A pharmaceutical composition for preventing and treating sow twinning syndrome and application thereof

Abstract

This invention discloses a pharmaceutical composition for preventing and treating sows with second-parity syndrome and its application. The pharmaceutical composition, which contains long-acting FSH fusion protein and recombinant hCG, is used to improve the estrus rate, mating frequency, conception rate, farrowing rate, utilization rate, total number of piglets and total number of healthy piglets in sows with second-parity syndrome. It can also eliminate follicular cysts in sows and reduce losses in the pig farming industry.

Description

Technical Field

[0001] This invention relates to the fields of veterinary drugs and bioengineering technology, specifically to a pharmaceutical composition for the prevention and treatment of sow second-parity syndrome and its application. Background Technology

[0002] In recent years, with the rapid development of large-scale and intensive pig farming in my country, the scale and quality of breeding pigs have become particularly important. However, excessive breeding of breeding pigs and mismatched feeding and management techniques have led to the occurrence of sow second-parity syndrome, the symptoms of which are quite complex (Guo Yaxin et al., Research progress on sow second-parity syndrome and prevention and control technology. China Animal Husbandry Journal, 2021 Pig Industry Science and Technology Special Issue, 7-10; Li Jiren, Hu Xialing. Systemic health care to reduce sow second-parity syndrome [J]. Pig Science, 2014, 31(9):96-98), and often there are no obvious pathological phenomena. It is mainly manifested as the phenomenon of "two highs and one low", namely, high culling rate of second-parity sows, high rate of non-estrus after weaning, and low number of piglets born in second-parity sows. Usually, the optimal parity age of sows is 5 parities, but sows with second-parity syndrome are often culled before reaching the optimal service life due to low estrus rate and unsatisfactory number of piglets born. The breeding value of sows is not fully utilized, which is not conducive to the sustainable development of large-scale pig farms. According to research, 73.4% of sows experiencing reproductive disorders in large-scale pig farms are in their first or second parity, with primiparous sows accounting for about 65% of these cases (Saito H, Sasaki Y, Hoshino Y, et al. The occurrence of decreased numbers of pigs bornalive in parity 2 sows does not negatively affect herd productivity in Japan[J].Livest Sci, 2010,128(1):189-192.). These sows exhibit symptoms such as weight loss after weaning and prolonged intervals between weaning and estrus. Second-parity syndrome severely reduces sow reproductive performance, causing significant economic losses to pig farming enterprises.

[0003] The causes of sow second-parity syndrome are complex, including feeding management, disease, and environment. For example, primiparous sows are not fully developed, and their reproductive organs and birth canals have not been subjected to compression and stimulation. Therefore, primiparous sows are very prone to congenital pelvic and birth canal stenosis, which is one of the possible causes of sow second-parity syndrome (Guo Yaxin et al., Research progress on sow second-parity syndrome and prevention and control technology. China Animal Husbandry Journal, 2021 Pig Industry Science and Technology Special Issue, 7-10). In addition, because primiparous sows often have insufficient feed intake, the embryo mortality rate increases (Hoving LL, Soede NM, Feitsma H, et al. Lactation weight loss in primiparous sows: consequences for embryosurvival and progesterone and relations with metabolic profiles[J]. ReprodDomest Anim,2012,47(6):1009-1016.), which can also lead to excessive weight loss at weaning and delayed estrus. This is also an important reason for the reduced litter size in sows with second-parity syndrome. Reports also indicate that the farrowing season and the scale of pig farms are risk factors for second-litter syndrome, and autumn is the season with a high number of culled sows due to second-litter syndrome. High temperatures in the dry season and high humidity in the rainy season also increase the incidence of second-litter syndrome in sows (Segura Correa JC, Alzina-López A, Santos-Ricalde R H. Risk factors associated with the occurrence of the second-litter syndrome in sows in southeastern mexico[J].Sci World J,2013,2013: 969620-969624.).

[0004] The prevention and treatment of second-parity syndrome in sows differs significantly from the conventional approach used in assisted reproduction in first-parity livestock, which focuses on promoting follicle development, oocyte maturation, and ovulation. Unlike sows in a normal, healthy physiological state, those with second-parity syndrome are in a more complex pathological condition. Therefore, applying conventional hormonal drugs used to assist reproduction in first-parity livestock to sows in this pathological state is uncertain in its effectiveness and prohibitively costly. Consequently, faced with such risks, livestock farms often cull affected sows. From a preventative perspective, the industry currently focuses on selecting suitable replacement gilts, appropriately delaying gilt mating, and strengthening the feeding and management of both replacement and first-parity sows. Emphasis is placed on breed selection, domestication, and the feeding and management of both types of sows to reduce the incidence of second-parity syndrome and extend the sow's productive lifespan. However, while scientific feeding and management can reduce the incidence of second-parity syndrome, it cannot completely eliminate the problem. In particular, the number of replacement gilts in farms is currently quite large, and primiparous sows account for a high proportion of the entire sow herd. For sows that have been significantly or potentially affected by second-parity syndrome, excessively high selection and culling standards will lead to their breeding value not being effectively realized, forcing the culling of breeding pigs and further increasing the economic losses of related pig farming enterprises.

[0005] Therefore, in order to effectively prevent and control sow second-parity syndrome, and at the same time minimize the resulting high culling rate and economic losses, it is urgent to develop an intervention method that can be directly applied to sows with a high incidence of second-parity syndrome or those already affected by it, in order to solve the problems of high anestrus rate after weaning and low litter size in second-parity sows. Summary of the Invention

[0006] In order to effectively prevent and control sow second-parity syndrome, reduce losses in the pig farming industry, and improve the application value of replacement gilts, this invention provides the following technical solution.

[0007] In a first aspect, the present invention provides a composition for preventing and treating sow second-parity syndrome, the composition comprising FSH (Follicle-Stimulating Hormone) drugs and CG (Chorionic Gonadotropin) drugs.

[0008] Preferably, the FSH-type drug is a long-acting FSH fusion protein, and the CG-type drug is human chorionic gonadotropin.

[0009] Preferably, the long-acting FSH fusion protein comprises two peptide chains conforming to the following equation: (pFSHα:pFSHβ-L-Fc)2, where pFSHβ refers to the β subunit of porcine FSH without the signal peptide; the colon represents the relationship between the β and α subunits of porcine FSH connected by van der Waals forces; pFSHα refers to the α subunit of porcine FSH without the signal peptide; L represents the connection relationship between the pFSHα or pFSHβ subunit and the Fc fragment; Fc refers to the Fc fragment of immunoglobulin or its mutant; the subscript 2 outside the parentheses indicates that the porcine FSH fusion protein is a divalent homodimer.

[0010] Preferably, the amino acid sequence of pFSHα is the sequence shown in SEQ ID NO.1 or a sequence that has more than 90% homology with the sequence shown in SEQ ID NO.1.

[0011] Specifically, the sequence shown in SEQ ID NO.1 is as follows: FPDGEFTMQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGNARVENHTECHCSTCYYHKS.

[0012] Preferably, the amino acid sequence of pFSHβ is the sequence shown in SEQ ID NO.2 or a sequence having more than 90% homology with the sequence shown in SEQ ID NO.2.

[0013] Furthermore, the sequence shown in SEQ ID NO.2 is as follows: CELTNITITVEKEECNFCISINTTWCAGYCYTRDLVYKDPARPNIQKTCTFKELVYETVKVPGCAHHADSLYTYPVATECHCGKCDSDSTDCTVRGLGPSYCSFSEMKE.

[0014] Furthermore, the aforementioned 90% or more homology includes at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0015] Preferably, the Fc may include an immunoglobulin hinge region as well as CH2 and CH3 regions.

[0016] Furthermore, the immunoglobulins are derived from humans, pigs, cattle, sheep, horses, or dogs. Immunoglobulins are classified into IgG, IgM, IgA, IgD, and IgE, and each immunoglobulin includes various subtypes, such as IgG1, IgG2, IgG3, and IgG4.

[0017] Preferably, the Fc is derived from porcine immunoglobulin, i.e., pFc, and includes the hinge region, CH2 and CH3 regions of porcine immunoglobulin.

[0018] Furthermore, the amino acid sequence of the pFc is as follows: ICPACESPGPSVFIFPPPKPKDTLMISRTPQVTCVVVDVSQENPEVQFSWYVDGVEVHTAQTRPKEEQFNSTYRVVSVLPIQHQDWLNGKEFKCKVNNKDLPAPITRIISKAK GQTREPQVYTLPPHAEELSRSKVSITCLVIGFYPPDIDVEWQRNGQPEPEGNYRTTPPQQDVDGTYFLYSKFSVDKASWQGGGIFQCAVMHEALHNHYTQKSISKTPGK (SEQ ID NO.3).

[0019] Preferably, the Fc mutant refers to an Fc variant containing one or more mutated amino acid sites on the Fc fragment, such as the human IgG2 Fc variant, which contains the Pro331Ser mutated human IgG2 hinge region, CH2 and CH3 region.

[0020] Preferably, the connection between the pFSHβ subunit and Fc is a direct splicing connection or a connection through a connector, preferably a connection through a connector.

[0021] Furthermore, the linker is a flexible polypeptide composed of 2-20 flexible amino acids, wherein the flexible amino acids are selected from at least one of Gly, Ser, Ala or Thr.

[0022] Furthermore, the connector is (Gly-Gly-Gly-Gly-Ser)n, where n is an integer between 2 and 5, preferably n=3.

[0023] Preferably, the amino acid sequence of pFSHβ-L-Fc is the sequence shown in SEQ ID NO.4 or a sequence having more than 90% homology with the sequence shown in SEQ ID NO.4.

[0024] Furthermore, the sequence shown in SEQ ID NO.4 is as follows: CELTNITITVEKEECNFCISINTTWCAGYCYTRDLVYKDPARPNIQKTCTFKELVYETVKVPGCAHHADSLYTYPVATECHCGKCDSDSTDCTVRGLGPSYCSFSEMKEGGGGSGGGGSGGGGSICPACESPGPSVFIFPPKPKDTLMISRTPQVTCVVVDVSQENPEVQFSW YVDGVEVHTAQTRPKEEQFNSTYRVVSVLPIQHQDWLNGKEFKCKVNNKDLPAPITRIISKAKGQTREPQVYTLPPHAEELSRSKVSITCLVIGFYPPDIDVEWQRNGQPEPEGNYRTTPPQQDVDGTYFLYSKFSVDKASWQGGGIFQCAVMHEALHNHYTQKSISKTPGK.

[0025] Preferably, the long-acting FSH fusion protein can also be modified.

[0026] Furthermore, the modification methods include, but are not limited to, glycosylation, polyethylene glycolation, acetylation, or combination with BSA.

[0027] Preferably, the human chorionic gonadotropin (hCG) includes naturally derived hCG (such as hCG extracted from the urine of pregnant women) or hCG recombinantly expressed through bioengineering technology, more preferably recombinant hCG.

[0028] Furthermore, the recombinant hCG comprises α and β subunits connected by van der Waals forces. Furthermore, the sequence of the α subunit in the recombinant hCG is as follows: MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS (SEQ ID NO. 5).

[0029] Furthermore, the sequence of the β subunit in the recombinant hCG is as follows: MEMFQGLLLLLLLSMGGTWASKEPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMTRVLQGVLPALPQVVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCALCRRSTDCGGPKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO. 6).

[0030] Preferably, in the composition, the ratio of long-acting FSH fusion protein to human chorionic gonadotropin in terms of medical potency units is 400-10000 U:100-2000 U.

[0031] Furthermore, each dose of the long-acting FSH fusion protein administered to the subject contains a medical potency unit of 400-10000 U, for example: 400 U, 800 U, 1000 U, 1800 U, 2500 U, 3000 U, 4000 U, 5000 U, 6000 U, 7000 U, 8000 U, 9000 U, 10000 U.

[0032] Furthermore, each dose of the human chorionic gonadotropin contained in the subject contains a medical potency unit of 100-2000 U, for example: 100 U, 200 U, 300 U, 500 U, 800 U, 1000 U, 1200 U, 1500 U, 1800 U, 2000 U.

[0033] Furthermore, the ratio of the long-acting FSH fusion protein to the hCG in medical potency units is 2:1 to 20:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1 or any ratio within the above range, preferably 5:1 to 10:1, more preferably 8:1 to 10:1, and most preferably 9:1.

[0034] In a second aspect, the present invention provides a pharmaceutical composition for preventing and treating sow second-parity syndrome, the pharmaceutical composition comprising the composition described in the first aspect.

[0035] Preferably, the pharmaceutical composition further comprises pharmaceutically permissible excipients.

[0036] Furthermore, the excipients include mannitol, sucrose, polysorbate 80, and phosphate.

[0037] Furthermore, the mass percentage concentration of mannitol is 0.1% to 1%, for example: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.

[0038] Furthermore, the sucrose has a mass percentage concentration of 1-3%, for example: 1%, 1.5%, 2%, 2.5%, 3%.

[0039] Furthermore, the mass percentage concentration of the polysorbate 80 is 0.001-0.05%, for example: 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%.

[0040] Furthermore, the concentration of the phosphate is 10-50 mM, for example: 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM.

[0041] Preferably, the dosage form of the drug includes, but is not limited to, powder, granule, solution or injection.

[0042] Thirdly, the present invention provides a product for improving the reproductive performance of sows, the product comprising the composition described in the first aspect.

[0043] Preferably, the product for improving sow reproductive performance also includes any auxiliary ingredients that help improve product properties (such as stability, solubility, shelf life, etc.), including but not limited to adjuvants, stabilizers, preservatives, antioxidants, such as diluents (including lactose, microcrystalline cellulose, starch, etc.), binders (starch paste, povidone (PVP), hydroxypropyl methylcellulose (HPMC), etc.), and disintegrants (crosslinked carboxymethyl cellulose sodium (CCNa), low-substituted hydroxypropyl cellulose (L-HPC), sodium carboxymethyl starch (CMSNa), etc.). Lubricants (magnesium stearate, talc, micronized silica gel, etc.), plasticizers (polyethylene glycol (PEG), tributyl citrate, etc.), preservatives (sodium benzoate, parabens (such as methylparaben, propylparaben, etc.), antioxidants (vitamin E, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), pH adjusters (citric acid, phosphate buffers, etc.), antistatic agents (magnesium stearate, polyethylene glycol, etc.), absorbents (silica gel, alumina, etc.) or flow aids (micronized silica gel, talc, etc.).

[0044] Fourthly, the present invention provides a method for preventing and treating sow second-parity syndrome, the method comprising the step of applying the composition of the first aspect, the pharmaceutical composition of the second aspect, or the product for improving sow reproductive performance of the first aspect to gilts.

[0045] Preferably, the method includes the following steps: (1) Add the long-acting FSH fusion protein, hCG or a mixture of long-acting FSH fusion protein and hCG to the prescription solution and mix them evenly to obtain the long-acting FSH protein preparation, hCG preparation or a mixture of long-acting FSH fusion protein and hCG.

[0046] (2) According to the predetermined dosage, the long-acting FSH fusion protein preparation or hCG preparation is injected into the sow separately, the long-acting FSH fusion protein preparation and hCG preparation are mixed and injected into the sow at the same time, or the mixture of long-acting FSH fusion protein and hCG is injected into the sow.

[0047] Preferably, the formulation of the prescription solution is: 10-50 mM PB, 0.1-1% mannitol, 1-3% sucrose, and 0.001-0.05% polysorbate 80.

[0048] Preferably, the concentration of the long-acting FSH fusion protein in the long-acting FSH fusion protein formulation or the mixture of the long-acting FSH fusion protein and hCG is 400-10000 U / mL; and the concentration of the hCG in the hCG formulation or the mixture of the long-acting FSH fusion protein and hCG is 100-2000 U / mL.

[0049] Furthermore, the ratio of the long-acting FSH fusion protein to the hCG in medical potency units is 2:1 to 20:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1 or any ratio within the above range, preferably 5:1 to 10:1, more preferably 8:1 to 10:1, and most preferably 9:1.

[0050] Preferably, the long-acting FSH fusion protein and the hCG are prepared into stock solutions, respectively. As stock solutions, the long-acting FSH fusion protein and the hCG can be prepared at higher concentrations, with the long-acting FSH fusion protein concentration ≤90,000 U / mL and the hCG concentration ≤200,000 U / mL. For example, the concentration of the long-acting FSH fusion protein is 90,000 U / mL, and the concentration of the hCG is 200,000 U / mL.

[0051] Preferably, the predetermined doses of the long-acting FSH fusion protein and hCG are set according to the sow's body weight.

[0052] Preferably, the dosage of the long-acting FSH fusion protein is 5-15 U / kg body weight, for example: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 U / kg body weight. More preferably, the predetermined dosage of the long-acting FSH fusion protein is 10-15 U / kg body weight, and more preferably about 12 U / kg body weight.

[0053] Preferably, the dose of hCG is 1-5 U / kg body weight, for example: 1, 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5 or 5 U / kg body weight. More preferably, the predetermined dose of the long-acting FSH fusion protein is 1-2 U / kg body weight, more preferably 1.2-1.8 U / kg body weight, and most preferably about 1.5 U / kg body weight.

[0054] Fifthly, the present invention provides the use of the composition described in the first aspect in the preparation of products for the prevention and treatment of sow second-parity syndrome.

[0055] Preferably, the symptoms of sow second-parity syndrome include at least one of the following: (1) The estrus rate of second-parity sows is low; (2) The conception rate of second-parity sows is low; (3) The farrowing rate of second-parity sows is low; (4) The total number of piglets born to second-parity sows is low; (5) The total number of healthy piglets in second-parity sows is low.

[0056] Preferably, the application includes at least one of the following: (1) Application in the preparation of products that improve the estrus rate of second-parity sows; (2) Application in the preparation of products that improve the conception rate of second-parity sows; (3) Application in the preparation of products that improve the farrowing rate of second-parity sows; (4) Application in the preparation of products that increase the total number of piglets born to second-parity sows; (5) Application in the preparation of products that increase the total number of healthy piglets in second-parity sows.

[0057] Preferably, the product includes a drug.

[0058] The beneficial effects of this invention are: 1. The pharmaceutical composition of the present invention can effectively prevent and treat sows with second-parity syndrome, promote estrus in second-parity sows, promote estrus synchronization and enhance pregnancy ability in second-parity sows, improve the continued application value of sows after weaning of first-parity sows, and effectively reduce losses in the pig farming industry.

[0059] 2. The long-acting FSH fusion protein and recombinant hCG in the pharmaceutical composition of the present invention can synergistically enhance the estrus mating rate, estrus conception rate, farrowing rate, utilization rate, total number of piglets, and total number of healthy piglets in sows, achieving technical effects that exceed industry expectations. Attached Figure Description

[0060] Figure 1 The image shows a timed insemination protocol for first-parity sows suffering from second-parity syndrome. Detailed Implementation

[0061] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0062] Example 1: Preparation of long-acting FSH fusion protein and recombinant hCG

[0063] 1.1 A long-acting FSH fusion protein (pFSH-Fc-2) composed of pFSHα and pFSHβ-L-Fc was prepared according to the method in Example 1 of patent CN107540748B.

[0064] The pFSHα sequence is as follows: FPDGEFTMQGCPECKLKENKYFSKLGAPIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGNARVENHTECHCSTCYYHKS (SEQ ID NO. 1).

[0065] The sequence of pFSHβ-L-Fc is as follows: CELTNITITVEKEECNFCISINTTWCAGYCYTRDLVYKDPARPNIQKTCTFKELVYETVKVPGCAHHADSLYTYPVATECHCGKCDSDSTDCTVRGLGPSYCSFSEMKEGGGGSGGGGSGGGGSICPACESPGPSVFIFPPKPKDTLMISRTPQVTCVVVDVSQENPEVQFSWY VDGVEVHTAQTRPKEEQFNSTYRVVSVLPIQHQDWLNGKEFKCKVNNKDLPAPITRIISKAKGQTREPQVYTLPPHAEELSRSKVSITCLVIGFYPPDIDVEWQRNGQPEPEGNYRTTPPQQDVDGTYFLYSKFSVDKASWQGGGIFQCAVMHEALHNHYTQKSISKTPGK (SEQ ID NO.4).

[0066] The artificially synthesized pFSHα and pFSHβ-L-pFc genes were cloned into the vector pcDNA3.1. The recombinant vectors of pFSHα and pFSHβ-L-pFc were electroporated into 293 cells to express pFSH-Fc-2. The transiently expressed protein was purified to verify its activity. After confirming activity, the recombinant vectors of pFSHα and pFSHβ-L-pFc were linearized and electroporated into CHO cells to obtain a stable cell line expressing pFSH-Fc-2.

[0067] Stable pFSH-Fc-2 cells were cultured in a fermenter. The fermentation broth was first filtered through a two-stage deep filtration membrane to remove cells and cell debris, and then filtered through a 0.22 μm filter to obtain a clear fermentation broth. The fermentation broth was first purified by affinity chromatography using Protein A (MabSelect SuRe™, GE Healthcare): it was first equilibrated to baseline with equilibration buffer (50 mM glycine, 0.15 M NaCl, pH 7.2), and then eluted with eluent (50 mM glycine, pH 3.0), and the eluent was collected. The collected Protein A solution was further purified by cation exchange Capto S (GE Healthcare) column chromatography: the pH of the collected solution was adjusted to 6.5 with 1 M NaOH, the conductivity was adjusted to 4.5–5.0 ms / cm with water, equilibrated with equilibration buffer (50 mM glycine, pH 6.5), loaded, and the flow-through eluent was collected. The Capto S collection solution was finely purified by anion exchange Capto Q (GE Healthcare) column chromatography: the pH of the collection solution was adjusted to 8.0 with 1 M NaOH, equilibrated to baseline with equilibration buffer (50 mM glycine, pH 8.0), and then eluted with elution buffer (50 mM glycine, 1 M KCl, pH 8.0) to obtain the purified protein.

[0068] The activity of pFSH-Fc-2 was determined using the rat ovarian weight gain method (Steelman-Pohley method). This product is intended to replace PMSG in animal reproduction; therefore, the activity of the samples was determined according to the "Bioassay of Serum Gonadotropins" in the 2015 edition of the Chinese Pharmacopoeia, with PMSG as the standard. The specific implementation is as follows: pFSH-Fc-2 (based on preliminary calculations) and PMSG were prepared into three doses (high, medium, and low) with a total activity unit of 40 IU, 20 IU, and 10 IU per animal. Female Sprague Dawley SD rats aged 21-23 days and weighing 40-55 g were randomly divided into 9 groups of 6 rats each. Each rat was subcutaneously injected with 0.5 mL of the corresponding drug. Six days later, the rats were sacrificed, weighed, dissected, and the ovaries were removed and weighed, converting the weight to ovarian weight per 100 g of body weight. The specific activity of pFSH-Fc-2 was calculated to be 45,000 U / mg using the Pharmacopoeia Bioassay Statistical BS2000 software from the National Institutes for Food and Drug Control.

[0069] 1.2 Preparation of recombinant hCG Recombinant hCG contains α and β subunits, which are connected by van der Waals forces.

[0070] The sequence of the α subunit in recombinant hCG is as follows: MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS (SEQ ID NO. 5).

[0071] The sequence of the β subunit in recombinant hCG is as follows: MEMFQGLLLLLLLSMGGTWASKEPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMTRVLQGVLPALPQVVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCALCRRSTDCGGPKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO. 6).

[0072] The nucleotide sequences of the artificially synthesized hCG α and β subunits were transferred into the expression vector pcDNA3.1, and the recombinant vector was linearized and then transferred into CHO cells to express recombinant hCG.

[0073] Stable cells were cultured in a fermenter. The fermentation broth was passed through a two-stage deep filtration membrane to remove cells and cell debris, and then filtered through a 0.22 μm filter to obtain a clear fermentation broth. The clear fermentation broth was first purified by weak cation exchange chromatography (e.g., CM FF, GE Healthcare): the sample was equilibrated with equilibration buffer (20 mM ammonium acetate, pH 5.0), eluted with eluent (50 mM PB, 3 M sodium chloride, pH 8.5), and the eluent was collected. The collected solution from the weak cation exchange chromatography was then purified by hydrophobic chromatography (e.g., Phenyl, GE Healthcare): the sample was equilibrated with equilibration buffer (50 mM PB, 3 M sodium chloride, pH 8.5), eluted with eluent (10 mM PB, pH 8.5), and the eluent was collected. Finally, the hydrophobic chromatography collection was further purified by strong anion exchange chromatography (such as QFF, GE Healthcare): the sample was equilibrated with equilibration buffer (50 mM PB, pH 8.5), then eluted with elution buffer (50 mM PB, 3M sodium chloride, pH 8.5) and collected.

[0074] The activity of recombinant hCG stock solution was determined by measuring its effect on uterine weight gain in immature mice. Following the "Bioassay Method for Human Chorionic Gonadotropin" in the Chinese Veterinary Pharmacopoeia, the method was as follows: hCG standard and recombinant hCG sample (estimated activity 22000 IU / mg) were prepared into three doses: 0.625 IU / mL, 0.375 IU / mL, and 0.225 IU / mL (high, medium, and low doses). Healthy, qualified female mice aged 17-23 days, weighing 9-13g, and from the same source were randomly divided into 6 groups of 15 mice each, based on their weight. Each mouse was subcutaneously injected with the corresponding concentration of standard or recombinant hCG 0.2 mL at approximately the same time each day, once daily for three consecutive days. The animals were sacrificed 24 hours after the last injection, weighed, dissected, the uterus removed, attached tissues peeled off, the ovaries removed, the uterine fluid squeezed dry, and the uterine weight directly weighed and converted to the uterine weight per 10 g of body weight. The specific activity of recombinant hCG was calculated to be approximately 20900 U / mg using the parallel line assay method in the bioassay, which is 4.6 times the potency of urinary hCG (uhCG) specified in the Veterinary Pharmacopoeia of 4500 U / mg.

[0075] Example 2: Preparation of a pharmaceutical composition containing long-acting FSH fusion protein and recombinant hCG 2.1 Prepare the formulation solution according to the following formula, then add the long-acting FSH fusion protein at 40 μg / mL (equivalent to 1800 U / mL) to the formulation solution and mix well. Then add the recombinant hCG at 200 U / mL to the formulation solution and mix well.

[0076] Prescription solution formulation: 10-50mM PB, 0.1-1% mannitol, 1-3% sucrose, 0.001-0.05% polysorbate 80.

[0077] 2.2 When used in combination as a pharmaceutical composition, the above-mentioned long-acting FSH fusion protein solution and recombinant hCG solution are mixed at a volume ratio of 1:1 to obtain a pharmaceutical composition containing long-acting FSH fusion protein and recombinant hCG.

[0078] Example 3: Effects of a pharmaceutical composition of long-acting FSH fusion protein + recombinant hCG on sows with dwarfism syndrome.

[0079] 3.1 The relevant information about the experimental pig farm is as follows: Pig farm location: Shimen County, Hunan Province.

[0080] Pig farm size: Number of pigs: 463 replacement sows, 6,000 breeding sows, and 65 boars.

[0081] Production mode: continuous production.

[0082] Equipment and facilities: automated material line, transfer tower, negative pressure ventilation.

[0083] Staffing: 1 technical manager, 2 technical supervisors, 16 technicians, 16 feeders, and 13 support staff, totaling 48 people.

[0084] Pig breeds: French-American + American.

[0085] Business model: Primarily selling weaned piglets.

[0086] In its routine operations, the pig farm had discovered that first-parity weaned sows were experiencing second-parity syndrome, characterized by unstable estrus and mating rates. The usual approach was to cull affected sows. A small-scale trial using an imported drug (Xideyun) was conducted, which restored the overall mating rate to approximately 88%-90%, but the overall utilization rate remained lower than that of healthy sows.

[0087] The following information pertains to the discovery of pregnancy: Generic name: Human chorionic gonadotropin (HCG) for injection. Brand name: GESTAVET. Main components: PMSG 2000 IU and hCG 200 IU Manufacturer information: Laboratorios Hipra SA, a Spanish biopharmaceutical company.

[0088] 3.2 Experimental Methods: First-parity weaned sows weighing 130-150 kg who have or are suspected of having second-parity syndrome or exhibiting related symptoms (including weight loss after weaning, insufficient milk production during lactation, indistinct estrus signs, no or indistinct response to boar induction, prolonged anestrus, or prolonged estrus intervals, etc.) were selected and divided into a blank control group, a long-acting FSH fusion protein + recombinant hCG group, and a GESTAVET control group. Approximately 40-50 sows were selected in each batch, and a total of 12 batches of experiments were conducted.

[0089] Each batch was processed according to the following procedure: Day 0 was the weaning date. 24 hours after weaning (Day 1), sows were intramuscularly injected with a fixed dose of a combination of long-acting FSH fusion protein and recombinant hCG (1800 U (40 μg) of long-acting FSH fusion protein + 200 U of recombinant hCG). Sows in the blank control group were injected with an equal volume of physiological saline, 2 mL / head. Sows in the GESTAVET control group were intramuscularly injected with 2 mL of GESTAVET preparation (PMSG 2000 IU + hCG 200 IU) behind the ears and in the neck. Starting 24 hours after administration, from Day 2 to Day 6, boars were used to induce estrus and check for estrus in the morning and afternoon, recording the estrus status of the sows. Upon the appearance of standing still, 200 U of recombinant hCG was injected, and artificial insemination was performed on sows in estrus as appropriate (see...). Figure 1 Pregnancy status of sows was assessed 25-28 days after insemination, and farrowing was recorded around day 114. Seven days after drug administration, abdominal ultrasound was used to monitor the ovarian status of sows and observe for the presence of ovarian cysts. The estrus mating rate, estrus conception rate, farrowing rate, utilization rate, total number of piglets, and total number of healthy piglets were recorded and statistically analyzed for different groups of sows.

[0090] 3.3 Experimental Results 3.3.1 Analysis of Ultrasound Results The ovarian status of sows after medication was monitored by scientific research-grade ultrasound in two batches. Seven sows in the GESTAVET control group developed follicular cysts, while no follicular cysts were found in the group using the long-acting FSH fusion protein and recombinant hCG drug combination (see Table 1).

[0091] Table 1. Ultrasound Examination Results

[0092] 3.2.2 Comparison Results of Reproductive Indicators The reproductive indicators of all experimental batches were summarized (see Table 2).

[0093] Table 2 Comparison of Reproductive Indicators

[0094] Note: ** indicates a significant difference compared to the GESTAVET group. Table 2 shows that, for first-parity weaned sows potentially suffering from second-parity syndrome, compared to the GESTAVET group, the use of the long-acting FSH fusion protein + recombinant hCG drug combination increased the estrus rate (i.e., mating rate) by 6%, conception rate by 7%, farrowing rate by 9%, average total number of piglets per litter by 1, and average number of healthy piglets per litter by 0.58. Compared to the blank control group, all indicators of first-parity sows using GESTAVET and those using the long-acting FSH fusion protein + recombinant hCG drug combination were improved, and the difference between the long-acting FSH fusion protein + recombinant hCG drug combination group was statistically significant. This indicates that the long-acting FSH fusion protein + recombinant hCG drug combination can effectively prevent or improve second-parity syndrome in sows, and its efficacy is significantly better than that of GESTAVET.

[0095] Example 4: Comparison of the application effects of long-acting FSH fusion protein, recombinant hCG, and a drug composition of long-acting FSH fusion protein + recombinant hCG. First-parity weaned sows weighing 130-150 kg that have or are suspected of having second-parity syndrome or exhibit related symptoms (including weight loss after weaning, insufficient milk production during lactation, indistinct estrus signs, no or indistinct response to boar induction, prolonged anestrus, or prolonged estrus intervals, etc.) were selected and divided into a blank control group, a long-acting FSH fusion protein monotherapy group, a recombinant hCG monotherapy group, and a long-acting FSH fusion protein + recombinant hCG group (as shown in Table 3).

[0096] Administer the drug on any day of the estrous cycle (Day 0) via intramuscular injection in the neck of sows according to the dosage and timing specified in Table 3. The control group sows were injected with an equal volume of physiological saline, 2 mL / sow. Three days after administration (Day 3), all sows in each group were injected with an equal volume of physiological saline, 2 mL / sow. Estrus was induced by boars in the morning and afternoon daily after administration. Estrus status was recorded within 7 days following administration of the long-acting FSH fusion protein. Artificial insemination was performed on sows exhibiting estrus. Conception was assessed in sows 25-28 days after insemination, and farrowing was recorded around day 114. The estrus-mating rate, estrus-conception rate, farrowing rate, utilization rate, total number of piglets born, and total number of healthy piglets were recorded for different groups (Table 4).

[0097] In the experiment, the "long-acting FSH fusion protein + recombinant hCG group" was administered in three ways: first, the long-acting FSH fusion protein was injected, followed immediately by the recombinant hCG injection at the same site; second, the recombinant hCG was injected first, followed immediately by the long-acting FSH fusion protein injection at the same site; and third, the long-acting FSH fusion protein and recombinant hCG were mixed and injected simultaneously. Since there was no time difference between the sequential injections, the experimental results of the three injection methods showed no significant difference.

[0098] Table 3. Dosage schedule for first-parity sows in different groups

[0099] Table 4 Comparison of reproductive performance of sows

[0100] As shown in Table 4, the inoculation rate of the long-acting FSH fusion protein + recombinant hCG group was 65% higher than that of the long-acting FSH fusion protein monotherapy group and 50% higher than that of the recombinant hCG monotherapy group. The utilization rate of the long-acting FSH fusion protein + recombinant hCG group was 40% higher than that of the long-acting FSH fusion protein monotherapy group and 30% higher than that of the recombinant hCG monotherapy group. Compared with the long-acting FSH fusion protein monotherapy group and the recombinant hCG monotherapy group, the total number of litters in the long-acting FSH fusion protein + recombinant hCG group increased by 262% and 72%, respectively, and the total number of healthy litters increased by 292% and 96%, respectively. The final total number of litters and the total number of healthy litters were also significantly improved.

[0101] Furthermore, regarding the data on the number of reference molecules, reference ratio, and utilization rate, theoretically, the mechanisms of action and targets of the long-acting FSH fusion protein and recombinant hCG are independent. Therefore, the synergistic relationship between the long-acting FSH fusion protein and recombinant hCG was analyzed using the Bliss independent model and the King's method. The details are as follows: (1) Number of references and reference rate Compared to the control group, the effective response rate of the long-acting FSH fusion protein monotherapy group was: EA = (Number of reference drugs in single-drug groups - Number of reference drugs in control groups) / (Total number - Number of reference drugs in control groups) × 100% = (4-3) / (20-3)×100% =5.88% Compared to the control group, the recombinant hCG monotherapy group showed the following effective response rate: EB = (Number of drug combinations in single-drug group - Number of drug combinations in control group) / (Total number - Number of drug combinations in control group) × 100% = (7-3) / (20-3)×100% =23.53% Theoretical effective remission rate of the long-acting FSH fusion protein + recombinant hCG group: EAB (theoretical) = EA + EB - EA × EB =5.88% + 23.53% - 5.88% × 23.53% =28.03% Actual effective remission rate in the long-acting FSH fusion protein + recombinant hCG group: EAB (Actual) = (Number of references in the combination group - Number of references in the control group) / (Total number - Number of references in the control group) × 100% =(17 - 3) / (20 - 3)×100% =82.35% On this basis: CI = EAB (actual) / EAB (theoretical) =82.35% / 28.03% =2.94 According to the conventional criteria of the Kim method: 0.85 ≤ CI ≤ 1.15: simple addition; 1.15 < CI ≤ 20: enhancement (synergy); CI > 20: significant enhancement; 0.55 ≤ CI < 0.85: antagonism; CI < 0.55: obvious antagonism.

[0102] It can be seen that in terms of the improvement of the parameter combination number and parameter combination rate, the CI of the long - acting FSH fusion protein and recombinant hCG is 2.94, indicating that a synergistic enhancement effect is achieved between the two.

[0103] (2) Utilization rate For the long - acting FSH fusion protein single - drug group compared with the control group, its effective remission rate: EA=(single - drug group utilization rate - control group utilization rate) / (1 - control group utilization rate)×100% =(15% - 15%) / (1 - 15%)×100% =0 For the recombinant hCG single - drug group compared with the control group, its effective remission rate: EB=(single - drug group utilization rate - control group utilization rate) / (1 - control group utilization rate)×100% =(25% - 15%) / (1 - 15%)×100% =11.76% The theoretical effective remission rate of the long - acting FSH fusion protein + recombinant hCG group: EAB (theoretical)=EA + EB - EA×EB =0 + 11.76% - 0×11.76% =11.76% The actual effective remission rate of the long - acting FSH fusion protein + recombinant hCG group: EAB (actual)=(combined - use group utilization rate - control group utilization rate) / (1 - control group utilization rate)×100% =(55% - 15%) / (1 - 15%)×100% =47.06% On this basis: CI = EAB (actual) / EAB (theoretical) =47.06% / 11.76% =4.00 According to the conventional criteria of the Jin's method: 0.85 ≤ CI ≤ 1.15: simple addition; 1.15 < CI ≤ 20: enhancement (synergy); CI > 20: significant enhancement; 0.55 ≤ CI < 0.85: antagonism; CI < 0.55: obvious antagonism.

[0104] It can be seen that in terms of the improvement of utilization rate, the CI of the long - acting FSH fusion protein and recombinant hCG is 4.00, indicating that a synergistic enhancement effect is achieved between the two.

[0105] Therefore, the above data indicate that the long - acting FSH fusion protein + recombinant hCG pharmaceutical composition in this embodiment can effectively prevent and treat or improve the second - parity syndrome of sows.

[0106] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention. All equivalent structural transformations made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, are equally included in the patent protection scope of the present invention.

Claims

1. The application of a composition comprising a long-acting FSH fusion protein and human chorionic gonadotropin in the preparation of a product for the prevention and treatment of sow second-parity syndrome, characterized in that, The long-acting FSH fusion protein comprises two peptide chains conforming to the following equation: (pFSHα:pFSHβ-L-Fc)2, wherein the amino acid sequence of pFSHβ is shown in SEQ ID NO.2; the colon represents the relationship between the porcine FSH β subunit and α subunit linked by van der Waals forces; the amino acid sequence of pFSHα is shown in SEQ ID NO.1; L represents the connection relationship between the pFSHα or pFSHβ subunit and the Fc fragment; Fc refers to the Fc fragment of an immunoglobulin or its mutant; the subscript 2 outside the brackets indicates that the porcine FSH fusion protein is a divalent homodimer; the ratio of the medical potency units of the long-acting FSH fusion protein to the human chorionic gonadotropin is 5:1 to 10:1; the long-acting FSH fusion protein and the human chorionic gonadotropin are prepared into a mixed preparation for simultaneous or sequential injection, with no time difference between sequential and sequential injection.

2. The application according to claim 1, characterized in that, The human chorionic gonadotropin is recombinant human chorionic gonadotropin, which contains α and β subunits. The amino acid sequence of the α subunit is shown in SEQ ID NO. 5, and the amino acid sequence of the β subunit is shown in SEQ ID NO.

6.

3. The application according to claim 1, characterized in that, The concentration of the long-acting FSH fusion protein is 400-10000 U / mL, and the concentration of the human chorionic gonadotropin is 100-2000 U / mL.

4. The application according to claim 3, characterized in that, The concentration of the long-acting FSH fusion protein is 1800 U / mL, and the concentration of the human chorionic gonadotropin is 200 U / mL.

5. The application according to any one of claims 1-4, characterized in that, The long-acting FSH fusion protein and human chorionic gonadotropin are pre-formulated in the same formulation.

6. The application according to any one of claims 1-4, characterized in that, The long-acting FSH fusion protein and human chorionic gonadotropin are pre-prepared in different formulations.

7. The application according to any one of claims 1-4, characterized in that, The long-acting FSH fusion protein and human chorionic gonadotropin were respectively prepared into stock solutions, wherein the concentration of the long-acting FSH fusion protein stock solution was ≤90000 U / mL and the concentration of the human chorionic gonadotropin stock solution was ≤200000 U / mL.

8. The application according to any one of claims 1-4, characterized in that, The application includes at least one of the following: (1) Application in the preparation of products that improve the estrus rate of second-parity sows; (2) Application in the preparation of products that improve the conception rate of second-parity sows; (3) Application in the preparation of products that improve the farrowing rate of second-parity sows; (4) Application in the preparation of products that increase the total number of piglets born to second-parity sows; (5) Application in the preparation of products that increase the total number of healthy piglets in second-parity sows.

9. The application according to any one of claims 1-4, characterized in that, The product also includes auxiliary materials.

10. The application according to claim 9, characterized in that, The excipients include mannitol, sucrose, polysorbate 80, and phosphate.

Images