Berberine tauroursodeoxycholate, compositions and methods thereof

Abstract

The present invention provides berberine tauroursodeoxycholic acid (BTUDC), its pharmaceutical composition, and its method of use, as monotherapy, in combination with other drugs, or as an adjuvant, for the treatment, alleviation, and / or prevention of Parkinson's disease or related diseases and disorders.The present invention further provides pharmaceutical compositions of berberine (BBR) and tauroursodeoxycholic acid (TUDCA), and its method of use, as monotherapy, in combination with other drugs, or as an adjuvant, for the treatment, alleviation, and / or prevention of Parkinson's disease or related diseases and disorders.

Description

[Technical Field]

[0001] Priority claims and related applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 446,004, filed February 15, 2023, and Chinese Patent Application No. 2024101662972, filed February 6, 2024, all of which are incorporated herein by reference in their entireties.

[0002] The present invention relates generally to pharmaceutical compositions and methods for their therapeutic use. In particular, the present invention relates to berberine tauroursodeoxycholate (BTUDC), pharmaceutical compositions thereof, and methods of use thereof, as monotherapy, in combination with other drugs, or as an adjuvant, for the treatment, amelioration, and / or prevention of central nervous system (CNS) diseases and disorders, such as Parkinson's disease and amyotrophic lateral sclerosis (ALS), or related diseases and disorders. The present invention further relates to pharmaceutical compositions of berberine (BBR) and tauroursodeoxycholic acid (TUDCA), as monotherapy, in combination with other drugs, or as an adjuvant, for the treatment, amelioration, and / or prevention of Parkinson's disease or related diseases and disorders, and methods of use thereof. [Background technology]

[0003] Degenerative neurological diseases (or neurodegenerative diseases) affect tens of thousands of people worldwide. These diseases include a wide range of illnesses that primarily affect neurons in the brain. Neurons are components of the nervous system, including the brain and spinal cord. Neurons do not normally regenerate or replace themselves when they are damaged or die. Examples of neurodegenerative diseases that affect the CNS include Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS. Currently, neurodegenerative diseases are incurable and debilitating illnesses that result in the progressive degeneration and / or death of nerve cells.

[0004] Parkinson's disease is a long-term degenerative disorder of the central nervous system that causes unintentional or uncontrollable movements and difficulties with balance and coordination. Symptoms usually begin gradually and worsen over time. Early symptoms include tremors, rigidity, slowness of movement, and difficulty walking. As the disease progresses, people may have difficulty walking and speaking. Cognitive and behavioral problems such as depression, anxiety, and numbness may also occur in many patients. Parkinson's disease dementia is common in the advanced stages of the disease. Parkinson's disease patients may also have problems with sleep and the sensory system. As the disease progresses, patients may have difficulty walking and speaking. (Sveinbjornsdottir 2016 “The clinical symptoms of Parkinson's disease” J.Neurochem.139 (Suppl 1): 318-324, “Parkinson's Disease Information Page Nat'l Inst.Neurol.Dis.&Stroke, https: / / www.ninds.nih.gov / health-information / disorders / parkinsons-disease.)

[0005] Currently, there is no cure or effective treatment for Parkinson's disease. Treatments are usually aimed at reducing the impact of symptoms. Initial treatment options include levodopa (L-DOPA), MAO-B inhibitors, and dopamine agonists. These medications become less effective as the disease progresses and simultaneously produce side effects characterized by involuntary muscle movements. Deep brain stimulation using surgically placed microelectrodes has been used to reduce motor symptoms in severe cases where medications are ineffective. Dietary therapy and certain forms of rehabilitation have shown some effectiveness in improving symptoms. (Samii, et al.2004 “Parkinson's disease” Lancet 363 (9423):1783-1793, Armstrong, et al.2020 “Diagnosis and Treatment of Parkinson Disease: A Review” JAMA 323(6):548-560, Barichella, et al.2009 “Major nutritional issues in the management of Parkinson's disease” Movement Disorders 24(13):1881-1892.)

[0006] Currently available therapeutic agents and methods for treating Parkinson's disease remain inadequate. New, safe, and effective treatments are urgently needed. Summary of the Invention

[0007] In one aspect, the present invention generally relates to a compound of formula (I):

[0008] [ka] The present invention relates to a salt having the formula:

[0009] In another aspect, the invention generally relates to a solid form of the compound of Formula (I), wherein the solid form is Form A, the X-ray powder diffraction (XRPD) pattern of which, using a Cu-Kα radiation source, comprises one or more characteristic diffraction peaks at the following 2θ angles: 4.62°, 9.32°, and 17.02°±0.2°.

[0010] In another aspect, the invention generally relates to pharmaceutical compositions comprising a BTUDC and a pharmaceutically acceptable excipient, carrier, or diluent.

[0011] In another aspect, the invention generally relates to pharmaceutical compositions comprising the solid forms disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0012] In another aspect, the invention generally relates to unit dosages containing pharmaceutical compositions of BTUDC.

[0013] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating a neurodegenerative disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BTUDC.

[0014] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating Parkinson's disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BTUDC.

[0015] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating a neurodegenerative disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BBR and TUDCA.

[0016] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating Parkinson's disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BBR and TUDCA.

[0017] In yet another aspect, the invention relates generally to the use of BTUDCs to treat neurodegenerative diseases or related diseases or disorders.

[0018] In yet another aspect, the invention relates generally to the use of BTUDCs to treat Parkinson's disease or a related disease or disorder.

[0019] In yet another aspect, the invention generally relates to the use of BTUDC for the manufacture of a medicament for preventing or treating a neurodegenerative disease or related disease or disorder.

[0020] In yet another aspect, the invention generally relates to the use of BTUDC for the manufacture of a medicament for preventing or treating Parkinson's disease or a related disease or disorder.

[0021] In another aspect, the invention generally relates to the use of BBR and TUDCA to treat neurodegenerative diseases or related diseases or disorders.

[0022] In another aspect, the invention generally relates to the use of BBR and TUDCA to treat Parkinson's disease or a related disease or disorder.

[0023] In another aspect, the invention generally relates to the use of BBR and TUDCA for the manufacture of a medicament for preventing or treating a neurodegenerative disease or related disease or disorder.

[0024] In another aspect, the invention generally relates to the use of BBR and TUDCA for the manufacture of a medicament for preventing or treating Parkinson's disease or a related disease or disorder.

[0025] In another aspect, the invention generally relates to methods for making the BTUDC salts disclosed herein.

[0026] In another aspect, the invention generally relates to methods for preparing the solid forms disclosed herein. [Brief explanation of the drawings]

[0027] [Figure 1] 1 shows an exemplary 1H NMR spectrum of BTUDC. [Figure 2] 1 shows an exemplary 1H NMR spectrum of TUDCA. [Figure 3] 1 shows an exemplary 1H NMR spectrum of BBR-Cl. [Figure 4] 1 shows a schematic representation of an asymmetric structural unit. [Figure 5] 1 shows a schematic representation of the single-cell structure of a crystal. [Figure 6] 1 shows exemplary results regarding the effect of test article on the number of ipsilateral rotations within 30 minutes of apomorphine injection in 6-hydroxydopamine (6-OHDA) stereotactic injection induced rats. [Figure 7] 10 shows exemplary results regarding the effect of test article on the time on beam test in 6-OHDA stereotaxic injection-induced rats. [Figure 8] 1 shows exemplary results regarding the effect of test article on the number of foot slips on the beam test in 6-OHDA stereotaxic injection-induced rats. [Figure 9] 1 shows exemplary results regarding the effect of test article on the time test on the rotarod in 6-OHDA stereotaxic injection induced rats. [Figure 10] 1 shows exemplary results regarding the effect of test article on peak grip strength testing in 6-OHDA stereotaxic injection induced rats. [Figure 11] 1 shows an exemplary XRPD pattern of Form A. [Figure 12] 1 shows exemplary DSC and TGA graphs of Form A. [Figure 13] 1 shows an exemplary XRPD comparison of the crystalline transitions of Form A. [Figure 14]1 shows an exemplary Fast DVS isotherm plot of Form A. [Figure 15] 1 shows exemplary XRPD of Form A before and after Fast DVS. [Figure 16] 1 shows an exemplary XRPD of a Form A single crystal sample. [Figure 17] 1 shows an exemplary XRPD pattern of Form A from a stability study. [Figure 18] 1 shows exemplary data for the effect of test article on ipsilateral rotation number within 30 minutes of apomorphine injection in 6-OHDA stereotaxic injection-induced rats. [Figure 19] 1 shows exemplary data on the effect of test article on peak grip strength testing in 6-OHDA stereotaxic injection-induced rats. [Figure 20] 1 shows exemplary data on the effect of test article on time on rod in the rotarod test in 6-OHDA stereotaxic injection induced rats. [Figure 21] 1 shows exemplary data for time to cross a balance beam. [Figure 22] 1 shows exemplary data for the number of foot slips on a balance beam. [Figure 23] Exemplary data are shown for immunofluorescence staining of tyrosine hydroxylase (TH) in the striatum (Str) and substantia nigra (SN) brain regions of PD model rats after administration of drugs for 21 days. [Figure 24] 1 shows exemplary data for fluorescence intensity analysis of tyrosine hydroxylase (TH) positive cells in the Str brain region of rats 21 days after intracerebral stereotaxic injection of 6-OHDA. [Figure 25] 1 shows exemplary data for fluorescence intensity analysis of tyrosine hydroxylase (TH) positive cells in the Str brain region of rats 21 days after intracerebral stereotaxic injection of 6-OHDA. [Figure 26] Exemplary data are shown for the results of immunofluorescence staining of microglia (Iba1) in the Str and SN brain regions of PD model rats after 21 days of drug administration. [Figure 27]1 shows an exemplary data analysis of microglia (Iba1) positive cell counts in the Str brain region of rats 21 days after stereotaxic injection of 6-OHDA. [Figure 28] 1 shows an exemplary data analysis of microglia (Iba1) positive cell counts in the SN brain region of rats 21 days after intracerebral stereotaxic injection of 6-OHDA. [Figure 29] 1 shows an exemplary data analysis of ELISA test results for IL-1β, IL-6, and TNF-α in rat cerebrospinal fluid after 21 days of administration. [Figure 30] 1 shows an exemplary 1H NMR spectrum of BTUDC prepared by Method 2. [Figure 31] 1 shows an exemplary XRPD pattern of BTUDC raw material prepared by Method 1. [Figure 32] 1 shows an exemplary TGA and DSC of BTUDC feedstock prepared by Method 1. [Figure 33] 1 shows an exemplary On-line Temperature Change Experiment of BTUDC feedstock prepared by Method 1.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas described herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0029] As used in this disclosure, the following words and phrases are generally intended to have the meanings set forth below unless expressly indicated otherwise or the context in which they are used dictates otherwise.

[0030] In this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0031] The term "and / or" is used in this disclosure to mean either "and" or "or," unless the context clearly dictates otherwise.

[0032] As used herein, "at least" a particular value is understood to be that value and all values ​​greater than that value.

[0033] Applicant's disclosure is described herein in preferred embodiments with reference to the drawings, wherein like numbers represent the same or similar elements. Throughout this specification, reference to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0034] The term "comprising," when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. The term "consisting essentially of," when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements and exclude any other elements of essential importance to the compositions and methods. For example, "consisting essentially of" refers to the administration of explicitly recited pharmacologically active agents and excludes pharmacologically active agents not explicitly recited. The term "consisting essentially of" does not exclude pharmacologically inactive or inert agents, such as pharmaceutically acceptable excipients, carriers, or diluents. The term "consisting of," when used to define compositions and methods, is intended to mean excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0035] Throughout the description where compositions and kits are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is contemplated that there are additionally compositions and kits of the invention that consist essentially of or consist of the recited compositions, and processes and methods of the invention that consist essentially of or consist of the recited processing steps.

[0036] In this application, when an element or component is said to be included in and / or selected from a recited list of elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0037] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to mean within normal tolerances in the art, for example, within two standard deviations of the mean. About can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein can be modified by the term about.

[0038] At various places in this specification, variables or parameters are disclosed in groups or ranges. The description is specifically intended to include any and all individual subcombinations of the elements of such groups and ranges. For example, a range of 1 to 16 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0039] As used herein, "XRPD" refers to X-ray powder diffraction. An XRPD pattern is an xy graph with 2θ (diffraction angle) plotted on the x-axis and intensity plotted on the y-axis. These are diffraction peaks that can be used to characterize crystalline materials. Because diffraction peak intensity can be particularly sensitive to sample orientation, diffraction peaks are usually represented and referenced by their position on the x-axis, rather than the intensity of the diffraction peak on the y-axis (see Pharmaceutical Analysis, Lee & Web, pp. 255-257 (2003)). Therefore, intensity is typically not used by those skilled in the art to characterize crystalline materials.

[0040] As used herein, the term "2θ value" or "2θ" refers to the peak position in degrees based on the experimental setup of an X-ray diffraction experiment, which is the usual abscissa unit in a diffraction pattern. The experimental setup requires that the reflected beam be recorded at an angle θ (2θ) when the reflection is diffracted when the incident beam makes an angle θ (θ) with a lattice plane. It should be understood that references herein to specific 2θ values ​​of a particular solid form are intended to mean the 2θ values ​​(in degrees) measured using the X-ray diffraction experimental conditions described herein.

[0041] As with any data measurement, XRPD data can be subject to variability. In addition to variability in diffraction peak intensities, there can also be variability in the position of diffraction peaks along the x-axis. However, this variability can typically be accounted for when reporting diffraction peak positions for characterization purposes. Such variability in the position of diffraction peaks along the x-axis can stem from several sources. One such source may be sample preparation. Samples of the same crystalline material prepared under different conditions may yield slightly different diffractograms. Factors such as particle size, water content, solvent content, temperature, and orientation can all affect how a sample diffracts X-rays. Another source of variability stems from instrument parameters. Different X-ray powder diffractometers operate using different parameters, which can result in slightly different diffraction patterns from the same crystalline material. Similarly, different software packages process XRPD data differently, which can also lead to variability. These and other sources of variability are known to those skilled in the art. Because of such sources of variation, each X-ray diffraction peak value may be preceded by the term "about" or with an appropriate range (e.g., ±0.1°, ±0.2°, ±0.3°, ±0.4°, ±0.5°, etc.) defining the experimental variation.

[0042] Crystalline forms, such as those of the compound of formula (I), are easily analyzed by XRPD. Data from X-ray powder diffraction can be used in multiple ways to characterize crystalline forms. For example, the entire X-ray powder diffraction pattern output from a diffractometer can be used to characterize a crystalline form (e.g., of a compound of formula (I)). However, a smaller subset of such data may also be suitable and can be used to characterize such a crystalline form. In fact, often, even a single X-ray powder diffraction peak can be used to characterize such a crystalline form. With respect to the crystalline form of the compound of formula (I), any one or more of the peaks in the X-ray powder diffraction pattern can be used to characterize the crystalline form of the compound of formula (I) disclosed herein.

[0043] The term "characteristic peaks," when referring to peaks in an XRPD pattern of a crystalline form of a given chemical substance (e.g., a crystalline form of a compound of Formula (I)), refers to a particular set of diffraction peaks having values ​​spanning a range of 2θ values ​​(e.g., 0° to 40°) that are collectively unique to that particular crystalline form.

[0044] Differential scanning calorimetry (DSC) profiles can be particularly sensitive to sample preparation and parameters. Therefore, there can be variability in DSC data (e.g., the location of the onset and peak maximum temperatures). Due to such sources of variability, each temperature value based on DSC data can be preceded by the term "about" or accompanied by an appropriate range (e.g., ±0.5°C, ±1°C, ±3°C, ±4°C, ±5°C, etc.) to define the experimental variation.

[0045] As used herein, the term "crystalline" refers to any solid material that exhibits three-dimensional order, giving a distinctive X-ray powder diffraction (XRPD) pattern with sharply defined peaks, in contrast to amorphous solid materials.

[0046] As used herein, the term "amorphous" refers to any solid material lacking three-dimensional order. In some cases, amorphous solids can be characterized by known techniques, including XRPD crystallography, solid-state nuclear magnetic resonance (ssNMR) spectroscopy, DSC, or some combination of these techniques. Amorphous solids typically give diffuse XRPD patterns consisting of one or two broad peaks (i.e., peaks with a base width of about 5° 2θ or greater).

[0047] As used herein, the term "polymorph" refers to different crystalline forms of the same compound and includes other solid state molecular forms, including, but not limited to, hydrates (e.g., bound water present in the crystalline structure) and solvates (e.g., bound solvents other than water) of the same compound.

[0048] As used herein, the term "essentially the same" with respect to X-ray powder diffraction peak positions and / or patterns means that typical peak position and intensity variability is taken into account. For example, those skilled in the art will understand that peak positions (2θ) will typically exhibit some variability, typically on the order of 0.1-0.2°, as well as some variability depending on the instrument used to measure the diffraction. Furthermore, those skilled in the art will understand that relative peak intensities will exhibit variability between instruments, as well as variability due to crystallinity, desired orientation, surface of the sample prepared, and other factors known to those skilled in the art, and should be viewed as qualitative indicators only. Similarly, as used herein, "essentially the same" with respect to DSC is intended to encompass the variability associated with these analytical techniques known to those skilled in the art.

[0049] As used herein, the term "stable" refers to a compound that does not change substantially when subjected to conditions that allow for its production, detection, recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable or chemically feasible compound is one that does not change substantially when kept at a temperature of 40° C. or less, in the absence of moisture or other chemically reactive conditions, for at least one week, preferably at least one month, more preferably at least six months, and even more preferably at least one year.

[0050] As used herein, the term "solvate" refers to a crystalline solid addition product containing a stoichiometric or non-stoichiometric amount of solvent incorporated into the crystalline structure. When the solvent is tightly bound to the drug, the resulting complex has a well-defined stoichiometry that is independent of humidity. However, when the solvent is weakly bound, such as in channel solvates and hygroscopic compounds, the solvent content depends on humidity and drying conditions. In such cases, the complex is often non-stoichiometric. When the incorporated solvent is water, such an adduct is called a "hydrate." Thus, the term "hydrate" describes a solvate containing a drug substance and a stoichiometric or non-stoichiometric amount of water.

[0051] As used herein, the term "anhydrous" or "anhydrous" when referring to a crystalline form (e.g., a crystalline form of a compound of Formula (I)) means that water molecules do not form part of the unit cell of the crystalline form. Nevertheless, an anhydrous crystalline form may contain water molecules that do not form part of the unit cell of the anhydrous crystalline form (e.g., left over from the production of the crystalline form as residual solvent molecules). In a preferred embodiment, water may comprise about 0.5% by weight of the total composition of a sample of the anhydrous form. In a more preferred embodiment, water may comprise about 0.2% by weight of the total composition of a sample of the anhydrous form. In some embodiments, a sample of an anhydrous crystalline form of a compound of Formula (I) does not contain water molecules, e.g., does not contain detectable amounts of water.

[0052] As used herein, a "pharmaceutical composition" refers to a combination of a therapeutically active agent with one or more pharmaceutically acceptable excipients, carriers, or diluents, making the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0053] As used herein, the term "pharmaceutically acceptable excipient, carrier, or diluent" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a pharmaceutical agent of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and refined sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; excipients such as tragacanth powder, malt, gelatin, talc, 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 glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate and polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0054] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, a human, a non-human primate, a rodent, etc., that will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject. "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., males or females of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or the elderly)) and / or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal.

[0055] As used herein, "administering" refers to administration to a subject orally, pulmonary, via suppository, intramuscular, intrathecal, intranasal, or subcutaneously, or via implantation of a sustained-release device, e.g., a mini-osmotic pump. Administration can be by any route, including transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or intramuscular). Parenteral administration includes, for example, intramuscular and subcutaneous. Other delivery modes include, but are not limited to, the use of liposomal formulations. "Coadministration" refers to administration of a composition described herein simultaneously with, immediately prior to, or immediately following administration of one or more additional therapies (e.g., therapeutic agents, chemotherapeutic agents, or treatments for neurodegenerative diseases). Compounds of Formula (I) can be administered alone or simultaneously to a patient. Coadministration refers to simultaneous or sequential administration of compounds, individually or in combination (multiple compounds or agents). Thus, preparations can optionally be combined with other active agents (e.g., to reduce metabolic degradation).

[0056] The terms "disease," "disorder," and "condition" are used interchangeably herein.

[0057] As used herein, the terms "treating," "alleviating," or "preventing" a disease or disorder refer to improving such a condition before or after it occurs. The degree of such reduction or prevention, as compared to an equivalent untreated control, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique. The terms "treat," "treating," and "treatment" contemplate actions that occur while a subject is afflicted with a particular disease, disorder, or condition and that reduce the severity of the disease, disorder, or condition or slow or delay the progression of the disease, disorder, or condition ("therapeutic treatment"), as well as actions that occur before a subject begins to suffer from a particular disease, disorder, or condition ("prophylactic treatment"). In some embodiments, provided herein is intended to be used in therapeutic treatment methods, which act while a subject is suffering from a specific disease, disorder or condition, and result in reducing the severity of the disease, disorder or condition, or delaying or slowing down the progression of the disease, disorder or condition.In another embodiment, provided herein is intended to be used in prophylactic treatment methods, which act before a subject begins to suffer from a specific disease, disorder or condition, and result in preventing the disease, disorder or condition, or one or more symptoms associated with the disease, disorder or condition, or preventing the recurrence of the disease, disorder or condition.

[0058] As used herein, the term "effective amount" of an active agent refers to an amount sufficient to induce a desired biological response.As will be understood by those skilled in the art, the effective amount of the compound of the present invention can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, and the patient.

[0059] Following their preparation, the compounds of the present invention are preferably isolated and purified to obtain compositions containing 95% by weight or greater ("substantially pure"), and then used or formulated as described herein. In certain embodiments, the compounds of the present invention are greater than 99% pure. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention is based in part on compositions and methods of use of BTUDC and combinations of BBR and TUDCA for the treatment of neurodegenerative diseases, particularly Parkinson's disease.

[0061] Berberine (5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium), an isoquinoline alkaloid isolated from plants such as Rhizoma Coptidis, has a long history of medicinal use in China to treat various gastrointestinal disorders.

[0062] [ka]

[0063] Berberine is found in various plants, including Berberis (Barberry), Hydrastis canadensis (Hydratis), Xanthorhiza simplicissima (Hollywood), Phellodendron amurense (Amuraceae), Coptis chinensis (Coptis), Tinospora cordifolia (Tinospora cordifolia), Argemone mexicana (Thistle poppy), and Eschscholzia californica (Calliflower). BBR has been used for digestive ailments, including traveler's diarrhea. It possesses broad-spectrum activity with multiple modes of action. Previous studies on berberine have reported its antiviral, anti-inflammatory, and hepatoprotective properties, as well as its ability to reduce oxidative stress. For example, berberine has demonstrated antiviral activity, including anti-influenza, anti-hepatitis C, anti-cytomegalovirus, and anti-alphavirus. (Neag,et al.2018 “Berberine: Botanical Occurrence,Traditional Uses,Extraction Methods,and Relevance in Cardiovascular,Metabolic,Hepatic,and Renal Disorders” Front.Pharmacol.,21 August Sec.Ethnopharmacology vol.9, “Berberine” Altern.Med.Rev.2000 Apr.5(2):175-7.)

[0064] Berberine is commercially available in the form of chloride, sulfate, or tannate, and berberine hydrochloride has been used in almost all previous studies. The low bioavailability of berberine in currently available forms makes its application for the treatment of chronic and systemic diseases very difficult.

[0065] Tauroursodeoxycholic acid (TUDCA), also known as ursodoxycotaurine or taursodiol, is a naturally occurring hydrophilic bile acid.

[0066] [ka]

[0067] TUDCA has been used to treat chronic cholestatic liver disease and gallstones. Studies have shown that TUDCA can inhibit apoptosis in different cell types by stabilizing mitochondrial membranes or regulating the expression of specific upstream targets of apoptosis. Recently, it was reported that glycine-conjugated TUDCA inhibited nitrite production and prevented the activation of matrix metallopeptidase 9 in a cellular model of superoxide dismutase 1 neurodegeneration. A study was conducted to collect preliminary safety and efficacy data on the long-term biological effects of TUDCA in patients with ALS. (Hofmann 1999 “The continuing importance of bile acids in liver and intestinal disease” Arch.Intern.Med.159:2647-2658, Rodrigues, et al.2001 “The therapeutic effects of ursodeoxycholic acid as an anti-apoptotic agent” Expert Opin.Investig.Drugs 10:1243-1253, Vaz, et al. al. “Glycoursodeoxycholic acid reduces matrix metalloproteinase‐9 and caspase‐9 activation in a cellular model of superoxide dismutase‐1 neurodegeneration” Mol.Neurobiol.2014, Elia, et al.2016 “Tauroursodeoxycholic acid in the treatment of patients with amyotrophic lateral sclerosis” Eur.J.Neurol.23(1):45-52.)

[0068] The BTUDC first discovered by the present applicant was

[0069] [ka] It is an ionic salt of BBR and TUDCA represented by the formula:

[0070] Without wishing to be bound by theory, it is believed that BTUDC and the combination of BBR and TUDCA can alleviate or ameliorate one or more symptoms of neurodegenerative diseases, particularly Parkinson's disease. Furthermore, it is believed that BTUDC and the combination of BBR and TUDCA can effectively alleviate, slow, and / or stop the progression of Parkinson's disease.

[0071] More specifically, the present invention relates to BTUDC, which synergistically combines the beneficial effects of TUDCA and BBR.Therefore, the present invention provides a unique approach for the treatment of neurodegenerative diseases, particularly Parkinson's disease, either alone or in combination with other available treatments or therapies.Therefore, the present invention provides a novel strategy for the treatment of neurodegenerative diseases, particularly Parkinson's disease.

[0072] In one aspect, the present invention generally relates to a compound of formula (I):

[0073] [ka] The present invention relates to a salt having the formula:

[0074] In certain embodiments, the BTUDC salt is in substantially pure form.

[0075] In certain embodiments, the BTUDC salts are characterized by a purity of about 90% or greater (eg, about 95%, about 98%, about 99%).

[0076] In certain embodiments, the BTUDC salt is made by an acid-base reaction between BBR (or a salt thereof) and TUDCA (or a salt thereof).

[0077] In another aspect, the invention generally relates to a solid form of the compound of Formula (I), wherein the solid form is Form A, the X-ray powder diffraction (XRPD) pattern of which comprises one or more characteristic diffraction peaks at the following 2θ angles using a Cu-Kα radiation source: 4.62°, 9.32°, and 17.02°±0.2°.

[0078] In certain embodiments, the XRPD pattern of the solid form further comprises one or more characteristic diffraction peaks at the following 2θ angles with a Cu-Kα radiation source: 5.96°, 6.23°, 15.19°±0.2°.

[0079] In certain embodiments, the XRPD pattern of the solid form comprises characteristic diffraction peaks at the following 2θ angles using a Cu-Kα radiation source: 4.62°, 5.96°, 6.23°, 9.32°, 15.19°, and 17.02°±0.2°.

[0080] In certain embodiments, the XRPD pattern of the solid form further comprises one or more characteristic diffraction peaks at 11.99°, 12.56°, 12.90°±0.2° 2θ angles from a Cu-Kα radiation source.

[0081] In certain embodiments, the XRPD pattern of the solid form comprises characteristic diffraction peaks at the following 2θ angles using a Cu-Kα radiation source: 4.62°, 5.96°, 6.23°, 9.32°, 11.99°, 12.56°, 12.90°, 13.32°, 14.25°, 14.88°, 15.19°, 17.02°, 17.51°, 17.73°, 18.02°, 21.39°, 24.25°, and 24.71°±0.2°.

[0082] In certain embodiments, the XRPD pattern of the solid form includes characteristic diffraction peaks at the following 2θ angles:

[0083] [Table 1-1] [Table 1-2]

[0084] In certain embodiments, the XRPD pattern of the solid form is essentially the same as that shown in FIG. 11 with a Cu-Kα radiation source.

[0085] In certain embodiments, the differential scanning calorimetry (DSC) curve of Form A comprises an endothermic peak having a peak value at about 280°C.

[0086] In some embodiments, the thermogravimetric analysis (TGA) curve for Form A comprises a weight loss of about 0.5% to about 3% from room temperature to about 150°C.

[0087] In some embodiments, the solid form is anhydrous.

[0088] In one embodiment, the solid form is a hydrate having the formula (II): In one embodiment, the solid form is a hydrate having up to two HO molecules per BTUDC molecule (i.e., BTUDC:HO = 1:x, where x is a number ranging from 0 to 2, preferably from 0.3 to 1.5, and more preferably from 0.5 to 1.2).

[0089] [ka]

[0090] In certain embodiments, solid forms include hydrates and anhydrates.

[0091] In some embodiments, the solid form is a crystalline form.

[0092] In another aspect, the invention generally relates to pharmaceutical compositions comprising a BTUDC and a pharmaceutically acceptable excipient, carrier, or diluent.

[0093] In another aspect, the invention generally relates to pharmaceutical compositions comprising the solid forms disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0094] In another aspect, the invention generally relates to unit doses comprising pharmaceutical compositions of BTUDCs disclosed herein.

[0095] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating a neurodegenerative disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BTUDC.

[0096] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating Parkinson's disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BTUDC.

[0097] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating a neurodegenerative disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BBR and TUDCA.

[0098] In another aspect, the invention generally relates to a method for alleviating, preventing, or treating Parkinson's disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising BBR and TUDCA.

[0099] In certain embodiments, the method results in a reduction or amelioration of one or more symptoms of Parkinson's disease.

[0100] In certain embodiments, the method results in amelioration of the effects of pathology of the substantia nigra.

[0101] In certain embodiments, the method results in amelioration of chronic neuroinflammation.

[0102] In certain embodiments, the method results in a slowing of the progression of Parkinson's disease.

[0103] In certain embodiments, the method results in the halting of the progression of Parkinson's disease.

[0104] In certain embodiments, the method results in a reversal of the progression of Parkinson's disease.

[0105] In certain embodiments, the method further comprises administering to the patient a second therapeutic agent.

[0106] In certain embodiments, the second therapeutic agent is selected from levodopa (L-DOPA), an MAO-B inhibitor, and a dopamine agonist.

[0107] In yet another aspect, the invention relates generally to the use of BTUDCs to treat neurodegenerative diseases or related diseases or disorders.

[0108] In yet another aspect, the invention relates generally to the use of BTUDCs to treat Parkinson's disease or a related disease or disorder.

[0109] In yet another aspect, the invention generally relates to the use of BTUDC for the manufacture of a medicament for preventing or treating a neurodegenerative disease or related disease or disorder.

[0110] In yet another aspect, the invention generally relates to the use of BTUDC for the manufacture of a medicament for preventing or treating Parkinson's disease or a related disease or disorder.

[0111] In another aspect, the invention generally relates to the use of BBR and TUDCA to treat neurodegenerative diseases or related diseases or disorders.

[0112] In another aspect, the invention generally relates to the use of BBR and TUDCA to treat Parkinson's disease or a related disease or disorder.

[0113] In another aspect, the invention generally relates to the use of BBR and TUDCA for the manufacture of a medicament for preventing or treating a neurodegenerative disease or related disease or disorder.

[0114] In another aspect, the invention generally relates to the use of BBR and TUDCA for the manufacture of a medicament for preventing or treating Parkinson's disease or a related disease or disorder.

[0115] In certain embodiments of the methods disclosed herein, BTUDC is administered at a daily dose ranging from about 25 mg to about 3,500 mg for a period ranging from about one week to about two years.

[0116] In certain embodiments of the methods disclosed herein, the total amount of BBR and TUDCA is administered in daily doses ranging from about 25 mg to about 3,500 mg for a period ranging from about 1 week to about 2 years.

[0117] In one embodiment, the weight ratio of BBR to TUDCA ranges from about 10:1 to about 1:10.

[0118] In another aspect, the invention generally relates to a method for making the BTUDC salt disclosed herein, comprising dissolving tauroursodeoxycholic acid in ethanol, adding an aqueous solution of NaHCO to obtain a sodium tauroursodeoxycholic acid solution, dissolving berberine hydrochloride in warm water to obtain a berberine hydrochloride solution, adding the berberine hydrochloride solution dropwise to the sodium tauroursodeoxycholate solution, stirring the combined solution at about 60°C to about 80°C, and cooling the combined solution to obtain berberine tauroursodeoxycholate. In one embodiment, the method further comprises crystallizing the berberine tauroursodeoxycholate.

[0119] In another aspect, the present invention generally relates to a method for preparing the solid forms disclosed herein, the method comprising the steps of adding an aqueous solution of sodium tauroursodeoxycholate to an aqueous solution of berberine chloride to form a combination solution, mixing the combination solution, and cooling the mixed combination solution to obtain the solid form.

[0120] In another aspect, the present invention generally relates to a method for preparing the solid forms disclosed herein, the method comprising the steps of adding an aqueous solution of tauroursodeoxycholic acid to an aqueous solution of berberine chloride to form a combination solution, mixing the combination solution, and cooling the mixed combination solution to obtain the solid form.

[0121] Possible formulations include those suitable for oral, sublingual, buccal, parenteral (e.g., subcutaneous, intramuscular, or intravenous), rectal, transdermal, intranasal, and topical use, including inhalation administration. The most suitable means of administration for a particular patient will depend on the nature and severity of the disease or condition being treated, or the nature of the therapy being used, and the nature of the active compound. [Example]

[0122] Characterization Methods nuclear magnetic resonance (NMR) NMR characterization was performed on a Bruker AVANCE NEO 400 (Bruker, Germany). Samples were prepared by dissolving 3–5 mg of solid in methanol-d4.

[0123] X-ray powder diffraction (XRPD) Standard XRPD patterns were collected using a Panalytical EMPYREAN (PANalytical, UK). The X-ray source was a Cu tube operated at 45 kV and 40 mA. Powder samples were prepared in a zero-background Si holder using manual light pressure to keep the sample surface level. Each sample was analyzed from 3 to 40° (2θ) with an effective step size of 0.013° 2θ. The measurement time for each sample was 3.5 min.

[0124] The thermal transformation was investigated using an online variable-temperature XRPD Malvern PANalytical Aeris (Malvern Panalytical, UK). The X-ray source was a Cu tube operated at 40 kV and 7.5 mA. Powder samples were prepared in a zero-background Si holder using manual light pressure to keep the sample surface level. Each sample was analyzed from 3 to 40° (2θ) with an effective step size of 0.02° 2θ. The measurement time for each sample was 13 min. The samples were placed on a BTS500 hot stage (Anton Paar, AT) and XRPD was collected at RT. They were heated to the target temperature at 20 °C / min and maintained for 10 min before XRPD analysis. The samples were then cooled to room temperature and characterized by XRPD.

[0125] Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) was performed on a TA Instruments Discovery 550 (TA, USA). Each sample was placed in a pre-tared platinum pan and heated from room temperature to the set temperature at a heating rate of 10 °C / min under a nitrogen atmosphere. The nitrogen purge was 40 mL / min for the balance and 60 mL / min for the furnace.

[0126] Differential scanning calorimetry (DSC) DSC analyses were performed on a TA Instruments Discovery 250 (TA, USA). Indium was used to calibrate the instrument temperature and cell constant. The DSC cell was maintained under a 50 mL / min nitrogen purge during each analysis. Samples were placed on a Tzero sealed pan with a pinhole and heated from 25°C to the set temperature at a rate of 10°C / min.

[0127] Polarized Light Microscopy (PLM) Polarized light microscopy images of the crystals were taken using a Nikon Ci-POL445 polarized light microscope (Nikon, Japan) under appropriate objective lenses. For some samples, oil was used for observation.

[0128] Single Crystal X-ray Diffraction (SCXRD) SCXRD was collected using an XtaLAB Synergy R, DW system, HyPix. The X-ray source was Cu Kα (λ = 1.54184 Å). A suitable single crystal was selected and mounted on a glass fiber. The crystal was maintained at a steady temperature of 296 K during data collection. Preliminary testing and data collection were performed and analyzed using the CrysAlisPro software package.

[0129] Cell parameters and orientation matrices for data collection were retrieved and refined by CrysAlisPro using set angles for 33656 reflections in the range 2.32° < θ < 76.01°. Final data completeness was 99.95% (θ = 66.97°).

[0130] Data Reduction Frames were integrated using CrysAlisPro 1.171.42.84a (Rigaku Oxford Diffraction, 2023). A total of 56,856 reflections were collected, of which 8,736 were unique. Multiscan absorption correction was performed using spherical harmonics as implemented in the SCALE3 ABSPACK scaling algorithm. The absorption coefficient μ of this material is 1.201 mm. -1 and the minimum and maximum transmittances are 0.7952 and 0.8893. The Rint value was 5.26% based on intensity.

[0131] Single crystal structure determination and refinement The structure was determined in space group P21 using the intrinsic phasing method in the SHELXS-97 (Sheldrick, 1990) structure determination program, using Olex2 as the graphical interface. 2 Refinement was performed with a version of SHELXS-97 (Sheldrick, 1990) using full-matrix least-squares based minimization. All non-hydrogen atoms were refined anisotropically. All hydrogen atom positions were calculated geometrically and refined using a riding model.

[0132] Single crystal structure diagram Crystal structure representations and thermal ellipsoid diagrams were generated by Olex2.

[0133] Example 1. Synthesis of BTUDC (Method 1) and Characterization Tauroursodeoxycholic acid (1 equivalent) was dissolved in ethanol, and an aqueous solution of NaHCO3 (0.95 to 1.5 equivalents) was added and stirred for 15 to 60 minutes to obtain a sodium tauroursodeoxycholate solution.

[0134] Berberine hydrochloride was dissolved in hot water. The berberine hydrochloride solution was added dropwise to the sodium tauroursodeoxycholate solution at 60-80°C and stirred for 10 minutes or more to allow the reaction to proceed at 60-80°C. The solution was cooled, filtered, and the precipitate was dried to obtain berberine tauroursodeoxycholate.

[0135] Resulting BTUDC example 1 The H NMR spectra are provided in Figure 1. For comparison, Figures 2 and 3 show the H NMR spectra of TUDCA and BBR chloride. 1 H NMR spectra are provided for each.

[0136] Single crystal growth and structure confirmation The resulting BTUDC sample was added to methanol / water (1 / 1, v / v) and stirred until the solid was completely dissolved. The mixture was then filtered, and the resulting clear solution was left at room temperature to evaporate, yielding a solid. PLM images showed the solid to be clustered needle-like crystals. The resulting needle-like crystals were examined by single-crystal X-ray diffraction (SCXRD), and the diffraction data were analyzed to obtain the crystal structure. The crystallographic data and refinement parameters of the crystal are detailed in Table 2. A schematic diagram of the asymmetric structural unit is shown in Figure 4. Figure 5 shows a schematic diagram of the unit cell of a BTUDC single crystal.

[0137] [Table 2]

[0138] The results indicate that the crystal is monoclinic and belongs to the P21 space group, with lattice parameters of {a = 15.42465(15) Å, b = 7.35920(6) Å, c = 19.9879(2) Å, α = 90°, β = 109.5641(12)°, γ = 90°, V = 2137.90(4) Å}. The single crystal structure indicates that the asymmetric structural unit of this crystal structure contains a 1:1 berberine salt of tauroursodeoxycholic acid forming one salt and two water molecules, with the occupancies of the two water molecules being 0.45 (O11) and 0.38 (O12), respectively. The chemical structure of this crystal is shown below. Analysis results indicate that the hydrate has a non-fixed stoichiometry, and the number of water molecules can vary within a certain range, for example, between 0 and 2.

[0139] [ka]

[0140] Example 2. Synthesis of BTUDC (Method 2) and Characterization Tauroursodeoxycholic acid (1 equivalent) was added to water and stirred until dissolved. NaHCO3 aqueous solution (0.95-1.5 equivalents) was added and stirred for at least 10 minutes to obtain a sodium tauroursodeoxycholic acid solution.

[0141] Berberine hydrochloride was dissolved in hot water. Sodium tauroursodeoxycholate solution was added at 60-80°C, and the mixture was stirred for 10 minutes or more to react at 60-80°C. The solution was cooled. The mixture was filtered. The filter cake was washed and dried to obtain berberine tauroursodeoxycholate.

[0142] Resulting BTUDC example 1 The 1 H NMR spectrum is provided in Figure 30.

[0143] Characterization of crystal morphology The synthesized BTUDC was designated crystalline form A (form A), and its XRPD diffraction and other relevant characterization data are shown in Figures 11-15. PLM images showed that form A consisted of short, rod-like particles generally less than 10 μm in size, and XRPD results indicated that it was a crystalline solid. TGA results indicated a 1.2% weight loss during the heating process to 150 °C, suggesting possible decomposition above 300 °C. DSC results indicated a broad endothermic signal from room temperature to 80 °C (peak at 43 °C) and an endothermic peak at approximately 280 °C. Thermal transcrystallization experiments showed that the crystalline form remained unchanged during heating to 150 °C and during the return to room temperature, and NMR results indicated that the sample was consistent with the reference pattern. Rapid dynamic vapor sorption (DVS) results showed an adsorption weight gain of approximately 0.74% at 95% RH, an adsorption weight gain of approximately 0.46% at 80% RH, and a desorption weight gain of approximately 0.49%. XRPD results showed that the sample did not undergo any changes in crystalline form after the DVS test compared to the preliminary test. KF titration results showed that the sample batch had a moisture content of 2.4%.

[0144] XRPD characterization of the BTUDC raw material and single crystal sample obtained from Example 1 was performed, and the results are shown in Figures 31 and 16, respectively. Based on a comparison of the XRPD results, it can be confirmed that the single crystal sample of Example 1 is of the same crystalline type as Form A. Therefore, from the characterization results of Form A, and combined with the structural analysis of the single crystal structure of BTUDC, it was determined that Form A is a hydrate with a non-fixed stoichiometry. Other relevant characterization data for the BTUDC raw material obtained from Example 1 are shown in Figures 32-33.

[0145] The stability of Form A was tested under high temperature (60°C), high humidity (25°C / 92.5% RH), light (25°C / 4500 Lux), and accelerated conditions (40°C / 75% RH). Samples were taken on days 7 and 15 for XRPD characterization and HPLC testing, respectively. The results are shown in Tables 3-4 and Figure 17. The XRPD results indicated that Form A was stable under high temperature, high humidity, light, and accelerated conditions for 15 days without undergoing a crystalline transition. The HPLC results indicated that there was no significant change in the chemical purity of Form A when it was exposed to high temperature, high humidity, and accelerated conditions for 15 days, and that there was a significant decrease in the chemical purity of Form A when it was exposed to light for 15 days.

[0146] [Table 3]

[0147] [Table 4]

[0148] Information on sample preparation for animal testing raw material

[0149] [Table 5]

[0150] Formulation information Preparation of 0.5% sodium carboxymethylcellulose (0.5% CMC-Na): 0.5 g of CMC-Na was transferred to purified water, stirred until a clear solution was obtained, and water was added to a final volume of 100 mL.

[0151] [Table 6]

[0152] Preparation of PD modeling agent: 20 μg of 6-hydroxydopamine (6-OHDA) was dissolved in 8 μL of 0.9% saline (containing 0.02% ascorbic acid).

[0153] Animal models The rats were anesthetized with isoflurane, the head was fixed to ensure no movement, the brain surface was adjusted to be flat, the head was shaved, a skin incision was made along the sagittal suture to expose the fontanel point, a glass electrode was placed on the fontanel, the axes of the coordinate monitor were zeroed, and the brain regions of the SN and Str were located based on the coordinates, where the SN was AP = -5.0 mm; ML = -1.9 mm; DV = -8.5 mm, and the Str was AP = The needle was slowly inserted into the SN and striatum (AP = +0.5 mm; ML = -3.0 mm; DV = -6.0 mm, respectively; Stria: AP = +0.5 mm; ML = -3.0 mm; DV = -6.0 mm). After waiting for 10 minutes, 6-OHDA was administered at a rate of 0.4 μL / min for 10 minutes. 4 μL of 6-OHDA was administered into the SN and striatum of each animal. After 10 minutes of administration, the needle was slowly removed. Information on surgical PD modeling methods can also be found in the literature, showing the injection sites in the SN and striatum (Sokoudi, et al. 2022 Physiol. Res. 71(4):551-560; Haddadi, et al. 2013 Neuroscience Letters 555, 106-111).

[0154] Test Method 1. Apomorphine-Asymmetric Rotation Test The animals were tested 3 weeks after administration. 0.5 mg / kg apomorphine was injected intraperitoneally. The number of rotations per rat was recorded for 30 minutes, and the rats appeared to rotate in place toward the contralateral side of the injury, using their forelimbs as a support point for rotation. Information on the apomorphine asymmetric rotation test can also be found in the literature (Ximenes, et al. 2015 J Neurodegener Dis. 2015:313702).

[0155] 2. Balance beam test Rats were placed on a balance beam. The time it took for the rat to cross the balance beam and the number of foot slips (when the foot left the top of the balance beam) were recorded. The average time between two successful crossings and the number of foot slips were used as the assessment criteria for the rat's motor balance. Information about the beam test can also be found in the literature (Allbutt, et al. 2007 J Neurosci Methods 159(2):195-202; Fine, et al. 2014 Brain Res. 1574:96-104).

[0156] 3. Grip strength test The animal was placed on the platform with both forelimbs on the gripping pole, and then the tail was grasped and pulled straight until the pulling force exceeded its grip strength. After the animal lost its grip, the preamplifier automatically recorded the maximum pulling force. After the measurements were completed, the average maximum pulling force for each animal group was calculated to evaluate the motor muscle strength of the rats. Information about the grip strength test can also be found in the literature (Jeyasingham, et al. 2001 Brain Res Bull. 55(4):541-8).

[0157] 4. Rotarod Test The rotation speed of the rotorod fatigue meter was set at 20 revolutions per minute for a 5-minute test. Animals were placed in batches on the rotorod for testing, and the time each animal spent on the rod was recorded to assess the rats' motor coordination. Information about the rotorod test can also be found in the literature (Bohlen, et al. 2009 J Neurosci Methods 178(1):10-4).

[0158] 6. Perfusion-fixed Sugar-precipitated Slices (Str / SN Brain Regions) After anesthetizing the rats, the abdominal cavity and chest were opened with straight scissors to expose the heart. Blood was first flushed out with normal saline perfusion, followed by paraformaldehyde for initial perfusion fixation. The head was removed, the braincase carefully opened with tweezers, and the brain tissue was removed and placed in paraformaldehyde solution for 24 hours. On day 2, the brain tissue was removed and placed in 20% sucrose solution for 24 hours. On day 3, the brain tissue was removed and placed in 30% sucrose solution for 24 hours. On day 4, the brain tissue was removed and placed in 35% sucrose solution for 24 hours (the time or concentration was increased appropriately depending on the state of sugar deposition in the brain tissue). The brain tissue was then removed, embedded in OCT embedding medium, and 16 μm-thick brain sections were cut using a freezing microtome.

[0159] 7. Immunofluorescence staining (TH, Iba-1) Immunohistochemical staining for TH and Iba-1 was performed on the above sections. The sections were rewarmed for 30 minutes, then sealed with 50 μL of 50% serum + 0.3% Triton X-100 per slice for 1 hour at room temperature. The slices were shaken to remove the liquid, and primary antibody (diluted in PBS) was added. The sections were then rewarmed for 30 minutes, rinsed three times with the primary antibody (PBS) for 5 minutes, and placed at 4°C overnight. Secondary antibody (diluted in PBS) was added under backlighting and placed at room temperature for 2 hours. The secondary antibody (diluted in PBS) was then rinsed three times with the secondary antibody (PBS) for 5 minutes. 4',6-diamidino-2-phenylindole (DAPI) was added and incubated at room temperature for 10 minutes. The DAPI was then rinsed three times with 5 minutes. 70% glycerol was added to prevent air bubbles from forming, and the slices were then sealed and observed under a microscope. The same target brain region was selected for each slice. The number or area of ​​signals across the entire slice was then counted using ImageJ.

[0160] 8. Elisa test (IL-6, IL-1β, TNF-α) For ELISA testing, cerebrospinal fluid was directly extracted by passing a needle through the large hole, and the supernatant was collected after centrifugation. The kit brand was Shanghai Enzyme Link. The product codes for IL-6, IL-1β, and TNF-α were ml064292, ml037361, and ml002859, respectively. Samples were processed according to the ELISA detection kit instructions, and blank wells were adjusted to zero. The absorbance (OD value) of each well was measured sequentially at a wavelength of 450 nm. Sample concentrations were calculated based on the standard curve and OD values.

[0161] 9. Statistics SPSS software was used for statistical analysis (one-way analysis of variance, p<0.05 was considered significant), and GraphPad software was used to draw images based on the SPSS analysis results. Adobe Photoshop software was used to generate histological examination results. Highly significant difference (p<0.001); significant difference (p<0.01); statistically significant difference (p<0.05).

[0162] Example 2. Tolerability and efficacy studies using a 6-OHDA injection-induced rat model of Parkinson's disease (PD) Animal model: The PD model is established by stereotactic injection of 6-OHDA into the brain of Sprague-Dawley rats.

[0163] 1. Method In this study, 16 male Sprague Dawley (SD) rats weighing 180–220 g were used to establish a PD model by stereotactic unilateral injection of 6-OHDA into the substantia nigra (SN) and striatum (Str) brain regions after 1 week of acclimation feeding. They were then randomly divided into two groups of 8 rats each. One group received the test compound BTUDC 500 mg / kg (QD, oral), while the other group, the model group, received the same volume of 0.5% CMC-Na (QD, oral) as the test compound group. Starting on day 2 after model establishment, the animals were administered BTUDC once daily for 21 consecutive days to evaluate the tolerability and therapeutic potential of the 6-OHDA PD model.

[0164] 2. General condition observation Body weights and food intake of the rats were collected and recorded daily, and cageside observations of the animals were performed.

[0165] 3. Behavioral Testing After 21 days of administration, the following tests were performed: apomorphine-asymmetric rotation test, balance beam test, grip strength test and rotarod test.

[0166] 4.Results All animals in each group remained in good condition throughout the study, with no abnormal or accidental deaths. Both the drug-treated and model groups showed weight loss on Day 1 after modeling due to surgical trauma, but steadily increased from Day 2 after gradual recovery from the trauma. Weight changes were similar between the two groups. Food intake steadily increased on Day 4 after modeling in both the drug-treated and model groups and stabilized from Day 5 onwards.

[0167] Tolerability and efficacy Figures 6-10 show exemplary results of various experiments, including ipsilateral rotation, time on beam, foot slip on beam, time on rotarod, and grip strength tests.

[0168] On the 21st day after the stereotaxic injection of 6-OHDA, all rats showed in situ rotational behavior, using their forelimbs as a support point to rotate toward the contralateral side of the injury after apomorphine (APO) induction, indicating successful establishment of the model. There was no statistically significant difference in the number of ipsilateral rotations within 30 minutes between the test compound group and the model group (p>0.05).

[0169] [Table 7]

[0170] There were no significant differences in body weight and food intake data between the model control group and the test compound-treated group throughout the entire test period. Furthermore, compared with the model group, the test compound-treated group showed no test compound-related deaths, moribundity, or abnormal animal conditions, indicating that the test compound was well tolerated in the PD rat model. Compared with the model group, after 21 days of continuous treatment with the test compound, rats in the test compound group showed a significant decrease in crossing time (p<0.001) and number of foot slips (p<0.001) in the balance beam test, a significant increase in rod time (p<0.001) in the rotarod test, and a significant increase in peak grip strength (p<0.001) in the grip strength test.

[0171] [Table 8]

[0172] This study demonstrated that the PD rat model tolerated the test compound well, and the test compound showed beneficial effects on Parkinson's disease model animals induced by stereotactic injection of 6-OHDA into the substantia nigra and striatum of rats.

[0173] Example 3. Pharmacodynamic studies in Parkinson's disease Animal Model: The PD model is established by stereotactic injection of 6-OHDA into the brain of Sprague-Dawley rats.

[0174] Experimental Method: Seventy male SD rats weighing 180-220 g were selected and fed for 1 week for accrual. PD models were established by stereotaxic unilateral injection of 6-OHDA in the substantia nigra (SN) and striatum (Str). They were randomly divided into five groups of 10 animals each, including Madopar (50 mg / kg), BTUDC-L (100 mg / kg), BTUDC-M (500 mg / kg), and BTUDC-H (1000 mg / kg). Another 10 SD male rats were used as a sham group (4 μL of 0.9% NS containing 0.02% ascorbic acid was administered to the SN and striatum during modeling). Drugs were administered by gavage once daily for 21 consecutive days, starting on the second day of modeling (day 1 of administration, D1). General observations were conducted throughout the administration period. At the end of administration, the test substances were evaluated for tolerability in this model. To assess the pharmacodynamic effects of the test substances, behavioral tests (apomorphine asymmetric rotation test, beam test, rotarod test, and grip strength test), immunofluorescence staining (TH, Iba1), and cerebrospinal fluid inflammatory factor assays were performed. The inflammatory factors tested included interleukin-6 (IL-6), inflammatory factors interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α). The inflammatory assays were performed in a total of four groups, including sham, model, Madopar, and BTUDC-H groups.

[0175] [Table 9]

[0176] 1. General observations: Body weight, food intake test, and cageside observations were performed every 3 days during the drug administration period.

[0177] 2. Behavioral test: After 21 days of administration, behavioral tests were conducted the next day.

[0178] 3. Histochemical test: After the behavioral test, cerebrospinal fluid was collected from each animal (N=10), centrifuged, and the supernatant was collected and stored at -80°C for ELISA tests (IL-6, IL-1β, TNF-α). Then, animals from each group (N=5) were randomly selected to collect the modeled side of the striatum for neurotransmitter detection. The remaining (N=5) were subjected to cardiac perfusion, and processed through the procedures of perfusion, fixation, sedimentation, and slicing for immunofluorescence staining, photographed, and the number or area of ​​signals was counted using ImageJ software.

[0179] 4. Data analysis: After data were tabulated and counted, they were analyzed using SPSS data statistical software (one-way analysis of variance, p<0.05 was considered significant), and images were plotted using Graph Pad software according to the SPSS analysis results. Histological examination results were generated using Adobe Photoshop software.

[0180] result The results of the rotation test for the APO-induced PD model rats are shown in Figure 18. After APO induction, rats in each treatment group and the model group exhibited in-place rotation toward the contralateral side of the injury using their forelimbs as a pivot point. Compared with the sham group, rats in the model group rotated in a circular pattern within 30 minutes after apomorphine injection. The number of rotations significantly increased (p<0.001), suggesting successful establishment of the model. Compared with the model group, rats in the BTUDC-H group that received apomorphine injection had significantly fewer circular rotations within 30 minutes, indicating that BTUDC has the potential to protect neurons in the brain.

[0181] [Table 10]

[0182] The results of the grip strength test of PD model rats are shown in Figure 19 and Table 10. In comparison, the peak grip strength of rats in each treatment group (G3 to G6) was significantly higher than that of the model group, and the difference was extremely significant (p<0.001). The groups administered different doses of BTUDC improved grip strength in a dose-dependent manner.

[0183] The results of the rotarod test in PD rats are shown in Figure 20. The rod time of rats in each administration group (G3 to G6) was significantly higher than that of the model group, and the effect of different BTUDC doses on improving rod time was dose-dependent. In particular, the rod time of the medium- and high-dose BTUDC groups was statistically higher than that of the model group (p<0.001).

[0184] In the balance beam test, the results of the time taken by PD rats to pass the balance beam are shown in Figure 21. Compared with the model group, the time taken by rats in each administration group (G3 to G6) to pass the balance beam was significantly shorter than that of the model group (p<0.01), and the improving effect of different doses of BTUDC on the time taken to pass the balance beam was dose-dependent.

[0185] The results of the number of foot slips in PD rats in the beam test are shown in Figure 22. Compared with the model group, the number of times rats in each treatment group (G3 to G6) slipped on the balance beam was significantly less than that in the model group (p<0.05), and the improving effect of different doses of BTUDC on the number of slips rats made on the balance beam was dose-dependent.

[0186] [Table 11]

[0187] The results of TH staining are shown in Figure 23. The results of TH staining for fluorescence intensity in the Str brain region of rats after 21 days of administration are shown in Table 11 and Figure 24. The results of TH staining for the number of positive cells in the SN brain region of rats after 21 days of administration are shown in Table 11 and Figure 25. Compared with the sham group, the TH staining for fluorescence intensity in the Str brain region and the number of TH-positive cells in the SN brain region were significantly lower in the model group rats, and the differences were highly significant (p<0.001). Compared with the model group, the TH staining fluorescence intensity in the Str brain region of rats in each treatment group (G4-G6) was significantly higher than that of the model group (p<0.05), and the number of TH-positive cells in the SN brain region of the BTUDC-H group was significantly higher than that of the model group (p<0.001). The ameliorative effects of BTUDC on TH fluorescence intensity in the Str brain region and the number of TH-positive cells in the SN brain region of 6-OHDA rats were dose-related. These findings suggest that BTUDC has a beneficial effect on improving substantia nigra lesions and has a neuroprotective effect.

[0188] The results of Iba-1 staining are shown in Figure 26. The results of Iba-1 staining for the number of positive cells in the Str brain region of rats after 21 days of treatment are shown in Figure 27. The results for the number of positive cells are shown in Figure 28. Compared with the model group, the number of Iba1-positive cells in the Str brain region of rats in each treatment group (G4-G6) was significantly lower than that in the model group (p<0.05). The number of Iba1-positive cells in the SN brain region of the BTUDC-H group was significantly lower than that in the model group (p<0.001). The ameliorative effect of BTUDC on the number of Iba1-positive cells in the Str and SN brain regions of 6-OHDA rats showed a clear dose-correlation, indicating that BTUDC has the potential to ameliorate chronic neuroinflammation and has neuroprotective effects.

[0189] [Table 12]

[0190] The results of the ELISA test for IL-1β in rat cerebrospinal fluid after 21 days of administration are shown in Table 12 and FIG.

[0191] The results of the ELISA test for IL-6 in the cerebrospinal fluid of rats after 21 days of administration are shown in Table 12 and Figure 30.

[0192] The results of the ELISA test for TNF-α in the cerebrospinal fluid of rats after 21 days of administration are shown in FIG.

[0193] In summary, BTUDC was well tolerated by animals up to 1000 mg / kg, with no obvious abnormalities in weight gain or food intake compared with animals in other groups. BTUDC demonstrated beneficial effects in various behavioral tests, such as grip strength, rotarod, and balance beam. The beneficial effects were dose-dependent, with the improvements in the medium and high-dose groups achieving statistical significance. Furthermore, BTUDC also increased the number and intensity of tyrosine hydroxylase-positive cells and reduced the number of microglia. BTUDC dose-dependently improved the fluorescence intensity of tyrosine hydroxylase (TH)-positive cells in the str brain region. The high-dose BTUDC group significantly increased the number of TH-positive cells in the sn brain region. All BTUDC-treated groups significantly reduced the number of microglia (Iba1) in the str brain region. The high-dose BTUDC group significantly reduced the number of microglia (Iba1) in the sn brain region.

[0194] Applicant's disclosure is described herein in preferred embodiments with reference to the drawings, wherein like numbers represent the same or similar elements. Throughout this specification, reference to "one embodiment," "one embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment," "in one embodiment," and similar language throughout this specification can, but do not necessarily, all refer to the same embodiment.

[0195] The described features, structures, or characteristics of Applicant's disclosure may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present invention. However, one skilled in the art will recognize that Applicant's compositions and / or methods may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the present disclosure.

[0196] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described here. The methods described herein can be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0197] Incorporation by Reference This disclosure makes references and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes. Any material or portion thereof that is incorporated herein by reference but that conflicts with existing definitions, descriptions, or other disclosure material explicitly set forth herein is incorporated only to the extent that no conflict arises between the incorporated material and the presently disclosed material. In the event of a conflict, the conflict shall be resolved in favor of the present disclosure as the preferred disclosure.

[0198] equivalent The representative examples are intended to aid in the explanation of the invention and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the examples and references to the scientific and patent literature contained herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. Formula (I) 【Chemistry 1】 A salt having the formula:

2. Formula (I) 【Chemistry 2】 The salt in substantially pure form is represented by:

3. The salt according to claim 2, characterized by a purity of 95% or more.

4. The salt according to any one of claims 1 to 3, prepared by an acid-base reaction between berberine (BBR) and tauroursodeoxycholic acid (TUDCA).

5. Formula (I) 【Transformation 3】 wherein said solid form is Form A, the X-ray powder diffraction (XRPD) pattern of which comprises one or more characteristic diffraction peaks at the following 2θ angles with a Cu—Kα radiation source: 4.62°, 9.32°, 17.02°±0.2°.

6. 6. The solid form of claim 5, wherein the XRPD pattern of the solid form further comprises one or more characteristic diffraction peaks at the following 2-theta angles with a Cu-Kα radiation source: 5.96°, 6.23°, 15.19°±0.2°.

7. 6. The solid form of claim 5, wherein the XRPD pattern of the solid form comprises characteristic diffraction peaks at the following 2-theta angles with a Cu—Kα radiation source: 4.62°, 5.96°, 6.23°, 9.32°, 15.19°, 17.02°±0.2°.

8. 8. The solid form of claim 7, wherein the XRPD pattern of the solid form comprises one or more characteristic diffraction peaks at the following 2-theta angles with a Cu-Kα radiation source: 11.99°, 12.56°, 12.90°±0.2°.

9. 6. The solid form of claim 5, wherein the XRPD pattern of the solid form comprises characteristic diffraction peaks at the following 2-theta angles with a Cu—Kα radiation source: 4.62°, 5.96°, 6.23°, 9.32°, 11.99°, 12.56°, 12.90°, 13.32°, 14.25°, 14.88°, 15.19°, 17.02°, 17.51°, 17.73°, 18.02°, 21.39°, 24.25°, 24.71°±0.2°.

10. 10. The solid form of any one of claims 5 to 9, wherein a differential scanning calorimetry (DSC) curve of said solid form comprises an endothermic peak having a peak value at about 280°C.

11. 11. The solid form of any one of claims 5 to 10, wherein a thermogravimetric analysis (TGA) curve of the solid form comprises a weight loss of about 0.5% to 3% from room temperature to about 150°C.

12. The solid form is a 1:X BTUDC:H ２ 12. A solid form according to any one of claims 5 to 11, characterized by a 0 ratio, where X is a number ranging from 0 to 2.

13. 13. The solid form of claim 12, which is anhydrous.

14. The solid form may contain up to two H per BTUDC molecule. ２ 13. The solid form of claim 12, which is a hydrate having an O molecule.

15. 15. The solid form of any one of claims 5 to 14, which is in a crystalline form.

16. A pharmaceutical composition comprising the salt of any one of claims 1 to 4 and a pharmaceutically acceptable excipient, carrier, or diluent.

17. A pharmaceutical composition comprising the solid form of any one of claims 5 to 15 and a pharmaceutically acceptable excipient, carrier, or diluent.

18. 18. A unit dose containing the pharmaceutical composition of claim 16 or 17.

19. 10. A method for alleviating, preventing, or treating a neurodegenerative disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising the salt of any one of claims 1 to 4.

20. 16. A method for alleviating, preventing or treating a neurodegenerative disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising the solid form of any one of claims 5 to 15.

21. 10. A method for alleviating, preventing, or treating Parkinson's disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising the salt of any one of claims 1 to 4.

22. 16. A method for alleviating, preventing, or treating Parkinson's disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising the solid form of any one of claims 5 to 15.

23. 23. The method of claim 21 or 22, wherein one or more symptoms of Parkinson's disease are reduced or ameliorated.

24. 24. The method of claim 23, wherein the progression of Parkinson's disease is slowed.

25. 24. The method of claim 23, wherein the progression of Parkinson's disease is halted.

26. 24. The method of claim 23, wherein the progression of Parkinson's disease is reversed.

27. 27. The method of any one of claims 19-26, wherein the subject is administered a daily dose of about 25 mg to about 3,500 mg.

28. 28. The method of any one of claims 19 to 27, further comprising administering to the subject a second therapeutic agent.

29. 29. The method of claim 28, wherein the second therapeutic agent is selected from levodopa (L-DOPA), an MAO-B inhibitor, and a dopamine agonist.

30. Use of berberine tauroursodeoxycholate (BTUDC) for treating a neurodegenerative disease or related disease or disorder.

31. Use of BTUDC to treat Parkinson's disease or a related disease or disorder.

32. Use of BTUDC for the manufacture of a medicament for preventing or treating a neurodegenerative disease or related disease or disorder.

33. Use of BTUDC for the manufacture of a medicament for preventing or treating Parkinson's disease or a related disease or disorder.

34. 34. The use of any one of claims 30 to 33, wherein the BTUDC is in a solid form as defined in any one of claims 5 to 15.

35. 1. A method for alleviating, preventing, or treating a neurodegenerative disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising berberine (BBR) and tauroursodeoxycholic acid (TUDCA).

36. 1. A method for alleviating, preventing, or treating Parkinson's disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising berberine (BBR) and tauroursodeoxycholic acid (TUDCA).

37. 37. The method of claim 36, wherein one or more symptoms of Parkinson's disease are reduced or ameliorated.

38. 37. The method of claim 36, wherein the progression of Parkinson's disease is slowed.

39. 37. The method of claim 36, wherein the progression of Parkinson's disease is halted.

40. 37. The method of claim 36, wherein the progression of Parkinson's disease is reversed.

41. 41. The method of any one of claims 35-40, wherein the subject is administered BBR and TUDCA at a dose of about 25 mg to about 3,500 mg daily.

42. 42. The method of any one of claims 35 to 41, further comprising administering to the subject a second therapeutic agent.

43. 43. The method of claim 42, wherein the second therapeutic agent is selected from levodopa (L-DOPA), an MAO-B inhibitor, and a dopamine agonist.

44. Use of berberine (BBR) and tauroursodeoxycholic acid (TUDCA) for treating a neurodegenerative disease or related disease or disorder.

45. Use of berberine (BBR) and tauroursodeoxycholic acid (TUDCA) for treating Parkinson's disease or a related disease or disorder.

46. Use of berberine (BBR) and tauroursodeoxycholic acid (TUDCA) for the manufacture of a medicament for preventing or treating a neurodegenerative disease or related disease or disorder.

47. Use of berberine (BBR) and tauroursodeoxycholic acid (TUDCA) for the manufacture of a medicament for preventing or treating Parkinson's disease or a related disease or disorder.

48. A method for preparing the salt of any one of claims 1 to 4, comprising the steps of: Dissolving tauroursodeoxycholic acid in ethanol; NaHCO ３ to obtain a sodium tauroursodeoxycholate solution; dissolving berberine hydrochloride in warm water to obtain a berberine hydrochloride solution; adding the berberine hydrochloride solution dropwise to the sodium tauroursodeoxycholate solution; stirring the combined solution at about 60°C to about 80°C; cooling the combined solution to obtain berberine tauroursodeoxycholate; A method comprising:

49. 49. The method of claim 48, further comprising the step of crystallizing berberine tauroursodeoxycholate.

50. A process for preparing the solid form of any one of claims 5 to 15, comprising the steps of: adding an aqueous solution of sodium tauroursodeoxycholate to an aqueous solution of berberine chloride to form a combination solution; mixing the combined solution; cooling the combined solution to obtain the solid form; A method comprising:

51. A process for preparing the solid form of any one of claims 5 to 15, comprising the steps of: adding an aqueous solution of tauroursodeoxycholic acid to an aqueous solution of berberine chloride to form a combination solution; mixing the combined solution; cooling the combined solution to obtain a solid form; A method comprising:

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