Serotonin receptors as therapeutic targets

Abstract

A serotonin receptor 5-HT2A inhibitor or a composition comprising a serotonin receptor 5-HT2A inhibitor is provided for use in the treatment of a proliferative disease in a subject in need thereof. Also disclosed are uses of a serotonin receptor 5-HT2A inhibitor in the preparation of a medicament for the treatment of proliferative diseases in a subject and methods of treatment thereof. Also disclosed are pharmaceutical compositions or combination therapies comprising a serotonin receptor 5-HT2A inhibitor and an immune checkpoint blocking / ICB therapy.

Description

Technical Field

[0001] The present disclosure broadly relates to methods of treating proliferative diseases. In particular, the present disclosure relates to 5-HT 2A serotonin receptor inhibitors. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common form of liver cancer (more than 800,000 deaths worldwide each year), with the Asia-Pacific region having the largest disease burden. In Singapore, HCC is the fourth most common cancer in men, with an age-standardized incidence of 17.7 per 100,000 men. Although early HCC can be treated by resection, local ablation therapy, or liver transplantation, the tumor recurs within 5 years in 60 - 80% of such cases, usually progressing to advanced HCC. Advanced HCC (aHCC) is a disease with poor prognosis (median survival < 1 year) and limited treatment options. Although combination immunotherapy using anti-PD-L1 + anti-VEGFA monoclonal antibodies has recently achieved a promising breakthrough by exceeding the efficacy of the historically systemic treatment drug sorafenib in the IMbrave150 clinical trial, there is still much room for improvement because two-thirds of aHCC patients remain refractory to any kind of treatment.

[0003] In addition, the molecular pathways targeted in all approved aHCC treatments are a very narrow range of molecular pathways (including the VEGF, PD-1, and CTLA-4 pathways). Therefore, there is a clinical need to search for and evaluate more potential molecular targets that can be rationally combined with existing approved immunotherapeutic agents. Considering that all three drugs approved by the FDA for first-line HCC treatment were initially approved for other non-HCC indications and then repurposed for HCC after successful clinical trials, there is a particular lack of HCC-specific therapeutic agents that target the liver immune microenvironment to improve treatment efficacy.

[0004] Accordingly, there is a need to provide alternative targeted treatments for proliferative diseases such as hepatocellular carcinoma.

[0005] Summary of the Invention

[0006] In a first aspect, there is provided a serotonin receptor 5-HT 2A inhibitor or a composition comprising a serotonin receptor 5-HT 2A inhibitor for treating a proliferative disease in a subject in need thereof.

[0007] In a second aspect, there is provided the use of a serotonin receptor 5-HT 2A inhibitor in the preparation of a medicament for treating a proliferative disease in a subject in need thereof.

[0008] In a third aspect, a method for treating a proliferative disease in a subject in need thereof is provided, wherein the method comprises administering to the subject an effective amount of a serotonin receptor 5-HT 2A inhibitor.

[0009] In some instances, the serotonin receptor 5-HT 2A inhibitor improves the suppression of the immune system, optionally improving the suppression of CD8 T cell function.

[0010] In some instances, the 5-HT 2A inhibitor is a selective antagonist of 5-HT 2A .

[0011] In some instances, the 5-HT 2A inhibitor is a reagent that targets the serotonin receptor 5-HT 2A by disrupting its function in enhancing CD8 T cell activation and / or a reagent that targets the serotonin receptor 5-HT 2A as a whole and thus disrupts its function in enhancing CD8 T cell activation. 2A

[0012] In some instances, the 5-HT 2A inhibitor is a compound having the following formula:

[0013]

[0014] its pharmaceutically acceptable acid addition salts and its possible stereoisomeric forms; wherein

[0015] R is hydrogen or C 1-6 alkyl;

[0016] Alk is C 1-4 alkanediyl;

[0017] Q is a group of the following formula:

[0018]

[0019] wherein Y 1 and Y 2 are each independently O or S;

[0020] R 2 is hydrogen, halogen, C 1-6 alkyl or C 1-6 alkoxy;

[0021] R 3 is hydrogen or halogen;

[0022] or

[0023] Q is a group of the following formula:

[0024]

[0025] wherein R 4 is hydrogen or C 1-6 alkyl;

[0026] Z is -S-, -CH2- or -CR 5 =CR 6 -; said R 5 and R 6 are each independently hydrogen or C 1-6 alkyl; and A is a divalent group -CH2-CH2-, -CH2-CH2-CH2- or -CR 7 =CR 8 -, said R 7 and R 8 are each independently hydrogen, halogen, amino or C 1-6 alkyl;

[0027] R1 is a group of the following formula:

[0028] -X-Ar(c),

[0029] wherein Ar is phenyl or substituted phenyl, said substituted phenyl being substituted with amino and / or 1, 2 or 3 halogen atoms; and

[0030] wherein X is >C=O, >CH-OH, >CH-O-C(O)-R 9 、>CH2、>C(OC 1-6 alkyl)2,

[0031]

[0032] >C=N-OH or >C=N-NH2;

[0033] said R 9 is hydrogen or C 1-6 alkyl and

[0034] said q is an integer 2 or 3;

[0035] or

[0036] R 1 is a group of the following formula:

[0037]

[0038] wherein R 10 is hydrogen or C 1-6 alkyl; R 11 、R 12 and R 13Each independently is hydrogen or a halogen;

[0039] or

[0040] R1 is a group of the following formula:

[0041]

[0042] wherein A is O or S; R 14 and R 15 each independently is hydrogen, a halogen, a hydroxyl group, a C 1-6 alkoxy group or a C 1-6 alkyl group.

[0043] In some instances, the 5-HT 2A inhibitor is a compound having the following formula:

[0044]

[0045] wherein

[0046] Z is a group selected from the following:

[0047]

[0048] R is hydrogen, a cyclic or straight-chain or branched acyclic organic group, a lower hydroxyalkyl group, a lower aminoalkyl group, or an aralkyl or heteroaralkyl group;

[0049] n is 0, 1 or 2;

[0050] X1 is a methylene group, a vinylene group or an NH or N(lower alkyl) group; and

[0051] X2 is a methylene group, or when X1 is a methylene group or a vinylene group, X2 is a methylene group or a bond; or when X1 is a methylene group, X2 is O, S, NH or N(lower alkyl) or a bond;

[0052] Y1 is a methylene group and Y2 is a methylene group, a vinylene group, an ethylene group, a propylene group or a bond; or

[0053] Y1 is a bond and Y2 is a vinylene group; or

[0054] Y1 is an ethylene group and Y2 is O, S, NH or N(lower alkyl);

[0055] Ar1 and Ar2 are independently an unsubstituted or substituted aryl or heteroaryl group;

[0056] W is oxygen or sulfur; or

[0057] its pharmaceutically acceptable salts, esters or prodrugs.

[0058] In some instances, the 5-HT2A The inhibitor is a compound having the following formula:

[0059]

[0060] wherein one of X and Y is CH2, and the other is selected from CH2, O and S;

[0061] The dashed line extending from Z represents an optional bond; when it does not represent a bond, Z is N, CH or COH; and when it represents a bond, Z is C;

[0062] Ar is phenyl, 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, 2-pyrimidinyl, 1-indolyl, 2-indolyl, 3-indolyl, 1-indolin-2-one, 3-indolin-2-one, 2- or 3-benzofuryl, 2- or 3-benzothienyl, 1-naphthyl or 2-naphthyl, each optionally substituted by halogen, lower alkyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, trifluoromethylsulfonyloxy, cycloalkyl, cycloalkyl-lower alkyl, nitro, amino, lower alkylamino, di-lower alkylamino, acylamino or C 1-2 alkylenedioxy;

[0063] R 1 is hydrogen, lower alkyl, lower alkenyl, lower alkynyl, cycloalk(en)yl, cycloalk(en)yl-lower alk(en / yne)yl, aryl, aryl-lower alkyl, acyl, thioacyl, lower alkylsulfonyl, trifluoromethylsulfonyl, arylsulfonyl,

[0064] R 1 is the group R 9 VCO-, where V is O or S, and R 9 is lower alkyl, cycloalkyl, cycloalkyl-lower alkyl or aryl, or R 1 is the group R 10 R 11 NCO- or R 10 R 11 NCS-, where R 10 and R 11 are independently hydrogen, lower alkyl, cycloalkyl, cycloalkyl-lower alkyl or aryl, or R 10 and R 11 together with the N atom to which they are attached form a pyrrolidinyl, piperidinyl or perhydroazepin group;

[0065] R 2 is hydrogen, lower alkyl, cycloalkyl or cycloalkyl-lower alkyl;

[0066] Or R 1 and R2 Together with the N atoms to which they are attached, form a group,

[0067] wherein Q is C═O, C═S or CH2; T is NH, S, O or CH2; and m is from 1 to 4, including the end values;

[0068] R 3 -R 5 are independently hydrogen, halogen, lower alkyl, lower alkylcarbonyl, phenylcarbonyl, halogen-substituted phenylcarbonyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, cycloalkyl, cycloalkyl-lower alkyl or nitro;

[0069] R 6 and R 7 are each hydrogen or lower alkyl, or they are joined together to form a 3- to 7-membered carbocyclic ring;

[0070] R 8 is hydrogen or lower alkyl;

[0071] Any alkyl, cycloalkyl or cycloalkylalkyl group present is optionally substituted by one or two hydroxy groups, which are again optionally esterified with an aliphatic or aromatic carboxylic acid; and any aryl substituent present is optionally substituted by halogen, lower alkyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, trifluoromethylsulfonyloxy, cycloalkyl, cycloalkyl-lower alkyl or nitro;

[0072] and pharmaceutically acceptable acid addition salts thereof.

[0073] In some instances, the 5-HT 2A inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), 2,3-dihydroxybutanedioic acid, 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (ketanserin tartrate), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (volinanserin) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

[0074] In some instances, the proliferative disease is a tumor and / or cancer.

[0075] In some instances, the proliferative disease is liver cancer.

[0076] In some instances, the proliferative disease is hepatocellular carcinoma.

[0077] In some instances, the serotonin receptor 5-HT used as described herein 2A inhibitor or the use of claim 2 as described herein or the method as described herein further comprises one or more combination therapies selected from immunotherapy, chemotherapy, ablation therapy, and transplantation.

[0078] In some instances, the serotonin receptor 5-HT used as described herein 2A inhibitor or the use as described herein or the method as described herein further comprises immunotherapy, such as, but not limited to, immune checkpoint blockade (ICB) therapy including the administration of monoclonal antibodies. Optionally, the immunotherapy may include the use of anti-PD-L1 (e.g., atezolizumab or nivolumab), anti-VEGFA (e.g., bevacizumab), anti-CTLA4 (e.g., ipilimumab), or anti-PD-1 (e.g., nivolumab) plus anti-CTLA4 (e.g., ipilimumab), and / or one or more of their combinations.

[0079] In some instances, the combination therapy is administered to a subject simultaneously, sequentially, or separately from the serotonin receptor 5-HT 2A inhibitor.

[0080] In some instances, the inhibitor will be administered intravenously, subcutaneously, orally, sublingually, or intraperitoneally.

[0081] In a fourth aspect, there is provided a pharmaceutical composition comprising a serotonin receptor 5-HT 2A inhibitor and an immune checkpoint blockade / ICB therapy.

[0082] In some instances, the serotonin receptor inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), 2,3-dihydroxybutanedioic acid, 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (tartrate ketanserin), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (florocserin), or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

[0083] In a fifth aspect, there is provided a pharmaceutical composition comprising a serotonin receptor 5-HT 2ACombination therapies of an inhibitor and an immune checkpoint blockade / ICB therapy, wherein the serotonin receptor inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), 2,3-dihydroxybutanedioic acid, 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (tartrate ketanserin), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (floridanserin) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

[0084] Definitions

[0085] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine; the term "C" 1-6 "Alkyl" refers to straight-chain and branched-chain saturated hydrocarbon groups having 1 to 6 carbon atoms, such as methyl, ethyl, 1-methylethyl, 1,1-dimethylethyl, propyl, 2-methylpropyl, butyl, pentyl, hexyl, etc.; "C" 1-4 alkanediyl" includes divalent straight-chain or branched-chain alkanediyls having 1 to 4 carbon atoms.

[0086] As used herein, the term "inhibitor" or "antagonist" refers to a molecule that reduces the amount or duration of the biological activity of serotonin, particularly the binding activity of the serotonin receptor 5-HT 2A . In some instances, the inhibitor or antagonist can include a compound, protein, nucleic acid, carbohydrate, antibody or small molecule that reduces the action of serotonin. For example, an antagonist can be used to inhibit the serotonin receptor 5-HT according to the present disclosure 2A .

[0087] The terms “treatment” and “therapy” and their synonyms refer to therapeutic treatment and prophylactic or preventative measures, where the aim is to prevent or slow down (mitigate) a medical condition, which includes but is not limited to diseases (e.g., proliferative diseases, including tumors and / or cancer), symptoms, and disorders. A medical condition also includes the body's response to a disease or disorder, such as dysregulated cell proliferation, dysregulated cell metabolism, and / or inflammation. Those in need of such treatment include those who already have a medical condition and those who are predisposed to developing a medical condition or in need of preventing a medical condition. In some instances, treatment as described herein delays tumor outgrowth and / or prolongs survival. In some instances, the present disclosure can provide a higher survival rate, delayed tumor outgrowth, and / or a lower lethality rate. In some instances, the present disclosure can provide a higher survival rate, delayed tumor outgrowth, and / or a lower lethality rate when used alone or in combination with therapies known in the art (e.g., immune checkpoint blockade (ICB) therapy).

[0088] As used herein, the term “subject” includes patients and non-patients. The term “patient” refers to an individual who has or may have a medical condition such as a proliferative disease (including tumors and / or cancer), while a “non-patient” refers to an individual who does not have or may not have a medical condition. “Non-patients” include healthy individuals, individuals without disease, and / or individuals without a medical condition. The term “subject” includes humans and animals. Animals can include but are not limited to mammals (e.g., non-human primates, canines, murine, rabbits, etc.). “Mouse” refers to any mammal from the murine family, such as mice, rats, etc.

[0089] The term “and / or,” e.g., “X and / or Y” should be understood to mean “X and Y” or “X or Y,” and should be regarded as providing explicit support for both meanings or either meaning.

[0090] In addition, in the specification of this document, whenever used, the term "substantially" should be understood to include, but not limited to, "entirely" or "completely", etc. Additionally, terms such as "comprising", "including", etc., whenever used, are intended to be non-restrictive descriptive languages, because in addition to other components not explicitly listed, they also broadly include the elements / components listed after these terms. For example, when using "comprising", referring to "a" feature also intends to refer to "at least one" feature. Terms such as "consisting of", etc., can be considered as subsets of terms such as "comprising", "including", etc. in appropriate contexts. Therefore, in the embodiments disclosed in this document using terms such as "comprising", "comprise", etc., it should be understood that these embodiments provide teachings of the corresponding embodiments using terms such as "consisting of", etc. Moreover, terms such as "about", "approximately", etc., whenever used, generally mean reasonable variations, such as a variation of + / -5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

[0091] In addition, in the specification of this document, certain values may be disclosed within a range. The values at the endpoints of the displayed range are intended to illustrate the preferred range. Whenever a range is described, it means that the range covers and teaches all possible sub-ranges and each individual value within the range. That is, the endpoints of the range should not be construed as inflexible limitations. For example, the description of the range from 1% to 5% means that there are specifically disclosed sub-ranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., and individually, the values within the range, such as 1%, 2%, 3%, 4%, and 5%. It should be understood that the individual values within the range also include integers, fractions, and decimals. Moreover, whenever a range has been described, it also means that the range covers and teaches values with up to 2 additional decimal places or significant figures (where appropriate) from the indicated numerical endpoints. For example, the description of the range from 1% to 5% means that there are specifically disclosed ranges of 1.00% to 5.00% and 1.0% to 5.0%, and all their intermediate values across the range (such as 1.01%, 1.02%...4.98%, 4.99%, 5.00% and 1.1%, 1.2%...4.8%, 4.9%, 5.0%, etc.). The meaning of the above specific disclosure applies to any depth / width of the range.

[0092] In addition, when describing some embodiments, the present disclosure may have disclosed a method and / or process as steps in a particular order. However, unless otherwise required, it should be understood that the method or process should not be limited to the steps in the particular order disclosed. Other orders of steps are possible. The steps in the particular order disclosed herein should not be construed as undue limitations. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to the steps performed in the written order. The order of the steps can vary and still remain within the scope of the present disclosure.

[0093] Furthermore, it should be understood that although the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be waived in other alternative embodiments, and the present disclosure provides support for such waivers and these related alternative embodiments.

[0094] Description of Embodiments

[0095] Exemplary non-limiting embodiments of serotonin receptor 5-HT 2A inhibitors and methods of treatment using the inhibitors are disclosed hereinafter.

[0096] 5-HT 2A receptor is one of three members of the 5-HT2 receptor family, is a typical 7-transmembrane G protein-coupled receptor, and is coupled to the Gq / 11 signal transduction protein. 5-HT 2A receptor is found postsynaptically in serotonergic neurons. 5-HT 2A receptor is mainly present in the frontal cortex or in regions connected to the visual cortex. Peripherally, 5-HT 2A receptor can be found to elicit responses in vascular smooth muscle and is a component of smooth muscle responses in the intestine.

[0097] The inventors of the present disclosure have found that serotonin receptor 5-HT 2A is a molecule that has a surprising inhibitory effect on CD8 T cells that activate NPC in the liver. Thus, the inventors of the present disclosure have found that serotonin receptor 5-HT 2A is a surprisingly effective target for adjuvant therapy in conjunction with HCC immunotherapy, which has the potential to meet the key clinical need of an HCC-specific molecular therapy that can be rationally combined with existing approved immunotherapeutic agents.

[0098] The hepatic immune microenvironment is inherently immunosuppressive because, under normal resting conditions, hepatocytes and non-parenchymal cells (NPCs) such as Kupffer cells and sinusoidal endothelial cells inhibit T cell activation. Thus, disrupting these immunosuppressive interactions between hepatic NPCs and tumor-specific T cells may potentially increase the efficacy of T cell-targeted immunotherapies in HCC.

[0099] Thus, in one aspect, there is provided a serotonin receptor 5-HT 2A inhibitor or a composition comprising a serotonin receptor 5-HT 2A inhibitor for treating a proliferative disease in a subject in need thereof.

[0100] In another aspect, there is provided the use of a serotonin receptor 5-HT 2A inhibitor in the manufacture of a medicament for treating a proliferative disease in a subject in need thereof.

[0101] In yet another aspect, there is provided a method of treating a proliferative disease in a subject in need thereof, wherein the method comprises administering to the subject an effective amount (e.g., a therapeutically effective amount) of a serotonin receptor 5-HT 2A inhibitor.

[0102] In some instances, there is provided a method of modulating the immune system of a subject, wherein the method comprises administering to the subject an effective amount of a serotonin receptor 5-HT 2A inhibitor.

[0103] As used herein, the immune system encompasses all types of cells, compounds, compositions, and / or proteins that are part of the body's self-defense mechanism against foreign objects and / or abnormal cells. In some instances, the immune system includes the innate immune system, such as NK cells, neutrophils, eosinophils, basophils, monocytes, etc. In some instances, the immune system includes the adaptive immune system, such as T lymphocytes, B lymphocytes, NK T cells, antigen-presenting cells, dendritic cells, etc. In some instances, the CD8 T cells in a subject are modulated by the methods disclosed herein.

[0104] In some instances, methods as disclosed herein eliminate the inhibition of immune cells by hepatocytes or liver-resident cells. In some instances, liver-resident cells can include, but are not limited to, liver non-parenchymal cells. In some instances, liver non-parenchymal cells include hepatic sinusoidal endothelial cells, hepatic stellate cells, and the like. In some instances, liver-resident cells can include, but are not limited to, liver-resident immune cells. In some instances, liver-resident immune cells include liver-resident T cells, liver-resident NK cells, and the like. In some instances, methods as disclosed herein remove the inhibition of T lymphocytes. In some instances, methods as disclosed herein remove the inhibition of CD8 T lymphocytes. In some instances, serotonin receptor 5-HT 2A inhibitors improve the inhibition of the immune system, optionally improving the inhibition of CD8 T cell function.

[0105] In some instances, serotonin receptor 5-HT 2A inhibitors serve as adjuvant therapy / immunotherapy for a subject in need thereof.

[0106] In some instances, serotonin receptor 5-HT 2A inhibitors enhance CD8 T cell activation. In some instances, serotonin receptor 5-HT 2A inhibitors increase the secretion of the pro-inflammatory cytokine IFNγ in CD8 T cell activation.

[0107] Without wishing to be bound by theory, it is believed that serotonin receptor 5-HT 2A inhibitors (such as ketanserin) disrupt the immunosuppressive interaction between hepatocytes and tumor-specific immune cells. In some instances, the inventors believe that serotonin receptor 5-HT 2A inhibitors (such as, ketanserin) surprisingly improve the inhibition of liver non-parenchymal cells (NPCs) on CD8 T cell effector function. The inventors have found that treatment with serotonin receptor 5-HT 2A inhibitors surprisingly results in a significant expansion and / or activation of CD8 T cell effectors (function). Thus, in some instances, serotonin receptor 5-HT 2A inhibitors increase the effectiveness of immune cells in immunotherapy. For example, serotonin receptor 5-HT 2A inhibitors increase the effectiveness of T cell-targeted immunotherapy.

[0108] In some instances, the functional phenotypes of activated immune cells (e.g., activated CD8 T cells) can include, but are not limited to, upregulation of CD25 expression, increased expression / production of IFNγ and / or TNFα, increased CD44 expression, increased expression / production of granzyme B, increased expression / production of perforin, increased cytotoxicity (e.g., as demonstrated by killing target cells in vitro), and the like.

[0109] Serotonin receptor 5-HT is also disclosed 2A Use of an inhibitor to enhance CD8 T cell activation, one embodiment of which is to reduce the inhibition of CD8 T cells by liver non-parenchymal cells (NPC). In some examples, a specific aspect of the enhanced CD8 T cell activation is the secretion of the pro-inflammatory cytokine IFNγ.

[0110] Without wishing to be bound by theory, targeting 5-HT 2A serotonin receptor disrupts the functions of liver non-parenchymal cells and CD8 T cells resulting in enhanced CD8 T cell activation. In some examples, this disruption is mediated by disrupting the gene encoding 5-HT 2A (Htr2a in mice, HTR2A in humans).

[0111] In some examples, the 5-HT 2A inhibitor is a selective antagonist of 5-HT 2A .

[0112] In some examples, the 5-HT 2A inhibitor is a reagent that targets serotonin receptor 5-HT by disrupting its function of enhancing CD8 T cell activation and / or a reagent that targets the overall function of serotonin receptor 5-HT 2A and thus disrupts its function of inhibiting CD8 T cell activation. 2A

[0113] In some examples, the 5-HT 2A inhibitor is a compound having the following formula:

[0114]

[0115] Its pharmaceutically acceptable acid addition salts and their possible stereoisomeric forms; wherein

[0116] R is hydrogen or C 1-6 alkyl;

[0117] Alk is C 1-4 alkanediyl;

[0118] Q is a group of the following formula:

[0119]

[0120] wherein Y1 and Y 2 are each independently O or S;

[0121] R 2 is hydrogen, halogen, C 1-6 alkyl or C 1-6 alkoxy;

[0122] R 3 is hydrogen or a halogen;

[0123] or

[0124] Q is a group of the formula:

[0125]

[0126] wherein R 4 is hydrogen or C 1-6 alkyl;

[0127] Z is -S-, -CH2- or -CR 5 =CR 6 -; said R 5 and R 6 are each independently hydrogen or C 1-6 alkyl; and A is a divalent group -CH2-CH2-, -CH2-CH2-CH2- or -CR 7 =CR 8 -, said R 7 and R 8 are each independently hydrogen, a halogen, an amino group or C 1-6 alkyl;

[0128] R 1 is a group of the formula:

[0129] -X-Ar(c),

[0130] wherein Ar is phenyl or substituted phenyl, said substituted phenyl being substituted with an amino group and / or 1, 2 or 3 halogen atoms; and

[0131] wherein X is >C=O, >CH-OH, >CH-O-C(O)-R 9 、>CH2、>C(OC 1-6 alkyl)2,

[0132]

[0133] >C=N-OH or >C=N-NH2;

[0134] said R 9 is hydrogen or C 1-6 alkyl, and

[0135] said q is an integer of 2 or 3;

[0136] or

[0137] R1 is a group of the formula:

[0138]

[0139] wherein R 10 is hydrogen or C 1-6 alkyl; R 11 , R 12 and R 13 are each independently hydrogen or halogen; or

[0140] R 1 is a group of the formula:

[0141]

[0142] wherein A is O or S; R 14 and R 15 are each independently hydrogen, halogen, hydroxy, C 1-6 alkoxy or C 1-6 alkyl.

[0143] In some instances, the compounds of formula (I) according to the present disclosure are those wherein R 1 is a group of formula (c).

[0144] In some instances, the compounds of formula (I) according to the present disclosure are those compounds of formula (I) wherein Q is a group of formula (a), wherein Y 1 and Y 2 are both oxygen atoms, and R 2 and R 3 are both hydrogen; or wherein Q is a group of formula (b), wherein R 4 is methyl, Z is -CR 5 =CR 6 -, wherein R 5 and R 6 are independently hydrogen or methyl, A is -CR 7 =CR 8 -, wherein R 7 and R 8 are independently hydrogen or methyl; and / or wherein the group (c) X is >C=O, and Ar is a halogen-substituted phenyl.

[0145] In some instances, the compounds according to the present disclosure are selected from 3-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2,4(1H,3H)-quinazolinedione (this compound is commonly known as ketanserin) and 3-[2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl]-2,7-dimethyl-4H-pyrido-[1,2-a]-pyrimidin-4-one, and pharmaceutically acceptable acid addition salts thereof.

[0146] The compound of formula (I) can be used as such or in the form of its acid addition salts. The latter can be conveniently obtained by treating the base form with a suitable acid, such as inorganic acids like hydrohalic acids, e.g., hydrochloric acid, hydrobromic acid, etc., and sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids, such as acetic acid, propionic acid, glycolic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, (Z)-2-butenedioic acid, (E)-2-butenedioic acid, 2-hydroxybutanedioic acid, 2,3-dihydroxybutanedioic acid, 2-hydroxy-1,2,3-propanetricarboxylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, cyclohexanesulfamic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, etc.

[0147] In some instances, the 5-HT 2A inhibitor is a compound having the following formula:

[0148]

[0149] wherein

[0150] Z is a group selected from:

[0151]

[0152] R is hydrogen, a cyclic or straight-chain or branched acyclic organic group, a lower hydroxyalkyl group, a lower aminoalkyl group, or an aralkyl or heteroaralkyl group;

[0153] n is 0, 1 or 2;

[0154] X1 is methylene, vinylene or NH or N(lower alkyl) group; and

[0155] X2 is methylene, or when X1 is methylene or vinylene, X2 is methylene or a bond; or when X1 is methylene, X2 is O, S, NH or N(lower alkyl) or a bond;

[0156] Y1 is methylene and Y2 is methylene, vinylene, ethylene, propylene or a bond; or

[0157] Y1 is a bond and Y2 is vinylene; or

[0158] Y1 is ethylene and Y2 is O, S, NH or N(lower alkyl);

[0159] Ar1 and Ar2 are independently unsubstituted or substituted aryl or heteroaryl groups;

[0160] W is oxygen or sulfur; or

[0161] its pharmaceutically acceptable salts, esters or prodrugs.

[0162] There is also provided a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof.

[0163] In some instances, the 5-HT 2A inhibitor is a compound having the following formula:

[0164]

[0165] wherein one of X and Y is CH2, and the other is selected from CH2, O and S;

[0166] The dotted line emanating from Z represents an optional bond; when it does not represent a bond, Z is N, CH or COH; and when it represents a bond, Z is C;

[0167] Ar is phenyl, 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, 2-pyrimidinyl, 1-indolyl, 2-indolyl, 3-indolyl, 1-indolin-2-one, 3-indolin-2-one, 2- or 3-benzofuranyl, 2- or 3-benzothienyl, 1-naphthyl or 2-naphthyl, each optionally substituted by halogen, lower alkyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, trifluoromethylsulfonyloxy, cycloalkyl, cycloalkyl-lower alkyl, nitro, amino, lower alkylamino, di-lower alkylamino, acylamino or C 1-2 alkylenedioxy;

[0168] R 1 is hydrogen, lower alkyl, lower alkenyl, lower alkynyl, cycloalk(en)yl, cycloalk(en)yl-lower alk(en / yne)yl, aryl, aryl-lower alkyl, acyl, thioacyl, lower alkylsulfonyl, trifluoromethylsulfonyl, arylsulfonyl,

[0169] R 1 is the group R 9 VCO-, where V is O or S, and R 9 is lower alkyl, cycloalkyl, cycloalkyl lower alkyl or aryl, or R 1 is the group R 10 R 11 NCO- or R 10 R 11 NCS-, where R 10 and R 11 are independently hydrogen, lower alkyl, cycloalkyl, cycloalkyl-lower alkyl or aryl, or R 10 and R 11 together with the N atom to which they are attached form a pyrrolidinyl, piperidinyl or perhydroazepin group;

[0170] R 2 is hydrogen, a lower alkyl, a cycloalkyl or a cycloalkyl-lower alkyl;

[0171] Or R 1 and R 2 together with the N atom to which they are attached form a group,

[0172] wherein Q is C=O, C=S or CH2; T is NH, S, O or CH2; and m is 1-4, including the end values;

[0173] R 3 -R 5 are independently hydrogen, halogen, lower alkyl, lower alkyl carbonyl, phenyl carbonyl, halogen-substituted phenyl carbonyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, cycloalkyl, cycloalkyl-lower alkyl or nitro;

[0174] R 6 and R 7 are each hydrogen or lower alkyl, or they are joined together to form a 3-7 membered carbocyclic ring;

[0175] R 8 is hydrogen or lower alkyl;

[0176] Any alkyl, cycloalkyl or cycloalkylalkyl group present is optionally substituted by one or two hydroxy groups, and said hydroxy groups are again optionally esterified with an aliphatic or aromatic carboxylic acid; and any aryl substituent present is optionally substituted by halogen, lower alkyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, trifluoromethylsulfonyloxy, cycloalkyl, cycloalkyl-lower alkyl or nitro;

[0177] and its pharmaceutically acceptable acid addition salts.

[0178] In some instances, 5-HT 2AThe inhibitors are [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), tartaric acid, 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (ketanserin tartrate), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (volinanserin), or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

[0179] As used herein, the term pimavanserin refers to a member of the ureas in which three of the four hydrogens are replaced by 4-fluorobenzyl, 1-methylpiperidin-4-yl, and 4-(isopropoxy)benzyl. Pimavanserin has the CAS number 706779-91-1 or 706782-28-7, or is also known as 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea, or the following formula:

[0180]

[0181] As used herein, the term refers to a selective 5-HT 2A receptor antagonist. Volinanserin has the CAS number 139290-65-6, or is also known as: (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol, or the following formula:

[0182]

[0183] As used herein, the term ketanserin refers to a quinazoline derivative that acts as a selective 5-HT 2A serotonin antagonist. Ketanserin has the CAS number 74050-98-9 or 83846-83-7, or is also known as: [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione], or the following formula:

[0184]

[0185] In some instances, ketanserin as described herein can be ketanserin tartrate, which has CAS: 83846-83-7, or is also known as 2,3-dihydroxybutanedioic acid; 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione, or the following formula:

[0186]

[0187] In some instances, based on the molecular structure of ketanserin, the 5-HT 2A specific inhibitor can exhibit the efficacy of enhancing CD8 T cell activation. In some instances, the 5-HT 2A inhibitor is ketanserin.

[0188] Pharmaceutically acceptable salts that can be mentioned include acid addition salts and base addition salts. Such salts can be formed by conventional means, for example, by reacting the compound of formula I in free acid or free base form with one or more equivalents of a suitable acid or base, optionally in a solvent or in a medium (wherein the salt is insoluble). Then, the solvent or the medium is removed using standard techniques (e.g., in vacuo, by freeze-drying, or by filtration). The salts can also be prepared by exchanging the counterion of a serotoninergic compound in salt form with another counterion, for example, using a suitable ion exchange resin.

[0189] Examples of pharmaceutically acceptable salts include acid addition salts derived from inorganic acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably potassium and calcium.

[0190] Examples of acid addition salts include those formed with acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, arylsulfonic acids (such as benzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, and p-toluenesulfonic acid), ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid (e.g., D-gluconic acid), glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxyethanesulfonic acid, lactic acid (e.g., (+)-L-lactic acid and (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid (e.g., (-)-L-malic acid), malonic acid, (±)-DL-mandelic acid, metaphosphoric acid, methanesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, tartaric acid (e.g., (+)-L-tartaric acid), thiocyanic acid, undecylenic acid, and valeric acid.

[0191] Specific examples of salts are salts derived from inorganic acids, organic acids, and metals, said inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid; said organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, arylsulfonic acids; said metals such as sodium, magnesium, or preferably potassium and calcium.

[0192] As described above, any solvates of the compounds and their salts are also included. Preferred solvates are those formed by incorporating molecules of a non-toxic pharmaceutically acceptable solvent (hereinafter referred to as the solvating solvent) into the solid state structure (e.g., crystal structure) of the compounds of the present invention. Examples of such solvents include water, alcohols (such as ethanol, isopropanol, and butanol), and dimethyl sulfoxide. Solvates can be prepared by recrystallizing the compounds of the present invention with a solvent or a solvent mixture containing the solvating solvent. Whether a solvate has been formed in any given case can be determined by analyzing the crystals of the compound using well-known methods and standard techniques, such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC), and X-ray crystal diffraction.

[0193] Solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, examples of hydrates including hemihydrates, monohydrates, and dihydrates.

[0194] The compounds of the present disclosure will generally be administered as pharmaceutical formulations admixed with pharmaceutically acceptable adjuvants, diluents, or carriers, which can be selected with due consideration of the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers can be chemically inert to the active compounds and can be without adverse side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations are known in the art. For parenteral administration, parenterally acceptable aqueous solutions can be used, which are pyrogen-free and have the required pH, isotonicity, and stability. Suitable solutions are well known to those skilled in the art, and many methods are described in the literature.

[0195] In addition, those skilled in the art can use conventional techniques and / or routinely achieve the preparation of suitable formulations in accordance with standard and / or recognized pharmaceutical practice.

[0196] The amount of the compound in any pharmaceutical formulation used in accordance with the present disclosure will depend on various factors such as the severity of the condition to be treated, the particular patient / subject to be treated, and the compound used. In any case, the amount of the compound in the formulation can be routinely determined by those skilled in the art.

[0197] In some instances, the proliferative disease is a tumor and / or cancer.

[0198] In some instances, the tumor / cancer is selected from: liver cancer (e.g., hepatocellular carcinoma), bone cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC), pancreatic adenocarcinoma, etc.), skin cancer (e.g., melanoma, including but not limited to cutaneous or intraocular malignant melanoma), head and neck cancer, breast cancer, lung cancer, kidney cancer, uterine cancer, bowel cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal area cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including cancers induced by asbestos), hematological malignancies, including for example multiple myeloma, B cell lymphoma, Hodgkin lymphoma / primary mediastinal B cell lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and precursor T lymphoblastic lymphoma and any combination of their cancers.

[0199] In some instances, the proliferative disease is liver cancer.

[0200] In some instances, the proliferative disease is hepatocellular carcinoma.

[0201] In some instances, the present disclosure also includes additional / combination therapies, including but not limited to immunotherapy (e.g., immune checkpoint blockade / ICB therapy / adoptive immune cell transfer therapy, e.g., CAR-T cell therapy), chemotherapy, ablation therapy (local ablation therapy) such as radiotherapy or surgery (e.g., resection), transplantation, etc.

[0202] In some instances, the present disclosure also includes immunotherapy, such as but not limited to immune checkpoint blockade (ICB) therapy, which includes administering a monoclonal antibody, optionally, the immunotherapy may include using anti-PD-L1 (e.g., atezolizumab or nivolumab), anti-VEGFA (e.g., bevacizumab), anti-CTLA4 (e.g., ipilimumab), or anti-PD-1 (e.g., nivolumab) plus anti-CTLA4 (e.g., ipilimumab) and / or one or more of their combinations.

[0203] Also disclosed are methods of treating HCC, which comprise administering a therapeutically effective amount of a serotonin receptor 5-HT 2A inhibitor in combination with an immune checkpoint blockade (ICB) therapy. In some instances, the immune checkpoint blockade therapy can take the form of a combination of anti-PD-L1 and anti-VEGFA mAbs.

[0204] In some instances, the methods disclosed herein provide higher survival rates, delayed tumor growth, and lower lethality compared to using ICB therapy alone. In one such embodiment, the ICB therapy can take the form of a combination of anti-PD-L1 and anti-VEGFA mAbs.

[0205] In some instances, the combination therapy is administered to a subject simultaneously, sequentially, or separately with a serotonin receptor 5-HT 2A inhibitor.

[0206] In some instances, the inhibitor will be administered intravenously, subcutaneously, orally, sublingually, or intraperitoneally.

[0207] Also disclosed are compositions comprising a serotonin receptor 5-HT 2A inhibitor and an immune checkpoint blockade / ICB therapy.

[0208] In another aspect, provided are pharmaceutical compositions comprising a serotonin receptor 5-HT 2A inhibitor and an immune checkpoint blockade / ICB therapy.

[0209] In some instances, the serotonin receptor inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), 2,3-dihydroxybutanedioic acid; 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (ketanserin tartrate), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (floridserin) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

[0210] Also disclosed is a combination therapy comprising a serotonin receptor 5-HT 2A inhibitor and an immune checkpoint blockade / ICB therapy.

[0211] In yet another aspect, provided is a composition comprising a serotonin receptor 5-HT 2ACombination therapies of an inhibitor and an immune checkpoint blockade / ICB therapy, wherein the serotonin receptor inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), tartaric acid; 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (ketanserin tartrate), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (florocerine) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

[0212] In some instances, a patient or subject receiving treatment / therapy may exhibit a decrease in the size or rate of tumor growth after administration of an inhibitor and / or combination therapy as described herein. In some instances, the decrease may be observed at week 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some instances, the reduction may be about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. For example, at about week 12 after weekly administration of an inhibitor and / or combination therapy as described herein, a patient may exhibit a reduction in tumor growth (size and / or growth rate) of about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

[0213] Also disclosed is the combination therapy as described herein.

[0214] Also disclosed is the therapy / composition / method as described herein. Brief Description of the Drawings

[0216] Based on the following discussion and if applicable, in conjunction with the drawings, the exemplary embodiments of the present disclosure will be better understood and apparent to those of ordinary skill in the art. It should be understood that other modifications to the serotonin inhibitor may be made without departing from the scope of the present invention. Exemplary embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more embodiments to form new exemplary embodiments. The exemplary embodiments should not be construed as limiting the scope of the present disclosure.

[0217] Figure 1In vitro screening of candidate small molecules that enhance the effector function of CD8 T cells activated by liver NPCs. Ovalbumin-specific OT-I CD8 T cells were co-cultured in 96-well plates in the presence of equal numbers of liver NPCs pulsed with ovalbumin peptide. Individual compounds from the Prestwick chemical library of FDA-approved drugs were diluted into separate wells of the co-culture to a final concentration of 10 μM. As a negative control, an equal volume of DMSO (to a final concentration of 0.5% volume / volume) was added to the co-culture (black symbols). As a positive control, OT-I CD8 T cells were activated with plate-bound αCD3ε (3 μg / mL) + soluble αCD28 antibody (1 μg / mL). Four days after activation, the functional phenotypes of the activated CD8 T cells were determined using high-throughput multiparameter flow cytometry. The perturbation of each drug on each parameter was evaluated by the direction and extent of the perturbation from the mean of the negative control for each parameter, and scored by the total number of positive perturbations. (A) Distribution of total positive perturbation scores among all 640 drugs screened. (B–F) Distribution of the extent and direction of perturbation of the 5 measured functional parameters (normalized by standard deviation from the mean of the negative control). Left panel—negative control, middle panel—50 preliminary hits, right panel—positive control. Each drug was tested in individual wells and evaluated against 80 negative and 80 positive control wells.

[0218] Figure 2 Shows the effect of increasing concentrations of ketanserin on CD8 T cell effector function markers during activation of CD8 T cells by liver NPCs. Ketanserin was added to the liver NPC–CD8 T cell co-culture at the indicated concentrations, as Figure 1 shown. Four days after activation, the effector function of CD8 T cells was determined by flow cytometry for the indicated parameters. Statistical analysis used the Kruskal–Wallis test and Dunn's multiple comparison test, *p < 0.05, **p < 0.01. Data represent 2 independent biological replicates.

[0219] Figure 3 Show 5-HT 2A Genetic and / or pharmacological ablation of 5-HT + / + activity on CD8 T cells activated by liver NPCs. OT-I Cas9 2A CD8 T cells were transduced with lentiviral vectors carrying gRNA sequences targeting Htr2a or LacZ for CRISPR gene editing. (A) 5-HT 2A immunoblot to assess the gene editing efficiency of each candidate gRNA. Red arrow (gRNA sequence #5) indicates the sequence used to generate CRISPR knockout of Htr2a in subsequent experiments. (B) Htr2a-KO or control gRNA (LacZ)-transduced OT-I cells were co-cultured with asFigure 1 Hepatic NPCs pulsed with ovalbumin were co-cultured in the presence of 5 μM ketanserin or an equivalent concentration of DMSO vehicle. On day 4 after activation, effector functions of CD8 T cells were measured by flow cytometry for the indicated parameters. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001.

[0220] Figure 4 Showing 5-HT 2A is upregulated in CD8 T cells after activation in mice and humans. Polyclonal CD8 T cells isolated from wild-type mice and healthy human PBMCs were activated in vitro with plate-bound α-CD3 and soluble α-CD28 antibodies for 4 days. (A) Immunoblot analysis of 5-HT 2A and GAPDH in whole cell lysates prepared from murine CD8 T cells sampled at the indicated time points. Lysates equivalent to 5 × 10 5 cells were loaded per lane. (B) Quantification of 5-HT 2A protein levels relative to the GAPDH loading control in the samples in (A). (C) Immunoblot analysis of 5-HT 2A and histone H3 in whole cell lysates prepared from human CD8 T cells sampled at the indicated time points. Lysates equivalent to 5 × 10 5 cells were loaded per lane. (D) Quantification of 5-HT 2A protein levels relative to the histone H3 loading control in the samples in (C). (E) Kinetics of 5-HT 2A protein expression relative to the loading control during activation of CD8 T cells isolated from mice and two healthy human donors.

[0221] Figure 5 Showing that 5-HT 2A inhibition using ketanserin enhanced the cytotoxic effector phenotype in murine and human CD8 T cells during T cell activation. Polyclonal CD8 T cells isolated from (A) wild-type mice and (B) healthy human PBMCs were activated in vitro with plate-bound α-CD3 and soluble α-CD28 antibodies for 4 days. On day 4 after activation, effector functions of CD8 T cells were measured by flow cytometry for the indicated parameters. Statistical analysis was performed using Welch's two-tailed t-test, *p < 0.05, **p < 0.01.

[0222] Figure 6 Showing targeting of 5-HT 2AFunction. Plasmids expressing oncogenes and firefly luciferase reporter were injected by hydrodynamic injection to induce HCC in immunocompetent mice. (a) Survival of mice bearing HCC tumors treated with 2.5 mg / kg ketanserin (red symbols) or an equal volume of 2.5% (v / v) DMSO vehicle (blue symbols) by intraperitoneal injection twice a week starting on day 25 after tumor induction. Data were pooled from two independent replicates. Statistics were performed using the Mantel-Cox logrank test. (b) Survival of mice induced with Htr2a knockout (+Htr2a gRNA) or Htr2a wild-type (+LacZ gRNA) HCC tumors. Data were pooled from two independent replicates. Statistical analysis was performed using the Mantel-Cox logrank test. (c) Log2 fold change in tumor burden of mice bearing HCC adoptively transferred with Htr2a-KO or hTR2A-WT tumor-specific CD8 T cells. Statistical analysis was performed using the Mann-Whitney test.

[0223] Experimental data

[0224] The inventors of the present disclosure identified ketanserin, a selective antagonist of a serotonin receptor, as a lead hit in an in vitro screen of the Prestwick library of small molecule drugs approved by the US FDA that can improve the inhibition of CD8 T cell effector function by liver non-parenchymal cells (NPCs). Figure 1 ) Ketanserin scored high in the number of positive perturbation parameters of CD8 T cell function, scoring 7 out of a maximum possible 9 points. Figure 1 A). Treatment of liver NPC-activated CD8 T cells with ketanserin increased their survival and proliferation Figure 1 B), and also led to increased expression of the activation marker CD25 Figure 1 C) and increased expression of the effector cytokines IFNγ and TNFα Figure 1 D and 1E), but did not lead to increased expression of the cytotoxic effector molecule granzyme B Figure 1 F).

[0225] The inventors then verified the hit molecule by culturing CD8 T cells activated in vitro with liver NPCs with increasing doses of ketanserin tartrate and found that ketanserin treatment showed a dose-dependent effect on the number of live cells, CD25 expression, and IFNγ and TNFα secretion. Figure 2 ) As a further verification, the inventors generated CRISPR-Cas9 edited to disrupt the Htr2a locus (encoding 5-HT 2A , Figure 3CD8 T cells of (A), and these Htr2a-KO cells were activated with liver NPCs in the presence or absence of ketanserin. The inventors found that genetic disruption of Htr2a recapitulated the increased proliferation, CD25 expression, and increased IFNγ secretion phenotypes that the inventors previously found under ketanserin-mediated 5-HT 2A inhibition ([ Figure 3 B).

[0226] To determine whether serotonin receptors are expressed by human CD8 T cells, the inventors outlined the kinetics of 5-HT 2A expression during mouse and human CD8 T cell activation by immunoblot analysis ([ 2A ). The inventors found that 5-HT Figure 4 expression increased consistently after in vitro activation of both mouse (consistent with data from Leon-Ponte et al. (Blood, 2007)) and human CD8 T cells (a new finding not previously described in the literature). These data suggest that the molecular target of the tool compound ketanserin indeed exists in activated CD8 T cells and can therefore be inhibited by the specific action of ketanserin. 2A

[0227] The inventors further compared the effect of inhibiting 5-HT 2A activity with ketanserin during activation in mouse and human CD8 T cells ([ Figure 5 ). In both mouse and human CD8 T cells, ketanserin treatment significantly increased the percentage of granzyme B + cells after in vitro activation. In human CD8 T cells, there was a trend towards increased cell number and IFNγ expression compared to mouse CD8 T cells, although these were not significant.

[0228] The inventors evaluated the therapeutic efficacy of ketanserin administration in our HCC-customized MITCH model (SIGN / TDF / 121). The inventors previously described that the MITCH model of HCC only partially responds to combined monoclonal antibody blockade of PD-L1 and VEGFR2, which mimics the resistance to treatment observed when treating HCC patients with the combination of atezolizumab (anti-PD-L1) + bevacizumab (anti-VEGFA), which is clinically approved as a first-line HCC therapy. Mice treated with ketanserin survived significantly longer than control mice, and tumor growth and lethality were generally delayed ([ Figure 6 A). The protective effect of ketanserin appears to be mediated by its action on cells other than HCC tumor cells, as mice with Htr2a-deficient HCC tumors did not show significant differences in survival or spontaneous tumor rejection compared to mice with Htr2a wild-type HCC tumors ([ Figure 6 ​B). Additionally, adoptive transfer of Htr2a-KO tumor-specific CD8 T cells also more strongly reduced tumor burden in mice bearing HCC tumors compared to mice treated with an equal number of Htr2a wild-type CD8 T cells ( Figure 6 C).

[0229] Thus, these data provide multiple lines of evidence that disruption of the serotonin receptor 5-HT 2A function may be an effective adjuvant for T cell-targeted immunotherapy of HCC.

[0230] Application

[0231] The embodiments of the serotonin receptor 5-HT 2A inhibitors disclosed herein provide alternative adjuvant immunotherapies for treating proliferative diseases.

[0232] Advantageously, specifically targeting the 5-HT 2A serotonin receptor provides specific activation of tumor-specific CD8 T cells by improving the inhibition of liver non-parenchymal cells on CD8 T cells.

[0233] Those skilled in the art should understand that other changes and / or modifications can be made to the embodiments disclosed herein without departing from the spirit or scope of the present disclosure as broadly described. For example, in the description herein, the features of different exemplary embodiments can be mixed, combined, interchanged, incorporated, adopted, modified, included, etc. in different exemplary embodiments. Therefore, this embodiment is considered illustrative in all aspects and not restrictive.

Claims

1. Serotonin receptor 5-HT 2A inhibitor or a composition comprising a serotonin receptor 5-HT 2A inhibitor for treating a proliferative disease in a subject in need thereof.

2. Serotonin receptor 5-HT 2A Use of an inhibitor in the preparation of a medicament for treating a proliferative disease in a subject in need thereof.

3. A method of treating a proliferative disease in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a serotonin receptor 5-HT 2A inhibitor.

4. The serotonin receptor 5-HT used in claim 1 2A inhibitor or the use of claim 2 or the method of claim 3, wherein the serotonin receptor 5-HT 2A inhibitor improves the inhibition of the immune system and optionally improves the inhibition of CD8 T cell function.

5. The serotonin receptor 5-HT used in claim 1 or 4 2A inhibitor, or the use of claim 2 or 4, or the method of claim 3 or 4, wherein said 5-HT 2A inhibitor is a 5-HT 2A selective antagonist.

6. The serotonin receptor 5-HT used in any one of claim 1 or claims 4 to 5 2A inhibitor or the use of any one of claim 2 or claims 4 to 5 or the method of any one of claim 3 or claims 4 to 5, wherein said 5-HT 2A inhibitor is a reagent that targets the serotonin receptor 5-HT by disrupting its function of enhancing CD8 T cell activation 2A and / or a reagent that targets the overall function of the serotonin receptor 5-HT 2A and thus disrupts its function of enhancing CD8 T cell activation.

7. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 6 2A inhibitor, or the use of any one of claims 2 or 4 to 6, or the method of any one of claims 3 or 4 to 6, wherein the 5-HT 2A inhibitor is a compound having the following formula: Its pharmaceutically acceptable acid addition salts and their possible stereoisomeric forms; wherein R is hydrogen or C 1-6 alkyl; Alk is C 1-4 alkanediyl; Q is a group of the formula: where Y 1 and Y 2 are each independently O or S; R 2 is hydrogen, halogen, C 1-6 alkyl or C 1-6 alkoxy; R 3 is hydrogen or a halogen; or Q is a group of the formula: wherein R 4 is hydrogen or C 1-6 alkyl; Z is -S-, -CH2- or -CR 5 =CR 6 -; said R 5 and R 6 are each independently hydrogen or C 1-6 alkyl; and A is a divalent group -CH2-CH2-, -CH2-CH2-CH2- or -CR 7 =CR 8 -, said R 7 and R 8 are each independently hydrogen, halogen, amino or C 1-6 alkyl; R 1 is a group of the formula: -X-Ar (c), wherein Ar is phenyl or substituted phenyl, said substituted phenyl being substituted with an amino group and / or one, two or three halogen atoms; and wherein X is >C=O, >CH-OH, >CH-O-C(O)-R 9 , >CH2, >C(OC 1-6 alkyl)2, >C=N-OH or >C=N-NH2; Said R 9 is hydrogen or C 1-6 alkyl, and said q is the integer 2 or 3; or R1 is a group of the formula: wherein R 10 is hydrogen or C 1-6 alkyl; R 11 , R 12 and R 13 are each independently hydrogen or a halogen; or R 1 is a group of the following formula: wherein A is O or S; R 14 and R 15 are each independently hydrogen, halogen, hydroxy, C 1-6 alkoxy or C 1-6 alkyl.

8. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 7 2A inhibitor, or the use of any one of claims 2 or 4 to 7, or the method of any one of claims 3 or 4 to 7, wherein the 5-HT 2A inhibitor is a compound having the following formula: wherein Z is a group selected from: R is hydrogen, a cyclic or straight-chain or branched acyclic organic group, a lower hydroxyalkyl group, a lower aminoalkyl group, or an aralkyl or heteroaralkyl group; n is 0, 1 or 2; X1 is a methylene group, a vinylene group, an NH or N(lower alkyl) group; and X2 is a methylene group, or when X1 is a methylene group or a vinylene group, X2 is a methylene group or a bond; or when X1 is a methylene group, X2 is O, S, NH or N(lower alkyl) or a bond; Y1 is a methylene group and Y2 is a methylene group, a vinylene group, an ethylene group, a propylene group or a bond; or Y1 is a bond and Y2 is a vinylene group; or Y1 is an ethylene group and Y2 is O, S, NH or N(lower alkyl); Ar1 and Ar2 are independently unsubstituted or substituted aryl or heteroaryl groups; W is oxygen or sulfur; or its pharmaceutically acceptable salts, esters or prodrugs.

9. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 8 2A inhibitor, or the use of any one of claims 2 or 4 to 8, or the method of any one of claims 3 or 4 to 8, wherein the 5-HT 2A inhibitor is a compound having the following formula: wherein one of X and Y is CH2 and the other is selected from CH2, O and S; The dotted line emanating from Z represents an optional bond; when it does not represent a bond, Z is N, CH or COH; and when it represents a bond, Z is C; Ar is phenyl, 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, 2-pyrimidinyl, 1-indolyl, 2-indolyl, 3-indolyl, 1-indolin-2-one, 3-indolin-2-one, 2- or 3-benzofuryl, 2- or 3-benzothienyl, 1-naphthyl or 2-naphthyl, each optionally substituted by halogen, lower alkyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, trifluoromethylsulfonyloxy, cycloalkyl, cycloalkyl-lower alkyl, nitro, amino, lower alkylamino, di-lower alkylamino, acylamino or C 1-2 substituted by alkylenedioxy; R 1 is hydrogen, lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl(ene), cycloalkyl(ene)-lower alkyl(ene / alkyne), aryl, aryl-lower alkyl, acyl, thioacyl, lower alkylsulfonyl, trifluoromethylsulfonyl, arylsulfonyl, R 1 is the group R 9 VCO-, where V is O or S, and R 9 is a lower alkyl, cycloalkyl, cycloalkyl-lower alkyl or aryl, or R 1 is the group R 10 R 11 NCO- or R 10 R 11 NCS-, where R 10 and R 11 are independently hydrogen, lower alkyl, cycloalkyl, cycloalkyl-lower alkyl or aryl, or R 10 and R 11 together with the N atom to which they are attached form a pyrrolidinyl, piperidinyl or perhydroazepinyl group; R 2 is hydrogen, a lower alkyl, a cycloalkyl or a cycloalkyl-lower alkyl; or R 1 and R 2 together with the N atom to which they are attached form a group wherein Q is C═O, C═S or CH2; T is NH, S, O or CH2; and m is from 1 to 4, inclusive; R 3 -R 5 independently is hydrogen, halogen, lower alkyl, lower alkylcarbonyl, phenylcarbonyl, halogen-substituted phenylcarbonyl, lower alkoxy, lower alkylthio, hydroxy, lower alkylsulfonyl, cyano, trifluoromethyl, cycloalkyl, cycloalkyl-lower alkyl or nitro; R 6 and R 7 each is hydrogen or a lower alkyl group, or they are joined together to form a 3- to 7-membered carbocyclic ring; R 8 is hydrogen or a lower alkyl group; Any alkyl, cycloalkyl or cycloalkylalkyl group present is optionally substituted with one or two hydroxy groups, said hydroxy groups being again optionally esterified with an aliphatic or aromatic carboxylic acid; and any aryl substituent present is optionally substituted with a halogen, a lower alkyl, a lower alkoxy, a lower alkylthio, a hydroxy, a lower alkylsulfonyl, a cyano, a trifluoromethyl, a trifluoromethylsulfonyloxy, a cycloalkyl, a cycloalkyl-lower alkyl or a nitro group; and its pharmaceutically acceptable acid addition salts.

10. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 9 2A inhibitor, or the use of any one of claims 2 or 4 to 9, or the method of any one of claims 3 or 4 to 9, wherein the 5-HT 2A inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), 2,3-dihydroxybutanedioic acid; 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (ketanserin tartrate), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (floridserin) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

11. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 10 2A inhibitor, or the use according to claim 2 or any one of claims 4 to 10, or the method according to claim 3 or any one of claims 4 to 10, wherein the proliferative disease is a tumor and / or cancer.

12. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 11 2A inhibitor, or the use of any one of claims 2 or 4 to 11, or the method of any one of claims 3 or 4 to 11, wherein the proliferative disease is liver cancer.

13. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 12 2A inhibitor, or the use of any one of claims 2 or 4 to 12, or the method of any one of claims 3 or 4 to 12, wherein the proliferative disease is hepatocellular carcinoma.

14. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 13 2A inhibitor, or the use according to claim 2 or any one of claims 4 to 13, or the method according to claim 3 or any one of claims 4 to 13, further comprising one or more combination therapies selected from immunotherapy, chemotherapy, ablation therapy, and transplantation.

15. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 14 2A inhibitor, or the use of any one of claims 2 or 4 to 14, or the method of any one of claims 3 or 4 to 14, which further comprises immunotherapy, such as but not limited to immune checkpoint blockade (ICB) therapy including the administration of monoclonal antibodies. Optionally, the immunotherapy may include the use of anti-PD-L1 (e.g., atezolizumab or nivolumab), anti-VEGFA (e.g., bevacizumab), anti-CTLA4 (e.g., ipilimumab), or anti-PD-1 (e.g., nivolumab) plus anti-CTLA4 (e.g., ipilimumab), and / or one or more of their combinations.

16. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 15 2A inhibitor, or the use of any one of claims 2 or 4 to 15, or the method of any one of claims 3 or 4 to 15, wherein the combination therapy is administered to the subject simultaneously, sequentially, or separately with the serotonin receptor 5-HT 2A inhibitor.

17. The serotonin receptor 5-HT used in claim 1 or any one of claims 4 to 16 2A inhibitor, or the use according to claim 2 or any one of claims 4 to 16, or the method according to claim 3 or any one of claims 4 to 16, wherein the inhibitor will be administered intravenously, subcutaneously, orally, sublingually or intraperitoneally.

18. A pharmaceutical composition comprising a serotonin receptor 5-HT 2A inhibitor and an immune checkpoint blockade / ICB therapy.

19. The pharmaceutical composition according to claim 17, wherein the serotonin receptor inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), 2,3-dihydroxybutanedioic acid; 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (tartrate ketanserin), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (floridanserin) or its pharmaceutically acceptable salt or solvate, or a pharmaceutically functional derivative of these compounds.

20. A combination therapy comprising a serotonin receptor 5-HT 2A inhibitor and an immune checkpoint blockade / ICB therapy, wherein the serotonin receptor inhibitor is [3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione] (ketanserin), 2,3-dihydroxybutanedioic acid; 3-[2-[4-(4-fluorobenzoyl)piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione (ketanserin tartrate), 1-[(4-fluorophenyl)methyl]-1-(1-methylpiperidin-4-yl)-3-[[4-(2-methylpropoxy)phenyl]methyl]urea (pimavanserin), (R)-(2,3-dimethoxyphenyl)-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (floridserin) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically functional derivative of these compounds.

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