Zinc transporter activator composition

Abstract

The present invention relates to a composition comprising alanine, threonine, and valine, wherein the composition may further comprise aspartic acid. The composition of the present invention can be useful for preventing or treating bone-related or prostate-related diseases, or for improving bone- or prostate-related health conditions, by increasing the expression of Zip proteins.

Description

Zinc transporter activator composition

[0001] The present invention relates to a novel composition comprising alanine, threonine, and valine. Furthermore, the composition may further comprise aspartic acid. Specifically, the composition of the present invention increases the expression of the Zip protein, a zinc transporter protein, and exhibits excellent effects in the prevention, treatment, and improvement of bone-related and prostate-related diseases.

[0002] Zinc is the second most abundant essential trace element after iron. Zinc deficiency is known to cause various symptoms, including immune disorders, skin development, and taste disturbances. Furthermore, zinc levels in the body can change dramatically under certain critical conditions, such as surgery, aging, and inflammation. Therefore, zinc plays a crucial role in various biological processes, and maintaining adequate zinc levels is crucial for regulating physiological responses.

[0003] Zinc content is primarily regulated by zinc transporters, which belong to the solute carrier (SLC) protein family and are expressed on the cell surface or in the plasma membrane of organelles. To date, two types of zinc transporters have been identified: the SLC30A family, which exports zinc ions to the extracellular space, and the SLC39A family, which imports zinc ions from the extracellular space. These transporters regulate zinc homeostasis, and zinc ions transported by zinc transporters are involved in specific cellular processes. Therefore, zinc ions transported by zinc transporters serve as signaling molecules that regulate various cellular functions, a phenomenon known as "zinc signaling." Interestingly, although zinc transporters share the common ability to transport zinc ions, they each exhibit specific physiological functions. Various studies have elucidated the physiological functions of each zinc transporter. In particular, knockout mouse and human genetic studies have revealed important interactions between zinc transporters and various human diseases. Therefore, the physiological functions of zinc transporters reveal the biological significance of zinc and zinc signaling, making them attractive therapeutic targets.

[0004] Zinc has been linked to a variety of physiological functions. Zinc deficiency can cause numerous symptoms, including skin diseases, taste disorders, immune disorders, and mental disorders. Excessive zinc levels can lead to toxicity, including nausea, vomiting, fever, and headaches. Therefore, maintaining adequate zinc levels in the body is crucial for maintaining good health.

[0005] Zinc transporters belong to the solute carrier (SLC) protein family, which are expressed in both the cell membrane and the membranes of intracellular organelles. There are two types of zinc transporters: the SLC30A / ZnT family and the SLC39A / ZIP family, with 10 and 14 transporters belonging to the ZnT and ZIP families, respectively. The SLC39A / ZIP family increases cytosolic zinc levels, whereas the SLC30A / ZnT family decreases cytosolic zinc levels. Zinc transporters have specific expression patterns for each organelle, and each molecule has been reported to regulate different physiological functions. Specifically, zinc transported by zinc transporters acts as a zinc signal, modulating intracellular organelles and thereby regulating specific cellular processes and resulting physiological responses. Over the past decade, human genetic analysis and studies of transgenic mice, knockout (KO), and conditional knockout (cKO) mice have revealed a strong association between zinc transporters and human diseases.

[0006] Zrt / Irt-like proteins 1, 2, and 3 (ZIP1, ZIP2, and ZIP3) are multipass membrane zinc transport proteins that mediate zinc uptake into the cytoplasm and are responsible for mammalian zinc homeostasis. Osteoblasts are known to be the primary functional cells for bone formation. ZIP1 was found to be expressed early in mouse embryonic development, in osteoblasts at E15.5, and later in ameloblasts and odontoblasts, indicating a close association of ZIP1 with bone formation.

[0007] Furthermore, ZIP1 is located at the cytoplasmic membrane and mediates zinc influx during osteoblast differentiation of mesenchymal stem cells (MSCs). ZIP1 expression was induced during osteogenic differentiation of pluripotent human MSCs and osteoblast progenitor MC3T3-E1 cells in vitro, accompanied by increased intracellular zinc uptake. Furthermore, ZIP1-mediated zinc influx led to higher expression of Runx2 and Osterix, master regulators of osteogenesis. The enhanced Runx2 and Osterix expression further regulated ZIP1 transcriptional expression by directly binding to response elements in their promoters. This process resulted in a series of intertwined feed-forward loops that induce zinc influx and osteogenic differentiation, and promote osteoapatite formation.

[0008] Citrate, a key intermediate in the TCA cycle, is produced from acetyl-CoA and oxaloacetate by citrate synthase. Because citrate homeostasis is crucial for energy production and survival, it is tightly controlled throughout the body. High concentrations of citrate are observed in bone and prostate tissue. Glucose, once incorporated into osteoblasts, is metabolized to generate energy, ultimately forming citric acid, which is then secreted. Furthermore, zinc inhibits aconitase, allowing citric acid to remain in the citrate form, further increasing intracellular citrate concentrations.

[0009] Citric acid produced by osteoblasts is essential for providing bone biochemical properties such as bone formation, bone stability, strength, and fracture resistance through citration, which combines inorganic components such as bone minerals and organic components such as collagen.

[0010] Increased ZIP1 expression and abundance increased zinc uptake by prostate cells, whereas downregulation of ZIP1 significantly reduced zinc uptake. Furthermore, the ZIP1 transporter coexists with high levels of zinc in normal epithelium. In prostate cancer, ZIP1 transporter activity is essentially absent, along with decreased zinc levels. These observations provide evidence that ZIP1 is a key functional transporter involved in zinc uptake and accumulation in the normal prostate, and that increasing ZIP1 transporter expression could be a therapeutic target for prostate cancer.

[0011] Meanwhile, alanine is a non-essential amino acid that can be synthesized in the body. It reduces toxic substances produced in the body, helps convert glucose into energy, and has a blood sugar control function.

[0012] Threonine is an essential amino acid that cannot be synthesized in the body, and is known to slow down cell aging and have the effect of increasing lifespan.

[0013] Valine is an essential amino acid that cannot be synthesized in the body, and has the function of regulating muscle protein synthesis and blood sugar control.

[0014] Although aspartic acid is a nonessential amino acid, it is known to be involved in both the TCA cycle and the ornithine cycle.

[0015] The present inventors have first discovered that a complex of alanine-threonine-valine and a complex of alanine-threonine-valine-aspartic acid, which contain nonessential amino acids and essential amino acids, have an excellent effect in enhancing Zip protein and in treating, improving and preventing bone diseases or prostate diseases, thereby completing the present invention.

[0016]

[0017] [Prior Art Literature]

[0018] [Non-patent literature]

[0019] (Non-patent literature 1) Kim et al., Nature Communications 13:6554 (2022).

[0020] (Non-patent Document 2) Renty B. Franklin and Leslie C. Costello, Zinc as an anti-tumor agent in prostate cancer and in other cancers, Arch Biochem Biophys, 463(2): 211-217 (2007).

[0021]

[0022] The present invention aims to provide a composition for preventing, improving or treating bone-related diseases or prostate-related diseases, comprising alanine, threonine and valine.

[0023] In addition, the present invention aims to provide a composition for preventing, improving or treating bone-related diseases or prostate-related diseases, comprising alanine, threonine, valine and aspartic acid.

[0024] The present invention aims to provide a method or composition for increasing the expression of Zip protein and citric acid.

[0025] The present invention relates to a composition for preventing, improving or treating bone-related diseases or prostate-related diseases, comprising alanine, threonine and valine.

[0026] In addition, the present invention relates to a composition for preventing, improving or treating bone-related diseases or prostate-related diseases, comprising alanine, threonine, valine and aspartic acid.

[0027] The composition of the present invention may be a pharmaceutical composition, a food composition or an animal feed composition for increasing the expression of Zip protein (Zrt-Irt-like protein) or the expression of citric acid.

[0028] In the composition of the present invention, the molar ratio of threonine and valine to alanine may be 0.9 to 1.1, respectively. In addition, the molar ratio of alanine, threonine and valine may preferably be about 1: about 1: about 1.

[0029] Because the composition of the present invention can increase the expression of Zip proteins, it can be usefully used to prevent, improve, or treat bone-related diseases or prostate-related diseases. In particular, the low-content composition of alanine, threonine, and valine of the present invention exhibits significantly superior effects compared to high-content single compositions of each of these compounds, thereby demonstrating excellent effects at low doses and reducing side effects.

[0030]

[0031] Hereinafter, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be implemented in various forms and is not limited to the embodiments and examples described herein.

[0032] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless otherwise stated.

[0033] The present invention provides a composition comprising alanine, threonine and valine.

[0034] The composition of the present invention may additionally contain aspartic acid.

[0035] The composition of the present invention may be a pharmaceutical composition for increasing the expression of Zip protein (Zrt-Irt-like protein).

[0036] The above Zip protein may be selected from the group consisting of Zip1, Zip2, Zip3, Zip4, Zip5, Zip6, Zip7, Zip8, Zip9, Zip10, Zip11, Zip12, Zip13 and Zip14, and is preferably Zip1.

[0037] The pharmaceutical composition according to the present invention may be a pharmaceutical composition for preventing or treating a disease state characterized by reduced expression of a Zip protein, preferably Zip1.

[0038] The pharmaceutical composition according to the present invention can preferably be used for the prevention or treatment of bone-related diseases or prostate-related diseases.

[0039] The bone-related diseases for which the pharmaceutical composition of the present invention can be used may be one or more selected from the group consisting of osteoporosis, Paget's bone disease, rickets, osteomalacia, alveolar bone loss, osteopenia, renal osteodystrophy, short stature, dwarfism, dwarfism, and growth hormone deficiency, but are not limited thereto.

[0040] The prostate-related diseases for which the pharmaceutical composition of the present invention can be used may be at least one selected from the group consisting of benign prostatic hyperplasia, prostate cancer, prostatitis, prostatic abscess, prostatic calcification, prostatic seminal vesicle inflammation, and prostatic effusion, but are not limited thereto.

[0041] The pharmaceutical composition of the present invention may include alanine, threonine and valine.

[0042] Preferably, the molar ratio of alanine, threonine, and valine contained in the pharmaceutical composition of the present invention may be 0.8 to 1.2 to 0.8 to 1.2 to 0.8 to 1.2, preferably 0.9 to 1.1 to 0.9 to 1.1 to 0.9 to 1.1. For example, the molar ratio of threonine and valine based on alanine may each independently be 0.9 to 1.1. The molar ratio may preferably be about 1 to about 1 to about 1. The above content ratio may be applied as is even if aspartic acid is added to the composition of the present invention.

[0043] The pharmaceutical composition of the present invention may include alanine, threonine, valine, and aspartic acid. Aspartic acid may be included in an amount (on a molar basis) of 50 to 200 times, 50 to 100 times, about 50 times, or about 100 times that of alanine, threonine, or valine. In addition, the molar ratio of alanine, threonine, valine, and aspartic acid may preferably be about 1 to about 1 to about 1 to about 100.

[0044] As used herein, the term "about" means an acceptable range of error for a value, determined by one skilled in the art, depending on the method by which the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean ± 10% of the stated value.

[0045] The pharmaceutical composition of the present invention can be administered parenterally or orally depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. In addition, the therapeutically effective amount of the composition may vary depending on the administration method, target site, and patient's condition, and when used in the human body, the dosage should be determined as an appropriate amount considering both safety and efficacy.

[0046] The composition of the present invention may be a food composition for increasing the expression of Zip protein (Zrt-Irt-like protein).

[0047] The above Zip protein may be selected from the group consisting of Zip1, Zip2, Zip3, Zip4, Zip5, Zip6, Zip7, Zip8, Zip9, Zip10, Zip11, Zip12, Zip13 and Zip14, and is preferably Zip1.

[0048] The food composition according to the present invention may be a food composition for preventing or treating a disease state characterized by reduced expression of a Zip protein, preferably Zip1, or for improving a related health condition.

[0049] The food composition according to the present invention can preferably be used to prevent or treat bone-related diseases or prostate-related diseases, or to improve health conditions or prostate-related health conditions.

[0050] The food composition according to the present invention may be a health functional food, and the "health functional food" refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body, and "functionality" refers to consumption for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0051] The composition according to the present invention may be an animal feed composition for increasing the expression of Zip protein (Zrt-Irt-like protein).

[0052] The above Zip protein may be selected from the group consisting of Zip1, Zip2, Zip3, Zip4, Zip5, Zip6, Zip7, Zip8, Zip9, Zip10, Zip11, Zip12, Zip13 and Zip14, and is preferably Zip1.

[0053] The animal feed composition according to the present invention may be a composition for preventing or treating a disease state characterized by reduced expression of a Zip protein, preferably Zip1, or for improving a related health condition.

[0054] In the present invention, “treatment” means partially or completely alleviating, improving, alleviating, inhibiting or delaying a symptom of a specific disease, disorder and / or condition or condition, reducing the severity or reducing the occurrence of one or more symptoms or characteristics.

[0055] In the present invention, “prevention” means any action that suppresses or delays the onset of a specific disease, and “improvement” means any action that at least reduces a parameter related to the condition being treated, for example, the degree of symptoms.

[0056] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0057]

[0058] [Example 1] Confirmation of increased Zip1 protein expression in bone metastatic prostate cancer.

[0059]

[0060] To confirm the effect of increasing Zip1 protein expression in bone metastatic prostate cancer according to treatment with a composition of alanine, threonine, and valine, the following experiment was performed.

[0061] LNCaP cells were seeded in RPMI1640 medium on a 60 mm plate at 1 x 10 6 cells / well, and when the cells grew to about 80% confluence, a control group that was not treated with the test substance in a bone metastatic prostate cancer cell line, a group treated with alanine, threonine, or valine alone in the same cell line, a group treated with aspartic acid alone, a group treated with alanine-threonine-valine at different concentrations, and a group treated with aspartic acid-alanine-threonine-valine were cultured for 24 hours.

[0062] Afterwards, cells were collected and ZIP1 protein was quantified through Western blot. The relative expression level of Zip1 protein was calculated for each group and is shown in Table 1 below.

[0063]

[0064] Group Expression Level Control 1.00 ± 0.16Alanine 10μM 1.56 ± 0.14Threonine 10μM 1.40 ± 0.36Valine 10μM 1.49 ± 0.17Aspartic Acid 1mM 1.90 ± 0.45Ala-Thr-Val (3μM:3μM:3μM) 1.84 ± 0.89Ala-Thr-Val (10μM:10μM:10μM) Complex 2.24 ± 0.54*AsP-Ala-Thr-Val (1mM:10μM:10μM:10μM) Complex 2.44 ± 0.19**

[0065] (Ala: alanine; Thr: threonine; Val: valine; AsP: aspartic acid)

[0066] As shown in Table 1 above, the alanine, threonine, and valine complex treatment group showed a significant increase in Zip1 protein expression in bone metastatic prostate cancer cell lines compared to the groups treated with alanine, threonine, and valine alone.

[0067] Additionally, in the complex group treated with alanine, threonine, and valine at a concentration of 10 μM each, the Zip1 protein expression level was higher than in the complex group treated with the above components at a concentration of 3 μM each.

[0068] In particular, the group treated with a combined amount of 9 μM of alanine, threonine, and valine (molar ratio of 1:1:1) showed a higher Zip1 protein expression level compared to the group treated with a high concentration of 10 μM of alanine, threonine, and valine alone.

[0069] Additionally, the alanine, threonine, valine, and aspartic acid complex treatment group showed a significant increase in Zip1 protein expression in bone metastatic prostate cancer cell lines compared to the groups treated with alanine, threonine, valine, and aspartic acid alone.

[0070]

[0071] [Example 2] Confirmation of increased Zip1 protein expression in osteoblasts

[0072]

[0073] To determine the effect of treatment with alanine, threonine, valine, and aspartic acid complex on increasing Zip1 protein expression in osteoblasts, the following experiment was performed.

[0074] Human osteosarcoma cell line SaOS-2 (Korea Cell Line Bank) was seeded in 100 mm plates in DMEM medium containing 10% FBS. When the cells reached approximately 80% confluence, the osteoblast cell line was divided into a control group not treated with test substances, a group treated with aspartic acid alone, a group treated with alanine-threonine-valine, and a group treated with aspartic acid-alanine-threonine-valine, and a group treated with aspartic acid-alanine-threonine-valine, and the groups were cultured for 24 hours.

[0075] Afterwards, cells were collected and ZIP1 protein was quantified through Western blot. The relative expression level of Zip1 protein was calculated for each group and is shown in Table 2 below.

[0076]

[0077] Group Expression Level Control 1 Alanine 10 μM 1.15 Threonine 10 μM 1.05 Valine 10 μM 1.07 Aspartic acid 1 mM 2.3 Ala-Thr-Val (3 μM:3 μM:3 μM) complex 2.3 Ala-Thr-Val (10 μM:10 μM:10 μM) complex 2.4 1 AsP-Ala-Thr-Val (1 mM:10 μM:10 μM:10 μM) complex 2.7

[0078] (Ala: alanine; Thr: threonine; Val: valine; AsP: aspartic acid)

[0079] As shown in Table 2 above, it was confirmed that each of the single treatment groups of alanine, threonine, and valine showed a similar expression level to the control group. On the other hand, it was confirmed that the complex treatment group of alanine, threonine, and valine showed a significantly superior expression level compared to each single treatment group. In addition, the complex treatment group of alanine, threonine, valine, and aspartic acid showed a significant increase in Zip1 protein expression in osteoblast cell lines compared to the single treatment group of aspartic acid and the complex treatment group of alanine, threonine, and valine.

[0080]

[0081] [Example 3] Confirmation of promotion of citrate production in osteoblasts

[0082]

[0083] To determine whether treatment with alanine, threonine, valine, and aspartic acid complex promotes citrate production in osteoblasts, the following experiment was performed to measure the amount of citrate produced using a citrate assay kit (abx298835, Cambridge, UK).

[0084] After culturing the control group that was not treated with the test substance in the medium, the group treated with only aspartic acid, the group treated with alanine-threonine-valine, and the group treated with aspartic acid-alanine-threonine-valine, the cells were disrupted. At this time, the assay buffer included in the kit was added and pipetted to mix the cells and buffer well, and the supernatant was collected after centrifugation at 16,000g x 5min x 4℃. The measured values ​​were corrected to the values ​​quantitatively determined using the Bradford protein assay method, and the results are shown in Table 3 below.

[0085]

[0086] Group Citric acid (%) Normal group 100 Alanine 10 μM 10 1.2 Threonine 10 μM 10 0.8 Valine 10 μM 10 0.5 Aspartic acid 1 mM 10 5.1 Ala-Thr-Val (3 μM:3 μM:3 μM) 10 1.1 Ala-Thr-Val (10 μM:10 μM:10 μM) complex 10 2.2 AsP-Ala-Thr-Val (1 mM:10 μM:10 μM:10 μM) complex 11 0.9

[0087] (Ala: alanine; Thr: threonine; Val: valine; AsP: aspartic acid)

[0088] As shown in Table 3 above, the single treatment groups of alanine, threonine, and valine were confirmed to be similar to the control group. In contrast, the complex treatment group of alanine, threonine, and valine (10 μM:10 μM:10 μM) was confirmed to show a significantly superior expression level compared to the individual single treatment groups. In addition, the complex treatment group of alanine, threonine, valine, and aspartic acid was confirmed to have a significantly increased amount of citrate production compared to the control group.

[0089] Therefore, it can be seen that the complex of alanine, threonine, valine, and aspartic acid can increase osteoblast differentiation and bone formation by promoting the production of citrate.

Claims

1. A pharmaceutical composition for preventing or treating bone-related diseases or prostate-related diseases, comprising alanine, threonine and valine.

2. A pharmaceutical composition according to claim 1, further comprising aspartic acid.

3. A pharmaceutical composition according to claim 1, wherein the bone-related disease is selected from the group consisting of osteoporosis, Paget's bone disease, rickets, osteomalacia, alveolar bone loss, osteopenia, renal osteodystrophy, short stature, dwarfism, dwarfism, and growth hormone deficiency.

4. A pharmaceutical composition according to claim 1, wherein the prostate-related disease is selected from the group consisting of benign prostatic hyperplasia, prostate cancer, prostatitis, prostatic abscess, prostatic calcification, prostatic seminal vesicle inflammation, and prostatic effusion.

5. A pharmaceutical composition in which the molar ratio of threonine and valine based on alanine is independently 0.9 to 1.1 in the first paragraph.

6. A pharmaceutical composition in which the molar ratio of alanine to threonine to valine in paragraph 1 is 1:1:

1.

7. A pharmaceutical composition in which the molar ratio of aspartic acid to alanine is 50 to 200 in the second paragraph.

8. A food composition for preventing or improving bone-related diseases or prostate-related diseases, comprising alanine, threonine and valine.

9. A food composition according to claim 9, further comprising aspartic acid.

10. A food composition in paragraph 8, wherein the molar ratio of threonine and valine based on alanine is independently 0.9 to 1.

1.

11. A food composition in claim 8, wherein the molar ratio of alanine to threonine to valine is 1 to 1 to 1.

12. A food composition in claim 9, wherein the molar ratio of aspartic acid to alanine is 50 to 200.

13. An animal feed composition for preventing or improving bone-related diseases or prostate-related diseases, comprising alanine, threonine and valine.

14. An animal feed composition according to claim 13, further comprising aspartic acid.

15. An animal feed composition in claim 13, wherein the molar ratio of threonine and valine based on alanine is independently 0.9 to 1.

1.

16. A food composition in claim 13, wherein the molar ratio of alanine to threonine to valine is 1 to 1 to 1.

17. An animal feed composition in claim 14, wherein the molar ratio of aspartic acid to alanine is 50 to 200.

18. A pharmaceutical composition according to claim 1, wherein the composition increases the expression of Zip protein or citric acid.