Inhibition of serotonin transporter (5-HTT)

Abstract

Compositions comprising mesembryanol, such as (-) mesembryanol or (-) 6-epi-mesembryanol, are disclosed. The disclosed compositions can be used in methods of inhibiting SERT. Also provided are methods of treatment against anxiety or depression, including methods comprising orally administering to a subject in need thereof a therapeutically effective amount of certain pharmaceutical compositions.

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 434,726, filed on December 22, 2022, the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present disclosure relates to the field of medicine, including the discovery of alkaloid compounds that are useful for inhibiting the serotonin transporter (5-HTT). Background Art

[0004] Plants in the genus Sceletium contain indole alkaloids, which have biological activity that can be used to treat mental health conditions such as mild to moderate depression. Natural extracts of Sceletium tortuosum (a South African native herb also known as "kougoed," "channa," or "kanna") can contain pharmacologically active alkaloids. Among the alkaloids listed below, mesembrine, mesembrenone, and mesembrenol are present in Sceletium tortuosum extracts used to treat anxiety, stress, and mental health disorders.

[0005]

[0006] Mesembranol has not been well characterized pharmacologically and is present in relatively low concentrations in plant extracts relative to other bioactive Mesembranol alkaloids. Mesembranol is virtually absent in extracts derived from the Mesembranol plant. Figures 1A-1C Liquid chromatography mass spectrometry (LC-MS) chemical fingerprints showing the alkaloids present in plants of Mesembryanthemum contortum taken from three different regions of South Africa, where Mesembryanthemum contortum is native (J. Zhao et al., Phytochemistry, 2018). In each chromatogram, the peak at 3.77 minutes is attributed to mesembryanthol. From the Northern Cape ( Figure 1A ) and Western Cape 1( Figure 1B ) showed only trace amounts of mesembryanthemum. Mesembryanthemum was not the main alkaloid present and was only moderately abundant in the Western Cape 2 sample (the 4th most abundant alkaloid in the Western Cape 2 sample; Figure 1C These data suggest that extracts of the Mesembryanthemum contortum plant are unlikely to produce sufficient amounts of mesembryol to provide therapeutic benefit.

[0007] In addition, extracts from Mesembryanthemum contortum contain a variety of bioactive alkaloids with a range of pharmacological effects. For example, the reported pharmacological activities of Mesembryanthemum contortum extracts include serotonin transporter inhibition (5-HTT), phosphodiesterase-4 inhibition (PDE4), monoamine oxidase A (MAO-A) inhibitory activity, acetylcholinesterase (AChE) inhibitory activity, GABA receptor binding activity, opioid receptor binding activity, dopamine transporter inhibition, AMPA receptor modulation, effects on vesicular monoamine transporter-2 (VMAT-2) and subsequent monoamine release.

[0008] An analysis of a standardized commercial extract of Mesembryanthemum contortum was reported in 2011 (as the trade name The product obtained from the method is a product having 0.35%-0.45% total alkaloids, of which mesembrine and mesembrenol account for ≥60% and mesembrine accounts for <20% (see Harvey et al., “Pharmacological actions of the South African medicinal and functional food plant Sceletium tortuosum and its principal alkaloids,” Journal of Ethnopharmacology 137 (2011) 1124-1129 and Murbach et al., “A toxicological safety assessment of a standardized extract of Sceletium tortuosum in rats,” Food and Chemical Toxicology 74 (2014) 190-199. The extract inhibited the serotonin (5-HT) transporter by >80%, with the potency of the isolated alkaloids on the 5-HT transporter reported in Table A below (Harvey et al., 2011). Referring to the data in Table A, concentration-dependent inhibition was found, with mesembrine being the more active compound in the 5-HT transporter assay (i.e., 20-fold more potent than mesembrine and 87-fold more active than mesembrenol). A toxicological safety assessment of the standardized extract was subsequently reported in 2014 (Murbach et al., 2014).

[0009] Table A. Summary of analysis of concentration response curves for alkaloid binding to the 5-HT transporter (Harvey et al., 2011)

[0010]

[0011] However, bioactive plant extracts for therapeutic consumption can vary widely seasonally and among different P. tortuous plants and have failed to provide a sufficiently reproducible and stable phytochemical profile of the desired bioactive components. Harmsia plants and their extracts can vary widely in terms of total alkaloid content as well as the chemical nature and relative concentration of individual Harmsia plant-derived alkaloids. Furthermore, it has been reported that the concentration of pinoresinol varies among different regions of South Africa in P. tortuous and that pinoresinol abundance is relatively low in most plant extracts tested. Finally, Harmsia alkaloids can be unstable under various conditions that can occur during extraction from plant material as well as during storage and formulation of the extract.

[0012] In P. tortuous extracts, pinoresinol and its isomer 6-epi-pinoresinol have low concentrations compared to other major alkaloids. As a result, pinoresinol and 6-epi-pinoresinol have not been fully characterized in the scientific literature. The pharmacological activity and selectivity of pinoresinol or 6-epi-pinoresinol have not been reported. Their therapeutic use is limited by the low abundance, variability, and instability of these alkaloids in natural extract products as well as the instability and pharmacokinetic profile of these compounds obtained from natural products. Naturally occurring pinoresinol and 6-epi-pinoresinol are serotonin transporter inhibitors with high specificity relative to other aforementioned pharmacological targets. P. tortuous alkaloid extracts do not produce sufficient levels of pinoresinol and 6-epi-pinoresinol relative to other components, and the extract can also exert other additional pharmacological effects that are not desirable for therapeutic regimens for anxiety or depression.

[0013] There remains an unmet need for pharmaceutical compositions comprising therapeutic alkaloid compounds such as pinoresinol and 6-epi-pinoresinol in a higher purity, predictable, stable, and reproducible form. Furthermore, there is a need for oral pharmaceutical compositions that provide pure therapeutic alkaloid compositions with desirable pharmacokinetic properties at the time of administration. Finally, there is an unmet need for pharmaceutical compositions comprising properties that are significantly different from naturally occurring compositions obtained from plant extracts. SUMMARY

[0014] Applicants have discovered new and useful methods for using pinoresinol (i.e., (-) pinoresinol or (-) 6-epi-pinoresinol). In some embodiments, pinoresinol (Compound 18) or (-) 6-epi-pinoresinol (Compound 19) can be used to selectively inhibit the serotonin transporter (5-HTT) at or near the levels found in other pharmacologically active alkaloids identified in P. tortuous extracts while avoiding inhibition of phosphodiesterase-4 (PDE4).

[0015]

[0016] In some embodiments, the methods of using (-)mecetin or (-)6-epi-mecetin are based in part on the discovery that (-)mecetin or (-)6-epi-mecetin can be used to provide a therapeutic mechanism of action similar to that of selective serotonin reuptake inhibitors (SSRIs).

[0017] In some embodiments, the methods using (-)6-epi-mecenitol are based in part on the finding of distinct pharmacokinetic data for (-)6-epi-mecenitol (Compound 019) and (-)mecenitol (Compound 018), including the finding that the half-life of Compound 019 is approximately three times greater than that of Compound 018 and approximately twice as long as the half-life measured for mesembrine (Compound 001) (as measured following intravenous administration to dogs according to Example 7).

[0018] In some embodiments, (-)6-epi-mecenitol (Compound 19) can be used to selectively inhibit the serotonin transporter (5-HTT) at or near levels found in other pharmacologically active alkaloids identified in Mesembryanthemum twist extracts, while avoiding inhibition of phosphodiesterase-4 (PDE4). In some embodiments, (-)6-epi-mecenitol (Compound 19) can be used to treat anxiety or depression. In certain embodiments, a method of treating anxiety and depression comprises orally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising Compound 019,

[0019]

[0020] or a pharmaceutically acceptable salt thereof. In certain embodiments, the method is a method of treating major depressive disorder (MDD). In certain embodiments, the method is a method of treating generalized anxiety disorder (GAD).

[0021] In certain embodiments, Compound 019 or a pharmaceutically acceptable salt thereof is administered in the form of a capsule or tablet. In certain embodiments, Compound 019 or a pharmaceutically acceptable salt thereof is administered once or twice daily. In certain embodiments, the pharmaceutical composition contains no more than about 1% of an alkaloid selected from (-) mesembrine, mesembrine, mesembrenol, or a combination thereof.

[0022] In certain embodiments, a method of inhibiting SERT comprises administering to a mammal in need thereof a therapeutically effective amount of

[0023]

[0024] or a pharmaceutically acceptable salt thereof.

[0025] In certain embodiments, the pharmaceutical composition comprises

[0026]

[0027] or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition contains no more than about 3% alkaloids selected from the group consisting of (-)pennsivyn, pennsivynol, pennsivynenol, or combinations thereof; and Compound 019 comprises at least 90% of the total alkaloid content in the pharmaceutical composition. In certain embodiments, the ratio of PDE4 / SERT inhibition values of the pharmaceutical composition is at least 10: 1. In certain embodiments, Compound 019 has a half-life of at least 30 minutes in a human hepatocyte assay of the pharmaceutical composition. In certain embodiments, the pharmaceutical composition has an IC 50 In certain embodiments, the pharmaceutical composition has less than about 6% inhibition % of at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2. In certain embodiments, the ratio of PDE4 / SERT inhibition values of the pharmaceutical composition is at least 10: 1.

[0028] In certain embodiments, a method of inhibiting SERT without inhibiting PDE4 comprises administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition comprising Compound 019, or a pharmaceutically acceptable salt thereof. In certain embodiments, the composition comprises Compound 019, or a pharmaceutically acceptable salt thereof, and less than about 0.5% pennsivyn or pennsivynol as measured by HPLC.

[0029] A method of treating anxiety or depression can comprise orally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising Compound 019,

[0030]

[0031]

[0032] or a pharmaceutically acceptable salt thereof. In some embodiments, the method is a method of treating major depressive disorder (MDD) or a method of treating generalized anxiety disorder (GAD).

[0033] In some embodiments, Compound 019, or a pharmaceutically acceptable salt thereof, is administered in the form of a capsule or tablet. In some embodiments, Compound 019, or a pharmaceutically acceptable salt thereof, is administered once or twice a day. In some embodiments, the pharmaceutical composition contains no more than about 1% alkaloids selected from the group consisting of (-)pennsivyn, pennsivynol, pennsivynenol, or combinations thereof.

[0034] In some embodiments, a method of inhibiting SERT comprises administering to a mammal in need thereof a therapeutically effective amount of

[0035]

[0036] or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, a pharmaceutical composition is provided, comprising

[0038]

[0039] or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition contains no more than about 3% of an alkaloid selected from (-)mecetin, mesembrine, mesembrenol, or a combination thereof; and Compound 019 accounts for at least 90% of the total alkaloid content in the pharmaceutical composition.

[0040] In some embodiments, the ratio of PDE4 / SERT inhibition values ​​of the pharmaceutical composition is at least 10: 1. In some embodiments, Compound 019 has a half-life of at least 30 minutes in a human hepatocyte assay of the pharmaceutical composition. In some embodiments, the pharmaceutical composition has an IC of less than about 30 nM for SERT. 50 In some embodiments, the pharmaceutical composition has a % inhibition of at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 of less than about 6%. In some embodiments, the ratio of PDE4 / SERT inhibition values ​​of the pharmaceutical composition is at least 10:1.

[0041] In some embodiments, a method of inhibiting SERT without inhibiting PDE4 comprises administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition comprising Compound 019 or a pharmaceutically acceptable salt thereof.

[0042] In some embodiments, provided is a composition comprising Compound 019 or a pharmaceutically acceptable salt thereof, wherein the composition comprises less than about 0.5% mesembrine or mesembrine-rine as measured by HPLC.

[0043] In some embodiments, a method of treating anxiety or depression comprises orally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising compound 018,

[0044]

[0045] or a pharmaceutically acceptable salt thereof.

[0046] Numerous other embodiments are further provided, which may be applied to any aspect of the invention described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A is a chromatogram of a Northern Cape sample as reproduced from J. Zhao et al., Phytochemistry, 2018.

[0048] Figure 1B is a chromatogram of the Western Cape 1 sample as reproduced from J. Zhao et al., Phytochemistry, 2018.

[0049] Figure 1C is a chromatogram of the Western Cape 2 sample as reproduced from J. Zhao et al., Phytochemistry, 2018.

[0050] Figure 2A Graph showing compound stability of (-)mesembrine (triangles) compared to (-)mesembrol (squares) in human hepatocytes.

[0051] Figure 2B Graph showing compound stability of (-)mesembrine (triangles) compared to (-)6-epi-mesembrol (circles) in human hepatocytes.

[0052] Figure 3 is a graph showing the pharmacokinetic profiles of Compound 018 and Compound 019 evaluated in dogs, showing plasma concentrations (nM) of Compound 018, Compound 019, and Compound 001 following intravenous (IV) administration of the compounds at 2 mg / kg.

[0053] Figure 4 is a graph showing the plasma concentrations (nM) of Compound 018, Compound 019, and Compound 001 following oral (PO) administration of the compounds at 10 mg / kg. DETAILED DESCRIPTION

[0054] Described herein are compositions comprising a compound selected from mesembryanthemum or 6-epi-mesembryanthemum, or a pharmaceutically acceptable salt thereof, and methods of using (-)mesembryanthemum and (-)6-epi-mesembryanthemum.

[0055] Applicants have discovered new and useful methods of using salviagenol (i.e., (-) salviagenol or (-) 6-epi-salviagenol). In some embodiments, salviagenol (Compound 18) or (-) 6-epi-salviagenol (Compound 19) can be used to selectively inhibit the serotonin transporter (5-HTT) at or near the levels found in other pharmacologically active alkaloids identified in extracts of Salvia divinorum, while avoiding inhibition of phosphodiesterase-4 (PDE4).

[0056]

[0057] In some embodiments, methods of using (-) salviagenol or (-) 6-epi-salviagenol are based in part on the discovery that (-) salviagenol or (-) 6-epi-salviagenol can be used to provide a therapeutic mechanism of action similar to selective serotonin reuptake inhibitors (SSRIs).

[0058] In some embodiments, a method of treating a mental health disorder includes administering to a mammal in need thereof an effective amount of a compound selected from (-) salviagenol and (-) 6-epi-salviagenol, or a salt thereof. In certain embodiments, the compound is (-) salviagenol. In certain embodiments, the compound is (-) 6-epi-salviagenol.

[0059] In certain embodiments, the present disclosure provides a method of inhibiting SERT, the method comprising administering to a mammal in need thereof a therapeutically effective amount of a compound selected from (-) salviagenol and (-) 6-epi-salviagenol, or a salt thereof. In certain embodiments, the salviagenol is (-) salviagenol. In certain embodiments, the compound is (-) salviagenol. In certain embodiments, the compound is (-) 6-epi-salviagenol.

[0060] In certain embodiments, the present disclosure provides a method of inhibiting SERT, the method comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound selected from (-) salviagenol and (-) 6-epi-salviagenol, or a salt thereof. In certain embodiments, the compound is (-) salviagenol. In certain embodiments, the compound is (-) 6-epi-salviagenol.

[0061] In certain embodiments, the pharmaceutical composition comprises (-) 6-epi-salviagenol and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises salviagenol and a pharmaceutically acceptable excipient. In certain embodiments, the present disclosure provides a method of treating a mental disorder, the method comprising administering to a subject a compound of the present disclosure. In certain embodiments, a method of inhibiting SERT without inhibiting PDE4 comprises administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition.

[0062] In certain embodiments, the composition comprises mesembryl (Compound 18) or a pharmaceutically acceptable salt thereof and less than about 0.5% mesembrine or mesembrine-based as measured by HPLC. In certain embodiments, the composition comprises (-)6-epi-mesembryl (Compound 19) or a pharmaceutically acceptable salt thereof and less than about 0.5% mesembrine or mesembrine-based as measured by HPLC.

[0063] In certain embodiments, a method of treating anxiety comprises administering to a subject in need thereof a pharmaceutical composition comprising mesembryanthemum (or a pharmaceutically acceptable salt thereof) and / or (-)6-epi-mesembryanthemum (or a pharmaceutically acceptable salt thereof). In certain embodiments, a method of inhibiting serotonin transporter (5-HTT) in the central nervous system of a subject comprises administering to a subject in need thereof a pharmaceutical composition comprising mesembryanthemum (or a pharmaceutically acceptable salt thereof) and / or (-)6-epi-mesembryanthemum (or a pharmaceutically acceptable salt thereof), wherein the pharmaceutical composition does not inhibit PDE4A1A, PDE4B2, PDE4C1, or PDE4D2 by more than 5% at 10 micromolar.

[0064] In some embodiments, pinolenic acid (Compound 18) or (-)6-epi-pinolenic acid (Compound 19) can be used to selectively inhibit the serotonin transporter (5-HTT) at or near the levels found in other pharmacologically active alkaloids identified in Pinus contorta extract while avoiding inhibition of phosphodiesterase-4 (PDE4). Applicants have discovered that pinolenic acid (Compound 18) and (-)6-epi-pinolenic acid (Compound 19) are potent inhibitors of the serotonin transporter (5-HTT) with no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar. Compounds 18 and 19 surprisingly exhibit substantially lower activity against PDE4 than other pharmacologically active alkaloid species identified in Pinus contorta extract. For example, as measured by % inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, alkaloid (+) pinocembrin (Compound 2) exhibits about 6 to 23 times greater PDE4 activity than either Compound 18 or Compound 19. As measured by % inhibition of PDE4A1A and PDE4B2 at 10 micromolar, isolated pinolenic acid enantiomers (Compounds 20, 21, 23, and 24) show about 3 to 13 times greater PDE4A1A activity and about 3 to 12 times greater PDE4B2 activity than Compound 18. In some embodiments, a pharmaceutical composition comprises an active pharmaceutical ingredient (API) consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) and / or Compound 19 (or a pharmaceutically acceptable salt thereof), wherein the API is free of pinocembrin and pinokinin.

[0065] In certain embodiments, the pharmaceutical composition comprises (-) mesembryant and / or (-) 6-epi-mesembryant. In certain embodiments, the pharmaceutical composition contains no more than about 10% of an alkaloid selected from (-) mesembryine, mesembryone, mesembrenol, alone or in combination. For example, the pharmaceutical composition may contain less than about 10% by weight, less than about 5% by weight, less than about 3% by weight, less than about 2% by weight, or less than about 1% by weight of a combination of alkaloids of (-) mesembryine and mesembrenol, or a combination of mesembrenol and mesembrenol, or a combination of (-) mesembryine, mesembrenol, and mesembrenol. In certain embodiments, the composition is substantially free of (-) mesembryine, mesembrenol, and mesembrenol. For example, the pharmaceutical composition may contain alkaloids, and less than about 10% by weight, less than about 5% by weight, less than about 3% by weight, less than about 2% by weight, or less than about 1% by weight of the total alkaloids are a combination of (-) mesembrine and mesembrine, or a combination of mesembrine and mesembrenol, or a combination of (-) mesembrine, mesembrine, and mesembrenol. In certain embodiments, the pharmaceutical composition may contain, for example, about 0.3% by weight to about 0.5% by weight of alkaloids, and mesembrine and mesembrenol account for less than about 60% by weight of the total alkaloid content. In certain embodiments, the pharmaceutical composition may contain, for example, about 0.3% by weight to about 0.5% by weight of alkaloids, and mesembrine accounts for less than about 20% by weight of the total alkaloid content. In certain embodiments, the pharmaceutical composition may contain, for example, about 0.3 wt% to about 0.5 wt% alkaloids, with (-)mesequinol and / or (-)6-epimesequinol comprising at least about 70%, at least 80%, or at least about 90% of the total alkaloids in the composition. In certain embodiments, the composition comprises less than about 70 micrograms of mesequinone and mesequinone combined. In certain embodiments, the composition comprises less than about 23 micrograms of (-)mesequinone.

[0066] In certain embodiments, the composition comprises less than about 70 micrograms of mesembrine and mesembrenol per 11.3 mg of total alkaloids. In certain embodiments, the composition comprises less than about 23 micrograms of (-)mesembrine per 11.3 mg of total alkaloids.

[0067] In some embodiments, the method for treating a patient with a disease comprises administering to the patient a composition comprising a compound disclosed herein, for treating or preventing a mental health disorder. In some embodiments, the method for treating a patient with a disease comprises administering to the patient a composition comprising a compound disclosed herein, for treating or preventing a diagnosed condition selected from anxiety, stress and depression. In some embodiments, the method comprises administering to a patient in need a therapeutically effective amount of a compound selected from (-) mesembranol and / or (-) 6-epimeriol, for treating depression. In some embodiments, the method comprises administering to a patient in need a therapeutically effective amount of a compound selected from (-) mesembranol and / or (-) 6-epimeriol, for treating a condition selected from the group consisting of: anxiety associated with depression, anxiety with depression, mixed anxiety and depressive disorders. In some embodiments, the method comprises administering to a patient in need a therapeutically effective amount of a compound selected from (-) mesembranol and / or (-) 6-epimeriol, for treating anxiety and hysteria or anxiety and depression.

[0068] In certain embodiments, the present disclosure provides a method of treating a psychiatric disorder, comprising administering to a subject a compound of the present disclosure (eg, a compound selected from (-)mesequinol and / or (-)6-epi-mesequinol).

[0069] In certain embodiments, the present disclosure provides a method of inhibiting SERT, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound selected from (-)mecetin and (-)6-epi-mecetin. In certain embodiments, the compound is (-)mecetin. In certain embodiments, the compound is (-)6-epi-mecetin. In certain embodiments, the compound (e.g., (-)mecetin or (-)6-epi-mecetin) is in the form of a composition according to the present disclosure.

[0070] In certain embodiments, the disclosure provides a method of inhibiting SERT, comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound selected from (-)pennsylvanin and (-)6-epi-pennsylvanin, or a salt thereof. In certain embodiments, the compound is (-)pennsylvanin. In certain embodiments, the compound is (-)6-epi-pennsylvanin. In certain embodiments, the pharmaceutical composition contains no more than about 10% of the alkaloids selected from (-)pennsylvanin, pennsylvanic acid, pennsylvanenol, either alone or in combination. For example, the pharmaceutical composition can contain less than about 10%, less than about 5%, less than about 3%, less than about 2%, or less than about 1% by weight of the combination of (-)pennsylvanin and pennsylvanic acid, or the combination of pennsylvanic acid and pennsylvanenol, or the combination of (-)pennsylvanin, pennsylvanic acid, and pennsylvanenol. In certain embodiments, the composition is substantially free of (-)pennsylvanin, pennsylvanic acid, and pennsylvanenol.

[0071] In some embodiments, the compounds disclosed herein are administered to a patient in a unit dose. In some embodiments, the compounds disclosed herein are prescribed to a patient in an oral unit dose (such as a capsule or tablet) once or more times per day. In some embodiments, the compounds disclosed herein are administered to a patient to treat a disease or condition for which pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) is safe and effective for the treatment. In some embodiments, the methods comprise administering to a patient in need thereof a therapeutically effective amount of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) for the treatment of anxiety. In some embodiments, the methods comprise administering to a patient in need thereof a therapeutically effective amount of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) for the treatment of a disease selected from the group consisting of mild to moderate depression and major depressive episode. In some embodiments, the methods comprise administering to a patient in need thereof a therapeutically effective amount of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) for the treatment of a disease selected from the group consisting of psychological and psychiatric disorders with presence of anxiety. In some embodiments, the methods comprise administering to a patient in need thereof a therapeutically effective amount of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) for the treatment of major depressive episode. In some embodiments, the methods comprise administering to a patient in need thereof a therapeutically effective amount of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) for the treatment of a disease selected from the group consisting of alcohol and drug dependence, bulimia nervosa, and obsessive-compulsive disorder. In some embodiments, an amount of 20 micrograms to 2 milligrams of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) is administered orally to a patient in need thereof. In some embodiments, an amount of 20 micrograms to 2 milligrams of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) is administered orally to a patient in need thereof.

[0072] In some embodiments, the methods comprise administering to a patient in need thereof a therapeutically effective amount of pinolenic acid (e.g., (-)pinolenic acid or (-)6-epi-pinolenic acid) for the treatment of a disease selected from the group consisting of acute and maintenance treatment of major depressive disorder (MDD), acute and maintenance treatment of obsessive-compulsive disorder (OCD), acute and maintenance treatment of bulimia nervosa, and acute treatment of panic disorder with or without agoraphobia.

[0073] In some embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of a royleanol (e.g., (-)royleanol or (-)6-epi-royleanol) for the treatment of a disease selected from the group consisting of major depressive disorder (MDD), obsessive-compulsive disorder (OCD), panic disorder (PD), social anxiety disorder (SAD), generalized anxiety disorder (GAD), and post-traumatic stress disorder (PTSD).

[0074] In some embodiments, the method comprises administering to a patient in need thereof a therapeutically effective amount of a royleanol (e.g., (-)royleanol or (-)6-epi-royleanol) for the treatment of a disease selected from the group consisting of major depressive disorder (MDD), obsessive-compulsive disorder (OCD), panic disorder (PD), post-traumatic stress disorder (PTSD), social anxiety disorder (SAD), and premenstrual dysphoric disorder (PMDD).

[0075] In certain embodiments, the (-)6-epi-royleanol of the pharmaceutical composition has a half-life in a human hepatocyte assay (e.g., a human hepatocyte assay performed according to Example 4) of at least 30 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes.

[0076] In certain embodiments, the (-)6-epi-royleanol has an AUC 最后 ratio to (-)royleucine of about 2 to about 2.5. In certain embodiments, the (-)6-epi-royleanol has an AUC 最后 ratio to (-)royleucine of about 4 to about 4.5.

[0077] In certain embodiments, for example in an assay performed according to Example 5, Compound 018 has about 3 times longer plasma half-life, about 9 times higher Cmax, and about 4 times greater AUC (last) compared to royleucine (Compound 001). In certain embodiments, for example in an assay performed according to Example 5, Compound 019 has about 2 times longer plasma half-life, 2 times higher Cmax, and about 2 times greater AUC (last) compared to royleucine (Compound 001).

[0078] In certain embodiments, a method of inhibiting SERT without inhibiting PDE4 comprises administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound selected from (-)royleanol and (-)6-epi-royleanol. In some embodiments, the pharmaceutical composition has an IC 50In certain embodiments, the pharmaceutical composition has an inhibition % of less than about 6% for at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 (e.g., as performed in Example 3B). In some embodiments, the pharmaceutical composition has an inhibition % of less than about 6% for at least two of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 (e.g., as performed in Example 3B). In some embodiments, the pharmaceutical composition has an inhibition % of less than about 6% for at least three of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 (e.g., as performed in Example 3B). In some embodiments, the pharmaceutical composition has an inhibition % of less than about 6% for each of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 (e.g., as performed in Example 3B).

[0079] Pharmaceutical composition

[0080] In certain embodiments, the present application relates to a pharmaceutical composition comprising an active pharmaceutical ingredient. In certain embodiments, the pharmaceutical composition comprises a compound as disclosed herein as an active pharmaceutical ingredient (API) and a pharmaceutically acceptable carrier comprising one or more excipients. In some embodiments, the pharmaceutical composition optionally further comprises an additional therapeutic compound (i.e., agent) with a pharmaceutically acceptable carrier. The pharmaceutical composition can be a medicament.

[0081] In some embodiments, the pharmaceutical composition comprises Compound 18 and / or Compound 19, or a pharmaceutically acceptable salt thereof, in the absence of one or more of the pharmacologically active alkaloid compounds in a St. John’s Wort extract. The pharmaceutical composition comprising Compound 18, Compound 19, or mixtures thereof, including pharmaceutically acceptable salts of Compound 18 and Compound 19, can be used to inhibit the serotonin transporter (5-HTT) at levels found in other pharmacologically active alkaloids identified in St. John’s Wort extracts, such as hypericin and hyperforin, while avoiding inhibition of phosphodiesterase-4 (PDE4).

[0082] In some embodiments, the pharmaceutical composition may comprise mesembryl (Compound 18) and / or (-)6-epi-mesembryl (Compound 19) or a pharmaceutically acceptable salt thereof, including a mixture of Compound 18 and Compound 19, and less than 0.5% mesembrine or mesembrylon-based detectable by HPLC. In some embodiments, the pharmaceutical composition may comprise mesembryl (Compound 18) or a pharmaceutically acceptable salt thereof, and less than 0.5% mesembrine or mesembrylon-based detectable by HPLC. In some embodiments, the pharmaceutical composition may comprise (-)6-epi-mesembryl (Compound 19) or a pharmaceutically acceptable salt thereof, and less than 0.5% mesembrine or mesembrylon-based detectable by HPLC.

[0083] In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) and / or Compound 19 (or a pharmaceutically acceptable salt thereof) for treating anxiety in a subject in need thereof. In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) for treating anxiety in a subject in need thereof. In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 19 (or a pharmaceutically acceptable salt thereof) for treating anxiety in a subject in need thereof. In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) and / or Compound 19 (or a pharmaceutically acceptable salt thereof) for treating anxiety in a subject in need thereof, in the absence of mesembrine or mesembrine.

[0084] In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) and / or Compound 19 (or a pharmaceutically acceptable salt thereof) for the treatment of generalized anxiety disorder (GAD). In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) for the treatment of generalized anxiety disorder (GAD). In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 19 (or a pharmaceutically acceptable salt thereof) for the treatment of generalized anxiety disorder (GAD). In some embodiments, the pharmaceutical composition comprises an API consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) and / or Compound 19 (or a pharmaceutically acceptable salt thereof) for the treatment of generalized anxiety disorder (GAD), in the absence of mesembrine or mesembrine.

[0085] Pharmaceutically acceptable carriers include those known in the art. The choice of a pharmaceutically acceptable carrier can depend, for example, on the desired route of administration of the composition. Pharmaceutical compositions (formulations) can be administered to a subject by any of a number of routes of administration including, for example, parenteral administration (e.g., intravenous, subcutaneous, or intramuscular), oral administration (e.g., tablets and capsules); absorption through the oral mucosa (e.g., sublingual); or transdermal administration (e.g., as a patch applied to the skin) or topical administration (e.g., as a cream, ointment, or spray applied to the skin).

[0086] In some embodiments, a pharmaceutical composition comprising Compound 18 and / or Compound 19, or a pharmaceutically acceptable salt thereof, can be formulated for oral administration. For example, the compounds provided herein can be combined with suitable pharmaceutically-acceptable excipients to form an oral unit dosage form, such as a capsule or tablet, containing a target dose of ambroin. The drug product can be manufactured by first manufacturing ambroin as the active pharmaceutical ingredient (API), then roller compaction / grinding with intragranular excipients and blending with extragranular excipients. The drug product can contain the desired dose strength of ambroin as the API and excipient components in a tablet. The blended material can be compressed to form tablets, then film coated. The excipients can be selected from materials suitable for inclusion in a pharmaceutical composition for the intended purpose and delivery route, including providing the drug composition with the desired manufacturing and stability properties and / or the desired in vivo characteristics or other properties. In some embodiments, the pharmaceutical composition can comprise ambroin as the API in combination with a filler (e.g., in the form of microcrystalline cellulose), a dry binder or disintegrant (e.g., a cross-linked polymer), a glidant (e.g., colloidal silicon dioxide), and / or a lubricant (e.g., magnesium stearate). In some embodiments, the pharmaceutical composition can comprise materials that participate in the passage or transport of the API agent from one organ or body part to another (such as sustained release agents or disintegrants), including materials that are desirable to control the absorption of the API in the intestine.

[0087] The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound which produces a therapeutic effect. For use in the methods of the application, the active compound can be administered alone, or as a pharmaceutical composition, containing, for example, from 0.1% to 99.5% (more preferably from 0.5% to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0088] The process of preparation of these formulations or compositions includes the step of bringing into association active compound (such as a compound of the present application) and the carrier and, optionally, one or more accessory ingredients. In general, the preparations are prepared by uniformly and intimately bringing into association the compound of the present application with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0089] For preparing solid dosage forms for oral administration, the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders, (2) binders, (3) humectants, (4) disintegrating agents, (5) dissolution retardants, (6) absorption accelerators, (7) wetting agents, (8) absorbents, (9) lubricants, (10) complexing agents, and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Similar types of solid compositions can also be used as fillers in soft and hard filled gelatin capsules. The pharmaceutical compositions according to the present application can contain conventional pharmaceutical carriers and / or adjuvants. In some embodiments, the pharmaceutical compositions according to the present application can contain conventional carrier agents, including binders, lubricants, and / or glidants, selected from those products and materials commonly used in the pharmaceutical industry to prepare pharmaceutical compositions for the intended route of administration.

[0090] Tablets can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form with the necessary binders, lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked carboxymethylcellulose sodium), surface-active or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with a liquid diluent.

[0091] Liquid dosage forms for oral administration can include a pharmaceutically acceptable carrier and the active ingredient in either a solid form or a liquid form (e.g., a solution, suspension, or emulsion) that is reconstituted prior to administration. In addition to the active ingredient, the liquid dosage form can contain inert diluents that are commonly used in the art. For example, the formulation of a pharmaceutically acceptable composition for injection can include an aqueous solution, such as water or physiologically buffered saline or other solvents or vehicles suitable for the intended route of administration. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0092] The therapeutically effective amount of the pharmaceutical composition can be determined by human clinical trials to determine a safe and effective dose for patients with relevant diagnoses. It is generally understood that the effective amount of the compound can vary according to the subject's weight, sex, age, and medical history. Other factors affecting the effective amount may include, but are not limited to, the severity of the patient's illness, the condition being treated, the stability of the compound, and, if necessary, another type of therapeutic agent administered together with the compound of the present invention. A larger total dose can be delivered by administering the pharmaceutical composition multiple times to determine a dose and dose interval that is safe and effective for the patient.

[0093] In certain embodiments, the pharmaceutical composition may contain less than about 10% by weight, less than about 5% by weight, less than about 3% by weight, less than about 2% by weight, or less than about 1% by weight of the combination of (-)mesembrine and mesembrine alkaloids, or the combination of mesembrine and mesembrenol, or the combination of (-)mesembrine, mesembrenol, and mesembrenol. In certain embodiments, the composition is substantially free of (-)mesembrine, mesembrenol, and mesembrenol.

[0094] In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of (-)mecetin or (-)6-epi-mecetin.

[0095] The present disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. Pharmaceutically acceptable salts include, for example, acid addition salts and base addition salts. The acid added to the compound to form the acid addition salt can be an organic acid or an inorganic acid. The base added to the compound to form the base addition salt can be an organic base or an inorganic base. In some embodiments, the pharmaceutically acceptable salt is a metal salt, and in some embodiments, the pharmaceutically acceptable salt is an ammonium salt. For example, pharmaceutically acceptable acid addition salts can exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates can also be prepared. The source of such solvates can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or insoluble in such solvent.

[0096] In some embodiments, the pharmaceutical composition comprises Compound 018 or a pharmaceutically acceptable salt thereof and an excipient, such as starch, gelatin, silicone, titanium dioxide, iron oxide, and other inactive ingredients, such as coloring dyes. In some embodiments, a tablet or capsule comprises Compound 018 or a pharmaceutical composition thereof and an inactive ingredient.

[0097] In some embodiments, the pharmaceutical composition comprises Compound 019 or a pharmaceutically acceptable salt thereof and an excipient, such as containing starch, gelatin, silicone, titanium dioxide, iron oxide, and other inactive ingredients, such as coloring dyes. In some embodiments, the tablet or capsule contains Compound 019 or a pharmaceutical composition thereof and inactive ingredients.

[0098] Selective inhibition of 5-HTT

[0099] In some embodiments, matatabiinol (Compound 18) or a pharmaceutically acceptable salt thereof can be used to inhibit serotonin transporter (5-HTT) without inhibiting phosphodiesterase-4 (PDE4) in the absence of matatabiine and matatabiinone. In some embodiments, (-)6-epi-matatabiinol (Compound 19) or a pharmaceutically acceptable salt thereof can be used to inhibit serotonin transporter (5-HTT) without inhibiting phosphodiesterase-4 (PDE4) in the absence of matatabiine and matatabiinone.

[0100] For a particular composition described herein, the ratio of PDE4 / SERT inhibition values can be calculated from (i) the SERT inhibition value (e.g., IC 50 ) of the composition determined via the assay described in Example 3A, and (ii) the PDE4 inhibition value (e.g., IC 50 ) of the same composition determined via the assay described in Example 3B. In certain embodiments, the ratio of PDE4 / SERT inhibition values is at least 10: 1, at least 100: 1, or at least 500: 1. In some embodiments, the pharmaceutical composition has an IC 50

[0101] ​In some embodiments, there is provided a method of inhibiting serotonin transporter (5-HTT) in the central nervous system (CNS) of a subject with an active pharmaceutical ingredient (API) having no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, wherein the method comprises administering an API consisting of pinosylvin (Compound 18) or a pharmaceutically acceptable salt thereof, (-)6-epi-pinosylvin (Compound 19) or a pharmaceutically acceptable salt thereof, a mixture of Compound 18 and Compound 19, or a pharmaceutically acceptable salt thereof. In some embodiments, the method of inhibiting serotonin transporter (5-HTT) in the central nervous system (CNS) of a subject with an active pharmaceutical ingredient (API) having no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar comprises administering an API consisting of pinosylvin (Compound 18) or a pharmaceutically acceptable salt thereof. In some embodiments, the method of inhibiting serotonin transporter (5-HTT) in the central nervous system (CNS) of a subject with an active pharmaceutical ingredient (API) having no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar comprises administering an API consisting of (-)6-epi-pinosylvin (Compound 19) or a pharmaceutically acceptable salt thereof. In certain embodiments, the API has less than about 6% inhibition of at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, at least two of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, at least three of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, or each of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0102] In some embodiments, the method of treatment comprises administering eriodicytol (Compound 18), (-)6-epi-eriodicytol (Compound 19), or a pharmaceutically acceptable salt thereof, in the absence of eriodicyclamine or eriodicyclonamine, to inhibit serotonin transporter (5-HTT) in a subject in need thereof without inhibiting phosphodiesterase-4 (PDE4). In some embodiments, the method of treatment comprises administering eriodicytol (Compound 18), or a pharmaceutically acceptable salt thereof, in the absence of eriodicyclamine or eriodicyclonamine, to inhibit serotonin transporter (5-HTT) in a subject in need thereof without inhibiting phosphodiesterase-4 (PDE4). In some embodiments, the method of treatment comprises administering (-)6-epi-eriodicytol (Compound 19), or a pharmaceutically acceptable salt thereof, in the absence of eriodicyclamine or eriodicyclonamine, to inhibit serotonin transporter (5-HTT) in a subject in need thereof without inhibiting phosphodiesterase-4 (PDE4). In certain embodiments, eriodicytol (Compound 18), (-)6-epi-eriodicytol (Compound 19), or a pharmaceutically acceptable salt thereof, has an inhibition % of less than about 6% for at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, for at least two of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, for at least three of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, or for each of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0103] In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject an API comprising abemaciclib (compound 1), or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject an API comprising abemaciclib (compound 1). In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject an API consisting of abemaciclib (compound 1). In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject an API comprising at least one of abemaciclib (compound 1) or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject an API consisting of at least one of abemaciclib (compound 1) or a pharmaceutically acceptable salt thereof. In certain embodiments, the API has less than about 6% inhibition of at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, at least two of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, at least three of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, or each of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0104] In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar comprises administering to the subject, in the absence of mescaline or mescaline ketone, mescaline alcohol (Compound 18), (-)6-epi-mescaline alcohol (Compound 19), or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject, in the absence of mescaline and mescaline ketone, mescaline alcohol (Compound 18), or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject, in the absence of mescaline, mescaline alcohol (Compound 18), or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject, in the absence of mescaline ketone, mescaline alcohol (Compound 18), or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject, in the absence of mescaline and mescaline ketone, (-)6-epi-mescaline alcohol (Compound 19), or a pharmaceutically acceptable salt thereof. In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) that has no more than about 5% inhibition of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 at 10 micromolar, the method comprising administering to the subject, in the absence of mescaline, (-)6-epi-mescaline alcohol (Compound 19), or a pharmaceutically acceptable salt thereof.In some embodiments, a method of inhibiting serotonin transporter (5-HTT) in a subject in need thereof with an active pharmaceutical ingredient (API) having no more than about 5% inhibition at 10 micromolar against PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, the method comprising administering to the subject (-)6-epi-manoyl oxide (Compound 19) or a pharmaceutically acceptable salt thereof in the absence of manoyl oxide base. In certain embodiments, the API has less than about 6% inhibition against at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, at least two of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, at least three of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2, or each of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0105] In some embodiments, there is provided a method of treatment comprising administering a pharmaceutical composition comprising an API consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) and / or Compound 19 (or a pharmaceutically acceptable salt thereof).

[0106] In some embodiments, a method for treating generalized anxiety disorder is provided. Generalized anxiety disorder (DSM-IV) is characterized by excessive anxiety and worry (anticipatory anxiety) that lasts for at least 6 months and that the patient finds difficult to control. It must be associated with at least 3 of the following symptoms: restlessness or feeling nervous or agitated, easy fatigue, difficulty concentrating or absent-mindedness, irritability, muscle tension, and sleep disorders. In some embodiments, the method for treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising compound 18 (or a pharmaceutically acceptable salt thereof) as an API. In some embodiments, the method for treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising compound 19 (or a pharmaceutically acceptable salt thereof) as an API. In some embodiments, the method for treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising a mixture of compound 18 (or a pharmaceutically acceptable salt thereof) and compound 19 (or a pharmaceutically acceptable salt thereof) as an API. In some embodiments, the method for treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising compound 18 (or a pharmaceutically acceptable salt thereof) and compound 19 (or a pharmaceutically acceptable salt thereof) as an API. In some embodiments, the method for treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising compound 18 (or a pharmaceutically acceptable salt thereof) as an API in the absence of mesembrine or mesembrine. In some embodiments, the method of treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising compound 18 (or a pharmaceutically acceptable salt thereof) as the API, wherein the pharmaceutical composition contains 0%-0.5% mesembrine or mesembrine-based as measured by HPLC. In some embodiments, the method of treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising compound 19 (or a pharmaceutically acceptable salt thereof) as the API, wherein the pharmaceutical composition contains 0%-0.5% mesembrine or mesembrine-based as measured by HPLC. In some embodiments, the method of treating generalized anxiety disorder (GAD) comprises administering a pharmaceutical composition comprising a mixture of compound 18 (or a pharmaceutically acceptable salt thereof) and compound 19 (or a pharmaceutically acceptable salt thereof) as the API, wherein the pharmaceutical composition contains 0%-0.5% mesembrine or mesembrine-based as measured by HPLC.

[0107] definition

[0108] Unless otherwise defined herein, the scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally speaking, the terms and techniques used in connection with chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0109] Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics", Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th Edition", WH Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th Edition", WH Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th Edition", Sinauer Associates, Inc., Sunderland, MA (2000).

[0110] All of the above and any other publications, patents, and published patent applications mentioned in this application are expressly incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0111] The term "agent" is used herein to refer to a compound (such as an organic or inorganic compound, a mixture of compounds), a biomacromolecule (such as a nucleic acid, an antibody, including portions thereof, and humanized, chimeric, and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, lipid, carbohydrate), or an extract made from biological material such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents of known structure and agents of unknown structure.

[0112] "Patient," "subject," or "individual" are used interchangeably and refer to a human or non-human animal. These terms include mammals, such as humans, primates, livestock animals (including cattle, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice and rats).

[0113] "Treatment" of a condition or patient refers to taking measures to achieve a beneficial or desired result, including clinical results. As used herein and as is well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0114] The term "prevention" is art-recognized and, as used with respect to a condition such as local recurrence (e.g., pain), a disease such as cancer, a sign such as heart failure, or any other medical condition, is well understood in the art and includes administration of a composition that reduces the frequency of, or delays onset of, symptoms of a medical condition in a subject relative to a subject not receiving the composition. Thus, prevention of cancer, for example, includes, e.g., reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population, in a statistically and / or clinically significant amount.

[0115] "Administering" or "administration" of a substance, compound, or agent to a subject can be carried out using one of a variety of methods known to those of skill in the art. For example, a compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin tube). A compound or agent can also be introduced, as appropriate, by a rechargeable or biodegradable polymeric device or other device (e.g., patches and pumps) or formulation that provides extended, slow, or controlled release of the compound or agent. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0116] An appropriate method of administering a substance, compound, or agent to a subject will also depend on, for example, the age and / or physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, a compound or agent is administered orally to a subject, e.g., by ingestion. In some embodiments, a compound or agent is administered orally in an extended release or slow release formulation, or using a device for such slow or extended release.

[0117] As used herein, the phrase "administered in combination" refers to any form of administration of two or more different therapeutic agents such that a second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., both agents are effective in the patient simultaneously, which may include a synergistic effect of the two agents). For example, different therapeutic compounds can be administered simultaneously or sequentially in the same formulation or in separate formulations. Thus, an individual receiving such treatment can benefit from the combined effects of the different therapeutic agents.

[0118] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that will have the intended therapeutic effect when administered to a subject. The full therapeutic effect does not necessarily occur with the administration of a single dose, but may only occur after a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount required for a subject will depend, for example, on the subject's size, health, and age, and the nature and extent of the condition being treated (such as cancer or MDS). A skilled artisan can readily determine the effective amount for a given situation by routine experimentation.

[0119] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted as well as instances where the alkyl group is not substituted.

[0120] It will be appreciated that substituents and substitution patterns on the compounds of the invention can be selected by one of ordinary skill in the art to yield chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art, as well as those described below. If a substituent is itself substituted with more than one group, it will be appreciated that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0121] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (such as cell proliferation) as well as enhancing a function or activity.

[0122] "Pharmaceutically acceptable salt" or "salt" as used herein refers to an acid addition salt or a base addition salt that is suitable for or compatible with the treatment of a patient.

[0123] The term "pharmaceutically acceptable acid addition salt," as used herein, denotes any non-toxic organic or inorganic salt of a base compound described herein. Illustrative inorganic acids useful for preparing the desired salt include hydrochloric, hydrobromic, sulfuric, and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids useful for preparing the desired salt include the mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic, and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Mono- or di-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts of the compounds described herein are more soluble in water and various hydrophilic organic solvents, and generally exhibit a higher melting point than their free base forms. The selection of an appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts can be useful, for example, in the preparation of compounds described herein for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0124] The term "pharmaceutically acceptable base addition salt," as used herein, denotes any non-toxic organic or inorganic base addition salt of an acid compound described herein, or any intermediate thereof. Illustrative inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium. Illustrative organic bases that form suitable salts include aliphatic, cycloaliphatic or aromatic organic amines such as methylamine, trimethylamine, and methylpyridine or ammonia. The selection of an appropriate salt will be known to one skilled in the art.

[0125] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without an excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0126] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

[0127] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more active compounds with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which combination may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0128] Many compounds that can be used in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter can exist in either R or S configuration, with the R and S symbols being used according to the rules described in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms, such as enantiomers and diastereomeric forms (including all possible mixtures of stereoisomers) of compounds, salts, prodrugs, or mixtures thereof. See, for example, WO 01 / 062726.

[0129] Additionally, certain alkenyl-containing compounds may exist as Z (ipsilateral) or E (heterolateral) isomers. In each case, the disclosure includes both the mixture and the individual isomers.

[0130] Some compounds may also exist as tautomeric forms. Although not explicitly indicated in the formulae described herein, such forms are intended to be encompassed within the scope of the present disclosure.

[0131] The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle suitable for formulating a medicament for medical or therapeutic use, such as a liquid or solid filter aid, diluent, excipient, solvent or encapsulating material.

[0132] As used herein, the term "logarithm of solubility," "LogS," or "logS" is used in the art to quantify the water solubility of a compound. The water solubility of a compound significantly affects its absorption and distribution characteristics. Low solubility is often associated with poor absorption. The LogS value is the unit exfoliation logarithm (base 10) of the solubility measured in moles / liter.

[0133] Additional embodiments

[0134] 1. A method for treating a mental health disorder, comprising administering to a mammal in need thereof an effective amount of a compound selected from (-)mecetin and (-)6-epi-mecetin or a pharmaceutically acceptable salt thereof.

[0135] 2. The method of embodiment 1, wherein the compound is (-) mesembryanthemum.

[0136] 3. The method of embodiment 1, wherein the compound is (-)6-epi-methenol.

[0137] 4. The method of any one of embodiments 1-3, wherein the mental health disorder is anxiety, stress, or depression.

[0138] 5. The method of embodiment 4, wherein the mental health disorder is anxiety.

[0139] 6. The method of embodiment 4, wherein the mental health disorder is stress.

[0140] 7. The method of embodiment 4, wherein the mental health disorder is depression.

[0141] 8. The method of any one of embodiments 1-7, wherein the mammal is a human.

[0142] 9. A method of inhibiting SERT, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound selected from (-)mesequinol and (-)6-epi-mesequinol; or a pharmaceutically acceptable salt thereof.

[0143] 10. The method of embodiment 9, wherein the compound is (-) mesembryanthemum.

[0144] 11. The method of embodiment 9, wherein the compound is (-)6-epi-methenol.

[0145] 12. A method for inhibiting SERT, comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound selected from (-)mesequinol and (-)6-epi-mesequinol, or a pharmaceutically acceptable salt thereof.

[0146] 13. The method of embodiment 12, wherein the compound is (-) mesembryanthemumol.

[0147] 14. The method of embodiment 12, wherein the compound is (-)6-epi-methenol.

[0148] 15. The method of any one of embodiments 12-14, wherein the pharmaceutical composition contains no more than about 10% of an alkaloid selected from (-)pennsivyn, pennserone, pennserenol, or a combination thereof.

[0149] 16. The method of any one of embodiments 12-14, wherein the pharmaceutical composition contains no more than about 5% of an alkaloid selected from (-)pennsivyn, pennserone, pennserenol, or a combination thereof.

[0150] 17. The method of any one of embodiments 12-14, wherein the pharmaceutical composition contains no more than about 3% of an alkaloid selected from (-)pennsivyn, pennserone, pennserenol, or a combination thereof.

[0151] 18. The method of any one of embodiments 12-14, wherein the pharmaceutical composition contains no more than about 2% of an alkaloid selected from (-)pennsivyn, pennserone, pennserenol, or a combination thereof.

[0152] 19. The method of any one of embodiments 12-14, wherein the pharmaceutical composition contains no more than about 1% of an alkaloid selected from (-)pennsivyn, pennserone, pennserenol, or a combination thereof.

[0153] 20. The method of any one of embodiments 15-19, wherein the alkaloid is (-)pennsivyn.

[0154] 21. The method of any one of embodiments 15-19, wherein the alkaloid is pennserone.

[0155] 22. The method of any one of embodiments 15-19, wherein the alkaloid is pennserenol.

[0156] 23. The method of any one of embodiments 15-19, wherein the pharmaceutical composition is substantially free of (-)pennsivyn, pennserone, and pennserenol.

[0157] 24. A pharmaceutical composition comprising (-)6-epi-pennserol and a pharmaceutically acceptable excipient.

[0158] 25. The pharmaceutical composition of embodiment 24, wherein the pharmaceutical composition contains no more than about 10% of an alkaloid selected from (-)pennsivyn, pennserone, pennserenol, or a combination thereof.

[0159] 26. The pharmaceutical composition of embodiment 24, wherein the pharmaceutical composition contains no more than about 5% of an alkaloid selected from (-)pennsivyn, pennserone, pennserenol, or a combination thereof.

[0160] 27. The pharmaceutical composition of embodiment 24, wherein the pharmaceutical composition contains no more than about 3% of alkaloids selected from the group consisting of (-) pukatol, pukateine, pukanol, or combinations thereof.

[0161] 28. The pharmaceutical composition of embodiment 24, wherein less than about 60% of the total alkaloid content is pukateine and pukanol.

[0162] 29. The pharmaceutical composition of embodiment 24, wherein less than about 20% of the total alkaloid content is (-) pukatol.

[0163] 30. The pharmaceutical composition of embodiment 24, wherein the composition comprises about 0.3 wt% to about 0.5 wt% of total alkaloids; and pukateine and pukanol comprise less than about 60 wt% of the total alkaloid content.

[0164] 31. The pharmaceutical composition of embodiment 24, wherein the composition comprises about 0.3 wt% to about 0.6 wt% of total alkaloids, and pukatol comprises less than about 20 wt% of the total alkaloid content.

[0165] 32. The pharmaceutical composition of embodiment 30 or 31, wherein (-) 6-epi-pukanol comprises at least 70% of the total alkaloid content in the composition.

[0166] 33. The pharmaceutical composition of embodiment 30 or 31, wherein (-) 6-epi-pukanol comprises at least 80% of the total alkaloid content in the composition.

[0167] 34. The pharmaceutical composition of embodiment 30 or 31, wherein (-) 6-epi-pukanol comprises at least 90% of the total alkaloid content in the composition.

[0168] 35. The pharmaceutical composition of embodiment 24, wherein the composition comprises less than about 70 micrograms of pukateine and pukanol per 11.3 mg of total alkaloid content.

[0169] 36. The pharmaceutical composition of embodiment 24, wherein the composition comprises less than about 23 micrograms of (-) pukatol per 11.3 mg of total alkaloid content.

[0170] 37. The pharmaceutical composition of any one of embodiments 24-36, wherein the ratio of PDE4 / SERT inhibition values is at least 10: 1.

[0171] 38. The pharmaceutical composition of any one of embodiments 24-36, wherein the ratio of PDE4 / SERT inhibition values is at least 100: 1.

[0172] 39. The pharmaceutical composition of any one of embodiments 24-36, wherein the ratio of PDE4 / SERT inhibition values is at least 500: 1.

[0173] 40. The pharmaceutical composition of any one of embodiments 24-39, wherein the (-)6-epi-pinosylvinol has a half-life of at least 30 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0174] 41. The pharmaceutical composition of any one of embodiments 24-39, wherein the (-)6-epi-pinosylvinol has a half-life of at least 60 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0175] 42. The pharmaceutical composition of any one of embodiments 24-39, wherein the (-)6-epi-pinosylvinol has a half-life of at least 90 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0176] 43. The pharmaceutical composition of any one of embodiments 24-39, wherein the (-)6-epi-pinosylvinol has a half-life of at least 120 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0177] 44. The pharmaceutical composition of any one of embodiments 24-43, wherein the AUC ratio of (-)6-epi-pinosylvinol to (-)pinosylvin is about 2 to about 2.5. 最后

[0178] 45. A pharmaceutical composition comprising (-)pinosylvinol and a pharmaceutically acceptable excipient.

[0179] 46. The pharmaceutical composition of embodiment 45, wherein the pharmaceutical composition contains no more than about 10% of an alkaloid selected from (-)pino sylvin, pino sylvinone, pino sylvinol, or a combination thereof.

[0180] 47. The pharmaceutical composition of embodiment 46, wherein the pharmaceutical composition contains no more than about 5% of an alkaloid selected from (-)pino sylvin, pino sylvinone, pino sylvinol, or a combination thereof.

[0181] 48. The pharmaceutical composition of embodiment 46, wherein the pharmaceutical composition contains no more than about 3% of an alkaloid selected from (-)pino sylvin, pino sylvinone, pino sylvinol, or a combination thereof.

[0182] 49. The pharmaceutical composition of embodiment 46, wherein the composition contains less than about 60% of pino sylvinone and pino sylvinol.

[0183] ​50. The pharmaceutical composition of embodiment 46, wherein the composition contains less than about 20% of (-) pukateine.

[0184] 51. The pharmaceutical composition of embodiment 46, wherein the composition comprises about 0.3 wt% to about 0.5 wt% total alkaloids, and pukateinine and pukateinol comprise less than about 60 wt% of the total alkaloid content.

[0185] 52. The pharmaceutical composition of embodiment 46, wherein the composition comprises about 0.3 wt% to about 0.6 wt% total alkaloids, and pukateine comprises less than about 20 wt% of the total alkaloid content.

[0186] 53. The pharmaceutical composition of embodiment 51 or 52, wherein (-) pukatoside comprises at least 70% of the total alkaloid content of the composition.

[0187] 54. The pharmaceutical composition of embodiment 51 or 52, wherein (-) pukatoside comprises at least 80% of the total alkaloid content of the composition.

[0188] 55. The pharmaceutical composition of embodiment 51 or 52, wherein (-) pukatoside comprises at least 90% of the total alkaloid content of the composition.

[0189] 56. The pharmaceutical composition of embodiment 46, wherein the composition comprises less than about 70 micrograms of pukateinine and pukateinol per 11.3 mg of total alkaloid content.

[0190] 57. The pharmaceutical composition of embodiment 46, wherein the composition comprises less than about 23 micrograms of (-) pukateine per 11.3 mg of total alkaloid content.

[0191] 58. The pharmaceutical composition of any one of embodiments 45-57, wherein the ratio of PDE4 / SERT inhibition values is at least 10: 1.

[0192] 59. The pharmaceutical composition of any one of embodiments 45-57, wherein the ratio of PDE4 / SERT inhibition values is at least 100: 1.

[0193] 60. The pharmaceutical composition of any one of embodiments 45-57, wherein the ratio of PDE4 / SERT inhibition values is at least 500: 1.

[0194] 61. The pharmaceutical composition of any one of embodiments 45-60, wherein the (-) 6-epi-pukatoside has a half-life of at least 30 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0195] 62. The pharmaceutical composition of any one of embodiments 45-60, wherein the (-)6-epi-mecetomyl alcohol has a half-life of at least 60 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0196] 63. The pharmaceutical composition of any one of embodiments 45-60, wherein the (-)6-epi-mecetomyl alcohol has a half-life of at least 90 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0197] 64. The pharmaceutical composition of any one of embodiments 45-60, wherein the (-)6-epi-mecetomyl alcohol has a half-life of at least 120 minutes in a human hepatocyte assay of the pharmaceutical composition.

[0198] 65. A pharmaceutical composition as described in any one of embodiments 45-64, wherein the AUC of (-) 6-epi-mecenitol and (-) mesembrine is 最后 The ratio is from about 2 to about 2.5.

[0199] 66. A method of inhibiting SERT without inhibiting PDE4, the method comprising administering to a mammal in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 24-65.

[0200] 67. The method of embodiment 66, wherein the pharmaceutical composition comprises (-) mesembryanthemum.

[0201] 68. The method of embodiment 66, wherein the pharmaceutical composition comprises (-)6-epi-mecetylcholine.

[0202] 69. The method of any one of embodiments 66-68, wherein the pharmaceutical composition has an IC50 for SERT of less than about 30 nM.

[0203] 70. The method of embodiment 66 or 68, wherein the pharmaceutical composition has an IC for SERT of less than about 15 nM. 50 .

[0204] 71. The method of any one of embodiments 66-70, wherein the pharmaceutical composition has a % inhibition of at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2 of less than about 6%.

[0205] 72. The method of any one of embodiments 66-70, wherein the pharmaceutical composition has a % inhibition of less than about 6% for at least two of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0206] 73. The method of any one of embodiments 66-70, wherein the pharmaceutical composition has an inhibition % of less than about 6% for at least three of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0207] 74. The method of any one of embodiments 66-70, wherein the pharmaceutical composition has an inhibition % of less than about 6% for each of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0208] 75. The method of any one of embodiments 66-74, wherein the ratio of PDE4 / SERT inhibition values is at least 10: 1.

[0209] 76. The method of any one of embodiments 66-74, wherein the ratio of PDE4 / SERT inhibition values is at least 100: 1.

[0210] 77. The method of any one of embodiments 66-74, wherein the ratio of PDE4 / SERT inhibition values is at least 500: 1.

[0211] 78. A method of inhibiting SERT without inhibiting PDE4, the method comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition.

[0212] 79. The method of embodiment 78, wherein the pharmaceutical composition is the composition of any one of embodiments 24-65.

[0213] 80. The method of embodiment 78 or 79, wherein the pharmaceutical composition has an IC 50 .

[0214] 81. The method of embodiment 78 or 79, wherein the pharmaceutical composition has an IC 50 .

[0215] 82. The method of any one of embodiments 78-81, wherein the pharmaceutical composition has an inhibition % of less than about 6% for at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0216] 83. The method of any one of embodiments 78-81, wherein the pharmaceutical composition has an inhibition % of less than about 6% for at least two of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0217] 84. The method of any one of embodiments 78-81, wherein the pharmaceutical composition has an inhibition % of less than about 6% for at least three of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0218] 85. The method of any one of embodiments 78-81, wherein the pharmaceutical composition has an inhibition % of less than about 6% for each of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

[0219] 86. The method of any one of embodiments 78-85, wherein the ratio of PDE4 / SERT inhibition values is at least 10: 1.

[0220] 87. The method of any one of embodiments 78-85, wherein the ratio of PDE4 / SERT inhibition values is at least 100: 1.

[0221] 88. The method of any one of embodiments 78-85, wherein the ratio of PDE4 / SERT inhibition values is at least 500: 1.

[0222] 89. A composition comprising miltirum ketone (Compound 18) or a pharmaceutically acceptable salt thereof; wherein the composition comprises less than about 0.5% miltirum base or miltirum ketone base as measured by HPLC.

[0223] 90. The composition of embodiment 89, further comprising (-)6-epi-miltirum ketone (Compound 19) or a pharmaceutically acceptable salt thereof.

[0224] 91. A composition comprising (-)6-epi-miltirum ketone (Compound 19) or a pharmaceutically acceptable salt thereof; wherein the composition comprises less than about 0.5% miltirum base or miltirum ketone base as measured by HPLC.

[0225] 92. The composition of any one of embodiments 89-91, wherein the composition is free of miltirum base or miltirum ketone base.

[0226] 93. The composition of any one of embodiments 89-92, wherein the composition is free of additional alkaloid compounds other than Compound 18 or Compound 19.

[0227] 94. The composition of embodiment 89, wherein the composition is a pharmaceutical composition comprising an active pharmaceutical ingredient (API) consisting of Compound 18 (or a pharmaceutically acceptable salt thereof).

[0228] 95. The composition of embodiment 91, wherein the composition is a pharmaceutical composition comprising an active pharmaceutical ingredient (API) consisting of Compound 19 (or a pharmaceutically acceptable salt thereof).

[0229] 96. The composition of any one of embodiments 89-93, wherein the composition is a pharmaceutical composition comprising an active pharmaceutical ingredient (API) consisting of Compound 18 (or a pharmaceutically acceptable salt thereof) and / or Compound 19 (or a pharmaceutically acceptable salt thereof).

[0230] 97. A method of treating anxiety, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising matatabi alcohol (or a pharmaceutically acceptable salt thereof) and / or (-)6-epi-matatabi alcohol (or a pharmaceutically acceptable salt thereof).

[0231] 98. The method of embodiment 97, wherein the active pharmaceutical ingredient (API) of the pharmaceutical composition consists of a mixture of matatabi alcohol (or a pharmaceutically acceptable salt thereof) and / or (-)6-epi-matatabi alcohol (or a pharmaceutically acceptable salt thereof).

[0232] 99. The method of embodiment 97, wherein the active pharmaceutical ingredient (API) of the pharmaceutical composition consists of matatabi alcohol (or a pharmaceutically acceptable salt thereof).

[0233] 100. The method of embodiment 97, wherein the active pharmaceutical ingredient (API) of the pharmaceutical composition consists of a mixture of (-)6-epi-matatabi alcohol (or a pharmaceutically acceptable salt thereof).

[0234] 101. The method of any one of embodiments 97-100, wherein the pharmaceutical composition does not comprise matatabiine.

[0235] 102. The method of any one of embodiments 97-101, wherein the pharmaceutical composition does not comprise matatabiketonine.

[0236] 103. The method of any one of embodiments 97-100, wherein the pharmaceutical composition contains less than 0.5% matatabiine or matatabiketonine in the pharmaceutical composition as detected by HPLC.

[0237] 104. The method of any one of embodiments 97-103, wherein the method is a method of treating generalized anxiety disorder (GAD) in a subject diagnosed with GAD.

[0238] 105. A method of inhibiting serotonin transporter (5-HTT) in the central nervous system of a subject, the method comprising the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising mesembryanthemum (or a pharmaceutically acceptable salt thereof) and / or (-)6-epi-mesembryanthemum (or a pharmaceutically acceptable salt thereof), wherein the pharmaceutical composition does not inhibit PDE4A1A, PDE4B2, PDE4C1, or PDE4D2 by more than 5% at 10 micromolar.

[0239] 106. The method of embodiment 105, wherein the pharmaceutical composition does not comprise mesembrine.

[0240] 107. The method of any one of embodiments 105-106, wherein the pharmaceutical composition does not comprise mesembryantrine.

[0241] 108. The method of embodiment 105, wherein the pharmaceutical composition comprises less than 0.5% mesembrine or mesembrine as detected by HPLC.

[0242] Example

[0243] LC / MS spectra were acquired using an Agilent 1200\G1956A or a SHIMADZU LCMS-2020. Standard LC / MS conditions were as follows (run time 1.55 minutes):

[0244] Acidic conditions: Mobile phase A: 0.0375% TFA (v / v) in water. Mobile phase B: 0.01875% TFA (v / v) in acetonitrile. Column: Kinetex EVO C18 30*2.1 mm, 5 μm.

[0245] Basic conditions: Mobile phase A: 0.025% NH3·H2O (v / v) in water; Mobile phase B: acetonitrile; Column: Kinetex EVOC18 2.1×30 mm, 5 μm.

[0246] Abbreviations

[0247] Ac Acetyl ACN Acetonitrile br Broad peak C degrees Celsius δ Chemical shift d Double Peaks DCM dichloromethane de Diastereomeric excess DIBAL-H Diisobutylaluminum hydride DMAP 4-Dimethylaminopyridine DMF N,N-Dimethylformamide ESI Electrospray ionization Et Ethyl FA Formic acid HPLC High-performance liquid chromatography hr Hour Hz hertz IPA Isopropyl alcohol J Coupling constant LC-MS Liquid chromatography-mass spectrometry m multiplets M Moore m / z mass-to-charge ratio Me methyl min minute mol Moore NMR Nuclear magnetic resonance imaging Psi Pound force per square inch Pr Propyl q Quadruple Peak s Single peak t Triple Peak t-Bu tert-butyl TEA Triethylamine THF Tetrahydrofuran TMS trimethylsilyl

[0248] Summary of the names of mesembryanthemum compounds

[0249] Compound (-) enantiomer Mesembryanthemum alcohol Compound 018 6-epi-meconyl alcohol Compound 019

[0250] Example 1: Synthesis of (3aS,6R,7aS)-3a-(3,4-dimethoxyphenyl)-1-methyloctahydro-1H-indol-6-ol (018, (-)-mesequinol)

[0251]

[0252] A mixture of 001 (200 mg, 691 umol) and Pt02(20.0 mg, 88.0 umol) in IPA (4 mL) was degassed and purged with N2three times. The mixture was stirred at 25 °C under N2atmosphere for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by recrystallization from EtOH (1 mL) at 25 °C to give (3aS,6R,7aS)-3a-(3,4-dimethoxyphenyl)-1-methyloctahydro-1H-indol-6-ol (018, (-)-mikanol) (100 mg, 49%) as a white solid. Melting point 145.5-146.5 °C. LC-MS (ESI + m / z 292.4 (M+H). 1 H NMR (400 MHz, CDC13) δ 6.86 - 6.78 (m, 2H), 6.77 - 6.71 (m, 1H), 3.86 (s, 1H), 3.81 (d, J = 6.8 Hz, 6H), 3.30 (dt, J = 6.8, 9.6 Hz, 1H), 2.83 (s, 1H), 2.40 (s, 3H), 2.33 - 2.20 (m, 1H), 2.09 (dd, J = 2.8, 14.8 Hz, 1H), 1.90 - 1.82 (m, 2H), 1.78 (dd, J = 6.8, 11.6 Hz, 1H), 1.67 - 1.62 (m, 2H), 1.57 (td, J = 2.8, 14.8 Hz, 1H), 1.39 - 1.30 (m, 2H).

[0253] Example 2: Synthesis of (3aS,6S,7aS)-3a-(3,4-dimethoxyphenyl)-1-methyloctahydro-1H-indol-6-ol (019, (-)-6-epi-mikanol)

[0254]

[0255] To a solution of 001 (2.00 g, 6.91 mmol) and CeCl 3.7H 2 O (3.09 g, 8.29 mmol, 788 uL) in MeOH (80 mL) was added NaBH 4 (1.57 g, 41.4 mmol). The mixture was stirred at 0 ° C for 2 hours. The reaction mixture was added to 50 mL of NH 4 Cl aqueous solution, the organic layer and the water layer were separated, and the aqueous solution was extracted with DCM (50 mL x 3). The organic solutions were combined, dried over anhydrous Na 2 SO 4, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Xtimate C18 150*25mm*5um; mobile phase:

[0256] [water (NH3H2O)-ACN]; B%: 28%-58%, 8 minutes) to give (3aS,6S,7aS)-3a-(3,4-dimethoxyphenyl)-1-methyloctahydro-1H-indol-6-ol (019, (-)-6-epi-methenol) (730 mg, 37%) as a white oil. 1 H NMR (400MHz, CDCl3) δ6.95-6.88(m,2H),6.86-6.80(m,1H),3.95(s,1H),3.90(d,J=6.8Hz,6H),3.46-3.35(m,1H),2.93(s,1H),2.50(s,3H) ),2.45-2.29(m,2H),2.19(dd,J=2.4,14.9Hz,1H),2.01-1.82(m,2H),1.79-1.72(m,1H),1.70-1.59(m,3H),1.44(tt,J=2.8,13.6Hz,1H).

[0257] Determination procedure

[0258] Example 3A: SERT inhibition assay

[0259] SERT inhibition was measured using neurotransmitter transport fluorescence assay. Briefly, stable 5HTT HEK293 cells (20,000 cells / 20 μL wells) were prepared in 384 microwell plates. The compound was prepared in assay buffer (20 mM HEPES in HBSS, 0.1% BSA) with a maximum concentration of 1 μM. 10 doses of test compound (3-fold serial dilution) were added to the plated cells and incubated at 37°C for 30 minutes. 25 μL dye solution (Molecular Devices neurotransmitter transporter uptake assay kit) was added to each well and incubated at 37°C for 30 minutes. The plate was then read on a plate reader, and the results are shown in Table 1 (n=6 ± SD).

[0260] Table 1

[0261] Compound name Compound ID SERT IC 50 (nM) (-)-Mesephyllin 018 21.9±8.0 (-)-6-Epimycelium 019 10.6±10.1

[0262] Example 3B: PDE4 inhibition assay

[0263] Assay conditions for PDE4 inhibition percentage determination:

[0264] Prepare a 100 mM dilution of test compound in assay buffer (10% DMSO concentration) and add 5 μΐ of the dilution to 50 μΐ of the reaction, such that the final concentration of DMSO is 1% in all reactions. Perform the enzymatic reaction at room temperature in a 50 μΐ mixture containing PDE assay buffer, 100 nM FAM-cAMP, PDE enzyme (Table 2.3.1), and test compound (Section 2.2) for 60 minutes. After the enzymatic reaction, add 100 μΐ of the binding solution (dilute the binding agent 1 : 100 with binding agent diluent) to each reaction and allow the reaction to proceed at room temperature for 60 minutes.

[0265] Measure the fluorescence intensity at 485 nm excitation and 528 nm emission using a Tecan Infinite M1000 microplate reader.

[0266] The PDE4 assay kit (BPS Bioscience, San Diego, CA) is described below.

[0267] Step 1:

[0268] 1) Dilute the 20 μΜ FAM-cyclic-3',5'-AMP stock 100-fold with PDE buffer to make a 200 nM solution.

[0269] 2) Add 25 μΐ of FAM-cyclic-3',5'-AMP (200 nM) to each well designated as "positive control," "test inhibitor," and "substrate control."

[0270] 3) Add 20 μΐ of PDE assay buffer to each well designated as "substrate control" and 45 μΐ of PDE assay buffer to each well designated as "blank."

[0271] 4) Add 5 μΐ of inhibitor solution to each well designated as "test inhibitor." For wells labeled "positive control," "substrate control," and "blank," add 5 μΐ of the same solution without inhibitor (inhibitor buffer).

[0272] 5) Thaw the PDE on ice. After the first thaw, briefly spin the tube containing the enzyme to recover the entire contents of the tube.

[0273] 6) Dilute PDE4 in PDE buffer to 7.5 pg / ul (0.15 ng / reaction)*. Initiate the reaction by adding 20 ul of PDE4 (7.5 pg / ul) to the wells designated as "positive control" and "test inhibitor".

[0274] 7) Incubate for 1 hour at room temperature.

[0275] Step 2:

[0276] 1) Mix the binding agent thoroughly and dilute the binding agent 1 : 100 with binding agent diluent.

[0277] 2) Add 100 ul of the diluted binding agent to each micro well. Incubate for 1 hour at room temperature while slowly shaking.

[0278] 3) Read the fluorescence polarization of the samples in a microtiter plate reader equipped for measuring fluorescence polarization, which is capable of exciting at a wavelength in the range of 485 ± 5 nm and detecting emission light in the range of 528 ± 10 nm. Subtract the blank value from all other values.

[0279] 4) Data analysis: The PDE activity assay was performed in duplicate at each concentration. The fluorescence intensity was converted to fluorescence polarization using the Tecan Magellan 6 software. The fluorescence polarization data was analyzed using computer software Graphpad Prism. The fluorescence polarization (FP t ) in the absence of compound in each data set was defined as 100% activity. The value of the fluorescence polarization (FP b ) in the absence of PDE and compound in each data set was defined as 0% activity. The percent activity in the presence of compound was calculated according to the following equation: % activity = (FP - FP b ) / (FP t - FP b ) x 100%, where FP = fluorescence polarization in the presence of compound. The % activity values versus a range of compound concentrations were then plotted using nonlinear regression analysis of the S-shaped dose-response curve generated by the equation Y = B + (T - B) / 1 + 10 ((LogEC50-X)×希尔斜率) , where Y = percent activity, B = minimum percent activity, T = maximum percent activity, X = log of compound, and Hill Slope = slope factor or Hill coefficient. IC 50 values were determined by the concentration that caused half-maximal percent activity. The results are shown in Table 2A and Table 2B.

[0280] Table 2A

[0281]

[0282] Table 2B

[0283]

[0284] Example 4: Hepatocyte Metabolism

[0285] Figure 2A is a graph showing compound stability of (-)matatabiaine (triangles) compared to (-)matatabial (squares) in human hepatocytes measured according to Example 4. Figure 2B is a graph showing compound stability of (-)matatabiaine (triangles) compared to (-)6-epi-matatabial (circles) in human hepatocytes measured according to Example 4.

[0286] A 2.5 uL aliquot of 100 uM test compound was incubated with 247.5 uL of cryopreserved hepatocytes suspended at 1 million viable cells / mL in serum-free incubation medium. The mixture was incubated at 37 °C with shaking at 500 rpm for the indicated time points (0.5, 5, 10, 15, 30, 60, 90, or 120 minutes). At each time point, a 25 uL aliquot of the incubation mixture was transferred to 125 uL of cold acetonitrile containing internal standard, then centrifuged at 3,220 g for 30 minutes. 100 uL of the supernatant was mixed with 100 uL of distilled water for analysis by LC-MS / MS. Peak area ratios were determined from extracted ion chromatograms, and percent recovery was calculated. In vitro half-life (t 1 / 2 ) was determined from the slope of the concentration versus time regression, with in vitro t 1 / 2 = 0.693 / k. In vitro t 1 / 2 (min) was converted to in vitro intrinsic clearance (in vitro CL int , in uL / min / 10 6 cells): in vitro CL int = kV / N. Results are also shown in Figure 2A and Figure 2B .

[0287] Table 3

[0288]

[0289] In human hepatocytes, matatabiaine (001) undergoes rapid metabolism (t 1 / 2 = 13 minutes). In contrast, both matatabial (018) and 6-epi-matatabial (019) are highly stable in human hepatocytes (t 1 / 2These data demonstrate that oral doses of marrubiin will undergo rapid first-pass hepatic metabolism in vivo, resulting in low drug concentrations in plasma and brain. In addition, the remaining marrubiin concentrations in plasma and brain will be rapidly cleared, limiting its duration of action. In contrast, oral delivery of marricine (018) and 6-epi-marricine (019) can have low hepatic clearance in humans and can achieve stable drug concentrations, allowing for a long duration of action suitable for therapeutic benefit.

[0290] In vitro intrinsic clearance data in human hepatocytes are a predictor of in vivo plasma clearance (drug stability) of a drug. When the extraction ratio is high for a given compound (theoretical maximum = 1.0), the drug will be rapidly cleared from the body by the liver. Compound 001 has a high extraction ratio (E ~ 0.9), while compounds 018 and 019 have a low extraction ratio (E ~ 0.1). As a predictor of drug clearance from plasma in vivo, these data demonstrate that 001 will be rapidly cleared from human plasma in vivo.

[0291] Example 5: Pharmacological effects of mesembryol, 6-epi-mesembryol and mesembrine after oral administration in male SD rats Metadynamics research

[0292] Three separate pharmacokinetic studies were performed for each compound. Typically, male SD rats were orally administered 10 mg / kg of compound (formulated in 1% NMP and 99% saline v / v). Plasma samples were collected at time points 0-4 hours post-dose (n=3 per time point) to measure drug concentrations in plasma. Plasma samples were mixed with acetonitrile containing internal standards. Samples were vortexed and then centrifuged for 15 minutes at 4°C. The supernatant was diluted with water (1:2 V / V) and analyzed by LC / MS / MS for quantitative analysis. Concentrations of compound in plasma were calculated for each time point.

[0293] Table 4

[0294] parameter 018:001 ratio 019:001 ratio plasma t 1 / 2 ]]> 3.4:1 1.8:1 <![CDATA[血浆C max ]]> 9.0:1 2.0:1 plasma AUC 最后 ]]> 4.2:1 2.2:1

[0295] The data presented in Table 4 are the ratios of values obtained from marricine (018): marrubiin (001) (“ratio of 018:001”) or (-)-6-epi-marricine (019): (-) marrubiin (001) (“ratio of 019:001”). Ratios > 1.0 indicate an increase in t 1 / 2 , C max or area under the curve (AUC) compared to compound 001 (marrubiin). Both compounds 018 and 019 exhibit an increase in plasma half-life (t 1 / 2 ), maximum concentration (C max) and a significant improvement in the area under the curve (AUC). These results demonstrate the improved pharmacokinetic profile of compound 018 and compound 019 (compared to compound 001), leading to higher plasma levels over a longer period of time. For example, compared to miltirum base (compound 001), compound 018 surprisingly exhibits about 3 times longer plasma half-life, about 9 times higher Cmax, and about 4 times greater AUC (last). Furthermore, compared to miltirum base (compound 001), compound 019 surprisingly exhibits about 2 times longer plasma half-life, about 2 times higher Cmax, and about 2 times greater AUC (last).

[0296] Example 6 HPLC method for detecting mesembrenol or mesembrenine:

[0297] Gericke et al. (Journal of Ethnopharmacology, 2022, 284, 114550) performed an alkaloid profiling and quantification of an ethanol extract of 50

[0298] Example 7: Pharmacokinetic characteristics of compounds 018 and 019 in dogs

[0299] The pharmacokinetic profile of compound 018 and compound 019 was evaluated in dogs. Figure 3 Comparison of plasma concentrations (nM) of compound 018, compound 019 and compound 001 after intravenous administration of the compounds at 2 mg / kg. Figure 4 Comparison of plasma concentrations (nM) of compound 018, compound 019 and compound 001 after oral administration of the compounds at 10 mg / kg.

[0300] ​​HPLC instrument: Shimadzu (DGU-20A5R, serial number: L20705621435IX; LC-30AD serial number: L20555611905AE and L20555611816AE; SIL-30AC, serial number: L20565605499AE; Rack Changer II serial number L20585601125SS; CTO-20A: serial number L20205620413CD; CBM-20A: serial number L20235636200CD)

[0301] MS: AB API 5500 LC / MS / MS instrument (serial number EF221221812)

[0302] Column: Phenomenex Synergi 2.5 pm Polar-RP 100A (50 x 3 mm)

[0303] Mobile phase: (A) 5% acetonitrile in water (0.1% formic acid); (B) 95% acetonitrile in water (0.1% formic acid)

[0304] Sample: 50 pL plasma sample + 5 pL blank solution + 200 pL acetonitrile for PPE (protein precipitation extraction).

[0305] Compounds 018 and 019:

[0306] Injection volume: 2 microliters

[0307] Flow rate: 0.6 mL / min

[0308]

[0309] Compound 001

[0310]

[0311] Sample preparation

[0312] Working solutions of the desired series of concentrations were obtained by diluting stock solutions of the analytes with 50% aqueous acetonitrile. Five μL of working solution (5, 10, 20, 50, 100, 500, 1000, 5000, 10000 ng / mL) was added to 50 μL of blank beagle dog plasma to obtain calibration standards of 0.5 to 1000 ng / mL (0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 55 μL. Five quality control samples of 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL and 800 ng / mL of plasma were prepared separately from those used for the calibration curve. These QC samples were prepared on the day of analysis in the same manner as the calibration standards.

[0313] Fifty-five μL of standard, 55 μL of QC sample and 55 μL of unknown sample (50 μL of plasma with 5 μL of blank solution) were added to 200 μL of acetonitrile containing the IS mixture to precipitate the proteins. The samples were then vortexed for 30 seconds. After centrifugation at 3900 rpm for 15 minutes at 4°C, the supernatant was diluted 3-fold with water. Two μL (for SNTX-004, SNTX-005) and 4 uL (for SNTX-001) of the diluted supernatant were injected into the LC / MS / MS system for quantitative analysis.

[0314] Compound Information Compound ID Compound 018

[0315] MW (free form) 291.39

[0316] FW (salt form) 291.39

[0317] Formulation Preparation Preparation of Compound 018 for oral (2 mg / kg, 5 mL / kg) dosing

[0318] 0.4 mg / mL "1% NMP and 99% v / v saline" solution 71.21 mg of SNTX-004 was dissolved in 1.780 mL of NMP with vortexing and sonication, then 176.245 mL of saline was added with vortexing and sonication to give the solution.

[0319] Dog PK data (IV) from Compound 018, 019 and 001 (data plotted in Figure 3 )

[0320]

[0321] Dog PK data (PO) from Compound 018, 019 and 001 (data plotted in Figure 4 )

[0322]

[0323] Compound 019 showed excellent oral bioavailability. Compound 018 was predicted to have poor oral bioavailability based on in vitro clearance data in dog hepatocytes.

[0324] Example 8: Antidepressant properties of Compound 018 and Compound 019 in mice using the Smartcube system (signature).

[0325] In order to confirm that compounds 018 and 019 have antidepressant effects, System (Psychogenics, Inc., Paramus, NJ). The system uses features derived from mouse behavioral data to classify compounds for their ability to treat neuropsychiatric symptoms by comparing the features with a proprietary reference database of behavioral feature sets associated with marketed drug classes known to treat neuropsychiatric symptoms. Therefore, the system can be used as a model to identify the psychotropic effects of compounds by comparing the effects of compounds with drugs with known validated effects. By comparing the responses of animals to known drugs, the test drugs can be classified according to their function; for example, hallucinogens, anxiogenics, analgesics, cognitive enhancers, psychostimulants, mood stabilizers, high-dose antipsychotics, antipsychotics, sedatives / hypnotics, anxiolytics, high-dose antidepressants, antidepressants.

[0326] Once all features are extracted from the raw data through an automated process, proprietary bioinformatics algorithms are used to decorrelate groups of features and find the combination of values ​​that best discriminates between different target groups. For each compound and dose, the system provides a probability that the drug is active and categorizes this putative activity into different target classes.

[0327] The system is designed to and can successfully measure many spontaneous behaviors and responses to challenges in the same test environment.

[0328] The hardware includes force sensors and some aversive stimuli to induce behavior. Three high-resolution cameras provide Constant 3D view of the mouse in the device (SC).

[0329] exist Mice were tested in a 45-minute test system in which they were exposed to a series of challenges during a 45-minute test period. The cubes were cleaned between each run.

[0330] For class and subclass analysis, a reference dataset has been established from hundreds of drug doses from multiple drug classes plus a control group. Each reference drug was tested in mice at multiple doses appropriate for that drug. The best-performing classifiers from our evaluation tests were selected and two different types of classifiers were established to make independent predictions at the drug class and subclass level. Classes include drugs that are currently on the market or have been clinically validated for a specific indication. Subclasses include marketed drugs and other compounds that have been validated by mechanistic means and are larger collections than classes.

[0331] The data was processed using proprietary computer vision and data mining algorithms and the results were compared to the features of reference compounds in our database. Multiple analyses were performed on the data to quantitatively produce independent predictions of drug class and drug subclass. The behavior of test drugs was evaluated using these classifiers to predict potential therapeutic utility.

[0332] Various analysis tools are available for follow-up analysis to answer different questions. For example, distinguishing typical and atypical antipsychotics or similarity to mood stabilizers can help to broaden the therapeutic indications of a lead compound. In a class analysis for screening compounds, a dozen pharmacological classes are evaluated at once (class analysis), whereas in a cloud analysis, only 2-4 target classes can be focused on. For example, a classifier can be trained using supervised training to distinguish between typical and atypical antipsychotics, then a new drug is used as a test set and the corresponding samples are classified in the space created by the training. In this way, not only can the similarity to one or the other class be quantified, but it can also be visualized. As mentioned above, The result of the run is a set of over 2000 features. By creating independent combinations of the original features, it is possible to reduce the data complexity and extract derived features that maximize the discrimination of the target groups. Similar to principal component analysis, the most important composite axes that best discriminate the groups under analysis can be found. To create a useful visualization tool, the Gaussian distributions representing the target groups in the two-dimensional space created by these principal axes can be plotted, where the width is given by the standard deviation through the inter-mouse variability. The separation between groups or classes is a measure of similarity or lack thereof (discrimination), and its statistical significance can be computed by estimating the probability that the result is due to chance.

[0333] Compound 018 was not behaviorally active (<40%) at 3 mg / kg. At higher doses (10, 30, and 100 mg / kg), the compound showed robust antidepressant-like features, with a small fraction showing psychostimulant-like features in both class and subclass. The subclass analysis indicated that the antidepressant feature was similar to an SSRI.

[0334] Compound 019 showed low behavioral activity at 3 mg / kg. At higher doses (10, 30, and 100 mg / kg), the compound showed mixed antidepressant and psychostimulant-like characteristics within both the class and subclass. At all doses, the antidepressant profile was similar to that of an SSRI, and at 100 mg / kg, it was a mixture of an SSRI and a SNRI.

Claims

1. A method for treating anxiety or depression, comprising orally administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising compound 019, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the method is a method of treating major depressive disorder (MDD).

3. The method of claim 1, wherein the method is a method of treating generalized anxiety disorder (GAD).

4. The method according to any one of claims 1 to 3, wherein Compound 019 or a pharmaceutically acceptable salt thereof is administered in the form of a capsule or a tablet.

5. The method of claim 4, wherein Compound 019 or a pharmaceutically acceptable salt thereof is administered once or twice daily.

6. The method of claim 4, wherein the pharmaceutical composition contains no more than about 1% of an alkaloid selected from (-) mesembrine, mesembrine, mesembrenol, or a combination thereof.

7. A method of inhibiting SERT, comprising administering to a mammal in need thereof a therapeutically effective amount of or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition comprising or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition contains no more than about 3% of an alkaloid selected from (-)mesembrine, mesembrine, mesembrenol, or a combination thereof; and Compound 019 accounts for at least 90% of the total alkaloid content in the pharmaceutical composition.

9. The pharmaceutical composition of claim 8, wherein the ratio of PDE4 / SERT inhibition value of the pharmaceutical composition is at least 10:

1.

10. The pharmaceutical composition of claim 8, wherein the compound 019 has a half-life of at least 30 minutes in a human hepatocyte assay of the pharmaceutical composition.

11. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition has an IC of less than about 30 nM for SERT. 50 .

12. The pharmaceutical composition of claim 8 or 11, wherein the pharmaceutical composition has a % inhibition of less than about 6% on at least one of PDE4A1A, PDE4B2, PDE4C1, and PDE4D2.

13. The method of claim 12, wherein the pharmaceutical composition has a PDE4 / SERT inhibition value ratio of at least 10:

1.

14. A method of inhibiting SERT without inhibiting PDE4, comprising administering to a mammal in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 8.

15. A composition comprising (-)6-epi-mecenitol or a pharmaceutically acceptable salt thereof, wherein the composition comprises no more than about 0.5% mesembrine or mesembrineine as measured by HPLC.

16. The composition of claim 15, wherein the composition comprises less than about 0.5% (-) mesembryanthemum as measured by HPLC.

17. A composition comprising (-)6-epi-mecetin or a pharmaceutically acceptable salt thereof, wherein the composition comprises no more than about 0.5% (-)mecetin as measured by HPLC.

18. A pharmaceutical composition comprising (-)6-epi-mecenitol or a pharmaceutically acceptable salt thereof, wherein the composition comprises no more than about 0.5% mesembrine, mesembrine, or (-)mecenitol as measured by HPLC.

19. The composition of claim 18, wherein the composition has no more than 0.5% of any other alkaloid compound as measured by HPLC.

20. A method for treating anxiety or depression, comprising orally administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of claims 17 to 19.

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