Long-acting sustained-release human recombinant growth hormone fusion protein, pharmaceutical composition and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a long-acting sustained-release human recombinant growth hormone fusion protein, a pharmaceutical composition and application thereof. The growth hormone fusion protein provided by the present application comprises a growth hormone and a temperature-sensitive elastin-like polypeptide. After subcutaneous injection of the fusion protein, phase transition occurs in situ at body temperature, and drug depots are formed. The drug is slowly released from the depots to the body circulation system, significantly prolonging the effective action time of a single injection of growth hormone, improving patient compliance, and improving pharmacokinetic parameters. The growth hormone fusion protein also has significantly improved biological stability and high biological activity, and has excellent application value and potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a long-acting sustained-release human recombinant growth hormone fusion protein, its pharmaceutical composition, and its applications. Background Technology

[0002] The discovery, research, and application of growth hormone have a history of over a century. In 1985, the FDA approved recombinant human growth hormone (rhGH) produced using gene recombination technology, and rhGH gradually became the first-line drug for treating various growth hormone deficiencies (GHD) and their complications. The therapeutic effect of rhGH on GHD patients depends mainly on accurate diagnosis, appropriate dosage, and timely treatment. GHD treatment requires long-term daily subcutaneous injections of rhGH, which leads to poor patient adherence, especially among children, who are the primary treatment group for rhGH. Studies have shown that missing 1-2 injections per week during rhGH treatment significantly reduces its effectiveness. Therefore, there is an urgent need to develop long-acting rhGH to improve patient adherence.

[0003] There has been some progress in research on long-acting rhGH. Nutropin was approved in 1999. Microsphere technology enabled sustained release of rhGH in vivo and extended the dosing cycle to over 14 days; however, due to difficulties in large-scale production, the drug was withdrawn from the market in 2004. Currently, there are two main commercially available long-acting growth hormones: Novo Nordisk's Somapacitan and Changchun Jinsai Pharmaceutical's... The dosing cycle is 7 days. Somapacitan prolongs the drug's circulating half-life and achieves long-lasting effect by modifying fatty acids to increase the binding of the drug to albumin. Long-acting growth hormones are typically achieved through polyethylene glycol (PEG) modification. Additionally, long-acting rhGHs utilizing PEGylation and protein fusion technologies are under development. However, these long-acting strategies generally only extend the drug's in vivo half-life to a maximum of 12 days in mouse models, limiting the duration of long-acting effects. Furthermore, the production cost of these long-acting growth hormones is high, often resulting in higher prices and limited market penetration. Therefore, it is necessary to overcome the limitations of commonly used technologies on the long-acting cycle of growth hormones while minimizing their production costs. Summary of the Invention

[0004] This invention provides a long-acting sustained-release human recombinant growth hormone fusion protein, its pharmaceutical composition, and its applications.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a growth hormone fusion protein, the fusion protein comprising growth hormone and a thermosensitive elastin-like polypeptide (ELP).

[0007] In response to growth hormone protein, this invention designs the sequence of an elastin-like polypeptide to enable the elastin-like polypeptide to better cooperate with growth hormone, prolong the half-life of the fusion protein, and improve the stability of the fusion protein.

[0008] Elastin-like polypeptides are composed of tandem repeat sequences of pentapeptides (X-Gly-Val-Pro-Gly).

[0009] Preferably, the amino acid sequence of the thermosensitive elastin-like polypeptide comprises (XGVPG)n, where X is any natural amino acid other than proline, and 30≤n≤150.

[0010] Specifically, X is selected from any one of valine, phenylalanine, tryptophan, tyrosine, alanine, glycine, methionine, threonine, serine, leucine, and isoleucine.

[0011] Preferably, X is valine, and 60 ≤ n ≤ 120.

[0012] More preferably, X is valine and n is 90.

[0013] In some embodiments of the present invention, the amino acid sequence of the thermosensitive elastin-like polypeptide is (VGVPG). 90 .

[0014] The present invention has found that the above-mentioned elastin-like polypeptide sequence can work well with growth hormone protein, significantly prolonging the half-life of the fusion protein and exerting an excellent sustained-release effect, while also significantly improving the stability of the fusion protein. In addition, the fusion protein can also ensure high growth hormone activity.

[0015] Preferably, the growth hormone is recombinant human growth hormone;

[0016] More preferably, the growth hormone is any one of the following 1) and 2):

[0017] 1) The amino acid sequence is shown in SEQ ID NO.1;

[0018] 2) The amino acid sequence is obtained by adding one or more of the following to the N-terminus or C-terminus of the sequence shown in SEQ ID NO.1: a protein tag, an enzyme cleavage site, or a linker peptide.

[0019] Adding a protein tag, restriction enzyme site, or linker peptide to the N-terminus or C-terminus of the sequence shown in SEQ ID NO.1 generally does not affect the structure and function of the protein with the sequence shown in SEQ ID NO.1. Therefore, the growth hormone shown in 2) above is also within the scope of protection of this invention.

[0020] There are no specific restrictions on the sequences of protein tags, enzyme digestion sites, and linker peptides. Protein tags include, but are not limited to, His-tag, Gst, MBP, Strep, and Flag tags. Enzyme digestion sites can be enzymes used to cleave protein tags, including but not limited to TEV proteases and transpeptidases. Linker peptides can be short peptides rich in glycine and serine.

[0021] In the aforementioned fusion protein, growth hormone is directly linked to a thermosensitive elastin-like polypeptide or linked through a linker peptide.

[0022] The aforementioned linker peptides can be polypeptides rich in glycine and serine.

[0023] Preferably, in the above-mentioned fusion protein, growth hormone is directly linked to a thermosensitive elastin-like polypeptide.

[0024] Among the aforementioned fusion proteins, the thermosensitive elastin-like polypeptide can be fused to the C-terminus or N-terminus of growth hormone.

[0025] Preferably, the thermosensitive elastin-like polypeptide is linked to the C-terminus of the growth hormone.

[0026] More preferably, the N-terminus of the thermosensitive elastin-like polypeptide is directly linked to the C-terminus of the growth hormone.

[0027] This invention has found that directly linking the N-terminus of a thermosensitive elastin-like polypeptide to the C-terminus of the growth hormone is more conducive to increasing the expression level of the fusion protein and better ensuring the stability of the fusion protein and the activity of the growth hormone.

[0028] In some embodiments of the present invention, the fusion protein is rhGH-ELP, wherein rhGH is recombinant human growth hormone and ELP is a thermosensitive elastin-like polypeptide. Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.2.

[0029] Secondly, the present invention provides a nucleic acid molecule that encodes the growth hormone fusion protein described above.

[0030] The nucleic acid molecule is either DNA or RNA.

[0031] Based on the amino acid sequence of the fusion protein described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the fusion protein. Due to the degeneracy of codons, the nucleotide sequence of the above nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the above fusion protein are within the scope of protection of this invention.

[0032] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the fusion protein is shown in SEQ ID NO.3.

[0033] Thirdly, the present invention provides a biomaterial comprising the nucleic acid molecules described above or expressing the growth hormone fusion protein described above;

[0034] The biomaterial is an expression cassette, vector, or host cell.

[0035] The expression cassette can be operatively linked to the nucleic acid molecule by a promoter. Depending on the expression requirements and the upstream and downstream sequences of the expression cassette, it may also contain other transcriptional and translational regulatory elements such as terminators and enhancers.

[0036] The vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, transposons, etc., wherein plasmid vectors include replicating vectors and non-replicating vectors.

[0037] The host cell includes microbial cells or animal cells, wherein the microorganisms include prokaryotic microorganisms (e.g., Escherichia coli) and eukaryotic microorganisms (e.g., yeast).

[0038] Fourthly, the present invention provides a method for preparing the fusion protein described above, the method comprising:

[0039] 1) The nucleic acid molecule encoding the fusion protein is ligated into the expression plasmid to obtain the recombinant expression plasmid;

[0040] 2) The recombinant expression plasmid from step 1) is introduced into host cells to obtain recombinant host cells;

[0041] 3) Cultivate the recombinant host cells from step 2) to express the fusion protein, and then purify the fusion protein to obtain the purified fusion protein.

[0042] In some embodiments of the present invention, the plasmid is selected from the pET series plasmids, preferably pET-24a(+).

[0043] In some embodiments of the present invention, the host cell is Escherichia coli, preferably Escherichia coli Rosetta-gami(DE3)pLysS.

[0044] Fifthly, the present invention provides the use of the above-described growth hormone fusion protein, the nucleic acid molecule, or the biomaterial in the preparation of a drug.

[0045] Preferably, the drug is used to prevent or treat growth hormone deficiency.

[0046] Preferably, the drug is for subcutaneous injection and is a subcutaneous injection preparation.

[0047] The present invention has verified through animal experiments that the growth hormone fusion protein has a significant therapeutic effect on growth hormone deficiency and can promote animal growth, weight gain and cartilage development.

[0048] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the growth hormone fusion protein described above.

[0049] Preferably, the pharmaceutical composition is used for the prevention or treatment of growth hormone deficiency. The active ingredient of the pharmaceutical composition may contain only the growth hormone fusion protein, or it may contain other active ingredients for the prevention or treatment of growth hormone deficiency.

[0050] The pharmaceutical composition may also contain one or more pharmaceutically suitable excipients.

[0051] The administration methods of the pharmaceutical compositions described in this invention include, but are not limited to, subcutaneous injection, and can also be used for local administration.

[0052] In practical applications, after subcutaneous injection of the growth hormone fusion protein rhGH-ELP, rhGH-ELP precipitates from the solution and forms a gel reservoir at the injection site. Because the phase transition temperature of ELP is concentration-dependent, the rhGH-ELP surrounding the reservoir gradually dissolves and diffuses into the bloodstream over a long period, resulting in a significantly prolonged duration of drug action and improved pharmacokinetic parameters. This achieves sustained-release of rhGH-ELP, effectively extending the drug's duration of action in the body and improving its bioavailability.

[0053] The beneficial effects of this invention are as follows:

[0054] 1. The growth hormone fusion protein provided by this invention is a novel thermosensitive, long-acting, sustained-release human recombinant growth hormone delivery system. After subcutaneous injection, it undergoes a phase transition in situ triggered by body temperature and precipitates to form a drug reservoir. The drug is slowly released from the reservoir into the circulatory system, achieving continuous and slow release of growth hormone in vivo without relying on microsphere formulations and chemical modification technology. This significantly prolongs the effective duration of action of a single injection of growth hormone (the half-life of rhGH is extended from 0.72h of free rhGH to 594.59h of rhGH-ELP, an extension of 825.8 times, with an effective duration of action of not less than 3 weeks, and even up to 4 weeks). It can extend the dosing cycle of human recombinant growth hormone, improve patient compliance, save treatment energy, improve pharmacokinetic parameters, and has significant advantages in improving drug metabolism performance.

[0055] 2. The growth hormone fusion protein provided by this invention significantly improves the biological stability of growth hormone (rhGH retains less than 10% of its biological activity after being stored in aqueous solution at 4°C for one week, while rhGH-ELP retains more than 80% of its biological activity after being stored under the same conditions for 3 months). This not only helps the drug exert its efficacy in vivo for a longer period of time, but also facilitates the storage and transportation of the drug, with relatively low requirements for transportation and storage conditions.

[0056] 3. The growth hormone fusion protein provided by this invention has high biological activity and good therapeutic effect, and can be used to prevent or treat growth hormone deficiency.

[0057] 4. The growth hormone fusion protein provided by this invention also has the advantages of simple equipment required for preparation, simple production and processing steps, low cost, and convenient process operation, and has excellent promotion and application value and potential. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in this invention 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0059] Figure 1 A schematic diagram of the method flow for constructing the rhGH-ELP plasmid in Embodiment 1 of the present invention is shown.

[0060] Figure 2 The results of obtaining rhGH-ELP by ITC purification and obtaining rhGH by nickel column affinity chromatography in Example 2 of the present invention are shown.

[0061] Figure 3The results of MALDI-TOF analysis of the molecular weights of rhGH-ELP and rhGH in Example 3 of this invention are shown.

[0062] Figure 4 The results of the hydration radius of rhGH-ELP and rhGH in Example 3 of the present invention are shown.

[0063] Figure 5 The results of the rhGH-ELP phase transition temperature and the concentration dependence of the phase transition temperature in Example 3 of the present invention are shown: where a is the relationship between different concentrations of rhGH-ELP and turbidity changes, and b is a function graph of rhGH-ELP concentration and phase transition temperature.

[0064] Figure 6 The in vitro bioactivity results of rhGH-ELP and rhGH in Example 4 of this invention are shown.

[0065] Figure 7 The results of rhGH-ELP biostability in Example 5 of this invention are shown.

[0066] Figure 8 The results show the changes in blood drug concentrations over time in rats after subcutaneous injection of the same molar amounts of rhGH-ELP and rhGH in Example 6 of the present invention.

[0067] Figure 9 The results of the area under the drug-time curves of rhGH-ELP and rhGH over time after subcutaneous injection of the same molar amount of rhGH-ELP and rhGH in Example 6 of the present invention are shown.

[0068] Figure 10 The results of a single injection of rhGH-ELP in Example 7 of this invention demonstrate the effect on weight gain in pituitary deprived rats. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0070] Example 1: Construction of rhGH-ELP fusion protein expression plasmid and its expression in Escherichia coli

[0071] This embodiment constructs an expression plasmid for the rhGH-ELP fusion protein. The plasmid construction process is as follows: Figure 1As shown in SEQ ID NO.2, the amino acid sequence of the expressed rhGH-ELP fusion protein is VGVPG, and the repeating unit amino acid sequence of ELP is VGVPG, with a repeat count of 90.

[0072] Gene fragments containing the aforementioned repeating units and BseRI / AcuI sticky ends were synthesized by Sangon Biotech (Shanghai, China).

[0073] Upstream segment:

[0074]

[0075] Downstream segment:

[0076]

[0077] The synthesized fragment was inserted into the pET-24a(+) vector via the BseRI / AcuI restriction site, and a plasmid with 18 repeating units as shown above was obtained by rolling circle plasmid construction.

[0078] The amino acid sequence of rhGH (Drugbank, DB00052) was synthesized and inserted by Sangon Biotech (Shanghai, China) after codon optimization. In the vector. Using PCR technology, from The rhGH coding sequence was amplified in the vector and inserted into the pET-24a(+) vector via a single NdeI restriction site. Plasmids containing the rhGH-ELP gene (sequence shown in SEQ ID NO.3) were then constructed. The rhGH gene sequence is as follows:

[0079]

[0080]

[0081] The primers for amplifying the above rhGH gene sequence are as follows:

[0082] Upstream primer:

[0083]

[0084] Downstream primer:

[0085]

[0086] After constructing the expression plasmid of the fusion protein rhGH-ELP, it was expressed in *E. coli* (Rosetta-gami(DE3)pLysS, Novagen). Before large-scale expression, the transformed single clones were inoculated into 10 mL of LB medium (containing 100 μg / mL kanamycin) and cultured overnight at 37°C and 180 rpm with shaking. The next day, they were transferred to 1 L of fresh LB medium (placed in a 2 L shake flask, kanamycin concentration 100 μg / mL) for large-scale culture and expression induction. The specific steps are as follows: First, the culture was carried out at 37°C and 200 rpm with shaking until the bacterial density reached OD0.05. 600 After adjusting the concentration to 0.6-0.8, the culture temperature was set to 20℃, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5mM. The cells were collected after culturing for 18 hours.

[0087] Example 2: Purification of rhGH-ELP fusion protein

[0088] This embodiment purifies the rhGH-ELP fusion protein expressed in Example 1.

[0089] 1. First, rhGH-ELP was purified using inverse transition cycling (ITC) technology. The specific method is as follows:

[0090] (1) Collect 1L of Escherichia coli culture medium prepared according to the method of Example 1 into a centrifuge bottle, centrifuge at 3000×g to collect the bacterial cells, and remove the upper culture medium.

[0091] (2) Resuspend the bacterial cells in 30 mL of ice-cold PBS, then break the cells with an ultrasonic instrument at 4°C, and then centrifuge the E. coli lysate at 4°C and 14000×g for 15 minutes.

[0092] (3) Add 2 mL of polyethyleneimine (PEI, 10%) to the supernatant collected in step (2), and centrifuge again for 15 minutes to remove nucleic acids and other negatively charged substances from the cell lysate. The resulting supernatant is then purified by ITC: add NaCl to a final concentration of 3 M, dissolve completely at 37 °C, centrifuge at 14000 × g for 15 minutes, discard the supernatant, dissolve the precipitate in pre-cooled 10 mM PBS, centrifuge after complete dissolution, and obtain the supernatant. Repeat this process 2-3 times to obtain the sample.

[0093] 2. The His-tagged rhGH-ELP fusion protein was purified using Ni affinity chromatography. The specific steps are as follows: The supernatant was filtered through a 0.22 μm filter membrane and loaded onto a Ni affinity column. Purification was then performed using an AKTAPurifier 10 system. The protein was then washed with a gradient of 0–100% buffer B (10 mM PBS, 500 mM imidazole, pH 7.4), and each elution peak was collected. Analysis was then performed using polyacrylamide gel electrophoresis (SDS-PAGE). After obtaining the target protein, imidazole was removed using a HiPrep 26 / 10 desalting column, and the buffer was replaced with 10 mM, pH 7.4 PBS solution. The protein was then stored at -80°C.

[0094] The purity of the purified samples was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein concentration was determined using a BCA protein assay kit (Solarbio). SDS-PAGE analysis samples were prepared with Laemmli sample buffer containing 5% β-mercaptoethanol at a concentration of 1 mg / mL. After heating at 95°C for 5 min, 10 μL of sample was loaded into a pre-prepared 10% SDS-PAGE gel and subjected to vertical electrophoresis at 80–100 V for 90 min (electrophoresis buffer: 25 mM Tris, 250 mM M Lycine, 0.1% SDS). The gel was stained with Coomassie Blue G-250 before observing the band positions. Figure 2 The expression and purification of rhGH-ELP and rhGH were shown (the vector and host cells used for rhGH expression were the same as those used for rhGH-ELP, and rhGH was purified using a nickel column). The results showed that expression in *E. coli* and purification yielded a protein with a purity >95%, which was identified as the rhGH-ELP fusion protein.

[0095] Example 3: Determination of physicochemical characterization parameters of rhGH-ELP fusion protein

[0096] 1. The molecular weight of the purified rhGH-ELP fusion protein obtained in Example 2 was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF). The instrument used was a 4800 Plus MALDI-TOF / TOF. TM The analyzer (AB SCIEX) produced the following results: Figure 3 As shown, the experimentally measured molecular weights of rhGH-ELP and rhGH are close to the theoretical values.

[0097] 2. The hydration radius of the rhGH-ELP fusion protein sample was determined using dynamic light scattering (DLS) on a Malvern Zetasizer Nano-zs90. The sample was diluted in PBS buffer and filtered through a 0.22 μm pore size membrane before testing. DLS analysis showed that the hydration radius of rhGH was 3 nm, while the hydration radius of the synthesized rhGH-ELP reached 10 nm. The renal filtration clearance size was approximately 5 nm, indicating that rhGH-ELP can prolong the in vivo circulating half-life of rhGH. Figure 4 The hydration radius of rhGH-ELP and rhGH is shown in the DLS analysis.

[0098] 3. The phase transition temperature (Tt) of rhGH-ELP was determined by turbidimetric method: the sample was diluted with PBS to 1 mg / mL, and the OD was measured using a microplate reader (Molecular Devices) in the temperature range of 4-50℃ (incrementing by 2℃ / min). 350 The ultraviolet absorption was measured, and then the temperature was lowered to 4°C at the same rate to determine the OD. 350 The ultraviolet absorption, where Tt refers to the temperature at which the sample turbidity reaches half of its maximum value. Figure 5 Figure a shows the phase transition temperature of rhGH-ELP, which exhibits rapid reversible phase transition behavior during both heating and cooling processes.

[0099] 4. To explore the concentration dependence of the rhGH-ELP phase transition temperature, samples were diluted with PBS to different concentrations, and OD values ​​were measured using a microplate reader (Molecular Devices) within the temperature range of 4-50℃ (incrementing at 2℃ / min). 350 The ultraviolet absorption results are as follows: Figure 5 As shown in b, rhGH-ELP can undergo a phase transition at body temperature when the concentration is above 5 μM. The results indicate that when rhGH-ELP is injected subcutaneously at a high concentration above 5 μM, it will precipitate from the solution and form a reservoir in situ. However, due to the low concentration dissolution effect, it will gradually be released from the reservoir and diffuse into the circulation system.

[0100] Example 4: In vitro bioactivity assay of rhGH-ELP fusion protein

[0101] This embodiment tests the in vitro activity of the rhGH-ELP fusion protein prepared in Example 2.

[0102] The biological activity of the rhGH-ELP fusion protein was detected by a rat Nb2-11 lymphoma cell proliferation assay. Specifically, 24 hours before the start of the proliferation assay, the cell culture medium was replaced with serum-free bovine medium, and an appropriate amount of cells were aliquoted into 96-well plates. Three groups were included: a control group (BSA), an rhGH drug group, and an rhGH-ELP drug group. At the start of the assay, gradient concentrations (0.01, 0.1, 1, 10, 100, 1000, 10000, 10000, 100000 ng / mL) of BSA, rhGH, and rhGH-ELP were added to the culture medium, and after incubation for 48 hours, cell proliferation activity was measured using a CCK-8 assay kit. Data were collected, and drug concentration versus cell proliferation curves were plotted to analyze the in vitro biological activity of the long-acting rhGH drug. Results are as follows: Figure 6 As shown in Table 1, the results indicate that the rhGH-ELP fusion protein still retains in vitro biological activity, with 26% of the activity retained compared to rhGH.

[0103] Table 1

[0104] sample EC 50 (ng / mL) rhGH 2.11 rhGH-ELP(V) 8.12

[0105] Example 5: Detection of the biological stability of the rhGH-ELP fusion protein

[0106] This embodiment tests the biological stability of the rhGH-ELP fusion protein prepared in Example 2.

[0107] The biostability of rhGH-ELP was assessed during a three-month storage period at 4°C, using rhGH as a control. The prepared rhGH-ELP fusion protein was concentrated to the same concentration, filtered for sterilization, and stored at 4°C. The bioactivity of rhGH-ELP stored at specific time points (1, 14, 30, and 90 days after purification) was determined by Nb2 cell proliferation assay. The bioactivity of rhGH and rhGH-ELP purified on day 1 was used as a reference value, and the results are expressed as the percentage of bioactivity retained by rhGH and rhGH-ELP. Results are as follows: Figure 7 As shown, the results indicate that rhGH-ELP retained high bioactivity (>80%) after 3 months of storage, while rhGH's activity was less than 10% after 1 day of storage, indicating that rhGH-ELP has a significant advantage in bioactivity stability compared to rhGH.

[0108] Example 6: Pharmacokinetics of rhGH-ELP fusion protein

[0109] This embodiment tests the pharmacokinetics of the rhGH-ELP fusion protein prepared in Example 2.

[0110] Using a rat model, the same dose of growth hormone rhGH-ELP and rhGH were injected subcutaneously, and the changes in blood growth hormone concentration over time were measured. Data analysis was performed using DAS software.

[0111] Before the drug treatment period, 12 six-week-old female Sprague-Dawley rats weighing approximately 200g (purchased from Vital River Pharmaceuticals, Beijing) were observed for a period of time and then randomly divided into three groups. rhGH-ELP and rhGH were subcutaneously injected at a dose of 3.8 μmol / kg body weight. At the designated time points, after anesthetizing the rats with isoflurane, 0.3-0.4 mL of blood was collected via the inner canthal vein. The blood was allowed to stand at room temperature for 1 hour, and the supernatant serum was collected by centrifugation at 3000×g at 4℃ and stored at -80℃. The serum GH content was determined using a human GHELISA Kit (Dogesce, China) according to the manufacturer's instructions. Pharmacokinetic parameters were calculated using DAS 3.0 pharmacokinetic analysis software. The pharmacokinetic parameters of rhGH-ELP and rhGH were analyzed using the compartment elimination model in DAS software.

[0112] The results are as follows Figure 8 As shown, after subcutaneous injection, compared to the rapid increase and decrease in plasma concentration in the rhGH group, the rhGH-ELP group maintained a higher plasma concentration for 4 weeks, exhibiting a slow release lasting for one month. This significant difference can be further evidenced by the pharmacokinetic parameters of rhGH-ELP and rhGH listed in Table 2. Moreover, from Figure 9 The results show that the area under the curve of rhGH-ELP accumulation in the blood circulation system is linearly correlated with time, confirming that rhGH-ELP has the characteristic of sustained release in vivo, while rhGH exhibits a logarithmic release curve. In summary, these results demonstrate that the ELP fusion strategy can achieve sustained release of rhGH-ELP in vivo, with a sustained release period of up to 4 weeks.

[0113] Table 2

[0114] parameter rhGH rhGH-ELP Half-life (h) 0.72±0.03 594.59±35.23 Area of ​​the curve during drug administration (μg / mL·h) 15.11±1.53 2065.35±222.71 Peak concentration (μg / mL) 8.26±1.06 4.53±0.36

[0115] Example 7: In vivo biological activity analysis of rhGH-ELP fusion protein

[0116] In this embodiment, the in vivo growth-promoting activity of the rhGH-ELP fusion protein prepared in Example 2 was tested using a pituitary-removed rat model, according to the Chinese Pharmacopoeia. Female Sprague Dawley rats were used in this embodiment. The pituitary gland was surgically removed 2-3 weeks prior to the experiment. Postoperatively, rat body weight was monitored. Rats whose body weight change was less than ±10% of preoperative weight were randomly divided into four groups of six each:

[0117] The solvent group received subcutaneous injections of PBS buffer daily for four consecutive weeks.

[0118] Growth hormone-1 group: single subcutaneous injection of 84 mg / kg rhGH;

[0119] Growth hormone-2 group, 3 mg / kg rhGH was injected subcutaneously daily for 28 consecutive days;

[0120] The rhGH-ELP group received a single subcutaneous injection of 84 mg / kg rhGH equivalent rhGH-ELP.

[0121] The rats' body weight was measured daily, and the changes in body weight were recorded weekly. Figure 10 The weight change curves of rats in each group during the treatment process are shown in Table 3. The average weight change of rats on days 7, 14, 21 and 28, as well as the statistical analysis results, are shown in Table 3. The statistical analysis was performed using the T-test for two-tailed unequal variances in Excel software.

[0122] The results showed that rats that received a single subcutaneous injection of rhGH-ELP could continue to gain body weight for more than three weeks, while a single injection of the same dose of rhGH did not achieve a similar effect.

[0123] Table 3

[0124]

[0125] After the experiment, the width of the epiphyseal plate was measured on the right leg of the rat. Table 4 summarizes the results of the epiphyseal plate width of each group of rats after treatment. Statistical analysis was performed using the T-test two-tailed unequal variance test in Excel software.

[0126] Table 4

[0127]

[0128] The results showed that a single subcutaneous injection of rhGH-ELP significantly increased the width of the epiphyseal plate in rats, while a single injection of the same dose of rhGH did not significantly increase the width of the epiphyseal plate in rats.

[0129] In summary, this invention innovatively proposes a protein-based sustained-release recombinant human growth hormone rhGH-ELP. Through protein fusion, the effective duration of action of a single subcutaneous injection is greatly improved. In rats, a single subcutaneous injection of rhGH-ELP showed an ultra-long duration of action of more than 3 weeks, while also improving protein stability.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A growth hormone fusion protein, characterized in that, The fusion protein is composed of growth hormone and a thermosensitive elastin-like polypeptide, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the growth hormone fusion protein of claim 1.

3. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid molecule of claim 2 or expresses the growth hormone fusion protein of claim 1; The biomaterial is an expression cassette, vector, or host cell.

4. The use of the growth hormone fusion protein of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in the preparation of a drug.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the growth hormone fusion protein of claim 1.

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