New retinoic acid compound, pharmaceutical composition, and its application

Abstract

FIELD: pharmaceutical composition.SUBSTANCE: compound or pharmaceutically acceptable salt thereof and pharmaceutical composition thereof, wherein the compound is isotretinoin conjugated to a carbohydrate via a -C3H6NH- linker, and wherein the carbohydrate comprises glucose, fructose, galactose, mannose or any combination thereof.EFFECT: use of this compound or a pharmaceutically acceptable salt thereof or this pharmaceutical composition in the manufacture of a medicament for inhibiting infection or replication of a virus, or for the treatment of cancer.13 cl, 12 dwg, 7 tbl, 1 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to a novel retinoic acid compound, a pharmaceutical composition comprising the same, and its use in the manufacture of a medicament for inhibiting viral infection or replication or for treating cancer.BACKGROUND OF THE INVENTION

[0002] Coronaviruses are a group of positive-sense, single-stranded RNA viruses belonging to the Coronaviridae family, which includes seven species / strains that infect humans, namely: severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), and human coronavirus HKU (HCoV-HKU1). Notably, SARS-CoV-2 is identified as the viral strain causing the coronavirus disease 2019 (COVID-19) pandemic.

[0003] The SARS-CoV-2 genome shares high sequence identity with the SARS-CoV genome. Both SARS-CoV-2 and SARS-CoV critically depend on the activity of two viral proteases, namely, 3C-like protease (3CLpro, also known as major protease (Mpro) or nonstructural protein 5 (nsp5)) and papain-like protease (PLpro, which is the protease domain of nonstructural protein 3 (nsp3)), to achieve the viral proliferation cycle and viral spread. It has been reported that all-trans retinoic acid (ATRA, also known as vitamin A acid or tretinoin) can be considered as a potential therapeutic agent against SARS-CoV-2 due to its ability to inhibit 3CLpro activity.

[0004] In addition to 3CLpro, PLpro is also a potential target, as this enzyme plays a key role in the cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. Although the primary function of Mpro and PLpro is the coordinated processing of the viral polyprotein, PLpro has the additional function of removing ubiquitin and IFN-stimulated gene 15 (ISG15) from host cell proteins, allowing coronaviruses to evade host innate immune responses. Thus, PLpro is not only associated with viral replication but also with dysregulation of signaling cascades in infected cells, leading to the death of surrounding uninfected cells. Therefore, drugs designed to inhibit PLpro function also have potential for combating SARS-CoV-2.Recently, in addition to ATRK, some researchers have reported that its derivative, 13-cis-retinoic acid (also called isotretinoin, which is a potential PLpro inhibitor), is a potential PLpro inhibitor and can be used to treat COVID-19 caused by SARS-CoV-2.

[0005] Furthermore, retinoic acids, including ATRA and 13-cis-retinoic acid, are promising compounds for the treatment of various types of cancer due to their specific effects on cell proliferation, differentiation, and apoptosis, as well as their low toxicity. Retinoic acid receptors in the nuclei of human cells were discovered by biochemists and were not found to be mutated in cancer cells, suggesting that retinoic acids have the potential to exert anticancer effects in many malignancies. For example, in children with high-risk neuroblastoma, treatment with 13-cis-retinoic acid was found to reduce the risk of cancer recurrence after high-dose chemotherapy and stem cell transplantation. ATRA has been studied in combination with other drugs for various types of cancer and precancerous lesions. A number of clinical trials using ATRA as part of combination therapy are currently underway.For example, ATRK with various interferons (IFNs) has been shown to enhance the effects of both drugs and lead to growth inhibition and cell death in tumor cell lines. However, to unlock the therapeutic potential of retinoic acids, many studies highlight the need for a better understanding of the mechanisms that block retinoic acid-mediated signaling and retinoic acid-regulated gene expression in cancers such as acute myeloid leukemia (AML). Clearly, combinatorial therapies targeting multiple gene silencing mechanisms may be the most effective strategy for reactivating ATRK-sensitive gene expression and AML cell differentiation, as well as for mediating the overall anticancer activity of ATRK.Currently, the identification of protein classes that control gene expression through histone and DNA modifications is driving the development of new therapeutic agents, so-called epigenetic drugs, that alter chromatin structure. However, these epigenetic-modifying drugs have been shown to be only partially effective against various types of cancer when used individually. SUMMARY OF THE INVENTION

[0006] Based on the above reasons, the present invention relates to a novel retinoic acid compound and a pharmaceutical composition including the same, which are capable of effectively inhibiting viral infection or replication or treating cancer.

[0007] In one aspect, the present invention relates to a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises retinoic acid conjugated to a carbohydrate.

[0008] Preferably, the compound is represented by formula (I): where R 1 represents a substituted or unsubstituted functional group of a carbohydrate.

[0009] Preferably, the carbohydrate is selected from the group consisting of a monosaccharide, a disaccharide, an oligosaccharide, and a polysaccharide.

[0010] Preferably, the oligosaccharide is a monooligosaccharide or a heterooligosaccharide; and the polysaccharide is a monopolysaccharide or a heteropolysaccharide.

[0011] Preferably, the carbohydrates include glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3 / 1,6-glucan oligosaccharides, raffinose, stachyose, verbascose, fructooligosaccharides, starch, glycogen, cellulose, or any combination thereof.

[0012] It is preferable that the compound or a pharmaceutically acceptable salt thereof is encapsulated in a liposome.

[0013] In another aspect, the present invention relates to a pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0014] Preferably, the pharmaceutically acceptable carrier includes a liposome, and the compound or a pharmaceutically acceptable salt thereof is encapsulated in the liposome.

[0015] Preferably, the pharmaceutical composition further includes a metal ion.

[0016] Preferably, the metal ion includes a monovalent ion, a divalent ion, or a combination of both. More preferably, the monovalent ion includes K + , Na + or a combination of these; and the divalent ion includes Zn 2+ , Mg 2+ , Cu 2+ , Mn 2+ , Ca 2+ , Fe 2+ or any combination of them.

[0017] Preferably, the pharmaceutically acceptable carrier includes a liposome, and the metal ion is encapsulated in the liposome.

[0018] It is preferable that the compound or its pharmaceutically acceptable salt and the metal ion are individually or simultaneously encapsulated in a liposome.

[0019] In another aspect, the present invention relates to the use of said compound or a pharmaceutically acceptable salt thereof or said pharmaceutical composition in the manufacture of a medicament for inhibiting infection or replication of a virus.

[0020] In another aspect, the present invention relates to the use of said compound or a pharmaceutically acceptable salt thereof or said pharmaceutical composition in the manufacture of a medicament for the treatment of cancer.

[0021] Therefore, the present invention provides at least the following advantages: 1. The compound of the present invention, which is a carbohydrate-conjugated retinoic acid, and the pharmaceutical composition thereof can enhance the inhibitory ability against viral infection and / or replication, compared with retinoic acid alone. 2. The compound of the present invention and the pharmaceutical composition thereof can serve as a potential drug against coronavirus, particularly SARS-CoV-2, which causes COVID-19. 3. The compound of the present invention and the pharmaceutical composition thereof can also have an enhanced ability to inhibit cancer cells and thus effectively treat various cancers such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, and liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Fig. 1 illustrates the result of liquid chromatography-mass spectrometry (LC-MS) analysis of compound (Ia) according to one embodiment of the present invention.

[0023] Fig. 2 illustrates an absorbance-time curve in the PLPro SARS-CoV-2 inhibition assay with a novel retinoic acid compound according to one embodiment of the present invention.

[0024] Fig. 3 shows the TCID diagram 50 , demonstrating the antiviral effect of a novel retinoic acid compound in an animal model of SARS-CoV-2 in accordance with one embodiment of the present invention.

[0025] Fig. 4 is a cell viability curve showing the cytotoxic effect of a novel retinoic acid compound on an AsPC-1 cancer cell line according to one embodiment of the present invention.

[0026] Fig. 5 is a cell viability curve showing the cytotoxic effect of a novel retinoic acid compound on an MDA-MB-231 cancer cell line according to one embodiment of the present invention.

[0027] Fig. 6 is a cell viability curve showing the cytotoxic effect of a novel retinoic acid compound on an HCT-116 cancer cell line according to one embodiment of the present invention.

[0028] Fig. 7 is a cell viability curve showing the cytotoxic effect of a novel retinoic acid compound on a Huh-7 cancer cell line according to one embodiment of the present invention.

[0029] Fig. 8 is a cell viability curve showing the cytotoxic effect of a novel retinoic acid compound on a SKOV-3 cancer cell line according to one embodiment of the present invention.

[0030] Fig. 9 is a cell viability curve showing the cytotoxic effect of a novel retinoic acid compound on an A549 cancer cell line according to one embodiment of the present invention.

[0031] Fig. 10 is a cell viability curve showing the cytotoxic effect of a novel retinoic acid compound on an H460 cancer cell line according to one embodiment of the present invention.

[0032] Fig. 11 illustrates an absorbance-time curve in the PLPro SARS-CoV-2 inhibition assay of the second pharmaceutical compositions according to one embodiment of the present invention.

[0033] Fig. 12 shows the TCID diagram 50 , demonstrating the antiviral effect of the second pharmaceutical compositions in an animal model of SARS-CoV-2 in accordance with one embodiment of the present invention.DETAILED DESCRIPTION

[0034] Although the present invention admits of various modifications and alternative forms, specific embodiments are shown by way of example in the drawings and described in detail. However, it should be understood that this description is not intended to limit the present invention to specific embodiments, but, on the contrary, the present invention should include all modifications, equivalents, and alternatives that fall within the spirit and scope of the present invention.

[0035] Definitions

[0036] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. It will further be understood that terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the related field and will not be interpreted in an idealized or overly formal sense unless expressly defined in this description.

[0037] The terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to limit the embodiments of the present invention. The singular forms used herein are intended to include plural forms as well, unless the context clearly dictates otherwise. It will further be understood that the terms "comprises," "comprising," "includes," and / or "including" as used herein mean the presence of the stated features, integers, steps, operations, elements, parts, and / or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts, and / or combinations thereof.

[0038] As used herein, the term "subject" refers to a mammal for whom a diagnosis, prognosis, or therapy is desired. Typically, the mammal is a human. In some embodiments, the mammal may be a non-human mammal used, for example, for screening, characterizing, and evaluating drugs and therapies, such as non-human primates, cows, horses, goats, sheep, dogs, cats, rabbits, pigs, mice, or rats.

[0039] As used herein, the term "administering" or "administered" refers to the introduction, provision, or delivery of a predetermined active ingredient to a subject by any suitable route to perform its intended function.

[0040] As used herein, the term "cancer" refers to leukemia, lymphoma, carcinoma, sarcoma, and other malignant tumors of potentially unlimited growth that can spread locally by invasion and systemically by metastasis. Examples of cancer include, but are not limited to, cancers of the ovary, adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid gland, skin, stomach, and thyroid gland. Some other examples of cancer include cholangiocarcinoma, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, epidermoid carcinomas, giant cell tumor, glioblastoma multiforme, hairy cell tumor, enteric ganglioneuroma, hyperplastic tumor of the corneal nerve,Islet cell carcinoma, Kaposi's sarcoma, leiomyoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, tumor of marfanoid appearance, medullary carcinoma, metastatic cutaneous carcinoma, mucosal neurinoma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, pheochromocytoma, polycythermia vera, primary brain tumor, small cell lung tumor, squamous cell carcinoma of both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, local skin lesion, veticulosarcoma cell sarcoma, and Wilms' tumor.

[0041] The term "oligosaccharide" as used herein refers to a carbohydrate composed of a small number of monosaccharides, typically approximately three to ten monosaccharide units. An oligosaccharide with one type of monosaccharide subunit is called a mono-oligosaccharide; an oligosaccharide with more than one type of monosaccharide subunit is called a hetero-oligosaccharide.

[0042] The term "polysaccharide" used here refers to a carbohydrate composed of a large number of monosaccharide units. A polysaccharide with one type of monosaccharide subunit is called a monopolysaccharide; a polysaccharide with more than one type of monosaccharide subunit is called a heteropolysaccharide.

[0043] The term "liposome" as used herein refers to a particle characterized by the presence of an aqueous interior isolated from the external environment by a membrane of one or more bilayers forming a vesicle. The main types of liposomes are multilamellar vesicles (MLVs, with multiple lamellar phase lipid bilayers), small unilamellar liposome vesicles (SUVs, with a single lipid bilayer), and large unilamellar vesicles (LUVs, with a single lipid bilayer). The bilayer membranes of unilamellar or multilamellar vesicles are usually formed by lipids, i.e., amphiphilic molecules of synthetic or natural origin that have spatially separated hydrophobic and hydrophilic domains.

[0044] Descriptive embodiments of the invention

[0045] Next, embodiments of the present invention, given as examples, will be described in more detail with reference to the accompanying drawings.

[0046] In one embodiment, the present invention relates to a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound (hereinafter referred to as the "first" pharmaceutical composition), wherein the compound comprises retinoic acid conjugated with a carbohydrate.

[0047] In an exemplary embodiment, the compound is represented by formula (I): ,where R 1 represents a substituted or unsubstituted functional group of a carbohydrate.

[0048] In some embodiments, the concentration of the compound comprising retinoic acid conjugated to a carbohydrate may be, but is not limited to, from 0.1 μM to 10 mM, from 0.1 μM to 1 mM, from 0.1 μM to 500 μM, from 0.1 μM to 250 μM, from 0.1 μM to 100 μM, from 0.1 μM to 50 μM, from 1 μM to 10 mM, from 1 μM to 1 mM, from 1 μM to 500 μM, from 1 μM to 250 μM, from 1 μM to 100 μM, from 1 µM to 50 µM, 10 µM to 10 mM, 10 µM to 1 mM, 10 µM to 500 µM, 10 µM to 250 µM, 10 µM to 100 µM, or 10 µM to 50 µM.

[0049] In another embodiment, the present invention relates to a pharmaceutical composition comprising retinoic acid and a carbohydrate (hereinafter referred to as a "second" pharmaceutical composition). In some embodiments, the retinoic acid is 1,3-cis-retinoic acid (also referred to as isotretinoin).

[0050] In some embodiments, the concentration of retinoic acid may be, but is not limited to, from 1 μM to 10 mM, from 1 μM to 1 mM, from 1 μM to 500 μM, from 1 μM to 250 μM, from 1 μM to 100 μM, from 1 μM to 50 μM, from 10 μM to 10 mM, from 10 μM to 1 mM, from 10 μM to 500 μM, from 10 μM to 250 μM, from 10 μM to 100 μM, or from 10 μM to 50 μM.

[0051] In some embodiments, the carbohydrate may be a monosaccharide, disaccharide, oligosaccharide, or polysaccharide. The oligosaccharide may be a mono-oligosaccharide or a hetero-oligosaccharide. The polysaccharide may be a mono-polysaccharide or a hetero-polysaccharide. The monosaccharide may be selected from, but not limited to, glucose, fructose, galactose, and mannose. The disaccharide may be selected from, but not limited to, sucrose, lactose, and maltose. The oligosaccharide may be selected from, but not limited to, β-1,3 / 1,6-glucan oligosaccharides, raffinose, stachyose, verbascose, and fructooligosaccharides. The polysaccharide may be selected from, but not limited to, starch, glycogen, and cellulose.

[0052] In some embodiments, the concentration of the carbohydrate may be, but is not limited to, from 0.1 μM to 200 mM, from 0.1 μM to 150 mM, from 0.1 μM to 100 mM, from 0.1 μM to 10 mM, from 0.1 μM to 1 mM, from 0.1 μM to 500 μM, from 0.1 μM to 250 μM, from 0.1 μM to 100 μM, from 0.1 μM to 50 μM, from 1 μM to 200 mM, from 1 μM to 150 mM, from 1 μM to 100 mM, from 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 200 mM, 10 μM to 150 mM, 10 μM to 100 mM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM or 10 μM to 50 μM.

[0053] In some embodiments, the molar ratio of retinoic acid to carbohydrate in the second pharmaceutical composition may be about 1:10 -3 -100, 1:10 -3 -20, 1:1-20 or 1:1-10.

[0054] In some embodiments, the first and second pharmaceutical compositions each optionally further comprise a metal ion. Preferably, the metal ion comprises a monovalent ion, a divalent ion, or a combination thereof. In some embodiments, the monovalent ion comprises K + , Na + or a combination of these; and the divalent ion includes Zn 2+ , Mg 2+ , Cu 2+ , Mn 2+ , Ca 2+ , Fe 2+ or any combination of them.

[0055] In some embodiments, the metal ion concentration may be from 1 μM to 300 mM, from 1 μM to 250 mM, from 1 μM to 200 mM, from 1 μM to 150 mM, from 1 μM to 100 mM, from 1 μM to 10 mM, from 1 μM to 1 mM, from 1 μM to 500 μM, from 1 μM to 250 μM, from 1 μM to 100 μM, from 1 μM to 50 μM, from 10 μM to 300 mM, from 10 μM to 250 mM, from 10 μM to 200 mM, 10 μM to 150 mM, 10 μM to 100 mM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0056] In some embodiments, the molar ratio of the compound to the metal ion in the first pharmaceutical composition may be about 1:10 -3 -10 3 , 1:0.1-20, 1:0.1-10 or 1:1-10.

[0057] In some embodiments, the molar ratio of retinoic acid, carbohydrate, and metal ion in the second pharmaceutical composition may be about 1:10-4 -20:10 -4 -10 3 , 1:10 -4 -20:10 -3 -10 3 , 1:10 -4 -20:10 -3 -20, 1:10 -4 -20:0,1-20, 1:10 -4 -20:1-10, 1:0,1-20:10 -4 -10 3 , 1:0.1-20:10 -3 -10 3 , 1:0.1-20:10 -3 -20, 1:0.1-20:0.1-20, 1:0.1-20:1-20 or 1:0.1-20:1-10, 1:0.1-1:10 -4 -10 3 , 1:0.1-1:10 -3 -10 3 , 1:0.1-1:10 -3 -20, 1:0.1-1:0.1-20, 1:0.1-1:1-20 or 1:0.1-1:1-10.

[0058] In some embodiments of the invention, the first and second pharmaceutical compositions each optionally further comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is one that is widely used in the field of drug production. Examples of a pharmaceutically acceptable carrier may include, but are not limited to, liposomes, excipients, adjuvants, solvents, buffers, emulsifiers, suspending agents, disintegrating agents, disintegrating agents, binders, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricating agents, absorption delaying agents, and the like. The selection and amount of a pharmaceutically acceptable carrier are within the competence of those skilled in the art.

[0059] In some embodiments, the pharmaceutically acceptable carrier is a liposome. The compound or a pharmaceutically acceptable salt thereof is encapsulated in the liposome. The compound or a pharmaceutically acceptable salt thereof and the metal ion in the first pharmaceutical composition can be individually or simultaneously encapsulated in the liposome. The retinoic acid and carbohydrate in the second pharmaceutical composition can be individually or simultaneously encapsulated in the liposome. All or at least two of the following components: retinoic acid, carbohydrate, and metal ion in the second pharmaceutical composition can be individually or simultaneously encapsulated in the liposome.

[0060] The liposomes cited as examples can be neutral, positively charged, or negatively charged. In general, lipids commonly used in liposomes typically include dialiphatic chain lipids such as phospholipids, diglycerides, and dialiphatic glycolipids; individual lipids such as sphingomyelin and glycosphingolipids; steroids such as cholesterol and its derivatives, and combinations thereof. Examples of phospholipids include, but are not limited to, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DM PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (DPPG), 1-palmitoyl-2-stearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (PSPG), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-seine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), 1,2-dipalmitoyl-9n-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (DSPI), 1,2-dioleoyl-sn-glycero-3-phospho-(1-myo-inositol) (ammonium salt) (DOPI), cardiolipin, L-alpha-phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), L-alpha-phosphatidylethanolamine (EPE), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and 3beta-[N'-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.,

[0061] A suitable lipid may be a lipid mixture of one or more of the above lipids or a mixture of one or more of the above lipids with one or more other lipids not listed above, membrane stabilizers or antioxidants.

[0062] The molar concentration of the lipid in the bilayer membrane may be equal to or less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or any value or range of values ​​therebetween (e.g., about 5-50%, about 5-45%, about 5-40%, about 5-35%, about 5-30%, about 5-25%, about 5-20%, about 5-15% or about 5-10%).

[0063] The lipid of the bilayer membrane may be a mixture of a first phospholipid and a second phospholipid. The first phospholipid may be selected from the group substantially consisting of PC, HSPC, DO PC, POPC, DSPC, DPPC, DMPC, PSPC, and combinations thereof, and the second phospholipid is selected from the group substantially consisting of PE, PG, DOPE, PEG-DSPE, DPPG, DOPG, DOTAP, DOTMA, DDAB, and combinations thereof.In other embodiments, the molar concentration of the first phospholipid in the bilayer membrane is about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or any value or range therebetween (e.g., about 5-50%, about 5-45%, about 5-40%, about 5-35%, about 5-30%, about 5-25%, about 5-20%, about 5-15%, or about 5-10%), and the molar concentration of the second phospholipid in the bilayer membrane is from 0.1% to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, or any value or range therebetween (e.g., about 0.1-15%, about 0.1-10%, about 0.5-15%, about 0.5-10%, or about 0.5-7%). In an exemplary embodiment of the invention, the first phospholipid (DSPC) and the second phospholipid (DOPE, DOPG, or DDAB) can be in a molar ratio of 4:1 to 6:1.

[0064] In an exemplary embodiment of the invention, the liposome bilayer membrane includes less than about 55 molar percent of steroids, preferably cholesterol. The molar concentration of the steroid (such as cholesterol) in the bilayer membrane can be about 15-55%, about 20-55%, about 25-55%, about 15-50%, about 20-50%, about 25-50%, about 15-45%, about 20-45%, about 25-45%, about 15-40%, about 20-40%, or about 25-40%. The molar concentration of phospholipid and cholesterol in the bilayer membrane may be approximately 25-50%:15-55%, 25-50%:20-55%, or 25-50%:15-50%. The phospholipid(s) and cholesterol may be in a molar ratio of 1:1 to 3:1. The molar percentage ratio of the first phospholipid, the second phospholipid, and cholesterol in the bilayer membrane may be approximately 25-50%:0.1-15%:15-55%, 5-50%:0.1%-15%:10-40%, or 25-50%:0.5-10%:5-20%.

[0065] The liposome encapsulating the entrapping agent can be produced by any method currently known or subsequently developed. For example, MLV liposomes can be directly formed from a hydrated lipid film, spray-dried powder, or lyophilized pellet of selected lipid compositions with the entrapping agent; SUV liposomes and LUV liposomes can be produced from MLV liposomes by sonication, homogenization, microfluidization, or extrusion.

[0066] In another embodiment of the invention, a compound comprising retinoic acid conjugated to a carbohydrate or a pharmaceutically acceptable salt thereof and the first and second pharmaceutical compositions can be used to inhibit infection or replication of a virus. The virus can be an RNA virus. The RNA virus can include a coronavirus, a human immunodeficiency virus (HIV), a hepatitis C virus (HCV), an influenza virus, or any combination thereof. The coronavirus can include severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HcoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU (HCoV-HKU1), or any combination thereof.In an exemplary embodiment of the invention, the coronavirus is SARS-CoV-2, which causes COVID-19.

[0067] In another embodiment of the invention, a compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and first and second pharmaceutical compositions can be used to treat cancer. Cancer can include leukemia, lymphoma, carcinoma, or sarcoma. In exemplary embodiments of the invention, the cancer can be lung cancer, ovarian cancer, breast cancer, liver cancer, pancreatic cancer, or cholangiocarcinoma.

[0068] In some embodiments, the compound and the metal ion in the first pharmaceutical composition may be administered separately, simultaneously, or sequentially. The retinoic acid and carbohydrate in the second pharmaceutical composition may be administered separately, simultaneously, or sequentially. All or at least two of the following components: retinoic acid, carbohydrate, and metal ion in the second pharmaceutical composition may be administered separately, simultaneously, or sequentially. In an exemplary embodiment, the metal ion and carbohydrate are administered simultaneously, followed by the retinoic acid.

[0069] In exemplary embodiments of the invention, the time intervals between successive administrations may be from 1 to 30 minutes, from 30 to 60 minutes, from 60 to 90 minutes, from 90 to 120 minutes, from 2 to 3 hours, from 3 to 12 hours, or from 12 to 24 hours.

[0070] In some embodiments of the invention, a compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and the first and second pharmaceutical compositions can each be prepared in dimethyl sulfoxide (DMSO), ethanol, a buffer, or water for administration. The dosage and frequency of administration can vary depending on the following factors: the severity of the viral infection (e.g., coronavirus infection) or the disease being treated (e.g., cancer) and the weight, age, physical condition, and response of the subject being treated. The daily dose of the aforementioned therapeutic agents can be administered as a single dose or in multiple doses.

[0071] In some embodiments of the invention, a compound comprising retinoic acid conjugated with a carbohydrate, a pharmaceutically acceptable salt thereof, and the first and second pharmaceutical compositions are administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, or topically.

[0072] Examples

[0073] The present invention will be further described by means of the following examples. However, it should be understood that the following examples are intended for illustrative purposes only and should not be construed as limiting the present invention in practice.

[0074] Materials and methods

[0075] A. Preparation of a new retinoic acid compound, galactose-modified isotretinoin, represented by the formula (Ia), based on the synthesis scheme below:

[0076] I. General Experimental Methods All reagents were commercially available and used without further purification. The yields refer to purified and spectroscopically pure compounds. Thin-layer chromatography (TLC) was performed using Merck TLC Aluminum sheets Silica gel 60 F254 plates and visualized by UV fluorescence quenching. Flash chromatography was performed using silica gel (Chrornatorex, MB 70-40 / 75, 40-75 μM) purchased from Fuji Silysia Chemicals. NMR spectra were recorded on a Varian-400MR instrument operating at 400 MHz for 1H. Chemical shifts are reported in ppm with solvent resonance as internal standard. Data are presented as follows: s=singlet, br=broad, d=doublet, t=triplet, q=quartet, m=multippet, dd=doublet of doublets; coupling constants in Hz; integration. Purity was determined on a Waters e2695 Separates Module / 2998 PDA Detector HPLC system (column: XBridge C18, 5 μM, 4.6 mM (ID) × 150 mM (L), eluent: a mixture of mobile phases A and B, mobile phase A: 100% acetonitrile; mobile phase B: pure water containing 0.1% formic acid and 10 mM NH4OAc, flow rate: 0.5 mL / min. Detection: UV, 254 nm).

[0077] II. Synthesis of (2R,3S,4,5R,6R)-2-(acetoxymethyl)-6-(3-bromopropoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2) A solution of galactose pentaacetate (compound (1), 5.0 g, 13 mmol), 3-bromo-1-propanol (1.73 mL, 19.0 mmol), and freshly dried molecular sieve in anhydrous dichloromethane (50 mL) was mixed with boron trifluoride diethyl etherate (8.04 mL, 64.0 mmol) at 0°C. The mixture was stirred at room temperature overnight. The solution was neutralized with triethylamine, the molecular sieve was removed by passing through celite, and the reaction mixture was washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The crude product was purified by silica gel column chromatography (EtOAc:hexane=1:2) to give the desired product (2) (0.625 g, 10%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 5.40 (dd, J=3.4, 1.2 Hz, 1H), 5.22-5.16 (m, 1H), 5.04-5.01 (m, 1H), 4.48 (d, J=8 Hz, 1H). 4.21-4.11 (m, 2H), 4.03-3.98 (rn, 1H), 3.92 (td, J=5.8, 0.8 Hz, 1H), 3.72-3.66 (m, 1H), 3.50-3.47 (m, 2H), 2.25-1.99 (m, 14H); LCMS (ESI) m / z calculated for C17 H 25 BrO 10 469.28; found, 491.04 [M+Na] + .

[0078] III. Synthesis of (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(3-azidopropoxy)tetrahydro-2H-pyran 3,4,5-triyl triacetate (3) A solution of compound (2) (0.62 g, 1.3 mmol) in DMF (4 mL) was added to sodium azide (0.43 g, 6.6 mmol). The mixture was stirred at 100°C for 12 h. The solution was concentrated to dryness. The crude product was purified by silica gel column chromatography (EtOAc:hexane=1:2) to afford the desired product (3) (458 mg, 80%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 5.39 (dd, J=3.4, 0.8 Hz, 1H), 5.22-5.18 (m, 1H), 5.03-5.00 (m, 1H), 4.48 (d, J=8 Hz, 1H), 4.21-4.10 (m, 2H), 3.99-3.89 (m, 1H), 3.63-3.58 (m, 1H), 3.39-3.35 (m, 2H), 2.15 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H), 1.92-1.77 (m, 2H); LCMS (ESI) m / z calculated for C l7 H 25 N3O 10431.40; 454.2 [M+Na] found + .

[0079] IV. Synthesis of the compound (2R,3R,4S,5R,6R)-2-(3-azidopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (4) A solution of compound (3) (0.45 g, 1 mmol) in dichloromethane (1.0 mL) and methanol (4.0 mL) was mixed with sodium methoxide (28 mg, 0.5 mmol). The mixture was stirred at room temperature for 10 h. The solution was neutralized with Dowex 50WX8 and evaporated to dryness to give the desired product (4), which was used in the next step without further purification. LCMS (ESI) m / z calculated for C9H 17 N3O6263.25; found, 286.1 [M+Na] + .

[0080] V. Synthesis of the compound (2R,3R,4S,5R,6R)-2-(3-aminopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (5) Crude compound (4) in methanol (10.5 mL) was treated with Pd / C (11 mg) and acetic acid (6.3 mg) under H2. The solution was filtered through a pad of celite, and the filtrate was then concentrated under reduced pressure to give (2R,3R,4S,5R,6R)-2-(3-aminopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (5), which was used in the next step without further purification. LCMS (ESI) m / z calculated for C9H 19 NO6237.25; found, 237.8 [M] + .

[0081] VI. Synthesis of perfluorophenyl (2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenoate (6) A solution of retinoic acid (compound (7), 0.10 g, 0.33 mmol) and triethylamine (0.093 mL, 0.67 mmol) in anhydrous DMF (1.0 mL) was mixed with pentafluorophenyl trifluoroacetate (compound (8), 0.081 mL, 0.47 mmol) at 0°C. The solution was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc and washed with 0.1 N aqueous HCl, then with aqueous NaHCO and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated to dryness to give the desired product (6) without further purification.

[0082] VII. Synthesis of the target compound (2Z,4E,6E,8E)-3,7-dimethyl-N-(3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide (Ia) A solution of crude compound (5) (30 mg, 0.13 mmol) and activated ester (6) (150 mg, 0.33 mmol) in DMF (1.0 mL) was added to triethylamine (0.049 mL, 0.33 mmol) at room temperature. The mixture was stirred overnight. The solution was concentrated to dryness. The crude product was purified by column chromatography on silica gel (methanol:dichloromethane = 1:9). LCMS (ESI) m / z calcd for C29H45NO7 519.68; found, 520.5 [M+H] + See Fig. 1.

[0083] B. Obtaining PLPro SARS-CoV-2

[0084] In order to determine whether the compound and pharmaceutical compositions of the present invention can inhibit SARS-CoV-2, their in vitro inhibitory ability against the activity of SARS-CoV-2 papain-like protease (PLpro) was first evaluated.

[0085] The codon-optimized gene sequence encoding the wild-type SARS-CoV-2 PLpro was synthesized by Biotools Co., Ltd. (New Taipei City, Taiwan) and subcloned into the pET-21a vector (Novagen) using Ndel and Xhol restriction sites, while maintaining the His-tag (-LEHHHHHH-) coding region at the C-terminus.

[0086] The vector with the inserted SARS-CoV-2 PLPro gene was transformed into E. coli BL21 (DE3) (Yeastern Biotech Co., Ltd., New Taipei City, Taiwan) to overexpress PLPro. Cultivation was carried out in LB medium (containing 1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with ampicillin (100 μg / mL) as an antibiotic marker. The resulting culture was initially incubated at 37°C with shaking at 200 rpm. At an optical density measured at a wavelength of 600 nm (OD 600), isopropyl β-D-1-thiogalactopyranoside (IPTG) was added between 0.6 and 0.8 to achieve a final concentration of 0.4 mM to induce PLpro expression. Incubation was continued at 18°C ​​and 200 rpm for 20 h. Cells were collected by centrifugation (5,000 × g) and disrupted by sonication in lysis buffer containing 50 mM sodium phosphate (pH 7.4), 1.0 mM DTT, 5% glycerol, and 100 mM NaCl. Cell debris was then removed by centrifugation at 20,000 × g for 50 min. The supernatant was loaded onto a 5 ml His-Trap HP column (GE Healthcare Life Sciences), and the protein was eluted using a gradient of 0-500 mM imidazole in 50 mM sodium phosphate (pH 7.4) and 100 mM NaCl. Fractions containing His-tagged SARS-CoV-2 PLpro were pooled and concentrated using a Centricon membrane (molecular weight cutoff 10 K, GE Healthcare Life Sciences).His-tagged SARS-CoV-2 PLpro was further purified by gel filtration chromatography using a Superdex 75 gel filtration column (GE Healthcare Life Sciences) in 50 mM sodium phosphate buffer (pH 7.4). The concentration of SARS-CoV-2 PLpro was determined by measuring the ultraviolet absorbance at a wavelength of 280 nm using an extinction coefficient (ε280) of 45,270 M. -1 cm -1 .

[0087] C. Analysis of PLPro SARS-CoV-2 activity

[0088] The enzymatic activity of PLpro SARS-CoV-2 obtained above was measured by a colorimetric-based peptide cleavage assay using a 6-mer peptide substrate, FRLKGG-p-nitroanilide (FG6-pNA) (97% purity by HPLC; GL Biochem Ltd., Shanghai, China). In the cleavage assay, the 6-mer peptide substrate was cleaved at the Gly-pNA bond to release free pNA, which changed the color of the solution to yellow. The enzymatic activity was determined by continuously monitoring the absorbance at a wavelength of 405 nm (A 405 ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek) at 30°C.

[0089] Specifically, the cleavage assay was carried out in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4), and FG6-pNA was added to the corresponding well to obtain substrate solutions with different FG6-pNA concentrations (0.1875 mM, 0.375 mM, 0.75 mM, 1.5 mM, 3.0 mM, 6.0 mM). The assay mixture (180 μL in each well) was pre-incubated for 10 minutes to accurately control the temperature, and the reaction was initiated by adding 20 μL of PLpro SARS-CoV-2 solution (1.75 μM) to the assay mixture. The PLpro SARS-CoV-2 solution was prepared by mixing the PLpro SARS-CoV-2 obtained above with 50 mM sodium phosphate buffer (pH 7.4). The concentration of pNA released during proteolysis was calculated by measuring A 405 , using the extinction coefficient (ε405) of 9800 M -1 cm -1 (A 405 =9.8 at 1 mM).

[0090] The steady-state kinetic parameters of the enzyme were obtained by fitting the empirical curve of the initial velocity (V0) data based on the Michaelis-Menten equation using OriginPro 8.0 software (OriginLab Corporation, USA). All measurements were performed in triplicate. The obtained data are expressed as the mean ± standard deviation.

[0091] Results: K values м and k cat were 2.50±0.03 mM and 0.85±0.01 s -1 Thus, it is confirmed that SARS-CoV-2 PLPro with protease activity was successfully obtained according to the above production method and can be used for subsequent analysis of SARS-CoV-2 PLPro inhibition.

[0092] D. Establishment of an animal model of SARS-CoV-2

[0093] Golden Syrian hamsters (5-6 weeks old, average weight approximately 100 g) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The hamsters were maintained in a specialized pathogen-free (SPF) animal facility, as is commonly used in the field. All hamsters had unlimited access to water and food. All experiments involving hamsters were submitted to, reviewed, and approved by the Institutional Animal Care and Use Committee (IACUC) of Academia Sinica, Taiwan.

[0094] Hamsters were infected with SARS-CoV-2 (obtained from P3 Lab, Genomics Research Center, Academia Sinica; wild type WuHan) in phosphate-buffered saline (PBS) by intranasal inoculation at a concentration of 1×10 4 plaque-forming units (PFU) at 12:00 to obtain an animal model of SARS-CoV-2. The production of an animal model of SARS-CoV-2 was confirmed (data not shown).

[0095] Example 1

[0096] PLPro SARS-CoV-2 Inhibition Assay for In vitro Evaluation of the Antiviral Effect of a Novel Retinoic Acid Compound, Galactose-Modified Isotretinoin, Against SARS-CoV-2

[0097] The enzyme inhibition assay was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4). The above-prepared SARS-CoV-2 PLPro (0.9 μM) was added to the corresponding well to form an enzyme solution. The enzyme solutions in the wells were divided into one control group (abbreviated as “Control”), one comparative group (abbreviated as “Comparative 1”), and one experimental group (abbreviated as “Experimental 1”). The corresponding inhibitory agent listed in Table 1 below was added to the enzyme solution of the corresponding group to form a test mixture (total volume of 180 μL). Preincubation was carried out for 30 minutes.

[0099] 20 μL of FG6-pNA (1.2 mM) described above was added to the test mixture of the corresponding group to initiate the enzymatic reaction. The enzymatic reaction was carried out at 30°C for 300 seconds. The enzymatic activity was determined by continuously monitoring the absorbance at a wavelength of 405 nm (A 405 ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). The reaction rate was calculated accordingly, where the reaction rate is the absorbance slope A 405 as a function of time (seconds) for a total reaction time of 300 seconds. The data are presented in Fig. 2.

[0100] The inhibition percentage was calculated using the following equation: A=[1-(B / C)]×100(I)where A=percentage inhibition B=reaction rate of the corresponding group C=reaction rate of the control group The obtained data are expressed as the mean ± standard deviation.

[0101] Results: The inhibition percentages of different treatment groups are shown below in Table 2.

[0103] As shown in Fig. 2 and Table 2, the inhibition rate in Experimental Group 1 was significantly higher than that in Comparative Group 1, indicating that galactose-modified isotretinoin was more effective than isotretinoin alone in inhibiting the enzymatic activity of PLpro. This result suggests that galactose-modified isotretinoin has a better inhibitory effect on papain-like protease activity.

[0104] Furthermore, it provides important insights into the biochemical properties of the corona virus PLpro family and paves the way for promising therapeutic strategies against SARS-CoV-2.

[0105] Example 2

[0106] Evaluation of the in vivo antiviral activity of a novel retinoic acid compound, galactose-modified isotretinoin, against SARS-CoV-2 in an animal model of SARS-CoV-2

[0107] Since the novel retinoic acid compound, galactose-modified isotretinoin, was proven to have an inhibitory effect against SARS-CoV-2 in vitro, an in vivo animal test was further conducted to evaluate the antiviral effect of the novel retinoic acid compound against SARS-CoV-2.

[0108] The infected hamsters obtained as described above were divided into the following four groups (n=5 per group): a control group (abbreviated as “Control”), two comparative groups (abbreviated as “Comparative 1” and “Comparative 2”, respectively), and one experimental group (abbreviated as “Experimental 1”). The therapeutic agents respectively used for these groups are listed below in Table 3.

[0110] Specifically, each hamster in Experimental Group 1 was administered the novel retinoic acid compound intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, Day 1) at a dose of 0.35 mg / kg and at 8:00 PM on the day of infection at a dose of 0.35 mg / kg. On the following two days (Days 2 and 3), the novel retinoic acid compound was administered intranasally twice a day at 8:00 AM and 8:00 PM each time at a dose of 0.35 mg / kg.

[0111] Each hamster in the comparative groups was administered isotretinoin in Comparison Group 1 or liposome-encapsulated isotretinoin in Comparison Group 2 intranasally at 8:00 a.m. on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) at a dose of 0.35 mg / kg and at 8:00 p.m. on the day of infection at a dose of 0.35 mg / kg. Isotretinoin in Comparison Group 1 or liposome-encapsulated isotretinoin in Comparison Group 2 was administered intranasally twice a day at a dose of 0.35 mg / kg at 8:00 a.m. and 8:00 p.m. on the next two days after the day of infection. Liposome-encapsulated isotretinoin in Comparative Group 2 was prepared at Taipei Medical University (Taiwan) using methods commonly used in the art and described in detail above. Because the key technical feature of the present invention is a novel retinoic acid compound, galactose-modified isotretinoin, details of liposome preparation are omitted here for brevity.

[0112] Each hamster in the control group was administered 100 μL of the buffer intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and 100 μL at 8:00 PM on the day of infection. The buffer was administered intranasally twice daily at 100 μL at 8:00 AM and 8:00 PM for two days after the day of infection.

[0113] After 3 days of treatment, hamsters were sacrificed and their lungs were collected to measure live viral load using TCID assay. 50 in Vero E6 cells. The virus titer was defined as the dose infecting 50% of cells in tissue culture (TCID 50 ) using the Reed-Muench method. All experiments with SARS-CoV-2 were conducted in a biosafety level 3 (BSL-3) laboratory and were approved by Academia Sinica (Taipei, Taiwan).

[0114] Experimental data were analyzed using Tukey's test to assess differences between groups. Statistical significance was defined as p<0.05.

[0115] Results: As shown in Fig. 3, TCID 50 in Experimental Group 1 was significantly lower than that in Comparative Group 1 and Comparative Group 2, indicating that the novel retinoic acid compound, galactose-modified isotretinoin, has higher in vivo efficacy against SARS-CoV-2 compared with isotretinoin alone or liposome-encapsulated isotretinoin alone.

[0116] Example 3

[0117] In vitro evaluation of the cytotoxic effect of a novel retinoic acid compound modified with galactose isotret and no and na on various cancer cell lines

[0118] In this example, seven cell lines were used, namely, the AsPC-1, MDA-MB-231, HCT-116, Huh-7, SKOV-3, A549, and H460 cancer cell lines, respectively. Each cell line was cultured in the specified growth medium with 10% FBS and maintained in a humidified incubator at 37°C in an atmosphere containing 5% CO2.

[0119] The day before treatment, cells in the logarithmic growth phase were collected, counted, and seeded in a 96-well plate at a density of 1×10 4 cells / 100 µl / well. After overnight cultivation, the culture medium from each well was carefully removed, and then fresh medium (200 µl / well) was added.

[0120] The test substance (TA) was a novel retinoic acid compound (abbreviated as “New Compound” in this example) freshly prepared on the day of treatment in 100% DMSO as a stock solution with a concentration of 96 mM. Isotretinoin was serially diluted 2-fold using 100% DMSO to obtain different isotretinoin concentrations from 7.5 mM to 96 mM. Cells were treated by adding 2.02 μL of the indicated isotretinoin concentrations to each well to obtain a final isotretinoin concentration range from 7.5 μM to 960 μM, with all wells having a DMSO concentration of 1%, including the DMSO control. All components were gently mixed and incubated for 24 hours.

[0121] Cells were treated by adding 2.02 μL of the indicated concentrations of Novel Compound A or DMSO to each well, then gently mixed and incubated for 24 hours.

[0122] On the day of cell viability determination, the culture medium of each well was replaced with freshly prepared Alamar blue dye (10% v / v) and incubated at 37°C for 2-3 hours. Spectrophotometric absorbance was recorded at wavelengths of 570 nm and 600 nm.

[0123] The percentage of cell viability was calculated by the following formula:% viability = ([ratio A 570 / A 600 ] TA / [ratio A 570 / A 600 ]DMSO)*100%where TA = test substance A 570 = absorbance at 570 nA 600 = absorbance at 600 nm

[0124] Results: Figures 4-10 show that the new compound can significantly reduce the cell viability of all seven cancer cell lines, indicating that the new retinoic acid compound, galactose-modified isotretinoin, may be a potential anticancer drug for the effective treatment of various cancers such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, and liver cancer. Table 4 shows the IC 50 .

[0126] Example 4

[0127] PLPro SARS-CoV-2 Inhibition Assay for In vitro Evaluation of Antiviral Effect of Second Pharmaceutical Compositions against SARS-CoV-2

[0128] The enzyme inhibition assay was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4). PLPro SARS-CoV-2 prepared as described above was added to the corresponding well to form a final concentration of 0.9 μM enzyme solution. The enzyme solutions in the wells were divided into one control group (abbreviated as “Control”), four comparative groups (abbreviated as “Comparative 1”, “Comparative 2”, “Comparative 3”, and “Comparative 4”, respectively), and two experimental groups (abbreviated as “Experimental 1” and “Experimental 2”, respectively). The corresponding inhibitory agent shown below in Table 5 was added to the enzyme solution of the corresponding group to form a test mixture (total volume 180 µl). Preincubation was carried out for 30 minutes.

[0130] 20 μL of FG6-pNA (1.2 mM) described above was added to the test mixture of the corresponding group to initiate the enzymatic reaction. The enzymatic reaction was carried out at 30°C for 300 seconds. The enzymatic activity was determined by continuously monitoring the absorbance at a wavelength of 405 nm (A 405 ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). The reaction rate was calculated accordingly, where the reaction rate is the absorbance slope A 405 as a function of time (seconds) for a total reaction time of 300 seconds. The data are presented in Fig. 11. The percentage inhibition was calculated as described in Example 3.

[0131] Results: The inhibition percentages of different treatment groups are shown below in Table 6.

[0133] As shown in Fig. 11 and Table 6, the inhibitory effect of each group in the experimental groups was significantly higher than that of each group in the comparative groups, indicating that in the two experimental groups: (1) isotretinoin and oligosaccharides and (2) isotretinoin, oligosaccharides, Zn2+ and Mg2+, were more effective than the single component isotretinoin, oligosaccharides only, Zn 2+ or Mg2 + These results suggest that isotretinoin, oligosaccharides, and divalent metal ions exert a synergistic inhibitory effect on papain-like protease activity. Furthermore, they provide important insights into the biochemical properties of the coronavirus PLpro family and pave the way for promising therapeutic strategies against SARS-CoV-2.

[0134] Example 5

[0135] In vivo evaluation of the treatment effect of a combination of isotretinoin, oligosaccharides, or divalent metal ions against SARS-CoV-2 in an animal model of SARS-CoV-2

[0136] The infected hamsters obtained in Part A of this case were divided into the following six groups (n=5 per group): a control group, three experimental groups (i.e., Experimental Groups 1, 2, and 3), and four comparative groups (i.e., Comparative Groups 1, 2, 3, and 4). The therapeutic agents respectively used for these groups are listed below in Table 7.

[0138] Therapeutic agents encapsulated in liposomes were prepared at Taipei Medical University using liposomes using methods commonly used in the art and described in detail above. Since the key technical feature of the present invention is the combination of retinoic acid and oligosaccharides with or without metal ions, the details of liposome preparation are omitted here for brevity.

[0139] Specifically, each hamster in the experimental groups was administered liposome-encapsulated isotretinoin intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, day 1) at a dose of 0.35 mg / kg and at 8:00 PM on the day of infection at a dose of 0.35 mg / kg. On the following two days (days 2 and 3), liposome-encapsulated isotretinoin was administered intranasally twice daily at 8:00 AM and 8:00 PM at a dose of 0.35 mg / kg each time.

[0140] In addition to liposome-encapsulated isotretinoin, 0.35 mg / kg was administered intranasally twice daily at 8:00 a.m. and 8:00 p.m., a liposome-encapsulated oligosaccharide mixture in a volume of 15 μl (i.e., in Experimental Group 1), or an oligosaccharide mixture (15 μl) and a combination of Zn 2+ (100 μM), Mg2 + (200 μM) and K +(200 μM) (15 μL), both encapsulated in liposome, in a total volume of 30 μL (i.e., in Experimental Group 2), were intranasally administered once a day from 7:30 PM to 8:00 PM (i.e., 0.5 hour to 1 hour before the administration of liposome-encapsulated isotretinoin at night) on the day of infection and the two following days in a volume of 15 μL in Experimental Group 1 or 30 μL in Experimental Group 2.

[0141] Each hamster in the comparative groups was administered isotretinoin in Comparative Group 1 or liposome-encapsulated isotretinoin in Comparative Group 2 intranasally at 8:00 a.m. on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) at a dose of 0.35 mg / kg and at 8:00 p.m. on the day of infection at a dose of 0.35 mg / kg; isotretinoin in Comparative Group 1 or liposome-encapsulated isotretinoin in Comparative Group 2 was administered intranasally twice a day at a dose of 0.35 mg / kg at 8:00 a.m. and 8:00 p.m. on the next two days after the day of infection.

[0142] Each hamster gets a combination of Zn 2+ (100 μM), Mg2 + (200 μM) and K + (200 μM) encapsulated in liposomes in Comparative Group 3 and the oligosaccharide mixture encapsulated in liposomes in Comparative Group 4 were administered intranasally once a day from 19:30 to 20:00 on the day of infection and two days thereafter in a volume of 15 μl each time.

[0143] Each hamster in the control group was administered 100 μL of buffer intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and 100 μL of buffer at 8:00 PM on the day of infection. The buffer was administered intranasally twice daily at 8:00 AM and 8:00 PM for two days after infection.

[0144] After 3 days of treatment, hamsters were sacrificed and their lungs were collected to measure live viral load using TCID assay. 50 in Vero E6 cells. The virus titer was defined as the dose infecting 50% of cells in tissue culture (TCID 50) using the Reed-Muench method. All experiments with SARS-CoV-2 were conducted in a biosafety level 3 (BSL-3) laboratory and were approved by Academia Sinica (Taipei, Taiwan).

[0145] Experimental data were analyzed using Tukey's test to assess differences between groups. Statistical significance was defined as p<0.05.

[0146] Results: As shown in Fig. 12, TCID 50 in each of Experimental Groups 1, 2 and 3 was significantly lower than in Comparative Groups 1, 2, 3 and 4, indicating that the combination of isotretinoin and oligosaccharide with / without metal ions has higher in vivo efficacy against SARS-CoV-2 compared with isotretinoin alone, metal ions alone or oligosaccharides alone.

[0147] Conclusion

[0148] As can be seen from the results of Examples 1 to 5, it is confirmed that the claimed new retinoic acid compound or its pharmaceutically acceptable salt, or the claimed pharmaceutical composition comprising this compound (i.e., the first pharmaceutical composition), or the claimed pharmaceutical composition comprising retinoic acid and a carbohydrate with or without metal ions (i.e., the second pharmaceutical composition), can provide an improved and / or synergistic effect in inhibiting the infection and replication of SARS-CoV-2 and treating a disease associated with SARS-CoV-2 infection or cancer. Therefore, a compound comprising retinoic acid conjugated to a carbohydrate, its pharmaceutically acceptable salt, as well as the first and second pharmaceutical compositions of the present invention can indeed serve as drug repurposing agents.

[0149] The present invention has been described herein broadly and generally. Each of the more specific species and subgeneric groups falling within the general disclosure is also part of the present invention. Furthermore, when features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will understand that the present invention is also thereby described in terms of any individual member or subgroup of members of a Markush group.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is isotretinoin conjugated to a carbohydrate via a -C3H6NH- linker, and wherein the carbohydrate comprises glucose, fructose, galactose, mannose, or any combination thereof.

2. A pharmaceutical composition for inhibiting infection or replication of a virus encoding a papain-like protease, or for treating cancer, comprising a compound or a pharmaceutically acceptable salt thereof according to claim 1 at a concentration of 0.1 μM to 10 mM and a pharmaceutically acceptable carrier thereof, wherein the cancer includes lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer or liver cancer.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable carrier comprises a liposome and the compound or its pharmaceutically acceptable salt is encapsulated in the liposome.

4. The pharmaceutical composition according to claim 2, additionally containing a metal ion.

5. The pharmaceutical composition of claim 4, wherein the metal ion comprises a monovalent ion, a divalent ion, or a combination thereof.

6. The pharmaceutical composition according to claim 5, wherein the monovalent ion comprises K + , Na + or a combination of these; and the divalent ion includes Zn 2+ , Mg 2+ , Cu 2+ , Mn 2+ , Ca 2+ , Fe 2+ or any combination of them.

7. The pharmaceutical composition of claim 4, wherein the pharmaceutically acceptable carrier comprises a liposome, and the metal ion is encapsulated in the liposome.

8. The pharmaceutical composition of claim 7, wherein the compound or its pharmaceutically acceptable salt and the metal ion are individually or simultaneously encapsulated in a liposome.

9. Use of a compound or a pharmaceutically acceptable salt thereof according to claim 1, or a pharmaceutical composition according to claim 2 in the manufacture of a medicament for inhibiting infection or replication of a virus encoding a papain-like protease.

10. The use according to claim 9, wherein the virus encoding the papain-like protease comprises a coronavirus.

11. The use according to claim 10, wherein the coronavirus includes severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HcoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU (HCoV-HKU1), or any combination thereof.

12. Use of a compound, or a pharmaceutically acceptable salt thereof, according to claim 1, or a pharmaceutical composition according to claim 2 in the manufacture of a medicament for the treatment of cancer, wherein the cancer includes lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer or liver cancer.

13. Use of a compound or a pharmaceutically acceptable salt thereof according to claim 1, or a pharmaceutical composition according to claim 2, in the manufacture of a medicament for inhibiting infection or replication of an RNA virus, wherein the RNA virus is an influenza virus or a hepatitis C virus (HCV).

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