Low-dose therapeutic adjuvant agents for cytokine regulation

Abstract

The present invention provides a novel and improved method for preparing stable amorphous pentacyclic triterpenoids, compositions prepared using such pentacyclic triterpenoids, and a method for mitigating the effects caused by pro-inflammatory cytokines in mammals (including humans) by providing effective doses of pentacyclic triterpenoids treated according to the present invention. Furthermore, the present invention provides effective low-dose therapeutic compositions comprising pentacyclic triterpenoids treated according to the present invention.

Description

Detailed Description of the Invention

[0001] [Cross - Reference to Related Applications] This application claims priority to U.S. Provisional Application No. 63 / 487,873, filed March 1, 2023; U.S. Provisional Application No. 63 / 487,887, filed March 2, 2023; U.S. Provisional Application No. 63 / 492,546, filed March 28, 2023; and U.S. Provisional Application No. 63 / 492,549, filed March 28, 2023. All provisional applications are hereby incorporated by reference in their entirety.

[0002] [Background of the Invention] Cytokines are a diverse group of small secreted proteins released for the purpose of cell - to - cell signaling and communication. Cytokines are produced in response to invading pathogens, stimulating, mobilizing, and proliferating immune cells. Cytokines can act on the cell that secreted them, nearby cells, or, in some cases, distant cells.

[0003] Inflammatory cytokines are mainly produced by activated macrophages and are involved in the up - regulation of the inflammatory response. Major inflammatory cytokines include interleukin 1 (IL - 1), interleukin 6 (IL - 6), and tumor necrosis factor α (TNF - α). These are essential for the regulation of the cellular immune response and play an important role in the regulation of the immune system. Inflammatory cytokines generally regulate the proliferation, activation, differentiation, and homing of immune cells to the site of infection, and aim to control and eradicate intracellular pathogens (including viruses).

[0004] An excessive or uncontrolled release of pro-inflammatory cytokines in the body can cause a 'cytokine storm'. Cytokine storms are associated with a wide variety of infectious and non-infectious diseases. Inflammation associated with a cytokine storm begins locally and spreads throughout the body via the systemic circulation. During viral infections (e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), parvovirus B19, and influenza virus), levels of pro-inflammatory cytokines increase and decrease after the virus is cleared.

[0005] In COVID-19 patients, elevated levels of interleukin-1β (IL-1β), IL-6, interleukin-10 (IL-10), interferon-gamma (IFN-γ), TNF-α, interferon-gamma-inducible protein (IP10), granulocyte colony-stimulating factor (GCSF), and monocyte chemotactic protein-1 (MCP1) were observed. In addition, patients admitted to the intensive care unit (ICU) showed higher plasma cytokine levels compared to patients not admitted to the ICU. This suggests that the severity of COVID-19 disease is amplified by cytokine levels. See: C. Huang, Y. et al., Clinical Features of Patients Infected with 2019 Novel Coronavirus in Wuhan, China, Lancet, 395, 497-506 (2020); P. Mehta et al., Covid-19: Consider Cytokine Storm Syndromes and Immunosuppression, Lancet, 395, 1033-1034 (2020); B. Diao, et al., Reduction and Functional Exhaustion of T Cells in Patients with Coronavirus Disease 2019 (COVID-19), medRxiv, 11, 827 (2020).

[0006] Allergies are the immune system's response to foreign substances that do not cause immunogenicity in a healthy state. Accumulated evidence indicates that T helper 2 cell-derived cytokines (such as interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13)) play a crucial role in orchestrating and amplifying allergic inflammation in asthma. See, for example, Nakajima H, Takatsu K., Role of Cytokines in Allergic Airway Inflammation, Int. Arch. Allergy Immunol., 142(4), 265-73 (2007). Vulvodynia is a chronic pain or discomfort around the vulva that lasts for at least three months. Vulvodynia syndrome (VPS) is a multifactorial disease that has a significant impact on the lifestyle of women. Eleven molecules specifically involved in pro-inflammatory pathways have been shown to be significantly regulated in VPS patients compared to healthy women, suggesting the presence of a persistent inflammatory process. See: Nunzia Zanotta et al., Cytokine Profiles of Women with Vulvodynia: Identification of a Panel of Pro-inflammatory Molecular Targets, Eur. J Obstet. Gynecol. Reprod. Biol., 226, 66-70 (2018).

[0007] Otitis media is inflammation or infection of the middle ear. Otitis media can occur as a result of a cold, sore throat, or respiratory infection. Acute otitis media (AOM) is an inflammatory response to microorganisms in the middle ear, sometimes accompanied by tympanic membrane perforation. Culture-positive middle ear effusions show significantly higher levels of IL-1β, tumor necrosis factor (TNF), interleukin-8 (IL-8), and interleukin-10 (IL-10) compared to culture-negative middle ear effusions. S. Skovbjerg et al., High Cytokine Levels in Perforated Acute Otitis Media Exudates Containing Live Bacteria, European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 1382-1388 (2010).

[0008] Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a group of chronic diseases characterized by inflammation of the gastrointestinal tract, typically involving a clinical course of relapses and remissions. Cytokines play a crucial role in IBD, determining the differentiation of T cells into Th1, Th2, T regulatory cells, and the newly identified Th17 cells. Cytokine levels in time and space modulate the onset, relapse, and exacerbation of the inflammatory process in IBD. Therefore, multiple cytokine therapies have been developed and tested for the treatment of IBD patients. Fausto Sanchez-Munoz et al., Role of Cytokines in Inflammatory Bowel Disease, World J. Gastroenterol 14(27), 4280-4288 (2008).

[0009] Myasthenia gravis (MG) is a chronic autoimmune disease in which antibodies disrupt nerve-muscle communication, resulting in skeletal muscle weakness. Inflammation can be an important factor in understanding the pathogenesis of myasthenia gravis. In MG patients, serum levels of proliferation-inducing ligand (APRIL), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-28A (IL-28A), and interleukin-35 (IL-35) were significantly elevated compared to the control group (p<0.05). Of these, IL-20, IL-28A, and IL-35 were significantly decreased after treatment (p<0.05). Uzawa, A. et al., Changes in Inflammatory Cytokine Networks in Myasthenia Gravis., Sci. Rep. 6, 25886, doi: 10.1038 / srep25886 (2016).

[0010] Multiple sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system, characterized by demyelination, axonal loss, gliosis, and inflammation. Immune system cells mistakenly attack myelin, which covers nerve fibers (axons) in the central nervous system (e.g., brain, optic nerve, and spinal cord). Pro-inflammatory cytokines (such as interleukin-17 (IL-17), interleukin-22 (IL-22), TNF-α, interleukin-1 (IL-1), interleukin-12 (IL-12), and interferon-γ (IFN-γ)) can trigger MS through multiple signaling pathways. Conversely, anti-inflammatory circulating cytokines (such as IL-4 and IL-10) are reduced, and these cytokines may exert a direct protective effect in the MS state. Kexin Wang et al., The Properties of Cytokines in Multiple Sclerosis: Pros and Cons, Am. J. Med. Sci., 356(6), 552-560 (2018).

[0011] Chronic alcohol consumption leads to hepatocyte damage and hepatitis. Alcohol consumption promotes hepatitis by increasing the transfer of intestinal endotoxins into the portal circulation. Inflammatory cytokines (such as TNF-α and IFN-γ) have been shown to induce liver damage in a rat model of alcoholic liver disease (ALD). Hepatoprotective cytokines (such as IL-6) and anti-inflammatory cytokines (such as IL-10) are also associated with ALD. Hideto Kawaratani et al., The Effect of Inflammatory Cytokines in Alcoholic Liver Disease, Mediators Inflamm., 2013:495156 (2013).

[0012] Mucositis is an adverse side effect of anticancer treatment using radiotherapy and chemotherapy. Pro-inflammatory cytokines are thought to be involved in the pathophysiology of chemotherapy-induced gastrointestinal mucositis. [Ong, ZY, Gibson, RJ, Bowen, JM et al. Pro-inflammatory cytokines play a key role in the development of radiotherapy-induced gastrointestinal mucositis. Radiat Oncol 5, 22 (2010)].

[0013] Sepsis is an excessive and life-threatening bodily response to infection, which can lead to tissue damage, organ failure, and death. The aforementioned pro-inflammatory cytokines are closely associated with the progression of coagulation processes in sepsis. Certain inflammatory skin diseases are associated with cytokine overproduction, alterations in cytokine receptors, or cytokine dysregulation. The most common chronic inflammatory skin diseases are atopic dermatitis, psoriasis, urticaria, lichen planus, and hidradenitis suppurativa, which are caused by complex interactions of genetic and environmental factors. Autoimmunity is also another significant cause of chronic skin inflammation.

[0014] Chronic pro-inflammatory states are a universal feature of aging, leading to degeneration of multiple organs. There is strong evidence that the progression of age-related multifactorial conditions (such as cancer, cardiovascular disease, Alzheimer's disease, type 2 diabetes, frailty, sarcopenia, and osteoporosis) is associated with a mild increase in circulating inflammatory mediators.

[0015] Compared to younger individuals, older adults consistently showed elevated levels of inflammatory cytokines (particularly interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). Singh T, Newman AB., Inflammatory Markers in Population Studies of Aging., Ageing Res. Rev. 10(3), 319-29 (2011).

[0016] Renal damage causes a dangerous accumulation of extracellular matrix, known as kidney (or renal) failure. If left untreated, this can be fatal.

[0017] There are two main types of kidney failure: acute (sudden onset), known as "acute kidney injury" (AKI); and chronic (progressing over time), known as chronic kidney disease (CKD). Chronic kidney disease (CKD) is defined as the presence of structural or functional abnormalities in one or both kidneys over a long period of time. CKD is a global public health problem, affecting approximately 8-10% of the population in Western countries. It is estimated that 30 million adults, or 15%, have CKD. The prevalence of CKD in people with type 2 diabetes is currently around 40% and continues to increase.

[0018] CKD is also common in cats. The prevalence of CKD increases with age. It is known that more than 10% of dogs and more than 30% of cats over 15 years of age are diagnosed with CKD.

[0019] The excessive accumulation of extracellular matrix (primarily composed of collagen) is called renal fibrosis, which typically leads to loss of function as normal tissue is replaced by scar tissue. Because unchecked renal fibrosis precedes irreversible kidney damage, stopping or slowing its insidious progression is an ideal therapeutic target to prevent CKD from progressing to end-stage renal disease.

[0020] While there is no definitive cure for CKD, treatment can improve the quality of life and extend the lifespan of mammals with this disease. Treatment aims to minimize the buildup of harmful waste products in the bloodstream, maintain adequate hydration, address electrolyte imbalances, support proper nutrition, control blood pressure, and slow the progression of the kidney disease.

[0021] Treatment for kidney disease may include antihypertensive drugs to maintain kidney function (AC inhibitors or angiotensin II receptor blockers), diuretics, anti-anemia drugs, the hormone preparation erythropoietin, and cholesterol-lowering drugs.

[0022] Despite ongoing research, there are currently no effective treatments that significantly slow the progression of renal fibrosis in humans or cats. Jack Lawson et al., Renal Fibrosis in Feline Chronic Kidney Disease: Known Mediators and Mechanisms of Injury, The Veterinary Journal, 203, 18-26 (2015).

[0023] The link between oxidative stress, inflammation, and the progression of chronic kidney disease (CKD) is well established. Reducing oxidative stress and inflammation are promising targets for slowing the progression of CKD.

[0024] Pentacyclic triterpenoids (such as ursolic acid and oleanolic acid) are thought to have a wide range of therapeutic effects, including antioxidant, anti-inflammatory, anti-cancer, anti-allergic, hepatoprotective, gastric protective, lipid-lowering, hypoglycemic, lipolytic anti-obesity, anti-atherosclerotic, and immunomodulatory effects.

[0025] Ursolic acid, which has antioxidant and anti-inflammatory properties, is a candidate substance suitable for managing kidney health.

[0026] In Mexico, the fruit of Randia echinocarpa (granjel) has traditionally been widely used to treat kidney ailments. The main components of Randia echinocarpa have been identified as ursolic acid and oleanolic acid. Robert Bye et al., Ethanobotanical and Phytochemical Investigation of Randia Echinocarpa, Anales Inst. Biol. Univ. Nac. Auton. Mexico. Ser. Bot., 62(1), 87-106 (1991).

[0027] Transforming growth factor beta 1 (TGF-β1) has been reported to be associated with various diseases including chronic glomerulonephritis and diabetic nephropathy. For example, in a previous in vitro study using Balb / c 3T3 cells reported by JP 2000159673 and Hiromitsu Yoshimura et al., the TGF-β1 inhibitory effect by an extract of Clerodendranthus spicatus containing ursolic acid and oleanolic acid was explained. Hiromitsu Yoshimura et al., In Vitro TGF-β1 Antagonistic Activity of Ursolic and Oleanolic Acids Isolated from Clerodendranthus Spicatus, Planta. Med. 69(7), 673-675 (2003). The concentration of ursolic acid that inhibits 50% of the specific binding of TGF-β1 was 3.0 μg / ml (see ibid.).

[0028] From the viewpoint of physicochemical properties, ursolic acid is an ursane-type pentacyclic triterpenoid, a white crystal, odorless and tasteless, and a compound insoluble in water.

[0029] Ursolic acid is a pentacyclic triterpene compound (molecular formula: C30H48O3, molecular weight: 456.68 g / mol) and has been found in the following plants: Salvia officinalis, Salvia miltiorrhiza L, Eriobotrya japonica (Loquat), Rosmarinus officinalis, Origanum onites, Origanum vulgare, Ocimum accessions, Ocimum sanctum, Oldenlandia diffusa, olive pulp, and Macrocarpium officinale. (officinale), Prunella vulgaris, Psychotria serpens, Hyptis capitata, Hedyotis corymbose, Swertia chirata, Hedyotis diffusa, and Vitex negundo. Triterpenes include lupane, oleanane, ursane, arjunolic acid, maslinic acid, lupenol, betulin, betulinic acid, asiatic acid, corosolic acid, boswellic acid, glycyrrhetinic acid, and madecassic acid.

[0030] Triterpenes isolated from the mushroom Ganoderma lucidum have been shown to significantly suppress the secretion of inflammatory cytokines (tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)). Shailesh Dudhgaonkar et al., Suppression of the Inflammatory Response by Triterpenes Isolated from the Mushroom Ganoderma Lucidum, Int. Immunopharmacol., 9:11, 1272-1280 (October 2009).

[0031] Ana Marquez-Martin et al. investigated the effect of trace components of olive pomace oil on the production of pro-inflammatory cytokines by human peripheral blood mononuclear cells. They reported that pentacyclic triterpenes in orujo oil exhibit pro-inflammatory and anti-inflammatory properties depending on their chemical structure and dosage, and concluded that they may be useful in modulating the immune response. Ana Marquez-Martin et al., Modulation of Cytokine Secretion by Pentacyclic Triterpenes from Olive Pomace Oil in Human Mononuclear Cells., Cytokine, 36:5-6, 211-217 (2006).

[0032] Research results showed that betulin and betulinic acid suppressed ethanol-induced hepatic stellate cell (HSC) activation at different levels by acting as antioxidants, cytokine production inhibitors, and TGF-β and NFκB / IκB signaling pathway inhibitors. Betulin and betulinic acid attenuate ethanol-induced hepatic stellate cell activation by suppressing the production of reactive oxygen species (ROS) and cytokines (TNF-α, TGF-β), and by influencing intracellular signaling. AgnieszkaSzuster-Ciesielska et al., Betulin and Betulinic acid Attenuate Ethanol-induced Liver Stellate Cell Activation by Inhibiting Reactive Oxygen Species (ROS), Cytokine (TNF-α, TGF-β) Production and by Influencing Intracellular Signaling, Toxicology, 280: 3, 152-163 (2011).

[0033] Yue Huang et al. reported that cranberry extract (containing ursolic acid) inhibits the NF-κB transcriptional response in human T lymphocytes, the release of IL-1β, IL-6, and TNF-α in PBMs stimulated by LPS, and the catalytic activity of COX-2. (Yue Huang et al., Effects of Cranberry Extracts and Ursolic acid Derivatives on P-fimbriated Escherichia Coli, COX-2 Activity, Pro-inflammatory Cytokine Release and the NF-κβ Transcriptional Response in Vitro, Pharmaceutical Biology, 47:1, 18-25 (2009)).

[0034] Hai Bing Peng et al. reported that the protective effect of oleanolic acid is (at least partially) due to its antioxidant activity and its ability to reduce cytokine and collagen expression by regulating the AKT / NF κB pathway. Peng HB et al., Protective Effects of Oleanolic Acid on Oxidative Stress and the Expression of Cytokines and Collagen by the AKT / NF κB Pathway in Silicotic Rats, Mol. Med. Rep., 15 3121-3128 (2017).

[0035] Ursolic acid and oleanolic acid have a wide range of therapeutic effects, including antioxidant, anti-inflammatory, anti-cancer, anti-allergic, hepatoprotective, gastric protective, lipid-lowering, hypoglycemic, lipolytic anti-obesity, anti-atherosclerotic, and immunomodulatory effects.

[0036] For example, a previous in vitro study using Balb / c 3T3 cells, reported by JP 2000159673 and Hiromitsu Yoshimura et al., described the TGF-β1 inhibitory effect of an extract of Clerodendranthus spicatus containing ursolic acid and oleanolic acid. (Hiromitsu Yoshimura et al., In Vitro TGF-β1 Antagonistic Activity of Ursolic and Oleanolic Acids Isolated from Clerodendranthus Spicatus, Planta. Med. 69(7), 673-675 (2003)). The concentration of ursolic acid that inhibited the specific binding of TGF-β1 by 50% was 3.0 μg / ml (see ibid.).

[0037] Mapanga et al. reported in vivo evidence that oleanolic acid extracted from the leaves of Syzygium cordatum improves kidney function in diabetic rats. The rats were housed in cages and administered oleanolic acid twice daily (60 mg / kg, orally) for 5 weeks. Oleanolic acid was added to the infusate at a rate of 90 μg / h for 1.5 hours (treatment period), with a total dose of 0.45 mg / kg (for 300 g rats). Non-diabetic and diabetic rats were housed separately and administered oleanolic acid twice every 3 days (60 mg / kg, orally) for 5 weeks. Mapanga et al. Renal Effects of Plant-Derived Oleanolic Acid in Streptozotocin-Induced Diabetic Rats., Renal Failure, 31, 481-491 (2009).

[0038] CN101732323 describes the use of 'low-dose' ursolic acid as a treatment for early diabetic nephropathy. The oral dose used in the aforementioned study was reported to be 50-70 mg / kg (body weight), which is described as a 'low dose'. This 'low dose' corresponds to approximately 4,000-5,600 mg for an average person weighing 80 kg, which is considered a very high dose.

[0039] The renal protective effect of ursolic acid has been reported in gentamicin-induced renal injury in Wistar albino rats. In this study, pure ursolic acid (Sigma Aldrich Chemicals Pvt. Ltd., UK) was dissolved in 14% DMSO and administered orally at doses of 2 mg / kg, 5 mg / kg, and 10 mg / kg. Preethi G. Pai et al., Nephroprotective Effect of Ursolic Acid in a Murine Model of Gentamicin-Induced Renal Damage, International Scholarly Research Network (ISRN) Pharmacology, Article ID 410902 (2012).

[0040] Pure ursolic acid (dosage: 30 mg / kg) suspended in 0.5% CMC solution was orally administered to rats for 28 days while simultaneously administering adenine. Richa Thakur et al. Ameliorative Effect of Ursolic Acid on Renal Fibrosis in Adenine-Induced Chronic Kidney Disease in Rats, Biomedicine & Pharmacotherapy, 101 972-980 (2018).

[0041] Other studies have also been conducted demonstrating the effects of ursolic acid. For example, see: Qi My et al., Study on the Protective Effect of Ursolic Acid on Alloxan-induced Diabetic Renal Injury and its Underlying Mechanisms, Chinese J. Applied Physiology, 30(5), 445-448 (2014), at https: / / europepmc.org / article / med / 25571638 (ursolic acid dose of 35 mg / kg / d, administered intragastricly, continued for 8 weeks); Zhaohui Jia, Ursolic Acid Treats Renal Tubular Epithelial Cell Damage Induced by Calcium Oxalate Monohydrate via Inhibiting Oxidative Stress and Inflammation, Bioengineered, 12:1, 5450-5461 (2021) (rats were administered ursolic acid at a dose of 40 mg / kg per day via gastric tube (gavage)).

[0042] Ursolic acid treatment has been shown to effectively reduce the urinary albumin / creatinine ratio in mice (p<0.05). Tian-Kui Ma et al., Ursolic Acid Treatment Alleviates Diabetic Kidney Injury by Regulating the ARAP1 / AT1R Signaling Pathway, Diabetes Metabolic Syndrome and Obesity, 12, 2597-2608 (2019). In this study, mice treated with ursolic acid (UA) were fed a diet containing 0.3% UA (0.3g UA per 100g of standard diet) for 10 weeks.

[0043] High-dose ursolic acid (50 mg / kg) and low-dose ursolic acid (25 mg / kg) have been shown to have significant protective effects in diabetic rats. Hui-lin Xu, Ursolic Acid Improves Diabetic Nephropathy via Suppression of Oxidative Stress and Inflammation in Streptozotocin-induced Rats, Biomedicine & Pharmacotherapy, 105, 915-921 (2017).

[0044] Some literature discloses studies claiming protective effects of ursolic acid, albeit at extremely high doses of 25-70 mg / kg (body weight). This would mean using 4-5 g of ursolic acid per day for an average person. Considering the extraction, concentration, and processing of ursolic acid, this is not commercially feasible. Furthermore, such high doses could cause unintended side effects on other systems of the body. The effective dose needs to be reduced.

[0045] Other reported studies have tested ursolic acid dissolved in DSMO, but this is not clinically feasible. Another study administered ursolic acid parenterally. Ursolic acid was dissolved in a solvent and injected directly into the body.

[0046] According to the Biopharmaceutical Classification System (BCS), drug substances are classified into four classes based on their solubility and permeability. Of these classes, compounds belonging to Class IV have low permeability and solubility, and therefore low bioavailability. Typically, these compounds are not adequately absorbed by the intestinal mucosa, and high variability is expected.

[0047] Due to its low water solubility (<5.64 μg / mL) (Lin. H. et al., Determination of Equilibrium Solubility and Apparent Oil / Water Partition Coefficient of Ursolic Acid. Chin. J. Mod. Appl. Pharm., 29, 635-637 (2012)) and low permeability (Papp=2.8 Å~10⁻⁶ cm / s, value in the direction from apical to batholateral at 20 μM) (Qiang. Z. et al. Permeability of Rosmarinic Acid in Prunella Vulgaris and Ursolic Acid in Salvia Officinalis Extracts Across Caco-2 Cell Monolayers., J. Ethnopharmacol. 137, 1107-1112 (2011)), ursolic acid belongs to BCS class IV.

[0048] After oral administration, only about 0.6% of the ingested ursolic acid was recovered in rat plasma. Liao. Q. et al., LC-MS Determination and Pharmacokinetic Studies of Ursolic Acid in Rat Plasma after Administration of the Traditional Chinese Medicinal Preparation Lu-Ying Extract, Yakugaku Zasshi, 125, 509-515 (2005).

[0049] Solid oral formulations are generally the preferred method for all therapeutic drugs due to their low manufacturing costs and high convenience for the user. Therefore, there is a need for a low-dose and effective form of ursolic acid administration.

[0050] Ursolic acid is insoluble in water and exists in a stable crystalline form. For better therapeutic effects, ursolic acid can be converted to an amorphous state. However, the amorphous state is thermodynamically unstable, and ursolic acid entropically tends to transition from the amorphous state to the stable crystalline state. The high internal energy and specific volume of the amorphous state can lead to improved solubility and bioavailability, but it also creates the possibility that the amorphous state may spontaneously revert to the crystalline state during processing or storage. See: Huttenrauch R., Molecular Pharmaceutics as a Basis for Modern Drug Formulation, Acta Pharmaceutica Technology Supplement, 24(6), 55-127 (1978); Yoshioka M et al., Crystallization of Indomethacin from the Amorphous State Below and Above its Glass Transition Temperature, J. Pharmaceutical Sciences, 83(12), 1700-5 (1994).

[0051] On the other hand, Danni Yu et al. reported that crystalline ursolic acid exhibits low physical stability and undergoes structural changes when stored for 30 days under conditions of 25°C / 75%RH (relative humidity) or 40°C / 75%RH.

[0052] Danni Yu et al., and CN102234304A, demonstrated that amorphous ursolic acid prepared by ball milling is almost completely converted to a stable crystalline form when stored for 8 days under both 25°C / 75%RH and 40°C / 75%RH conditions. (Danni Yu et al., Triple Strategies to Improve Oral Bioavailability by Fabricating Co-amorphous Forms of Ursolic Acid with Piperine: Enhancing Water-Solubility, Permeability and Inhibiting Cytochrome P450 Isozymes, Mol. Pharmaceutics, 17(12):4443-4462 (2020)). The presence of piperine limits the therapeutic applications of ursolic acid and therefore makes it unsuitable for all practical purposes.

[0053] Other techniques have also been applied to improve the pharmacological properties of ursolic acid. These techniques include liposomal formulations, encapsulation with hydroxypropyl β-cyclodextrin, nanostructured lipid carriers, self-nanoemulsifying formulations, phospholipid complexes, and solid dispersions. Challenges in scaling up these laboratory processes, as well as the use of solvents in specific processes, pose practical problems.

[0054] There is no universal and reliable method for selecting a technology or polymer that has guaranteed amorphous stability and improved bioavailability.

[0055] Although ursolic acid showed significant biological activity in in vitro assays and some animal models, it has been difficult to confirm its efficacy in vivo in humans. Some of the reasons for this include its low biological activity, low solubility, low absorption rate, low permeability, poor first-pass metabolism and metabolism, and pre-systemic excretion from the intestines or liver before entering the systemic circulation.

[0056] A practical method is needed to improve the therapeutic effects of ursolic acid.

[0057] [Summary of the Invention] The present invention provides a novel and improved method for preparing stable amorphous pentacyclic triterpenoid compounds, compositions prepared using such amorphous pentacyclic triterpenoids, and a method for mitigating the effects caused by pro-inflammatory cytokines in mammals (including humans) by administering an effective dose of a stable amorphous pentacyclic triterpenoid. Furthermore, the present invention provides an effective low-dose therapeutic composition prepared using a stable amorphous pentacyclic triterpenoid.

[0058] In some embodiments, the present invention provides pharmaceutical compositions for mammals (including humans and companion animals), particularly for treating kidney injury.

[0059] In certain embodiments, the present invention provides a method for treating kidney injury in mammals (including companion animals). The method comprises administering to a subject in need of such treatment a pharmaceutical composition prepared using an amorphous pentacyclic triterpenoid (such as ursolic acid).

[0060] In some embodiments, the pharmaceutical composition is prepared by a process comprising the following steps: mixing a pentacyclic triterpenoid with a carrier (such as an inert water-soluble polymer having a glass transition temperature lower than 170°C); melt-extruding the mixture at a temperature of about 60°C to about 180°C; grinding the extruded material; and mixing the ground extruded material with an excipient to form the pharmaceutical composition.

[0061] In some embodiments, the pharmaceutical composition is a capsule, tablet, chewable tablet, soft chew, gummy, lozenge, or powder, and the composition contains no residual solvent. In some embodiments, the process according to the present invention irreversibly converts a pentacyclic triterpenoid into an amorphous form with improved water solubility.

[0062] [Brief explanation of the drawing] [Figure 1] Figure 1 shows the results of X-ray diffraction measurements: [A] Untreated ursolic acid (shows crystalline properties); [B] Treated ursolic acid sample (shows amorphous properties; active ingredient 1); [C] Treated ursolic acid sample (shows amorphous properties; active ingredient 2).

[0063] [Figure 2] Figure 2 shows the amorphous form of ursolic acid samples stored for 3 to 15 months. The fact that ursolic acid remained stable for the 15 months tested and that no inversion from amorphous to crystalline form was observed demonstrates the superiority of this invention.

[0064] [Detailed description of the invention] In one embodiment, the present invention provides a dispersion composition in the form of a solid dispersion comprising an amorphous pentacyclic triterpenoid and a support. In some embodiments, the pentacyclic triterpenoid is ursolic acid, and the support is a water-soluble polymer having a glass transition temperature lower than 170°C.

[0065] In another embodiment, the present invention provides a method for preparing a solid dispersion containing an amorphous pentacyclic triterpenoid (such as ursolic acid) by mixing the pentacyclic triterpenoid with a support (such as a water-soluble polymer having a glass transition temperature lower than 170°C). In some embodiments, the dispersion is prepared by melt extrusion.

[0066] In some embodiments, the pentacyclic triterpenoids used according to the present invention include ursolic acid, oleanolic acid, argunolic acid, maslinic acid, lupenol, betulin, betulinic acid, asiatic acid, corosolic acid, boswellic acid, glycylretinic acid, madecasic acid, or combinations thereof. The pentacyclic triterpenoids may be in the form of pharmaceutically acceptable salts, isomers, solvates, and / or extracts, or combinations thereof. In certain embodiments, the pentacyclic triterpenoid is ursolic acid.

[0067] Ursolic acid is a naturally derived lipophilic pentacyclic triterpenic acid (chemical formula: C 30 H 48 Ursolic acid is O3 (melting point: 283-285°C). In the present invention, ursolic acid may be in the form of a pharmaceutically acceptable salt, isomer or solvate, or an extract containing ursolic acid.

[0068] [ka]

[0069] In some embodiments, the present invention provides a pharmaceutical composition as a final composition prepared with a solid dispersion of an amorphous pentacyclic triterpenoid (such as ursolic acid) as an active ingredient, and a method for producing the same. The solid dispersion of the amorphous pentacyclic triterpenoid can be formulated into a pharmaceutical composition alone or with one or more excipients. In certain embodiments, the pentacyclic triterpenoid as the active ingredient is ursolic acid.

[0070] In some embodiments, the pentacyclic triterpenoid in an amorphous solid dispersion is therapeutically effective at a dose of about 0.05 mg / kg (body weight) to about 20 mg / kg (body weight), preferably about 0.5 mg / kg (body weight) to about 10 mg / kg (body weight), and more preferably 1 mg / kg (body weight) to about 5 mg / kg (body weight).

[0071] In some embodiments, the pharmaceutical composition according to the present invention is used in a method for treating a medical condition caused by overactive pro-inflammatory cytokines. It is used. In certain embodiments, such conditions include kidney disease, acute kidney injury, or chronic kidney disease, morbidity due to Covid-19 virus infection, morbidity due to viral infection, morbidity due to bacterial infection, tuberculosis, diabetic nephropathy, inflammatory arthritis, allergy or allergic asthma, periodontal disease, vulvodynia, otitis, ulcerative colitis, Crohn's disease, myasthenia gravis, multiple sclerosis, liver disease, alcoholic liver disease, mucositis, major depressive disorder, anxiety-related disorder, aging, or chronic inflammatory skin disease.

[0072] In some embodiments, the pharmaceutical composition according to the present invention is administered orally and taken as a therapeutic adjunct or as an additive to food or feed.

[0073] In some embodiments, the solid dispersion of amorphous pentacyclic triterpenoids according to the present invention improves the water solubility of the pentacyclic triterpenoids and / or enhances their therapeutic effect. As a result, the effective dose of the pentacyclic triterpenoids is reduced compared to conventional formulations of the pentacyclic triterpenoids. In some embodiments, such low-dose formulations contribute to reducing renal damage.

[0074] In some embodiments, the pharmaceutical compositions according to the present invention are used in a method to mitigate renal damage in a subject who has a renal condition such as CKD and / or is at risk of developing such a renal condition. Herein, renal damage refers to oxidative stress and / or inflammation in the subject. Oxidative stress and inflammation can be measured by methods known in the art.

[0075] In some embodiments, the amorphous solid dispersion according to the present invention contains a pentacyclic triterpenoid as an active ingredient, preferably in an amount of 10 to 70% by weight based on the total weight of the final composition. Due to its low density, it is not difficult to achieve uniformity of the contents in the mixture at the 10% by weight level. In some embodiments, the pentacyclic triterpenoid is ursolic acid.

[0076] In some embodiments, the amorphous solid dispersion according to the present invention further comprises a carrier. The carrier may contain a water-soluble polymer having a glass transition temperature of less than 170°C, and in some embodiments, the water-soluble polymer includes polyvinyl caprolactam, polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus®), polyvinylpyrrolidone-vinyl acetate copolymer (PVP VA64), polyethylene glycol (PEG), Eudragit® EPO, and / or hypromellose acetate succinate (HPMCAS). In some embodiments, the water-soluble polymer is contained in an amount of 30% by weight or more, more preferably 30-90% by weight, based on the total weight of the final composition. In some embodiments, polyvinylpyrrolidone having any molecular weight can be used, but those with a molecular weight of 45,000-70,000 are particularly preferred because they can produce the viscosity necessary for melt extrusion. A more preferred polymer is vinylpyrrolidone-vinyl acetate copolymer. Other polymers can also be used according to the present invention.

[0077] In some embodiments, the amorphous solid dispersion according to the present invention further comprises a plasticizer. The use of plasticizers (e.g., D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), polyethylene glycol 400, and other common plasticizers) is not limited to the extent that it does not affect the amorphous nature of the pentacyclic triterpenoid (e.g., ursolic acid). In some embodiments, the amorphous solid dispersion according to the present invention does not contain a plasticizer.

[0078] In some embodiments, the amorphous solid dispersion according to the present invention comprises, with respect to the final composition, 5 to 50% by weight of a pentacyclic triterpenoid (such as ursolic acid) and 5 to 95% by weight of a water-soluble polymer (such as polyvinylpyrrolidone).

[0079] In some embodiments of the method according to the present invention, a mixture comprising a pentacyclic triterpenoid (such as ursolic acid) and a water-soluble polymer is melted as it passes through at least four (4) heating zones, the temperature of which is continuously decreasing. Specifically, the solid dispersion of the present invention is prepared by introducing the mixture, which has been pre-mixed as a powder, into an extruder and performing melt extrusion. The extruder consists of a plurality of heating zones connected in series, which are designed to be distinct from each other. In some embodiments, the distinct heating zones are controlled to have a temperature lower than the melting point of the pentacyclic triterpenoid (e.g., ~284°C).

[0080] The melting of the mixture is carried out at a temperature lower than the melting point of the pentacyclic triterpenoid, but the extrusion is carried out by specific sustained cooling and continuous melting. This continuous melting is achieved by passing the mixture through multiple heating zones in which the set temperature decreases continuously. This causes a change in the active ingredient, resulting in a conversion from the crystalline structure to the amorphous form in the pentacyclic triterpenoid. In some embodiments, the melt extrusion is carried out at a temperature of about 60°C to about 180°C. As illustrated in Figure 1, the active ingredient ursolic acid processed according to the method of the present invention described herein clearly shows the conversion to the amorphous form.

[0081] The melt-extruded product is ground into a fine powder using a mill and mixed with additives to form a pharmaceutical composition (for example, in the form of tablets, capsules, chewable tablets, soft chews, gummies, bars, oral pastes, lozenges, or powders). In some embodiments, such a pharmaceutical composition does not contain a solvent.

[0082] Excipients that can be used to prepare the pharmaceutical compositions according to the present invention include, but are not limited to, fluidizing agents for oral capsule filling, tableting agents, disintegrants for forming tablets, flavoring agents, sweeteners for forming chews, and solidified fats for forming suppositories.

[0083] Table 1 below summarizes the processing conditions and measurement parameters for the dispersion composition according to the present invention.

[0084] [Table 1]

[0085] [Exemplary methods for carrying out the present invention] Manufacturing method: An extract containing ursolic acid is mixed with a vinylpyrrolidone-vinyl acetate copolymer in a 10:90 ratio to form a homogeneous physical mixture (PM). This PM is passed through a hot melt extruder (Thermofisher, Steer) at a temperature of 180°C. The extruded material is recovered from the die, cooled to ambient temperature, and ground to 80 mesh to obtain the extruded material as an amorphous solid dispersion.

[0086] The extruded material was tested, and it was confirmed that the crystalline structure of ursolic acid had been converted to an amorphous form. This amorphous state was maintained during storage as described herein.

[0087] When the solubility of the extruded material was compared with that of an untreated material containing ursolic acid, it was found to be significantly improved compared to the untreated material.

[0088] The extruded material is formulated into a pharmaceutical composition (for example, in the form of capsules, tablets, chewable tablets, soft chews, gummies, or lozenges) to supply a desired amount of ursolic acid as an active ingredient in the pharmaceutical composition prepared according to the present invention.

[0089] [Product testing] 1. Transformation into amorphous form: Figure 1: X-ray diffraction measurement results: [A] Untreated ursolic acid (shows crystalline properties); [B] Treated ursolic acid (shows amorphous properties; active ingredient 1); [C] Treated ursolic acid (shows amorphous properties; active ingredient 2).

[0090] 2. Stability of amorphous form during shelf-life study: Figure 2 shows the amorphous morphology of ursolic acid samples stored for 3 to 15 months. The stability of ursolic acid during the 15-month test period, and the absence of inversion from amorphous to crystalline morphology, demonstrates the superiority of this invention.

[0091] 3. Therapeutic effects at low doses: A sprinkler formula for cats was prepared using a sage extract containing ursolic acid and oleanolic acid processed according to the present invention, and a powdered palatant as a filler. One 0.625 CC scoop dispenses approximately 400 mg of the mixture, which provides 5 mg of ursolic acid. This is the dosage for a 5 kg cat. Administering it twice a day results in a total dose of 2 mg / kg, and this administration was shown to be therapeutically effective in managing the renal health of the cat.

[0092] Ursolic acid, the active ingredient processed by the method of the present invention, (1) is thermally stable as an amorphous solid dispersion, (2) is encapsulated at the molecular level and forms a stable amorphous form without recrystallization during storage, (3) has improved solubility, and (4) shows consistent results in repeated experiments. This demonstrates the value of this process for preparing a stable and highly water-soluble form of pentacyclic triterpenoids, and therefore has therapeutic effects at low doses.

[0093] In this specification, the terms “subject” or “patient” refer to living organisms, including mammals (primates, etc.). Mammalian species that may benefit from the inventions of this disclosure include, but are not limited to, great apes, chimpanzees, orangutans, humans, and monkeys; domesticated animals (dogs, cats, etc.); livestock (horses, cattle, pigs, sheep, goats, and chickens, etc.); and other animals (mice, rats, guinea pigs, and hamsters, etc.).

[0094] In this specification, the terms “effective amount” or “effective dose” are used to represent the amount of something (e.g., compound, composition, time) that is capable of producing the desired result (e.g., improved consistency of the final composition).

[0095] The transitional term "comprising" is synonymous with "including" or "containing," and is comprehensive or open, not excluding additional, undescribed components or method steps. In contrast, the transitional phrase "consisting of" excludes any components, steps, or ingredients not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that "do not substantially affect the fundamental and novel properties" of the present invention (e.g., the ability to improve the bioavailability of a substance). The use of the term "comprising" implies other embodiments of components that are described as "consisting of" or "consisting essentially of."

[0096] Unless otherwise stated or the context makes clear, the term “or” is understood to be inclusive in this specification. Unless otherwise stated or the context makes clear, the terms “a,” “an,” and “the” are understood to be singular or plural in this specification.

[0097] Unless otherwise specified or made clear from the context, the term “about” in this specification is understood to mean within the normal acceptable range in the art (e.g., within two standard deviations of the mean). Further examples include “about” being understood to mean within the range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[0098] Where ranges (such as dosage ranges), combinations of ranges, and partial combinations are used herein (for example, a subrange within the disclosed range), specific embodiments within that range are expressly included.

[0099] In this specification, when a definition of a variable includes an enumeration of chemical groups, such enumeration includes defining the variable as a single group or as a combination of the enumerated groups. Where embodiments for a variable or aspect are described herein, such description includes any single embodiment or any combination of any other embodiment or part thereof.

[0100] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein. [Brief explanation of the drawing]

[0101] [Figure 1] Figure 1 shows the results of X-ray diffraction measurements: [A] Untreated ursolic acid (shows crystalline properties); [B] Treated ursolic acid sample (shows amorphous properties; active ingredient 1); [C] Treated ursolic acid sample (shows amorphous properties; active ingredient 2). [Figure 2] Figure 2 shows the amorphous morphology of ursolic acid samples stored for 3 to 15 months. The fact that ursolic acid remained stable for the 15 months tested and that no reversal from amorphous to crystalline morphology was observed demonstrates the superiority of this invention.

Claims

1. a) A step of obtaining a mixture by mixing a pentacyclic triterpenoid with a water-soluble polymer having a glass transition temperature lower than 170°C; b) A step of melting and extruding the mixture at a temperature of about 60°C to about 180°C to obtain an extruded product; c) A step of grinding the extruded material to obtain a ground extruded material containing a solid dispersion of the pentacyclic triterpenoid in an amorphous form; and d) A step of formulating the solid dispersion to obtain a pharmaceutical composition, A process for preparing the pharmaceutical composition containing the pentacyclic triterpenoid, including the pentacyclic triterpenoid.

2. The process according to claim 1, wherein the pentacyclic triterpene is selected from the group consisting of ursolic acid, oleanolic acid, argunolic acid, maslinic acid, lupenol, betulin, betulinic acid, asiatic acid, corosolic acid, boswellic acid, glycylretinic acid, and madecasic acid.

3. The process according to claim 1, wherein the pentacyclic triterpenoid is ursolic acid or oleanolic acid.

4. The process according to claim 1, wherein the pentacyclic triterpenoid is present in an amount of about 5% to about 50% of the weight of the pharmaceutical composition.

5. The process according to claim 1, wherein the water-soluble polymer is selected from the group consisting of polyvinyl caprolactam, polyvinyl acetate-polyethylene glycol graft copolymer, polyvinylpyrrolidone-vinyl acetate copolymer, polyethylene glycol, Eudragit® EPO, and hypromellose acetate succinate.

6. The process according to claim 1, wherein the water-soluble polymer is a polyvinylpyrrolidone-vinyl acetate copolymer.

7. The process according to claim 1, wherein the amount of the water-soluble polymer is about 5% to 95% of the weight of the pharmaceutical composition.

8. The process according to claim 1, wherein the mixture of the pentacyclic triterpenoid and the water-soluble polymer in step b) is melted as it passes through at least four heating zones, the temperature of the heating zones decreasing in succession.

9. A pharmaceutical composition comprising a pentacyclic triterpenoid, wherein the pharmaceutical composition is prepared according to the process described in claim 1.

10. The pharmaceutical composition according to claim 9, wherein the pentacyclic triterpenoid is therapeutically effective at a dose of about 0.05 mg / kg (body weight) to about 20 mg / kg (body weight).

11. The pharmaceutical composition according to claim 9, wherein the pentacyclic triterpenoid is therapeutically effective at a dose of about 0.5 mg / kg (body weight) to about 10 mg / kg (body weight).

12. The pharmaceutical composition according to claim 9, which is in the form of a capsule, tablet, chewable tablet, soft chew, gummy, bar, oral paste, lozenge, or powder.

13. A pharmaceutical composition according to claim 9, which does not contain a solvent.

14. A method for treating a condition caused in a subject by an overactive pro-inflammatory cytokine, the method comprising the step of administering the pharmaceutical composition described in claim 9 to a subject in need of treatment.

15. The method according to claim 14, wherein the aforementioned pathological condition is kidney disease, acute kidney injury, or chronic kidney disease.

16. The method according to claim 14, wherein the aforementioned pathological condition is a pathological state caused by a viral infection.

17. The method according to claim 14, wherein the aforementioned pathological condition is a pathological state caused by a bacterial infection.

18. The method according to claim 14, wherein the disease state is tuberculosis.

19. The method according to claim 14, wherein the aforementioned pathological condition is diabetic nephropathy.

20. The method according to claim 14, wherein the aforementioned pathological condition is inflammatory arthritis.

21. The method according to claim 14, wherein the aforementioned pathological condition is an allergy or allergic asthma.

22. The method according to claim 14, wherein the aforementioned pathological condition is periodontal disease.

23. The method according to claim 14, wherein the aforementioned pathological condition is vulvar pain.

24. The method according to claim 14, wherein the aforementioned pathological condition is otitis.

25. The method according to claim 14, wherein the aforementioned pathological condition is ulcerative colitis or Crohn's disease.

26. The method according to claim 14, wherein the aforementioned pathological condition is myasthenia gravis.

27. The method according to claim 14, wherein the aforementioned pathological condition is multiple sclerosis.

28. The method according to claim 14, wherein the aforementioned pathological condition is liver disease or alcoholic liver disease.

29. The method according to claim 14, wherein the aforementioned pathological condition is mucositis.

30. The method according to claim 14, wherein the aforementioned pathological condition is sepsis.

31. The method according to claim 14, wherein the aforementioned pathological condition is major depressive disorder.

32. The method according to claim 14, wherein the aforementioned pathological condition is an anxiety-related disorder.

33. The method according to claim 14, wherein the aforementioned pathological condition is a chronic inflammatory skin disease.

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