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WO2025068602A2 - Production et utilisation médicale de psilocybine et de composés apparentés - Google Patents

Production et utilisation médicale de psilocybine et de composés apparentés Download PDF

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Publication number
WO2025068602A2
WO2025068602A2 PCT/EP2024/077495 EP2024077495W WO2025068602A2 WO 2025068602 A2 WO2025068602 A2 WO 2025068602A2 EP 2024077495 W EP2024077495 W EP 2024077495W WO 2025068602 A2 WO2025068602 A2 WO 2025068602A2
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yeast cell
cell
gene
recombinant yeast
psilocybin
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WO2025068602A3 (fr
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Evaldas ČIPLYS
Laura Martinkute KORSAKOVA
Eimantas ŽITKUS
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Psylink Uab
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Psylink Uab
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Definitions

  • the present disclosure relates to novel production process for psilocybin and related compounds as well as products related to the production process, including recombinant cells in which these compounds can be produced.
  • the present disclosure relates to novel compounds related to psilocybin, which may be produced by the production processes, and their use in medicine.
  • Psilocybin is a naturally occurring compound of the alkaloid class, found in some species of fungi, in particular members of the genus Psilocybe, e.g. P. mexicana, P. cubensis, P.
  • Psilocybin is a prodrug; in the body psilocybin (3-[2-dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate is dephosphorylated to psilocin (4-hydroxy-N,N-dimethyltryptamine), which is a hallucinogenic compound.
  • psilocybin 3-[2-dimethylamino)ethyl]-1H-indol-4-yl dihydrogen phosphate is dephosphorylated to psilocin (4-hydroxy-N,N-dimethyltryptamine), which is a hallucinogenic compound.
  • the hallucinogenic effects of mushrooms containing psilocybin have long been known, and psilocybin was isolated from mushrooms in the 1950s.
  • Psilocin has a similar chemical structure to serotonin (5-hydroxytryptamine), an important neurotransmitter in the human body, and is known to bind to different human serotonin receptors including the 5-hydroxytryptamine 2A (5-HT2A) receptor.
  • 5-HT2A 5-hydroxytryptamine 2A
  • the present invention provides a method for producing a target alkaloid from a precursor via a metabolic pathway in a recombinant yeast cell, wherein the metabolic pathway comprises a psiH enzyme, a psiK enzyme and a psiM enzyme, the method comprising culturing the recombinant yeast cell in a culture medium comprising the precursor under conditions suitable to allow the precursor to enter the recombinant yeast cell such that the recombinant yeast cell produces the target alkaloid, wherein the precursor is a substrate for the psiH enzyme.
  • the present invention further provides a recombinant yeast cell comprising a psiH gene, a psiK gene, and a psiM gene, wherein the recombinant yeast cell: (i) does not comprise an L- tryptophan decarboxylase gene; and/or (ii) comprises an exogenous CPR gene and an adenosylhomocysteinase (SAH1) gene.
  • the present invention provides a yeast cell culture produced by the method of the invention described above and comprising the recombinant yeast cell and the target alkaloid. Further provided is a medical formulation comprising the recombinant yeast cells containing the target alkaloid and at least one excipient.
  • the present invention provides a method for extracting a produced target alkaloid comprising an amine group and an -HPO 4 group from a recombinant yeast cell, the method comprising: (i) contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell; (ii) centrifuging the extraction mixture to form a supernatant comprising the target alkaloid; (iii) ensuring the supernatant has a pH between 10 and 14 and has a low ionic strength solution such that the charge of N in the amine group is neutral; (iv) applying the supernatant to an anion exchange column; and (v) using a hydroxyl ion gradient to elute at least one fraction comprising the target alkaloid.
  • the amine group is selected from -N(CH 3 ) 2 and an -NHCH 3 group.
  • a yeast cell extract prepared by this method is also provided.
  • the present invention provides a method for increasing methylation capacity of a psiM enzyme in a recombinant yeast cell, the method comprising increasing expression of an adenosylhomocysteinase (SAH1) enzyme in the recombinant yeast cell so as to increase the capacity of the recombinant yeast cell to hydrolyse S-adenosyl-l-homocysteine.
  • SAH1 adenosylhomocysteinase
  • the present invention provides a compound according to Formula (I), or a pharmaceutically acceptable salt thereof, wherein: X is S, O, C, or N, wherein when X is S or O, R 8a and R 8b are absent, and wherein when X is N, R 8b is absent, Z is C or N, wherein when Z is N, R 2 is absent, and wherein when X is N, Z is N, R 4 is -HPO 4 , or -OH, Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3 , R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3 , R 2 , R 3a , R 3b , R 3c , R 3d , R 8a and R 8b are independently selected from H, or D.
  • X is S, O, C, or N
  • R 8a and R 8b are absent
  • Z is C or N
  • the present invention provides a compound according to Formula (IA): wherein: R 4 is -HPO4, or -OH, Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3 , R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3 , R 2 , R 3a , R 3b , R 3c , R 3d are independently selected from H, or D.
  • R 2 , R 3a , R 3b , R 3c , R 3d , R 5 , R 6 , R 7 are all H.
  • R 4 is -HPO 4 , or -OH
  • Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3
  • R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3
  • R 2 , R 3a , R 3b , R 3c , R 3d are independently selected from H, or D.
  • R 2 , R 3a , R 3b , R 3c , R 3d are all H.
  • the target alkaloids according to formula IA and IB may also be produced by the methods of the invention and may be used in the products and compositions of the invention.
  • the present invention further provides a pharmaceutical composition comprising the compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the present invention provides the compound of Formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, for use in medicine.
  • the present invention provides improved methodology for the production of target alkaloids, and especially psilocybin, psilocin and related compounds in recombinant yeast cells.
  • the method described herein advantageously avoids the need to rely on endogenously generated L-tryptophan as the first substrate for the metabolic pathway and instead begins with a later substrate (also referred to herein as precursor) that is the substrate for the psiH enzyme.
  • a later substrate also referred to herein as precursor
  • suitable conditions can be obtained that allow this later substrate in the pathway to be efficiently taken up by the cells.
  • the methods described herein can be used to obtain superior conversion rates and titres of the target alkaloid.
  • the present invention provides purification methods that advantageously lead to the efficient and effective purification of the target alkaloids.
  • the purification methods can be used to avoid the need for cell disruption and chromatographic separation steps.
  • Figure 1 provides a schematic of one example of the biosynthetic route of psilocybin production according to present invention.
  • Figure 2 is a schematic showing the vector map for pPpGAP-KanS-HIS4-psiM (P. cyan.)-psiK (P. cub.)-psiH (P. cyan.)-CPR (P. cyan.)-SAH1 (P. p.).
  • Figure 3 is a picture showing SDS-PAGE analysis of selected P.pastoris strains having psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus and an additional copy of endogenous P. pastoris SAH1 (Adenosylhomocysteinase) gene integrated into the genome.
  • Figure 4 provides results of HPLC analysis of an extract of yeast cells converting tryptamine to psilocybin.
  • Figure 5 provides a chromatogram of the first step of psilocybin purification using 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 6 provides a chromatogram of the second step of psilocybin purification using 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 7 provides HPLC analysis of psilocybin product at 230 nm of combined 22-32 fractions after second step purification using Sepharose HP.
  • Figures 8A and 8B provide analysis showing production of Substance (4) from Example 2.
  • Figure 8A provides a chromatogram from HPLC analysis showing production of Substance (4).
  • Figure 8B provides a mass spectrum for Substance (4).
  • Figures 9A and 9B provide analysis showing production of Substance (5) from Example 2.
  • Figure 9A provides a chromatogram from HPLC analysis showing production of Substance (5).
  • Figure 8B provides a mass spectrum for Substance (5).
  • Figure 10 provides a chromatogram of Substance (5) from Example 2 purification, using 150 ml volume (XK 26/40) SepharoseQ HP.
  • Figures 11A-C provide chromatograms from HPLC analysis of whole cell broth (WCB) extract after addition of 2mM substrate – 2- (Benzofuran-3-yl) ethylamine (Substance (1) Example 3).
  • Figure 11A shows result 5 mins after addition.
  • Figure 11B shows result 4 hours after addition.
  • Figure 11C shows result 20 hours after addition.
  • Figure 12 provides a chromatogram of the first step of O-Psilocybin purification (in Example 3) using a 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 13 provides a chromatogram of the second step of O-Psilocybin purification (in Example 3) using a 150 ml volume (XK 26/40) SepharoseQ HP column.
  • Figure 14 provides HPLC analysis of combined 11-13 fractions after the second step purification of O-Psilocybin (in Example 3) using Sepharose HP.
  • Figure 15 provides bar charts showing the dose-response for compounds tested in the 5-HT2A serotonin receptor agonist assay. Cells were treated with the test compounds at serial concentrations. Data points represent the mean ⁇ SD for each condition for a single experiment performed in triplicate. Results are expressed as the fluorescence intensity (arbitrary units, a.u.) of Fluo-4. C- represents vehicle.
  • Figure 16 provides the dose response curve for PSY-1185 tested in the 5-HT2A serotonin receptor agonist assay. Data is presented as the percentage of activity of the HTR2A normalized against the zero control (vehicle) that represents the 0% of activity and the maximum concentration of each compound that represents the 100%. DETAILED DESCRIPTION As noted above, the biosynthetic pathway for the production of psilocybin was discovered in 2017 by a group of scientists at the Leibniz Institute for Natural Product Research and Infection Biology (Fricke et al., September 25, 2017, Angewandte Chemie, 56(40): 12352-12355).
  • This route (which is shown in Figure 1) uses the enzymes psiD, psiH, psiK, and psiM, to produce psilocybin from the precursor tryptophan; tryptophan being produced in the cell via other metabolic pathways, e.g. the Shikimate-Chorismate pathway as described in Milne et al., (Metabolic Engineering, 60 (2020): 25-36). Further work in this area has been done by Vogan et al., (US 11,441164 B2, published 13 September 2022), which suggested the construction of S. cerevisiae yeast strains that have been genetically engineered to increase the metabolic flux towards L-tryptophan.
  • the present inventors have found that it is possible to improve the production of the target alkaloid by starting the metabolic pathway with a precursor that is a substrate for the psiH enzyme (as shown in Figure 1), since suitable conditions can be created in the culture media that allow the precursor to be efficiently taken up by the yeast cells.
  • This route is advantageous as it is not limited to cell-produced tryptophan.
  • the target alkaloid may be according to formula (I): in which X is N, S, O or C, wherein when X is N, R 8b is absent, and wherein when X is S or O, R 8a and R 8b are absent, Z is C or N, wherein when Z is N, R 2 is absent, R 4 is -HPO 4 , or OH, Y 1 is -N + H(CH 3 ) 2 , or -N + H 2 CH 3 , R 5 , R 6 , R 7 are independently selected from H, D, -CFH 2 , -CHF 2 or -CF 3 , R 2 , R 3a , R 3b , R 3c , R 3d , R 8a and R 8b are independently selected from H or D.
  • formula (I) in which X is N, S, O or C, wherein when X is N, R 8b is absent, and wherein when X is S or O, R 8a and R 8b are absent, Z is
  • the precursor can be selected based on the target alkaloid and the reactions that the enzymes psiH, psiK and psiM catalyse in the metabolic pathway. While these enzymes have been shown to produce psilocybin (as shown in one example of the invention in Figure 1), the examples provided herein surprisingly show that the enzymes are able to accommodate variations in their substrates (in particular in locations on the substrate molecules that are away from positions involved in the enzymatic reaction), allowing the method described herein to be used to produce compounds that are related to psilocybin.
  • X is N and Z is C, and more preferably the target alkaloid is psilocybin.
  • the precursor can be tryptamine.
  • the precursors can be 2-(benzothien-3-yl)ethylamine, 2- (Benzofuran-3-yl)ethylamine and 2-(1H-inden-3-yl)ethanamine, respectively.
  • the experimental work described herein shows that the alteration of the substrates of psiH, psiK and psiM enzymes does not prevent these from catalysing the reactions.
  • X is N
  • Z is N and R 2 is absent
  • R 3a , R 3b , R 3c , R 3d , R 5 , R 6 , R 7 ,R 8a and R 8b are all H.
  • the precursor can be 2-(1H-indazol-3- yl)ethanamine hydrochloride.
  • R 5 , R 6 , R 7 are independently selected from H, and D, and are more preferably H.
  • Y 1 is -N + H(CH 3 ) 2 .
  • the enzymes that may be used in the method of the present invention are as follows: PsiH Enzyme -
  • the psiH enzyme is a tryptamine 4-monooxygenase, which is able to catalyse the conversion of tryptamine to 4-hydroxytryptamine.
  • the psiH enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens.
  • the psiH enzyme may be produced from a cDNA sequence having SEQ ID NO: 7.
  • the psiH enzyme comprises an amino acid sequence having SEQ ID NO: 8, or an amino acid sequence having at least 85% identity to SEQ ID NO: 8. More preferably the psiH enzyme comprises an amino acid sequence having SEQ ID NO: 8, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 8.
  • PsiK Enzyme – The psiK enzyme is a 4-hydroxytrptamine kinase, which is able to catalyse the conversion of 4-hydroxytryptamine to norbaeocystin.
  • the psiK enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens.
  • the psiK enzyme may be produced from a cDNA sequence having SEQ ID NO: 3.
  • the psiK enzyme comprises an amino acid sequence having SEQ ID NO: 4, or an amino acid sequence having at least 85% identity to SEQ ID NO: 4. More preferably the psiK enzyme comprises an amino acid sequence having SEQ ID NO: 4, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 4.
  • the psiM enzyme is a methyltransferase, which is able to catalyse the methylation of norbaeocystin to form baeocystin, and the subsequent methylation of baeocystin to form psilocybin.
  • the psiM enzyme may be derived from Psilocybe cubensis or Psilocybe cyanesens.
  • the psiM enzyme may be produced from a cDNA sequence having SEQ ID NO: 5.
  • the psiM enzyme comprises an amino acid sequence having SEQ ID NO: 6, or an amino acid sequence having at least 85% identity to SEQ ID NO: 6.
  • the psiM enzyme comprises an amino acid sequence having SEQ ID NO: 6, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 6.
  • Cytochrome P450 reductase (CPR) – a CPR enzyme may also be used in the method of the invention.
  • yeast endogenous P450 reductases are not compatible with psiH. Accordingly, it is preferred to use a recombinant yeast cell expressing an exogenous CPR gene in order to increase psiH efficiency.
  • the CPR enzyme may be derived from Psilocybe cyanesens, and may be produced from a nucleotide sequence having SEQ ID NO: 1.
  • the CPR enzyme comprises an amino acid sequence having SEQ ID NO: 2, or an amino acid sequence having at least 85% identity to SEQ ID NO: 2. More preferably the CPR enzyme comprises an amino acid sequence having SEQ ID NO: 2, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO: 2.
  • Adenosylhomocysteinase (SAH1) – the SAH1 enzyme may also be used in the method of the invention to improve the efficiency of the psiM methylation steps.
  • the present inventors have surprisingly found that the S-adenosyl-L-homocysteine (SAH), the molecule that is formed when a methyl group is transferred from SAM to a target molecule, acts as competitive inhibitor of the SAM-dependent methyl transferase reactions catalysed by psiM.
  • SAH S-adenosyl-L-homocysteine
  • the yeast cell may already comprise an endogenous SAH1 gene. However, one or more (additional) copies of a SAH1 gene may be incorporated into the recombinant yeast cell.
  • the gene may be derived from P. pastoris, and may comprise the nucleotide sequence of SEQ ID NO: 9.
  • the SAH1 enzyme comprises an amino acid sequence having SEQ ID NO: 10, or an amino acid sequence having at least 85% identity to SEQ ID NO: 10. More preferably the SAH1 enzyme comprises an amino acid sequence having SEQ ID NO: 10, or an amino acid sequence having at least 90% identity or at least 95% identity to SEQ ID NO:
  • the recombinant yeast cell used in the method of the invention does not comprise a psiD gene or other gene encoding a tryptophan decarboxylase.
  • a further aspect of the invention relates to a method for increasing methylation capacity of a psiM enzyme in a recombinant yeast cell.
  • the method comprises increasing expression of an adenosylhomocysteinase (SAH1) enzyme in the recombinant yeast cell so as to increase the capacity of the recombinant yeast cell to hydrolyse S-adenosyl-l-homocysteine.
  • SAH1 adenosylhomocysteinase
  • expression may be increased by transforming the yeast cell with one or more copies of an SAH1 gene encoding the SAH1 enzyme; and/or increasing transcription from an SAH1 gene encoding the SAH1 enzyme using a promoter or an enhancer.
  • a method may be used as part of a method for producing a target alkaloid from a precursor as described above. Alternatively, it may be used in combination with the psilocybin production methods already described in the prior art.
  • the psiM enzyme carries out two methylation reactions to produce a target alkaloid in which Y 1 is -N + H(CH 3 ) 2 , however, by adding more substrate and/or by restricting the methylation capacity of the psiM enzyme a target alkaloid in which Y 1 is -N + H 2 CH 3 can be produced.
  • the present disclosure provides recombinant yeast cells that can be used in the methods described herein to produce the target alkaloid. Types of yeast cell that are suitable for performing the method of the invention are those already know in the art for recombinant protein production.
  • the yeast may be from the genus Pichia, such as Pichia pastoris, from the genus Sacchromyces, such as S. cerevisiae or S. pombe, or from the genus Yarrowia such as Y. lipolytica.
  • the recombinant yeast cell is from the genus Pichia, and most preferably the yeast cell is Pichia pastoris.
  • Recombinant vectors carrying the genes for the enzymes described above can be transformed into the yeast cell by methods known in the art. It is preferred that the DNA constructs are integrated into the yeast genome in order to provide stable expression of the genes.
  • the genes for the enzymes described above can be placed under the control of inducible or constitutive promoters or gene expression control elements.
  • Suitable promoters are known in the art for the production of recombinant proteins in yeast. However, where the yeast is Pichia, it is preferred that the promoters AOX1 or GAP are used. The GAP promoter is most preferred.
  • the recombinant yeast cell is cultured in a culture medium comprising the precursor under conditions that are suitable to allow the precursor for the enzyme psiH to enter the recombinant host cells.
  • the precursors for the enzyme psiH are charged molecules in normal yeast growth conditions, which do not effectively cross the yeast cell membrane.
  • the present inventors have surprisingly found that they are able to culture the recombinant yeast cells at a pH of 6 to 7.8, preferably at a pH of 7.5 to 7.8 and most preferably at a pH of 7.8.
  • these pH values are not particularly close to the pKa of the precursor molecules (with the pKa of tryptamine being approximately 10.2 at which yeast cannot be readily cultivated)
  • the present inventors have observed rapid diffusion of the precursor into the yeast cell, as a first step to efficient production of the target alkaloid.
  • a suitable phosphate source in the culture media is therefore sodium hexametaphosphate.
  • the precursor may be added to the culture media in batches, and one batch may be added after the full conversion of the previously added precursor has been achieved.
  • the constant addition of precursor may lead to an accumulation of intermediates.
  • the precursor is added to the culture media at a rate of 4 to 6 ⁇ M every 2.5 to 3.5 hours per 1 gram wet cell weight.
  • the method according to the present invention can achieve advantageous production rates of the target alkaloid.
  • the target alkaloid can be produced at a rate of at least 1 g/litre of culture every 24 hours, preferably at least 3 g/litre of culture every 24 hours, more preferably at least 5g/litre of culture every 24 hours.
  • the present invention provides a method for extracting a produced target alkaloid comprising an amine group and an -HPO 4 group from a recombinant yeast cell, the method comprising: (i) contacting the recombinant yeast cell containing the target alkaloid with a solution comprising 15 to 75% (v/v) acetonitrile to form an extraction mixture in which the target alkaloid has been extracted from the yeast cell; (ii) centrifuging the extraction mixture to form a supernatant comprising the target alkaloid; (iii) ensuring the supernatant has a pH between 10 and 14 and has a low ionic strength solution such that the charge of N in the -N(CH 3 ) 2 group is neutral; (iv) applying the supernatant to an anion exchange column; and (v) using a hydroxyl ion gradient to elute at least one fraction comprising the target alkaloid.
  • the amine group may be an -N(CH 3 ) 2 group or an -NHCH 3 group.
  • step (iii) would normally include a step of buffer exchange, e.g. via nanofiltration, to transfer the target alkaloid into a suitable buffer so that it can bind to the anion exchange column.
  • the whole cell broth may be centrifuged to separate the yeast cells from the growth media and the yeast cells washed in order to remove the media and placed the cells in a suitable media prior to contact with the acetonitrile.
  • the solution for extraction in (i) may comprise 15 to 75% (v/v) acetonitrile, preferably 20 to 60% (v/v) acetonitrile, more preferably 25 to 35% (v/v) acetonitrile.
  • the supernatant has a pH between 10 and 14 and a low ionic strength such that the charge of the N in the -N(CH 3 ) 2 group is neutral.
  • Such low ionic strength solution is a solution containing 0.05mM to 50 mM ions wherein the ions have a lower selectivity towards the positively charged groups of the anion exchange column than the -HPO 4 group.
  • the anion exchange column may be a Sepharose column, e.g. Sepharose Q HP, Sepharose Q, or Sepharose DEAE.
  • a hydroxyl ion gradient is used to elute at least one fraction comprising the target alkaloid.
  • a hydroxyl ion gradient has been found by the inventors to produce the best selectivity for the target alkaloid.
  • the at least one fraction may be two or more fractions and the method may comprise combining the two or more fractions, diluting the combined fractions and reapplying to the anion exchange column, and repeating step (v).
  • the present invention provides novel compounds related to psilocybin and to psilocin. These compounds can be prepared by the methods described above.
  • Substance (6) (A dephosphorylated form of the product is shown as Substance (6) (“S-psilocin”) below.) Substance 1 Name (IUPAC) 2-(benzothien-3-yl)ethylamine hydrochloride Substance 2 Substance 5 Formula C12H16NO4PS MW 301.2988 Fermentation technology a) Collection of yeast P. pastoris strains with integrated into the genome psiH, psiK, psiM and CPR (cytochrome P450 reductase) genes from Psilocybe genus having different S-tryptamine (Substance (1)) to S-psilocybin (Substance (5)) conversion properties were screened to find a strain with the best conversion rate.
  • Substance 1 Name
  • IUPAC 2-(benzothien-3-yl)ethylamine hydrochloride
  • Substance 2 Substance 5 Formula C12H16NO4PS MW 301.2988 Fermentation technology a) Collection of yeast
  • Target genes were placed under strong constitutive GAP promoter (enzymes are produced constantly) or very strong inducible AOX1 promoter (enzymes are produced only when methanol is added). Our research has shown that currently the best option is P. pastoris strain, where target genes are placed under GAP promoter.
  • CBS7435 strain CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1; CBS7435_pPpGAP- HIS4-psiM-psiK-psiH-CPR_2, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH- CPR_5, CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_8. II.
  • Glycerol 98% 20 g Milli Q ultrapure water (UPW) 912 g Calcium sulfate dihydrate 0.46 g Magnesium sulfate heptahydrate 5.84 g Potassium sulfate 7.34 g Ammonium sulfate 9.0 g Hexametaphosphate 300g/L solution 84 ml Trace metal solution (PTM) 4 ml Table 3 – BSM media, 1 Litre Copper (II) sulfate 3.0 g Sodium iodide 0.040 g Manganese (II) sulfate 1.50 g Sodium molybdate 0.100 g Boric acid 0.010 g Cobalt (II) chloride 0.250 g Zinc chloride 10.0 g Iron (II) sulfate heptahydrate 32.5 g Biotin 0.100 g Sulfuric acid, conc.
  • pastoris fermentation conditions that allow uptake of S-tryptamine to the cell, and efficient conversion to S-psilocybin were developed, that allow complete conversion of 2mM of S-tryptamine to S-psilocybin resulting in 600 mg of S-psilocybin in 1L WCB.
  • the process uses BSM media as shown below in Table 5.
  • Glycerol 98% 63 g Milli Q ultrapure water (UPW) 912 g Calcium sulfate dihydrate 0.46 g Magnesium sulfate heptahydrate 5.84 g Potassium sulfate 7.34 g Ammonium sulfate 9.0 g Hexametaphosphate 300g/L solution 84 ml Trace metal solution (PTM) 4 ml Table 5 – BSM media, 1L Bioreactors: EDF-1.2 bioreactors (Biotechniskais centrs, AS) Starting fermentation conditions: 28 ⁇ C, 1 vvm air, pH 5.0, 400 rpm.
  • S-psilocybin 600 mg S-psilocybin in 1L of WCB. About 20-25% of S-psilocybin is in the culture media and the rest is inside the yeast cells.
  • pH 5.0 S-tryptamine is relatively uncharged molecular that can’t freely cross yeast cells plasma membrane.
  • S- tryptamine is partly charged and can cross plasma membrane.
  • Most of the phosphate salts used for yeast fermentation precipitate under pH 7 – 7.8.
  • HMP sodium hexametaphosphate
  • S-tryptamine conversion to S-psilocybin is 10 ⁇ M of S- tryptamine to S-psilocybin in 6-7 hours per 1 gram of wet cell weight (WCW) by CBS7435_pPpGAP-HIS4-psiM-psiK-psiH-CPR_1 strain. Average amount of WCW during conversion is about 200-240 g/L, so conversion rate is 2 mM of S-tryptamine to 2 mM S- psilocybin per 6-7 hours (600 mg of S-psilocybin is synthesized in 6-7 hours).
  • S-psilocybin Different pH values, glycerol feeding profiles, amount and addition time of substrate Substance (1) - (S-tryptamine) were analyzed to develop the optimal fermentation conditions for efficient and complete conversion of substrate to final product - Substance (5) (S-psilocybin). Purification technology Testing was performed to achieve efficient and cost-effective extraction and purification of S- psilocybin. Materials and methods used for S-psilocybin extraction optimization: I. Yeast cell biomass after fermentation containing 1,5-2 mg of S-psilocybin per gram of WCW. II. S-psilocybin was extracted by resuspending cells in 1:1 to 1:5 g of WCW to ml of solution ratio using different solutions. Mixture was vortexed for 2 min.
  • Extracts were analyzed by HPLC. Materials and methods used for S-psilocybin chromatographic purification optimization: I. Extract of yeast cells, containing S-psilocybin, using 30% of acetonitrile in 1mM NaOH. II. Classical chromatography techniques were used to determine optimal S-psilocybin binding conditions, binding capacity and elution profiles. III. Fractions were analyzed by HPLC. It was noted that the findings described above in Example 1 in relation to the extraction and purification of psilocybin also applied to S-psilocybin.
  • Example 1 the purification methodology points 1 to 17 set out in Example 1 can also be used.
  • S-psilocybin elutes as a single peak at about 70-75 mM NaOH 12.7-12.9 mS/cm ( Figure 10, fractions 33-34).
  • This Example demonstrates the ability of the method of the invention to produce target alkaloids that are related to psilocybin.
  • Example 3 Biosynthesis of 3-(N,N-dimethylaminoethyl)benzo[b]furan-4-yl phosphate (O-Psilocybin) in the yeast P.
  • Substance 6 (A dephosphorylated form of the product is shown as Substance 6 (“O-psilocin”).)
  • Substance 1 Name (IUPAC): 2-(benzofuran-3-yl)ethylamine hydrochloride Formula: C10H11NO*ClH MW: 197.664 CAS no. 27404-32-6
  • Substance 2 Name 3-(2-aminoethyl)-1-benzofuran-4-ol Formula: C10H11NO2 MW: 177.20 CAS no. 1890736-76-1
  • Substance 4 Substance 5
  • Substance 6 Fermentation technology a) Yeast P.
  • Example 4 Steps for biosynthesis of further compounds in the yeast P. pastoris cells using enzymes from fungi of Psilocybe genus
  • Example 4A To provide a compound according to formula I described herein where X is N and Z is C and W is N (a benzimidazole derivative).
  • the substrate can be 2- (1H-Benzimidazol-1-yl)ethylamine hydrochloride and the following synthesis pathway can be used: 1. Substrate (starting material) 2-(1H-Benzimidazol-1- yl)ethylamine hydrochloride 2. 3. 4. 5. 6. Example 4B — To provide a compound according to formula I described herein where X is O and Z is N and W is C (a benzisoxazole derivative).
  • the substrate (starting material) can be 1,2-Benzisoxazole-3-ethanamine and the following synthesis pathway can be used: Example 4C – To provide a compound according to formula I described herein where X is O and Z is C and W is N (a benzo[d]oxazole derivative).
  • the substrate (starting material) can be 2-(benzo[d]oxazol-3(2H)-yl)ethan-1-amine and the following synthesis pathway can be used: FUNCTIONAL STUDIES
  • the functional activity of the psilocybin related compounds described herein can be shown in vitro using radioligand binding assays and in vivo using head twitch response studies.
  • Competition Assays can be used to show binding to 5-HT receptors, e.g. 5-HT1A.
  • a suitable example assay is described by Gifford Bioscience Limited (www.giffordbioscience.com - Radioligand Binding Assay Protocols). This assay is as follows: Membrane preparation: Frozen tissue or washed cells are homogenized in 20 volumes of cold lysis buffer (50mM Tris-HCl, 5 mM MgCl2, 5 mM EDTA, protease inhibitor cocktail). After a low speed spin (100 x g for 3 minutes) to remove large tissue chunks (tissue homogenates), the homogenate is centrifuged at 17,000 x g for 10 minutes at 4 °C to pellet the membranes.
  • the pellet is resuspended in fresh buffer and centrifuged at the same speed for a second time, again at 4 °C.
  • the pellet is then resuspended into buffer (15 ml) containing 10% sucrose as a cryoprotectant, divided into 1 ml aliquots and stored at -80 °C.
  • a sample of the homogenate is analyzed for protein content.
  • the membrane preparation is thawed and the pellet resuspended in final assay binding buffer (50 mM Tris, 5 mM MgCl2, 0.1 mM EDTA, pH 7.4).
  • Incubation and filtration The filtration binding assay is carried out in 96-well plates in a final volume of 250 ⁇ L per well. To each well is added 150 ⁇ L membranes (3 - 20 ⁇ g protein for cells or 50 - 120 ⁇ g protein for tissue), 50 ⁇ L of the competing test compound and 50 ⁇ L of radioligand solution in buffer. The plate is incubated at 30 °C for 60 minutes with gentle agitation. The incubation is stopped by vacuum filtration onto 0.3% PEI presoaked GF/C filters using a 96-well FilterMateTM harvester followed by four washes with ice-cold wash buffer. Filters are then dried for 30 minutes at 50 °C.
  • Saturation assays can also be used to show binding to 5-HT receptors, e.g. 5-HT1A.
  • a suitable example assay is described by Gifford Bioscience Limited (www.giffordbioscience.com - Radioligand Binding Assay Protocols). This assay is as follows: Membrane preparation: as described above for the competition assay. Incubation and filtration: The filtration binding assay is carried out in 96-well plates in a final volume of 250 ⁇ L per well.
  • membranes (3 - 20 ⁇ g protein for cells; 50 - 120 ⁇ g protein for tissue), 50 ⁇ L of the unlabeled compound (non-specifics) or buffer and 50 ⁇ L of radioligand solution in binding buffer.
  • the radioligand is added at up to 8 different concentrations (e.g. 0.2 - 20 nM).
  • the plate is incubated at 30 °C for 60 minutes with gentle agitation. The incubation is stopped by vacuum filtration onto 0.3% PEI presoaked GF/C filters using a 96-well FilterMateTM harvester followed by four washes with ice-cold wash buffer. Filters are then dried for 30 minutes at 50 °C.
  • the filter is sealed in polyethylene, scintillation cocktail (Betaplate Scint; PerkinElmer) added and the radioactivity counted in a Wallac® TriLux 1450 MicroBeta counter. Data analysis: For each radioligand concentration, non-specific binding is subtracted from total binding to give specific binding. Bound CPM values are converted to fmoles per mg protein. Data is fitted using the saturation analysis non-linear curve fitting routines in Prism® (Graphpad Software Inc). The Kd (in nM) and Bmax (fmol/mg or sites/cell) are derived from the saturation curve.
  • mice C57BL/6J male mice aged 8-12 weeks are used in the study. Water and food are provided ad libitum throughout the entire study. The holding room maintains a temperature of 21 ⁇ 1°C, humidity at 55 ⁇ 10%, and operates on a 12-hour light/dark cycle. All efforts are made to minimize animal suffering and reduce the number of animals used. Each animal are weighed at the start of the experiment.
  • Treatment Groups Mice are randomly assigned to different treatment groups, with each group receiving either a PSY series compound or a control substance (saline).
  • Test Substances Stock solutions of test substances are prepared on the same day of the experiment and are protected from direct light.
  • Administration The compounds are administered via intraperitoneal injections (i.p.), and the dosages administered are individually determined based on each animal's body weight.
  • Behavioural Assessment Immediately following the intraperitoneal injection of the drug, a mouse is placed in the open field, and video recording is initiated for 30 minutes. A camera is positioned directly above the mouse to capture its behaviour. The environment is kept quiet to minimize disturbances during the recording period.
  • Data Collection Review the recorded videos and count the number of head twitch responses displayed by each mouse during the observation period. A head twitch response is characterized by a rapid, involuntary, rotational movement of the head.
  • the aim of the present example was to screen the agonist effect of 4 test compounds on the 5- HT2A serotonin receptor activity using the U2OS 5-HTR2A Serotonin receptor Hitseeker cell line.
  • HiTSeeker 5-HTR2A/U2OS contains U2OS cells stably expressing human 5-HT2A Serotonin receptor with no tag.
  • the HiTSeeker HTR2A cell line has been designed to assay compounds or analyse their capability to modulate 5-HT2A Serotonin receptor.
  • a G protein is activated, which in turn, triggers a cellular response mediated by second messengers (Calcium). This cellular response can be measured quantifying calcium increase inside the cell determining the intensity of Fluo4.
  • Fluorescence intensity acquisition was performed in the SynergyTM 2 Multi-Detection microplate reader from Biotek. Vehicle (NaCl or H 2 O) was used as negative control; 5-HT was used as positive control. Compounds were tested at 200, 100, 50, 25, 10, 5, 2.5, 1, 0.5 ⁇ M in triplicates.
  • the U2OS 5-HTR2A Hitseeker cell line was thawed (2x10 6 cells per T25).
  • the cells were maintained in DMEM-F12 supplemented with 10% FBS at 37oC in a humidified 5% CO 2 atmosphere.
  • Day 3 The cells were plated at a concentration of 20.000 cells/well (+/-1000 cells) in 96- well plates.
  • 5-HT at 10 ⁇ M was used as positive control and vehicle (water for PSY- 1185 and PSY-1186 and NaCl for PSY-1342 and PSY-1134) as negative control.
  • Figure 15 shows the results obtained after treating the cells with the compounds. Data are presented as fluorescence intensity of Fluo-4 representing calcium increase after HTR2A stimulation. For each compound also negative control (vehicle) and 5-HT are presented. Two compounds showed an increase in intensity compared to the control: compound PSY- 1185 is the most active, exhibiting an increase of 2.76 fold compared to the control and demonstrating a clear dose-dependent activity; and compound PSY-1343 exhibit a smaller increase, 2.4 fold, and only at higher concentrations, but in a concentration-dependent manner.
  • PSY-1185 emerged as the most potent activator, showing a clear dose-response relationship across the concentrations tested, with an EC 50 value of 2.88 x 10 ⁇ 6 M. This indicates that PSY-1185 has a relatively high affinity for the 5-HT2A receptor, making it the strong agonist. PSY- 1343 exhibited measurable activity, and this was only observed at the two highest concentrations tested. These findings demonstrate the capabilities of PSY-1185 and PSY-1343.
  • SEQUENCES The sequences allocated SEQ ID Nos. herein have the following sequences: SEQ ID No: 1 Description: cDNA sequence of cytochrome P450 reductase CPR (P.

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Abstract

La présente invention concerne un procédé de production d'un alcaloïde cible à partir d'un précurseur par l'intermédiaire d'une voie métabolique dans une cellule de levure recombinante, la voie métabolique comprenant une enzyme psiH, une enzyme psiK et une enzyme psiM, le procédé comprenant la culture de la cellule de levure recombinante dans un milieu de culture comprenant le précurseur dans des conditions appropriées pour permettre au précurseur d'entrer dans la cellule hôte recombinante de telle sorte que la cellule de levure recombinante produit l'alcaloïde cible, le précurseur étant un substrat pour l'enzyme psiH. L'invention concerne également des procédés associés et des cellules recombinantes destinées à être utilisées dans ceux-ci. La présente invention concerne en outre des composés associés à la psilocybine, des compositions pharmaceutiques les comprenant, et leur utilisation en médecine.
PCT/EP2024/077495 2023-09-29 2024-09-30 Production et utilisation médicale de psilocybine et de composés apparentés Pending WO2025068602A2 (fr)

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US11441164B2 (en) 2019-11-15 2022-09-13 Cb Therapeutics, Inc. Biosynthetic production of psilocybin and related intermediates in recombinant organisms

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CA3191102A1 (fr) * 2020-09-01 2022-03-10 Jillian M. HAGEL Derives de psilocybine halogenes et leurs procedes d'utilisation
CA3208152A1 (fr) * 2021-02-12 2022-08-18 Jillian M. HAGEL Derives de psilocybine a substituants multiples et leurs procedes d'utilisation
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WO2021052989A1 (fr) 2019-09-16 2021-03-25 Danmarks Tekniske Universitet Cellules de levure et procédés de production de dérivés de tryptophane
US11441164B2 (en) 2019-11-15 2022-09-13 Cb Therapeutics, Inc. Biosynthetic production of psilocybin and related intermediates in recombinant organisms

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