A pharmaceutical composition containing recombinant human growth hormone and low-dose estradiol and its use

Abstract

The present application relates to the field of biological medicine, and particularly relates to application of PEG-modified long-acting recombinant human growth hormone combined with natural 17beta-estradiol transdermal preparation in preparation of a medicine for treating obesity girl pubertal growth retardation and improving adult lifelong height. The medicine composition is PEG-modified long-acting recombinant human growth hormone combined with low-dose 17beta-estradiol transdermal preparation; the PEG-modified long-acting recombinant human growth hormone is 30 kDa U-shaped PEG covalently modified recombinant human growth hormone; the pubertal growth retardation is a disease that girls still do not have breast development at the age of 13. The medicine composition of the present application simultaneously realizes three pharmacological effects of activating obesity girl hypothalamus-pituitary-gonadal axis, improving pubertal development, delaying premature differentiation of epiphyseal cartilage to improve lifelong height, reducing internal fat and improving insulin resistance, and provides a new scheme for treatment of pubertal growth retardation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of PEG-modified long-acting recombinant human growth hormone combined with a natural 17β-estradiol transdermal formulation in the preparation of a drug for treating pubertal developmental delay in obese girls and improving their final adult height. Background Technology

[0002] In the overall obese child population, the vast majority of children have elevated levels of estrogen due to increased aromatase activity in adipose tissue, leading to increased conversion of androgens to estrogens and premature epiphyseal closure. Current guidelines and clinical expert consensus recommend avoiding exogenous estrogen supplementation in obese children, and no literature suggests that ultra-low doses of transdermal estradiol can conversely delay bone age.

[0003] This invention, through a retrospective case study, has identified a specific subtype of obese girls: long-term obesity can lead to hyperinsulinemia, adipokines disorder, and inhibition of the hypothalamic-pituitary-gonadal axis. Despite their obesity, insufficient estrogen synthesis results in delayed puberty; the onset of puberty is delayed, the growth plate window is shortened, and their final height falls short of their genetic target height. Furthermore, visceral fat accumulation and insulin resistance further exacerbate endocrine disorders. According to the "Guidelines for the Diagnosis and Treatment of Abnormal Pubertal Development in Children" issued by the Chinese Medical Association's Pediatrics Branch and the European ESPE guidelines, delayed puberty (DP) is defined as the absence of breast development in girls by age 13. This retrospective case analysis suggests that PEG-long-acting recombinant human growth hormone combined with transdermal low-dose 17β-estradiol may have a therapeutic effect on delayed puberty in obese girls and improve their final height, warranting further research and verification. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical composition that can simultaneously achieve the triple pharmacological effects of activating the hypothalamic-pituitary-gonadal axis in obese girls to improve pubertal development, delaying premature differentiation of epiphyseal cartilage to increase final height, reducing visceral fat, and improving insulin resistance, thus providing a new treatment option for dysplasia (DP).

[0005] The use of a pharmaceutical composition containing recombinant human growth hormone and low-dose estradiol in the preparation of a drug for treating pubertal growth retardation in obese girls and improving final adult height; the pharmaceutical composition is a PEG-modified long-acting recombinant human growth hormone and low-dose 17β-estradiol transdermal formulation; The PEG-modified long-acting recombinant human growth hormone is a 30 kDa U-type PEG covalently modified recombinant human growth hormone; the 17β-estradiol transdermal formulation is a natural 17β-estradiol sustained-release transdermal patch; the delayed puberty is a condition in which a girl has not developed breasts by the age of 13.

[0006] The active component of the drug is composed of PEG-modified long-acting recombinant human growth hormone and natural 17β-estradiol, without the addition of androgens or aromatase inhibitors.

[0007] The steady-state release dose of the low-dose 17β-estradiol transdermal patch is 3-6 μg / 24 h.

[0008] The pharmaceutical composition simultaneously achieves a triple pharmacological effect: activating the hypothalamic-pituitary-gonadal axis in obese girls to improve pubertal development, delaying premature differentiation of epiphyseal cartilage to increase final height, reducing visceral fat, and improving insulin resistance.

[0009] The pharmaceutical composition achieves its application in the preparation of a drug for treating delayed pubertal development and improving final adult height by activating the hypothalamic-pituitary-gonadal axis in obese girls, delaying premature differentiation of epiphyseal cartilage to increase final height, and reducing visceral fat and improving insulin resistance.

[0010] A drug kit for treating delayed puberty in obese girls and improving final adult height: containing PEG-modified long-acting recombinant human growth hormone injection and a 17β-estradiol transdermal patch with a steady-state release dose of 3-6 μg / 24 h.

[0011] The PEG-modified long-acting recombinant human growth hormone is a 30 kDa U-type PEG covalently modified recombinant human growth hormone.

[0012] The beneficial effects of this invention are: (1) Overcoming inherent technical biases in the industry: It is generally believed in the field that supplementing obese girls with exogenous estrogen will accelerate epiphyseal closure; This invention proves that for obese girls with delayed puberty due to suppression of the HPG-axis, the use of continuously released ultra-low dose transdermal 17β-estradiol, combined with PEG-rhGH, can activate the gonadal axis while moderately inhibiting Runx-2 expression, delaying premature differentiation of epiphyseal cartilage, breaking the inherent perception, providing a new treatment option for delayed puberty (DP) and improving final adult height; (2) Unexpected synergistic therapeutic effects were produced: PEG-rhGH steadily increased IGF-1 levels and promoted growth plate cartilage proliferation; transdermal 17β-estradiol improved the function of the hypothalamus-pituitary-gonadal axis. The combination of the two not only improved pubertal development and delayed bone age progression, but also reduced visceral fat and improved insulin resistance, resulting in a synergistic effect. (3) Transdermal formulations are safer: Transdermal administration avoids the first pass metabolism in the liver, and compared with oral estrogen, it will not cause elevated blood lipids or abnormal liver function; ultra-low dose administration results in lower systemic estrogen exposure. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is a comparative chart of bone age progression in a retrospective case study analysis, where A represents incomplete closure and B represents near-closure. Detailed Implementation

[0015] According to the "Guidelines for the Diagnosis and Treatment of Abnormal Pubertal Development in Children" issued by the Chinese Medical Association's Pediatrics Branch and the European ESPE guidelines, delayed puberty (DP) is defined as the condition in which girls have not developed breasts by the age of 13. The existing treatment options have obvious shortcomings: (1) The existing conventional intervention methods are limited: For obese girls with delayed puberty, the clinical priority is to use weight loss and lifestyle adjustment; when the weight loss effect is not ideal, doctors generally postpone the timing of estrogen initiation, and only start oral estradiol valerate sequential treatment after the age of 14-15; oral estrogen has the first-pass effect of the liver, which can easily raise blood lipids, and conventional doses of estrogen will still accelerate epiphyseal closure, resulting in limited height benefits; (2) Existing technology has technical bias: Influenced by the inherent perception that estrogen levels are high in obese children, pediatric endocrinologists are still hesitant to use exogenous estrogen in obese girls with delayed puberty; and the conventional indications for growth hormone are limited to growth hormone deficiency, Turner syndrome, and idiopathic short stature, and PEG-rhGH is rarely used in clinical practice for children with delayed puberty; those skilled in the art generally believe that estrogen supplementation in obese girls will inevitably accelerate epiphyseal closure, which is not conducive to height growth. Therefore, this invention explores a new treatment option through retrospective case study analysis, animal experiment verification, and prospective case study clinical verification.

[0016] Example 1: Retrospective Case Analysis 1.1 Case screening criteria Female children who visited Hunan Provincial Maternal and Child Health Hospital between January 1, 2023 and December 31, 2024 were selected.

[0017] Inclusion criteria: ①Female, aged 13-14 years old; ②BMI ≥ P95 for the same age and sex, meeting the diagnostic criteria for childhood obesity; ③Tanner stage 1 breast development, no spontaneous breast development, clinically diagnosed with delayed puberty; ④ The annual growth rate of height in the past 12 months is <4.4 cm / year; ⑤ The baseline bone age of the enrolled patients was 0.6 to 0.9 years ahead of their actual age (congenital condition at the time of consultation), indicating a risk of premature epiphyseal aging and incomplete epiphyseal closure.

[0018] Exclusion criteria: Turner syndrome, hypothyroidism, central precocious puberty, hereditary bone disease, long-term use of glucocorticoids; missing follow-up data, change of treatment plan midway, lost to follow-up.

[0019] A total of 52 eligible children were selected and included according to the above criteria. The initial age, BMI, baseline bone age, fasting insulin and serum IGF-1 levels of all children were extracted. One-way ANOVA was used to compare the baselines between groups. The baseline indicators of each group were P>0.05, indicating no statistical difference. The baseline data were balanced and comparable.

[0020] This study was a retrospective real-world study. No treatment plans were artificially assigned. The children were divided into four groups based on the long-term treatment plans actually implemented in clinical practice during the 12-month follow-up period. There was no experimental artificial intervention.

[0021] Group A (5 cases): Long-term use of PEGylated long-acting recombinant human growth hormone injection (Jinsai Zeng, Changchun Jinsai Pharmaceutical, 30 kDa PEG modified) combined with steady-state release dose of 3-6 μg / 24 h 17β-estradiol transdermal patch; Group B (23 cases): PEGylated long-acting recombinant human growth hormone injection was used alone throughout the entire course; Group C (16 cases): Long-term treatment with daily injections of short-acting recombinant human growth hormone combined with oral estradiol valerate sequential replacement; Group D (8 cases): Long-term use of PEGylated long-acting recombinant human growth hormone injection combined with conventional high-dose 17β-estradiol transdermal patch (steady-state release 8 μg / 24 h).

[0022] 1.2 Observation Indicators: The observation period was from enrollment to 12 months of follow-up. Four core retrospective indicators were extracted: ① cumulative annual growth rate over 12 months; ② new bone age progression value within 12 months; ③ percentage of children who reached Tanner stage 2 or above of breast development at the end of the follow-up period (puberty initiation rate); ④ percentage of children whose fasting insulin returned to the normal range after follow-up (insulin resistance improvement rate).

[0023] Quantitative data are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used to compare multiple groups of quantitative data, and the χ² test was used for categorical data. P < 0.05 was considered statistically significant between groups. The statistical results of each group are shown in Table 1.

[0024] Table 1. Results of retrospective case study analysis (x±s)

[0025] Note: * indicates that cases with incomplete fasting insulin data during the follow-up period were excluded when calculating the insulin improvement rate. Only complete test data was used to calculate the percentage. For example, (2 / 3) means that there were actually 3 cases with complete insulin test data, of which 2 cases showed significant insulin improvement.

[0026] 1.3 Retrospective Case Analysis like Figure 1 The image shows a comparison of the degree of epiphyseal closure in representative children from groups A, C, and D at the end of the follow-up period. Figure 1 In the image, A represents the epiphyseal image of a typical case in group A. Figure 1 B in the table represents epiphyseal images of typical cases from groups C and D. A comprehensive analysis based on the data in Table 1 is as follows: Group C and Group D data and Figure 1 As shown in B: Under the routine oral estrogen or 8 μg / 24h high-dose transdermal estradiol regimen, the bone age progression of the children at 12 months exceeded 1.2 years, and the rate of bone age increase was significantly accelerated; bone age X-ray showed narrowing of the epiphyseal cartilage band and the tendency of the epiphysis to close prematurely, which is consistent with the general understanding in the field: supplementing obese children with conventional doses of estrogen will accelerate epiphyseal aging and compress the growth cycle.

[0027] Comparing the data of Group A with the other three groups: Children who received long-term combined use of 3-6 μg / 24h ultra-low dose transdermal 17β-estradiol + PEG-rhGH showed completely different clinical characteristics: (1) Their annual growth rate was significantly higher than that of Groups B, C, and D, and their height catch-up effect was the best; (2) Their bone age progression in 12 months was only 0.89 years, which effectively inhibited the rapid growth of bone age. Figure 1In A, the width of the growth plate was moderately visible, and there was no tendency for premature epiphyseal closure; (3) The puberty initiation rate reached 100% (all 5 girls in group A initiated puberty), which could effectively improve the delayed puberty development in obese girls; (4) After excluding patients with missing insulin data, more than 60% of the children had significantly alleviated insulin resistance, and the obesity-related metabolic disorders were improved synchronously.

[0028] Conclusion: The results of the above retrospective case analysis are contrary to the long-existing technical biases in the pediatric endocrinology industry. It is generally believed in this field that obese children have high activity of adipose aromatase and high levels of endogenous estrogen, and additional supplementation of exogenous estrogen will necessarily accelerate epiphyseal closure and damage the adult final height; however, through retrospective case analysis, the present invention suggests that for obese pubertal girls with HPG axis inhibition, the combination of ultra-low-dose sustained-release transdermal 17β-estradiol and PEG-rhGH can simultaneously achieve multiple benefits including promoting growth, delaying bone age, initiating puberty, and improving metabolism.

[0029] Example 2 Animal experiment verification Example 1 was a single-center, non-interventional retrospective analysis. The grouping was based on the actual previous medications in clinical practice, and the confounding interference factors such as children's diet, exercise, and daily routine were not uniformly controlled, reflecting the relevant clinical trends and preliminarily proving the synergistic effect of the combination of the two drugs. To exclude the interference of confounding factors such as diet, exercise, and individual compliance in clinical retrospective studies, clarify the synergistic mechanism, dose-effect characteristics, and action targets of the combination of PEG-rhGH and low-dose transdermal sustained-release 17β-estradiol, the present invention established a model of HPG axis inhibition-obese epiphyseal premature aging in female mice induced by high-fat diet, and carried out standardized animal intervention and mechanism detection experiments to systematically clarify the scientificity and effectiveness of the combination regimen.

[0030] 2.1 Experimental animals and model construction Sixty 3-week-old SPF-grade C57-BL6 female mice with a body weight of 18 - 20 g were selected, provided by Hunan Slack Jingda Experimental Animal Co., Ltd., and the animal license number was: SCXK(Xiang)2025-0003. Animal rearing environment: temperature 22 - 25 °C, humidity 50% - 60%, 12 h light-dark cycle, free access to food and water. After 1 week of adaptive feeding, the mice were modeled: The mice in the model group were continuously fed with a high-fat diet (45% fat energy supply) for 8 weeks to construct a pathological model of obesity combined with HPG axis inhibition and epiphyseal premature aging; the blank control group was fed with a normal standard diet. After the modeling was completed, through verification of body weight, serum sex hormones, insulin, and growth plate pathology, the model was stable and reliable, meeting the core pathological characteristics of pubertal development retardation in clinical obese girls.

[0031] 2.2 Experimental drugs and grouping 2.2.1 Experimental drug: Corresponding to the clinical investigational drug, the laboratory equivalent dose was converted to simulate the long-acting, sustained-release steady-state dosing characteristics in humans, with a dosing cycle of 6 weeks.

[0032] (1) PEGylated long-acting recombinant human growth hormone (PEG-rhGH): 30 kDa U-type PEG-modified recombinant human growth hormone (Changchun Jinsai Pharmaceutical Co., Ltd., batch number JSC20240912), mouse equivalent dose: 0.8 mg / kg, subcutaneous injection once a week, to simulate the clinical long-acting sustained release and steady-state blood drug concentration characteristics.

[0033] (2) 17β-estradiol sustained-release formulation: Natural 17β-estradiol (Sigma, E8875) was prepared by encapsulation with medical sustained-release microspheres to simulate the continuous steady-state release characteristics of human transdermal patches; it was divided into low-dose and high-dose groups. The release rate of the low-dose sustained-release microspheres was equivalent to the steady-state blood drug level of 3-6 μg / 24h transdermal patches in humans, while the high-dose microspheres were equivalent to 8 μg / 24h high-dose patches in humans. The sustained-release microspheres were implanted subcutaneously once and released at a constant rate for 6 weeks without fluctuations due to frequent dosing.

[0034] 2.2.2 Experimental grouping (12 mice per group): Model group: Obesity model + saline intervention; GH group: Obesity model + PEG-rhGH alone; High-E2 group: Obesity model + PEG-rhGH + high-dose 17β-estradiol sustained-release microspheres; Low-E2 group: Obesity model + PEG-rhGH + low-dose 17β-estradiol sustained-release microspheres; Blank group: normal wild-type mice + conventional feeding.

[0035] 2.3 Research Methods 2.3.1 General growth and body fat indicators: The mice were weighed every 7 days; at the end of the experiment, the abdominal visceral fat was dissected and the visceral fat coefficient was calculated (visceral fat wet weight / body weight × 100%); the bilateral femurs were dissected and the total length of the femurs was accurately measured with calipers.

[0036] 2.3.2 Serum endocrine and metabolic indicators: Blood was collected from the orbital cavity of mice, and serum was separated by centrifugation. The levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17β-estradiol (E2), and fasting insulin (FINS) were detected using a commercial ELISA kit. The kit was operated strictly in accordance with the instructions, and three replicates were set up for each sample.

[0037] 2.3.3 Uterine development level: The bilateral uteruses of mice were dissected and separated, and the wet weight of the uterus was measured using a precision electronic balance to evaluate the degree of gonadal development.

[0038] 2.3.4 Pathological morphological examination of growth plates: The distal femur of mice was fixed, decalcified, embedded in paraffin, and serially sectioned. It was stained with hematoxylin and eosin (HE), photographed under an optical microscope, and the total thickness of the growth plate and the width of the cartilage proliferation zone were measured using ImageJ software to evaluate the degree of epiphyseal growth and aging.

[0039] 2.3.5 Detection of key pathway protein expression (Western-Blot): Total protein was extracted from the cartilage tissue of the distal femoral growth plate. Protein quantification was performed using the BCA method. SDS-PAGE electrophoresis, membrane transfer, and blocking were then performed. The tissue was incubated with primary antibodies against IGF-1R, Runx2, IL-6, and TNF-α, and their corresponding secondary antibodies. ECL chemiluminescence imaging was performed, and ImageJ grayscale quantitative analysis was conducted. GAPDH was used as an internal control to calculate the relative expression levels of each protein.

[0040] 2.4 Experimental Results 2.4.1 Growth and body fat: The results are shown in Table 2: The obese model group mice showed significantly shortened femur length, severe visceral fat accumulation, and delayed uterine development, confirming successful model establishment. PEG-rhGH alone could only slightly improve body length and had limited effect on gonadal development, insulin resistance, and fat inflammation; high-dose estradiol combined with PEG could partially activate the HPG axis, but could not effectively improve premature epiphyseal aging; the Low-E2 group showed femur length close to the normal control group, significantly reduced visceral fat, and substantial correction of insulin resistance, demonstrating a triple benefit of promoting growth, reducing fat, and improving metabolism.

[0041] Table 2 Comparison of growth, organ and metabolic indicators of mice in each group (x±s)

[0042] Note: * indicates a significant difference between the model group and the control group (* means P < 0.05, ** means P < 0.01); # indicates a significant difference between the treatment group and the model group (# means P < 0.05, ## means P < 0.01). The same applies below.

[0043] 2.4.2 Gonadal axis activation effect: As shown in Table 3, low-dose sustained-release 17β-estradiol can steadily increase LH and FSH levels, precisely repair the HPG axis inhibition caused by obesity, and achieve physiological puberty initiation; while high-dose estradiol will cause excessive fluctuations in estrogen levels, which does not conform to the physiological replacement pattern of children.

[0044] Table 3 Comparison of sex hormone levels in mice of different groups (x±s)

[0045] 2.4.3 Verification of the growth plate mechanism: The results are shown in Table 4.

[0046] Obese mouse models exhibit significant growth plate inflammation and premature cartilage aging (high expression of Runx2 and inflammatory factors, and inhibition of IGF-1R). PEG-rhGH alone only slightly upregulated the proliferation pathway. The combination therapy of this invention significantly upregulated IGF-1R to promote cartilage proliferation, while significantly downregulated Runx2, IL-6, and TNF-α. Mechanistically, this demonstrates that ultra-low dose steady-state sustained-release estradiol can repair obesity-induced growth plate inflammatory damage and inhibit premature epiphyseal closure, contrary to the finding that "estrogen supplementation in obese children accelerates bone age closure."

[0047] Table 4. Comparison of relative expression levels of key proteins in the growth plate (x±s)

[0048] 2.5 Conclusion (1) Obesity causes adipokines disorder and inhibits the HPG-axis, while increasing Runx-2 and promoting premature aging of the epiphysis, which may be the core mechanism of delayed puberty development in obese girls in clinical practice; PEG-rhGH alone cannot effectively improve gonadal development; (2) The combination of sustained-release ultra-low dose 17β-estradiol and PEG-rhGH can simultaneously activate the HPG-axis, promote growth plate proliferation, inhibit Runx-2 expression, and improve insulin resistance, which is consistent with the results of the retrospective case analysis in Example 1; verifying that the combination of low dose 17β-estradiol and PEG-rhGH produces a synergistic effect.

[0049] In summary, the experimental results revealed that the core pathology of delayed puberty in obese girls is HPG axis inhibition combined with chronic inflammation of the growth plate and premature epiphyseal aging, rather than simply growth hormone deficiency. PEG-rhGH alone cannot repair gonadal developmental defects and metabolic disorders, and conventional high-dose estradiol cannot avoid the risk of premature epiphyseal aging. Neither drug alone can simultaneously achieve the aforementioned multiple effects. Only the proposed combination of PEG-rhGH and ultra-low-dose steady-state sustained-release 17β-estradiol can produce a synergistic pharmacological effect that is not simply additive: stable activation of the gonadal axis, promotion of height growth, inhibition of premature epiphyseal aging, and improvement of insulin resistance in obesity. The mechanism is clear and the effect is stable, providing a solid theoretical and experimental basis for previous clinical retrospective trends and future prospective clinical efficacy.

[0050] Example 3: Prospective Clinical Case Validation Building upon previous retrospective real-world case trend findings and animal efficacy mechanism verification, this study conducts prospective clinical case validation to further verify the reproducibility, clinical safety, and practical application value of the drug combination efficacy of this invention, and to eliminate confounding factors from retrospective studies. The drug information used in this invention is as follows: PEGylated long-acting recombinant human growth hormone injection: Generic name: Polyethylene glycol recombinant human growth hormone injection; Trade name: Jinsai Zeng; Manufacturer: Changchun Jinsai Pharmaceutical Co., Ltd.; National Drug Approval Number: S20140001; Specification: 54 IU / 9.0 mg / 1.0 ml / vial; Recommended dose: 0.2 mg / kg / dose, once a week.

[0051] 17β-estradiol transdermal patch: Generic Name: Hemihydrated Estradiol Patch (Natural 17β-estradiol transdermal sustained-release formulation); Trade Name: Songqi; Manufacturer: LTS Lohmann Therapie-Systeme AG; Registration Certificate Number: H20130627; Dosage Form: Transdermal sustained-release patch; Suitable Specifications: Steady-state sustained-release dose of 3–6 μg / 24h after trimming and adjustment; Pharmacological Characteristics: Transdermal release of natural 17β-estradiol without first-pass metabolism in the liver, suitable for long-term low-dose intervention in children.

[0052] This study consecutively included three obese girls with delayed puberty who met the diagnostic criteria of the "Guidelines for the Diagnosis and Treatment of Pediatric Developmental Abnormalities in Children" issued by the Chinese Medical Association's Pediatrics Branch. They underwent standardized intervention using the aforementioned compliant drugs and the PEG-rhGH combined with a low-dose 17β-estradiol transdermal formulation of this invention. They were followed up for 6 months to systematically evaluate the initiation of puberty, growth benefits, bone age progression, and metabolic improvements. The specific case reports are as follows: 3.1 Inclusion criteria and general baseline data Inclusion criteria: Female children aged 13.0–13.7 years; BMI ≥ P95 for age and sex, meeting the diagnostic criteria for childhood obesity; Tanner stage 1 breast development, without spontaneous puberty onset, meeting the diagnostic criteria for delayed puberty; bone age 0.7–0.9 years ahead of chronological age, showing a trend of premature epiphyseal aging; annual height growth rate <4.0 cm in the past year, with a significant decrease in growth rate; laboratory tests showing elevated fasting insulin levels and concurrent insulin resistance; exclusion criteria included Turner syndrome, thyroid dysfunction, central precocious puberty, hereditary skeletal diseases, chronic liver and kidney diseases, and a history of long-term hormone use.

[0053] General intervention protocol: PEGylated long-acting recombinant human growth hormone was used at the standard clinical dose, combined with a steady-state release dose of 3-6 μg / 24h of natural 17β-estradiol transdermal patch for continuous external application, for 6 consecutive months of standardized intervention, with unified dietary and exercise guidance throughout the process, and no other growth-promoting, puberty-regulating, or blood sugar-lowering adjuvant drugs were used for intervention.

[0054] 3.2 Detailed Case Information Case 1: The patient was a 13.0-year-old female who presented with no breast development, no pubic or axillary hair growth, and no onset of puberty. Physical examination revealed a BMI of 97 for her age and sex, classifying her as severely obese. Her baseline height was stable, with a growth rate of only 3.6 cm in the past year, significantly lower than normal. Imaging studies showed a bone age 0.7 years ahead of her chronological age, indicating premature epiphyseal aging and a risk of a shortened growth window. Laboratory tests revealed elevated fasting insulin levels, indicating insulin resistance; liver and kidney function and baseline blood lipid levels were normal.

[0055] Intervention and follow-up outcomes: After 6 months of standardized intervention, the child successfully developed Tanner stage 2 breast development, and puberty was successfully initiated; the corrected annual growth rate increased to 7.4 cm, and the growth rate was significantly improved; the bone age progression at 6 months was 0.43 years, and the annualized bone age progression was controlled within 0.90 years, effectively inhibiting premature epiphyseal aging; the fasting insulin level decreased significantly from the baseline, and insulin resistance was significantly improved; no adverse reactions such as excessive breast development, dyslipidemia, abnormal liver function, or local skin allergies were observed during the entire follow-up, indicating good safety.

[0056] Case 2: The patient was a 13.4-year-old female. At presentation, she was in Tanner stage 1 of breast development and showed no signs of puberty onset. Physical examination revealed a BMI of 96 for her age and sex, indicating obesity. Her height had increased by 3.4 cm in the past year, showing a clear trend of growth retardation. Bone age assessment showed a bone age 0.8 years ahead of her chronological age, indicating a high risk of premature epiphyseal closure. Laboratory tests revealed elevated fasting insulin levels and associated metabolic disorders; other baseline indicators were normal.

[0057] Intervention and follow-up outcomes: After 6 months of continuous intervention, the child's breast development began, entering the normal puberty development process; the corrected annual growth rate reached 7.6 cm, with significant growth benefits; the annualized bone age progression was 0.88 years, with no accelerated bone age progression, effectively delaying premature epiphyseal aging; visceral fat accumulation improved, fasting insulin levels decreased, and metabolic disorders were corrected; no adverse events occurred throughout the course, and the child was well tolerated.

[0058] Case 3: The patient was a 13.7-year-old female who had not yet entered puberty at the time of presentation, and had no breast development. Physical examination revealed a BMI ≥ 95 for her age and sex, confirming a diagnosis of obesity. Her height had increased by 3.8 cm in the past year, indicating a slow growth rate. Bone age examination showed a bone age 0.9 years ahead of her chronological age, a typical phenotype of obesity-related premature epiphyseal aging. Laboratory tests revealed elevated fasting insulin levels and mild to moderate insulin resistance.

[0059] Intervention and follow-up outcomes: Six months after the intervention, the child successfully initiated puberty and achieved the expected breast development; the annual growth rate increased to 7.7 cm, showing a significant catch-up effect; the annualized bone age progressed by 0.89 years, with stable bone age progression, successfully avoiding the risk of rapid epiphyseal closure caused by obesity; insulin resistance significantly improved, and the metabolic state tended to normalize; there were no adverse reactions such as hormonal imbalances, organ damage, or developmental abnormalities throughout the entire process, demonstrating excellent clinical safety.

[0060] 3.3 Results Three prospectively enrolled children underwent 6 months of intervention with the drug combination of this invention, all of whom achieved 100% effective initiation of puberty, significantly improving the problem of delayed puberty development in obese girls. All children experienced a significant increase in growth rate compared to baseline, reversing growth retardation and low growth rate. Bone age progression was stably controlled within a reasonable range, breaking the inherent pattern of rapid epiphyseal closure in obese children. Simultaneously, insulin resistance and obesity-related metabolic disorders were improved to varying degrees in all children. No serious adverse reactions were observed during the follow-up period, demonstrating good drug safety.

[0061] 3.4 Conclusion The proposed combination of PEG-rhGH and a low-dose 17β-estradiol transdermal formulation demonstrates stable and reproducible clinical efficacy in treating delayed puberty in obese girls. It simultaneously provides multiple benefits, including initiating puberty, promoting healthy growth, delaying premature epiphyseal aging, and improving obesity-related insulin resistance. Prospective independent case studies have validated its reliability, definite clinical efficacy, and excellent safety profile, demonstrating significant clinical application value and translational potential, further supporting the synergistic effect of the drug combination.

Claims

1. The use of a pharmaceutical composition containing recombinant human growth hormone and low-dose estradiol in the preparation of a drug for treating pubertal growth retardation in obese girls and improving final adult height; said pharmaceutical composition is a transdermal formulation of PEG-modified long-acting recombinant human growth hormone and low-dose 17β-estradiol; The PEG-modified long-acting recombinant human growth hormone is a 30 kDa U-type PEG covalently modified recombinant human growth hormone; the delayed puberty refers to a condition in girls who have not developed breasts by the age of 13.

2. The application according to claim 1, characterized in that: The active component of the drug is composed of PEG-modified long-acting recombinant human growth hormone and natural 17β-estradiol, without the addition of androgens or aromatase inhibitors.

3. The application according to claim 1, characterized in that: The steady-state release dose of the low-dose 17β-estradiol transdermal patch is 3-6 μg / 24 h.

4. The application according to claim 1, characterized in that: The pharmaceutical composition achieves its application in the preparation of a drug for treating delayed pubertal development and improving final adult height by activating the hypothalamic-pituitary-gonadal axis in obese girls, delaying premature differentiation of epiphyseal cartilage to increase final height, and reducing visceral fat and improving insulin resistance.

5. A pharmaceutical kit for treating delayed puberty in obese girls and improving final adult height, characterized in that: It includes PEG-modified long-acting recombinant human growth hormone injection and 17β-estradiol transdermal patch with a steady-state release dose of 3-6 μg / 24 h, as well as instructions for treating pubertal growth retardation in obese girls and improving final adult height.

6. The drug kit according to claim 5, characterized in that: The PEG-modified long-acting recombinant human growth hormone is a 30 kDa U-type PEG covalently modified recombinant human growth hormone.

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