Active super-antigen compound biological injection preparation NF for treating male and female dysfunctions and preparation method of active super-antigen compound biological injection preparation NF

Abstract

The invention relates to an active super antigen compound biological injection preparation NF for treating male and female dysfunctions and a preparation method thereof, and the compound biological injection preparation NF comprises a general basic formula NF, male NF-M and female NF-F, the general basic formula NF comprises the following components and parameters: 0.1 to 0.5 mg / mL of super antigen mutant, 50 to 100 [mu] g / mL of neurotrophic factor, 10 to 20 [mu] g / mL of hormone regulating peptide and 8 to 10 mg / mL of pH-responsive lipid nanoparticles. According to the active super antigen compound biological injection preparation NF for treating male and female dysfunctions and the preparation method, by activating TCR-V beta chains on the surfaces of T cells and MHC-II molecules of APC, local immune regulation instead of systemic inflammatory reaction is induced, the microcirculation and neuroendocrine functions of a reproductive system are improved in a targeted manner, and the effect of treating male and female dysfunctions is achieved. Superantigen is adopted to activate local immune cells to secrete vascular endothelial growth factors and NO, blood perfusion is improved, neurotrophic factors repair cavernous nerve injury, hormone regulating peptides indirectly regulate the sex hormone level through hypothalamus-pituitary-gonad axis, and the composition is suitable for hormone-related dysfunction.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and specifically to a compound biological injection preparation NF for treating male and female sexual dysfunctions with active superantigen and a preparation method thereof. Background Art

[0002] Sexual function is a complex physiological process. The maintenance of normal sexual function depends on the cooperation of multiple systems in the human body, involving the coordination of the nervous system, cardiovascular system, endocrine system and reproductive system. In addition, a good mental state and healthy psychology are also required. When abnormal changes occur in the above systems or in terms of mental psychology, it will affect the normal progress of sexual life, affect the quality of sexual life, and show sexual dysfunction. Male sexual dysfunctions mainly include sexual desire disorders, penile erection disorders and ejaculation disorders, etc. The incidence of female sexual dysfunctions is also very high, among which sexual desire and orgasm disorders are the most common.

[0003] However, existing compound biological injection preparations for treating male and female sexual dysfunctions, such as compound glycyrrhizin injection, injection of hydrochloric acid alkali, alprostadil injection and other drugs, although they can improve male and female sexual dysfunctions to a certain extent, their effects are often limited, and there are significant individual differences; different treatment regimens need to be adopted for patients of different genders, and the differences in treatment regimens are large. Therefore, a compound biological injection preparation NF for treating male and female sexual dysfunctions with active superantigen and a preparation method thereof are proposed. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a compound biological injection preparation NF for treating male and female sexual dysfunctions with active superantigen and a preparation method thereof, which have the advantages of superantigen immune regulation and multi-target synergistic effects, and solve the problems that existing compound biological injection preparations for treating male and female sexual dysfunctions, such as compound glycyrrhizin injection, injection of hydrochloric acid alkali, alprostadil injection and other drugs, although they can improve male and female sexual dysfunctions to a certain extent, their effects are often limited, and there are significant individual differences; different treatment regimens need to be adopted for patients of different genders, and the differences in treatment regimens are large.

[0005] To achieve the above object, the present invention provides the following technical solution: A compound biological injection preparation NF for treating male and female sexual dysfunctions with active superantigen, comprising a general basic formula NF, NF-M for male and NF-F for female. The general basic formula NF contains the following components and their parameters:

[0006] Superantigen mutant 0.1 - 0.5 mg / mL, neurotrophic factor 50 - 100 μg / mL, hormone regulatory peptide 10 - 20 μg / mL, and pH-responsive lipid nanoparticles 8 - 10 mg / mL;

[0007] The male NF-M contains the following components and their parameters:

[0008] The PDE5 inhibitor prodrug is 5 - 10 mg / mL, the L-arginine complex is 40 - 50 mM, and the glutathione antioxidant is 2 - 5 mM;

[0009] The female NF-F contains the following components and their parameters:

[0010] The TGF-β3 activating peptide is 20 - 40 μg / mL, the hyaluronic acid cross-linked gel is 1.0 - 1.5%, and the DHEA microspheres are 0.1 - 0.3 mg / mL.

[0011] Furthermore, the pH-responsive lipid nanoparticles contain the following components and their molar ratios:

[0012] The ionizable lipid is 45 - 50%, phosphatidylcholine is 8 - 10%, cholesterol is 28.3 - 38.5%, and PEG-DMG is 1.5 - 2.0%.

[0014] Furthermore, it also includes excipient sustained-release microspheres. The particle size of the sustained-release microspheres is 10 - 20 μm, the encapsulation efficiency is ≥85, the molecular weight is 24 kDa, and the degradation period is 3 - 7 days.

[0015] Furthermore, the excipient also includes 0.2 - 0.3% phosphate buffer, 0.01 - 0.15% polysorbate, and 3 - 4% mannitol. The pH value of the phosphate buffer is 7.4 ± 0.2.

[0016] Furthermore, the pH-responsive lipid nanoparticles are prepared by the microfluidic mixing method, with a flow rate ratio of 1:3, a total flow rate of 12 mL / min, and ultrafiltration and concentration to the target concentration.

[0017] Furthermore, the sustained-release microspheres are prepared by the W / O / W double emulsion method, with a homogenization speed of 10,000 rpm, stabilized by 5% PVA, and sieved to 20 μm after lyophilization.

[0018] Furthermore, the hormone regulatory peptide is Kisspeptin-10, and the superantigen mutant is a low-toxic superantigen mutant modified by genetic engineering, the modified staphylococcal enterotoxin SEA / D227A, which activates the TCR-Vβ chain on the surface of T cells and the MHC-II molecule of APC.

[0019] Furthermore, the injection method of the active superantigen for treating male and female sexual dysfunction compound biological injection preparation NF: For males, it is injected into the corpus cavernosum of the penis, and for females, it is injected subcutaneously at multiple points under the vaginal mucosa.

[0020] The preparation method of the active superantigen for treating male and female sexual dysfunction compound biological injection preparation NF is as follows:

[0021] Step 1. Superantigen production

[0022] Gene construction:

[0023] Synthesize the SEA mutant gene sequence, clone it into the pET-28a(+) vector, and transfer it into BL21(DE3) Escherichia coli;

[0024] Fermentation and purification:

[0025] Adopt high-density fermentation, fed-batch culture, OD600 = 60, induce expression with IPTG, and perform purification through Ni-NTA affinity chromatography → molecular sieve chromatography Superdex75 → endotoxin removal column EndoTrapHD, with a purity > 99% and endotoxin < 0.1 EU / mg;

[0026] Step 2. Preparation of compound preparation

[0027] Nano-carrier construction:

[0028] Encapsulate the superantigen in lipid nanoparticles by microfluidic technology, control the particle size at 80 - 100 nm, and PDI < 0.2;

[0029] Load neurotrophic factors on PLGA microspheres:

[0030] Adopt the W / O / W double emulsion method, with PLGA50:50, MW24 kDa, dissolved in DCM, encapsulate neurotrophic factors, with a concentration of 2 mg / mL, and the particle size after freeze-drying is 10 - 20 μm, and the encapsulation efficiency > 85%;

[0031] Final preparation configuration:

[0032] Mix the superantigen, PLGA microspheres loaded with neurotrophic factors, and Kisspeptin-10 lyophilized powder in a mass ratio of 5:3:2, redissolve in phosphate buffer pH7.4 containing 0.01% polysorbate 80, and perform sterile filtration through 0.22 μm to obtain the injection preparation NF.

[0033] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0034] 1. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen and its preparation method adopt a low-toxic superantigen mutant modified by genetic engineering. By activating the TCR-Vβ chain on the surface of T cells and the MHC-II molecule of APC, it induces local immune regulation rather than a systemic inflammatory response, targets and improves the microcirculation of the reproductive system and neuroendocrine function. The superantigen activates local immune cells to secrete vascular endothelial growth factor and NO, improves blood perfusion, and neurotrophic factors repair cavernous nerve damage, enhances nerve signal conduction. Hormone regulatory peptides indirectly regulate sex hormone levels through the hypothalamus-pituitary-gonadal axis, and are applicable to hormone-related sexual dysfunction.

[0035] 2. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen and its preparation method use pH-responsive lipid nanoparticles to encapsulate the superantigen, and through surface modification, target the surface markers of vascular endothelial cells in the reproductive system to achieve precise release and reduce systemic toxicity. The neurotrophic factors are loaded by microsphere embedding technology, forming a time-gradient synergy with the rapid onset of the superantigen, and extending the efficacy to more than 72 hours. The male preparation focuses on vasodilation and adds a PDE5 inhibitor prodrug, while the female preparation focuses on pelvic floor muscle repair and integrates a TGF-β3 collagen synthesis activating peptide. Through multi-pathway intervention of immunity-nerve-blood vessels, it has greater potential for psychological and organic mixed sexual dysfunction. Description of the Drawings

[0036] Figure 1 It is a flow chart of the preparation method of the compound biological injection preparation NF for treating male and female sexual dysfunction with the active superantigen of the present invention. Detailed Embodiments

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0038] Example 1

[0039] Please refer to Figure 1 , the compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen in this embodiment includes a general basic formula NF, NF-M for men, and NF-F for women. The general basic formula NF contains the following components and their parameters:

[0040] Superantigen mutant 0.1 mg / mL, neurotrophic factor 50 μg / mL, hormone regulatory peptide 10 μg / mL, and pH-responsive lipid nanoparticles 8 mg / mL;

[0041] NF-M for men contains the following components and their parameters:

[0042] 5 mg / mL of PDE5 inhibitor prodrug, 40 mM of L-arginine complex, and 2 mM of glutathione antioxidant;

[0043] NF-F for women contains the following components and their parameters:

[0044] 20 μg / mL of TGF-β3 activating peptide, 1.0% of hyaluronic acid cross-linked gel, and 0.1 mg / mL of DHEA microspheres.

[0045] In this example, the pH-responsive lipid nanoparticles contain the following components and their molar ratios:

[0046] 45% of ionizable lipid, 8% of phosphatidylcholine, 28.3% of cholesterol, and 1.5% of PEG-DMG. The pH-responsive lipid nanoparticles are prepared by the microfluidic mixing method with a flow rate ratio of 1:3 and a total flow rate of 12 mL / min, and ultrafiltered and concentrated to the target concentration.

[0047] In this example, the excipient sustained-release microspheres have a microsphere particle size of 10 μm, an encapsulation efficiency of ≥85%, a molecular weight of 24 kDa, and a degradation period of 3 - 7 days. The sustained-release microspheres are prepared by the W / O / W double emulsion method with a homogenization speed of 10,000 rpm, stabilized with 5% PVA, and sieved to 20 μm after lyophilization.

[0048] In this example, the excipient also includes 0.2% of phosphate buffer solution, 0.01% of polysorbate, and 3% of mannitol, and the pH value of the phosphate buffer solution is 7.2.

[0049] The preparation method of the active superantigen for treating male and female sexual dysfunction compound biological injection preparation NF is as follows:

[0050] Step 1. Superantigen production

[0051] Gene construction:

[0052] Synthesize the SEA mutant gene sequence, clone it into the pET-28a(+) vector, and transfer it into BL21(DE3) Escherichia coli;

[0053] Fermentation and purification:

[0054] Adopt high-density fermentation, fed-batch culture, OD600 = 60, IPTG induction expression, through Ni-NTA affinity chromatography → molecular sieve chromatography Superdex75 → endotoxin removal column EndoTrapHD, with a purity > 99% and endotoxin < 0.1 EU / mg;

[0055] Step 2. Compound preparation

[0056] Construction of nano - carriers:

[0057] Lipid nanoparticles encapsulate superantigen through microfluidic technology, with the particle size controlled at 80 nm and PDI < 0.2;

[0058] PLGA microspheres loaded with neurotrophic factors:

[0059] Using the W / O / W double - emulsion method, PLGA 50:50, MW 24 kDa, dissolved in DCM, encapsulating neurotrophic factors, with a concentration of 2 mg / mL, and the particle size is 10 - 20 μm after lyophilization, and the encapsulation efficiency > 85%;

[0060] Configuration of the final preparation:

[0061] Mix the superantigen, PLGA microspheres loaded with neurotrophic factors, and Kisspeptin - 10 lyophilized powder in a mass ratio of 5:3:2, and redissolve in phosphate - buffered saline pH 7.4 containing 0.01% polysorbate 80, and sterile - filter through 0.22 μm to obtain the injection preparation NF.

[0062] Example 2

[0063] Please refer to Figure 1 , the compound biological injection preparation NF for treating male and female sexual dysfunctions with active superantigen in this example includes the general basic formula NF, NF - M for men, and NF - F for women. The general basic formula NF contains the following components and their parameters:

[0064] Superantigen mutant 0.5 mg / mL, neurotrophic factor 100 μg / mL, hormone - regulating peptide 20 μg / mL, and pH - responsive lipid nanoparticles 10 mg / mL;

[0065] NF - M for men contains the following components and their parameters:

[0066] PDE5 inhibitor prodrug 10 mg / mL, L - arginine complex 50 mM, and glutathione antioxidant 5 mM;

[0067] NF - F for women contains the following components and their parameters:

[0068] TGF - β3 activation peptide 40 μg / mL, hyaluronic acid cross - linked gel 1.5%, and DHEA microspheres 0.3 mg / mL.

[0069] In this example, the pH - responsive lipid nanoparticles contain the following components and their molar ratios:

[0070] Ionizable lipid 50%, phosphatidylcholine 10%, cholesterol 38.5%, and PEG-DMG 2.0%. The pH-responsive lipid nanoparticles were prepared by the microfluidic mixing method with a flow rate ratio of 1:3 and a total flow rate of 12 mL / min, and concentrated by ultrafiltration to the target concentration.

[0071] In this example, the excipient sustained-release microspheres have a microsphere diameter of 20 μm, an encapsulation efficiency of ≥85%, a molecular weight of 24 kDa, and a degradation period of 3 - 7 days. The sustained-release microspheres were prepared by the W / O / W double emulsion method with a homogenization speed of 10,000 rpm, stabilized with 5% PVA, and sieved to 20 μm after lyophilization.

[0072] In this example, the excipients also include 0.3% phosphate buffer, 0.15% polysorbate, and 4% mannitol, and the pH value of the phosphate buffer is 7.6.

[0073] Preparation method of the compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen: The preparation method is as follows:

[0074] Step 1: Superantigen production

[0075] Gene construction:

[0076] Synthesize the SEA mutant gene sequence, clone it into the pET-28a(+) vector, and transfer it into BL21(DE3) Escherichia coli.

[0077] Fermentation and purification:

[0078] High-density fermentation was used, fed-batch culture was carried out, OD600 = 60, IPTG was used for induction expression, and through Ni-NTA affinity chromatography → molecular sieve chromatography Superdex75 → endotoxin removal column EndoTrapHD, the purity > 99%, and the endotoxin < 0.1 EU / mg.

[0079] Step 2: Compound preparation

[0080] Nanocarrier construction:

[0081] The lipid nanoparticles encapsulate the superantigen by microfluidic technology, with the particle size controlled at 100 nm and PDI < 0.2.

[0082] PLGA microspheres loaded with neurotrophic factors:

[0083] The W / O / W double emulsion method was used, with PLGA 50:50, MW 24 kDa, dissolved in DCM, encapsulated with neurotrophic factors at a concentration of 2 mg / mL, and the particle size was 10 - 20 μm after lyophilization, and the encapsulation efficiency > 85%.

[0084] Final preparation configuration:

[0085] Mix superantigen, PLGA microspheres loaded with neurotrophic factors, and Kisspeptin-10 lyophilized powder in a mass ratio of 5:3:2, redissolve them in phosphate buffer pH 7.4 containing 0.01% polysorbate 80, and sterile filter through 0.22 μm to obtain the injection preparation NF.

[0086] By adopting the above technical solution, the injection method of the active superantigen for treating male and female sexual dysfunction compound biological injection preparation NF: for males, penile cavernous injection is used, and for females, multiple-point submucosal vaginal injection is used. NF-M is used for males to enhance the targeting of the penile cavernous body, and NF-F is used for females to prolong the retention time in the vaginal mucosa.

[0087] Specifically, pH-responsive lipid nanoparticles are used to encapsulate superantigen, and surface modification is carried out to target the surface markers of vascular endothelial cells in the reproductive system, realizing precise release and reducing systemic toxicity. Neurotrophic factors are loaded through microsphere embedding technology, forming a time-gradient synergy with the rapid onset of superantigen, and prolonging the efficacy to more than 72 hours. The male preparation focuses on vasodilation and adds a PDE5 inhibitor prodrug, while the female preparation focuses on pelvic floor muscle repair and integrates a TGF-β3 collagen synthesis activating peptide. Through multi-pathway intervention of immunity-nerves-blood vessels, it has greater potential for psychogenic and organic mixed sexual dysfunction.

[0088] In summary, the present invention uses a low-toxic superantigen mutant modified by genetic engineering. By activating the TCR-Vβ chain on the surface of T cells and the MHC-II molecule of APC, it induces local immune regulation rather than a systemic inflammatory response, targets and improves the microcirculation and neuroendocrine function of the reproductive system. Superantigen is used to activate local immune cells to secrete vascular endothelial growth factor and NO, improve blood perfusion, neurotrophic factors repair cavernous nerve damage, enhance nerve signal conduction, and hormone regulatory peptides indirectly regulate sex hormone levels through the hypothalamus-pituitary-gonadal axis, being applicable to hormone-related sexual dysfunction. It solves the problems of existing compound biological injection preparations for treating male and female sexual dysfunction, such as compound glycyrrhizin injection, injection of hydrochloric acid alkali, alprostadil injection, etc. Although they can improve male and female sexual dysfunction to a certain extent, their effects are often limited and there are significant individual differences; different treatment regimens need to be adopted for patients of different genders, and the differences in treatment regimens are large.

[0089] It should be noted that, in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variation thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device that includes a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device that includes the element.

[0090] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen includes a general basic formula NF, NF-M for male use, and NF-F for female use, and is characterized in that: The general basic formula NF contains the following components and their parameters: Superantigen mutant 0.1 - 0.5 mg / mL, neurotrophic factor 50 - 100 μg / mL, hormone regulatory peptide 10 - 20 μg / mL, and pH-responsive lipid nanoparticles 8 - 10 mg / mL; The NF-M for men contains the following components and their parameters: PDE5 inhibitor prodrug 5 - 10 mg / mL, L-arginine complex 40 - 50 mM, and glutathione antioxidant 2 - 5 mM; The NF-F for women contains the following components and their parameters: TGF-β3 activation peptide 20 - 40 μg / mL, hyaluronic acid cross-linked gel 1.0 - 1.5%, and DHEA microspheres 0.1 - 0.3 mg / mL.

2. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen as claimed in claim 1, wherein: The pH-responsive lipid nanoparticles contain the following components and their molar ratios: Ionizable lipid 45 - 50%, phosphatidylcholine 8 - 10%, cholesterol 28.3 - 38.5%, and PEG-DMG 1.5 - 2.0%.

3. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen as claimed in claim 1, wherein: It also includes excipient sustained-release microspheres. The particle size of the sustained-release microspheres is 10 - 20 μm, the encapsulation efficiency is ≥85, the molecular weight is 24 kDa, and the degradation period is 3 - 7 days.

4. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen according to claim 3, wherein: The excipient also includes phosphate buffer 0.2 - 0.3%, polysorbate 0.01 - 0.15%, and mannitol 3 - 4%. The pH value of the phosphate buffer is 7.4 ± 0.

2.

5. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen as claimed in claim 2, wherein: The pH-responsive lipid nanoparticles are prepared by the microfluidic mixing method, with a flow rate ratio of 1:3 and a total flow rate of 12 mL / min, and ultrafiltration and concentration to the target concentration.

6. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen according to claim 3, characterized in that: The sustained-release microspheres are prepared by the W / O / W double emulsion method, with a homogenization speed of 10,000 rpm, stabilized by 5% PVA, and sieved to 20 μm after freeze-drying.

7. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen as claimed in claim 1, wherein: The hormone regulatory peptide is Kisspeptin-10. The superantigen mutant is a low-toxic superantigen mutant modified by genetic engineering, the modified staphylococcal enterotoxin SEA / D227A, which activates the TCR-Vβ chain on the surface of T cells and the MHC-II molecule of APC.

8. The compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen according to claim 1, wherein: The injection method of the active superantigen for treating male and female sexual dysfunction compound biological injection preparation NF: For men, it is injected into the corpus cavernosum of the penis, and for women, it is injected subcutaneously at multiple points under the vaginal mucosa.

9. Preparation method of compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen, including the preparation method of compound biological injection preparation NF for treating male and female sexual dysfunction with active superantigen described in any one of claims 1-8, characterized in that: The preparation method is specifically as follows: Step 1. Superantigen production Gene construction: Synthesize the SEA mutant gene sequence, clone it into the pET-28a(+) vector, and transfer it into BL21(DE3) Escherichia coli; Fermentation and purification: Adopt high-density fermentation, fed-batch culture, OD600 = 60, IPTG induction expression, through Ni-NTA affinity chromatography → molecular sieve chromatography Superdex75 → endotoxin removal column EndoTrapHD, with a purity > 99% and endotoxin < 0.1 EU / mg; Step 2. Compound preparation Nanocarrier construction: The lipid nanoparticles encapsulate the superantigen by microfluidic technology, with the particle size controlled at 80 - 100 nm and PDI < 0.2; PLGA microspheres loaded with neurotrophic factor: Using the W / O / W double emulsion method, PLGA 50:50, MW 24 kDa, dissolved in DCM, encapsulating neurotrophic factors, with a concentration of 2 mg / mL, the particle size after lyophilization is 10 - 20 μm, and the encapsulation efficiency > 85%; Final preparation configuration: Mix the superantigen, PLGA microspheres loaded with neurotrophic factors, and Kisspeptin-10 lyophilized powder in a mass ratio of 5:3:2, redissolve in phosphate buffer pH 7.4 containing 0.01% polysorbate 80, and sterile filter through 0.22 μm to obtain the injection preparation NF.

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