EA042927B1 - BERBERINE SALTS, URSODEOXYCHOLIC ACID SALTS AND THEIR COMBINATIONS, METHODS FOR PRODUCTION AND APPLICATIONS - Google Patents

Description

Priority claim and related applications

This Application Claims Priority Based on U.S. Provisional Applications

62/030 140 and 62/030 147, filed July 29, 2014, and U.S. Provisional Application 62/128,077, filed March 4, 2015, the contents of each of which are incorporated herein by reference in their entirety.

Technical field

The present invention generally relates to new therapeutic compounds, pharmaceutical compositions, methods for their preparation and therapeutic use. In particular, the present invention relates to new compositions of berberine in combination with pharmaceutically active organic acids and methods for their use. In particular, the present invention also relates to new salts of berberine and organic acids and new salts of ursodeoxycholic acid and organic bases, pharmaceutical compositions and methods of their use. The compounds and pharmaceutical compositions of the present invention can be used to treat and/or prevent various diseases or disorders, including metabolic diseases or disorders such as pre-diabetic metabolic disorders, diabetes, complications of diabetes, dyslipidemia, dyslipidemia in statin intolerant patients, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, diabetic dyslipidemia, or obesity. In addition, the compounds and pharmaceutical compositions of the present invention can be used to treat and/or prevent atherosclerosis, heart disease, neurodegenerative diseases, sarcopenia, muscle wasting, inflammation, cancer, and various liver diseases or disorders such as fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cholestatic liver disease, or graft-versus-host liver disease. In addition, the compounds and pharmaceutical compositions of the present invention can be used to improve liver function in chronic viral diseases and alcoholic liver diseases.

State of the art

Diabetes mellitus is a metabolic disorder that has become a pandemic - today there are more than 300 million people worldwide with diabetes. In the absence of an effective way to prevent diabetes, by 2030 this number will increase to 500 million. There are three main types of diabetes: type 1 diabetes, type 2 diabetes, and gestational diabetes. Among them, type 2 diabetes is the most common form of diabetes, accounting for 9095% of all cases. Type 2 diabetes is characterized by impaired insulin secretion, increased production of glucose in the liver, and a reduced response of peripheral tissues to insulin, i.e. insulin resistance. Many therapeutic drugs are available for the treatment of type 2 diabetes, but their use is often associated with various side effects. Optimal therapeutic treatment should be safe and involve early combinations of drugs with complementary mechanisms of action.

Despite strenuous efforts and significant progress in understanding and managing diabetes over the past decades, people with diabetes continue to be at increased risk of developing, and many suffer from, a range of serious complications affecting the heart and blood vessels, eyes, kidneys, and nerves due to high blood glucose, high cholesterol and high blood pressure. Cardiovascular disease is the most common cause of death in people with diabetes. Diabetic nephropathy, caused by damage to the small blood vessels in the kidney, leads to reduced kidney function or eventually kidney failure. Diabetic neuropathy is caused by damage throughout the body associated with too high blood glucose and blood pressure. Most people with diabetes develop diabetic retinopathy, leading to decreased vision or blindness. Persistently elevated blood glucose, together with high blood pressure and high cholesterol, is the main cause of diabetic retinopathy. Despite the development of a number of antidiabetic agents, there is a significant unmet need for therapeutic drugs that can be effectively used to treat and manage the complications of diabetes.

Metabolic syndrome is a term that refers to risk factors that occur together (for example, abdominal (central) obesity, high blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein (HDL) cholesterol) ). Metabolic syndrome has been shown to increase the risk of developing cardiovascular disease, in particular heart failure, and diabetes. Studies have shown that the prevalence of metabolic syndrome in the US is about 34% in the adult population. Despite the availability of therapeutic drugs, lifestyle changes are the first line of treatment for metabolic syndrome. High-dose statin use, recommended to reduce the risk of cardiovascular disease, has been associated with accelerated progression to diabetes, especially in patients with metabolic syndrome.

Dyslipidemia is a disorder of lipoprotein metabolism, including increased production of lipoproteins (hyperlipidemia) or deficiency of lipoproteins. Dyslipidemia can be manifested by an increase in total cholesterol levels, an increase in the concentration of bad LDL cholesterol and triglycerides, and a decrease in the concentration of good HDL cholesterol in the blood. Dyslipidemia requires attention in many situations, including diabetes, a common cause of dyslipidemia. Hyperlipidemia is an increase in plasma levels of cholesterol (hypercholesterolemia) or triglycerides (hypertriglyceridemia), or an increase in both cholesterol and triglycerides, or a decrease in high-density lipoprotein, which contributes to the development of atherosclerosis. Causes can be primary (genetic) or secondary. The diagnosis is established by the results of measuring the total cholesterol level, the level of triglycerides and individual lipoproteins in the blood plasma. Treatment includes dietary changes, exercise, and lipid-lowering drugs.

The term cardiovascular disease (CVD) is often used interchangeably with heart disease and refers to a range of conditions that affect the heart, such as coronary artery disease, arrhythmia, congestive heart failure, cerebrovascular disease, and so on. Many forms of CVD can be prevented or treated by adopting a healthy lifestyle and by controlling conditions such as atherosclerosis, high blood pressure, diabetes, or obesity with various medications such as antiplatelet drugs, anticoagulants, digitalis, angiotensin-converting enzyme inhibitors ( ACE), beta blockers and LDL cholesterol lowering agents, etc. Due to comorbidities, patients often need to take multiple medications, so it is desirable to provide a single tablet that addresses multiple disorders.

Statins are one of the most commonly prescribed drugs due to their proven ability to prevent cardiovascular disease. Although statins are generally well tolerated, statin intolerance occurs in some patients and requires careful consideration. In addition, patients are concerned about the potential risk of statin-induced diabetes mellitus, cancer, and memory loss, and are often concerned about the need to continue treatment with these drugs. Patients with statin intolerance can be treated with non-statin drugs that lower LDL cholesterol; however, until PCSK9 inhibitors are approved, none of the approved drugs are even close to being as effective as statins. There is an urgent need to develop alternative and effective therapeutic agents for these patients.

Neurodegenerative diseases are an umbrella term for a number of conditions that primarily affect neurons in the human brain. Neurons are the building blocks of the nervous system, which includes the brain and spinal cord. Neurons normally do not divide and do not regenerate when damaged or killed. Examples of neurodegenerative diseases include Parkinson's disease, Alzheimer's disease and Huntington's disease. Neurodegenerative diseases are incurable and severe conditions that lead to progressive degeneration and/or death of nerve cells. The unfulfilled medical need for methods of treating neurodegenerative diseases creates an urgent need for the development of effective therapeutic drugs. Muscular atrophy is a decrease in muscle mass, which may include partial or complete muscle wasting. Muscle atrophy occurs as a result of changes in the balance between protein synthesis and degradation. Muscle atrophy can significantly impair a patient's quality of life because said patient is unable to perform specific tasks or is at risk of an accident (eg, a fall). Muscle wasting is associated with aging and can be a serious consequence of various diseases, including cancer, AIDS, and diabetes. Compared with non-diabetic older adults, older people with type 2 diabetes have reduced skeletal muscle strength, often associated with excessive muscle loss. To date, there are no drugs approved for the treatment of skeletal muscle atrophy. Sarcopenia is characterized primarily by muscle atrophy, along with a decrease in muscle tissue quality, which is characterized by factors such as replacement of muscle fibers by adipose tissue, progression of fibrosis, changes in muscle metabolism, oxidative stress, and degeneration of neuromuscular junctions, and leads to a progressive loss of muscle strength and function. To date, there are no approved therapeutic drugs for the treatment of sarcopenia.

Cancer is a group of diseases that involve abnormal cell growth and are capable of invading or spreading to other parts of the body. In 2012, there were about 14 million new cases of cancer worldwide. The most common types of cancer include lung cancer, prostate cancer, colorectal and stomach cancer for men, and breast, colorectal, lung and cervical cancer for women. Despite having many

- 2 042927 cancer treatment options, including surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy and palliative care, cancer remains a major health threat and is responsible for approximately 15% of all human deaths.

Fatty liver is a reversible condition in which, as part of the process of steatosis, large fat-containing vacuoles (triglycerides) accumulate in the liver cells. Despite the many causes that cause it, fatty liver can be considered as one disease that occurs throughout the world in people with excess alcohol consumption and obesity. Non-alcoholic fatty liver disease (NAFLD) is a form of fatty liver disease that occurs when excess fat is deposited in the liver in patients without excessive alcohol intake. Non-alcoholic fatty liver disease (NAFLD) is generally considered to be associated with metabolic syndrome such as insulin resistance, high blood pressure, and obesity. Non-alcoholic fatty liver disease (NAFLD) affects approximately one third of the adult population in developed countries. Non-alcoholic steatohepatitis (NASH) is an extreme form of non-alcoholic fatty liver disease (NAFLD) characterized by chronic inflammation that can lead to progressive fibrosis (scarring), cirrhosis, and ultimately liver failure and death. Non-alcoholic steatohepatitis (NASH) resembles alcoholic liver disease but occurs in people who drink little or no alcohol. The main symptom of non-alcoholic steatohepatitis (NASH) is the appearance of fat in the liver along with inflammation and damage. Most people with non-alcoholic steatohepatitis (NASH), which is often a silent liver disease, do well and are unaware of liver problems. However, non-alcoholic steatohepatitis (NASH) can be acute and lead to cirrhosis, in which there is permanent damage and scarring of the liver and the loss of its ability to function normally.

To date, there are no drugs approved for the treatment of non-alcoholic steatohepatitis (NASH), a condition that occurs in about a quarter of patients with non-alcoholic fatty liver disease (NAFLD). The current standard of care for non-alcoholic steatohepatitis (NASH) includes weight loss procedures and increased physical activity. Non-alcoholic steatohepatitis (NASH) affects 2-5% of Americans and is becoming more common, possibly due to an increase in the number of obese Americans. Over the past 10 years, the incidence of obesity has doubled in adults and tripled in children.

Therapeutic drugs and methods currently available for the treatment of diseases or disorders such as diabetes, diabetic complications, dyslipidemia, obesity, metabolic syndromes, pre-diabetic metabolic disorders, heart disease, neurodegenerative diseases, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), muscle wasting, inflammation and cancer are not enough. Thus, there remains a strong need for new and improved therapeutic drugs and methods for the treatment of such diseases or disorders.

Brief description of the invention

The present invention is based in part on various novel compositions of berberine in combination with pharmaceutically active organic acids and related uses for the treatment and/or prevention of various diseases or disorders.

The present invention is also based in part on various novel compounds derived from berberine and pharmaceutically active organic acids, various novel compounds derived from ursodeoxycholic acid and pharmaceutically active organic bases, and their pharmaceutical compositions and methods for the preparation and therapeutic use for the treatment and/or prevention of various diseases or disorders.

Compounds and pharmaceutical compositions according to the invention can be used to treat various diseases or disorders such as diabetes, complications of diabetes, dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, diabetic dyslipidemia, obesity, metabolic syndromes, metabolic disorders pre-diabetes , heart disease, neurodegenerative disease, sarcopenia, muscle atrophy, inflammation and cancer, and various liver diseases or disorders such as fatty liver disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cholestatic liver disease, or graft-versus-host liver disease. The compounds of the present invention can also be used to improve liver function in chronic viral liver diseases and alcoholic liver diseases.

According to one aspect, the invention generally relates to a composition comprising:

(a) berberine or a derivative or analogue thereof;

(b) one or more pharmaceutically active organic acids; and (c) optionally a pharmaceutically acceptable excipient, carrier or diluent.

Berberine and pharmaceutically active organic acid(s) are present in an amount

- 3 042927 which, when administered to a subject, are sufficient to treat, prevent, or ameliorate one or more diseases or disorders selected from metabolic disorders, heart disease, neurodegenerative diseases, muscle atrophy, inflammation, and cancer, or a related disease or disorder in a mammal, including a person.

According to another aspect, the invention generally relates to a method for treating, alleviating or preventing a metabolic disorder. The method includes administering to a subject in need thereof a pharmaceutical composition comprising:

(a) berberine or a derivative or analogue thereof;

(b) one or more pharmaceutically active organic acids in a therapeutically effective amount; and (c) optionally a pharmaceutically acceptable excipient, carrier or diluent.

According to another aspect, the invention generally relates to a kit that contains:

(i) a first agent that is berberine or a derivative or analogue thereof;

(ii) one (or more) second agent(s) selected from R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid, cholic acid , obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, Sallylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein, nicotinic acid , biotin, and other organic acids generally recognized by those skilled in the art as pharmaceutically active for the treatment of one or more diseases or disorders selected from metabolic disorders, heart disease, neurodegenerative diseases, muscle atrophy, inflammation, and cancer, or a disease or disorder associated therewith in mammal, including humans. The first and second agents can be presented as purified active pharmaceutical ingredients or as active ingredients in natural extracts, for example: bile acids (cholic acid, deoxycholic acid, etc.), rhubarb extracts (rhein), cinnamon extract (cinnamic acid) , banana extract (corosolic acid), etc.; and (iii) instructions for administering the combination of agents to a patient suffering from or at risk of developing one or more diseases or disorders selected from metabolic disorders, heart disease, neurodegenerative diseases, muscle wasting, and cancer.

According to another aspect, the invention generally relates to an acid-base addition salt in substantially pure form having the following formula:

(X*)m(U-)n (I).

where (a) U ^- represents an anionic fragment of ursodeoxycholic acid or its derivative or analogue;

(b) X ⁺ is a cationic fragment of a pharmaceutically active organic base; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained.

According to another aspect, the invention generally relates to a pharmaceutical composition containing an acid-base addition salt having the following formula:

(W). (I), where (a) U ^- represents an anionic fragment of ursodeoxycholic acid or its derivative or analogue;

(b) X+ is a cationic fragment of a pharmaceutically active organic base; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained in an amount effective to treat, prevent, or ameliorate one or more diseases. or disorders selected from fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), cholestatic liver disease, graft-versus-host liver disease, chronic viral liver disease, alcoholic liver disease, metabolic diseases or disorders such as metabolic disorders pre-diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, obesity, or related diseases or disorders in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

According to another aspect, the invention relates generally to a method for treating, alleviating, or preventing a disease or disorder. The method includes administering to a subject in need

- 4 042927 a pharmaceutical composition containing an acid-base addition salt having the following formula:

(X ⁺ ) _m (U-) " (I), where (a) U ^- represents an anionic fragment of ursodeoxycholic acid or its derivative or analogue;

(b) X ⁺ is a cationic fragment of a pharmaceutically active organic base; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained in an amount effective to treat, prevent, or ameliorate one or more diseases. or disorders selected from fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), cholestatic liver disease, graft-versus-host liver disease, chronic viral liver disease, alcoholic liver disease, metabolic diseases or disorders such as metabolic disorders pre-diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in statin intolerant patients, hyperlipidemia, obesity, or a related disease or disorder in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

According to another aspect, the invention generally relates to an acid-base addition salt in substantially pure form having the following formula:

(B ⁺ ) _m (Y-)n (II), where (a) B ⁺ is a cationic fragment of berberine or its derivative or analogue;

(b) Y ^- is an anionic moiety of a pharmaceutically active organic acid; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained.

According to another aspect, the invention generally relates to a pharmaceutical composition containing an acid-base addition salt having the following formula:

(B ⁺ ) _m (Y-)n (II), where (a) B ⁺ is a cationic fragment of berberine or its derivative or analogue;

(b) Y ^- is an anionic moiety of a pharmaceutically active organic acid; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained in an amount effective to treat, prevent, or ameliorate one or more diseases. or disorders selected from metabolic disorders, cardiac diseases, neurodegenerative diseases, sarcopenia, muscle wasting, inflammation and cancer, or a related disease or disorder in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

According to another aspect, the invention relates generally to a method for treating, alleviating, or preventing a disease or disorder. The method includes administering to a subject in need thereof a pharmaceutical composition containing an acid-base addition salt having the following formula:

(B ⁺ ) _m (Y-)n (II), where (a) B ⁺ is a cationic fragment of berberine or its derivative or analogue;

(b) Y ^- is an anionic moiety of a pharmaceutically active organic acid; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained in an amount effective to treat, prevent, or ameliorate one or more diseases. or disorders selected from metabolic disorders, cardiac diseases, neurodegenerative diseases, sarcopenia, muscle wasting, inflammation and cancer or a related disease or disorder in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier or diluent.

Brief description of graphic materials

Fig. 1. Body weight for each treatment group on days 0 and 14.

Fig. 2. Change in body weight in each treatment group after 14 days of treatment.

Fig. 3. Blood glucose levels for each treatment group on days 2 and 15.

Fig. 4. Change in blood glucose levels for each treatment group on days 2 and 15.

Fig. 5. 1H NMR spectrum of metformin ursodeoxycholate in DMSO-D ₆ .

Fig. 6. IR spectrum of metformin ursodeoxycholate.

Fig. 7. 1H NMR spectrum of berberine ursodeoxycholate (purified product).

Fig. 8. 1H NMR spectrum of a mixture of berberine hydrochloride (1.0 eq.) and ursodeoxycholic acid

- 5 042927 (1.0 eq.) in DMSO-O6.

Fig. 9. IR spectrum of ursodeoxycholate berberine (crude product). The C=O stretching band of ursodeoxycholic acid in the region of about 1721 cm- ¹ disappeared in the IR spectrum of berberine ursodeoxycholate.

Fig. 10. IR spectrum of a mixture of berberine hydrochloride (1.0 eq.) and ursodeoxycholic acid (1.0 eq.). The C=O stretching band of ursodeoxycholic acid carbonyl appeared in the region of about 1719 cm- ¹ .

Fig. 11. Mass spectrometric analysis of berberine ursodeoxycholate in negative ion detection mode: determined molecular weight of UDCA [M-H] 391.28.

Fig. 12. Mass spectrometric analysis of berberine ursodeoxycholate in positive ion detection mode: determined molecular weight of BBR ⁺ 336.14.

Fig. 13. (A) Oral glucose tolerance test (OGTT) plasma glucose concentration and (B) OGTT glucose area under the curve. Data are presented as mean ± standard error of the mean (n=7~13). ** p<0.01 for G2 versus G1; #p<0.05 for G4, G5, G6 or G7 compared to G2.

Fig. 14. Photos of the results of liver staining with Sultan III dye in different groups (n=7~13).

Fig. 15. 1H NMR spectrum of the ursolic acid salt of berberine (400 MHz, DMSO-D ₆ ).

Fig. 16. Effect of UDCA on LDL-C, Serum HDL-C, TC/HDL-C and AFI in Hyperlipidemic Hamsters.

Fig. 17. Effect of UDCCB on serum ACT levels in hyperlipidemic hamsters.

Fig. 18. Effect of UDCCB on serum ALT levels in hyperlipidemic hamsters.

Fig. 19. Effect of UDCCB on liver mass and liver index in hyperlipidemic hamsters.

Fig. 20. General assessment of lipid deposition in the liver tissue.

Fig. 21. Influence of UDCCB on the content of TC and TG in the liver of hamsters with hyperlipidemia.

Fig. 22. Influence of UDCCB on inflammation score and area of oil red O staining.

Definitions

Unless otherwise indicated, all technical and scientific terms used in the present invention have such conventional meaning as understood by those skilled in the art to which the present invention pertains. General principles of organic chemistry as well as specific functional moieties and reactive groups are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 2006.

Particular compounds according to the present invention may exist, in particular, in the form of geometric isomers or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, R and S enantiomers, diastereomers, (D) isomers, (b) isomers, racemic and other mixtures thereof, within the scope of the present invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in the present invention.

Mixtures of isomers containing any of the various ratios of isomers can be used in accordance with the present invention. For example, when only two isomers are combined, according to the present invention, mixtures containing these isomers in the ratio of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97 are considered: 3, 98:2, 99:1 or 100:0. It will be apparent to those skilled in the art that similar ratios are contemplated for more complex mixtures of isomers. If, for example, a particular enantiomer of a compound of the present invention is desired, it can be obtained by asymmetric synthesis or by formation with a chiral auxiliary element, whereby the resulting diastereomeric mixture is separated and the auxiliary element group is cleaved off to obtain the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as an amine or an acidic functional group such as a carboxyl, diastereomeric salts are prepared with the appropriate optically active acid or base, followed by separation of the diastereomers thus formed using fractional crystallization or chromatography techniques well known in the art. of the art, and subsequent recovery of the pure enantiomers.

In view of the advantages of the present invention, it will be apparent to one skilled in the art that various protecting groups may be used in the synthetic methods described in the present invention. The term protecting group as used in the present invention means that a particular functional moiety, such as O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another functional site in a compound containing multiple functional groups. According to preferred embodiments of the invention, the protecting group reacts selectively (with good intensity) to obtain a protected substrate that is resistant to the intended reactions; the protecting group should be selectively removable (with good intensity) using preferably readily available non-toxic reagents that do not attack other functional groups; the protecting group forms an easily separable derivative (preferably without the formation of new stereogenic

- 6 042927 centers); and the protecting group has a minimum number of additional functional groups to exclude additional reaction sites. You can use protective groups for oxygen, sulfur, nitrogen and carbon. Examples of various protecting groups can be found in Protective Groups in Organic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999.

It should be understood that the compounds described in the present invention may contain any number of substituents or functionally active fragments. Throughout the description, groups and their substituents can be chosen to provide stable moieties and compounds.

When used in the present invention, the term effective amount of an active agent refers to an amount sufficient to provide the desired biological response. As will be appreciated by those skilled in the art, an effective amount of a compound of the present invention may vary depending on such factors as the desired biological end point, the pharmacokinetic properties of the compound, the disease being treated, the route of administration, and the individual patient.

When used in the present invention, the term treatment, amelioration or prevention of a disease or disorder refers to the improvement of such a condition before or after it has occurred. Compared to an equivalent untreated control, this reduction or prevention rate is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% based on the results of evaluation using any standard method.

As used in the present invention, the term pharmaceutically acceptable excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid excipient, diluent, excipient, solvent, or encapsulating material involved in the delivery or transfer of the pharmaceutical agent of interest from one organ or body part to another organ or body part. Each carrier must be acceptable in terms of its compatibility with the other ingredients of the dosage form and not harmful to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include:

sugars such as lactose, glucose and sucrose;

starches such as corn starch and potato starch;

cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;

powdered tragacanth;

malt;

gelatin;

talc;

excipients such as cocoa butter and suppository waxes;

oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;

glycols such as propylene glycol;

polyols such as glycerol, sorbitol, mannitol and polyethylene glycol;

esters such as ethyl oleate and ethyl laurate;

agar;

buffering agents such as magnesium hydroxide and aluminum hydroxide;

alginic acids;

pyrogen-free water;

isotonic saline;

Ringer's solution;

ethanol;

phosphate buffer solutions and other non-toxic compatible substances used in pharmaceutical dosage forms.

Wetting agents, emulsifiers and lubricants, such as sodium laryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer, as well as colorants, release agents, coating agents, sweeteners, flavors and fragrances, preservatives and antioxidants may also be present in the compositions. As used in the present invention, the term isolated or purified refers to a material that is substantially or substantially free of components that normally co-localize with it in its native state. Purity and homogeneity are typically determined using analytical chemical methods such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

As used herein, the term subject refers to any animal (eg, mammal), including, but not limited to, humans, non-human primates, rodents, and the like, that is the intended recipient of a particular treatment. Generally, the terms subject and patient are used interchangeably in the present invention with reference to a human subject.

- 7 042927

As used herein, the term "sufficient amount" refers to the amount of a compound (when used alone or in combination with another therapeutic regimen) required to clinically meaningfully treat, prevent, or ameliorate metabolic disorders such as diabetes. The sufficient amount of active compound used to practice the present invention for the therapeutic treatment of conditions caused by or contributing to diabetes varies with the route of administration, age, body weight, and general health of the mammal or patient. The appropriate amount and dosage schedule is ultimately determined by the healthcare professional. In addition, an effective amount may be the amount of a compound in the combination of the present invention that is safe and effective in treating a patient suffering from metabolic disorders such as diabetes, as compared to the individual use of each agent as determined and approved by a regulatory authority (such as the US Food and Drug Administration). As used in the present invention, the term low dose refers to a dose that is at least 5% (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) less than the lower standard recommended dose. the specific compound in the dosage form for a given route of administration, for the treatment of any human disease or condition. For example, a low dose of a glucose lowering agent in an inhalation dosage form is different from a low dose of the same agent in an oral dosage form.

As used in the present invention, the term high dose refers to a dose that is at least 5% (e.g., at least 10%, 20%, 50%, 100% , 200% or even 300%). Isotopically labeled compounds are also included in the present invention. When used in the present invention, the term isotopically labeled compound refers to a compound described in the present invention, including its pharmaceutical salts and prodrugs described in the present invention, in which one or more atoms are replaced by an atom whose atomic mass or mass number differs from that normally found in nature of atomic mass or mass number. Examples of isotopes that can be included in the compounds described in the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O , ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ Cl, respectively.

The isotopically labeled compounds described herein can be used in drug and/or substrate tissue distribution assays. Tritium ( ³ H) and carbon-14 ( ¹⁴ C) labeled compounds are particularly preferred for ease of preparation and detection. In addition, substitution with heavier isotopes such as deuterium ( ² H) may provide certain therapeutic benefits resulting from greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and thus may be preferred in some conditions. Isotopically labeled compounds described in the present invention, including their pharmaceutical salts, esters and prodrugs, can be obtained using any methods known in the art.

In addition, the replacement of the normally ubiquitous hydrogen ( ¹ H) with heavier isotopes such as deuterium may provide certain therapeutic benefits, for example resulting from improved absorption, distribution, metabolism and/or excretion (APMB) properties, drug production with improved efficacy, safety and/or tolerability parameters. Beneficial effects can also be achieved by replacing the normally ubiquitous ¹² C with ¹³ C (see international application publications WO 2007/005643, WO 2007/005644, WO 2007/016361 and WO 2007/016431).

Stereoisomers (eg cis and trans isomers) and all optical isomers of the compound described herein (eg R and S enantiomers) as well as racemic, diastereomeric and other mixtures of such isomers are within the scope of the present invention.

Compounds of the present invention, once prepared, are preferably isolated and purified to provide a composition containing said compound in an amount equal to or greater than 95% by weight (substantially pure compound), which is then used or formulated into a dosage form as described in the present invention. . According to specific embodiments of the invention, the compounds of the present invention are greater than 99% pure. Also provided are solvates and polymorphs of the compounds of the present invention. Solvates of the compounds of the present invention include, for example, hydrates.

Possible dosage forms include forms suitable for oral, sublingual, buccal, parenteral administration (eg, subcutaneous, intramuscular or intravenous administration), rectal, topical administration, including transdermal administration, intranasal and inhalation administration. The choice of the most appropriate routes of administration for a particular patient depends on the nature and severity of the disease or condition being treated, or the nature of the therapeutic agent used and the nature of the active compound.

- 8 042927

Detailed description of the invention

The present invention provides various novel compositions of berberine in combination with pharmaceutically active organic acids and methods of using them to treat and/or prevent various diseases or disorders.

An important feature of the invention is the unique and synergistic effect brought about by the combination of berberine and selected pharmaceutically active organic acids.

The present invention also provides novel salts of ursodeoxycholic acid and organic bases, pharmaceutical compositions thereof, as well as methods for their preparation and use in the treatment and/or prevention of various diseases or disorders of the liver. Salts of ursodeoxycholic acid include salts with organic bases such as berberine, metformin, carnitine, coptisine, palmatin, yatrorrizine.

The present invention also provides salts of berberine and organic acids, pharmaceutical compositions thereof, as well as methods for their use in the treatment of various diseases or disorders. Berberine salts include salts with organic acids such as R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid, cholic acid, obeticcholic acid, ursodeoxycholic acid , oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, Sallylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein, nicotinic acid and biotin.

The main feature of the invention is the unique and synergistic effect that is provided by each of the two parts of the new salts, i.e. a pharmaceutically active cationic moiety and a pharmaceutically active anionic moiety that work together and synergistically to treat a disease or disorder through complementary mechanisms of action and thus provide enhanced efficacy.

Diseases and disorders that can be treated and/or prevented using the compounds, pharmaceutical compositions and methods described in the present invention include diseases and disorders such as diabetes, complications of diabetes, dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, diabetic dyslipidemia, obesity, metabolic syndromes, metabolic disorders preceding diabetes, atherosclerosis, heart disease, neurodegenerative diseases, sarcopenia, muscle atrophy, inflammation, cancer, and liver diseases and conditions such as fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis , cholestatic liver disease, or graft-versus-host liver disease. The compounds of the present invention can also be used to improve liver function in chronic viral and alcoholic liver diseases.

Combinations of berberine or its derivative(s) and pharmaceutically active organic acids

The present invention provides various novel compositions of berberine in combination with pharmaceutically active organic acids and methods of using them to treat various diseases or disorders. The invention thus embodies a unique approach in which berberine is used in synergistic combination with selected pharmaceutically active organic acids.

According to one aspect, the invention generally relates to a composition comprising:

(a) berberine or a derivative or analogue thereof;

(b) one or more pharmaceutically active organic acids; and (c) optionally a pharmaceutically acceptable excipient, carrier or diluent.

The berberine and the pharmaceutically active organic acid(s) are present in an amount which, when administered to a subject, is sufficient to treat, prevent, or ameliorate one or more diseases or disorders selected from metabolic disorders, heart disease, neurodegenerative disease, muscle atrophy, liver disease, inflammation and cancer, or a related disease or disorder in a mammal, including a human.

Berberine (5,6-dihydro-9,10-dimethoxybenzo[d]-1,3-benzodioxolo[5,6-a]quinolisinium), an isoquinoline alkaloid isolated from the rhizome of the Chinese coptis (Rhizoma Coptidis), has a long history of medicinal applications in China for the treatment of various gastrointestinal diseases. Berberine is found in a variety of plants such as barberry, goldenseal, xanthorhiza protozoan, amur barzat, Chinese coptis, tinospora cordifolia, Mexican argemona, and Californian escholcia. Over the past two decades, in vitro and in vivo studies have demonstrated the efficacy of berberine, alone or in combination, for the treatment of diabetes, dyslipidemia, cancer, and cardiovascular disease, as well as for neuroprotection. Currently, berberine is commercially available as chloride, sulfate, or tannate, with almost all previous studies using berberine hydrochloride. The low bioavailability of berberine in

- 9 042927 currently available forms makes it very difficult to use it for the treatment of chronic and systemic diseases.

Berberine

R-(+)-α-Lunoic acid ((R)-6,8-dithiooctanoic acid, (R)-6,8-thioctacid, (R)-(+)-1,2dithiolan-3-pentanoic acid) was identified as a catalytic agent for the oxidative decarboxylation of pyruvate and α-ketoglutarate. In humans, R-(+)-α-lipoic acid exists as part of several multi-enzyme complexes involved in energy production and is an important component of mitochondrial respiratory enzymes. R-(+)-α-lipoic acid is best known for its strong antioxidant properties and has been used to treat diabetic neuropathy, degenerative neuronal diseases, atherosclerosis, and other abnormalities associated with oxidative stress.

7?-(+)-a-Lipoic acid

Hydroxycitric acid (1,2-dihydroxypropane-1,2,3-tricarboxylic acid) is a citric acid derivative found in various tropical plants, including Garcinia cambogia and Hibiscus subdariffa. Hydroxycitric acid is the active ingredient in Garcinia Cambogia extract, which is widely used as a dietary supplement for weight loss. The effects of hydroxycitric acid in improving glucose tolerance, protecting the liver from ethanol and dexamethasone-related toxicity, and controlling blood pressure have been described. In addition, it was found that the specified connection leads to a decrease in inflammatory markers in the brain, intestines, kidneys and serum.

Hydroxycitric acid

Eicosapentaenoic acid (EPA or (5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-eicosapentaenoic acid) and docosahexaenoic acid (DHA, 4Z,7Z,10Z,13Z,16Z,19Z)-docose -4,7,10,13,16,19-hexaenoic acid) are the most studied omega-3 polyunsaturated fatty acids. EPA is the active molecule in two FDA-approved antihypertriglyceridemic agents. It has been shown that EPA and DHA can reduce the synthesis of free fatty acids and triglycerides and increase their utilization. EPA and DHA have also been shown to reduce chronic inflammation, improve insulin resistance, support heart and vascular health, and reduce the risk of coronary heart disease. There are many more omega-3 fatty acids in nature (besides EPA and DHA) with a range of therapeutic benefits, including, but not limited to, docosapentaenoic acid (DPA), α-linolenic acid (ALA), eicosatrienoic acid (ETE). ) etc.

\ .He

EPC

ABOUT

Dtk

- 10 042927

Ursolic acid ,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid) and corosolic acid ((1S,2R,4aS,6aR,6aS,6bR,8aR,10R , 11aR,12aR,14bS)-10,11-dihydroxy-1,2,6a,6b,9,9,12-heptamethyl-2,3,4,5,6,6a,7,8,8a,10, 11, 12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid) are members of the class of pentacyclic triterpene acids, compounds widely distributed in the plant kingdom. They have been shown to exhibit beneficial pharmacological effects both in vivo and in vitro, including glucose lowering, anti-obesity effect, anti-inflammatory effect, muscle atrophy reduction, anti-cancer effect, liver protection, anti-oxidative stress effect.

2¾

Ursolic acid, R= H

Corosolic acid, R=OH

Cinnamic acid, cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, S-allylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein, nicotinic acid, and biotin in purified form or as active extracts (Table 1) are additional organic acids shown to have pharmacological effects in the treatment or prevention of diabetes, diabetic complications, dyslipidemia, obesity, metabolic syndromes, disorders pre-diabetes metabolism, heart disease, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), muscle atrophy, inflammation and cancer.

Examples of pharmaceutically active organic acids are listed in table. 1.

- 11 042927

Table 1

Examples of pharmaceutically active organic acids

Name IUPAC name Structure Cinnamic acid (E)-3-phenylprop-2-enoic acid ABOUT Cholic acid (D)-4- ((37?,55.77?,87?,95, 105,125,13R, 14 5.177?)- 3,7,12-trihydroxy-10,13dimethylhexadecahydro- 177cycle openta[a]phenanthren-17yl)pentanoic acid About | ί N G / G J n T n but' ⁴ v _oh η Obeticholic acid (3a,53,6a,7a)-6-ethyl-3,7dihydroxycholan-24-oic acid o HjC, /on CHA snT n I \ LAJ n HO' x/γ _Ό Η ^x cn ₃ Ursodeoxycholic acid 3 a, 7 3-dihydroxy-5 3-cholan-24oic acid OR (R)-4- ((3R,5S,7S,8R,9S,10S,13R,14S,l 7R)-3,7-dihydroxy- 10,13-dimethylhexadecahydro- 1 H-cyclopenta[a]phenanthren-17yl)pentanoic acid O VyAoh X ⁿ 11/ G yTa n Oleanolic acid (4a5,6a7?,6a5,6b7?,8a7?, 105.12aR, 1 4b5)-10-gi droxy- 2,2,6a, 6b, 9,9,12a-heptamethyl1,3,4,5,6,6a,7,8,8a,10,I,12,13,14 b-tetradecahydropicene-4a- carboxylic acid gAE<,on 1 1 | 1 H J η /ΑΑΨΑ/ o | 1 n J ^{Σ but} ^ -' n

- 12 042927

Salicylic acid 2-hydroxybenzoic acid 0^-0 Η Ι,ΟΗ Betulinic acid (3 β)-3-hydroxylup-20(29)-en28-oic acid X ο Ζ™χ \ χ Δ—/„„„ '---/„„χ \ -4—6"χ ΟX Chlorogenic acid QSARAR,5R\3-{[(2Z)-3-(3Adihydroxyphenyl)prop-2enoyl]oxy}-1,4,5trihydroxycyclohexane carboxylic acid HQ CO ₂ H ΓΊ ⁰ on' 0Η Ή Αχ ΌΗ he caffeic acid 3-(3,4-dihydroxyphenyl)-2propenoic acid 3,4-dihydroxycinnamic acid~trans-caffeate 3,4-dihydroxy-trans-cinnamate (E)-3-(3,4-dihydroxyphenyl)-2propenoic acid 3,4-dihydroxybenzolacrylic acid 3-(3,4-dihydroxyphenyl)-2propenoic acid 0 H.O. JA γ' ήη Bassic acid (4aR, 6bS, 9R, 1 OR, 11S, 12a/?, 14bS)10,11-dihydroxy-9- (hydroxymethyl)-2,2,6b, 9,12apentamethyl- 1,2,3,4,4a, 5,6,6a,6b,7,9,10,11,12, 12a,12b,13,14b- octadecahydropicene-4acarboxylic acid 1 1 m ^soon ^but ^ -ζ. 1 i J HO^^ ^S

- 13 042927

Acetyl-L-carnitine (R)-3-acetyl hydroxy-4trimethylammonium butanoate \ / ζγ \.....Ό^Ο ABOUT, S-allyl-Ecysteine sulfoxide (27?)-2-Amino-3-[(5)-prop-2enylsulfinyl]propanoic acid 0 n „ / -he -2A nh ₂ H ₂ C \u003d / ^X b S-methyl-ecysteine sulfoxide 3-(methylsulfinyl)-E-alanine X O\cm \ X gl --- P O JV X Pantothenic acid 3-[(2,4-dihydroxy-3,3 dimethylbutanoyl)amino]propanoic acid n rn _n / \ / ^{he but} r γ Ά n ₃ with CH ₃ O 0 Ascorbic acid (5R)-[(15)-1,2-digidroxyethyl] 3,4-dihydroxyfuran-2(5R)-one ⁿ 9 n Η0 ^Ζ he Retinoic acid (2E,4E,6E,8E)-3,7-dimethyl-9(2,6,6-trim ethyl ring oxen-1 yl)nona-2,4,6,8-tetraenoic acid H ₃ C CH ₃ CH ₃ CH ₃ 0 Vhj Rhine 4,5-dihydroxy-9,10dioxoanthracene-2-carboxylic acid OH 0 he γ from I g oh he A nicotinic acid pyridine-3-carboxylic acid 0 Biotin 5-[(3a5,45,6aR)-2- oxohexahydro-177-thieno[3,4<7]imidazol-4-yl]pentanoic acid 0 J HN ^/X NH Η Δ - An o

Examples of derivatives or analogues of berberine are listed in table. 2.

- 14 042927

table 2

Derivatives or analogues of berberine

- 15 042927

Ri = OH, carbonyl; R _2j R ₃ = H, carbonyl; η = 2-6; Χ = Ο

Ri = OH, carbonyl; R _2j R3 = H, OH, carbonyl; η = 2 - 6; X=NH

Ri, R ₃ , R ₂ , R ₄ \u003d OH, Ci-Cb -alkoxy, OCH ₂ O

G = Z-Ar, Y- Ar ₂

Z = O(CH ₂ ) _m , CONH(CH ₂ ) _m , NHCO(CH ₂ ) _m

Y = O(CH ₂ ) _m CH, CONH(CH ₂ ) _m CH, NHCO(CH ₂ ) _m CH n = 1-5; m = 1-3; Ar = 5-15 membered unsaturated or aromatic ring

Ri, R ₃ , R ₂ , R ₄ \u003d OH, Ci-Ce -alkoxy, OCH ₂ O

G = Z-Ar, Y-Ar ₂

Z = O(CH ₂ ) _m , CONH(CH ₂ ) _m , NHCO(CH ₂ ) _m

Y = O(CH ₂ ) _t CH, CONH(CH ₂ ) _m CH, NHCO(CH ₂ ) _m CH η = 1-5; m = 1-3; Ar = 5-15 membered unsaturated or aromatic ring

Ri, R ₃ , R ₂ , R ₄ \u003d OH, Ci-Cb -alkoxy, OCH ₂ O

G = Z-Ar, Y-Ar ₂

Z \u003d O (CH ₂ ) _t , CONH (CH ₂ ) _m , NHCO (CH ₂ ) _m

Y = O(CH ₂ ) _t CH, CONH(CH ₂ ) _m CH, NHCO(CH ₂ ) _m CH η = 1-5; w = 1-3; Ar = 5-15 membered unsaturated or aromatic ring

Ri, R ₃ , R ₂ , R ₄ \u003d OH, Ci-Cb -alkoxy, OCH ₂ O

G = Z-Ar, Y-Ar ₂

Z \u003d O (CH ₂ ) _t , CONH (CH ₂ ) _m , NHCO (CH ₂ ) _m

Y = O(CH ₂ ) _t CH, CONH(CH ₂ ) _m CH, NHCO(CH ₂ ) _m CH η = 1-5; w = 1-3; Ar = 5-15 membered unsaturated or aromatic ring

- 16 042927

- 17 042927

- 18 042927

- 19 042927

R,

r.

= glucosyl, mannosyl, maltosyl, lactosyl, lactosyl, fructosyl, xylosyl, arabinosyl

X \u003d C1, Br, I

Ri, R ₂ \u003d H, Cx-Cr-alkoxy, 0CH ₂ 0

R ₂ = Ci-C3-alkyl

R4, R5 \u003d C1-C ₂ -alkoxy

Ri, R ₂ = H, C1-C4 alkoxy, 0CH ₂ 0 R3 = CN, COOR; (Rs = C1-C ₂ -alkyl)

R4, R5 \u003d C1-C ₂ -alkoxy

Ri, R ₂ \u003d H, C1-C4-alkoxy, 0CH ₂ 0

R3 = Ci-C3-alkyl, phenyl

R4, R5 \u003d C1-C ₂ -alkoxy

R.

EN', C(O)R, OR', NR,,, C(O)NR _;i R, , alkyl ,. R„ \u003d H, OH, Cl. Br, F, I, CN, NH _; , (:O)NH,. CO ,H, alkylH/ = H; R _? R,' = O: R. _t = H _r halo, OR', OSO-.R. OC(:O)R. OCO2R OC(O)NR'R, O-alkylene-NR'R, O-alkylene-OSO R. O-alkylene-NR'SO.-R'. 0 lmpen-NH C>.>R lmp; R5, Rs = halogen, OH, alkoxy

R ₍ R,- \u003d OCH O, R _t R \u003d OCH-O: R ₇ \u003d H. OH, halogen, alkyl or alkoxy

R,, R, I = H, CO R, alkyl

- 20 042927

- 21 042927

PS.N _Ch ο-χ-γ

X = (CH y. (CHA,CO; η = 2-10; m = 1-9

Y=NR. Ar. OAr: Ar - substituted aryl ' A R, = H, Me. Et Pr, i-Pr; Z = F.Cl. Br. I

R = 2-acetic acid methyl ester, 3-acetic acid methyl ester, 4-acetic acid methyl ester; 2-acetic acid methyl ethyl ester, 3-acetic acid methyl ethyl ester, 4-acetic acid methyl ethyl ester, 2-acetate, 3-acetate, 4-acetate, 2-sodium acetate, 3-potassium acetate, 4-potassium acetate; η = 2-6

According to specific embodiments of the invention, the pharmaceutically active organic acid(s) is one or more agents selected by a person skilled in the art from the group consisting of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid , docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid, cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-p-carnitine, S-allylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein, nicotinic acid, biotin and other organic acid generally recognized as pharmaceutically active for the treatment of one or more diseases or disorders selected from metabolic disorders, heart disease, neurodegenerative diseases, liver diseases, muscle atrophy , inflammation and cancer, or a related disease or disorder in a mammal, including a human.

According to specific embodiments of the invention, the composition further comprises one or more additional agent(s) selected from the group consisting of vitamin D, vitamin C, vitamin E, vitamin B12, vitamin A, benfotiamine, chromium and vanadium picolinate.

In specific embodiments, the disease or disorder is a metabolic disorder and is selected from diabetes, diabetic complications, dyslipidemia, obesity, metabolic syndromes, pre-diabetic metabolic disorders, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) . According to specific embodiments of the invention, the disease or disorder is a heart disease. According to specific embodiments of the invention, the disease or disorder is a neurodegenerative disease. In specific embodiments, the disease or disorder is cancer. In specific embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, gastric carcinoma, colorectal cancer, leukemia, multiple myeloma, melanoma, and glioblastoma. According to specific embodiments of the invention, the disease or disorder is muscular atrophy. According to specific embodiments of the invention, the muscle atrophy disease or disorder is selected from skeletal muscle atrophy.

- 22 042927

According to specific embodiments of the invention, the composition further comprises a pharmaceutically acceptable excipient, carrier or diluent.

According to specific preferred embodiments of the invention, the composition contains berberine and R-(+)-α-lipoic acid. According to specific preferred embodiments of the invention, the composition contains berberine, R-(+)-α-lipoic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, R(+)-a-lipoic acid and vitamin B12. According to specific preferred embodiments of the invention, the composition contains berberine, R-(+)-α-lipoic acid, vitamin B12 and benfotiamine. According to specific preferred embodiments of the invention, the composition contains berberine, R-(+)-α-lipoic acid, vitamin B12, benfotiamine and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and hydroxycitric acid. According to specific preferred embodiments of the invention, the composition contains berberine, hydroxycitric acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, hydroxycitric acid, vitamin D and omega-3 polyunsaturated fatty acids. According to specific preferred embodiments of the invention, the composition contains berberine, extracts from garcinia cambogia or hibiscus sabdariffa (hydroxycitric acid), vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and one or both of EPA and DHA. According to specific preferred embodiments of the invention, the composition contains berberine, one or both of EPA and DHA, and vitamin D.

According to specific preferred embodiments of the invention, the composition contains berberine and ursolic acid. According to specific preferred embodiments of the invention, the composition contains berberine, ursolic acid and/or corosolic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, ursolic acid and/or corosolic acid, vitamin D and omega-3 polyunsaturated fatty acids. According to specific preferred embodiments of the invention, the composition contains berberine, extracts of banaba (corosolic acid), extracts of holy basil or extracts of apple peel (ursolic acid), vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and cinnamic acid. According to specific preferred embodiments of the invention, the composition contains berberine, cinnamic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, cinnamic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition comprises berberine and bile acid(s) (eg cholic acid, obeticholic acid and/or ursodeoxycholic acid). According to specific preferred embodiments of the invention, the composition contains berberine, bile acid(s) (for example, cholic acid, obeticholic acid and/or ursodeoxycholic acid) and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, bile acid(s) (eg cholic acid, obeticholic acid and/or ursodeoxycholic acid), vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and oleanolic acid. According to specific preferred embodiments of the invention, the composition contains berberine, oleanolic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, oleanolic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and salicylic acid. According to specific preferred embodiments of the invention, the composition contains berberine, salicylic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, salicylic acid, vitamin D and omega-3-polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and betulinic acid. According to specific preferred embodiments of the invention, the composition contains berberine, betulinic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, betulinic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and chlorogenic acid. According to specific preferred embodiments

- 23 042927 of the invention, the composition contains berberine, chlorogenic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, chlorogenic acid, vitamin D and omega-3-polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and caffeic acid. According to specific preferred embodiments of the invention, the composition contains berberine, caffeic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, caffeic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and bassic acid. According to specific preferred embodiments of the invention, the composition contains berberine, bassic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, bassic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and acetyl-b-carnitine. According to specific preferred embodiments of the invention, the composition contains berberine, acetyl-L-carnitine and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, acetyl-L-carnitine, vitamin D and omega-3-polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and S-allylcysteine sulfoxide and/or S-methylcysteine sulfoxide. According to specific preferred embodiments of the invention, the composition contains berberine, Sallylcysteine sulfoxide and/or S-methylcysteine sulfoxide and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, Sallylcysteine sulfoxide and/or S-methylcysteine sulfoxide, vitamin D and omega-3 polyunsaturated fatty acids. acids.

According to specific preferred embodiments of the invention, the composition contains berberine and pantothenic acid. According to specific preferred embodiments of the invention, the composition contains berberine, pantothenic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, pantothenic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and ascorbic acid. According to specific preferred embodiments of the invention, the composition contains berberine, ascorbic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, ascorbic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and retinoic acid. According to specific preferred embodiments of the invention, the composition contains berberine, retinoic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, retinoic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and rhein. According to specific preferred embodiments of the invention, the composition contains berberine, rhein and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, rhein, vitamin D and omega-3 polyunsaturated fatty acids. According to specific preferred embodiments of the invention, the composition contains berberine, extracts of rhubarb (rhein), vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and nicotinic acid. According to specific preferred embodiments of the invention, the composition contains berberine, nicotinic acid and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, nicotinic acid, vitamin D and omega-3 polyunsaturated fatty acids.

According to specific preferred embodiments of the invention, the composition contains berberine and biotin. According to specific preferred embodiments of the invention, the composition contains berberine, biotin and vitamin D. According to specific preferred embodiments of the invention, the composition contains berberine, biotin, vitamin D and omega-3 polyunsaturated fatty acids. According to another aspect, the invention generally relates to methods for treating, alleviating or preventing metabolic disorders. The method includes administering to a subject in need thereof a pharmaceutical composition that contains:

(a) berberine or a derivative or analogue thereof;

(b) one or more pharmaceutically active organic acids in a therapeutically effective amount; and (c) optionally a pharmaceutically acceptable excipient, carrier or diluent.

- 24 042927

According to specific embodiments of the invention, metabolic disorders are selected from diabetes, complications of diabetes, dyslipidemia, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, obesity, metabolic syndromes, metabolic disorders preceding diabetes, fatty liver, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

According to specific preferred embodiments of the invention, the metabolic disorder is type 2 diabetes.

According to specific preferred embodiments of the invention, the complication of diabetes is diabetic neuropathy, diabetic retinopathy or diabetic nephropathy.

According to specific preferred embodiments of the invention, hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or hypercholesterolemia and hypertriglyceridemia.

According to specific preferred embodiments of the invention, the pharmaceutically active organic acid is selected from the group consisting of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid, cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, Sallylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein, nicotinic acid acids and biotin. According to specific embodiments of the invention, the pharmaceutical composition further comprises a third agent selected from the group consisting of vitamin D, vitamin C, vitamin E, vitamin B12, vitamin A, benfotiamine, chromium and vanadium picolinate.

According to specific preferred embodiments of the method, the subject suffers from diabetes and complications of diabetes, and the pharmaceutical composition contains berberine and R-(+)-α-lipoic acid. According to specific preferred embodiments of the method, the subject suffers from diabetic nephropathy and the pharmaceutical composition contains berberine and rhein (or rhubarb extracts). In specific preferred embodiments of the method, the subject is diabetic and obese and the pharmaceutical composition contains berberine and hydroxycitric acid (or garcinia cambogia extracts). According to specific preferred embodiments of the method, the subject suffers from diabetes and dyslipidemia, and the pharmaceutical composition contains berberine and one or more of EPA, DHA and DPA.

According to specific preferred embodiments of the method, the subject suffers from diabetes and muscle wasting, and the pharmaceutical composition contains berberine and one or both of ursolic acid and corosolic acid. In specific preferred embodiments of the method, the subject suffers from diabetes and muscle atrophy and the pharmaceutical composition comprises berberine and one or both of holy basil extract or apple peel extract (ursolic acid) and banaba extract (corosolic acid).

According to specific preferred embodiments of the method, the subject suffers from fatty liver, non-alcoholic fatty liver disease (NAFLD) and NASH, and the pharmaceutical composition contains berberine and one or more of cholic acid, obeticholic acid and ursodeoxycholic acid. According to specific preferred embodiments of the method, the subject suffers from fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and NASH, and the pharmaceutical composition contains berberine and bile acids.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises vitamin D.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises vitamin E.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises vitamin B12.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises benfotiamine.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises vitamin C.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises vitamin A.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises benfotiamine.

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises chromium picolinate.

- 25 042927

According to specific preferred embodiments of the method, the pharmaceutical composition further comprises vanadium.

According to specific preferred embodiments of the method, the treatment, amelioration or prevention of metabolic disorders is provided by lowering the blood glucose level of the subject.

According to specific preferred embodiments of the method, the treatment, amelioration or prevention of metabolic disorders is provided by reducing the total cholesterol (TC), triglycerides (TG) and low density lipoprotein cholesterol (LDL cholesterol), increasing the level of high density lipoprotein cholesterol (HDL cholesterol) in the subject . According to specific preferred embodiments of the method, the treatment, amelioration or prevention of a metabolic disorder is provided by normalizing the level of liver enzymes in the subject. According to specific preferred embodiments of the method, the treatment, amelioration, or prevention of a metabolic disorder is provided by altering the insulin signaling pathway, which results in a decrease in glucose levels. According to specific preferred embodiments of the method, the treatment, amelioration or prevention of a metabolic disorder is provided by regulation of multiple metabolic pathways, for example, by increasing insulin secretion, increasing insulin sensitivity, reducing hepatic gluconeogenesis, reducing glucose absorption, reducing dyslipidemia, anti-inflammatory action, to achieve desired pharmacological effects.

According to another aspect, the invention generally relates to a kit that includes:

(i) an agent that is berberine or a derivative or analogue thereof;

(ii) one (or more) agent(s) selected from pharmaceutically active organic acids; and (iii) instructions for administering the combination of agents to a patient suffering from or at risk of developing one or more diseases or disorders selected from metabolic disorders, heart disease, neurodegenerative disease, liver disease, muscle wasting, and cancer.

According to specific embodiments of the invention, a derivative or analogue of berberine is selected from the table. 2. According to specific embodiments of the invention, the additional agent is selected from any one or more of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid, corosolic acid, cholic, ursodeoxycholic acid or other agents listed in the table. 1.

Salts of ursodeoxycholic acid or its derivatives

The present invention also provides novel salts of ursodeoxycholic acid and their organic bases, pharmaceutical compositions, and methods for their preparation and use in the treatment and/or prevention of various diseases or disorders of the liver and metabolic disorders. Salts of ursodeoxycholic acid include salts with organic bases such as berberine, metformin, carnitine, coptisine, palmatin, yatrorrizine.

According to another aspect, the present invention relates generally to an acid-base addition salt in substantially pure form, having the following formula:

(X ⁺ ) _m (U-)n (I), where (a) U ^- represents an anionic fragment of ursodeoxycholic acid or its derivative or analogue;

(b) X+ is a cationic fragment of a pharmaceutically active organic base; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained.

U ^- may be an anionic fragment of any suitable derivative or analogue of ursodeoxycholic acid, for example, selected from the table. 3.

X ⁺ may be a cationic fragment of any suitable pharmaceutically active organic base. According to specific embodiments of the invention, the pharmaceutically active organic base may be selected, for example, from the group consisting of berberine, metformin, carnitine and coptisine, palmatin and jatrorrizine. According to specific embodiments of the invention, X ⁺ may also be a cationic fragment of another organic base, generally recognized as pharmaceutically active for the treatment of one or more diseases or disorders selected from various diseases or disorders of the liver, such as fatty liver, non-alcoholic fatty liver disease (NAFLD) , non-alcoholic steatohepatitis (NASH), cholestatic liver disease, graft-vs. or a related disease or disorder in a mammal, including a human.

- 26 042927

ABOUT

Ursodeoxycholic acid

Ursodeoxycholic acid (UDCA or ursodiol, with the chemical names 3a,7b-dihydroxy5b-cholan-24-oic acid or (R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)- 3,7-dihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoic acid) is a secondary bile acid, a substance that is normally produced in the body and stored in the gallbladder. Ursodiol is used to dissolve gallstones in patients as an alternative to surgery. Ursodiol is also used to prevent the formation of gallstones in overweight patients who lose weight too quickly. Ursodiol works by reducing cholesterol production and by dissolving cholesterol in bile so that it cannot form stones. Ursodiol is also a first-line therapy for the treatment of primary biliary cirrhosis, primary sclerosing cholangitis, and cholestatic liver disease. There have been a limited number of studies on the effects of ursodiol on non-alcoholic steatohepatitis (NASH), but the results have been inconsistent and inconclusive. Thus, the effect of ursodiol on non-alcoholic steatohepatitis (NASH) remains unexplored.

Metformin (R,R-dimethylimidodicarbonimide diamide) is an active hypoglycemic agent currently recommended as first-line oral therapy for the treatment of type 2 diabetes (D2T). The main effect of this drug is a sharp decrease in glucose production in the liver, mainly due to moderate and temporary inhibition of complex 1 of the mitochondrial respiratory chain. In addition, the achieved decrease in the energy status of the liver leads to the activation of AMP-activated protein kinase (AMPK), a cell metabolism sensor that provides the generally recognized mechanism of action of metformin on the program of gluconeogenesis in the liver. In addition to the effect of metformin on glucose metabolism, metformin has been described to restore ovarian function in polycystic ovary syndrome, reduce fatty liver and micro- and macrovascular complications associated with type 2 diabetes. It has also recently been proposed as an adjuvant therapy for cancer or gestational diabetes, as well as for the prevention of diabetes in a population with a predisposition to diabetes. Over the past few years, there has been an increase in research on metformin in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), but its efficacy in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) remains unconfirmed.

NH NH

Metformin

Coptisine [6,7-dihydro-bis(1,3)benzodioxolo(5,6-a:4',5'-g)quinolisinium], palmatin [2,3,9,10tetramethoxy-5,6-dihydroisoquinoline[2 ,1-b]isoquinoline-7-th] and yatrorrizine [2,9,10-trimethoxy-5,6dihydroisoquinoline[2,1-b]isoquinoline-7-th-3-ol] are natural alkaloids for which previous studies have shown similar pharmacological properties to berberine.

- 27 042927

L-Carnitine is a natural amino acid. Its biosynthesis occurs in the liver and kidneys from lysine and methionine. L-Carnitine plays an important role in fat metabolism by acting as a carrier of fatty acids into the mitochondria.

L-Carnitine

Examples of derivatives or analogues of ursodeoxycholic acid are listed in table. 3.

Table 3

Derivatives or analogues of ursodeoxycholic acid

- 28 042927

04: - (II. < 11.- PI < 11,( (1 AND ( H,- 1 12 -II (H;<<"- (II,- 013 ( II,( (I < 11.( o <11, I 1- PM()- " ( AND,- I H. ry o- WSJ (II,- eleven ire eft < ufo,- AND (II- 1 IS (H.M),- I 1130.- telly' t2" <II><>- I n,( o < I,-

» w < 11,- i ¹ < II < II, I I n fill-

- 29 042927

- 30 042927

According to specific preferred embodiments of the invention, U-is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of berberine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of berberine are listed in table. 2. According to specific preferred embodiments of the invention, U is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of berberine, m=1, and n=1.

According to specific preferred embodiments of the invention, U- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of metformin or its derivative or analogue, m=1, and n=1.

Examples of derivatives or analogues of metformin are listed in table. 4. According to specific preferred embodiments of the invention, U ^- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of metformin, m=1, and n=1.

According to specific preferred embodiments of the invention, U-is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of carnitine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of carnitine are listed in table. 5. According to specific preferred embodiments of the invention, U is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of carnitine, m=1, and n=1.

According to specific preferred embodiments of the invention, U- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of coptisin or its derivative or analogue, m=1, and n=1. According to specific preferred options

- 31 042927 implementation of the invention, U-is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of palmatin or its derivative or analogue, m=1, and n=1. According to specific preferred embodiments of the invention, U- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of jatrorrhizin or its derivative or analogue, m=1, and n=1. According to specific preferred embodiments of the invention, U- is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of berberine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of berberine are listed in table. 2. According to specific preferred embodiments of the invention, U- is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of berberine, m=1, and n=1.

According to specific preferred embodiments of the invention, U- is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of metformin or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of metformin are listed in table. 4. According to specific preferred embodiments of the invention, U- is an anionic moiety of obeticholic acid, X ⁺ is a cationic moiety of metformin, m=1, and n=1.

According to specific preferred embodiments of the invention, U' is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of carnitine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of carnitine are listed in table. 5. According to specific preferred embodiments of the invention, U- is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of carnitine, m=1, and n=1.

According to specific preferred embodiments of the invention, U' is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of coptisin or its derivative or analogue, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of palmatin or its derivative or analogue, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of jatrorrizin or its derivative or analogue, m=1, and n=1.

Table 4 Metformin derivatives or analogues

NH NH

NH NH

L ¹ and L ² independently represent a bond or -NH-C(NH)-;

R ¹ is -NR ^1A R ^1B , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where R ^1a u R ^1b are optionally combined together with the formation of a substituted or unsubstituted heterocycloalkyl;

R ² is -NR ^2A R ^2B , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where R ^2A and R ^2B are optionally combined together with the formation of a substituted or unsubstituted heterocycloalkyl;

R ^1A , R ^1b , R ^2A , and R ^2b are independently hydrogen, -OR ⁴ , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl ;

R ³ is hydrogen or unsubstituted C ₁ -C ₅ alkyl; And

R ⁴ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted hete

- 33 042927

Formula 1

where A is selected from the group consisting of a single bond, 'Of, or iCH2i;

m and n vary independently and are integers from 1 to 15;

p and q vary independently from 0 to 1;

B is iCR3R4;

D is selected from the group consisting of iCOZRs, ADR6, ADCOR7, iSO3R8, iSO2NH2, iOPO(OR9)(OR10), A)PO(OR9)(NH2), iOPO(OR9)i

OiPO(OR10)(OR11), where R1 - R4 are independently selected from C ₁ -C ₆ -alkyl; And

R5-R11 are independently selected from the group consisting of hydrogen, C1- _C6 alkyl, _C3 - _C6 cycloalkyl, _C2 - _C6 alkenyl, _C6 alkynyl, _C5 - _C10 aryl, whether or not containing as a substituent C1-C ₆ -alkyl, hydroxyl, C1-C ₆ -alkoxy, 1,3-dioxolanyl, cyano, halogen, nitro, trihaloalkyl, carboxyl, C1-C ₆ -acyl, C1-C ₆ -hydroxyalkyl, amino, C1- _C6 alkylamino, C1-C6 dialkylamino, C1- _C6 acylamino, C1- _C6 alkoxylcarbonyl; C ₅ -C ₆ -aryllyl, containing or not containing as a substituent C1-C ₆ -alkyl, hydroxyl, C1-C ₆ -alkoxy, 1,3-dioxolanyl, cyano, halogen, trihaloalkyl, carboxyl, C1-C ₆ - acyl, C1- _C6 hydroxyalkyl, amino, C1-C6 alkylamino, C1- _C6 dialkylamino, C1- _C6 alkoxylcarbonyl; C1- _C6 carboxyalkyl; C1- _C6 -acylamino; C1- _C6 sulfonatoalkyl; C1- _C6 sulfamilalkyl and C1- _C6 phosphonatoalkyl.

where X is an integer between about 0 and 5

- 34 042927

According to another aspect, the invention generally relates to a pharmaceutical composition containing an acid-base addition salt having the following formula:

(X ⁺ ) _m (U-)n (I), where (a) U ^- represents an anionic fragment of ursodeoxycholic acid or its derivative or analogue;

(b) X ⁺ is a cationic fragment of a pharmaceutically active organic base; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained in an amount effective to treat, prevent, or ameliorate one or more diseases. or disorders selected from fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), cholestatic liver disease, graft-versus-host liver disease, chronic viral liver disease, alcoholic liver disease, metabolic diseases or disorders such as metabolic disorders pre-diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, obesity, or related diseases or disorders in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier, or diluent.

According to specific preferred embodiments of the invention, the pharmaceutical composition of the invention is used to treat, prevent, or alleviate non-alcoholic steatohepatitis (NASH). According to specific preferred embodiments of the invention, the pharmaceutical composition of the invention is used to treat, prevent, or alleviate non-alcoholic fatty liver disease (NAFLD). According to specific preferred embodiments of the invention, the pharmaceutical composition of the invention is used to treat, prevent, or alleviate hepatic steatosis. According to specific preferred embodiments of the invention, the pharmaceutical composition of the invention is used to treat, prevent, or ameliorate a disease or disorder selected from cholestatic liver disease, graft-versus-host liver disease, chronic viral liver disease, alcoholic liver disease, metabolic disease or disorder, such as metabolic disorders preceding diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia or obesity. In the context of the pharmaceutical composition according to the invention, U ^- may be an anionic fragment of any suitable derivative or analogue of ursodeoxycholic acid, for example, selected from the table. 3. X ⁺ may be a cationic fragment of any suitable pharmaceutically active organic bases. According to specific embodiments of

- 35 042927 shave, the pharmaceutically active organic base may be selected, for example, from the group consisting of berberine, metformin, carnitine, coptisine, palmatin and jatrorrizine. According to specific embodiments of the invention, X ⁺ may also be a cationic fragment of other organic bases, generally recognized as pharmaceutically active for the treatment of one or more diseases or disorders selected from fatty liver, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cholestatic liver disease or graft-versus-host liver disease, chronic viral liver disease, alcoholic liver disease, metabolic diseases or disorders such as metabolic disorders preceding diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, obesity, or associated them diseases or disorders in a mammal, including humans. In the context of a pharmaceutical composition according to the invention, according to specific preferred embodiments of the invention, U- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of berberine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of berberine are listed in table. 2. According to specific preferred embodiments of the invention, U- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of berberine, m=1, and n=1. According to specific preferred embodiments of the invention, U- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of metformin or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of metformin are listed in table. 4. According to specific preferred embodiments of the invention, U- is an anionic fragment of ursodeoxycholic acid, X ⁺ is a cationic fragment of metformin, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic moiety of ursodeoxycholic acid, X ⁺ is a cationic moiety of coptisine, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic moiety of ursodeoxycholic acid, X ⁺ is a cationic moiety of palmatin, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic moiety of ursodeoxycholic acid, X ⁺ is a cationic moiety of jatrorrhizin, m=1, and n=1.

In the context of a pharmaceutical composition according to the invention, in accordance with specific preferred embodiments of the invention, U' is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of berberine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of berberine are listed in table. 2. According to specific preferred embodiments of the invention, U' is an anionic moiety of obeticholic acid, X ⁺ is a cationic moiety of berberine, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of metformin or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of metformin are listed in table. 4. According to specific preferred embodiments of the invention, U' is an anionic moiety of obeticholic acid, X ⁺ is a cationic moiety of metformin, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic moiety of obeticholic acid, X ⁺ is a cationic moiety of coptisine, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic moiety of obeticholic acid, X ⁺ is a cationic moiety of palmatin, m=1, and n=1. According to specific preferred embodiments of the invention, U' is an anionic fragment of obeticholic acid, X ⁺ is a cationic fragment of jatrorrizin, m=1, and n=1.

According to specific preferred embodiments of the invention, the pharmaceutical composition further comprises a compound selected from the group consisting of vitamin E, omega-3 fatty acids, S-adenosylmethionine, N-acetylcysteine, silymarin, polyenylphosphatidylcholine, resferatrol, or vitamin D.

According to another aspect, the invention relates generally to a method for treating, alleviating, or preventing a disease or disorder. The method includes administering to a subject in need thereof a pharmaceutical composition containing an acid-base addition salt having the following formula:

(X*)n.(U-)n (I), where (a) U' is an anionic moiety of ursodeoxycholic acid or a derivative or analog thereof;

(b) X ⁺ is a cationic fragment of a pharmaceutically active organic base; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6 such that

- 36 042927 to obtain a salt with a neutral charge, in an amount effective for the treatment, prevention or amelioration of one or more diseases or disorders selected from fatty liver, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cholestatic liver diseases, graft-versus-host liver disease, chronic viral liver disease, alcoholic liver disease, metabolic diseases or disorders such as metabolic disorders preceding diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, obesity, or a related disease, or disorders in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier or diluent.

According to specific preferred embodiments of the invention, the method is a method of treating, preventing or alleviating non-alcoholic steatohepatitis (NASH). According to specific preferred embodiments of the invention, the method is a method of treating, preventing or alleviating non-alcoholic fatty liver disease (NAFLD). According to specific preferred embodiments of the invention, the method is a method of treating, preventing or alleviating fatty liver. According to specific preferred embodiments of the invention, the method is a method of treating, preventing or attenuating a disease or disorder selected from cholestatic liver diseases, graft-versus-host liver disease, chronic viral liver diseases, alcoholic liver diseases, metabolic diseases or disorders such as metabolic disorders pre-diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hyperlipidemia, obesity, or an associated disease or disorder.

In the context of the method according to the invention, U^- may be an anionic fragment of any suitable derivative or analogue of ursodeoxycholic acid, for example, selected from the table. 3. X⁺ may be a cationic fragment of any suitable pharmaceutically active organic bases. According to specific embodiments of the invention, the pharmaceutically active organic base may be selected, for example, from the group consisting of berberine, metformin, carnitine and coptisine, palmatin, and jatrorrizine. According to specific embodiments of the invention, X⁺ may also be a cationic fragment of other organic bases generally recognized as pharmaceutically active for the treatment of one or more diseases or disorders selected from fatty liver, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), cholestatic liver disease, graft-versus-host liver disease , chronic viral liver diseases, alcoholic liver diseases, metabolic diseases or disorders such as metabolic disorders preceding diabetes, diabetes, diabetic dyslipidemia, dyslipidemia in statin intolerant patients, hyperlipidemia, obesity, or a related disease or disorder in a mammal, including person. In the context of the method according to the invention, according to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of berberine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of berberine are listed in table. 2. According to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of berberine, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of metformin or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of metformin are listed in table. 4. According to specific preferred embodiments of the invention, U is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of metformin, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of carnitine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of carnitine are listed in table. 5. According to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺is a cationic fragment of carnitine, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic moiety of coptisine, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of palmatin, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of ursodeoxycholic acid, X⁺ is a cationic fragment of jatrorrizin, m=1, and n=1. According to specific preferred embodiments of the invention, the pharmaceutical composition further comprises a compound selected from the group consisting of vitamin E, omega-3 fatty acids, S-adenosylmethionine, N-acetylcysteine, silymarin, polyenylphosphatidylcholine, resferatrol, or vitamin D. According to specific preferred embodiments implementation of the invention, the treatment, amelioration, or prevention of the disease or disorder is provided by normalizing the level of liver enzymes in the subject.

In the context of the method according to the invention, according to specific preferred embodiments of the invention, U- is an anionic moiety of obeticholic acid, X⁺ is a cationic fragment of berberine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of berberine are listed in table. 2. According to specific preferred embodiments of the invention, U^- is an anionic fragment of obeticholic acid, X⁺ is a cationic fragment of berberine, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of obeticholic acid, X⁺ is a cationic fragment of metformin or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of metformin are listed in table. 4. According to specific preferred embodiments of the invention, U^- is an anionic fragment of obeticholic acid, X⁺ is a cationic fragment of metformin, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of obeticholic acid, X⁺ is a cationic fragment of carnitine or its derivative or analogue, m=1, and n=1. Examples of derivatives or analogues of carnitine are listed in table. 5. According to specific preferred embodiments of the invention, U^- is an anionic fragment of obeticholic acid, X⁺ is a cationic fragment of carnitine, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of obeticholic acid, X⁺ is a cationic moiety of coptisine, m=1, and n=1. According to specific preferred embodiments of the invention, U^- is an anionic fragment of obeticholic acid, X⁺ is a cationic fragment of palmatin, m=1, and n=1. According to specific preferred embodiments of the invention, U is an anionic fragment of obeticholic acid, X⁺ is a cationic fragment of jatrorrizin, m=1, and n=1. According to specific preferred embodiments of the invention, the pharmaceutical composition further comprises a compound selected from the group consisting of vitamin E, omega-3 fatty acids, S-adenosylmethionine, N-acetylcysteine, silymarin, polyenylphosphatidylcholine, resferatrol, or vitamin D. According to specific preferred embodiments implementation of the invention, the treatment, mitigation or prevention of the disease or disorder is provided by the normalization of the level of liver enzymes in the subject.

Berberine salts or their derivatives

The present invention also provides salts of berberine and organic acids, their pharmaceutical compositions, as well as methods of their use for the treatment of various diseases or disorders.

Berberine salts include salts with organic acids such as R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, ursolic acid, corosolic acid, cinnamic acid, cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid , salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, S-allylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, nicotinic acid and biotin.

According to another aspect, the invention generally relates to an acid-base addition salt in substantially pure form having the following formula:

where (a) B ⁺ is a cationic fragment of berberine or a derivative or analogue;

(b) Y ^- is an anionic moiety of a pharmaceutically active organic acid; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 such that a charge-neutral salt is obtained.

In specific embodiments of the acid-base addition salt, the berberine derivative or analog is selected from Table 1. 2.

In particular embodiments of the acid-base addition salt, the pharmaceutically active organic acids are selected from the group consisting of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid, cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, Sallylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, nicotinic acid, biotin, and another organic acid generally recognized by those skilled in the art as pharmaceutically active for the treatment of one or more diseases or disorders selected from metabolic disorders, heart disease, atherosclerosis, neurodegenerative diseases, liver disease, sarcopenia, muscle atrophy , inflammation and cancer, or a related disease or disorder in a mammal, including a human.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of R-(+)-α-lipoic acid, and m=1, and n=1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of hydroxycitric acid, and m=1, and n=1, or m=2, n=1, or m=3, n= 1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of EPA, and m=1 and n= 1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of DHA, and m=1, and n=1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of duodenum, and m=1, and n=1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of ursolic acid, and m=1, and n=1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine. and Y ^- is an anionic moiety of corosolic acid, and m=1 and n=1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of cholic acid, and m=1, and n=1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of ursodeoxycholic acid, and m=1, and n=1.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and Y ^- is an anionic fragment of obeticholic acid, and m=1, and n=1.

According to another aspect, the invention generally relates to a pharmaceutical composition containing an amount of an acid-base addition salt having the following formula:

(B.W.). (ii).

where (a) B ⁺ is a cationic fragment of berberine or its derivative or analogue;

(b) Y ^- is an anionic moiety of a pharmaceutically active organic acid; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 so as to produce a charge-neutral salt effective for treating, preventing, or ameliorating one or more diseases or disorders, selected from metabolic disorders, heart disease, atherosclerosis, neurodegenerative diseases, liver disease, sarcopenia, muscle wasting, inflammation and cancer, or a related disease or disorder in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier or diluent.

According to specific embodiments of the invention, the disease or disorder is a metabolic disorder which is selected from diabetes, complications of diabetes, dyslipidemia, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, hypercholesterolemia, hypertriglyceridemia, metabolic syndromes, and metabolic disorders preceding diabetes. According to specific embodiments of the invention, the metabolic disorder is type 1 or type 2 diabetes.

In specific embodiments, the disease or disorder is cancer. In specific embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, gastric carcinoma, colorectal cancer, leukemia, multiple myeloma, melanoma, and glioblastoma.

According to specific embodiments of the invention, the disease or disorder is a heart disease.

According to specific embodiments of the invention, the disease or disorder is atherosclerosis.

According to specific embodiments of the invention, the disease or disorder is sarcopenia.

According to specific embodiments of the invention, the disease or disorder is muscular atrophy.

According to specific embodiments of the invention, the disease or disorder is muscle atrophy, which is selected from skeletal muscle atrophy.

According to specific embodiments of the pharmaceutical composition, the berberine derivative or analog is selected from Table 1. 2.

According to specific embodiments of the pharmaceutical composition, the pharmaceutically active organic acid is selected from the group consisting of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid, and corosolic acid, cinnamic acid, cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, Sallylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid , nicotinic acid, biotin, and another organic acid generally recognized by those skilled in the art as pharmaceutically active for the treatment of one or more diseases or disorders selected from metabolic disorders, heart disease, atherosclerosis, neurodegenerative diseases, liver disease, sarcopenia, muscle atrophy, inflammation, and cancer, or a related disease or disorder in a mammal, including a human.

According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and X ^- is an anionic fragment of R-(+)-α-lipoic acid, and m=1, and n=1. According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and X ^- is an anionic fragment of hydroxycitric acid, and m=1, and n=1, or m=2, n=1, or m=3, n= 1. According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and X ^- is an anionic fragment of EPA, and m=1, and n=1. According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and X ^- is an anionic fragment of DHA, and m=1, and n=1. According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and X ^- is an anionic fragment of duodenum, and m=1, and n=1. According to specific embodiments of the invention, B ⁺ is a cationic fragment of berberine, and X ^- is an anionic fragment of ursolic acid, and m=1, and n=1.

According to another aspect, the invention relates generally to a method for treating, alleviating, or preventing a disease or disorder. The method includes administering to a subject in need thereof a pharmaceutical composition containing an amount of an acid-base addition salt having the following formula:

(BW)" (II).

where (a) B ⁺ is a cationic fragment of berberine or its derivative or analogue;

(b) Y ^- is an anionic moiety of a pharmaceutically active organic acid; and (c) m and n are integers independently selected from 1, 2, 3, 4, 5, and 6 so as to produce a charge-neutral salt effective for treating, preventing, or ameliorating one or more diseases or disorders, selected from metabolic disorders, heart disease, atherosclerosis, neurodegenerative diseases, liver disease, sarcopenia, muscle wasting, inflammation and cancer, or a related disease or disorder in a mammal, including a human, and a pharmaceutically acceptable excipient, carrier or diluent.

According to specific embodiments of the invention, the metabolic disorder is selected from diabetes, complications of diabetes, dyslipidemia, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, obesity, metabolic syndromes, metabolic disorders preceding diabetes, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). According to specific embodiments of the invention, the metabolic disorder is type 1 or type 2 diabetes. According to specific embodiments of the invention, a derivative or analogue of berberine is selected from the table. 2.

According to specific embodiments of the invention, the pharmaceutically active organic acid is selected from the group consisting of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid and corosolic acid, cinnamic acid, cholic acid , obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetyl-L-carnitine, S-allylcysteine sulfoxide, S-methylcysteine sulfoxide, pantothenic acid, ascorbic acid, retinoic acid, nicotinic acid, biotin and another organic acid generally recognized by those skilled in the art as pharmaceutically active for the treatment of one or more diseases or disorders selected from metabolic disorders, heart disease, atherosclerosis, neurodegenerative diseases, liver disease, sarcopenia, muscle atrophy, inflammation, and cancer, or associated with them diseases or disorders in a mammal, including humans.

In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y- is an anionic moiety of R-(+)-α-lipoic acid, and m=1 and n=1 and the subject has diabetes and a complication of diabetes.

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In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y- is an anionic moiety of hydroxycitric acid, and m=1 and n=1, or m=2, n=1, or m=3, n= 1 and the subject is diabetic and obese.

In specific embodiments of the method, B ⁺ is a cationic berberine fragment and Y ^- is an anionic fragment of EPA, and m=1 and n=1, and the subject suffers from diabetes and dyslipidemia or heart disease, atherosclerosis, or neurodegenerative diseases.

In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y ⁻ is an anionic moiety of DHA, and m=1 and n=1, and the subject suffers from diabetes and dyslipidemia or heart disease, atherosclerosis, or neurodegenerative diseases.

In specific embodiments of the method, B ⁺ is a cationic berberine fragment and Y ^- is an anionic fragment of duodenum, and m=1 and n=1, and the subject suffers from diabetes and dyslipidemia or heart disease, atherosclerosis, or neurodegenerative diseases.

In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y ⁻ is an anionic ursolic acid moiety, and m=1 and n=1, and the subject suffers from diabetes and sarcopenia or muscle atrophy.

In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y ⁻ is an anionic corosolic acid moiety, and m=1 and n=1, and the subject suffers from diabetes and sarcopenia or muscle atrophy.

In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y ⁻ is an anionic cholic acid moiety, and m=1 and n=1, and the subject suffers from dyslipidemia, fatty liver, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y ⁻ is an anionic moiety of obeticholic acid, and m=1 and n=1, and the subject suffers from dyslipidemia, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

In specific embodiments of the method, B ⁺ is a cationic berberine moiety and Y ⁻ is an anionic moiety of ursodeoxycholic acid, and m=1 and n=1, and the subject suffers from dyslipidemia, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

According to specific preferred embodiments of the method, the treatment, amelioration or prevention of a metabolic disorder is provided by lowering the blood glucose level of the subject. According to specific preferred embodiments of the method, the treatment, amelioration or prevention of a metabolic disorder is provided by reducing the total cholesterol (TC), triglycerides (TG) and low density lipoprotein cholesterol (LDL cholesterol), increasing the level of high density lipoprotein cholesterol (HDL cholesterol) in the subject . According to specific preferred embodiments of the method, the treatment, amelioration or prevention of a metabolic disorder is achieved by normalizing the level of liver enzymes in the subject. According to specific preferred embodiments of the method, the treatment, amelioration or prevention of a metabolic disorder is provided by normalizing the level of liver lipids in the subject. According to specific preferred embodiments of the method, the treatment, amelioration or prevention of a metabolic disorder is achieved by altering the insulin signaling pathway to reduce glucose levels. According to specific preferred embodiments of the method, treatment, amelioration or prevention of a metabolic disorder is provided by regulation of multiple metabolic pathways, for example, increasing insulin secretion, improving insulin sensitivity, reducing hepatic gluconeogenesis, reducing glucose absorption, attenuating dyslipidemia, anti-inflammatory action to achieve the desired pharmacological effects.

The following examples are given as an illustration of the practical implementation of the invention and in no way limit the present invention.

Examples

Example 1 Efficacy of a combination of berberine and epcosapentaenoic acid (EPA), docosahexaenoic acid (DHA); or berberine and ursolic acid (UA) in a mouse model of high fat diet/streptozocin-induced diabetes.

This example describes an in vivo efficacy study of the combination of the present invention in a mouse model of high-fat diet-induced diabetes (HFA) and streptozotocin (STZ).

Sixty male NIH mice four weeks old were purchased from Guangzhou Institute of Laboratory Animal. After acclimatization for a week, five mice were selected as a normal control group (Group 1), the remaining 55 mice were given a single dose of STZ at 40 mg/kg and provided with DAF (40% of calories are fat) for 7 days to establish an animal model of diabetes. similar to the pathophysiology of type 2 diabetes in humans. Mice of the control group were not injected with STZ and were provided with a normal diet.

Of the 55 mice stimulated with STZ and DEVF, forty animals with fasting blood glucose levels greater than 12.0 mmol/L on the seventh day after STZ administration were selected and randomized into 4 groups (n=10 per group).

Group 2: vehicle control (normal saline).

Group 3: positive control (metformin, 300 mg/kg).

Group 4: combination of berberine (150 mg/kg) and UA (150 mg/kg).

Group 5: combination of berberine (150 mg/kg), EPA (75 mg/kg) and DHA (75 mg/kg).

Mice from groups 2-5 were treated with the respective test articles above once daily by intragastric feeding. DIC was maintained throughout the 28-day treatment. Mice from the normal group (group 1) received a normal saline solution by intragastric feeding. Fasting blood glucose, total cholesterol (TC) and triglyceride (TG) levels, food intake, water intake, and body weight were measured throughout the study.

On the 28th day of treatment, an oral glucose tolerance test (OGTT) was performed in animals fasted for 12 hours. For OGTT, after measuring the initial glucose concentration (T = -30 min), mice underwent an oral glucose load at a concentration of 2.5 g / kg and determined the glucose level using a glucometer (ACCU-CHEK Active, Roche) after 0, 30, 60 and 120 min.

After OGTT, blood samples were collected to measure the level of glucose, TC and TG in the blood. Mice were sacrificed and pancreas, liver, kidney and adipose tissue were collected for histopathological analysis.

The results of the experiment are listed in table. 6 and tab. 7.

Table 6

Mean body weight, food and water intake in different treatment groups*________

Group Body weight (g) Food intake (g) Water consumption (ml) 0 day Day 27 3 day Day 27 3 day Day 6 Day 27 No. 1 Norm 27.80±1.45 (n=5) 34.20±2.84 (n=5) 3.69 (n=5) 4.88 (n=5) 7.60 (n=5) 8.40 (n=5) 7.20 (n=5) #2 Media control 27.20±1.47 (n=10) 34.70±3.32 (n=10) 7.46 (n=10) 13.78 (n=10) 19.20 (n=10) 24.40 (n=10) 32.00 (n=10) No. 3 Metformin 26.30±2.54 (n=10) 36.99±3.90 (n=10) 8.66 (n=10) 9.37 (n=10) 19.40 (n=10) 23.60 (n=10) 34.40 (n=10) #4 Berberine+ UK 26.20±1.77 (n=10) 26.13±2.95 ( ⁿ = ⁸ ) 9.08 (n=10) 6.59 (u=8G 19.40 (n=10) 19.00 (n=10) 13.25 (n=8) ^{d' L} No. 5 Berberine + epk/dhk 25.90±2.04 (n=10) 29.76±4.89 (η=10ν 8.35 (n=10) 5.79 (u=10/ 19.40 (n=10) 20.00 (n=10) 14.20 (n^oV

*1) Body weight was measured on days 0 and 27 (the day before sacrifice) to minimize differences caused by a 12-hour fast

2) Food intake and water intake were measured twice a week throughout the study, the table shows representative data

3) Two animals from group 4 died during the entire study. ^{Autopsy findings} indicated mishandling when dosed by ^intragastric feeding G2 (p<0.05) ^f significant difference between G5 and G2 (p<0.01)

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Table 7

Mean values of fasting glucose, total cholesterol and blood triglycerides in different treatment groups

Group Fasting blood glucose (mmol/l) Total cholesterol (mg/dL) Triglycerides (mmol/L) 0 day Day 28 0 day Day 28 0 day Day 28 No. 1 Norm 11.59± 1.12 (n=5) 3.84±0.26 (n=5) 222.93± 16.17 (n=5) 234.63± 57.65 (n=5) 1.02± 0.31 (n=5) 1.94± 0.33 (n=5) #2 Media control 24.74± 8.47 (n=10) 24.58± 6.01 (n=10) 303.90± 65.51 (n=10) 335.49± 103.95 (n=10) 2.16± 0.78 (n=10) 4.99± 6.01 (n=10) No. 3 Metformin 24.08± 5.44 (n=10) 21.66± 4.71 (n=10) 297.96± 67.09 (n=10) 436.99± 159.73 (n=10) 2.40± 1.03 (n=10) 6.06± 6.71 (n=10) #4 Berberine+ UK 23.78± 8.56 (n=10) 14.70± 7.22 ( ⁿ =8) 327.52± 55.60 (n=10) 264.30± 81.49 (n=8/ 2.15± 0.87 (n=10) 2.87 ± 1.28 (n=8) ^d No. 5 Berberine + EPA/DHA 24.36± 7.43 (n=10) 18.20± 8.71 (n=10) 303.07± 47.27 (n=10) 242.39± 53.82 (n=10) ^s 2.56± 1.01 (n=10) 4.16± 3.66 (n=10)

^a significant difference between G4 and G2 (p<0.01) ^b,c significant difference between G4, G5 and G3 (p<0.001) ^d significant difference between G4 and G3 (p<0.05)

These results showed that combinations of berberine and EPA/DHA and berberine and UA were effective in reducing the symptoms of diabetes in a mouse model of diabetes induced by DVA/STZ. However, metformin (an oral anti-diabetic drug used as first-line therapy for type 2 diabetes) showed no apparent benefit in this study despite its use at the recommended therapeutic dose. Additional studies are being conducted to confirm the observations of this study.

Example 2 Synergistic effects of a combination of berberine and hydroxycitric acid in a mouse model of obesity induced by a high fat diet.

This example describes an in vivo efficacy study of the combination described in the present invention in a mouse model of high-fat diet-induced obesity (HFA).

Fifty 4-week-old male NIH mice were purchased from Guangzhou Institute of Laboratory Animal. After acclimatization for a week, eight mice were selected as normal controls; the remaining 42 mice were provided with DIAF (40% of calories are fat) for 14 days to establish a mouse model of DIAF-induced obesity similar to the pathophysiology of the human metabolic syndrome. Normal control mice received a normal diet. From forty-two mice treated with DIC for 14 days, thirty-two animals weighing 15-20% more than normal control mice were selected and randomized into 4 groups (n=8 per group).

Group 1: vehicle control (1% solution of carboxymethyl cellulose (CMC)).

Group 2: berberine (50 mg/kg in 1% CMC solution).

Group 3: hydroxycitric acid (50 mg/kg in 1% CMC solution).

Group 4: combination (berberine (50 mg/kg) and hydroxycitric acid (50 mg/kg) in 1% CMC solution).

Mice from groups 1-4 were treated with the respective test articles above once daily by intragastric feeding. DIC was maintained throughout the 28-day treatment. Blood glucose levels (fasting and non-fasting), total cholesterol (TC) and triglycerides (TG), food intake, water intake, and body weight were measured throughout the study.

On the 28th day of treatment, an oral glucose tolerance test (OGTT) was performed in animals fasted for 12 hours. For OGTT, after measuring the initial glucose concentration (T = -30 min), mice underwent an oral glucose load at a concentration of 2.5 g / kg and glucose levels were determined using a glucometer (ACCU-CHEK Active, Roche) after 0, 30, 60 and 120 min.

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After OGTT, blood samples were collected to measure the level of glucose, TC and TG in the blood. Mice were sacrificed and pancreas, liver, kidney and adipose tissue were collected for histopathological analysis.

The study was carried out for 15 days, the intermediate results of the experiment are shown in Fig. 1-4.

These results showed a trend towards a synergistic effect of the combination of berberine and hydroxycitric acid. In particular, for berberine (Group 2) and hydroxycitric acid (Group 3), when used alone at a dose of 50 mg/kg, no pharmacological effects were observed compared to the vehicle control group (Group 1); however, when they were used together (Group 4), there was a decrease in body weight gain and normalization of blood glucose levels.

Additional data will be received at the completion of the study.

Example 3. Synthesis and analysis of salt, which is metformin ursodeoxycholate.

mmol of metformin hydrochloride was dissolved in an aqueous solution of NaOH and the reaction was allowed to proceed at room temperature until a clear colorless solution was obtained. The solvent was evaporated to give a white powder. The white powder was added to absolute ethanol and the resulting suspension was then filtered to remove the white precipitate (NaCl). The filtrate was rotary evaporated and then dried in vacuo to give a white Met-OH powder. Met-OH was dissolved in absolute alcohol and reacted with UDCA at room temperature to give a clear light yellow solution. The solution was evaporated using a rotary evaporator and the residue was dried in vacuo (at room temperature). The resulting white powder was then characterized by 1H NMR and IR spectra (FIGS. 5-6) which showed the formation of a metformin ursodeoxycholate salt with a Met:UDCA stoichiometric ratio of 1:1.

Example 4 Synthesis and analysis of a berberine ursodeoxycholate salt.

Berberine ursodeoxycholate

BBR-Cl (1.0 eq.) was dissolved in hot distilled water, then the reaction mixture was cooled to room temperature. Simultaneously, ursodeoxycholic acid (0.9-1.5 eq.) was dissolved in anhydrous ethanol. An aqueous solution of NaCO ₃ (0.9-1.5 eq.) was added dropwise to the resulting ethanol solution of ursodeoxycholic acid. Then the resulting reaction mixture was stirred for 15-45 min and a solution of the sodium salt of ursodeoxycholic acid was obtained.

The BBR-Cl solution was added dropwise to the above-described solution of the sodium salt of ursodeoxycholic acid at a temperature of 60-80°C. The mixture was allowed to stir at the same temperature for 2 hours and then said mixture was cooled to room temperature. The precipitated solid was filtered and the wet filter cake was collected and dried in vacuo below 40° C. to give crude berberine ursodeoxycholate.

The crude ursodeoxycholate berberine was purified by crystallization with ethanol and ethyl acetate. The mixture was allowed to stir for 7-8 hours and then centrifuged to remove the solvent and collect the yellow powder. The yellow powder was washed again with ethyl acetate and the above procedures were repeated twice. The resulting yellow powder was dried under vacuum at 40° C. to give purified berberine ursodeoxycholate.

The resulting yellow powder was then characterized by Ή-NMR, IR spectra and MS (FIGS. 7-12). Ή-NMR (FIGS. 7-8) and IR spectra (FIGS. 9-10) showed clear differences between a simple mixture of berberine and UDCA (1:1) and berberine ursodeoxycholate, indicating the formation of a berberine ursodeoxycholate salt with a stoichiometric ratio BBR:UDCA, making 1:1. Mass spectrometric analysis (Fig. 11-12) showed that the molecular weight of UDCA [M-H] ^- determined using MS in the mode of registration of negative ions was 391.28; the molecular weight of BBR+ determined by MS in the positive ion detection mode was 336.14.

An alternative synthesis method is based on the high solubility of berberine ursodeoxycholate in ethanol, coupled with the solubility of BBR in MeOH and the solubility of sodium UDCA in EtOH.

For example.

1) Dissolve BBR (1.5 eq.) in MeOH at room temperature. (Solution A).

2) Dissolve UDCA (0.9 - 1.5 eq.) in EtOH at room temperature, add sodium ethoxide solution (Solution B).

3) Add Solution A to Solution B at room temperature and stir for 2-5 hours.

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Remove NaCl by vacuum filtration and concentrate the filtrate (T<40°C).

4) Purify the crude ursodeoxycholate berberine by dissolving the crude product in

EtOH (or other suitable solvent) and remove residual NaCl by filtration. Alternatively, the crude product may be purified by crystallization from a suitable solvent.

Example 5 Efficacy of ursodeoxycholate berberine (UDCKB) in a mouse model of high fat diet-induced non-alcoholic fatty liver disease.

This example describes an in vivo efficacy study of the UDCA described in accordance with the present invention in a mouse model of high-fat diet-induced non-alcoholic fatty liver disease (HAFA). Ninety-one 4-week-old male NIH mice were obtained from Vital River Laboratories (Beijing, China). After acclimatization for a week, 13 mice were selected as a control group (Group 1, G1) that received a normal diet. The remaining 78 mice were provided with DAF (40% of calories were fat) for 4 weeks to establish an animal model similar to the pathophysiology of non-alcoholic fatty liver disease in humans.

After 4 weeks of stimulation with a high fat diet, 78 mice were divided into 6 groups according to body weight (n=13 per group).

Group 2, G2: vehicle control group (0.5% CMC-Na solution).

Group 3, G3: group treated with a low dose of UDCCB (30 mg/kg).

Group 4, G4: group receiving the average dose of UDCCB (100 mg/kg).

Group 5, G5: group treated with a high dose of UDCCB (300 mg/kg).

Group 6, G6: BBR control group (berberine HCl, 150 mg/kg).

Group 7, G7: UDCA control group (ursodeoxycholic acid, 150 mg/kg).

Mice from Groups 2-7 were administered the respective test articles mentioned above once daily by intragastric feeding. DIC was maintained throughout treatment for 6 weeks. Normal mice (G1) were treated with vehicle (0.5% CMC-Na solution) via intragastric feeding. At the end of the experiment, the following biochemical parameters were measured and the following tests were performed.

• Body weight, relative liver mass • Total cholesterol (TC) and triglycerides (TG), high density lipoprotein cholesterol (HDL cholesterol) and low density lipoprotein cholesterol (L1SH11 cholesterol) • Alanine aminotransferase (ALT) and aspartate aminotransferase (ACT) levels • Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level • Oral glucose tolerance test (OGTT) • Liver histopathology (Sultan III stain)

After 6 weeks of treatment, each animal fasted for 12 hours was bled by retro-orbital phlebotomy. The liver was surgically removed for histopathological analysis after weighing. Serum was then isolated for determination of TC, TG, HDL cholesterol, LDL cholesterol, ALT, ACT, SOD, and MDA.

One week prior to sacrifice (week 5 of treatment), an oral glucose tolerance test (OGTT) was performed in animals fasted for 12 h. For OGTT, all mice underwent an oral glucose load of 2.0 g/kg and a blood glucose concentration was determined using a glucometer (ACCU-CHEK Active, Roche) after 0, 30, 60, 90 and 120 min.

Histopathological assessment of the liver tissue was performed by staining with Sultan III dye after freezing the sections.

The results of the experiment were presented as follows.

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Table 8

Body weight and biochemical parameters in different groups

Group Body weight (g) Relative weight of the liver (%) OH (mmol/l) TG (mmol/l) HDL cholesterol (mmol/l) LDL cholesterol (mmol/l) P 42.6±0.82 3.77±0.12 4.76±0.18 0.75±0.07 2.74±0.11 0.61±0.05 G2 45.7±0.68** 4.41±0.15** 7.60±0.76** 2.03±0.19** 2.65±0.26 1.79±0.24** GZ 40.3±1.18## 4.33±0.13 5.49±0.25# 1.18±0.20## 2.33±0.15 1.05±0.09## G4 41.7±1.59# 4.15±0.09 5.28±0.50# 0.92±0.06## 2.26±0.22 1.06±0.11# G5 41.5±1.16## 4.27±0.10 5.95±0.54 0.85±0.06## 2.48±0.15 1.03±0.22# GB 42.4± 1.37# 4.26±0.12 5.97±0.47 1.00±0.08## 2.47±0.20 1.42±0.21 G7 42.0±1.16# 4.77±0.26 4.77±0.70# 1.20±0.22# 2.13±0.33 1.37±0.26

Data are expressed as mean±SEM (n=7~13) *p<0.05, **p<0.01 for G2 versus sP #p<0.05, ##p<0.01 for GZ , D4, D5, GB, or G7 vs. D2

Table 9

Liver function in different groups

Group ALT (U/L) ACT (U/L)

P 32.7±2.88 154.6±10.01 G2 37.4±7.28 250.4±36.73* GZ 30.6±4.37 148.3±7.15# G4 29.0±3.95 140.2±16.32# G5 27.8±3.08 163.5±11.63# GB 37.1±4.08 198.7±18.93

G7 30.6±5.73 162.86±29.42

Data are expressed as mean±SEM (n=7~13) *p<0.05 for G2 versus cP #p<0.05 for GZ, G4 or G5 versus G2

Table 10

Oxidative stress coefficient in different groups

MDA

SOD group (U/ml) (mmol/l)

P 84.53±5.64 5.67±0.70 G2 38.23±11.61** 24.11±6.50** GZ 61.05±11.59 12.34±2.89 G4 91.83±4.90## 8.02±1.08# G5 97.54±4.88## 7.78±1.66# GB 77.03±8.98# 9.30±2.14# G7 44.75±11.99 18.94±4.42

Data are expressed as mean ± standard error of the mean (n=7~13) **p<0.01 for G2 versus P #p<0.05, ##p<0.01 for G4, G5, or Gb versus with G2

These results showed that the ionic compound ursodeoxycholate berberine resulted in a dose-dependent reduction in the symptoms of non-alcoholic fatty liver disease caused by DIAF in a mouse model. At the same time, monotherapy with the use of berberine or ursodeoxycholic acid, despite their use in the standard recommended therapeutic dose, was not such

-46042927 effective as ursodeoxycholate berberine. It was also noted that no gastrointestinal adverse reactions were observed in animals treated with berberine ursodeoxycholate at the time of sacrifice, while 50% of animals in the berberine treated group experienced gastrointestinal adverse reactions.

Example 6 Efficacy of UDCA in a high-fat diet-induced fatty liver model in golden hamsters.

This example describes an in vivo efficacy study of the UDCCB salt of the present invention in a high fat diet induced steatosis (HFA) model in golden hamsters.

SPF golden hamsters weighing 90-100 g were purchased from Vital River Laboratory Animal Technology Co., Ltd. After acclimating for one week, eight hamsters were selected as the normal control group (Group 1), which received the normal diet. The remaining 34 hamsters were provided with DIAF for two weeks to establish an animal model of fatty liver disease similar to the pathophysiology of non-alcoholic fatty liver disease (NAFLD) in humans.

From 34 hamsters stimulated with DRVF, on the fourteenth day of maintaining DRVF, 24 animals were selected, in which the TC level was 17.96±1.70 mmol/l, and randomly distributed into three groups (n=8 per group):

Group 2: model control (0.5% tragacanth solution, 10 ml/kg);

Group 3: low dose (UDCCB, 50 mg/kg);

Group 4: high dose (UDCCB, 200 mg/kg).

Hamsters from Groups 2-4 were treated with the respective test articles above once daily by intragastric feeding. DIC was maintained throughout the administration period for 7 weeks. Normal hamsters from group 1 were administered 0.5% tragacanth solution (10 ml/kg) by intragastric feeding. Serum lipid and blood glucose levels, liver function score, food intake and body weight were measured throughout the study. After administration of the compounds for 7 weeks, hamsters were sacrificed and dissected for general evaluation and histopathological analysis of liver tissue. The results of the experiment were presented as follows.

Food intake and body weight: There were no significant differences in food intake between the model control group and drug-treated groups (P>0.05). The body weight of the animals in the high dose group decreased significantly in the first week of treatment (P<0.05), while no significant changes were observed during the remainder of the treatment (P>0.05). In the low dose group, there were no significant changes in body weight (P>0.05) throughout the study.

Serum lipids and blood glucose: in the model control group, the levels of TC, TG, LDL cholesterol, TC/HDL cholesterol and arterial stiffness index (AI) were significantly increased compared to the normal control group (P<0.01) , there was also a significant compensatory increase in HDL cholesterol levels (P<0.01). There were no significant differences in blood glucose levels between the model control group and the normal control group (P>0.05). The TC level in both the low and high dose groups was significantly reduced (P<0.01) compared to the model control group, and the rate of decline was higher in the high dose group compared to the low dose group ( Table 11).

Table 11

Effect of UDCCB on serum TC in hyperlipidemic hamsters (mmol-L ^-1 , X±s, n=8)

Group Dose OH level in Treatment period serum on day 0 1 Week 3 week 5 week 7 week Group 1 10 mlkg' ¹ , 0.5% tragacanth solution 4.17+0.30 3.03+0.94 4.14+0.32 4.21+0.34 4.05+0.33 Group 2 10 ml kg' ¹ , 0.5% tragacanth solution 17.56±2.59ΔΔ 15.98±2.93ΔΔ 14.21±4.56ΔΔ 17.01±4.65ΔΔ 21.74±6.44ΔΔ Group 3 50 mgkg' ¹ , UDHKB 18.14+1.13 9.33+1.52** 7.58+2.01** 10.50+2.89** 9.78+2.58** Group 4 200 mgkg' ¹ , UDHCB 18.18+1.10 7.51+0.71** 6.75+1.00** 5.38+1.24** 4.95+0.84**

- 47 042927

Note: Compared with the normal control group:

^δ =P<0.05, ^δδ =P<0.01;

compared to the model control group:

*=P<0.05, **=P<0.01.

The TG level at week 1 in the low dose group and the TG level at weeks 1 and 7 in the high dose group were significantly reduced (P<0.01) compared with the model control group, and the intensity of the decrease in the group the high dose group was also higher compared to the low dose group (Table 12).

Table 12

Effect of UDCA on serum TG levels in hyperlipidemic hamsters (mmol-L- ¹ , X±s, n=8)

Group Dose Serum TG level on day 0 1 Week Treatment period 3 week 5 week 7 week Group 1 10 ml kg' ¹ , 0.5% tragacanth solution 2.21±0.27 2.04±0.85 1.13±0.27 1.47±0.47 1.07±0.20 Group 2 10 ml kg' ¹ , 0.5% tragacanth solution 5.87±1.38ΔΔ 5.77±1.17ΔΔ 2.74±0.94Δ 3.98±1.35ΔΔ 4.79±2.21ΔΔ Group 3 50 mg kg' ¹ UDHCB 5.97±1.19 3.60±0.78** 2.98±1.31 4.51±3.10 3.88±1.21 Group 4 200 mgkg' ¹ UDHCB 6.31±1.75 3.00±0.67** 2.68±1.09 3.04±1.68 1.90±0.66**

Note: Compared with the normal control group:

^δ =P<0.05, ^δδ =P<0.01;

compared to the model control group:

*=P<0.05, **=P<0.01

Serum LDL-cholesterol, TC/HDL-cholesterol, and AFI were significantly reduced (P<0.01) in both the high-dose and low-dose groups compared to the model control group; the HDL-cholesterol level in the high dose group was also significantly reduced (P<0.01). Moreover, serum LDL-cholesterol, TC/HDL-cholesterol, and AFI in the high-dose group after dosing for 7 weeks were very close to those in the normal control group (FIG. 16).

Liver function score: in the model control group, serum ACT and ALT levels were significantly elevated compared to the normal control group (P<0.01), and serum alkaline phosphatase (AP) also tended to increase (P>0, 05). After dosing for 7 weeks, ACT levels were significantly reduced (P<0.01) in both drug-treated groups compared to the model control group (FIG. 17). After dosing for 7 weeks, ALT levels were significantly reduced (P<0.01) in both drug-treated groups compared to the model control group (FIG. 18).

Overall assessment of liver tissue, liver index and fat index: there was a clear increase in liver volume in model control hamsters, in addition, the surface of the liver was oily. The liver also had an abnormal grayish yellow or grayish white color. The shape of the liver was rounded. A clear lipid deposition could be observed. Moreover, relative liver mass and liver index increased significantly (P<0.01).

Both the low dose group and the high dose group showed no significant change in body weight compared to the model control group (P>0.05). However, the relative liver mass in both drug-treated groups decreased significantly (P<0.01). Detailed results are shown in Fig. 19.

Compared to the model control group, liver color improved in both groups, sex

- 48 042927 who have been treated with drugs. In particular, in the high dose group, the liver had a reddish color, similar to the color of the liver in the normal control group. Detailed results are shown in Fig. 20. According to the results of pathological examination, the content of TC and TG in the liver, the liver tissue inflammation score and the area of oil red staining were significantly increased in the control model group compared with the normal control group (P<0.01). TC and TG, liver tissue inflammation score, and oil red area were significantly reduced in both drug-treated groups compared to the model control group (P<0.01). Detailed data is shown in Fig. 21-22.

The above experimental results indicate that UDCCB significantly reduced the level of TC, TG, LDL cholesterol in serum and reduced the ratio of TC/HDL cholesterol and arterial stiffness index (AI). It reduced the risk of developing atherosclerosis and significantly reduced body fat and led to a decrease in inflammation in the liver. The effects of UDCA were relatively dose dependent. UDCA may be a potential candidate for use in the treatment or prevention of non-alcoholic fatty liver disease (NAFLD)/NASH and hyperlipidemia.

Example 7 Synthesis schemes for examples of berberine salts.

(1) R-(+)-α-lipoic acid salt of berberine.

I / or ROH I /

R = Na, K

X=C1-, HSO ₄

- 49 042927 (2) Ursolic acid salt of berberine.

R = Na, K

X=C1-, HSO ₄

- 50 042927 (3) Hydroxycitric acid salt of berberine (m=1, n=1).

(3)

R = Na, K

X=C1-, HSO ₄ (4) EPA salt of berberine.

R = Na, K

X \u003d C1-, HSO4

- 51 042927 (5) Berberine DHA salt.

R = Na, K

X =C1-, HSO4- (6) Method for obtaining ursolic acid salt of berberine.

A solution of ursolic acid (0.9-1.5 eq.) in methanol was treated with a solution of sodium bicarbonate (0.9-1.5 eq.) in water. The solution was stirred for 30 minutes at room temperature and then added dropwise to an aqueous solution of berberine chloride (1.0 eq.). A yellow solid precipitated immediately upon addition. The mixture was stirred for 1 hour and then cooled to room temperature. A yellow solid was obtained by filtration in 30% yield (NMR results are shown in Fig. 15).

Example 8 Animal models for determining the pharmacological effects of berberine salts.

(1) Diabetes activity study.

Healthy male Sprague-Dawley rats of 8 weeks of age were placed in a room with controlled lighting (12-hour light-dark cycle) and controlled temperature (18°C-25°C) and humidity. All rats were fed normal diet (protein -21%, carbohydrates - 55%, fat - 6%, total calories - 15.36 kJ/g) for 1 week to adapt to the environment. Six rats were randomly selected as the normal control group (NC), which received a normal diet throughout the study. The remaining rats were fed a high fat diet (protein 16%, carbohydrates 38%, fat 46%, total calories 20.54 kJ/g). After maintaining a high-fat diet for 8 weeks, animals were induced to develop diabetes with a single intraperitoneal injection of freshly prepared streptozotocin (STZ, 30 or 50 mg/kg bw) (Sigma, St. Louis, MO, USA) in citrate buffer (pH 4.5) to rats fasting overnight. Two weeks after STZ administration, animals with fasting blood glucose levels >11.1 mM were selected for study and assigned randomly to the following groups: vehicle (water) group, low, medium, and high

- 52 042927 a dose of berberine salts, respectively, by intragastric administration once a day for 28 consecutive days. Fasting glucose, inulin, total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides in animals were recorded the day before the first dose (day 0) and on days 7, 14, 21, and 28 of dosing.

(2) Study of activity against complication of diabetes (diabetic nephropathy).

Five week old male Sprague-Dawley rats weighing 120 to 130 g were housed in wire bottom cages with a 12 hour light/dark cycle. Room temperature was maintained at about 25° C. with relatively constant humidity. Animals were provided free access to standard laboratory animal pellets and water. After 1 week of adaptation, the rats underwent resection of half of the left kidney, followed by complete removal of the right kidney after 10 days. After that, the animals were injected intraperitoneally with STZ (25 mg/kg body weight) in citrate buffer, pH 4.5. The level of glucose in blood and the level of urea nitrogen were determined after the recovery of animals after injection in order to avoid any intergroup differences in these blood parameters and rats were divided into four groups (control and three treatment groups). Also included was a normal group of rats that were subjected to a sham operation. Each experimental group included 10 rats. During the experiment, normal and control groups received water for 50 days. The other three groups received low, medium and high dose berberine salts, respectively, by intragastric feeding. At the end of this experiment, 24-hour urea samples were collected using metabolic cells, and blood samples were obtained by heart puncture. Serum was immediately separated from the blood samples by centrifugation. After the kidney was perfused through the renal artery with ice-cold saline, the remaining kidney was removed from each rat and a piece of tissue was immersed in formalin for histological evaluation. The other part was frozen at -80°C until the analysis. Serum glucose, total protein, albumin, total cholesterol, triglycerides, urea nitrogen, and creatinine were tested using commercial reagents.

(3) Study of activity against dyslipidemia and/or obesity.

A murine model of alimentary obesity (AO) was established by maintaining a high fat diet (40% Kcal was fat) in 4-week-old healthy NIH mice. Mice were kept in polycarbonate cages, three animals per group, while maintaining normal temperature (22±4°C) and normal humidity with a 12-hour light-dark cycle. After maintaining a high fat diet for 2 weeks, mice were weighed and randomly assigned to groups of 10 mice: control groups, groups receiving low, medium and high dose of berberine salt by intragastric feeding once a day for a total of 4 weeks while maintaining a high-fat diet throughout the treatment period. In addition, six normal mice were included as a normal group receiving a standard diet. Food and water intake, body weight and non-fasting glucose levels were studied in all animals on the day before the first dose (day 0) and on days 7, 14, 21 and 28 of dosing. Glucose levels after fasting for 6 hours, insulin levels, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides were studied in all animals on the day before the first dose (day 0) and on day 28 of the dose. An oral glucose tolerance test (OGTT) was performed after fasting for 12 hours on day 28. After OGTT, all animals were sacrificed and pancreas, liver, kidneys and adipose tissue were removed, weighed and collected for histological analysis.

(4) Efficacy study in a skeletal muscle atrophy model.

8-week-old male Sprague-Dawley rats were housed individually at 25±1° C. with light from 8:00 am to 8:00 pm and free access to water and standard commercially available rat chow. After acclimatization for a week, the rats were divided randomly into 4 groups. The control group (n=8) was injected with 2 ml/kg/day of saline, the other three groups were injected with 2 mg/kg/day of prednisolone, a glucocorticoid obtained from SIGMA-Aldrich (Missouri, USA). The three glucocorticoid treated groups received water, low or high dose berberine salts, respectively, via intragastric feeding (n=8 per group) for a total of 4 weeks. All animals were assessed for food and water intake, body weight and glucose levels on the day before the first dose (day 0) and on days 7, 14, 21 and 28 of the dose. At the end of the experiment, rats that had been starving overnight were sacrificed by decapitation. Blood was collected and centrifuged at 3000 rpm for 15 minutes to obtain serum. Serum was stored at -20°C. Liver, heart, and skeletal muscles (soleus, plantar, gastrocnemius, tibialis anterior, and extensor digitorum longus) were rapidly removed, weighed, and stored at -80° C. until analysis.

(5) Efficacy Study for Attenuating Non-Alcoholic Fatty Liver Disease (NAFLD).

Healthy female Sprague-Dawley rats were randomly assigned to two groups:

- 53 042927 high fat diet group (n=56, maintained high fat diet) and normal group (n=10, received standard diet). At the end of the 12th week, 6 rats from the high fat diet group were randomly selected for histopathological evaluation of the liver and the successful establishment of the rat model of non-alcoholic fatty liver disease (NAFLD) was confirmed. The remaining 50 model rats were divided into 5 equal subgroups: low, medium and high dose berberine salts via intragastric feeding, vehicle control and recovery group. Rats in the vehicle control group received water by force feeding. After 20 weeks, all rats were anesthetized with 3% sodium pentobarbital by intraperitoneal injection. The level of insulin in blood plasma and the content of TG, TC, LDL cholesterol, ACT and ALT in blood serum were determined. After sacrifice, liver tissue was collected for histopathological evaluation.

(6) Efficacy Study for Reducing Non-Alcoholic Steatohepatitis (NASH).

In the present study, male Sprague-Dawley rats of six weeks of age with a body weight of 160-170 g were used. Animals were kept in a temperature-controlled room (22±1°C) at normal humidity with a 12-hour light-dark cycle.

Rats were fed a standard diet (control group, n=8) or a high fat choline deficient diet (HFDC) throughout the experiment for 10 weeks. Fatty hepatosis was induced by the HIGH diet for 4 weeks. At the fifth week, the rats fed the HVHD diet were randomized into six groups. The HFHD group (n=8) received the HFHD diet only; rats in the non-alcoholic steatohepatitis (NASH) group (n=8) received an IVH diet followed by daily intraperitoneal administration of sodium nitrite (NaNO2) - an oxidizing agent - at a dose of 50 mg/kg/day (Nacalai Tesque Inc., Kyoto, Japan) to induce methemoglobinemia ( unsustainable hypoxic stress) starting from the 5th week of maintenance of the HIGH diet for 6 weeks; non-alcoholic steatohepatitis (NASH) group with low, medium and high dose berberine salts (n=8 per group) via intragastric feeding during the period of parallel nitrite administration.

At the end of the 10 week experimental period, the animals were sacrificed by anesthesia with diethyl ether. Blood samples were obtained by puncture from the inferior vena cava using a syringe containing heparin, before tissue samples were obtained through the left ventricle, the whole body was perfused with 0.1 M potassium solution containing 5 mM benzamidine. Blood plasma was obtained by centrifugation at 1,000xg for 10 min at 4°C and used for biochemical analysis. Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (ACT) were determined using commercial kits.

Freshly isolated livers were used for fractionation and lipid peroxidation assessment, a portion of the liver was placed in 10% formalin for 3 days and then embedded in paraffin for histopathological examination. The rest of the liver was quickly frozen with liquid nitrogen and stored at -80°C for further analysis.

(7) Study of action against atherosclerosis.

A mouse model of atherosclerosis (AC) was established by feeding healthy C57/BL 6J mice starting at 4 weeks of age with a composition containing 15% fat and 4.5% cholesterol. Mice were kept in polycarbonate cages, three animals per group, while maintaining normal temperature (22±4°C) and normal humidity with a 12-hour light-dark cycle.

After maintaining a high fat diet for 16 weeks, histopathological examination of hearts from 3-5 mice from the model group was performed to assess its establishment. In mice with a cholesterol-induced predisposition to atherosclerosis, the most pronounced atherosclerotic lesions were observed in the ascending aortic arch, where the aortic valves attach to the sinus wall. In control animals, one layer of endothelial cells was detected, covering the elastic membrane and a thin layer of connective tissue. No lipid droplets were observed. Model mice were weighed and randomized into groups of 10 mice: control groups, groups receiving low, medium and high dose berberine salt by intragastric feeding once a day for a total of 8 weeks while maintaining a high fat diet throughout treatment period; and six normal mice were included as a normal group receiving a standard diet. Evaluation of food and water intake and body weight was performed in all animals on the day before the first dose (day 0) and on 14, 28, 42 and 56 days of dosing. The day before the first dose (day 0) and on day 56 of the dose, total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides were examined in all animals. All animals were sacrificed, aorta, heart, liver and adipose tissue were removed, which were weighed and collected for histological analysis.

(8) Efficacy study for the treatment of heart failure.

The efficacy of the berberine salt for the treatment of heart failure was evaluated in a rat model of adriamycin-induced dialated cardiomyopathy. Male Wistar rats received 5 intraperitoneal injections of adriamycin at a dose of 2 mg/kg every 3 days, then 5 injections at a dose of

- 54 042927 mg/kg every 7 days to establish a pattern of heart failure. Animals from the group of the model receiving the vehicle were injected with 0.9% saline using the same method. Rats were kept in polycarbonate cages, three animals per group, while maintaining normal temperature (22±4°C) and normal humidity, providing a 12-hour light-dark cycle and standard food. At the end of week 10, four rats were randomly selected to evaluate cardiac function by transthoracic echocardiography and myocardial morphology by electron microscopy. The parameters of left ventricular end diastolic size (LV EDD) and left ventricular end systolic size (LV ESD), left ventricular ejection fraction (LV EF) and left ventricular shortening fraction (LV FU) showed that a model of heart failure was established by the type of dilated cardiomyopathy .

Rats were weighed and randomly assigned to groups of 6 animals: control groups, groups receiving low, medium and high dose of berberine salt by intragastric feeding once a day in total for 8 weeks of treatment. In addition, six vehicle control model rats were included as the normal group. On the day before the first dose (day 0) and on days 14, 28, 42 and 56 of dosing, food and water intake and body weight were assessed for all animals. On day 56, parameters of left ventricular end diastolic size (LVED) and left ventricular end systolic size (LV ESR), left ventricular ejection fraction (LV EF), and left ventricular shortening fraction (LV FU) were studied. After the study, all animals were sacrificed, removed the heart, liver and kidneys, which were weighed and collected for histological analysis.

(9) Efficacy study for the treatment of neurodegenerative diseases.

The efficacy of the berberine salt for the treatment of Parkinson's disease was evaluated in C57/BL6 mice stimulated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mice were kept in polycarbonate cages, three animals per group, while maintaining normal temperature (22±4°C) and normal humidity, providing a 12-hour light-dark cycle and standard food. For 7 consecutive days, 8-week-old mice were injected intraperitoneally with MPTP at a dose of 20 mg/kg/day, while vehicle control model rats were injected with the same volume of saline using the same method. Mice were weighed and randomly assigned to groups of 6 animals: control groups, groups receiving low, medium and high dose of berberine salt by intragastric feeding once a day for a total of 8 weeks of treatment. In addition, six vehicle control model rats were included as the normal group. MPTP administration caused dopaminergic death of neurons in the substantia nigra and loss of nerve fibers in the striatum, which led to impaired motor balance and coordination (assessed based on the elevated bar walking test). At the same time, treatment with berberine increased motor balance and coordination by preventing dopaminergic damage to neurons. Berberine treatment also improved short-term memory by suppressing cell apoptosis in the hippocampus.

In the present invention and the appended claims, reference to an item in the singular includes the plural of the specified item, unless otherwise clearly follows from the context.

Unless otherwise indicated, all technical and scientific terms used in the present invention have the meanings generally accepted by those skilled in the art. The present invention describes the preferred methods and materials, although any method and material similar or equivalent to the method or material described in the present invention can also be used in the practice or testing of the present invention. The methods set forth in the present invention can be performed in any logically possible order in addition to the specific order described.

Inclusion by reference

The present invention cites and links to other documents such as patents, patent applications, patent publications, magazines, books, articles, and Internet site content. All such documents are hereby incorporated by reference in their entirety in all respects. Any material or part thereof which is said to be incorporated by reference into the present invention, but which is inconsistent with the inventive definitions, statements or other material of the invention specifically set forth in the present invention, is included in the present invention solely to the extent that eliminates the occurrence of a conflict between the specified included material and the material of the present invention. In the event of a conflict, the present invention takes precedence.

Equivalents

The exemplary examples described in the present invention are provided for the purpose of illustrating the present invention and are not intended to limit or be considered as limiting the scope of the present invention. In fact, various modifications of the invention and many additional embodiments beyond those shown and described in the present invention will become apparent to those skilled in the art upon consideration of the entire contents of this document, including the following examples and references to the scientific and patent literature cited in the present invention. . The following examples contain important additional information, illustrations, and guidelines that can be adapted for the practical implementation of various embodiments of the invention and their equivalents.

Claims (15)

1. Pharmaceutical composition containing: (a) berberine or a salt thereof; (b) a pharmaceutically active organic acid selected from the group consisting of hydroxycitric acid and ursodeoxycholic acid or a salt thereof; and (c) a pharmaceutically acceptable excipient, carrier or diluent, wherein the berberine and the pharmaceutically active organic acid are present in an amount which, when administered to a subject, is sufficient to treat or ameliorate one or more diseases or disorders selected from metabolic disorders, atherosclerosis, heart disease , neurodegenerative diseases, liver diseases, sarcopenia, muscle atrophy, inflammation and cancer, in a mammal, including humans. 2. Pharmaceutical composition according to claim 1, characterized in that said pharmaceutically active organic acid is ursodeoxycholic acid. 3. Pharmaceutical composition according to claim 1, characterized in that said pharmaceutically active organic acid is hydroxycitric acid. 4. Pharmaceutical composition according to any one of claims 1 to 3, further comprising a third agent selected from the group consisting of vitamin D, vitamin C, vitamin E, vitamin B12, vitamin A, benfotiamine, chromium and vanadium picolinate. 5. Pharmaceutical composition according to any one of claims 1 to 4, wherein said disease or disorder is selected from diabetes, complications of diabetes, obesity, dyslipidemia, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, metabolic syndromes, metabolic disorders preceding diabetes, fatty liver , cholestatic liver disease, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). 6. The pharmaceutical composition of claim 5, wherein said disease or disorder is fatty liver, cholestatic liver disease, NAFLD, or NASH. 7. Pharmaceutical composition according to any one of claims 1 to 6, containing a pharmaceutically acceptable excipient, carrier or diluent. 8. The use of a composition for the treatment or amelioration of a disease or disorder selected from a metabolic disorder, heart disease, neurodegenerative disease, and liver disease, wherein said composition contains: (a) berberine or a salt thereof; (b) a pharmaceutically active organic acid selected from the group consisting of hydroxycitric acid and ursodeoxycholic acid or a salt thereof, in a therapeutically effective amount, and (c) a pharmaceutically acceptable excipient, carrier or diluent. 9. Use according to claim 8 for the treatment or amelioration of a metabolic disorder selected from diabetes, complications of diabetes, obesity, dyslipidemia, diabetic dyslipidemia, dyslipidemia in patients with statin intolerance, metabolic syndromes, metabolic disorders preceding diabetes, fatty liver, cholestatic liver disease , non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). 10. Use according to claim 8 or 9, characterized in that said disease or disorder is a liver disease. 11. Use according to claim 8 or 9, characterized in that said disease or disorder is fatty liver disease, non-alcoholic steatohepatitis (NASH), or non-alcoholic fatty liver disease (NAFLD). 12. Use according to claim 8 or 9, characterized in that said disease or disorder is cholestatic liver disease. 13. Use according to any one of claims 8 to 12, characterized in that said pharmaceutical composition further comprises one or more agents selected from the group consisting of vitamin D, vitamin C, vitamin E, vitamin B12, vitamin A, benfotiamine, picolinate chromium and vanadium. 14. Use according to claim 9, characterized in that said pharmaceutically active organic acid is ursodeoxycholic acid and said disease or disorder is dyslipidemia, diabetic dyslipidemia, fatty liver, cholestatic liver disease, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). - 56 042927 15. Set containing: (i) a first agent which is berberine or a salt thereof; (ii) a pharmaceutically active organic acid selected from hydroxycitric acid and ursodeoxycholic acid or a salt thereof; (iii) one (or more) third agent(s) selected from the group consisting of vitamin D, vitamin C, vitamin E, vitamin B12, vitamin A, benfotiamine, chromium and vanadium picolinate; and (iv) instructions for administering the first agent, second agent(s) and third agent(s) to a patient suffering from or at risk of developing one or more diseases or disorders selected from metabolic disorders, atherosclerosis, heart disease, sarcopenia, muscle atrophy, neurodegenerative diseases, liver disease and cancer.