TWI816323B - Pharmaceutical composition including retinoic acid and carbohydrate and use thereof - Google Patents

Abstract

A pharmaceutical composition including a retinoic acid and a carbohydrate is provided. The pharmaceutical composition may further include a metal ion. Use of the pharmaceutical composition in the manufacture of a medicament for inhibiting infection or replication of a virus or for treating a cancer is also provided. The pharmaceutical composition can enhance the inhibition ability of virus infection and/or or replication in comparison with the retinoic acid used only.

Description

Pharmaceutical compositions including retinoic acid and carbohydrates and their uses

The present invention relates to a pharmaceutical composition including retinoic acid and carbohydrates and its use in manufacturing drugs for inhibiting viral infection or replication or for treating cancer.

Coronaviruses are a group of positive-positive, single-stranded RNA viruses belonging to the family Coronaviridae, which include 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), human coronavirus Virus HKU (HCoV-HKU1). Notably, SARS-CoV-2 has been identified as the strain responsible for the coronavirus disease 2019 (COVID-19) pandemic.

The genomes of SARS-CoV-2 and SARS-CoV have a high degree of sequence identity. Both SARS-CoV-2 and SARS-CoV are highly dependent on the activity of two viral proteases, namely 3C-like protease (3CLpro, also known as main protease (Mpro) or non-structural protein 5 (nsp5)) and papain-like Protease (PLpro, which is the protease domain of nonstructural protein 3 (nsp3)) to achieve the virus proliferation cycle and virus transmission. It has been reported that all-trans retinoic acid (ATRA, also known as retinoic acid or retinoic acid (Tretinoin)) can be considered as a potential therapeutic agent against SARS-CoV-2 by inhibiting 3CLpro activity.

In addition to 3CLpro, PLpro is also a potential target, so this enzyme plays an essential role in the cleavage and maturation of viral polyprotein, the assembly of the replicase-transcriptase complex, and the destruction of the host response. Although the main function of Mpro and PLpro is to process viral polyproteins in a cooperative manner, PLpro also has the additional function of stripping ubiquitin and interferon-stimulatory gene factor 15 (ISG15) from host cell proteins to prevent coronavirus infection. Viruses evade the host's innate immune response. In other words, PLpro is not only related to virus replication, but also to the dysregulation of the signaling cascade in infected cells, which increases the cell death of surrounding uninfected cells. Therefore, drugs designed to inhibit PLpro function also have potential against SARS-CoV-2. Recently, in addition to ATRA, some researchers have reported that its derivative 13-cis-retinoic acid (also known as isotretinoin) is a potential PLpro inhibitor and can be used to treat COVID-19 caused by SARS-CoV-2. COVID-19.

In addition, retinoic acid (including ATRA and 13-cis-retinoic acid) has become a promising compound for the treatment of various cancers due to its specific effects on cell proliferation, differentiation and apoptosis and low toxicity. Retinoic acid receptors in human cell nuclei have been found by biochemists and found to not mutate in cancer cells, so retinoic acid may exert its anti-cancer effect in many malignant tumors. 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 used in combination with other drugs to treat various cancers and precancerous lesions. Several clinical trials using ATRA as part of combination therapy are currently ongoing. For example, ATRA combined with different interferons (IFNs) has been shown to enhance the effects of the two drugs and lead to growth inhibition and cell death of tumor cell lines. Nonetheless, to unlock the therapeutic potential of retinoic acid, many studies have highlighted the need to better understand the mechanisms by which retinoic acid signaling is blocked and retinoic acid regulates gene expression in cancers such as acute myeloid leukemia (AML). . Clearly, combinatorial therapies targeting multiple gene silencing mechanisms may be the most effective strategy to reactivate ATRA-sensitive gene expression and AML cell differentiation and mediate the general anticancer activity of ATRA. The identification of protein classes that control gene expression through histone and DNA modifications is now enabling the development of novel therapeutics, so-called epigenetic drugs that alter chromatin structure. However, these epigenetic-modifying drugs have been shown to be only partially effective against different cancers when used alone.

In view of the above description, the present invention provides a pharmaceutical composition including retinoic acid and carbohydrate, which can effectively inhibit viral infection or replication, or treat cancer.

In one aspect, the present invention provides a pharmaceutical composition including retinoic acid and carbohydrate.

Preferably, the retinoic acid includes isoretinoic acid.

Preferably, the carbohydrates are selected from the group consisting of monosaccharides, disaccharides, oligosaccharides and polysaccharides.

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

Preferably, carbohydrates include glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3/1,6-glucan oligosaccharide, raffinose, stachyose, verbascose, Fructooligosaccharides, starch, glycogen, cellulose or any combination thereof.

Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and/or metal ions.

Preferably, the pharmaceutically acceptable carrier includes liposomes.

Preferably, retinoic acid, carbohydrates and metal ions are individually coated with liposomes, or at least two of retinoic acid, carbohydrates and metal ions are simultaneously coated with liposomes.

Preferably, the metal ions include monovalent ions, divalent ions or combinations thereof. More preferably, the monovalent ions include K ⁺ , Na ⁺ or combinations thereof; and the divalent ions include Zn ²⁺ , Mg ²⁺ , Cu ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ or any combination thereof.

In yet another aspect, the present invention provides a use of the pharmaceutical composition in the manufacture of a medicament for inhibiting viral infection or replication.

In yet another aspect, the present invention provides a use of the pharmaceutical composition in the manufacture of a medicament for treating cancer.

Therefore, the present invention provides at least the following advantages: 1. Compared with the use of retinoic acid alone, the pharmaceutical composition including retinoic acid and carbohydrates requested in this case can enhance the ability to inhibit viral infection and/or replication. 2. The pharmaceutical composition requested in this case provides a synergistic effect in inhibiting coronavirus in vitro and in vivo. Therefore, such combinations of the claimed pharmaceutical compositions can be expected to serve as potential drug-repurposing agents against coronaviruses, particularly SARS-CoV-2, which causes COVID-19. 3. The pharmaceutical composition requested in this case can also effectively treat various cancers.

While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and described in detail. It should be understood, however, that the description is not intended to limit the invention to the particular embodiments, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

definition

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that, unless expressly so defined herein, terms defined in commonly used dictionaries shall be construed to have meanings consistent with their equivalents in the context of the relevant technology and not in an idealized or overly formal sense. .

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "includes," "includes," and/or "includes," when used herein, designate recited features, integers, steps, operations, elements, portions, and / or the existence of combinations thereof, but does not exclude the existence or addition of one or more other features, integers, steps, operations, elements, parts and / or combinations thereof.

The term "individual" as used herein means a mammal for which diagnosis, prognosis or treatment is desired. Generally speaking, mammals are humans. In certain embodiments, mammals may refer to non-human mammals, such as non-human primates, cattle, horses, goats, sheep, dogs, cats, rabbits, used, for example, to screen, characterize, and evaluate drugs and therapies. Pig, mouse or rat.

The term "administered" or "administered" as used herein means introducing, providing or delivering the intended active ingredient to an individual by any suitable route to perform its intended function.

The term "cancer" as used herein means leukemias, lymphomas, carcinomas, sarcomas and other malignant tumors that may grow indefinitely, expand locally by invasion and systemically by metastasis. Examples of cancer include (but are not limited to) ovarian cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colorectal cancer and/or rectal cancer, gallbladder cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer , nervous tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, gastric cancer and thyroid cancer. Some other examples of cancer include cholangiocarcinoma, acute and chronic lymphocytic and granulosa cell tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysepithelial differentiation and carcinoma in situ, Ewing's sarcoma, epidermoid Carcinoma, giant cell tumor, glioblastoma multiforma, trichome tumor, intestinal ganglioneuroma, proliferative corneal neuroma, islet cell carcinoma, Kaposi's sarcoma, smooth Myoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, nerve Blastoma, osteosarcoma, pheochromocytoma, polycythermia vera, primary brain tumors, small cell lung tumors, ulcerative and papillary squamous cell carcinoma, cellular hyperplasia, seminoma, Soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, reticulum cell sarcoma and Wilm's tumor.

The term "oligosaccharide" as used herein means a carbohydrate composed of a small number of monosaccharides, typically about three to ten monosaccharide units. Among them, oligosaccharides with one type of monosaccharide subunit are called monooligosaccharides; oligosaccharides with more than one type of monosaccharide subunit are called heterooligosaccharides.

The term "polysaccharide" as used herein means a carbohydrate composed of a large number of monosaccharide units. Among them, polysaccharides with one monosaccharide subunit are called monopolysaccharides; polysaccharides with more than one monosaccharide subunit are called heteropolysaccharides.

The term "liposome" as used herein means a particle characterized by having an aqueous interior space separated from the external medium by a membrane forming one or more bilayers of the vesicle. The main types of liposomes are multilamellar vesicles (MLV, with several lamellar phase lipid bilayers), small unilamellar vesicles (SUV, with a single lipid bilayer), and large unilamellar vesicles (LUV, has a single lipid bilayer). The bilayer membranes of unilamellar or multilamellar vesicles are typically formed from lipids (ie, synthetic or naturally derived amphipathic molecules that include spatially separated hydrophobic and hydrophilic domains).

Description of embodiments

Hereinafter, exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

In one embodiment, a compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition including the compound (hereinafter referred to as the "first" pharmaceutical composition) is provided, wherein the compound includes conjugate with a carbohydrate Retinoic acid.

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

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

In another embodiment, a pharmaceutical composition is provided, which includes retinoic acid and carbohydrate (hereinafter referred to as the "second" pharmaceutical composition). In certain embodiments, the retinoic acid is 13-cis-retinoic acid (also known as isoretinoic acid).

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

In certain embodiments, the carbohydrate may be a monosaccharide, disaccharide, oligosaccharide, or polysaccharide. Oligosaccharides can be mono-oligosaccharides or hetero-oligosaccharides. Polysaccharides can be monopolysaccharides or heteropolysaccharides. Monosaccharides may be selected from, but are not limited to, glucose, fructose, galactose and mannose. The disaccharide may be selected from, but is not limited to, sucrose, lactose and maltose. Oligosaccharides may be selected from, but are not limited to, beta-1,3/1,6-glucan oligosaccharides, raffinose, stachyose, verbascose and fructo-oligosaccharides. Polysaccharides may be selected from, but are not limited to, starch, glycogen, and cellulose.

In certain embodiments, the concentration of carbohydrates may be, but is not limited to, 0.1 μM to 200 mM, 0.1 μM to 150 mM, 0.1 μM to 100 mM, 0.1 μM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 500 μM, 0.1 μM to 250 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 200 mM, 1 μM to 150 mM, 1 μM to 100 mM, 1 μM to 10 mM, 1 μM to 1mM , 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 1mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM or 10 μM to 50 μM.

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.

In certain embodiments, each of the first pharmaceutical composition and the second pharmaceutical composition optionally further includes metal ions. Preferably, the metal ions include monovalent ions, divalent ions or combinations thereof. In specific embodiments, the monovalent ions include K ⁺ , Na ⁺ or combinations thereof; and the divalent ions include Zn ²⁺ , Mg ²⁺ , Cu ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ or any of them combination.

In certain embodiments, the concentration of the metal ion may be 1 μM to 300 mM, 1 μM to 250 mM, 1 μM to 200 mM, 1 μM to 150 mM, 1 μM to 100 mM, 1 μM to 10 mM, 1 μM to 1mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 300 mM, 10 μM to 250 mM, 10 μM to 200 mM, 10 μM to 150 mM, 10 μM to 100 mM, 10 μM to 10 mM, 10 μM to 1mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM or 10 μM to 50 μM.

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.

In some embodiments, the molar ratio of retinoic acid, carbohydrates and metal ions 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, 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.

In certain embodiments, each of the first pharmaceutical composition and the second pharmaceutical composition optionally further includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is widely used in the field of pharmaceutical manufacturing. Examples of pharmaceutically acceptable carriers may include (but are not limited to) liposomes, excipients, adjuvants, solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrants, dispersants, binders agents, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents and the like. The selection and amount of pharmaceutically acceptable carriers are within the expertise of those of ordinary skill in the art.

In certain embodiments, the pharmaceutically acceptable carrier is liposomes. The compound or its pharmaceutically acceptable salt is coated with liposomes. The compound or its pharmaceutically acceptable salt and the metal ion in the first pharmaceutical composition can be coated by microliposomes individually or simultaneously. The retinoic acid and carbohydrate in the second pharmaceutical composition can be coated with liposomes individually or simultaneously. All or at least two of the retinoic acid, carbohydrates and metal ions in the second pharmaceutical composition can be coated with microliposomes individually or simultaneously.

Exemplary liposomes may be neutral, positively charged, or negatively charged liposomes. Generally speaking, lipids commonly used in liposomes typically include dialiphatic chain lipids, such as phospholipids, diglycerides, and dialipid glycolipids; single lipids, such as sphingomyelin and glycosphingomyelin Glycosphingolipid; 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-dilauryl-sn- Glyceryl-3-phosphocholine (DLPC), 1,2-dimyristyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate Choline (DPPC), 1-palmitoyl-2-stearyl-sn-glyceryl-3-phosphocholine (PSPC), 1-palmitoyl-2-oleyl-sn-glycerol-3- Phosphatidylcholine (POPC), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleyl-sn-glycero-3-phosphocholine (DSPC) DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dimyristyl-sn-glycerol-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG) , 1,2-dipalmitoyl-sn-glycerol-3-phosphorus (1'-rac-glycerol) (sodium salt) (DPPG), l-palmitoyl-2-stearyl-sn-glycerol- 3-Phospho-(1'-rac-glycerol) (sodium salt) (PSPG), 1,2-distearyl-sn-glycerol-3-phosphorus-(1'-rac-glycerol) (sodium salt) (DSPG), 1,2-dioleyl-sn-glycerol-3-phosphorus-(1'-rac-glycerol) (DOPG), 1,2-dimyristyl-sn-glycerol-3-phosphorus -L-serine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycerol-3-phospho-L-serine (sodium salt) (DPPS), 1,2-disulfide Diacyl-sn-glycerol-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleyl-sn-glycerol-3-phospho-L-serine (DOPS) , 1,2-dimyristyl-sn-glycerol-3-phosphate (sodium salt) (DMPA), 1,2-dimyristol-sn-glycerol-3-phosphate (sodium salt) (DPPA ), 1,2-distearyl-sn-glycerol-3-phosphate (sodium salt) (DSPA), 1,2-distearyl-sn-glycerol-3-phosphate (sodium salt) ( DOPA), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphoethanolamine (POPE), 1, 2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl- sn-glyceryl-3-phospho-(1'-myo-inositol) (ammonium salt) (DPPI), 1,2-distearyl-sn-glycerol-3-phosphoinositol (ammonium salt) (DSPI ), 1,2-dioleyl-sn-glycerol-3-phospho-(1-myo-inositol) (ammonium salt) (DOPI), cardiolipin (cardiolipin), L-α-phosphatidylcholine ( EPC), 1,2-dioleyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), L-α-phosphotidylethanolamine (EPE), dimethyldioctadecyl ammonium (DDAB), 1,2-dioleyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 3β- [N-(N',N'-dimethylaminoethane)aminoformyl]cholesterol hydrochloride.

Suitable lipids may be lipid mixtures of one or more of the aforementioned lipids, or mixtures of one or more of the aforementioned lipids with one or more other lipids, membrane stabilizers or antioxidants not previously listed.

The molar percentage of lipids 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 therebetween (e.g., about 5 to 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% ).

The lipid of the bilayer membrane may be a mixture of the first phospholipid and the second phospholipid. The first phospholipid may be selected from the group consisting essentially of PC, HSPC, DOPC, POPC, DSPC, DPPC, DMPC, PSPC and combinations thereof, and the second phospholipid may be selected from the group consisting essentially of Groups formed: PE, PG, DOPE, PEG-DSPE, DPPG, DOPG, DOTAP, DOTMA, DDAB and their combinations. In other embodiments, the molar percentage of the first phospholipid in the bilayer membrane is about 50, 45, 40, 35, 30, 25, 20, 15, 10, or any value or range of values therebetween. (For example, 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 percentage of the second phospholipid in the bilayer membrane is between 0.1 and about 15, 14, 13, 12, 11, 10, 9, 8, 7, or the like Any value or range of values (for example, 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, the molar ratio of the first phospholipid (DSPC) to the second phospholipid (DOPE, DOPG, or DDAB) may be from 4:1 to 6:1.

In an exemplary embodiment, the bilayer membrane of liposomes includes less than about 55 molar percent of steroid, preferably cholesterol. The molar percentage of steroids (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 percentage of phospholipids and cholesterol in the bilayer membrane can be about 25-50%, 15-55%, 25-50%, 20-55% or 25-50%, 15-50%. The molar ratio of phospholipids to cholesterol can range from 1:1 to 3:1. The molar percentages of the first phospholipid, the second phospholipid and cholesterol in the bilayer membrane can be about 25~50%, 0.1~15%, 15~55%, 5~50%, 0.1~15%, 10~ 40% or 25~50%, 0.5~10%, 5~20%.

The liposomes coating the capture agent can be prepared by any technique currently known or subsequently developed. For example, MLV liposomes can be directly formed by hydrated lipid films, spray-dried powders or freeze-dried blocks of selected lipid compositions and capture agents; SUV liposomes and LUV liposomes can be formed by ultrasound. Create to size from MLV liposomes by shaking, homogenizing, microfluidizing or extruding.

In yet another embodiment, the compound comprising retinoic acid conjugated to a carbohydrate or a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition and the second pharmaceutical composition can be used to inhibit viral infection or replication. . The virus may be an RNA virus. RNA viruses may include coronaviruses, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza viruses, or any combination thereof. Coronaviruses may 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, the coronavirus is SARS-CoV-2, which causes COVID-19.

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

In specific embodiments, the compound and the metal ion in the first pharmaceutical composition can be administered separately, simultaneously, or sequentially. The retinoic acid and carbohydrate in the second pharmaceutical composition can be administered separately, simultaneously or sequentially. All or at least two of the retinoic acid, carbohydrates and metal ions in the second pharmaceutical composition can be administered separately, simultaneously or sequentially. In an exemplary embodiment, the metal ions are administered simultaneously with the carbohydrate, followed by retinoic acid.

In exemplary embodiments, the time interval between sequential administrations can be 1 to 30 minutes, 30 to 60 minutes, 60 to 90 minutes, 90 to 120 minutes, 2 to 3 hours, 3 to 12 hours, or 12 to 24 hours.

In certain embodiments, the compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition and the second pharmaceutical composition may each be prepared in dimethylsulfoxide ( DMSO), ethanol, buffer, or water prepared for administration. The dosage and frequency of administration may vary depending on 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 individual being treated . The daily dosage of the above therapeutic agents may be administered as a single dose or in several doses.

In certain embodiments, the compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition and the second pharmaceutical composition are administered orally, intravenously, respectively. , intramuscular, subcutaneous, intraperitoneal, intranasal or topical administration.

Example

The invention will be further illustrated by the following examples. However, it should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the invention in its practice.

Materials and methods

A. Prepare a novel retinoic acid compound represented by formula ( Ia ), that is, galactose-modified isoretinoic acid, according to the following synthesis flow chart:

I. General Experimental Methods All reagents are commercially available and can be used without further purification. Yield means a compound that is purified and spectroscopically identified as being of high purity. Thin layer chromatography (TLC) was performed using Merck TLC aluminum silica 60 F ₂₅₄ plates and visualized by fluorescence quenching under UV light. Flash chromatography was performed using silica gel (Chromatorex, MB 70-40/75, 40~75 μm) purchased from Fuji Silysia Chemicals. NMR spectra were recorded on a Varian-400MR operating at 400 MHz for ¹ H analysis. Chemical shifts are reported in ppm with the solvent resonance as the internal standard. Data are reported as follows: s = singlet, br = broadt, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet; coupling constants in Hz; integration performed. Purity was recorded on the Waters e2695 separation module/2998 PDA detector HPLC system (column: XBridge C18, 5 μm, 4.6 mm (ID) x 150 mm (L), eluate: 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 NH ₄ OAc, flow rate: 0.5 mL/min. Detection: UV, 254 nm).

II. Compound (2R, 3S, 4S, 5R, 6R)-2-( acetyloxymethyl )-6-(3- bromopropoxy ) tetrahydro -2H- piran -3,4,5- Triyl triacetate ( (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(3-bromopropoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate ) ( 2 ) synthesis Boron trifluoride-diethyl etherate (8.04 mL, 64.0 mmol) was added to the solution containing galactose pentaacetate (compound ( 1 ) , 5.0 g, 13 mmol), 3 -A solution of bromo-1-propanol (1.73 mL, 19.0 mmol) and freshly dried molecular sieves in anhydrous dichloromethane (50 mL). The resulting 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 column chromatography (EtOAc: hexane = 1:2) to obtain the desired product ( 2 ) (0.625 g, 10%) as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ): 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 (m, 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 C ₁₇ H ₂₅ BrO ₁₀ 469.28; found 491.04 [M + Na] ⁺ .

III. Compound (2R, 3S, 4S, 5R, 6R)-2-( acetyloxymethyl )-6-(3- azidopropoxy ) tetrahydro -2H- pyran 3,4,5 -Triyl triacetate ( (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(3-azidopropoxy)tetrahydro-2H-pyran 3,4,5-triyl triacetate ) ( 3 ) synthesis Sodium azide (0.43 g, 6.6 mmol) was added to a solution containing compound ( 2 ) (0.62 g, 1.3 mmol) in DMF (4 mL). The resulting mixture was stirred at 100°C for 2 hours. The solution was concentrated to dryness. The crude product was purified by silica column chromatography (EtOAc:hexane = 1:2) to obtain the desired product ( 3 ) (458 mg, 80%) as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ): 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 as C ₁₇ H ₂₅ N ₃ O ₁₀ 431.40; found as 454.2 [M + Na] ⁺ .

IV. Compound ( 2R,3R,4S,5R,6R)-2-(3- azidopropoxy )-6-( hydroxymethyl ) tetrahydro -2H- pyran -3,4,5- tri Synthesis of alcohol ( (2R,3R,4S,5R,6R)-2-(3-azidopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol ) ( 4 ) Sodium methoxide (28 mg, 0.5 mmol) was added to a solution containing compound ( 3 ) (0.45 g, 1 mmol) in dichloromethane (1.0 mL) and methanol (4.0 mL). The resulting mixture was stirred at room temperature for 10 hours. 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 C ₉ H ₁₇ N ₃ O ₆ 263.25; found 286.1 [M + Na] ⁺ .

V. Compound ( 2R,3R,4S,5R,6R)-2-(3- aminopropoxy )-6-( hydroxymethyl ) tetrahydro -2H- pyran -3,4,5- triol ( (2R,3R,4S,5R,6R)-2-(3-aminopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol ) ( 5 ) Synthesis The crude compound ( 4 ) in methanol (10.5 mL) was treated with Pd/C (11 mg) and acetic acid (6.3 mg) under _H2 environment. 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 (5) tetrahydro- 2H -pyran-3,4,5-triol ( 5 ), which was used in the next step without further purification. LCMS (ESI) m/z calculated for C ₉ H ₁₉ NO ₆ 237.25; found 237.8 [M] ⁺ .

VI. Compound perfluorophenyl (2Z,4E,6E,8E)-3,7- dimethyl - 9-(2,6,6 -trimethylcyclohex-1-en - 1 - yl ) non- 2,4,6,8 -tetraenoate ( perfluorophenyl (2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona -Synthesis of -2,4,6,8-tetraenoatee ) ( 6 ) Pentafluorophenyl trifluoroacetate (compound ( 8 ), 0.081 mL, 0.47 mmol) was added to a solution containing retinoic acid (compound ( 7 ) , 0.10 g, 0.33 mmol) and triethylamine at 0°C. (0.093 mL, 0.67 mmol) in anhydrous DMF (1.0 mL). The solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with EtOAc and washed with 0.1 N aqueous HCl, followed by aqueous _NaHCO3 and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated to dryness to obtain the desired product ( 6 ) without further purification.

VII. 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 ) nonan -2,4,6,8- tetraenamide ( (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, Synthesis of 4,6,8-tetraenamide ) ( Ia ) Triethylamine (0.049 mL, 0.33 mmol) was added to DMF (1.0 mL) containing crude compound ( 5 ) (30 mg, 0.13 mmol) and active ester ( 6 ) (150 mg, 0.33 mmol) at room temperature. in solution. The resulting mixture was stirred overnight. The solution was concentrated to dryness. The crude product was purified by silica column chromatography (methanol: dichloromethane = 1:9). LCMS (ESI) m/z calculated for C ₂₉ H ₄₅ NO ₇ 519.68; found to be 520.5 [M+H] ⁺ . See Figure 1 .

B , SARS-CoV-2 PLPro Preparation

In order to determine whether the claimed compounds and pharmaceutical compositions can inhibit SARS-CoV-2, their in vitro inhibitory effects on the activity of SARS-CoV-2 papain-like protease (PLpro) were first evaluated.

The codon-optimized gene sequence encoding wild-type SARS-CoV-2 PLpro was synthesized by Biotools Co., Ltd. (New Taipei City, Taiwan) and cloned into pET-21a (Novagen) using NdeI and XhoI restriction enzyme sites. ) in the vector, while the His tag coding region (-LEHHHHHH-) is retained at the C terminus.

The vector inserting the SARS-CoV-2 PLPro gene was transformed into E. coli BL21 (DE3) strain (Yeastern Biotech Co., Ltd., New Taipei City, Taiwan) to express a large amount of PLPro in it. Culture was performed 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 grown at 37°C with shaking at 200 rpm. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce the expression of PLpro at an optical density at 600 nm (OD ₆₀₀ ) between 0.6 and 0.8 . Incubation was continued for 20 hours at 18°C and 200 rpm. Cells were harvested by centrifugation (5,000 xg) and disrupted by sonication in a lysis buffer containing 50 mM sodium phosphate (pH 7.4), 1.0 mM DTT, 5% glycerol, and 100 mM NaCl. Subsequently, centrifuge at 20,000 xg for 50 minutes to remove cell debris. The supernatant was loaded onto a 5 mL His-Trap HP column (GE Healthcare Life Sciences), and a gradient of 50 mM sodium phosphate (pH 7.4) and 100 mM NaCl containing 0 to 500 mM imidazole was used. Protein dissolution. Fractions containing His-tagged SARS-CoV-2 PLpro were pooled and concentrated using Centricon membranes (10 K cutoff, 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 UV absorbance at 280 nm (using an extinction coefficient (ε280) of 45270 M ^-1 cm ^-1 ).

C , SARS-CoV-2 PLPro activity test

Through a colorimetry-based peptide cleavage assay and using a 6-mer peptide substrate, namely FRLKGG-p-nitroaniline (FG6-pNA) (HPLC purity 97%; GL Biochem Ltd., Shanghai, China) to measure the enzyme activity of the SARS-CoV-2 PLpro obtained above. In the cleavage test, the 6-monomer unit peptide receptor was cleaved at the Gly-pNA bond to release free pNA, which changed the color of the solution to yellow. Enzyme activity was determined by continuously monitoring the absorbance at 405 nm (A ₄₀₅ ) at 30°C using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek).

Specifically, the cutting test was performed in a 96-well microplate. Each well of the microplate contains 50 mM phosphate buffer (pH 7.4), and FG6-pNA is added to each well to produce various FG6-pNA concentrations (0.1875 mM, 0.375 mM, 0.75 mM, 1.5 mM , 3.0 mM, 6.0 mM) substrate solution. The test mixture (180 μL per well) was preincubated for 10 min to precisely control the temperature, and the reaction was initiated by adding 20 μL of SARS-CoV-2 PLpro solution (1.75 μM) to the test mixture. Prepare a SARS-CoV-2 PLpro solution by mixing the SARS-CoV-2 PLpro obtained above and 50 mM sodium phosphate buffer (pH 7.4). The concentration of pNA released by proteolysis was calculated by measuring A ₄₀₅ and using the extinction coefficient (ε405) of 9800 M ⁻¹ cm ⁻¹ (A ₄₀₅ = 9.8 at 1 mM).

Steady-state enzyme kinetic parameters were obtained by fitting the initial velocity (V ₀ ) data based on the Michaelis-Menten equation using OriginPro 8.0 software (OriginLab Corporation, USA). All measurements were performed in triplicate. The data obtained are expressed as mean ± standard deviation.

Results: K _M and k _cat values were 2.50±0.03 mM and 0.85±0.01 s ^-1 respectively. Therefore, it was verified that SARS-CoV-2 PLPro with protease activity was successfully prepared according to the above manufacturing method and can be used to conduct subsequent SARS-CoV-2 PLPro inhibition tests.

D. SARS-CoV-2 Creation of animal model

Golden Syrian hamsters (age 5–6 weeks old, average weight approximately 100 g) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Hamsters were housed in animal rooms under specific pathogen free (SPF) conditions commonly used in the field. In addition, provide all hamsters with ad libitum access to water and feed. All experiments involving hamsters were reviewed and approved by the Laboratory Animal Care and Use Committee (IACUC) of Academia Sinica (Taiwan).

Hamsters were inoculated intranasally with 1x10 ⁴ plaque-forming units (PFU) in phosphate containing SARS-CoV-2 (obtained from P3 Laboratory, Genome Research Center, Academia Sinica; Wuhan strain wild type) at 12:00 PM Salt-buffered saline (PBS) for infection in order to establish SARS-CoV-2 animal models. Confirmation of the established SARS-CoV-2 animal model (data not shown).

Example 1

For in vitro evaluation of novel retinoic acid compounds (Galactose-modified isoretinoic acid) Yes SARS-CoV-2 antiviral effect SARS-CoV-2 PLPro suppression test

Enzyme inhibition assays were performed in 96-well microplates. Each well of the microplate contains 50 mM phosphate buffer (pH 7.4). The SARS-CoV-2 PLPro (0.9 μM) obtained above was added to each well to form an enzyme solution. The enzyme solution in each well was divided into a control group (referred to as "Control"), a comparison group (referred to as "Comparative Example_1"), and an experimental group (referred to as "Experimental Example_1"). The corresponding inhibitors shown in Table 1 below were added to each group of enzyme solutions to form a test mixture (total volume: 180 μL). Perform pre-incubation for 30 minutes.

surface 1 Group Components control PLpro Comparative example_1 PLpro + 50 μM Isoretinoic Acid Experimental example_1 PLpro + 50 μM galactose-modified isoretinoic acid

Add 20 μL of the above FG6-pNA (1.2 mM) to each set of test mixtures to start the enzyme reaction. Allow the enzyme reaction to proceed at 30°C for 300 seconds. Enzyme activity was determined by continuously monitoring the absorbance at 405 nm (A ₄₀₅ ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). Calculate the reaction rate based on this, where the reaction rate is the slope of the change of absorbance A ₄₀₅ with time (seconds), and the total reaction time is 300 seconds. The data are presented in Figure 2 .

Calculate the inhibition percentage using the following formula: A = [1-(B/C)]x100 ( I ) where A = inhibition percentage B = reaction rate of each group C = reaction rate of the control group The data obtained are average ± Standard deviation is expressed.

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

surface 2 Group percent inhibition ( % ) Contrast: PLpro 0 Comparative example_1: PLpro + Isoretinoic Acid 20.90 ± 1.24 Experimental example_1: PLpro + galactose-modified isoretinoic acid 34.21 ± 0.17

As shown in Figure 2 and Table 2 , the inhibition rate of Experimental Example_1 is significantly higher than that of Comparative Example_1, which illustrates that galactose-modified isoretinoic acid is better than isoretinoic acid alone in inhibiting PLpro enzyme activity. Acid is more effective. This result shows that galactose-modified isoretinoic acid has a better inhibitory effect on the activity of papain-like protease.

Furthermore, it provides important insights into the biochemical properties of the PLpro family of coronaviruses and leads the way to promising therapeutic strategies against SARS-CoV-2.

Example 2

Novel retinoic acid compounds (galactose-modified isoretinoic acid) in SARS-CoV-2 pair in animal mode SARS-CoV-2 In vivo assessment of antiviral efficacy

Since the novel retinoic acid compound galactose-modified isoretinoic acid has been confirmed to have an inhibitory effect on SARS-CoV-2 in vitro, the antiviral effect of the novel retinoic acid compound on SARS-CoV-2 will be further evaluated in in vivo animal tests. .

The infected hamsters obtained above were divided into the following four groups (n=5 in each group): one group of control groups (referred to as "Control"), and two groups of comparison groups (referred to as "Comparative Example_1" and "Comparative Example_" respectively). 2") and an experimental group (referred to as "Experimental Example_1"). The series of therapeutic agents used in each of these groups are shown in Table 3 below.

surface 3 Group therapeutic agent control Buffer containing 40% ethanol, 40% Span ^® 80 (sorbitol monooleate) (Sigma-Aldrich), and 20% peanut oil Comparative example_1 isoretinoic acid Comparative example_2 Liposome-coated isoretinoic acid Experimental example_1 Galactose modified isoretinoic acid

Specifically, for each hamster in Experimental Example_1, at 8:00 AM on the day of infection (that is, 4 hours before SARS-CoV-2 infection, day 1), a dose of 0.35 mg/kg was administered on the day of infection. Novel retinoic acid compounds were administered intranasally at 8:00 PM at a dose of 0.35 mg/kg. On the next two days (Days 2 and 3), novel retinoic acid compounds were administered intranasally twice daily at 8:00 AM and 8:00 PM at doses of 0.35 mg/kg each.

Each hamster in the comparison group was dosed with 0.35 mg/kg at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and 0.35 mg/kg at 8:00 PM on the day of infection. The isoretinoic acid in Comparative Example_1 or the liposome-coated isoretinoic acid in Comparative Example_2 was administered intranasally. For the next two days after the day of infection, isoretinoic acid in Comparative Example_1 or Comparative Example_2 was intranasally administered twice daily at 8:00 AM and 8:00 PM at a dose of 0.35 mg/kg. Isoretinoic acid coated in microliposomes. The liposome-coated isoretinoic acid in Comparative Example_2 was prepared by Taipei Medical University (Taiwan) using techniques commonly used in this field and described in detail above. Since the main technical feature of the present invention lies in the novel retinoic acid compound galactose-modified isoretinoic acid, for the sake of simplicity, the details of the liposome are omitted here.

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

After 3 days of treatment, the hamsters were sacrificed and their lungs collected for measurement of viable viral load in Vero E6 cells by TCID ₅₀ assay. Viral titers were determined based on the 50% tissue culture infective dose (TCID ₅₀ ) using the Reed-Muench method. All experiments on SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by Academia Sinica (Taipei, Taiwan).

Experimental data were analyzed using Tukey's test to assess differences between groups. Statistical significance is expressed as p<0.05.

Results: Refer to Figure 3. The TCID ₅₀ of Experimental Example_1 is significantly lower than the TCID ₅₀ of Comparative Example_1 and Comparative Example_2. This shows the difference compared with only isoretinoic acid or only microliposome coating. Retinoic acid, a novel retinoic acid compound galactose-modified isoretinoic acid, has higher in vivo efficacy against SARS-CoV-2.

Example 3

Novel retinoic acid compounds Evaluation of the in vitro cytotoxic effect of (galactose-modified isoretinoic acid) on different cancer cell lines

In this example, seven cell lines were used, namely AsPC-1, MDA-MB-231, HCT-116, Huh-7, SKOV-3, A549 and H460 cancer cell lines. Each cell line was cultured in the designated growth medium containing 10% FBS and cultured in a 37°C humidified incubator containing 5% _CO2 .

The day before treatment, cells in the logarithmic growth phase were harvested, counted, and seeded in a 96-well plate at a density of 1x10 ⁴ cells/100 μL/well. After overnight incubation, the medium from each well was gently removed, and fresh medium (200 μL/well) was added.

The test article (TA) was a novel retinoic acid compound (referred to as "novel compound" in this example) that was freshly prepared as a 96 mM stock solution in 100% DMSO on the day of processing. Isoretinoic acid was serially diluted 2-fold using 100% DMSO to obtain different concentrations of isoretinoic acid from 7.5 mM to 96 mM. Cells were treated by adding 2.02 μL of the specified concentration of isoretinoic acid to each well to obtain a final concentration range of 7.5 μM to 960 μM isotretinoic acid, maintaining 1% DMSO in all wells, including the DMSO control. All components were mixed gently and incubated for 24 hours.

Cells were treated by adding 2.02 μL of the indicated concentrations of novel compounds or DMSO to each well, followed by gentle mixing and incubation for 24 h.

On the day of measuring cell viability, replace the culture medium in each well with freshly prepared Alamar blue dye (10% v/v) and incubate at 37°C for 2 to 3 hours. Spectrophotometric absorbance was recorded at wavelengths of 570 nm and 600 nm.

Calculate the percentage of cell viability using the following formula: % viability = (TA of [A ₅₇₀ /A ₆₀₀ ratio ]/DMSO of [A ₅₇₀ /A ₆₀₀ ratio ]) * 100% where TA = test item A ₅₇₀ = 570 Absorbance at nm A ₆₀₀ = Absorbance at 600 nm

Results: See Figures 4 to 10 , which show that the novel compound can significantly reduce the cell survival rate of all seven cancer cell lines. This shows that the novel retinoic acid compound galactose-modified isoretinoic acid can be an effective treatment for various cancers ( Potential anti-cancer drugs such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer and liver cancer). _IC50 is shown in Table 4 .

surface 4 Tumor type cell lines IC ₅₀ (μM) non-small cell lung cancer NCI-H460 22.95 non-small cell lung cancer A549 21.88 liver cancer Huh-7 77.46 pancreatic cancer AsPC-1 23.00 colorectal cancer HCT-116 123.44 ovarian cancer SK-OV-3 13.82 breast cancer MDA-MB-231 115.88

Example 4

For in vitro evaluation of a second pharmaceutical composition pair SARS-CoV-2 antiviral effect SARS-CoV-2 PLPro suppression test

Enzyme inhibition assays were performed in 96-well microplates. Each well of the microtiter plate contains 50 mM phosphate buffer (pH 7.4). The SARS-CoV-2 PLPro obtained above was added to each well to form an enzyme solution with a final concentration of 0.9 μM. The enzyme solution in each well was divided into one group of control groups (referred to as "Control") and four groups of comparison groups (referred to as "Comparative Example_1", "Comparative Example_2", "Comparative Example_3" and "Comparative Example_3"). Comparative Example_4") and two experimental groups (referred to as "Experimental Example_1" and "Experimental Example_2" respectively). The corresponding inhibitors shown in Table 5 below were added to each group of enzyme solutions to form a test mixture (total volume: 180 μL). Perform pre-incubation for 30 minutes.

surface 5 Group Components control PLpro Comparative example_1 PLpro + oligosaccharide mixture [(β-1,3/1,6-glucan oligosaccharide) (25 μM) + raffinose (25 μM) + stachyose (25 μM) + verbascose (25 μM) ] Comparative example_2 PLpro + Mg ²⁺ (0.9 μM) Comparative example_3 PLpro + Zn ²⁺ (0.09 μM) Comparative example_4 PLpro + Isoretinoic acid (50 μM) Experimental example_1 PLpro + isoretinoic acid (50 μM) + oligosaccharide mixture [(β-1,3/1,6-glucan oligosaccharide) (25 μM) + raffinose (25 μM) + stachyose (25 μM) + verbascose (25 μM)] Experimental example_2 PLpro + isoretinoic acid (50 μM) + oligosaccharide mixture [(β-1,3/1,6-glucan oligosaccharide) (25 μM) + raffinose (25 μM) + stachyose (25 μM) + verbascose (25 μM)] + Mg ²⁺ (0.9 μM) + Zn ²⁺ (0.09 μM))

Add 20 μL of the above FG6-pNA (1.2 mM) to each set of test mixtures to start the enzyme reaction. Allow the enzyme reaction to proceed at 30°C for 300 seconds. Enzyme activity was determined by continuously monitoring the absorbance at 405 nm (A ₄₀₅ ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). Calculate the reaction rate based on this, where the reaction rate is the slope of the change of absorbance A ₄₀₅ with time (seconds), and the total reaction time is 300 seconds. The data are presented in Figure 11 . The percent inhibition was calculated as described in Example 3.

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

surface 6 Group percent inhibition ( % ) Contrast: PLpro 0 Comparative example_1: PLpro + Oligosaccharide 12.43 ± 0.37 Comparative Example_2: PLpro + Mg ²⁺ 14.21 ± 0.15 Comparative example_3: PLpro + Zn ²⁺ 19.22 ± 0.21 Comparative example_4: PLpro + Isoretinoic Acid 20.90 ± 1.24 Experimental example_1: PLpro + Isoretinoic acid + Oligosaccharide 26.61 ± 0.49 Experimental example_2: PLpro + isoretinoic acid + oligosaccharide + Mg ²⁺ + Zn ²⁺ 72.16 ± 0.92

As shown in Figure 11 and Table 6 , the inhibitory effect of each group in the experimental group is significantly higher than that of each group in the comparison group, which illustrates the two experimental groups: (1) isoretinoic acid and oligosaccharide, and ( 2) Isoretinoic acid, oligosaccharides, Zn ²⁺ and Mg ²⁺ are more effective than the individual components of isoretinoic acid, oligosaccharides, Zn ²⁺ or Mg ²⁺ alone. These results indicate that isoretinoic acid, oligosaccharides and divalent metal ions have a synergistic inhibitory effect on papain-like protease activity. Furthermore, it provides important insights into the biochemical properties of the PLpro family of coronaviruses and leads the way to promising therapeutic strategies against SARS-CoV-2.

Example 5

The combination of isoretinoic acid, oligosaccharides or divalent metal ions is SARS-CoV-2 pair in animal mode SARS-CoV-2 Evaluation of therapeutic effects in vivo

The infected hamsters obtained in Part A of this experiment were divided into the following six groups (n=5 for each group): one control group, two experimental groups (i.e., Experimental Example_1 and Experimental Example_2), and four groups Comparative groups (i.e., Comparative Example_1, Comparative Example_2, Comparative Example_3, and Comparative Example_4). The series of therapeutic agents used in each of these groups are shown in Table 7 below.

surface 7 Group therapeutic agent control group Buffer containing 40% ethanol, 40% Span® 80 (sorbitol monooleate) (Sigma-Aldrich), and 20% peanut oil Comparative example_1 isoretinoic acid Comparative example_2 Liposome-coated isoretinoic acid Comparative example_3 Combination of liposome-coated Zn ²⁺ , Mg ²⁺ and K ⁺ Comparative example_4 Liposome-coated oligosaccharide mixture Experimental example_1 ​ Microliposome-coated isoretinoic acid + Liposome-coated oligosaccharide mixture Experimental example_2 Combination of liposome-coated isoretinoic acid + liposome-coated oligosaccharide mixture + liposome-coated Zn ²⁺ , Mg ²⁺ and K ⁺

The liposome-coated therapeutic agent was prepared from liposomes by Taipei Medical University using techniques commonly used in the field and described in detail above. Since the main technical feature of the present invention lies in the combination of retinoic acid and oligosaccharides with/without metal ions, for the sake of simplicity, the details of liposomes are omitted here.

Specifically, each hamster in the experimental group was given a dose of 0.35 mg/kg at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, day 1) and at 8:00 AM on the day of infection: Administer liposome-coated isoretinoic acid intranasally at 00 PM at a dose of 0.35 mg/kg. For the next two days (Days 2 and 3), liposome-coated isoretinoic acid was administered intranasally at 0.35 mg/kg twice daily at 8:00 AM and 8:00 PM. .

In addition to intranasal administration of liposome-coated isoretinoic acid twice daily at 8:00 AM and 8:00 PM at doses of 0.35 mg/kg each, on the day of infection from 7:30 PM to 8:00 PM ( That is, a volume of 15 μL of the liposome-coated oligosaccharide mixture was administered once a day through the nasal cavity (i.e., 0.5 to 1 hour before the administration of liposome-coated isoretinoic acid) in the evening (i.e., Experimental Example _1), or a combination of oligosaccharide mixture (15 μL) and Zn ²⁺ (100 μM), Mg ²⁺ (200 μM), and K ⁺ (200 μM) (15 μL), both coated in microlipids In vivo, the total volume was 30 μL (i.e., Experiment_2), and over the next two days, a 15 μL volume of Experiment_1 or a 30 μL volume of Experiment_2 was administered.

Each hamster in the comparison group was dosed with 0.35 mg/kg at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and 0.35 mg/kg at 8:00 PM on the day of infection. The isoretinoic acid in Comparative Example_1 or the liposome-coated isoretinoic acid in Comparative Example_2 was administered intranasally. For the next two days after the day of infection, isoretinoic acid in Comparative Example_1 or Comparative Example_2 was intranasally administered twice daily at 8:00 AM and 8:00 PM at a dose of 0.35 mg/kg. Isoretinoic acid coated in microliposomes.

For each hamster, the liposome-coated Zn ²⁺ (100 μM) and Mg ²⁺ (200 μM) in Comparative Example_3 were administered intranasally once a day from 7:30 PM to 8:00 PM on the day of infection. and K ⁺ (200 μM) and the liposome-coated oligosaccharide mixture in Comparative Example 4, and in the next two days, a volume of 15 μL each was administered.

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

After 3 days of treatment, the hamsters were sacrificed and their lungs collected for measurement of viable viral load in Vero E6 cells by TCID ₅₀ assay. Viral titers were determined based on the 50% tissue culture infective dose (TCID ₅₀ ) using the Reed-Muench method. All experiments on SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by Academia Sinica (Taipei, Taiwan).

Experimental data were analyzed using Tukey's test to assess differences between groups. Statistical significance is expressed as p<0.05.

Results: Referring to Figure 12 , the TCID ₅₀ of each of Experimental Example_1 and Experimental Example_2 is significantly higher than that of each of Comparative Example_1, Comparative Example_2, Comparative Example_3 and Comparative Example_4. TCID ₅₀ is low, which shows that the combination of isoretinoic acid and oligosaccharides with/without metal ions has higher efficacy against SARS-CoV-2 than isoretinoic acid alone, metal ions only, or oligosaccharides only. In vivo effects.

Conclusion

In view of the results of Examples 1 to 5, it is verified that the claimed novel retinoic acid compound or a pharmaceutically acceptable salt thereof, or the claimed pharmaceutical composition including the compound (i.e., the first pharmaceutical composition), Or it is claimed that a pharmaceutical composition including retinoic acid and carbohydrates with/without metal ions (i.e., the second pharmaceutical composition) can inhibit the infection and replication of SARS-CoV-2 and treat SARS-CoV-2. 2. Provide improved and/or synergistic effects on infections or cancer-related diseases. Therefore, the compounds including retinoic acid conjugated with carbohydrates, their pharmaceutically acceptable salts, and the first and second pharmaceutical compositions of the present invention can indeed be used as drug replacement agents.

The invention has been described broadly and throughout this document. Each narrower form and subgroup falling within the disclosure throughout this document also forms part of this invention. In addition, where the features or aspects disclosed in the present invention are described in accordance with the Markush group, those of ordinary skill in the art will understand that the present invention is also described in accordance with any individual member of the Markush group. Description of the member or subgroup of the member.

without

Figure 1 illustrates the liquid chromatography-mass spectrometry (LC-MS) analysis results of compound (Ia) according to embodiments of the present invention.

Figure 2 illustrates a graph of absorbance versus time in the SARS-CoV-2 PLPro inhibition assay of novel retinoic acid compounds according to embodiments of the present invention.

Figure 3 illustrates a graph of TCID ₅₀ showing the antiviral effect of novel retinoic acid compounds in SARS-CoV-2 animal models according to embodiments of the present invention.

Figure 4 illustrates a cell viability graph showing the cytotoxic effect of novel retinoic acid compounds on AsPC-1 cancer cell lines according to embodiments of the present invention.

Figure 5 illustrates a cell viability graph showing the cytotoxic effect of novel retinoic acid compounds on MDA-MB-231 cancer cell lines according to embodiments of the present invention.

Figure 6 illustrates a cell viability graph showing the cytotoxic effect of novel retinoic acid compounds on HCT-116 cancer cell lines according to embodiments of the present invention.

Figure 7 illustrates a cell viability graph showing the cytotoxic effect of novel retinoic acid compounds on Huh-7 cancer cell lines according to embodiments of the present invention.

Figure 8 illustrates a cell viability graph showing the cytotoxic effect of novel retinoic acid compounds on SKOV-3 cancer cell lines according to embodiments of the present invention.

Figure 9 illustrates a cell viability graph showing the cytotoxic effect of novel retinoic acid compounds on A549 cancer cell lines according to embodiments of the present invention.

Figure 10 illustrates a cell viability graph showing the cytotoxic effect of novel retinoic acid compounds on H460 cancer cell lines according to embodiments of the present invention.

Figure 11 illustrates a graph of the absorbance changing with time in the SARS-CoV-2 PLPro inhibition test of the second pharmaceutical composition according to the embodiment of the present invention.

Figure 12 illustrates a graph of TCID ₅₀ showing the antiviral effect of a second pharmaceutical composition according to an embodiment of the present invention in a SARS-CoV-2 animal model.

Claims (15)

A pharmaceutical composition comprising retinoic acid and carbohydrate, wherein the retinoic acid is isoretinoic acid. The pharmaceutical composition of claim 1, wherein the carbohydrate is selected from the group consisting of: monosaccharides, disaccharides, oligosaccharides and polysaccharides. Such as the pharmaceutical composition of claim 2, wherein the oligosaccharide is a mono-oligosaccharide or a hetero-oligosaccharide; and the polysaccharide is a mono-polysaccharide or a hetero-polysaccharide. Such as the pharmaceutical composition of claim 2, wherein the carbohydrate includes glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3/1,6-glucan oligosaccharide, raffinose, Stachyose, verbascose, fructooligosaccharides, starch, glycogen, cellulose or any combination thereof. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier and/or metal ions. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable carrier includes liposomes. The pharmaceutical composition of claim 6, wherein the retinoic acid, the carbohydrate and the metal ion are individually coated by the liposome, or at least two of the retinoic acid, the carbohydrate and the metal ion are simultaneously coated Coated by this liposome. Such as the pharmaceutical composition of claim 5, wherein the metal ion includes monovalent ions, divalent ions or a combination thereof. Such as the pharmaceutical composition of claim 8, wherein the monovalent ions include K ⁺ , Na ⁺ or a combination thereof; and ^the divalent ions include Zn ²⁺ , Mg 2+ , Cu ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ or any combination thereof. The use of a pharmaceutical composition according to any one of claims 1 to 9 in the manufacture of a drug for inhibiting viral infection or replication. Such as the use of claim 10, wherein the virus is an RNA virus. Such as the use of claim 11, wherein the RNA virus includes coronavirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus or any combination thereof. For example, the use of request item 12, 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. The use of a pharmaceutical composition according to any one of claims 1 to 9 in the manufacture of a drug for treating cancer. The use of claim 14, wherein the cancer includes leukemia, lymphoma, carcinoma or sarcoma.