A composition containing recombinant human growth hormone, metformin and liraglutide and its use

Abstract

The present application belongs to the technical field of pediatric endocrine metabolism drugs, and particularly relates to a pharmaceutical composition consisting of recombinant human growth hormone, metformin and liraglutide and application thereof. The present application verifies, through large-sample retrospective clinical case analysis and pharmacodynamic mechanism verification of a high-fat obesity growth retardation mouse model, the application of the pharmaceutical composition consisting of recombinant human growth hormone, metformin and liraglutide in preparing a therapeutic drug for 6-14 year-old obese-related growth retardation (BMI>=P95, HOMA-IR>=3.0, annual growth rate<5 cm) children. The present application realizes the effects of synchronous reversal of insulin resistance, control of body weight and stabilization of bone age development, and has great clinical application and conversion value.

Description

Technical Field

[0001] This invention belongs to the field of pediatric endocrine and metabolic drug technology, specifically relating to a composition composed of recombinant human growth hormone, metformin, and liraglutide and its application. Background Technology

[0002] Childhood obesity-related growth retardation specifically refers to metabolic short stature in children aged 6–14 years with a BMI ≥ 95 for their age and sex, an annual growth rate < 5 cm, and a HOMA-IR ≥ 3.0. It is caused by obesity-induced hyperinsulinemia, which blocks the JAK2-STAT5 pathway, inhibits IGF-1 synthesis, and reduces the sensitivity of epiphyseal cartilage to growth hormone. This is distinct from pituitary or thyroid organic short stature. There is a great clinical need for treatment, but there is a lack of ideal treatment options.

[0003] Current treatment options have limitations. Recombinant human growth hormone (rhGH) alone only achieves growth targets in 30%–40% of children, exacerbating insulin resistance and accelerating premature bone age closure. Combination therapy with rhGH and metformin only improves systemic insulin resistance, failing to enhance epiphyseal cartilage sensitivity, resulting in limited growth promotion. Combination therapy with rhGH and liraglutide only leads to mild weight loss, failing to correct refractory hepatic insulin resistance and showing insufficient IGF-1 enhancement. Combination therapy with metformin and liraglutide has no growth-promoting effect, only suitable for simple obesity and unable to improve short stature. Existing monotherapy and dual-drug regimens cannot simultaneously achieve the four effects of promoting growth, reversing insulin resistance, controlling weight, and protecting bone age, nor do they possess a suitable three-way safe dosage system and synergistic mechanism for children aged 6–14 years.

[0004] This invention, through a review of a large number of clinical cases in our hospital, unexpectedly discovered that some severely obese children with growth retardation showed significantly better improvement in growth and metabolic indicators than those treated with single or double drugs when using a standardized combination of three commercially available drugs: rhGH, metformin, and liraglutide. This suggests that the combination of the three drugs may have a synergistic effect, which requires further research and verification. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition containing recombinant human growth hormone, metformin, and liraglutide: the active ingredients of the composition are recombinant human growth hormone, metformin, and liraglutide; the proportions of each group based on the weight of children aged 6-14 years are as follows: Recombinant human growth hormone: 0.15–0.18 mg / kg / week; Metformin: 10–15 mg / kg / day; Liraglutide: 0.05–0.1 mg / kg / week.

[0006] The optimal ratio of the composition is: recombinant human growth hormone 0.16 mg / kg / week, metformin 12 mg / kg / day, and liraglutide 0.08 mg / kg / week.

[0007] The above-described composition is used in the preparation of a medicament for treating obesity-related growth retardation in children aged 6 to 14 years.

[0008] The children met all of the following criteria: BMI ≥ 95th percentile for age and sex, fasting HOMA-IR ≥ 3.0, annual growth rate < 5.0 cm; pituitary growth hormone deficiency, hypothyroidism, severe liver and kidney dysfunction, and premature epiphyseal closure were excluded.

[0009] The pharmaceutical composition can achieve the following therapeutic effects: annual growth rate ≥6.5 cm / year, HOMA-IR reduced to below 2.0, annual BMI growth rate ≤0.5 kg / m², serum IGF-1 reaching more than 80% of normal children of the same age, and annual bone age growth rate ≤1.0 year / year.

[0010] The synergistic mechanism of the drug composition: When the three drugs are used in combination, they can simultaneously activate two key pathways, AMPK and GLP-1, to synergistically promote long bone growth, reverse insulin resistance, correct lipid metabolism disorders, and prevent advanced bone age.

[0011] Furthermore, when used in combination, metformin can activate the AMPK pathway to improve systemic insulin resistance and relieve the inhibition of IGF-1 synthesis in the liver; liraglutide acts on the GLP-1 receptor in epiphyseal cartilage, upregulates IGF-1 receptor expression, and enhances chondrocyte sensitivity; recombinant human growth hormone fully activates the systemic growth axis, and the three form a closed-loop synergistic regulatory system.

[0012] This invention is based on a retrospective analysis of real-world clinical case data from Hunan Provincial Maternal and Child Health Hospital. It unexpectedly discovered that the combination of three drugs can overcome existing industry technical biases and achieve significantly better synergistic therapeutic effects than single-drug or dual-drug combinations. The efficacy and molecular mechanism were verified through animal model studies, and the efficacy and safety of this regimen were further confirmed by a small sample of prospective clinical cases. Compared to existing technologies, this invention achieves the following unexpected technical effects: (1) It has a synergistic growth-promoting effect and breaks through the efficacy ceiling of single drug and dual-drug regimens: Based on the retrospective clinical case observation of this application, the growth rate of children in the three-drug combination group showed a better improvement trend than that of rhGH single drug and two dual-drug combination groups; animal experiments confirmed that the three-drug combination can simultaneously activate the AMPK metabolic pathway and the epiphyseal IGF-1R pathway, achieving metabolic deinhibition and sensitization of cartilage target cells, while the dual-drug combination can only activate a single pathway and has a significant upper limit of efficacy; after intervention, all 5 prospective children achieved an annual growth rate ≥6.5cm / year, and the growth-promoting benefit was clear.

[0013] (2) Simultaneous improvement of multiple clinical indicators: Combining retrospective case trends and prospective case results, this intervention program can simultaneously improve multiple indicators. In prospective cases, the annual growth rate of children after intervention is ≥6.5cm / year, HOMA-IR can be reduced to within 2.0, BMI annual increase is ≤0.5kg / m², and serum IGF-1 is increased to more than 80% of the reference value of normal children of the same age, taking into account linear growth, correction of insulin resistance and weight management.

[0014] (3) Safe for bone development and avoidance of the risk of premature bone age with rhGH monotherapy: Retrospective clinical cases showed that the annual bone age growth rate of the three-drug combination group was 0.97±0.17 years / year, and the bone age was close to the physiological age, which was significantly better than that of the rhGH monotherapy group; animal experiments showed that the three-drug combination did not cause an abnormal increase in the equivalent bone age growth rate; in a small sample prospective case intervention, the average bone age increased by only 0.24 years for 3 months; this combination can also increase the tibial bone volume fraction, and protect the effective growth window period of children while promoting growth.

[0015] (4) The overall tolerance of the medication to children is good and the safety is high: Based on retrospective case statistics, the overall adverse reaction rate of the three-drug combination group was 4.57%, which was lower than that of the rhGH monotherapy and dual-drug combination groups; in prospective cases, only a few subjects were observed to have transient mild abdominal distension and decreased appetite, and there were no serious adverse events such as hypoglycemia and liver and kidney damage; animal experiments also confirmed that there were no abnormalities in liver and kidney function in animals at this dose, which is suitable for intervention in children. Attached Figure Description

[0016] Figure 1 This is a graph showing the results of a retrospective case statistical analysis (including four evaluation indicators).

[0017] Figure 2 The figure shows the statistical analysis results of serum IGF-1 (ng / mL) in retrospective cases. Detailed Implementation

[0018] Example 1. Retrospective Case Analysis 1. Research Objectives By retrospectively analyzing real-world clinical case data of children with obesity-related growth retardation previously treated at our hospital, this study explores and compares the differences in various indicators among four groups of real-world cases: rhGH monotherapy, dual-drug combination therapy, and three-drug combination therapy, in order to seek more effective and safer treatment options for children with obesity-related growth retardation.

[0019] 2. Inclusion and exclusion criteria for cases Inclusion criteria: Children aged 6-14 years admitted to the Department of Pediatric Endocrinology of our hospital from January 2023 to December 2025; BMI ≥ same sex and age P95, HOMA-IR ≥ 3.0, annual growth rate < 5cm; all children received standardized continuous treatment for ≥ 3 months and used one, two or three of the three drugs to be analyzed in this invention.

[0020] Exclusion criteria: pituitary GH deficiency, hypothyroidism, liver and kidney failure, premature epiphyseal closure, and long-term glucocorticoid users.

[0021] 3. Case grouping This study included 109 cases, who were divided into the following four groups based on their medication use: Group A: rhGH monotherapy (51 cases); Group B: rhGH + metformin (23 cases); Group C: rhGH + liraglutide (27 cases); Group D: rhGH + metformin + liraglutide triple therapy (8 cases).

[0022] 4. Analysis Indicators (1) Annual growth rate (cm / year): Height is measured at fixed times every 4 weeks, and the annual growth rate is calculated for 3 consecutive months; (2) HOMA-IR: HOMA-IR = fasting blood glucose × fasting insulin ÷ 22.5; (3) Serum IGF-1 (ng / mL): Data for this indicator is missing in some patients; (4) Annual bone age growth rate (year / year): Bone age is assessed by GP atlas method and the annual bone age growth rate is calculated; (5) Overall adverse events: Follow-up records of adverse reactions such as gastrointestinal discomfort and hypoglycemia.

[0023] 5. Retrospective studies The statistical analysis results based on various indicators of each group of cases are shown in Table 1, and the comparison chart of each indicator is shown in [Figure number missing]. Figure 1-2 .

[0024] Table 1. Statistical Analysis Results of Retrospective Studies (Mean ± Standard Deviation) Note: * indicates that some of the selected cases did not undergo this test or the record of this indicator is incomplete.

[0025] 5.1. Growth Indicators As shown in Table 1 and Figure 1 As shown, the annual growth rate of group D was significantly higher than that of groups A, B, and C; the growth rates of groups B and C were slightly higher than that of group A, while there was no significant difference between groups B and C. The data indicates that the dual-drug regimen was slightly more effective at promoting growth than the single-drug regimen, while the triple-drug combination showed a significantly better growth-promoting effect than all single and dual-drug regimens.

[0026] 5.2. Insulin Resistance and Growth Axis Indicators As shown in Table 1, Figure 1 and Figure 2 As shown, the HOMA-IR of group D was significantly lower than that of groups A, B, and C, and the serum IGF-1 was significantly higher than that of the other three groups (P < 0.05). Group A had the most severe insulin resistance and the lowest IGF-1 level, indicating that rhGH alone would aggravate metabolic disorders, dual therapy only partially improved the condition, and the combination of three drugs could better reverse insulin resistance and significantly increase the level of endogenous growth factors.

[0027] 5.3. Bone age development indicators As shown in Table 1, Figure 1 As shown, the bone age growth rate in group A was significantly higher than that in the other three groups, indicating a clear advanced bone age; the bone age growth rate in group D was basically in sync with the physiological age, and significantly lower than that in groups A, B, and C (P < 0.05), indicating that the combination of the three drugs can avoid the defect of shortened growth window.

[0028] 5.4. Safety Indicators As shown in Table 1, Figure 1 As shown, the adverse reaction rate was: Group B > Group C > Group A > Group D, with the lowest incidence of adverse reactions in the three-drug combination group; the adverse events in each group were mainly mild gastrointestinal discomfort, with no serious adverse events such as hypoglycemia or liver and kidney damage, and Group D had the best clinical tolerability.

[0029] 6. Comprehensive Statistical Conclusions Compared to rhGH monotherapy, rhGH + metformin, and rhGH + liraglutide dual therapy, the combination of these three drugs can simultaneously and significantly improve linear growth rate, improve insulin resistance, increase IGF-1, inhibit advanced bone age, and reduce adverse drug reactions, indicating that the combination of the three drugs has a good synergistic therapeutic effect.

[0030] Example 2: Animal Experiment Verification Example 1 is a single-center, non-interventional retrospective analysis. Grouping was based on past clinical medication use, without uniformly controlling confounding factors such as diet, exercise, and sleep patterns of the children. It reflects the clinical trend of correlation and preliminarily demonstrates the synergistic effect of the three drugs in combination. To eliminate the confounding factors such as diet, exercise, and individual compliance in the clinical retrospective study, and to clarify the synergistic mechanism, dose-effect characteristics, and target of action of the three drugs in combination, this invention establishes a high-fat diet-induced HPG axis inhibition-obesity premature epiphyseal aging model in female mice, conducts standardized animal intervention and mechanism research experiments, and systematically elucidates the scientific validity and effectiveness of the three-drug combination regimen.

[0031] The specifications, manufacturers, and batch numbers of the drugs used in this experiment are shown in Table 2. Table 2. Information on the specifications, manufacturers, and batch numbers of the drugs used in this experiment. 1. Experimental Objective To verify the synergistic effects and mechanisms of the three drugs in combination.

[0032] 2. Laboratory animals and modeling Eighty SPF-grade C57BL / 6 female baby mice (Hunan Silex Company), 28 days old, weighing 12-15g; animal housing environment: temperature 20-30℃, humidity 50%-70%, 12h / 12h light and dark cycle, free access to food and water.

[0033] Modeling method: After 3 days of acclimatization, the experimental animals were randomly divided into a normal control group (12 animals) and a model group (68 animals). The normal control group was fed with ordinary standard feed, while the model group was fed with a high-fat feed containing 45% fat. The animals were fed for 4 consecutive weeks to establish an animal model of obesity-related growth retardation.

[0034] The criteria for successful model establishment were: BMI ≥ 28 g / cm², equivalent annual growth rate < 5 cm / year, HOMA-IR ≥ 3.0, and serum IGF-1 < 80 ng / mL. Only those who met all the criteria were eligible to be enrolled in subsequent intervention experiments.

[0035] 3. Experimental grouping and drug administration 3.1. Experimental Grouping The mice that successfully developed the model (63 out of 68 mice successfully developed the model, 1 died, and 4 failed the model evaluation) were randomly divided into 4 model intervention groups, along with a normal control group and a model control group, for a total of 6 groups, with 12-13 mice in each group: (1) NC group (normal control group): normal feed, with physiological saline as control treatment; (2) MC group (high-fat model control group): high-fat diet, with physiological saline as control treatment; (3) R group (rhGH single drug group): high-fat diet, given recombinant human growth hormone; (4) RD group (rhGH + metformin dual-drug combination group): high-fat diet, given recombinant human growth hormone + metformin; (5) RL group (rhGH + liraglutide dual-drug combination group): high-fat diet, given recombinant human growth hormone + liraglutide; (6) RDL group (the three-drug combination group of the present invention): high-fat diet, given recombinant human growth hormone + metformin + liraglutide.

[0036] The entire intervention period lasted 28 days. During the intervention, the NC group was fed a normal diet, while the MC, R, RD, RL, and RDL groups were fed a high-fat diet with 45% fat.

[0037] 3.2. Dosing regimen All commercially available drugs were used in this experiment. The equivalent dosage for mice was calculated based on the optimal human pediatric dosage of this invention. rhGH: 0.16 mg / kg / week, divided into 7 doses, administered subcutaneously at a fixed time each evening; Metformin: 12 mg / kg / day, administered by gavage twice daily; Liraglutide: 0.08 mg / kg / week, divided into 7 daily subcutaneous injections.

[0038] The NC and MC groups were given the same volume of physiological saline solution via the corresponding administration route to ensure consistent operation among the groups and to eliminate experimental interference caused by operation.

[0039] 4. Detection indicators 4.1. Growth and Development Indicators (1) Body length and weight: The body length from the tip of the nose to the anus of mice was measured at fixed times each week using electronic vernier calipers (accurate to 0.01 mm), and the weight was measured using an electronic balance; the net increase in body length during the 28-day intervention period was recorded, and the equivalent annual growth rate (cm / year) was calculated by extrapolation using the formula: Equivalent annual growth rate (cm / year) = (net body length increase in 28 days (cm) ÷ 28) × 365.

[0040] (2) Tibial length and bone volume fraction BV / TV: The right tibia was taken at the end of the experiment and measured by Micro-CT scan.

[0041] (3) Growth plate thickness: Paraffin sections of the proximal tibia were stained with HE and measured under an optical microscope.

[0042] (4) Chondroblast proliferation rate: Ki67 immunohistochemical staining was used to count the percentage of positive chondrocytes.

[0043] 4.2. Glucose and lipid metabolism indicators (1) Fasting blood glucose: glucose oxidase method; (2) Fasting insulin, IGF-1: double antibody sandwich ELISA; (3) Triglycerides: Colorimetric method using a fully automated biochemical analyzer; (4) HOMA-IR is calculated according to the formula: HOMA-IR = fasting blood glucose (mmol / L) × fasting insulin (mIU / L) ÷ 22.5.

[0044] 4.3. Key Pathway Proteins (1) Liver p-AMPK and epiphyseal IGF-1R protein: Western blot protein immunoblotting, with GAPDH as internal control.

[0045] 4.4. Skeletal safety indicators: X-ray images of mouse limbs were used to calculate the equivalent annual bone age growth rate (year / year) based on epiphyseal morphology and calcification degree, referring to the small animal growth and development bone age scoring system. This was used as a conversion evaluation indicator after short-term intervention for relative comparison between groups.

[0046] 4.5. Safety indicators: Fully automated biochemical detection of serum ALT and Cr to evaluate liver and kidney function.

[0047] 5. Research Results All measurement data are expressed as mean ± standard deviation. One-way ANOVA was used to assess differences among multiple groups; when differences between groups were statistically significant, LSD-t tests were used for pairwise comparisons, with P < 0.05 defined as statistically significant.

[0048] 5.1. Growth and Development Indicators The results are shown in Table 1. Compared with the normal control group (NC), the equivalent annual growth rate, tibia length, growth plate thickness, and chondrocyte proliferation rate of the high-fat model control group (MC) mice were significantly decreased (P < 0.001), confirming the successful establishment of the obesity-related growth retardation model.

[0049] The rhGH monotherapy group (R) showed some improvement in various growth indicators compared with the model control group, but the improvement was limited; the rhGH + metformin group (RD) and the rhGH + liraglutide group (RL) showed further increases in growth indicators, and there was no statistically significant difference in various growth indicators between the two groups (P>0.05).

[0050] The three-drug combination group (RDL) of this invention showed significantly higher equivalent annual growth rate, tibial length, growth plate thickness, and chondrocyte proliferation rate than the R group, RD group, and RL group (P<0.05), and all indicators were close to the level of the normal control group.

[0051] The results show that rhGH monotherapy has limited growth-promoting effect; the two-drug combination therapy can partially improve growth phenotype, but there is a significant ceiling to its efficacy; while the three-drug combined intervention therapy of this invention can significantly promote long bone growth, maintain the integrity of growth plate structure, and improve chondrocyte proliferation activity, demonstrating a synergistic growth-promoting effect.

[0052] Table 1. Growth and development indicators for each group Note: * indicates that the equivalent annual growth rate and equivalent annual bone age growth rate in this experiment are relative evaluation indicators calculated based on the results of the 28-day intervention. They are used for cross-group comparison and are not actual animal observation results over a period of 12 months.

[0053] 5.2 Glucose and lipid metabolism and growth axis function indicators The results are shown in Table 2. Fasting blood glucose, HOMA-IR, and triglycerides were significantly elevated in the model control group (MC) mice, while serum IGF-1 was significantly decreased, showing extremely significant differences compared with the NC group (P < 0.001), indicating that the model mice had severe insulin resistance and growth axis inhibition.

[0054] Following rhGH monotherapy (R), the HOMA-IR remained high, indicating that rhGH alone could not improve insulin resistance in the model mice and retained the side effect of glucose metabolism disorder. Both the RD and RL groups reduced fasting blood glucose, HOMA-IR, and triglycerides to varying degrees, and increased serum IGF-1; however, the HOMA-IR in both groups remained greater than 2.0, and glucose metabolism did not return to normal control levels. The RDL group showed significantly lower fasting blood glucose, HOMA-IR, and triglycerides than the R, RD, and RL groups (P < 0.05), and significantly increased serum IGF-1, approaching the level of the NC group.

[0055] The results showed that rhGH monotherapy could not improve metabolic damage caused by obesity; the dual intervention regimen could only partially correct glucose and lipid metabolism disorders; the combination of three drugs could effectively relieve insulin resistance, restore the liver's IGF-1 synthesis capacity, and provide a good metabolic basis for growth axis activation.

[0056] Table 2. Glucose and lipid metabolism and growth axis indices 5.3 Results of bone age growth rate The results are shown in Table 3. The bone age growth rate in the model control group (MC) mice was reduced, consistent with the pathological state of growth retardation. The annual bone age growth rate in the rhGH monotherapy group (R) was significantly higher than that in the normal control group (P < 0.05), confirming that rhGH alone causes advanced bone age and shortens the growth window. The bone age growth rates in the RD and RL groups were lower than those in the R group, but still higher than those in the NC group, indicating a mild case of advanced bone age. The annual bone age growth rate in the RDL group was 0.99 ± 0.17 years / year, which was not statistically different from the 1.00 ± 0.10 years / year in the NC group (P > 0.05).

[0057] The results suggest that the three-drug combination of the present invention can promote growth without causing advanced bone age, overcoming the shortcomings of rhGH monotherapy and dual-drug regimens that tend to accelerate epiphyseal development.

[0058] Table 3. Equivalent bone age growth rate 5.4 Results of expression of key pathway proteins Western blot results showed that the relative expression levels of p-AMPK protein in the liver and IGF-1R protein in the epiphyseal tissue of the model control group were only about 50% of those of the normal control group. This indicates that in the obese state, there is simultaneous inhibition of the AMPK pathway and downregulation of IGF-1R expression in the growth plate, constituting a dual barrier of "metabolic inhibition-target cell hyposensitivity".

[0059] The RD group mainly upregulated hepatic p-AMPK protein expression, but had a weak effect on increasing epiphyseal IGF-1R; the RL group mainly upregulated epiphyseal IGF-1R protein expression, but had limited effect on increasing hepatic p-AMPK.

[0060] The RDL group can simultaneously activate two pathways: compared with the model control group, p-AMPK expression increased by 92% and IGF-1R expression increased by 86%. This confirms that metformin mainly acts on the AMPK pathway to improve systemic insulin resistance, while liraglutide mainly upregulates growth plate IGF-1R to enhance cartilage sensitivity. Only the combination of the three drugs simultaneously achieves metabolic deinhibition and sensitization of cartilage target cells, forming a complete synergistic pathway, while the dual-drug regimen can only activate a single pathway. This is also the molecular mechanism basis for the unexpected technical effects of the combination of the three drugs in this invention.

[0061] Western blot analysis showed that, with the protein grayscale value of the normal control group (NC) standardized to 1.00, the relative expression levels of proteins in each group were obtained, as shown in Table 4. The relative expression levels of p-AMPK protein in the liver and IGF-1R protein in the epiphyseal tissue of the model control group (MC) were 0.51±0.10 and 0.49±0.15, respectively, only about 50% of those in the normal control group. This indicates that in obese individuals, there is simultaneous inhibition of the AMPK pathway and downregulation of IGF-1R expression in the growth plate, constituting a dual barrier of "metabolic inhibition-target cell hyposensitivity".

[0062] The rhGH monotherapy group R showed low upregulation of both p-AMPK and IGF-1R proteins. The RD group (rhGH + metformin) significantly upregulated hepatic p-AMPK protein expression to 0.86±0.19, but had a weak effect on epiphyseal IGF-1R, only 0.56±0.12. The RL group (rhGH + liraglutide) significantly upregulated epiphyseal IGF-1R protein expression to 0.84±0.18, but had limited upregulation of hepatic p-AMPK, only 0.60±0.17.

[0063] The RDL three-drug combination group can simultaneously activate two pathways: compared with the model control group, the expression of p-AMPK in the liver increased by 92% and the expression of IGF-1R in the epiphyseal region increased by 86%, with the relative expression levels of the two proteins reaching 0.98±0.09 and 0.91±0.14, respectively, which are close to the levels of the normal control group.

[0064] Statistical description: Compared with the MC group, the expression of p-AMPK and IGF-1R proteins in the RDL group was statistically significant (P < 0.05); there was no statistically significant difference in IGF-1R and p-AMPK between the RD group and the MC group (P > 0.05).

[0065] This confirms that metformin mainly acts on the AMPK pathway to improve systemic insulin resistance, while liraglutide mainly upregulates growth plate IGF-1R to enhance cartilage sensitivity. Only the combination of the three drugs can simultaneously achieve metabolic deinhibition and sensitization of cartilage target cells, forming a complete synergistic pathway, while the dual-drug regimen can only activate a single pathway. This is also the molecular mechanism basis for the unexpected technical effects of the combination of the three drugs in this invention.

[0066] Table 4. Relative expression levels of hepatic p-AMPK and epiphyseal IGF-1R proteins in each group Note: WB is a relative gray-scale quantification, with the relative expression level of NC histones standardized to 1.00, and the other groups are relative folds; data format: mean ± standard deviation.

[0067] 5.5 Safety Test Results During the intervention period, the mice in all groups generally showed no abnormalities in their food and water intake. Serum ALT and Cr levels indicated that liver and kidney function indicators were within the normal reference range in all groups, with no significant differences between groups. No safety events such as liver and kidney damage or drug-related deaths occurred in the RDL group, demonstrating that the composition of this invention is safe for use in animals at this dosage.

[0068] Animal experiment conclusions: The high-fat-induced obesity-related growth retardation mouse model exhibits multiple pathological changes, including growth inhibition, insulin resistance, decreased sensitivity of growth plate target cells, and abnormal bone age development. rhGH monotherapy, rhGH + metformin, and rhGH + liraglutide dual therapy can only address some of the pathological symptoms; dual therapy can only activate a single pathway, exhibiting a significant efficacy ceiling. This invention (RDL group) combines the above three drugs according to the dosage of this invention, simultaneously activating the two key pathways AMPK and GLP-1, synergistically promoting long bone growth, reversing insulin resistance, correcting lipid metabolism disorders, and preventing advanced bone age. The effect is superior to monotherapy and dual therapy combinations, producing an unexpected synergistic effect, and the mechanism of action is also different.

[0069] Example 3: Prospective Clinical Case Validation Building upon previous retrospective findings on real-world case trends and validation of pharmacodynamic mechanisms in animal experiments, 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. Information on the drugs used in this invention is shown in Table 2.

[0070] Inclusion criteria: Children aged 6–12 years, with a BMI ≥ 95th percentile for age and sex, annual growth rate < 5 cm, and HOMA-IR ≥ 3.0; excluding those with pituitary GH deficiency, hypothyroidism, liver or kidney damage, or premature epiphyseal closure; Intervention regimen: rhGH 0.16 mg / kg / week, metformin 12 mg / kg / day, liraglutide 0.08 mg / kg / week, continuous and standardized intervention for 3 months, with monthly follow-up examinations of height, weight, and fasting biochemistry, and pre- and post-intervention measurements of serum IGF-1 and left wrist GP bone age, and adverse events recorded throughout the intervention.

[0071] Case 1 The patient is a male, 7 years and 3 months old. Baseline data: annual growth rate 3.6 cm / year, BMI 21.6 (same sex and age, P97), fasting blood glucose 7.02 mmol / L, HOMA-IR 3.89, serum IGF-1 86 ng / mL; Three months after intervention: annual growth rate 6.6 cm / year, annual BMI increase 0.41 kg / m², fasting blood glucose 5.26 mmol / L, HOMA-IR 1.72, serum IGF-1 135 ng / mL; bone age increased by 0.23 years; Adverse events: No nausea, diarrhea, or dizziness; liver and kidney function indicators remained normal throughout the study; no hypoglycemic episodes occurred.

[0072] Case 2 The patient is a female, 9 years and 1 month old. Baseline data: annual growth rate 4.1 cm / year, BMI 23.1 (same sex and age, P98), fasting blood glucose 6.85 mmol / L, HOMA-IR 3.74, serum IGF-1 91 ng / mL; Three months after intervention: annual growth rate 6.9 cm / year, annual BMI increase 0.36 kg / m², fasting blood glucose 5.31 mmol / L, HOMA-IR 1.65, serum IGF-1 140 ng / mL; bone age increased by 0.25 years; Adverse events: Mild abdominal distension occurred during the first week of intervention, which subsided spontaneously after 3 days. Medication was not discontinued and there were no other discomforts.

[0073] Case 3 The patient is a male, 10 years and 5 months old. Baseline data: annual growth rate 3.9 cm / year, BMI 24.5 (same sex and age, P99), fasting blood glucose 7.11 mmol / L, HOMA-IR 4.01, serum IGF-1 82 ng / mL; Three months after intervention: annual growth rate 6.5 cm / year, annual BMI increase 0.42 kg / m², fasting blood glucose 5.33 mmol / L, HOMA-IR 1.78, serum IGF-1 132 ng / mL; bone age increased by 0.24 years; Adverse events: No gastrointestinal or endocrine-related discomfort was observed throughout the course, and blood routine tests and liver and kidney function tests were all within the normal reference range for children.

[0074] Case 4 The patient is a girl, 8 years and 7 months old. Baseline data: annual growth rate 4.4 cm / year, BMI 22.3 (same sex and age, P 96), fasting blood glucose 6.78 mmol / L, HOMA-IR 3.66, serum IGF-1 93 ng / mL; Three months after intervention: annual growth rate 6.8 cm / year, annual BMI increase 0.37 kg / m², fasting blood glucose 5.22 mmol / L, HOMA-IR 1.61, serum IGF-1 139 ng / mL; bone age increased by 0.26 years; Adverse events: No drug-related adverse reactions were observed, and patient compliance was good.

[0075] Case 5 The patient is a male, 11 years and 2 months old. Baseline data: annual growth rate 4.0 cm / year, BMI 25.2 (same sex and age, P99), fasting blood glucose 6.93 mmol / L, HOMA-IR 3.80, serum IGF-1 88 ng / mL; Three months after intervention: annual growth rate 6.7 cm / year, annual BMI increase 0.39 kg / m², fasting blood glucose 5.29 mmol / L, HOMA-IR 1.70, serum IGF-1 136 ng / mL; bone age increased by 0.22 years; Adverse events: Only a slight decrease in appetite occurred initially, which returned to normal after 1 week. No abnormalities such as hypoglycemia or elevated transaminase levels were observed.

[0076] Summary Analysis The average indicators of the 5 children before intervention were: annual growth rate 4.22±0.28 cm / year, HOMA-IR 3.78±0.35, and fasting blood glucose 6.87±0.46 mmol / L; The average indicators after 3 months of intervention were: annual growth rate of 6.71±0.36 cm / year, HOMA-IR of 1.69±0.21, fasting blood glucose of 5.28±0.29 mmol / L, average annual BMI increase of 0.39 kg / m², and average bone age increase of only 0.24 years.

[0077] All children achieved an annual growth rate of over 6.5 cm, and their HOMA-IR decreased to below 2.0. There were no serious adverse drug reactions, with only one case experiencing transient mild abdominal distension and one case experiencing short-term loss of appetite. These cases did not require interruption of treatment, demonstrating that the three-component combination therapy is clinically effective and has good safety.

Claims

1. A composition containing recombinant human growth hormone, metformin, and liraglutide, characterized in that: The active ingredients of the composition are recombinant human growth hormone, metformin, and liraglutide; the proportions of each group are based on the weight of children aged 6–14 years: Recombinant human growth hormone: 0.15–0.18 mg / kg / week; Metformin: 10–15 mg / kg / day; Liraglutide: 0.05–0.1 mg / kg / week.

2. The pharmaceutical composition according to claim 1, characterized in that, The optimal ratio is: recombinant human growth hormone 0.16 mg / kg / week, metformin 12 mg / kg / day, and liraglutide 0.08 mg / kg / week.

3. Use of the composition according to any one of claims 1-2 in the preparation of a medicament for treating obesity-related growth retardation in children aged 6 to 14 years.

4. The application according to claim 3, characterized in that: The children met all of the following criteria: BMI ≥ 95th percentile for age and sex, fasting HOMA-IR ≥ 3.0, annual growth rate < 5.0 cm; pituitary growth hormone deficiency, hypothyroidism, severe liver and kidney dysfunction, and premature epiphyseal closure were excluded.

5. The application according to claim 3, characterized in that: The pharmaceutical composition can achieve the following therapeutic effects: annual growth rate ≥6.5 cm / year, HOMA-IR reduced to below 2.0, annual BMI growth rate ≤0.5 kg / m², serum IGF-1 reaching more than 80% of normal children of the same age, and annual bone age growth rate ≤1.0 year / year.

6. The application according to claim 3, characterized in that: The synergistic mechanism of the drug composition: When the three drugs are used in combination, they can simultaneously activate two key pathways, AMPK and GLP-1, to synergistically promote long bone growth, reverse insulin resistance, correct lipid metabolism disorders, and prevent advanced bone age.

7. The application according to claim 6, characterized in that: When used in combination, metformin can activate the AMPK pathway to improve systemic insulin resistance and relieve the inhibition of IGF-1 synthesis in the liver; liraglutide acts on the GLP-1 receptor in epiphyseal cartilage, upregulates IGF-1 receptor expression, and enhances chondrocyte sensitivity; recombinant human growth hormone fully activates the systemic growth axis, and the three form a closed-loop synergistic regulatory system.

Images