1990;56: 51:45–51. 327–328. 70. Agurell S, Nilsson JLG. Biosynthesis of psilocybin. Part II. Incorporation 95. Keller T, Schneider A, Regenscheit P, et al. Analysis of psilocybin and of labeled tryptamine derivatives. Acta Chem Scand. 1968;22:1210–1218. psilocin in Psilocybe subcubensis GUZMAN by ion mobility spectrom- 71. Gartz J. Biotransformation of tryptamine derivatives in mycelia cultures etry and gas chromatography–mass spectrometry. Forensic Sci Int. of Psilocybe. J Basic Microbiol. 1989;29:347–352. 1999;99:93–105. 72. Gross ST. Psychotropic drugs in developmental mushrooms: a case 96. Sottolano SM, Lurie IS. The quantitation of psilocybin in hallucino- study review. J Forensic Sci. 2002;47:1–5. genic mushrooms using high performance liquid chromatography. 73. Bigwood J, Beug MW. Variation of psilocybin and psilocin levels with J Forensic Sci. 1983;28:929–935. repeated flushes (harvests) of mature sporocarps of Psilocybe cubensis 97. Pedersen-Bjergaard S, Sannes E, Rasmussen KE, et al. Determination of (Earle) Singer. J Ethnopharmacol. 1982;5:287–291. psilocybin in Psilocybe semilanceata by capillary zone electrophoresis. 74. Catalfomo P, Tyler VE. The production in submerged culture by J Chromatogr B. 1997;694:375–381. Psilocybe cubensis. Lloydia. 1964;27:53–63. 98. Vanhaelen-Fastré R, Vanhaelen M. Qualitative and quantitative deter- 75. Wang WW. Aspects of Secondary Metabolism in Basidiomycetes I minations of hallucinogenic components of Psilocybe mushrooms by Biological and Biochemical Studies on Psilocybe cubensis [master of reversed-phase high-performance liquid chromatography. J Chroma- science thesis]. Department of Botany, The University of British togr. 1984;312:467–472. Columbia, Vancouver, British Columbia, Canada; 1977. 99. Cepas J, Silva M, Pérez-Bendito D. Sensitive peroxyoxalate chemilu- 76. Kysilka R, Wurst M. High-performance liquid chromatographic deter- minescence determination of psychotropic indole derivatives. Analyst. mination of some psychotropic indole derivatives. J Chromatogr. 1989; 1996;121:49–54. 464:434–437. 100. Sarwar M, McDonald JL. A rapid extraction and GC/MS methodology 77. Wurst MM, Semerdzieva M, Vokoun J. Analysis of psychotropic com- for the identification of psilocin in mushroom/chocolate concoctions. pounds in fungi of the genus Psilocybe by reversed-phase high perfor- Microgram J. 2003;1:177–183. mance liquid chromatography. J Chromatogr. 1984;286:229–235. 101. Thomson BM. Analysis of psilocybin and psilocin in mushroom 78. Gartz J. Extraction and analysis of indole derivatives from fungal biomass. extracts by reversed-phase high performance liquid chromatography. J Basic Microbiol. 1994;34:17–22. J Forensic Sci. 1980;25:779–785. 79. Gartz J. Observations on the Psilocybe cyanescens complex of Europe 102. Björnstad K, Beck O, Helander A. A multi-component LC–MS/MS and North America. Sez Arch St Sc Nat. 1998;12:209–218. method for detection of ten -derived psychoactive substances in 80. Durand R, Jacques R. Action Spectra for fruiting of the mushroom urine. J Chromatogr B. 2009;877:1162–1168. Coprinus congregates. Arch Microbiol. 1982;132:131–134. 103. Grieshaber AF, Moore KA, Levine B. The detection of psilocin in 81. Badham ER. The effect of light upon initiation in Psilocybe human urine. J Forensic Sci. 2001;46:627–630. cubensis. Mycologia. 1980;72:136–142. 104. Kamata T, Nishikawa M, Katagi M, et al. Optimized glucuronide 82. Koçak A, De Cotiis LM, Hoffman DB. Comparative study of ATR and hydrolysis for the detection of psilocin in human urine samples. transflection IR spectroscopic techniques for the analysis of hallucino- J Chromatogr B. 2003;796:421–427. genic mushrooms. Forensic Sci Int. 2010;195:36–41. 105. Sticht G, Käferstein H. Detection of psilocin in body fluids. Forensic 83. Lee RE. A technique for the rapid isolation and identification of psilocin Sci Int. 2000;113:403–407. from psilocin/psilocybin-containing mushrooms. J Forensic Sci. 1985; 106. Alnajjar A, Idris AM, Multzenberg M, et al. Development of a capillary 30:931–941. electrophoresis method for the screening of human urine for multiple 84. Lindenblatt H, Krämer E, Holzmann-Erens P, et al. Quantitation of drugs of abuse. J Chromatogr B. 2007;856:62–67. psilocin in human plasma by high-performance liquid chromatography 107. Bocks S. Fungal Metabolism—IV. The oxidation of psilocin by p-diphenol and electrochemical detection: comparison of liquid–liquid extraction oxidase (laccase). Phytochemistry. 1967;6:1629–1631. with automated on-line solid-phase extraction. J Chromatogr B Biomed 108. Darby PW, Ford-Kirkpatrick W, Lewis DT, et al. Microanalysis in Sci Appl. 1998;709:255–263. forensic and industrial investigations. Proc Soc Anal Chem. 1967;4: 85. Weber HP, Petcher TJ. Crystal structures of the teonanácatl hallucino- 169–175. gens. Part I. Psilocybin C12H17N2O4P. J Chem Soc Perkin Trans 2. 1973; 109. Jensen N. as Ligands and Modulators of the Serotonin 2443:942–946. 5-HT2A Receptor and the Isolation of Aeruginascin from the Halluci- 86. Petcher TJ, Weber HP. Crystal structures of the teonanhcatl hallucino- nogenic Mushroom Inocybe aeruginascens [PhD thesis].Göttingen: gens. Part II. Psilocin, C12H15N2O. J Chem Soc Perkin Trans 2. 1973; Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August- 2444:946–948. Universität zu Göttingen; 2004. 87. Beug MW, Bigwood J. Quantitative analysis of psilocybin and psilocin 110. Gartz J. Quantitative bestimmung der indolderivate von Psilocybe semi- in Psilocybe baecystis (Singer and Smith) by high-performance liquid lanceata (Fr.) Kumm. Biochem Physiol Pflanzen. 1986;181:117–124. chromatography and thin-layer chromatohraphy. J Chromatogr. 1981; 111. Gartz J, Allen JW, Merlin MD. , biochemistry, and 207:379–385. cultivation of Psilocybe samuiensis Guzmk, Bandala and Allen, 88. Laussmann T, Meier-Giebing S. Forensic analysis of hallucinogenic a new psychoactive fungus from Koh Samui, Thailand. J Ethnophar- mushrooms and khat (Catha edulis FORSK) using cation-exchange macol. 1994;43:73–80. liquid chromatography. Forensic Sci Int. 2010;195:160–164. 112. Stead AH, Gill R, Wright T, et al. Standardised thin-layer chromato- 89. Saito K, Toyo’oka T, Fukushima T, et al. Determination of psilocin in graphic systems for the identification of drugs and poisons. Analyst. magic mushrooms and rat plasma by liquid chromatography with fluo- 1982;107:1106–1168. rimetry and electrospray ionization mass spectrometry. Anal Chim Acta. 113. Jensen N, Gartz J, Laatsch H. Aeruginascin, a trimethylammonium 2004;527:149–156. analogue of psilocybin from the Inocybe 90. Koike Y, Wada K, Kusako G, et al. Isolation of psilocybin from aeruginascens. Planta Med. 2006;72:665–666. Psilocybe argentipes and its determination in specimens of some mush- 114. Hasler F, Bourquin D, Brenneisen R, et al. Determination of psilocin rooms. J Nat Prod. 1981;44:362–365. and 4-hydroxyindole-3-acetic acid in plasma by HPLC-ECD and phar- 91. Casale J. An aqueous-organic extraction method for the isolation and macokinetic profiles of oral and intravenous psilocybin in man. Pharm identification of Psilocin from hallucinogenic mushrooms. J Forensic Acta Helv. 1997;72:175–184. Sci. 1985;30:247–250. 115. Bogusz MJ. Liquid chromatography-mass spectrometry as a routine 92. Stenzel JR, Jiang G, Loran C. Identification of psychotropic substances method in forensic sciences: a proof of maturity. JChromatogr in mushrooms and chocolate by UHPLC/MS. Thermo Scientific, 2008; B Biomed Sci Appl. 2000;748:3–19. application note 428. 116. Pihlainen K, Sippola E, Kostiainen R. Rapid identification and quantitation 93. Christiansen AL, Rasmussen KE, Tønnesen F. Determination of psilo- of compounds with forensic interest using fast liquid chromatography-ion cybin in Psilocybe semilanceata using high-performance liquid chro- trap mass spectrometry and library searching. J Chromatogr A. 2003;994: matography on a silica column. J Chromatogr. 1981;210:163–167. 93–102.

2013 Lippincott Williams & Wilkins 439 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

117. Bogusz MJ, Maier RD, Schafer AT, et al. Honey with Psilocybe mush- 143. Trulson ME, Jacobs BL, Morrison AR. Raphe unit activity during REM rooms: a revival of a very old preparation on the drug market? Int sleep in normal cats and in pontine lesioned cats displaying REM sleep J Legal Med. 1998;111:147–150. without atonia. Brain Res. 1981;226:75–91. 118. Pedersen-Bjergaard S, Rasmussen KE, Sannes E. Strategies for the 144. Ursin R. Serotonin and sleep. Sleep Med Rev. 2002;6:55–69. capillary electrophoretic separation of indole alkaloids in Psilocybe 145. Monti JM. The role of dorsal raphe nucleus serotonergic and non- semilanceata. Electrophoresis. 1998;19:27–30. serotonergic neurons, and of their receptors, in regulating waking and 119. Walker JA, Marché HL, Newby N. A free zone capillary electrophoresis rapid eye movement (REM) sleep. Sleep Med Rev. 2010;14:319–327. method for the quantitation of common illicit drug samples. J Forensic 146. Fukuda K. Possible mechanisms of panic attack and schizophrenia via Sci. 1996;41:824–829. APUD system. Med Hypotheses. 2002;58:123–126. 120. Horita A. Some biochemical studies on psilocybin and psilocin. J Neu- 147. Aghajanian GK, Marek GJ. Serotonin and hallucinogens. Neuropsycho- ropsychiatry. 1963;4:270–273. pharmacology. 1999;21:16S–23S. 121. Eivindvik K, Rasmussen KE, Sund RB. Handling of psilocybin and 148. Freedman DX, Gottlieb R, Lovell RA. drugs and psilocin by everted sacs of rat jejunum and colon. Acta Pharm Nord. brain 5-hydroxytryptamine metabolism. Biochem Pharmacol. 1970; 1989;1:295–302. 19:1181–1188. 122. Kalberger F, Kreis W, Rutschmann J. The fate of psilocin in the rat. 149. Lewis JJ, Ritchie AP, van Pettent GR. The influence of hallucinogenic Biochem Pharmacol. 1962;11:261–269. drugs upon in vivo brain levels of adenine nucleotides, 123. Hopf A, Eckert H. Distribution patterns of 14C-psilocin in the of and inorganic phosphate in the rat. Br J Pharmacol. 1965;25:631–637. various animals. Acta Nerv Super. 1974;16:64–66. 150. Hasler F, Grimberg U, Benz MA, et al. Acute psychological and phys- 124. Passie T, Seifert J, Schneider U, et al. The pharmacology of psilocybin. iological effects of psilocybin in healthy humans: a double-blind, Addict Biol. 2002;7:357–364. placebo-controlled dose–effect study. Psychopharmacology. 2004; 125. Hasler F, Bourquin D, Brenneisen R, et al. Renal excretion profiles of 172:145–156. psilocin following oral administration of psilocybin: a controlled study 151. Studerus E, Kometer M, Hasler F, et al. Acute, subacute and long-term in man. J Pharm Biomed Anal. 2002;30:331–339. subjective effects of psilocybin in healthy humans: a pooled analysis of 126. Manevski N, Kurkela M, Höglund C, et al. Glucuronidation of psilocin experimental studies. J Psychopharmacol. 2011;25:1434–1452. and 4-hydroxyindole by the human UDP-glucuronosyltransferases. 152. Gouzoulis-Mayfrank E, Thelen B, Habermeyer E, et al. Psychopatho- Drug Metab Dispos. 2010;38:386–395. logical, neuroendocrine and autonomic effects of 3,4-methylenedioxye- 127. Nichols DE. Hallucinogens. Pharmacol Ther. 2004;101:131–181. thylamphetamine (MDE), psilocybin and d-methamphetamine in 128. Carter OL, Burr DC, Pettigrew JD, et al. Using psilocybin to investigate healthy volunteers. Psychopharmacology. 1999;142:41–50. the relationship between attention, working memory, and the serotonin 153. Wittmann M, Carter O, Hasler F, et al. Effects of psilocybin on time 1A and 2A receptors. J Cogn Neurosci. 2005;17:1497–1508. perception and temporal control of behaviour in humans. J Psychophar- 129. Carter OL, Hasler F, Pettigrew JD, et al. Psilocybin links binocular macol. 2007;21:50–64. rivalry switch rate to attention and subjective arousal levels in humans. 154. Johnson MW, Sewell RA, Griffiths RR. Psilocybin dose-dependently Psychopharmacology (Berl.). 2007;195:415–424. causes delayed, transient headaches in healthy volunteers. J Drug Alc 130. McKenna DJ, Repke DB, Peroutka SJ. Differential interactions of Dep 2012;123:132–140. indolealkylamines with 5-hydroxytryptamine receptor subtypes. Neuro- 155. Dittrich A. The standardized psychometric assessment of altered states pharmacology. 1990;29:193–198. of consciousness (ASCs) in humans. Pharmacopsychiatry. 1998;31: 131. Sard H, Kumaran G, Morency C, et al. SAR of psilocybin analogs: 80–84. discovery of a selective 5-HT2C agonist. Bioorg Med Chem Lett. 156. Fisher R, Hill RM, Warshay D. Effects of the psychotodysleptic drug 2005;15:4555–4559. psilocybin on visual perception. Changes in brightness preferences. 132. Bray JK, Goddard WA 3rd. The structure of human serotonin Expertientia. 1969;25:166–169. G-protein-coupled receptor bound to agonists and antagonists. J Mol 157. Fanciullacci M, Franchi G, Sicuteri F. Hypersensitivity to lysergic acid Graph Model. 2008;27:66–81. diethylamide (LSD-25) and psilocybin in essential headache. Experien- 133. Lee H-M, Roth BL. Hallucinogen actions on human brain revealed. tia. 1974;30:1441–1443. Proc Natl Acad Sci U S A. 2012;109:1820–1821. 158. Penden NR, Bissett AF, Macaulay KEC, et al. Clinical toxicology of 134. Nichols CD, Sanders-Bush E. Serotonin receptor signaling and halluci- ‘magic mushrooms’ ingestion. Postgrad Med J. 1981;57:543–545. nogenic drug action. Heffter Rev Psychedelic Res. 2001;2:73–79. 159. Gerault A, Picard T. Intoxication mortelle a la suite de la consommation 135. Filip M, Bader M. Overview on 5-HT receptors and their role in phys- volontaire et en groupe de champignons hallucinogens. Bull Soc Mycol iology and pathology of the central nervous system. Pharmacol Rep. France. 1996; 112:1–14. 2009;61:761–777. 160. Hartman AI, Hollister L. Effect of , lysergic acid diethylamide and 136. González-Maeso J, Yuen T, Ebersole BJ, et al. Transcriptome finger- psilocybin on color perception. Psychopharmacologia. 1963;4:441–451. prints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryp- 161. Carter OL, Pettigrew JD, Burr DC, et al. Vollenweider, Psilocybin tamine 2A receptor agonist effects in mouse somatosensory cortex. impairs high-level but not low-level motion perception. Neuroreport. J Neurosci. 2003;23:8836–8843. 2004;15:1947–1951. 137. Rabin RA, Regina M, Doat M, et al. 5-HT2A receptor-stimulated phos- 162. Wackermann J, Wittmann M, Hasler F, et al. Effects of varied doses of phoinositide hydrolysis in the effects of hallucinogens. Pharmacol psilocybin on time interval reproduction in human subjects. Neurosci Biochem Behav. 2002;72:29–37. Lett. 2008;435:51–55. 138. Kurrasch-Orbaugh DM, Watts VJ, Barker EL, et al. Serotonin 163. Studerus E, Gamma A, Kometer M, et al. Prediction of psilocybin 5-hydroxytryptamine 2A receptor-coupled and phos- response in healthy volunteers. PLoS ONE. 2012;7:e30800. pholipase A2 signaling pathways have different receptor reserves. 164. Espiard M-L, Lecardeur L, Abadie P, et al. Hallucinogen persisting J Pharmacol Exp Ther. 2003;304:229–237. perception disorder after psilocybin consumption: a case study. Eur 139. González-Maeso J, Ang R, Yuen T, et al. Identification of a novel Psychiatry. 2005;20:458–460. serotonin/glutamate receptor complex implicated in psychosis. Nature. 165. Gable RS. Comparison of acute lethal toxicity of commonly abused 2008;452:93–97. psychoactive substances. Addiction. 2004;99:686–696 140. Moldavan MG, Grodzinskaya AA, Solomko EF, et al. The effect of 166. Majdanik S, Borowiak K, Brzezinska M, et al. Concentration of Psilocybe cubensis extract on hippocampal neurons in vitro. Fiziol Zh. selected microelements in blood serum of rats exposed to the action 2001;47:15–23. of psilocin and phenylethylamine. Ann Pomeranian Med Univ. 2007;53 141. Aghajanian GK, Haigler HJ. Hallucinogenic indoleamines: preferential (suppl 2):153–158. action upon presynaptic serotonin receptors. Psychopharmacology. 167. Ghuran A, Nolan J. Recreational drug misuse: issues for the cardiologist. 1975;1:619–629. Heart. 2000;83:627–633. 142. Fischman LG. Dreams, hallucinogenic drug states, and schizophrenia: 168. Nef HM, Möllmann H, Hilpert P, et al. Apical regional wall motion a psychological and biological comparison. Schizophr Bull. 1983;9: abnormalities reminiscent to Tako-Tsubo cardiomyopathy following 73–94. consumption of psychoactive fungi. Int J Cardiol. 2009;134:e39–e41.

440 2013 Lippincott Williams & Wilkins Ther Drug Monit  Volume 35, Number 4, August 2013 Psilocin, Ibotenic Acid, and Muscimol

169. Spengos K, Schwartz A, Hennerici M. Multifocal cerebral demyelin- 197. Filer CN, Lacy JM, Peng CT. Ibotenic acid decarboxylation to muscimol: ation after magic mushroom abuse. J Neurol. 2000;247:224–225. dramatic solvent and radiolytic rate acceleration. Synth Commun. 2005; 170. Shalev H, Serlin Y, Friedman A. Breaching the blood-brain barrier as 35:967–970. a gate to psychiatric disorder. Cardiovasc Psychiatry Neurol. 2009; 198. Beutler JA, der Marderosian AH. Chemical variation in Amanita. J Nat 2009:278531. Prod. 1981;44:422–431. 171. Leary T, Metzner R. Use of psychedelic drugs in prisoner rehabilitation. 199. Chilton WS, Ott J. Toxic metabolites of Amanita pantherina, A. cothur- Br J Soc Psychiatry. 1968;2:27–51. nata, A. muscaria and other Amanita species. Lloydia. 1976;39:150–157. 172. Doblin R. Dr. Leary’s Concord Prison experiment: a 34-year follow-up 200. Stríbrný J, Sokol M, Merová B, et al. GC/MS determination of ibotenic study. J Psychoactive Drugs. 1999;30:419–426. acid and muscimol in the urine of patients intoxicated with Amanita 173. Leonard HL, Rapport JL. Relief of obsessive-compulsive symptoms by pantherina. Int J Legal Med. 2012;126:519–524. LSD and psilocin. Am J Psychiatry. 1987;144:1239–1240. 201. Fowler LJ, Lovell DH, John RA. Reaction of muscimol with 174. Moreno FA, Delgado PL. Hallucinogen-induced relief of obsessions 4-aminobutyrate aminotransferase. J Neurochem. 1983;41:1751–1754. and compulsions. Am J Psychiatry. 1997;154:1037–1038. 202. Benedict RG, Tyler VE, Brady LR. Chematoxanomic significance of 175. Sugrue MF. A study of the role of noradrenaline in behavioral changes isoxazole derivatives in Amanita species. Lloydia. 1966;29:333–342. produced in the rat by psychotomimetic drugs. Br J Pharmacol. 1969; 203. Gennaro MC, Giacosa D, Gioannini E, et al. Hallucinogenic species in 35:243–252. Amanita muscaria determination of muscimol and ibotenic acid by 176. Matsuhima Y, Shirota O, Kikura-Hanajiri R, et al. Effects of Psilocybe ion-interaction HPLC. J Liq Chromatogr Relat Technol. 1997;20: argentipes on marble-burying behavior in mice. Biosci Biotechnol 413–424. Biochem. 2009;73:1866–1868. 204. Lund U. Estimation of muscimol and ibotenic acid in Amanita muscaria 177. Sewell RA, Halpern JH, Pope HG. Response of cluster headache to using high-performance liquid chromatography. Arch Pharm Chem Sci psilocybin and LSD. Neurology. 2006;66:1920–1922. Ed. 1979;7:115–118. 178. Halpern JH, Sewell RA. Hallucinogenic botanicals of America: a growing 205. Merova B, Ondra P, Stankova M, et al. Isolation and identification of need for focused drug education and research. Life Sci. 2005;78:519–526. the Amanita muscaria and Amanita pantherina toxins in human urine. 179. Krieglsteiner GJ. Heim 1983 ss. restr. In: Krieglsteiner GJ, Neuro Endocrinol Lett. 2008;29:744–748. ed. Die Großpilze Baden-württembergs. 4. Ständerpilze: Blätterpilze. II. 206. Johnston GAR, Kennedy SME, Lodge D. Muscimol uptake, release and Stuttgart: Verlag Eugen Ulmer GmbH & Co.; 2003:8–47. binding in rat brain slices. J Neurochem. 1978;31:1519–1523. 180. Michelot D, Melendez-Howell LM. Amanita muscaria: chemistry, , 207. Magdalan J, Antonczyk A. Zatrucie muchomorem plamistym czy udar toxicology, and ethnomycology. Mycol Res. 2003;107:131–146. mózgu? Przeg Lek. 2007;64:4–5. 181. Döpp H, Musso H. Isolierung und chromophore der faus Amanita 208. Olpe HR, Koella WP. The action of muscimol on neurons of the sub- muscaria. Chem Ber. 1973;106:3473–3482. stantia nigra of the rat. Experientia. 1978;34:235. 182. Stintzing F, Schliemann W. Pigments of fly agaric (Amanita muscaria). 209. DeFeudi S. Physiological and behavioral studies with muscimol. Neuro- Z Naturforsch. 2007;62:779–785. chem Res. 1980;5:1047–1068. 183. Tsujikawa K, Mohri H, Kuwayama K, et al. Analysis of hallucinogenic 210. Coloma FM, Niles LP. In vitro effects of melatonin on [3H]muscimol constituents in Amanita mushrooms circulated in Japan. Forensic Sci binding in rat brain. Prog Neuropsychopharmacol Biol Psychiatry. Int. 2006;164:172–178. 1984;8:669–672. 184. Tsujikawa K, Kuwayama K, Miyaguchi H, et al. Determination of musci- 211. Chandra D, Halonen LM, Anni-Maija Linden AM, et al. Prototypic mol and ibotenic acid in Amanita mushrooms by high-performance liquid GABAA receptor agonist muscimol acts preferentially through fore- chromatography and liquid chromatography-tandem mass spectrometry. brain high-affinitybinding sites. Neuropsychopharmacology. 2010;35: J Chromatogr B. 2007;852:430–435. 999–1007. 185. Tsunoda K, Inoue N, Aoyagi Y, et al. Simultaneous of ibotenic acid and 212. Baraldi M, Grandison L, Guidotti A. Distribiution and metabolism of muscimol on toxic mushroom, Amanita muscaria, and analytical survey muscimol in the brain and other tissues of the rat. Neuropharmacology. on edible mushrooms. J Food Hyg Soc Jpn. 1989;34:12–17. 1979;18:57–62. 186. Størmer FC, Koller GEB, Janak K. Ibotenic acid in Amanita muscaria 213. Curtis DR, Lodge D, McLennan H. The excitation and depression of spores and caps. Mycologist. 2004;18:114–117. spinal neurones by ibotenic acid. J Physiol. 1979;291:19–28. 187. Narahashi T, Zhao X, Ikeda T, et al. Differential actions of on 214. Dunn SMJ, Thuynsma RP. Reconstitution of purified GABAA receptors: target sites: basis for selective toxicity. Hum Exp Toxicol. 2007;26:361–366. ligand binding and chloride transporting properties. Biochemistry.1994; 188. Cully DF, Paress PS, Liu KK, et al. Identification of a Drosophila 33:755–763. melanogaster glutamate-gated chloride channel sensitive to the antipar- 215. Baur R, Sigel E. On high- and low-affinity agonist sites in GABAA asitic agent . J Biol Chem. 1996;271:20187–20191. receptors. J Neurochem. 2003;87:325–332. 189. Wafford KA, Sattelle DB. L-Glutamate receptors on the cell body 216. Jones MV, Sahara Y, Dzubay JA, et al. Defining affinity with the membrane of an identified insect motor neurone. J Exp Biol. 1989;144: GABAA receptor. J Neurosci. 1998;18:8590–8604. 449–462. 217. Krogsgaard-Larsen P, Johnston GAR. Inhibition of GABA uptake in rat 190. Yokoi I, Takeuchi H, Sakai A, et al. Effects of ibotenic acid, quisqualic brain slices by , various isoxazoles and related compounds. acid and their relatives on the excitability of an identifiable giant neurone J Neurochem. 1975;25:797–802. of an African giant snail (Achatina fulica Férussac). Experientria.1977; 218. Krogsgaard-Larsen P, Frolund B, Frydenvang K. GABA uptake inhibi- 33:363–366. tors. Design, molecular pharmacology and therapeutic aspects. Curr 191. Lea TJ, Usherwood PNR. Effect of ibotenic acid on chloride perme- Pharm Des. 2000;6:1193–1209. ability of insect muscle-fibres. Comp Gen Pharmacol. 1973;4:351–363. 219. Zinkand WC, Moore WC, Thompson C, et al. Ibotenic acid mediates 192. Lea TJ, Usherwood PNR. The site of action of ibotenic acid and the neurotoxicity and phosphoinositide hydrolysis by independent receptor identification of two populations of glutamate receptors on insect muscle- mechanisms. Mol Chem Neuropathol. 1992;16:1–10. fibres. Comp Gen Pharmacol. 1973;4:333–350. 220. Schwarcz R, Hökfelt T, Fuxe K, et al. Ibotenic acid-induced neuronal 193. Liu HP, Lin SC, Lin CY, et al. Glutamate-gated chloride channels degeneration: a morphological and study. Exp Brain inhibit juvenile hormone biosynthesis in the cockroach Diploptera Res. 1979;37:199–216. punctata. Insect Biochem Mol Biol. 2005;35:1260–1268. 221. Tamminga C, Crayton JW, Chase TN. Muscimol: GABA agonist therapy 194. Chiang AS, Lin WY, Liu HP, et al. Insect NMDA receptors mediate in schizophrenia. Am J Psychiatry. 1978;135:746–747. juvenile hormone biosynthesis. Proc Natl Acad Sci U S A. 2002;99:37–42. 222. Nowacka A. Udzia1 jądra konarowo-mostowego nakrywki w regulacji 195. Chiang AS, Pszczolkowski MA, Liu HP, et al. Ionotropic glutamate snu paradoksalnego. Sen. 2002;2:109–119. receptors mediate juvenile hormone synthesis in the cockroach Diploptera 223. Nitz D, Siegel JM. GABA release in the dorsal raphe nucleus: role in punctata. Insect Biochem Mol Biol. 2002;32:669–678. the control of REM sleep. Am J Physiol. 1997;273:R451–R455. 196. Tuno N, Takahashi KH, Yamashita H, et al. Tolerance of Drosophila 224. Hobson JA, Lydic R, Baghdoyan HA. Evolving concepts of sleep cycle flies to ibotenic acid poisons in mushrooms. J Chem Ecol. 2007;33: generation: from brain centers to neuronal populations. Behav Brain Sci. 311–317. 1986;9:371–448.

2013 Lippincott Williams & Wilkins 441 Stebelska Ther Drug Monit  Volume 35, Number 4, August 2013

225. Koenig-Bersin P, Waser PG, Langemann H, et al. Monoamines in the 248. Conrad CD, Jackson JL, Wise LS. Chronic stress enhances ibotenic brain under the influence of muscimol and ibotenic acid, two psychoactive acid -induced damage selectively within the hippocampal CA3 region principles of Amanita muscaria. Psychopharmacologia. 1970;18:1–10. of male but not female rats. . 2004;125:759–767. 226. van der Heyden JA, Venema K, Korf J. In vivo release of endogenous 249. Orze1-Gryglewska J. in the rat with lesions of GABA from rat striatum: effects of muscimol, oxotremorine and mor- the . Sen. 2001;1:13–31. phine. J Neurochem. 1980;36:1648–1652. 250. Heo H, Shin Y, Cho W, et al. Memory improvement in ibotenic acid 227. Naik SR, Kelkar MR, Amladi SR, et al. Effect of muscimol, a central induced model rats by extracts of baicalensis. J Ethnopharmacol. GABA receptor agonist, on the catalepsy, striatal homovanillic acid 2009;122:20–27. increase, and analgesia induced by pilocarpine in rats. Psychopharma- 251. Chebib M, Johnston GAR. The ‘ABC’ of GABA receptors: a brief cology. 1981;74:393–394. review. Clin Exp Pharmacol Physiol. 1999;26:937–940. 228. Scotti de Carolis A, Lipparini F, Longo VG. Neuropharmacological 252. Johnston GAR. GABA receptors: as complex as ABC? Clin Exp Phar- investigations on muscimol, a psychotropic drug extracted of Amanita macol Physiol. 1994;21:521–526. muscaria. Psychopharmacologia. 1969;15:186–195. 253. Johnston GAR. GABAA receptor channel pharmacology. Curr Pharm 229. Kelly PA, McCulloch J. Effects of the putative GABAergic agonists, Des. 2005;11:1867–1885. muscimol and THIP, upon local cerebral glucose utilization. J Neurochem. 254. de Menezes RC, Zaretsky DV, Sarkar S, et al. Microinjection of muscimol 1982;39:613–624. into the periaqueductal gray suppresses cardiovascular and neuroendocrine 230. Kelly PA, McCulloch J. The effects og the GABAergic agonist muscimol response to air jet stress in conscious rats. Am J Physiol Regul Integr Comp upon the relationship between local cerebral blood flow and glucose Physiol. 2008;295:R881–R890. utilization. Brain Res. 1983;258:338–342. 255. Biggio G, Bella DD, Frigeni V, et al. Potentiation of morphine analgesia 231. Ito K, Sawada Y, Sugiyama Y, et al. Linear relationship between by muscimol. Neuropharmacology. 1977;16:149–150. GABAA receptor occupancy of muscimol and glucose metabolic 256. Christensen AV, Arnt J, Scheel-Krüger J. Muscimol antagonizes mor- response in the conscious mouse brain. Clinical implication based on phine hypermotility without potentiation of analgesia. Eur J Parmacol. comparison with receptor agonist. Drug Metab Dispos. 1978;48:459–462. 1994;22:50–54. 257. Aanonsen LM, Wilcox GL. Muscimol, gamma-aminobutyric acid A 232. Naik SR, Guidotti A, Costa E. Central GABA receptor agonists: compar- receptors and excitatory amino acids in the mouse spinal cord. J Phar- ison of muscimol and . Neuropharmacology. 1976;15:479–484. macol Exp Ther. 1989;248:1034–1038. 233. Curtis DR, Duggan AW, Felix D, et al. Bicuculline, an antagonist of 258. Pahapill PA, Levy R, Dostrovsky JO, et al. Tremor arrest with thalamic GABA and synaptic inhibition in the spinal cord of the cat. Brain Res. microinjections of muscimol in patients with essential tremor. Ann Neurol. 1971;132:69–96. 1999;46:249–252. 234. Peng Y, Frank E. Activation of GABAA receptors causes presynaptic 259. Pahapill PA, Levy R, Dostrovsky JO, et al. Tremor arrest with thalamic and postsynaptic inhibition at between muscle spindle affer- microinjections of muscimol in patients with essential tremor. Ann Neurol. ents and motoneurons in the spinal cord of bullfrogs. J Neurosci. 1989; 1999;46:249–252. 9:1516–1522. 260. Collins RC. effects of muscimol. Neurology. 1980;30: 235. Kerwin RW, Olpe HR, Schmutz M. The effect of sodium-n-dipropyl 575–581. acetate on g-aminobutyric acid-dependent inhibition in the rat cortex 261. Ludvig N, Baptiste SL, Tang HM, et al. Localized transmeningeal and sustantia nigra in relation to its anticonvulsant activity. Br J Phar- muscimol prevents neocortical seizures in rats and nonhuman primates: macol. 1980;71:545–551. therapeutic implications. Epilepsia. 2009;50:678–693. 236. Curtis DR, Lodge D, McLennan H. The excitation and depression of 262. Cunha C, Monfils M-H, LeDoux JE. GABAC receptors in the lateral spinal neurones by ibotenic acid. J Physiol. 1979;291:19–28. amygdala: a possible novel target for the treatment of fear and anxiety 237. Waser FG. The pharmacology of Amanita muscaria. In: Efron DH, disorders? Front Behav Neurosci. 2010;4:Article 6. Holmstedt B, Kline NS, eds. Ethnopharmacoplogical Search for Psy- 263. Shuaib A, Mazagri R, Ijaz S. GABA agonist "muscimol" is neuropro- choactive Drugs. Vol. l645. U.S. Public Health Service Publication, tective in repetitive transient forebrain ischemia in gerbils. Exp Neurol. , DC; 1979:419–439. 1993;123:284–288. 238. Tamminga CA, Crayton JW, Chase TN. Improvement in tardive dyski- 264. Ouyang C, Guo L, Lu Q, et al. Enhanced activity of GABA receptors nesia after muscimol therapy. Arch Gen Psychiatry. 1979;36:595–598. inhibits glutamate release induced by focal cerebral ischemia in rat 239. Cassady SL, Thaker GK, Moran M, et al. GABA agonist-induced striatum. Neurosci Lett. 2007;420:174–178. changes in motor, oculomotor, and attention measures correlate in schiz- 265. Xu J, Liu Y, Zhang G- Y. of GluR5-containing kainate ophrenics with tardive dyskinesia. Biol Psychiatry. 1992;32:302–311. receptor activation against ischemic brain injury through decreasing 240. Tamminga CA, Neophytides A, Chase TN, et al., Stimulation of prolactin phosphorylation of N-methyl-d-aspartate receptors mediated and growth hormone secretion by muscimol, a gamma-aminobutyric acid by src kinase. J Biol Chem. 2008;283:29355–29366. agonist. J Clin Endocrinol Metab. 1978;47:1348–1351. 266. Tuttolomondo A, Di Sciacca R, Di Raimondo D, et al. Neuron pro- 241. Durso R, Tamminga CA, Denaro A, et al. Plasma growth hormone and tection as a therapeutic target in acute ischemic stroke. Curr Top Med prolactin response to dopaminergic GABAmimetic and cholinergic stim- Chem. 2009;9:1317–1334. ulationinHuntington’s disease. Neurology. 1983;33:1229–1232. 267. Kurjak M, Fichna J, Harbarth J, et al. Effect of GABA-ergic mecha- 242. Yamahura Y, Komiyama S, Fukuhara M, et al. Biochemical effects of nisms on synaptosomal NO synthesis and the nitrergic component of Amanita muscaria extract in mice. J Food Hyg Soc Jpn. 1983;24: NANC relaxation in rat ileum. Neurogastroenterol Motil. 2011;23: 459–464. e181–e190. 243. Benjamin DR. in infants and children: the Amanita 268. Gharedaghi MH, Javadi-Paydar M, Yousefzadeh-Fard Y, et al. Muscimol pantherina/muscaria group. J Toxicol Clin Toxicol. 1992;30:13–22. delays -induced preterm delivery in mice: role of 244. Chapman AG. Glutamate and epilepsy. JNutr.2000;130:1043S–1045S. GABAA receptors and . J Matern Fetal Neonatal Med. 2013; 245. Johnston GAR. Convulsion induced in 10-day-old rats by intraperito- 26:36–43. neal injection of and related excitant amino 269. Hsu D-Z, Li Y-H, Chu P-Y, et al. Muscimol increases the survival rate acids. Biochem Pharmacol. 1973;22:137–140. and inhibits the inflammatory response in endotoxemic mice. Crit Care. 246. Theobald W, Büch O, Kinz HA, et al. Pharmacological and experimental 2009;13(suppl 4):P19. investigations of two components of the flyagaric.Arzneimittelforsch. 270. Silberstein SD, McCrory DC. Ergotamine and : history, 1968;18:311–315. pharmacology, and efficacy. Headache. 2003;43:144–166. 247. Antoniadis EA, Winslow JT, Davis M, et al. The non-human primate 271. Jhee SS, Shiovitz T, Crawford AW, et al. Pharmacokinetics and phar- amygdale is necessary for the acquisition but not the retention of fear macodynamics of the antimigraine agents: a comparative review. potentiated startle. Biol Psychiatry. 2009;65:241–248. Clin Pharmacokinet. 2001;40:189–205.

442 2013 Lippincott Williams & Wilkins