HK40060507B - Use of composition for preventing, ameliorating, or treating bone loss disorders, comprising cyclo-hispro (chp) and parathyroid hormone - Google Patents

Description

Use of compositions containing cyclic (histidine-proline) dipeptides and parathyroid hormone for the prevention, improvement, or treatment of bone loss disorders.

Technical Field

This invention relates to the use of a composition comprising a cyclic (histidine-proline) dipeptide (CHP) and parathyroid hormone for the prevention, improvement or treatment of bone loss diseases.

Background Technology

Bone modeling and remodeling play crucial roles in bone development, growth, and metabolism. Bone modeling begins at birth and continues until bone maturity and the end of growth during young adulthood, with the largest bone mass formed between the ages of 20 and early 30. For the next 30 years, a continuous cycle of bone loss and replenishment occurs, during which bone formation and resorption maintain a balance. After this period, bone formation is insufficient to keep up with the bone loss caused by resorption, resulting in a bone mass loss of approximately 0.3–0.5% per year. This is particularly pronounced in women, who experience a significant bone loss of 2–3% per year in early menopause.

Bone tissue constitutes the cartilage and skeletal system, serving as a support for mechanical functions and an attachment point for muscles. It is responsible for protecting biological organs and bone marrow, and storing calcium and phosphorus ions to maintain homeostasis. Bone tissue is composed of the cellular matrix, including collagen and glycoproteins, and various cell types such as osteoblasts, osteoclasts, and osteocytes.

Furthermore, bone tissue is a dynamic tissue formed by osteoblasts and constantly absorbed and destroyed by osteoclasts. Osteoporosis is a disease caused by an imbalance between osteoblasts and osteoclasts, resulting in increased bone resorption than bone formation. As bone calcification decreases, bone density thins, and the medullary cavity widens. As the disease progresses, the bones become increasingly fragile, making them prone to fractures even from minor impacts.

Osteoporosis is known to cause various fractures, often due to weakened bones rather than the symptoms themselves, particularly femoral or vertebral fractures. These fractures restrict long-term activity, preventing people from leading healthy lives and ultimately contributing to 15% of deaths in the elderly. Bone mass is influenced by a variety of factors, including genetics, nutritional intake, hormonal changes, and differences in exercise and lifestyle. Known causes of osteoporosis include old age, lack of exercise, low body weight, smoking, low-calcium diets, menopause, and oophorectomy. Women, in particular, continue to experience bone loss after age 30, and this loss accelerates during menopause due to hormonal changes.

As such, although the degree varies, osteoporosis is an unavoidable symptom for the elderly, especially postmenopausal women. With an aging population, developed countries are showing increasing interest in osteoporosis and its treatments. Furthermore, the global market related to bone disease treatment is known to be worth approximately $130 billion, and is expected to grow further in the future. Therefore, research institutions and pharmaceutical companies around the world are investing heavily in the development of bone disease treatments, and the research and development of bone resorption inhibitors is currently underway.

In recent years, parathyroid hormone (PTH) has become a popular treatment for osteoporosis. Unlike other therapies that reduce bone resorption, PTH increases bone mass and further increases bone mineral density (BMD). PTH has multiple direct and indirect effects on bone. PTH increases the rate of calcium release from bones into the blood. The chronic effect of PTH is to increase the number of osteoblasts and osteoclasts on both sides of the bone and to increase bone remodeling. Administration of PTH to patients with osteoporosis produces net stimulation of bone formation, particularly in the trabecular bone of the spine and hip joint, thus significantly reducing fractures. Bone formation is thought to occur through PTH stimulation of osteoblasts, just as osteoblasts have PTH receptors.

Against this backdrop, the inventors sought a substance that could enhance the therapeutic effect on bone loss diseases when used in combination with parathyroid hormone as a compound. When parathyroid hormone was used with cyclic (histidine-proline) dipeptide (CHP), it was confirmed that it exhibited a synergistic effect on osteoblast differentiation and bone formation, thus completing the present invention.

Existing technical documents

Patent documents

Patent Document 1: Korean Patent Publication No. 10-2008-0059430

Summary of the Invention

Technical issues

The purpose of this invention is to provide a pharmaceutical composition for the prevention or treatment of bone loss diseases, comprising: a cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof; and parathyroid hormone.

Another object of the present invention is to provide a pharmaceutical composition for enhancing the therapeutic effect of parathyroid hormone on bone loss diseases, comprising a cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof.

Furthermore, the object of the present invention is to provide a method for the prevention or treatment of bone loss disease, comprising the step of administering an effective amount of a composition to an individual in need of it, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and parathyroid hormone (PTH).

Another object of the present invention is to provide a method for enhancing the therapeutic effect of parathyroid hormone on bone loss diseases, comprising the steps of administering cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and administering parathyroid hormone (PTH) to an individual in need of it.

Another object of the present invention is to provide the use of a composition in the preparation of a medicament for the prevention or treatment of bone loss diseases, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and parathyroid hormone (PTH).

Another object of the present invention is to provide the use of a composition in the preparation of a medicament for enhancing the preventive or therapeutic effect of parathyroid hormone (PTH) on bone loss diseases, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof.

Problem-solving methods

To address the aforementioned problems, the present invention provides a pharmaceutical composition for the prevention or treatment of bone loss diseases, comprising: a cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof; and parathyroid hormone.

Furthermore, the present invention provides a pharmaceutical composition for enhancing the therapeutic effect of parathyroid hormone on bone loss diseases, comprising a cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof.

The present invention provides a method for the prevention or treatment of bone loss disease, comprising the step of administering an effective amount of a composition to an individual in need of the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and parathyroid hormone (PTH).

Furthermore, the present invention provides a method for enhancing the therapeutic effect of parathyroid hormone on bone loss diseases, comprising the steps of administering cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and administering parathyroid hormone (PTH) to an individual in need of it.

Furthermore, the present invention provides the use of a composition in the preparation of a medicament for the prevention or treatment of bone loss diseases, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and parathyroid hormone (PTH).

Furthermore, the present invention provides the use of a composition in the preparation of a pharmaceutical agent for enhancing the preventive or therapeutic effect of parathyroid hormone (PTH) on bone loss diseases, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof.

According to a preferred embodiment of the present invention, the above-mentioned parathyroid hormone may be parathyroid hormone _1-34 .

According to another preferred embodiment of the present invention, the aforementioned bone loss disease may be one or more selected from the group consisting of osteoporosis, Paget's disease, alveolar bone loss, osteomalacia, and renal osteodystrophy.

According to another preferred embodiment of the present invention, the aforementioned osteoporosis may be caused by a decrease in female hormones, destruction of osteoblasts, or inhibition of their activity.

According to another preferred embodiment of the invention, the above-mentioned cyclic (histidine-proline) dipeptide (CHP) or its pharmaceutically acceptable salt, along with parathyroid hormone (PTH), can be administered simultaneously, separately, or sequentially.

The effects of the invention

The composition of the present invention, comprising a cyclic (histidine-proline) dipeptide and parathyroid hormone for the prevention, improvement or treatment of bone loss diseases, exhibits a synergistic effect on osteoblast differentiation and bone formation promotion, thereby being effective in treating bone loss diseases. Since the cyclic (histidine-proline) dipeptide enhances the osteoblast differentiation and bone formation promotion effects of parathyroid hormone, the cyclic (histidine-proline) dipeptide can be used as a synergistic adjuvant when parathyroid hormone is used for the prevention, improvement or treatment of bone loss diseases.

Attached Figure Description

Figures 1a and 1b are graphs illustrating the effect of combined treatment with cyclic (histidine-proline) dipeptide (CHP) and parathyroid hormone _1-34 (human parathyroid hormone _1-34 ) on increasing alkaline phosphatase activity during MC3T3-E1 osteoblast differentiation.

Figure 2a is a graph illustrating the effect of combined treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on increasing alkaline phosphatase gene expression during primary osteoblast differentiation.

Figure 2b is a graph showing the effect of combined treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on increasing bone morphogenetic protein 2 (BMP2) gene expression during primary osteoblast differentiation.

Figure 2c is a graph showing the effect of combined treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on increasing osteocalcin gene expression during primary osteoblast differentiation.

Figure 2d is a graph showing the effect of combined treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on increasing the expression of the osteogenic-associated transcription factor antibody (Osterix) gene during primary osteoblast differentiation.

Figure 2e is a graph showing the effect of combined treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on increasing the expression of the core binding factor α1 (Runx2) gene during primary osteoblast differentiation.

Figure 2f is a graph showing the effect of combined treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on increasing collagen 1a1 (Col1a1) gene expression during primary osteoblast differentiation.

Figure 2g is a graph showing the effect of combined treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on increasing osteoprotegerin (OPG) gene expression during primary osteoblast differentiation.

Detailed Implementation

The present invention will now be described in more detail.

As described above, the inventors sought a substance that could enhance the therapeutic effect on bone loss diseases when used in combination with parathyroid hormone as a compound. When parathyroid hormone was used with cyclic (histidine-proline) dipeptide (CHP), it was confirmed that it exhibited a synergistic effect on osteoblast differentiation and bone formation, thus completing the present invention.

Therefore, the present invention provides a pharmaceutical composition for the prevention or treatment of bone loss diseases, comprising: a cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof; and parathyroid hormone.

Furthermore, the present invention provides a pharmaceutical composition for enhancing the therapeutic effect of parathyroid hormone on bone loss diseases, comprising a cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof.

In this invention, "Cyclo-HisPro (CHP)" is a naturally occurring cyclic dipeptide or a physiologically active dipeptide synthesized in vivo through TRH metabolism and de novo synthesis. It is widely distributed throughout the brain, spinal cord, and gastrointestinal tract. The aforementioned cyclic dipeptide is composed of histidine-proline, a metabolite of thyrotropin-releasing hormone (TRH).

In the compositions of the present invention, the aforementioned cyclic (histidine-proline) dipeptide can be used either by synthesis or by using commercially available alternatives. Furthermore, it can be used by purification from substances containing the cyclic (histidine-proline) dipeptide, such as prostate extracts.

The term "purified" is intended to indicate that the cyclic (histidine-proline) dipeptide is in a concentrated form compared to its form, which is available from natural sources such as prostate extract. Purified components can be obtained from their natural sources through concentration or chemical synthesis.

The main components of the aforementioned "prostate extract" are zinc, cyclic (histidine-proline) dipeptide, prostaglandin precursor, and arachidonic acid. Due to the high concentration of cyclic (histidine-proline) dipeptide, it is reasonable to expect that cyclic (histidine-proline) dipeptide will induce an increase in bone mineral density and bone volume ratio, as well as promote osteoblast differentiation and bone formation.

In the composition of the present invention, the above-mentioned "prostate extract" may be bovine or porcine prostate powder, etc. Preferably, in order to increase the content of cyclic (histidine-proline) dipeptide, it can be used in a fat-free form, but is not limited thereto.

In the compositions of the present invention, the aforementioned "parathyroid hormone (PTH)" can be a secretory 84-amino acid residue polypeptide or fragment thereof having the following amino acid sequence: Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Val-Ala -Leu-Gly-Ala-Pro-Leu-Ala-Pro-Arg-Asp-Ala-Gly-Ser-Gln-Arg-Pro-Arg-Lys-Lys-Glu-Asp-Asn-Val-Leu-Val-Glu -Ser-His-Glu-Lys-Ser-Leu-Gly-Glu-Ala-Asp-Lys-Ala-Asn-Val-Asp-Val-Leu-Thr-Lys-Ala-Lys-Ser-Gln (SEQ ID NO: 1).

According to a preferred embodiment of the present invention, the above-mentioned parathyroid hormone (PTH) may be parathyroid hormone _1-34 composed of the following N-terminal 34 amino acids of bovine or human hormone: Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe (Sequence Number 2).

The parathyroid hormone (1-34), also known as teriparatide, is currently marketed by Eli Lilly under the brand name FORTEO in Indianapolis, Indiana, USA, for the treatment of postmenopausal women with osteoporosis who are at high risk of fractures.

In this invention, the term "bone loss" refers to the symptoms of bone loss caused by an imbalance between osteoclasts and osteoblasts, and "bone loss disease" refers to all diseases related to the aforementioned symptoms. Therefore, it includes all diseases caused by decreased bone density due to excessive osteoclast activity leading to bone loss, or by decreased osteoblast activity leading to poor bone formation. Specific examples of the aforementioned bone loss diseases include, but are not limited to, osteoporosis, Paget's disease, alveolar bone loss, osteomalacia, and renal osteodystrophy.

In this case, the aforementioned osteoporosis is one of the symptoms of menopause or postmenopause, which may be caused by a decrease in female hormones or by damage to osteoblasts or inhibition of their activity.

In this invention, the term "menopause" generally refers to the period of transition from fertility to infertility. The term "menopause" primarily refers to the female menopause, but it encompasses not only the period of menopause but also the period surrounding menopause, typically corresponding to the age range of 40 to 60 years.

The composition of the present invention comprising a cyclic (histidine-proline) dipeptide or its salt and parathyroid hormone can promote bone formation by promoting osteoblast differentiation, thereby preventing, improving or treating bone loss diseases.

As shown in Figures 1a and 1b, the increase in alkaline phosphatase activity observed when cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 (hPTH _1-34 ) were administered in combination was significantly higher than the sum of the increases in alkaline phosphatase activity observed when either was administered alone. This confirms the synergistic effect of the combined administration of cyclic (histidine-proline) dipeptide and parathyroid hormone in promoting osteoblast differentiation. Alkaline phosphatase (ALP) is an early marker of osteoblast differentiation; increased ALP activity promotes osteoblast differentiation, thereby promoting bone formation.

In the compositions of the present invention for the prevention, improvement, or treatment of bone loss diseases, the term "prevention" refers to all actions that inhibit or delay the onset of a disease or condition. In the present invention, it refers to delaying the onset of bone loss diseases or inhibiting their onset by promoting osteoblast differentiation.

In the compositions of the present invention for the prevention, improvement or treatment of bone loss diseases, the term "improvement" refers to all behaviors that improve or beneficially alter the state of a disease or condition, and in the present invention refers to improving symptoms such as osteoporosis or alveolar bone loss by promoting osteoblast differentiation.

In the compositions of the present invention for the prevention, improvement or treatment of bone loss diseases, the term "treatment" refers to all actions that delay, stop or reverse the progression of a disease or condition, and in the present invention refers to stopping, reducing, alleviating, eliminating or reversing alveolar bone or bone loss by promoting osteoblast differentiation.

The term "synergy effect" refers to the effect produced when each component is administered in combination (as a single component) being greater than the sum of the effects produced when each component is administered alone (Chou and Talalay, Adv. Enzyme. Regul., 22:27-55, 1984). The cyclic (histidine-proline) dipeptide or its salt thereof, along with parathyroid hormone, of the present invention, enhances the preventive, ameliorative, or therapeutic effects on bone loss diseases through combined administration.

The term "administered in combination" refers to administering a compound or ingredient to a patient together. Administering each compound or ingredient together means that each ingredient can be administered at the same time, in any order, or sequentially at different times to achieve the desired therapeutic effect.

The term "patient" refers to any single individual requiring treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, etc. Furthermore, the subject includes any individual participating in a clinical research trial but without any clinical manifestations of disease, or any individual participating in an epidemiological study or used as a control group.

In this invention, the term "pharmaceutical acceptable" means physiologically acceptable and generally does not cause allergic reactions or similar reactions when administered to humans, and the salt is preferably an acid addition salt formed from a pharmaceutically acceptable free acid.

The pharmaceutically acceptable salts described above can be acid addition salts formed using organic or inorganic acids. The organic acids include, for example, formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, or methanesulfonic acid. The inorganic acids include, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, or boric acid. The acid addition salts are preferably in the form of hydrochloride or acetate, more preferably in the form of hydrochloride.

In addition, the salt can also be gaba salt, gabapentin salt, pregabalin salt, nicotinate, adipate, hemimalonic acid salt, cysteine salt, acetylcysteine salt, methionine salt, arginine salt, lysine salt, ornithine salt, or aspartate salt.

Furthermore, the pharmaceutical compositions of the present invention may also comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may also include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, suitable oils, saline solutions, glucose, and aqueous solutions of ethylene glycol, etc. Furthermore, stabilizers and preservatives may also be included. Suitable stabilizers include antioxidants, such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. Other pharmaceutically acceptable carriers can be found in the carriers described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical compositions of the present invention can be administered to mammals, including humans, by any method. For example, they can be administered orally or parenterally, and parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intraenteral, local, sublingual, or rectal administration.

Based on the above-described route of administration, the pharmaceutical compositions of the present invention can be formulated into preparations for oral or parenteral administration. In formulation, one or more buffer solutions (e.g., saline or phosphate-buffered saline (PBS)), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), antioxidants, antibacterial agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants or surfactants, diluents, or excipients can be used.

Solid dosage forms for oral administration include tablets, pills, acid preparations, granules, liquids, gels, syrups, pastes, suspensions, or capsules, etc. These solid dosage forms are formulated by mixing at least one excipient in the pharmaceutical composition of the present invention, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, glucose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose or gelatin, etc. For example, tablets or sugar-coated pills can be obtained by mixing the active ingredient with the solid excipient, grinding, adding suitable adjuvants, and processing them into a granular mixture.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solutions, emulsions, or syrups, and may include various excipients such as wetting agents, sweeteners, flavorings, or preservatives, in addition to water or liquid paraffin, which are commonly used as simple diluents.

Furthermore, depending on the circumstances, cross-linked polyvinylpyrrolidone, agar, alginate, or sodium alginate can be added as disintegrants, and may further include anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

When administered parenterally, the pharmaceutical compositions of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation, according to methods known in the art. In the case of the aforementioned injections, sterilization and protection against contamination by microorganisms such as bacteria and fungi are necessary. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and/or solvents or dispersion media containing vegetable oils. More preferably, suitable carriers may be Hanks' solution, Ringer's solution, phosphate-buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% glucose. To protect the injections from microbial contamination, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc., may also be added. Furthermore, in most cases, the injections may also contain sugar or sodium chloride isotonic agents.

In the case of transdermal drug delivery, these include forms such as ointments, creams, lotions, gels, topical solutions, pastes, liniments, and air-forming agents. In the context of this text, "transdermal delivery" refers to the delivery of an effective amount of the active ingredient contained in a pharmaceutical composition to the skin by applying the drug composition topically to the skin.

In the case of inhaled administration, the compounds used according to the invention can be conveniently delivered from a pressurized pack or nebulizer in the form of an aerosol spray using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve for delivering the amount of the metering. For example, the gelatin capsules and cartridges used in inhalers or blowpipes can be formulated as powder mixtures containing the compound and a suitable powder matrix such as lactose or starch. Formulations for parenteral administration are described in the literature (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour), which are generally well-known formulations in all pharmaceutical chemistry.

When the pharmaceutical compositions of the present invention contain an effective amount of cyclic (histidine-proline) dipeptide or its salt and parathyroid hormone, they can provide preferred effects for the prevention, improvement, or treatment of bone loss diseases. In this invention, the term "effective amount" refers to an amount that exhibits a response greater than that of the negative control group, and preferably an amount sufficient to prevent, improve, or treat bone loss diseases. The pharmaceutical compositions of the present invention may contain 0.01% to 99.9% of cyclic (histidine-proline) dipeptide or its salt and parathyroid hormone, with the remainder occupied by a pharmaceutically acceptable carrier. The effective amount of cyclic (histidine-proline) dipeptide or its salt and parathyroid hormone contained in the pharmaceutical compositions of the present invention will vary depending on the product form of the composition, etc.

The total effective amount of the pharmaceutical composition of the present invention can be administered to the patient as a single dose, or via a fractionated treatment protocol using multiple doses over a prolonged period. The content of the active ingredient in the pharmaceutical composition of the present invention can be varied according to the severity of the disease. For example, based on a cyclic (histidine-proline) dipeptide, it can be administered once or several times, preferably at a dose of 0.001 mg to 100 mg per kg of body weight per day, more preferably 0.01 mg to 10 mg. However, the dosage of the aforementioned cyclic (histidine-proline) dipeptide or its salts and parathyroid hormone is determined based on various factors such as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, as well as the route of administration and number of treatments of the pharmaceutical composition, and the effective dosage. Considering the above, those skilled in the art can determine the appropriate effective dosage of the aforementioned cyclic (histidine-proline) dipeptide or its salts and parathyroid hormone for a specific purpose of preventing, treating, or improving bone loss diseases. The pharmaceutical compositions according to the present invention are not particularly limited in terms of dosage form, route of administration, and method of administration, as long as they exhibit the effects of the present invention.

The pharmaceutical composition of the present invention for the prevention or treatment of bone loss diseases can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers.

The pharmaceutical compositions of the present invention for the prevention or treatment of bone loss diseases can also be provided in dosage forms containing a cyclic (histidine-proline) dipeptide or a salt thereof and parathyroid hormone for topical application. In this respect, the compositions of the present invention can be quasi-pharmaceutical compositions for the prevention or improvement of bone loss diseases and quasi-pharmaceuticals comprising the above compositions.

The above-mentioned topical agents can be applied directly to the skin or oral cavity. When the pharmaceutical composition of the present invention for the prevention or treatment of bone loss diseases is used as a topical agent, it may further contain fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emulsifiers, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, metal ion chelating agents, complexing agents, preservatives, vitamins, retardants, wetting agents, essential oils, dyes, pigments, hydrophilic surfactants, lipophilic surfactants, or may contain any other ingredients commonly used in dermatological adjuvants, such as lipoves. Furthermore, the above-mentioned ingredients can be introduced in amounts commonly used in dermatological fields.

When the compositions of the present invention are provided as topical agents, they may be in dosage forms such as liquids, ointments, patches, gels, creams, or sprays, but are not limited thereto. According to an example of the present invention, the quasi-pharmaceuticals of the present invention may include oral care products, ointments, masks, wet dressings, patches, and transdermal absorbents, etc., wherein the oral care products include toothpaste, mouthwash, and oral sprays.

When treated with the cyclic (histidine-proline) dipeptide of the present invention, osteoblast differentiation is promoted. When the above-mentioned active ingredient is applied to oral care products, it can effectively prevent or improve alveolar bone diseases by promoting osteoblast differentiation. Therefore, the above-mentioned quasi-pharmaceutical composition can be an oral care composition for preventing or improving bone loss.

When the compositions of the present invention are used as quasi-pharmaceutical compositions, they can be directly added to cyclic (histidine-proline) dipeptides or their salts and parathyroid hormone, or appropriately used together with other quasi-pharmaceutical ingredients according to conventional methods. The mixing amount of the active ingredients can be appropriately determined according to the purpose of use (prevention, health care, or therapeutic treatment).

The contents of the pharmaceutical compositions of the present invention can be compared with those of the quasi-pharmaceutical compositions and quasi-pharmaceuticals of the present invention.

The present invention also provides a method for the prevention or treatment of bone loss disease, comprising the step of administering an effective amount of a composition to an individual in need of it, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and parathyroid hormone (PTH).

Furthermore, a method for enhancing the therapeutic effect of parathyroid hormone on bone loss diseases is provided, comprising the steps of administering cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and administering parathyroid hormone (PTH) to an individual in need of it.

In the methods of this invention, the term "individual" includes, but is not limited to, any animal (e.g., human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent). These terms do not indicate a specific age or sex. Therefore, it is intended to include female/female, male/male, adult/adult, newborn subject, and fetus. "Patient" refers to a subject suffering from a disease or disorder. The term "patient" includes both human and veterinary subjects.

In the method of the present invention, the description of the structure including the effects of the cyclic (histidine-proline) dipeptide and parathyroid hormone, as well as its route of administration, frequency of administration, dosage, etc., is the same as above, and therefore its description will be omitted.

In the method of this invention, cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof, and parathyroid hormone (PTH) can be administered simultaneously, separately, or sequentially. For example, cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof and parathyroid hormone can be co-formulated and administered simultaneously as a combined unit dosage form, or they can be administered simultaneously or sequentially as separate formulations.

In the case of sequential administration, each active ingredient can be administered as a separate formulation at time intervals, and the order of administration can be determined by a physician or a person skilled in the art.

The present invention also provides the use of a composition in the preparation of a medicament for the prevention or treatment of bone loss diseases, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof; and parathyroid hormone (PTH).

The present invention also provides the use of a composition in the preparation of a pharmaceutical agent for enhancing the preventive or therapeutic effect of parathyroid hormone (PTH) on bone loss diseases, the composition comprising: a cyclic (histidine-proline) dipeptide (CHP) or a pharmaceutically acceptable salt thereof.

The present invention will now be described in more detail through embodiments. However, since the present invention can have many variations and forms, the specific embodiments and descriptions described below are only for the purpose of helping to understand the present invention, and the present invention is not limited to the specific disclosed form. It should be understood that the scope of the present invention includes all modifications, equivalent technical solutions, and alternative technical solutions within the spirit and scope of the present invention.

Embodiments of the present invention

Preparation Example 1

The cyclic (histidine-proline) dipeptide (CHP) used in the following examples was purchased from Bachem, and human parathyroid hormone _1-34 (hPTH _1-34 ) was purchased from Tocris. Gibco's MEMα, ascorbic acid-free medium, and Hyclone's fetal bovine serum (FBS) were purchased for the culture of MC3T3-E1 preosteoblasts. β-glycerophosphate and ascorbic acid for differentiation were purchased from Sigma-Aldrich. Cell lysis was performed using Thermo's RIPA lysis buffer. To measure alkaline phosphatase (ALP) activity, protein quantification was performed using Sigma-Aldrich's p-nitrophenyl phosphate (pNPP) substrate solution and Thermo's BCA quantitative kit. To extract ribonucleic acid (RNA) from cells, MAHEREY-NAGEL's NucleoZOL was purchased. For the synthesis of complementary deoxyribonucleic acid (cDNA) and real-time polymerase chain reaction (PCR) analysis, Bio-Rad's iScript cDNA synthesis kit and iQ SYBR Green Supermix were purchased.

Preparation Example 2

C57BL/6pup cells were purchased from Coretech on day one and used to culture primary osteoblasts derived from the skull. Collagenase and dispersase used for cell isolation were purchased from Gibco and Roche, respectively.

Preparation Example 3

Primers used for real-time polymerase chain reaction (PCR) were synthesized by Bioneer with the base sequences shown in Table 1.

Table 1

Example 1

Measurement of the effects of combined administration of cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on MC3T3-E1 osteoblast differentiation and bone formation promotion.

1-1. Culture conditions of MC3T3-E1 osteoblasts

MC3T3-E1 cells were cultured in MEMα medium (no ascorbic acid) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a 5% _CO2 incubator. To differentiate into osteoblasts, 10 mM β-glycerophosphate and 50 μg/μl ascorbic acid were added to the basal medium when the cells were confluent.

1-2. Treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 , alone or in combination, promoted the differentiation and induction of MC3T3-E1 osteoblasts.

In 12-well plates, 2.5 x ^10⁵ MC3T3-E1 cells were seeded at 1 ml and cultured for 48 hours until confluence. To differentiate into osteoblasts, the medium was replaced with differentiation medium, and each group was treated with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 at the appropriate concentrations, as shown in Table 2. In this case, the undifferentiated group, serving as the differentiation-negative control, was treated with basal medium instead of differentiation medium. After 6 hours of treatment, the medium in all groups was removed, washed once with phosphate-buffered saline (PBS), and replaced with differentiation medium. Treatment with each concentration of cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 was performed every 72 hours, for a total of 14 days.

Table 2

1-3. Measurement of Alkaline Phosphatase Activity

On day 14 of differentiation, the culture medium was removed, and the cells were washed once with phosphate-buffered saline (PBS). 100 μl of RIPA lysis buffer was added to each well, and cells were separated using a cell scraper. The cells were then transferred to 1.5 ml tubes using pipettes and sonicated using a sonicator (amplitude 10, 0.5 sec pulses, 10 times). After centrifugation at 15,000 rpm for 10 minutes at 4°C, only the supernatant was separated. 10 μl of each lysate sample was added to a 96-well plate, along with 200 μl of pNPP substrate solution, and incubated at room temperature for 1 hour. The absorbance was measured at 405 nm.

To correct the alkaline phosphatase activity value using the protein concentration of the lysate, each lysate was quantified using the BCA quantification method, and the alkaline phosphatase activity absorbance value at 405 nm was divided by the total protein content (Abs. OD 405 nm/μg of analyzed).

For statistical significance, one-way ANOVA was used, and Tukey's post-hoc test was used to compare the significance with the control group (*p<0.05).

The experimental results, as shown in Figures 1a and 1b, confirmed that alkaline phosphatase activity was increased in the group treated with human parathyroid hormone _1-34 in combination with cyclic (histidine-proline) dipeptide compared to the group treated with human parathyroid hormone _1-34 alone. This result can be explained by the fact that the osteoblast differentiation and bone formation promoting effects of human parathyroid hormone _1-34 are further enhanced by the combined administration of cyclic (histidine-proline) dipeptide. Therefore, it is confirmed that the combined administration of human parathyroid hormone _1-34 and cyclic (histidine-proline) dipeptide exhibits a synergistic effect on bone health and the treatment of osteoporosis.

Example 2

Measurement of the effects of combined administration of cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 on the differentiation of primary osteoblasts and the expression of osteogenic-related genes.

2-1. Culture of primary osteoblasts

Only C57BL/6pup calvaria skulls were isolated from natal cells on day 1. The isolated skulls were placed in digestion media (MEMα containing 0.1% collagenase and 0.2% dispersant) and digested five times at 37°C with shaking, each time for 15 minutes. The first digested solution was discarded, and the second through fifth digested solutions were collected. Cells were centrifuged at 1600 rpm for 5 minutes, and then resuspended in MEMα media (containing 10% fetal bovine serum (FBS)). Cells were then passed through a 0.2 μm strainer to remove debris. Cells obtained from the media were released and cultured at 37°C with 5% _CO2 for 3 days before use. Then, to differentiate into osteoblasts, 10 mM β-glycerophosphate and 50 μg/μl ascorbic acid were added to the culture medium composition when the cells were confluent.

2-2. Treatment with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 , alone or in combination, to promote the differentiation induction of primary osteoblasts.

In 12-well plates, 1 x ^10⁵ primary osteoblasts were seeded at 1 ml and cultured for 48 hours. To differentiate into osteoblasts, the medium was replaced with differentiation medium, and each group was treated with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 at the corresponding concentrations, as shown in Table 3. In this case, the undifferentiated group, serving as the differentiation-negative control, was treated with basal medium instead of differentiation medium. After 6 hours of treatment, the medium in all groups was removed, washed once with phosphate-buffered saline (PBS), and replaced with differentiation medium. The cells were treated with each concentration of cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 every 48 hours for a total of 6 days.

Table 3

2-3. Extraction of cellular ribonucleic acid (RNA)

On day 6 of differentiation, the culture medium was removed, and the cells were washed once with phosphate-buffered saline (PBS) and then placed in 500 μL of NucleoZOL for cell lysis. Next, ribonucleic acid (RNA) was extracted according to the NucleoZOL Total RNA isolation protocol. Using the iScript complementary deoxyribonucleic acid (cDNA) synthesis kit, 1 μg of RNA was synthesized into complementary deoxyribonucleic acid (cDNA) via reverse transcription polymerase chain reaction.

2-4. Analysis of osteoblast differentiation and osteogenic-related gene expression

Synthetic complementary deoxyribonucleic acid (cDNA) was polymerase chain reaction (PCR) performed in real-time using an iQ SYBR Green Supermix with corresponding forward/reverse primer sets for each gene. The expression value of each gene was corrected by dividing it by the expression value of the housekeeping gene β-actin. For statistical significance, one-way ANOVA was used, and Tukey's post-hoc test was used to compare significance with the control group (*p<0.05, **p<0.01).

The experimental results, as shown in Figure 2a, confirmed that the expression of alkaline phosphatase (ALP), a representative marker of osteoblast differentiation, was significantly increased in the group treated with the combined treatment of human parathyroid hormone _1-34 and cyclic (histidine-proline) dipeptide compared to the control group, and was also significantly increased compared to the groups treated with cyclic (histidine-proline) dipeptide and human parathyroid hormone _1-34 alone. Therefore, the combined administration of human parathyroid hormone _1-34 and cyclic (histidine-proline) significantly increased the differentiation of primary osteoblasts and promoted bone formation.

As shown in Figures 2b and 2c, the genes for bone morphogenetic protein 2 (BMP2) and osteocalcin, secreted concurrently with osteoblast differentiation, were confirmed to be significantly increased in the group treated with the combined human parathyroid hormone _1-34 and cyclic (histidine-proline) dipeptide. Therefore, compared with the control group, the combined administration of human parathyroid hormone _1-34 and cyclic (histidine-proline) dipeptide significantly promoted the differentiation of primary osteoblasts.

As shown in Figures 2d and 2e, compared with the control group, the expression of osteoblast-related transcription factor antibody (Osterix) and core-binding factor α1 (Runx2) genes were significantly increased in the human parathyroid hormone _1-34 and cyclic (histidine-proline) dipeptide combined treatment group. These genes are transcription factors involved in osteoblast differentiation and bone formation. Furthermore, as shown in Figures 2f and 2g, compared with the control group, the expression of collagen _1a1 (Col1a1) and osteoprotein (OPG) genes, which are increased through signal transcription related to osteoblast differentiation and bone formation, also showed a trend towards near-significant increases in the human parathyroid hormone 1-34 and cyclic (histidine-proline) dipeptide combined treatment group.

The above results confirm that the combined administration of human parathyroid hormone _1-34 and cyclic (histidine-proline) dipeptide exhibits a synergistic effect in promoting primary osteoblast differentiation and bone formation, exceeding the expected effects of either drug alone. Therefore, the combined administration of human parathyroid hormone _1-34 and cyclic (histidine-proline) dipeptide demonstrates surprisingly excellent efficacy in treating bone loss diseases, including bone health and osteoporosis.

sequence list

<110> Nometta Pharmaceutical Co., Ltd.

<120> Use of compositions containing a cyclic (histidine-proline) dipeptide and parathyroid hormone for the prevention, improvement, or treatment of bone loss disorders.

<130> PCT5040486

<150> KR10-2019-0058369

<151> 2019-05-17

<160> 18

<170> PatentIn version 3.2

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Claims (6)

1. A pharmaceutical composition for the prevention or treatment of bone loss diseases, characterized in that it comprises: Cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof; and Parathyroid hormone _1-34 . 2. The pharmaceutical composition according to claim 1, wherein the bone loss disease is selected from one or more of the group consisting of osteoporosis, Paget's disease, alveolar bone loss, osteomalacia, and renal osteodystrophy. 3. The pharmaceutical composition according to claim 2, wherein the osteoporosis is caused by a decrease in female hormones or by destruction or inhibition of osteoblast activity. 4. Use of a composition in the preparation of a medicament for the prevention or treatment of bone loss diseases, characterized in that the composition comprises: a cyclic (histidine-proline) dipeptide or a pharmaceutically acceptable salt thereof; and parathyroid hormone _1-34 . 5. The use according to claim 4, wherein the bone loss disease is selected from the group consisting of osteoporosis, Paget's disease, alveolar bone loss, osteomalacia, and renal osteodystrophy. 6. The use according to claim 5, wherein the osteoporosis is caused by a decrease in female hormones or by destruction or inhibition of osteoblast activity.