Anti-inflammatory, antioxidant, anti-aging and immunomodulatory injectable formulations containing human placental extract

Abstract

This invention discloses an injectable formulation containing human placental extract that has anti-inflammatory, antioxidant, anti-aging, and immunomodulatory effects, belonging to the fields of regenerative medicine and drug development technology. The formulation of this invention uses sterile water for injection as a solvent and contains the following components by weight and volume percentage: 1.0%–1.5% human placental low-temperature extraction active mixture, 0.25%–0.35% phosphate buffer, 1.0%–2.0% glycerol, 0.30%–0.55% sodium chloride, 3.0%–5.0% trehalose, and 0.02% EDTA-2Na. The formulation has a pH of 7.2±0.1 and an osmotic pressure of 280–320 mOsm / kg. It abandons the pH 2.5–3.0 and 80°C high-temperature acid hydrolysis and adopts low-temperature static extraction at 0–4°C throughout the process, which does not destroy the natural conformation of growth factors and immune peptides. All active components are complete peptides naturally released by placental cells, rather than hydrolyzed short peptides interrupted by strong acids. The immune regulation and dermal repair bioactivity are superior to hydrolyzed products. Pre-viral inactivation and low-temperature multi-stage ultrafiltration are used for dual impurity control, resulting in <1% residual large molecular weight sensitizing proteins, reducing the risk of redness and swelling and allergies after injection.

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine and drug development, and particularly to injectable formulations containing human placental extracts that have anti-inflammatory, antioxidant, anti-aging, and immunomodulatory effects. Background Technology

[0002] Skin aging is a comprehensive pathophysiological process involving the synergistic effects of endogenous and exogenous aging. Endogenous aging is characterized by a decline in the proliferative capacity of skin cells, a decrease in the function of dermal fibroblasts, an accumulation of reactive oxygen free radicals, and a reduction in collagen synthesis capacity. Exogenous aging is mainly caused by ultraviolet radiation and external stimuli, which can induce photoaging damage to the skin, release a large number of inflammatory factors, and accelerate aging problems such as skin laxity, wrinkle formation, dullness, and sensitivity.

[0003] Currently, mainstream facial rejuvenation injectable preparations on the market can be divided into three categories, all of which have significant technical shortcomings: The first category consists of filler preparations such as hyaluronic acid and collagen, which only provide physical filling and support, and cannot regulate skin cell activity or reverse the aging process at the cellular level. Their effects are singular and their duration of effect is limited. The second category consists of neuromodulators such as botulinum toxin, which can only improve dynamic facial wrinkles, but cannot repair dermal matrix damage or regulate skin immune inflammation, and have no root cause repair effect against aging. The third category consists of novel preparations such as platelet-rich plasma and exosomes. Although they incorporate the concept of growth factor repair, they suffer from problems such as complex and uncontrollable ingredients and large batch-to-batch activity differences. At the same time, they cannot effectively regulate chronic skin inflammation, resulting in poor product efficacy and overall effectiveness, which limits their clinical application.

[0004] Human placental tissue, after acid hydrolysis, yields a mixed component rich in various growth factors and immunomodulatory peptides, possessing excellent potential for tissue repair and cell activation, and has been preliminarily applied in the field of skin repair. However, traditional placental acid hydrolysis preparations have fatal technical defects: the hydrolysis products have a wide molecular weight distribution and a high residual amount of large molecular weight proteins, which easily triggers skin immune rejection reactions; active peptides and growth factors are extremely prone to inactivation in liquid storage environments, exhibiting extremely poor stability, and cannot meet the stringent industry standards for safety, stability, efficacy, and long-term efficacy of injectable preparations, greatly limiting their application research in the development of anti-inflammatory and immunomodulatory drug candidates.

[0005] In summary, there is a need for a placental injectable formulation with precise and controllable ingredients, simplified excipients, safety and non-immunogenicity, long-lasting stability, and multiple effects including anti-inflammatory, anti-aging, and immune regulation. Summary of the Invention

[0006] Purpose of the invention: The invention provides a low-temperature extraction injection formulation of human placenta, which is extracted gently at 0-4℃ throughout the process, without the destruction caused by strong acids or high temperatures, and fully preserves the original natural active peptides and growth factors in the placenta; combined with multi-stage ultrafiltration to remove large molecular weight allergenic proteins, it also has anti-inflammatory, antioxidant, anti-aging, and skin immune homeostasis regulation effects. The injection safety and activity retention rate are significantly better than traditional acid-hydrolyzed placenta preparations.

[0007] Technical solution: To solve the above-mentioned technical problems, according to one aspect of the present invention, more specifically, an injectable preparation containing human placental extract with anti-inflammatory, antioxidant, anti-aging, and immunomodulatory effects is provided, as follows: 1. Formulation (mass / volume percentage) Using sterile water for injection as the solvent, the components are as follows: The active mixture extracted from human placenta at low temperature contains 1.0%–1.5%, phosphate buffer 0.25%–0.35%, glycerol 1.0%–2.0%, sodium chloride 0.30%–0.55%, trehalose 3.0%–5.0%, and EDTA-2Na 0.02%. The formulation has a physiological pH of 7.2±0.1 and an osmotic pressure of 280~320mOsm / kg, matching the physiological environment for human dermal injection.

[0008] 2. Preferred formulation (per 100 mL injection solution) Human placenta low-temperature extraction active mixture 1.2g, sodium dihydrogen phosphate 0.23g, disodium hydrogen phosphate 0.08g, glycerol 1.5g, sodium chloride 0.48g, trehalose 4.0g, EDTA-2Na 0.02g, diluted to 100mL with sterile water for injection.

[0009] 3. Preparation process of low-temperature extraction active mixture of human placenta The raw material is freshly collected, full-term human placenta that has tested negative for all pathogens. The extraction and separation temperature is strictly controlled between 0 and 4°C throughout the process. Process steps: Pretreatment and impurity removal: Remove umbilical cord, fascia, and blood clots; rinse repeatedly with sterile water for injection at 0-4℃ until no blood contamination remains; Low-temperature virus inactivation: Placental tissue was immersed in a low-temperature citrate buffer solution with a pH of 3.5 to 4.0 and then incubated in a 60°C water bath for 10 hours to inactivate the virus. After inactivation, the temperature was rapidly reduced to 0 to 4°C to block the continuous damage of high temperature to active substances. Low-temperature homogenization: 0-4℃ ice-water bath environment, 1.5 times the volume of pre-cooled water for injection for low-temperature homogenization (10000rpm, 3×30s). Low-temperature static extraction at 0-4℃: The homogenate is sealed and protected from light, and statically extracted at 0-4℃ for 8-12 hours. It relies on the cell osmotic pressure to gently release intracellular native growth factors and complete immune peptides. The whole process is free of strong acids and high temperatures, and does not destroy the natural conformation of active proteins. Low-temperature solid-liquid separation: Centrifuge at 0-4℃ and 3000g for 20 minutes, collect the supernatant crude extract, and discard cell precipitate and lipid impurities; Multi-stage low-temperature ultrafiltration purification: The sample is sequentially filtered through 10kDa, 3kDa, and 0.5kDa ultrafiltration membranes at a low temperature of 0–4℃, precisely retaining native active components of 500–3000 Da; the retentate removes sensitizing proteins with macromolecular sizes above 3000 Da, leaving less than 1% of macromolecular sizes above 3000 Da remaining. Molecular weight distribution: 25% of the products are in the range of 500-1000 Da and 60% are in the range of 1000-3000 Da, which is consistent with the molecular weight range of the original hydrolysate, but all of them are natural placental polypeptides, not artificially hydrolyzed fragments. Low-temperature freeze drying: The low-temperature active filtrate obtained by ultrafiltration is directly freeze-dried to obtain a powder of human placental low-temperature extract active mixture.

[0010] Active ingredient index (per 1mg of low-temperature extracted powder, native natural components) Epidermal growth factor (EGF) ≥ 50 pg, basic fibroblast growth factor (bFGF) ≥ 30 pg, insulin-like growth factor (IGF-1) ≥ 80 pg, transforming growth factor (TGF-β1) ≥ 20 pg; the process fully preserves the natural thymosin β4 fragment and interleukin-1 receptor antagonist-like native immunomodulatory peptides; due to the absence of strong acid and high temperature destruction, the activity retention rate of each growth factor is increased by more than 35% compared to the acid hydrolysis process.

[0011] 4. Complete preparation steps of the injection solution, S1 to S5 S1. Pre-dissolution of excipients: Take 80% of the total amount of sterile water for injection, dissolve trehalose and EDTA-2Na in sequence at room temperature, stir until completely clear, and prepare a stable protective excipient solution; S2, Low-temperature active compound: Dissolve phosphate buffer salt, glycerol and sodium chloride in the excipient solution in sequence; keep the system temperature ≤20℃ and avoid light throughout the process; add placental powder extracted at low temperature and stir gently for 30 minutes to avoid denaturation of native active peptides caused by high temperature and light. S3. Adjust pH and make up volume: Add the remaining sterile water for injection to make up the volume, and finely adjust the pH to 7.20±0.05 with 0.1M dilute hydrochloric acid / 0.1M sodium hydroxide; S4. Sterilization Filtration and Dispensing: The drug solution is sterilized by terminal filtration through a 0.22μm PES filter membrane, dispensed into a neutral pre-filled syringe / ampoule in a Class A clean laminar flow environment, and sealed with nitrogen to isolate oxygen. S5. Finished product storage: Store at 2-8℃ in a sealed container away from light. Nitrogen lockout significantly delays the oxidative inactivation of native peptides.

[0012] Furthermore, the functions and synergistic mechanisms of each component. Low-temperature extraction of active mixture from human placenta The entire process is carried out at 0-4℃ without strong acid or high temperature treatment, which fully preserves the original natural polypeptides and growth factors of the placenta and avoids the problems of active fragment breakage and active site damage caused by acid hydrolysis.

[0013] EGF, bFGF, IGF-1, and TGF-β1 primary growth factors specifically activate the dermal fibroblast pathway, upregulate the synthesis of type I / III collagen and elastin, and repair the aging dermal matrix; Natural thymosin β4 and IL-1 receptor antagonist-like peptides directly antagonize skin pro-inflammatory receptors, inhibit M1 type inflammatory macrophage polarization, and alleviate redness, sensitivity, and inflammatory photoaging. Multi-stage ultrafiltration removes large molecular immunogenic proteins, reducing the risk of injection redness and rejection from the source, while small molecular native peptides penetrate the dermis faster.

[0014] Phosphate buffer system + glycerol + sodium chloride osmotic pressure regulator It stabilizes the body's physiological pH and osmotic pressure, reduces injection pain and local edema, and improves clinical injection comfort.

[0015] Trehalose + EDTA-2Na composite stabilizer Trehalose forms a hydration protective layer on the surface of native peptides, locking in the natural tertiary structure and preventing denaturation of fragile native active substances obtained from low-temperature extraction; EDTA-2Na chelates trace metal ions, blocking oxidative degradation and significantly improving the activity stability of liquid formulations during storage. Beneficial effects

[0016] Original activity fully preserved Instead of using pH 2.5–3.0 and 80℃ high-temperature acid hydrolysis, we use low-temperature static extraction at 0–4℃ throughout the process. This method does not damage the natural conformation of growth factors and immune peptides, and increases the retention rate of active substances by more than 30%, resulting in stronger efficacy and longer-lasting repair.

[0017] Artificially hydrolyzed fragmented peptides All active components are complete polypeptides naturally released by placental cells, rather than short hydrolyzed peptides interrupted by strong acids. Their immune regulation and dermal repair bioactivity are superior to those of hydrolyzed products.

[0018] Security synchronization protection Pre-inactivation of viruses and low-temperature multi-stage ultrafiltration for dual impurity control result in less than 1% residual large molecular weight allergenic proteins, significantly reducing the risk of redness and allergies after injection; the formula and excipients are simplified and there are no additional irritating additives.

[0019] Improved storage stability The native peptides are combined with a trehalose metal chelate stabilization system and stored in nitrogen at low temperature and protected from light. The activity decay rate after 6 months is much lower than that of traditional acid-hydrolyzed placental injection.

[0020] The process is mild and controllable. Low-temperature extraction conditions are easy to standardize, resulting in smaller fluctuations in the content of native growth factors and peptides between batches and stronger consistency in product quality. Attached Figure Description

[0021] Figure 1 This is a flowchart of the preparation method of the present invention. Detailed Implementation

[0022] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Example

[0023] refer to Figure 1 The specific steps for preparing an injectable formulation containing human placental extract for anti-inflammatory, anti-aging, and immunomodulatory effects are as follows: Step 1: Preparation of active powder from low-temperature extraction of human placenta Raw materials: Placenta from full-term vaginal deliveries, all tested negative for hepatitis B, hepatitis C, HIV, syphilis, and cytomegalovirus, immediately transported via 0-4℃ cold chain; umbilical cord and fascia removed, repeatedly rinsed with sterile water for injection at 0-4℃ until no blood remains; Virus inactivation: Placental tissue was immersed in pH 3.8 citrate buffer and incubated in a 60°C water bath for 10 hours for inactivation; immediately after inactivation, the tissue was rapidly cooled to 2°C in an ice-water bath. Low-temperature homogenization: In a 2°C ice-water bath, add 1.5 times the volume of pre-cooled water for injection at 2°C, and homogenize 3 times at 10000 rpm for 30 seconds each time; Low-temperature extraction at 0-4℃: The homogenate is sealed and protected from light, and extracted by standing at a constant temperature of 2℃ for 10 hours to gently release the intracellular native active substances. Low-temperature centrifugation: Centrifuge at 2℃ and 3000g for 20 min, and collect the clear crude extract in the upper layer; Multi-stage low-temperature ultrafiltration: The sample is sequentially passed through 10kDa, 3kDa, and 0.5kDa ultrafiltration membranes at a low temperature of 2℃, and the 500-3000Da cutoff fraction is collected. Molecular weight distribution analysis shows that 500-1000Da accounts for 25.3%, 1000-3000Da accounts for 60.1%, and macromolecules above 3000Da account for only 0.8%. Freeze-drying: The filtrate from low-temperature ultrafiltration was freeze-dried at -50℃ to obtain a white placental low-temperature extract powder; Activity assay (per 1 mg powder): EGF 65 pg, bFGF 41 pg, IGF-1 98 pg, TGF-β 1 28 pg; mass spectrometry detected intact natural thymosin β4 and IL-1 receptor antagonist-like peptides, with no hydrolyzed fragments.

[0024] Step 2: Preparation of the injection formulation (100mL system) Weigh the raw materials: 1.2g of placental powder extracted at low temperature, 0.23g of sodium dihydrogen phosphate, 0.08g of disodium hydrogen phosphate, 1.5g of glycerol, 0.48g of sodium chloride, 4.0g of trehalose, 0.02g of EDTA-2Na, and bring the volume to 100mL with sterile water for injection.

[0025] Take 80 mL of sterile water for injection cooled to room temperature, add trehalose and EDTA-2Na, and stir at room temperature until completely clear; Add phosphate, glycerol, and sodium chloride sequentially and stir to dissolve; cool the system to 18°C ​​and protect from light, add the low-temperature extraction powder, and gently stir at 150 rpm for 30 minutes until completely dispersed; Add water for injection to 100 mL, and adjust the pH to 7.19 with dilute hydrochloric acid. 0.22μm PES filter membrane positive pressure sterilization filtration, Class A laminar flow dispensing 1mL neutral pre-filled syringe, filled with high-purity nitrogen and sealed; The finished product should be refrigerated at 2-8℃ away from light. Batch number: PEP-C01.

[0026] It should be noted that the formulation of this invention is only a research candidate for regenerative medicine drug development and is strictly prohibited from use in the field of medical aesthetics. Using human placental extract injections in medical aesthetics is prohibited under Chinese law, and this invention is not intended to develop medical aesthetic injection products. Although the process of this invention improves safety through virus inactivation and ultrafiltration to remove highly immunogenic components, the anti-aging efficacy of placental extract has not been fully clinically verified, and potential risks such as allergies, infections, and even shock still exist when used on humans. This application complies with the provisions of the "Drug Administration Law of the People's Republic of China" and Article 5, Paragraph 1 of the Patent Law. The invention itself is not intended to engage in illegal activities, will not harm the public interest, and is a patentable technical solution.

[0027] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An injectable preparation containing human placental extract, characterized by its anti-inflammatory, antioxidant, anti-aging, and immunomodulatory properties. Using sterile water for injection as a solvent, the formulation contains the following components by weight and volume percentage: 1.0%–1.5% human placental low-temperature extract active mixture, 0.25%–0.35% phosphate buffer, 1.0%–2.0% glycerol, 0.30%–0.55% sodium chloride, 3.0%–5.0% trehalose, and 0.02% EDTA-2Na; the formulation has a pH of 7.2±0.1 and an osmotic pressure of 280–320 mOsm / kg.

2. The injectable preparation containing human placental extract with anti-inflammatory, antioxidant, anti-aging, and immunomodulatory effects according to claim 1, characterized in that, Each 100mL injection solution consists of the following components: 1.2g of human placental low-temperature extract active mixture, 0.23g of sodium dihydrogen phosphate, 0.08g of disodium hydrogen phosphate, 1.5g of glycerol, 0.48g of sodium chloride, 4.0g of trehalose, 0.02g of EDTA-2Na, and sterile water for injection to a final volume of 100mL.

3. The injectable preparation containing human placental extract with anti-inflammatory, antioxidant, anti-aging, and immunomodulatory effects according to claim 1, characterized in that, The method for preparing the human placenta low-temperature extraction active mixture is as follows: After health screening qualified human placenta is cleaned, virus inactivated, and rapidly cooled, it is homogenized at low temperature of 0-4℃ throughout the process and statically extracted at low temperature in the dark. After multi-stage low-temperature ultrafiltration to retain the original active components of 500-3000Da, it is freeze-dried at low temperature. The molecular weight distribution is: 500-1000Da accounts for 25%, 1000-3000Da accounts for 60%, and macromolecules above 3000Da remain <1%.

4. The injectable preparation containing human placental extract with anti-inflammatory, antioxidant, anti-aging, and immunomodulatory effects according to claim 3, characterized in that, In each milligram of the low-temperature extracted active mixture, the natural native activity indicators stably meet the following requirements: EGF≥50pg, bFGF≥30pg, IGF-1≥80pg, TGF-β1≥20pg; the natural complete immune peptides include thymosin β4 fragments and interleukin-1 receptor antagonist-like peptides, and there are no peptide fragments that are broken by strong acid hydrolysis.

5. The injectable preparation containing human placental extract with anti-inflammatory, antioxidant, anti-aging, and immunomodulatory effects according to claim 1, characterized in that, The preparation steps include: S1. Pre-dissolution of excipients: Take 80% of the total amount of sterile water for injection, dissolve trehalose and EDTA-2Na in sequence at room temperature, stir until completely clear, and prepare a stable protective excipient solution; S2, Low-temperature active compound: Dissolve phosphate buffer salt, glycerol and sodium chloride in the excipient solution in sequence; keep the system temperature ≤20℃ and avoid light throughout the process; add placental powder extracted at low temperature and stir gently for 30 minutes to avoid denaturation of native active peptides caused by high temperature and light. S3. Adjust pH and make up volume: Add the remaining sterile water for injection to make up the volume, and finely adjust the pH to 7.20±0.05 with 0.1M dilute hydrochloric acid / 0.1M sodium hydroxide; S4. Sterilization Filtration and Dispensing: The drug solution is sterilized by terminal filtration through a 0.22μm PES filter membrane, dispensed into a neutral pre-filled syringe / ampoule in a Class A clean laminar flow environment, and sealed with nitrogen to isolate oxygen. S5. Finished product storage: Store at 2-8℃ in a sealed container away from light. Nitrogen lockout significantly delays the oxidative inactivation of native peptides.

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