A use for promoting elongation of long bone during childhood to adolescence by gper-1 activation

Abstract

This invention provides a promoter that increases the proliferation of growth plate chondrocytes in children from infancy to puberty by activating the action of G protein-coupled estrogen receptor 1 (GPER-1), thereby promoting children's growth.

Description

Technical Field

[0001] This invention relates to the application of molecular biology and pharmacology, and in particular to the use of activating GPER-1 to promote the proliferation of growth plate cartilage in children during puberty. Prior Technology

[0002] The growth plate, also known as the epiphyseal plate, located at the top and bottom of bones, is crucial in influencing an individual's height and is the key area responsible for skeletal growth. Cell proliferation in the growth plate is related to the elongation of the bone shaft. The cartilage cells in the epiphyseal plate continuously proliferate and ossify, causing the bone length to increase. During human development, cartilage cell proliferation and ossification continue until early adulthood. The onset of menstruation in girls and the beginning of voice change in boys mark the beginning of puberty. The appearance of secondary sexual characteristics signifies the end of puberty and the final stage of height growth. At this point, the growth plate is replaced by bone tissue and closes, and bone elongation ceases, thus stopping height growth.

[0003] The regulation of the entire bone growth process involves many physiological responses. Besides exercise and dietary habits, the influence of the endocrine system is also crucial, with sex hormones playing a significant role in height growth during puberty. In early development, the secretion of trace amounts of estrogen and androgens initiates rapid bone growth, contributing to the development of pubertal secondary sexual characteristics. As puberty progresses, estrogen levels rise, and these high concentrations, through estrogen receptor alpha, cause the growth plates to close, ending the child's growth spurt. Therefore, a new method is needed to effectively and specifically promote the proliferation of chondrocytes in the growth plates of adolescents.

[0004] Height growth is a major focus of attention during childhood development, and there are still ways to intervene in regulating it from a physiological perspective. Currently, methods for regulating growth plate proliferation to promote height growth include growth hormone therapy and GnRH analogue therapy. Growth hormone therapy promotes bone growth plate proliferation by administering synthetic growth hormone; while GnRH analogues achieve the same goal by inhibiting the puberty process. However, these methods also have significant impacts on systemic physiological responses, such as the potential for side effects. Therefore, there is still a need for more effective, safe, and economical methods to promote growth plate proliferation in adolescents and provide more specific results for increasing individual height. Summary of the Invention

[0005] Other features and advantages of the invention will be revealed in the following non-limiting detailed description and examples.

[0006] To facilitate a better understanding of the invention, certain terms are first defined, and additional definitions are set forth throughout the detailed description.

[0007] G protein-coupled estrogen receptor 1 (GPER-1, GPR30) is an estrogen receptor identified in 2005. It is expressed in the growth plates of both boys and girls, and studies show that its physiological effects during puberty affect both sexes. Unlike other estrogen receptors, the expression of GPER-1 in the growth plate decreases with age. Research indicates that GPER-1 primarily affects the growth plate during puberty, and because bone growth is time-limited, appropriate intervention timing is crucial. Therefore, administering compounds or agonists that activate GPER-1 during childhood (approximately 6-12 years old) and puberty, before the growth plates close, can promote the proliferation of chondrocytes in the growth plates and increase their thickness, thereby promoting height growth.

[0008] In addition to being physiologically regulated by estrogen, GPER-1 can be deactivated by drugs or promoters that target GPER-1 alone. These drugs do not affect other estrogen receptors when regulating GPER-1, thereby reducing side effects on other physiological responses.

[0009] The terms “a,” “the,” “the,” “at least one,” and “one or more” used in this document may be used interchangeably.

[0010] The present invention provides the use of a composition for preparing a medicament that increases the thickness of an individual's growth plate, wherein the composition is a GPER-1 promoter.

[0011] In one embodiment, the GPER-1 promoter activates GPER-1 to promote chondrocyte proliferation in the growth plate of the individual and increase the thickness of the growth plate.

[0012] In one embodiment, the GPER-1 accelerator system comprises (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzobismuth) -5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl-ethenone ((±)-1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethenone), estrogen, tamoxifen, raloxifene, or selective estrogen receptor modulators (SERMs). In a preferred embodiment, the GPER-1 promoter is (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethenone. -5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-yl-ethenone.

[0013] In another embodiment, the GPER-1 promoter promotes the individual's height growth.

[0014] In another embodiment, the system consists of individuals with unclosed or underdeveloped growth plates.

[0015] In another embodiment, the individual's age range is 6-16 years old; in a preferred embodiment, the individual's age range is 8-14 years old; in a more preferred embodiment, the individual's age range is 10-12 years old.

[0016] In another embodiment, the method of administration of the GPER-1 promoter includes subcutaneous injection, oral administration, or intravenous injection.

[0017] In another embodiment, the effective dose range of the GPER-1 promoter administered to the individual is 10⁻⁸ g / kg to 10⁻² g / kg. In another preferred embodiment, the effective dose range of the GPER-1 promoter is 10⁻⁶ g / kg to 10⁻⁴ g / kg. In yet another preferred embodiment, the effective dose of the GPER-1 promoter is 10⁻⁴ g / kg. Simple Explanation of the Diagram

[0018] Figure 1 shows the tibial growth rate analysis of mice aged 4 to 8 weeks after receiving subcutaneous injections of different concentrations of GPER-1 promoters (agonist) to activate GPER-1 at 2 weeks of age. The control group consisted of mice injected with physiological saline.

[0019] Figure 2 shows the tibial length analysis of mice at 6 weeks of age after subcutaneous injection of 10⁻⁴ g / kg GPER-1 promoter to activate GPER-I. The control group consisted of mice injected with physiological saline.

[0020] Figure 3 shows the composition of growth plate cartilage and the thickness ratio of different components in mice after subcutaneous injection of different concentrations of GPER-1 promoters to activate GPER-1 at 2 weeks of age, analyzed by cell staining. Figure 3A shows the cartilage tissue staining of mice after subcutaneous injection of different concentrations of GPER-1 promoters to activate GPER-1 at 2 weeks of age, using Safranin O stain. Figure 3B shows the composition analysis of cartilage tissue in mice after subcutaneous injection of different concentrations of GPER-1 promoters to activate GPER-1 at 2 weeks of age. Abbreviations: RZ is the resting zone; PZ is the proliferation zone; HZ is the hypertrophy zone. The control group was a blank control group, consisting of mice injected with physiological saline.

[0021] Figure 4 shows the analysis of the proportion of proliferating cells in the in vitro cultured rat epiphyseal cartilage tissue after different concentrations of GPER-1 promoter were administered to activate chondrocytes. The proliferating cells were detected by detecting the proportion of proliferating cells in the living cells using deoxyuridine bromide (5-bromo-2'-deoxyuridine, BrdU). Implementation

[0022] The following examples are non-limiting and only represent various aspects and features of the present invention.

[0023] Example 1: Effect of GPER-1 on tibial growth rate

[0024] Starting at 2 weeks of age, mice were injected subcutaneously with 10⁻⁸ g / kg, 10⁻⁶ g / kg, and 10⁻⁴ g / kg of GPER-1 promoter (agonist) to activate GPER-1. Tibial length was analyzed by computed tomography to examine changes in tibial length between 4 and 8 weeks of age.

[0025] The GPER-1 accelerator system is (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxane). -5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl-ethenone ((±)-1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethenone,GPR30 Agonist;G-1).

[0026] The tibial growth rate of mice from 4 to 8 weeks of age is calculated as (tibial length at 8 weeks of age (L8W) - tibial length at 4 weeks of age (L4W)) / tibial length at 4 weeks of age.

[0027] The results, as shown in Figure 1, indicate that a dose of GPER-1 promoter at 10⁻⁴ g / kg significantly increased the tibial growth rate in mice aged 4-8 weeks.

[0028] Example 2: Effect of GPER-1 on tibial length

[0029] Starting at 2 weeks of age, mice were activated by subcutaneous injection of 10⁻⁴ g / kg of GPER-1 promoter, and tibial length at 6 weeks of age was analyzed by computed tomography.

[0030] As shown in Figure 2, the GPER-1 promoter can significantly promote tibial lengthening.

[0031] Example 3: Effect of GPER-1 on growth plate cartilage composition

[0032] Starting at 2 weeks of age, GPER-1 in mice was activated by subcutaneous injection of GPER-1 promoters at doses of 10⁻⁸ g / kg, 10⁻⁶ g / kg, and 10⁻⁴ g / kg. Cartilage tissue was then marked with Safranin O stain to analyze the composition of growth plate cartilage. The growth plate cartilage was divided into a resting zone (RZ), a proliferation zone (PZ), and a hypertrophy zone (HZ).

[0033] As shown in Figure 3, the GPER-1 promoters of 10⁻⁶ g / kg and 10⁻⁴ g / kg effectively promoted the thickening of the growth plate proliferation zone and the overall growth plate thickness, indicating that the growth plate chondrocytes were affected by the GPER-1 promoters of 10⁻⁶ g / kg and 10⁻⁴ g / kg, thereby promoting chondrocyte proliferation.

[0034] Example 4: Effects of GPER-1 on chondrocytes

[0035] After in vitro culture of epiphyseal cartilage tissue from four-day-old rats, the cartilage tissue was given 0.1, 0.5, 1, and 2 μM of GPER-1 promoter to activate GPER-1 in chondrocytes. Then, the number of proliferating cells in the living cells was detected by bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU).

[0036] As shown in Figure 4, 0.1 and 0.5 μM GPER-1 promoters significantly promoted the proliferation of epiphyseal chondrocytes.

[0037] Subcutaneous injection of a GPER-1 promoter activated GPER-1. A dose of 10⁻⁴ g / kg of the GPER-1 promoter promoted tibial growth rate and bone length in mice aged 4-8 weeks. Furthermore, doses of 10⁻⁶ g / kg and 10⁻⁴ g / kg of the GPER-1 promoter increased growth plate thickness and the proliferation layer in mice. Epiphyseal cartilage tissue from four-day-old rats was cultured in vitro (ex vivo), and different concentrations of the GPER-1 promoter were administered to activate GPER-1. The results showed that 0.1–1 μM concentrations of the GPER-1 promoter promoted cartilage tissue proliferation in rats.

[0038] While the invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use it, various alternatives, modifications and improvements should be apparent without departing from the spirit and scope of the invention.

[0039] Those skilled in the art will readily understand that the present invention is highly suitable for achieving the above-described objects and obtaining the aforementioned objects and advantages, as well as those inherent therein. The processes and methods described above for producing them represent preferred embodiments, are exemplary, and do not limit the scope of the invention. Modifications and other uses will be apparent to those skilled in the art. These modifications are contained within the spirit of the invention and are defined by the scope of the claims.

Claims

1. Use of a composition for preparing a medicament for increasing the thickness of a human growth plate, wherein the composition comprises an effective dose of a highly specific G protein-coupled estrogen receptor 1 (GPER-1) activating medicament or promoter, wherein the composition is a substituted dihydroquinoline small molecule compound having high morphological similarity but lacking hydrogen bonding patterns and spatial collision structural features that make it highly specific for GPER-1 and not binding to conventional estrogen receptors (ERα and ERβ), wherein the effective dose is 24.25 pmol / kg to 242.54 nmol / kg.

2. The use according to claim 1, wherein the composition comprises (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzobis-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-yl-ethenone.

3. As claimed in claim 1, wherein the GPER-1 promoter promotes the individual's height growth.

4. Individuals in the system whose growth plates are not closed or whose growth plates are underdeveloped, according to the purpose of request item 1.

5. According to the use of claim 1, wherein the method of administration of the GPER-1 promoter includes subcutaneous injection, oral administration or intravenous injection.

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