CN114848664B - Pharmaceutical composition and application thereof - Google Patents
Pharmaceutical composition and application thereof Download PDFInfo
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Abstract
The invention discloses a pharmaceutical composition and application thereof, wherein the pharmaceutical composition comprises 1, 6-Fructose Diphosphate (FDP) or pharmaceutically acceptable salt thereof and nicotinamide mononucleotide NMN/nicotinamide adenine dinucleotide NAD + Or a pharmaceutically acceptable salt thereof; and the application thereof in preparing medicines for preventing and/or treating primary dysmenorrhea and infertility. FDP and NMN/NAD are disclosed + Mechanism of action of co-administration on a model for treating primary dysmenorrhea in mice, FDP and NMN/NAD were found + Combination dosing ratio FDP or NMN/NAD + The single administration has better curative effect, can obviously reduce the frequency of torsion reaction of female mice, increase the latency period of the occurrence of the torsion reaction, reduce the gene expression level of COX-2 and PGF2α of uterine tissues, increase the gene expression level of PGE2, reduce the infiltration of inflammatory cells, and improve the edema and inflammatory pathological degree of uterine tissues; in addition, FDP and NMN/NAD + Combination dosing ratio FDP or NMN/NAD + Administration alone can also significantly increase conception rates in female mice.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a pharmaceutical composition and application thereof, wherein the pharmaceutical composition is prepared from 1, 6-Fructose Diphosphate (FDP) or pharmaceutically acceptable salt thereof and nicotinamide mononucleotide NMN/nicotinamide adenine dinucleotide NAD + Or a pharmaceutically acceptable salt thereof; and the application thereof in preparing medicines for preventing and/or treating primary dysmenorrhea and infertility.
Background
Primary dysmenorrhea (primary dysmenorrhea, PD) refers to menstrual cramping pain in the lower abdomen of women without obvious pelvic organic lesions, and is accompanied by symptoms such as lumbago, skelalgia, diarrhea, nervous tension, debilitation, anorexia, nausea and emesis. At present, the medicine for treating primary dysmenorrhea is mainly a non-steroidal anti-inflammatory medicine, but the inefficiency of the medicine is 20% -30%, so that the clinical urgent need is to find a low-risk and effective medicine for relieving dysmenorrhea.
Disclosure of Invention
The pathogenesis of primary dysmenorrhea is mainly related to the overproduction of Prostaglandins (PG) by uterine tissues, which are synthesized under physiological conditions mainly via the arachidonic acid pathway, where catalysis by cyclooxygenase is required. Prostaglandins synthesized by uterine tissue are mainly pgf2α and PGE2, pgf2α being capable of contracting uterine smooth muscle and PGE2 being capable of smoothing and dilating uterus, both being maintained in equilibrium under physiological conditions. When women get menstrual, the PGF2α content increases, causing the uterine smooth muscle to shrink cramps, resulting in ischemia and hypoxia of uterine tissue, and thus dysmenorrhea. And PGE2 antagonizes pgf2α, dilating uterine smooth muscle. Thus, detection of changes in uterine tissue pgf2α, PGE2 expression may be indicative of the mechanism of action of the drug.
The trisodium salt of 1, 6-fructose diphosphate, or D-fructose-1, 6-trisodium diphosphate, is a salt form of 1, 6-Fructose Diphosphate (FDP), is an intermediate product of glycolysis, and can activate phosphofructokinase, improve the metabolic level of cells, and provide energy for cells and organisms. The clinical FDP is mainly used for improving sugar metabolism, and has obvious functions of resisting anoxia, resisting fatigue and providing energy. Nicotinamide mononucleotide (nicotinamide mononucleotide, NMN) is a naturally occurring substance in the human body that provides energy to cells, improving the energy state of cells. NMN is nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide, NAD + ) Can directly and effectively supplement NAD + . NMN has anti-aging effect, and also has effects of relieving inflammation and improving oxidative stress injury. Primary dysmenorrhea can lead to ischemia and hypoxia of the uterus of mice, cause a series of inflammatory reactions of uterine tissues, and generate oxidative stress reactions.
To date, no related paper has described FDP and NMN/NAD + The invention has the core innovative content of FDP, FDP and NMN/NAD + The combination can be used for treating primary dysmenorrheaThe gene expression of uterine tissue PGs is reduced, and the conception rate of female mice is improved, which is a novel application which has not been proved so far.
Based on the above circumstances, the present invention provides a pharmaceutical composition and its use, thereby providing a new therapeutic agent for the treatment of primary dysmenorrhea.
For this purpose, the invention provides the following technical scheme:
a pharmaceutical composition comprising fructose-1, 6-diphosphate or a pharmaceutically acceptable salt thereof and nicotinamide mononucleotide NMN/nicotinamide adenine dinucleotide NAD + Or a pharmaceutically acceptable salt thereof; the 1, 6-fructose diphosphate or the pharmaceutically acceptable salt thereof and NMN/NAD + Or a pharmaceutically acceptable salt thereof in a molar ratio of 1:1 to 1:10, wherein the 1, 6-fructose diphosphate or a pharmaceutically acceptable salt thereof is based on 1, 6-fructose diphosphate, NMN/NAD + Or a pharmaceutically acceptable salt thereof as NMN/NAD + And (5) counting.
In some embodiments, the fructose-1, 6-bisphosphate or pharmaceutically acceptable salt thereof is mixed with NMN/NAD + Or pharmaceutically acceptable salts thereof in a molar ratio of 1:3 to 1:5.
In some embodiments, the pharmaceutically acceptable salt of fructose-1, 6-bisphosphate is selected from the trisodium salt of fructose-1, 6-bisphosphate.
In a second aspect, a pharmaceutical formulation is provided comprising a therapeutically effective amount of the pharmaceutical composition and a pharmaceutically acceptable carrier, adjuvant or vehicle.
In some embodiments, the pharmaceutical formulation is in the form of a gastrointestinal administration, an injection administration, a vaginal gel administration, or a dermal administration, including a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, cream, ointment, suppository, or patch.
In a third aspect, the application of the pharmaceutical composition and the pharmaceutical preparation in preparing medicines for preventing and/or treating primary dysmenorrhea and infertility is provided.
The pharmaceutical composition can reduce the number of times of torsion reaction after dysmenorrhea and increase the time of first torsion reaction; the pharmaceutical composition has the effects of treating primary dysmenorrhea by reducing the expression of COX-2 genes of uterine tissues, reducing the expression of PGF2 alpha, increasing the expression of PGE2 and obviously reducing the ratio of PGF2/PGE 2.
The pharmaceutical composition can reduce hypertrophy of uterine tissue, reduce inflammatory cell infiltration and reduce pathological degree of uterine tissue.
The pharmaceutical composition is capable of increasing conception rate in female individuals.
Different doses of FDP and NMN/NAD are provided + The combined application is used for preparing the mice primary dysmenorrhea model drug, and the treatment of the mice primary dysmenorrhea means that FDP can reduce the number of torsion reaction times after dysmenorrhea and increase the latency period of torsion reaction.
Based on the above regimen, it is preferred that the FDP daily lavage dose is 400mg/kg and the NMN daily lavage dose is 1.2g/kg.
The FDP and NMN combination has the following effects on a primary dysmenorrhoea model of a mouse:
experiments show that: the FDP and NMN combined can reduce the expression of COX-2 in uterine tissues, reduce the expression of PGF2α, increase the expression of PGE2 and obviously reduce the ratio of PGF2α/PGE 2; the combination of FDP and NMN can reduce the swelling of uterine tissue and reduce the infiltration of inflammatory cells through H & E staining.
The FDP and NMN combination of the invention can increase the conception rate of female mice.
In a second aspect, the invention provides a pharmaceutical preparation comprising FDP and NMN/NAD as active ingredients + The medicine composition is prepared into clinically acceptable gastrointestinal tract administration dosage forms, injection administration dosage forms, vaginal gel administration dosage forms and the like by adding conventional auxiliary materials.
The invention combines FDP with NMN/NAD + The combination is used for preparing the medicine for preventing primary dysmenorrhea and infertility, wherein FDP can be used in the absence ofEnhancing glycolysis of cells under oxygen and oxygen deficient conditions, rapidly converting glucose to energy, providing the uterine tissue with the required energy to enhance uterine repair while NAD + The method can accelerate metabolic pathway, increase glycolysis rate and relieve insufficient regeneration of intracellular NAD in the process of generating energy by FDP, thereby rapidly converting glucose into energy required by uterine tissue under the conditions of ischemia and hypoxia, or providing more energy for fertilization of oocyte in uterus, and the two are combined and mutually cooperated to prevent or improve primary dysmenorrhea and improve conception rate, and has unexpected effects of 1 plus 1 to more than 2.
Drawings
Figure 1 effect of fdp intragastric administration on primary dysmenorrhea model in mice. Effects of FDP intragastric administration on the number of writhing responses in female mice; the effect of FDP on time to first torsion response in female mice. Ibuprofen is a positive control. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum ** P<0.01 compared to model group, n=8.
FIG. 2 effects of FDP (400 mg/kg) on the expression of COX-2, PGF2α, PGE2, PGF2α/PGE2 genes in uterine tissues. Effects of FDP on COX-2 Gene expression; effects of FDP on PGF2α Gene expression; effects of FDP on PGE2 gene expression; FDP effect on PGF2α/PGE 2. Ibuprofen is a positive control. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 and 0 ** P<0.01 compared to model group, n=8.
FIG. 3 influence of FDP (400 mg/kg) on the pathological morphology of uterine tissue. Compared to Sham group, the vecle group had increased inflammatory cell number and edema, cell hypertrophy, arrangement disorder, FDP and ibuprofen group interstitial tightness, improved pathological extent of uterine tissue edema and inflammation in mice, n=3.
FIG. 4 effect of FDP (400 mg/kg) on conception rate in female mice. Compared with a blank group, the conception rate of the molding group is obviously reduced, and the conception rate of female mice can be increased after FDP gastric lavage administration. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum of ** P<0.01 compared to model group, n=3.
FIG. 5 effects of FDP (400 mg/kg) and NMN combination on primary dysmenorrhea model in mice. Effects of FDP (400 mg/kg) and NMN combination on the number of writhing responses in female mice; effects of FDP (400 mg/kg) and NMN combination on time to first-time torsion response in female mice. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum ** P<0.01 in comparison with the model set, & P<0.05 compared to FDP (400 mg/kg) group, n=6-8.
FIG. 6 effects of FDP (400 mg/kg) and NMN co-administration on COX-2, PGF2α, PGE2, PGF2α/PGE2 gene expression in uterine tissue. Effects of FDP (400 mg/kg) and NMN co-administration on COX-2 gene expression; effects of FDP (400 mg/kg) and NMN co-administration on PGF2α gene expression; effects of FDP (400 mg/kg) and NMN co-administration on PGE2 gene expression; effects of FDP (400 mg/kg) and NMN co-administration on PGF2α/PGE 2. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum ** P<0.01 in comparison with the model set, & P<0.05 N=6 compared to FDP (400 mg/kg) group.
FIG. 7 effects of FDP (400 mg/kg) and NMN co-administration on pathological morphology of uterine tissue. Compared with the Sham group, the Vehicle group has the advantages that the inflammatory cell number is increased, the cells are enlarged, the arrangement is disordered, the combined administration of FDP (400 mg/kg) and NMN (1.2 g/kg) is compact, the pathological degrees of the uterine tissue edema and inflammation of mice are improved, and N=3.
FIG. 8 effects of FDP (400 mg/kg) and NMN co-administration on conception rate in female mice. Compared with the blank group, the conception rate of the model group is obviously reduced, and the conception rate of female mice can be increased after FDP (400 mg/kg) and NMN (1.2 g/kg) are subjected to intragastric administration. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum ** P<0.01 in comparison with the model set, & P<0.05 And && P<0.01 compared to the FDP (400 mg/kg) group, n=3.
Detailed Description
In order to further illustrate the invention, the following examples are set forth which are purely illustrative and are intended to be a detailed description of the invention and should not be taken as limiting the invention.
The present application will be described in detail with reference to specific examples. 1, 6-Difructose diphosphate or a pharmaceutically acceptable salt thereof is exemplified by trisodium salt (molecular weight 406.06) of fructose-1, 6-diphosphate, abbreviated as FDP, NMN/NAD + Or a pharmaceutically acceptable salt thereof, NMN (molecular weight 334.22) is selected as an example in the examples.
Example 1
A pharmaceutical composition comprises 1, 6-fructose diphosphate trisodium salt and nicotinamide mononucleotide NMN; the molar ratio of the 1, 6-fructose diphosphate trisodium salt FDP to the nicotinamide mononucleotide NMN is 1:1.
Example 2
A pharmaceutical composition comprises 1, 6-fructose diphosphate trisodium salt and nicotinamide mononucleotide NMN; the molar ratio of the 1, 6-fructose diphosphate trisodium salt FDP to the nicotinamide mononucleotide NMN is 1:2.
Example 3
A pharmaceutical composition comprises 1, 6-fructose diphosphate trisodium salt and nicotinamide mononucleotide NMN; the molar ratio of the 1, 6-fructose diphosphate trisodium salt FDP to the nicotinamide mononucleotide NMN is 1:3.
Example 4
A pharmaceutical composition comprises 1, 6-fructose diphosphate trisodium salt and nicotinamide mononucleotide NMN; the molar ratio of the 1, 6-fructose diphosphate trisodium salt FDP to the nicotinamide mononucleotide NMN is 1:5.
Example 5
A pharmaceutical composition comprises 1, 6-fructose diphosphate trisodium salt and nicotinamide mononucleotide NMN; the molar ratio of the 1, 6-fructose diphosphate trisodium salt FDP to the nicotinamide mononucleotide NMN is 1:10.
Example 6
A pharmaceutical preparation comprises 1, 6-fructose diphosphate trisodium salt, nicotinamide mononucleotide NMN and carrier; the molar ratio of the 1, 6-fructose diphosphate trisodium salt FDP to the nicotinamide mononucleotide NMN is 1:3-1:5.
Preferably, the daily gastric lavage dose of 1, 6-fructose trisodium diphosphate FDP is 400mg/kg and the daily gastric lavage dose of NMN is 1.2g/kg.
Example 7 effects of fdp intragastric administration on primary dysmenorrhea model in mice.
Experimental materials: female ICR mice, weighing 20-22g, were purchased from the university of Yangzhou comparative medical center. FDP, ibuprofen, estradiol benzoate, oxytocin were purchased from Sigma, USA.
Experimental grouping: female ICR mice, 40 in each group, were evenly divided into a normal group, a model group, an ibuprofen group and a different dose of fructose-1, 6-bisphosphate trisodium salt group (200 mg/kg and 400 mg/kg). Each of the other groups except the normal group (normal saline subcutaneous injection) was subcutaneously injected with estradiol benzoate (10 mg/kg) for 10 consecutive days. The model group, the fructose-1, 6-bisphosphate trisodium salt group and the ibuprofen group were orally administered with equal amounts of solvent, fructose-1, 6-bisphosphate trisodium salt group (200 mg/kg and 400 mg/kg) and ibuprofen (0.1 g/kg), respectively, on day 4 for 7 consecutive days (1 time per day). After the last administration of 10min, oxytocin (10U/kg) is injected into the abdominal cavity, and the animal has a torsion reaction, namely the abdomen is concave, the trunk and the hind limbs are elongated, and the buttocks are raised.
As shown in table 1 and fig. 1, FDP (400 mg/kg) significantly reduced the increase in the number of torsion reactions caused by the primary dysmenorrhea model, significantly increased the time to first torsion reaction, and the ibuprofen group showed successful molding. ### P<0.001 And #### P<0.0001 in comparison to the blank group, ** P<0.01 and *** P<0.001 compared to model set.
TABLE 1
| Group of | Writhing number | Writhing latency |
| Sham | 0 | |
| Vehicle | 21.55±5.85 ### | 47.33±25.99 |
| FDP(200mg/kg) | 20.5±8.73 | 107.37±51.34 |
| FDP(400mg/kg) | 13.25±3.32 ** | 227.62±122.83 *** |
| Ibuprofen(100mg/kg) | 2.87±2.29 *** | 231.87±82.24 *** |
Example 8 effect of FDP (400 mg/kg) on the expression of COX-2, PGF2α, PGE2, PGF2α/PGE2 genes in uterine tissue.
Animal modelling and administration methods were the same as in example 1. Taking uterine tissue, weighing about 30mg on an electronic balance, adding Trizol for cleavage, extracting total RNA of the uterine tissue, performing reverse transcription to obtain cDNA, preparing a reaction system, and amplifying to detect the change of COX-2, PGF2α and PGE2 gene expression of the uterine tissue.
The results are shown in Table 2 and FIG. 2, where mRNA expression levels of COX-2 and PGF2α were elevated in mice following primary dysmenorrhea model, FDP and ibuprofen groups were able to be identifiedReducing mRNA expression levels of COX-2 and PGF2α in uterine tissue; the mRNA expression level of the PGE2 in the uterine tissue of the mice is reduced after the primary dysmenorrhea model, the PGF2alpha/PGE 2 ratio is increased, and the FDP and ibuprofen groups can increase the mRNA expression level of the PGE2 in the uterine tissue and reduce the PGF2alpha/PGE 2 ratio. These results suggest that FDP treats primary dysmenorrhea by reducing pgf2α/PGE2 ratio in uterine tissue. # P<0.05、 ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05、 ** P<0.01 and *** P<0.001 compared to model set.
TABLE 2
| Group of | COX-2 | PGF2α | PGE2 | PGF2α/PGE2 |
| Sham | 1±0.21 | 1±0.38 | 1±0.38 | 1.06±0.46 |
| Vehicle | 1.19±0.08 # | 1.58±0.48 # | 0.39±0.10 ## | 5.01±1.78 ### |
| FDP(400mg/kg) | 0.94±0.09 ** | 0.53±0.14 *** | 0.87±0.05 * | 0.63±0.19 *** |
| Ibuprofen(100mg/kg) | 0.89±0.06 ** | 0.22±0.10 *** | 0.99±0.14 ** | 0.24±0.10 *** |
Example 9 effect of FDP (400 mg/kg) on pathological morphology of uterine tissue.
Animal modelling and administration methods were the same as in example 1. Uterine tissue was taken and fixed with 4% paraformaldehyde. The fixed tissue was dehydrated with fractionated ethanol and then embedded in paraffin. The uterine tissue was then cut into 5 μm thick sections, morphologically assessed by hematoxylin and eosin (H & E) staining, followed by observation of the stained sections under an optical microscope (Olympus BX 53) and analysis using a pre-installed software program.
The results are shown in FIG. 3, and compared with the blank group, the model group mice have increased secretion of the lamina propria gland, with increased inflammatory cell number and edema, thickened smooth muscle of uterus, hypertrophy of cells, disorder of arrangement and uterine edema; both the FDP and ibuprofen groups reduced the extent of pathological changes in uterine tissue edema and inflammation in mice of the model group.
Example 10 effect of FDP (400 mg/kg) on conception rate in female mice.
The implementation materials are as follows: female ICR mice, weighing 20-22g, were purchased from the university of Yangzhou comparative medical center. FDP, cyclophosphamide were purchased from Sigma, USA.
Grouping animals: female ICR mice were divided into 3 groups of 10 female mice each of 5 weeks of age, and into physiological saline group, model group, FDP (400 mg/kg) group, physiological saline group was injected with physiological saline, and the remaining 2 groups were intraperitoneally injected with cyclophosphamide (20 mg/kg) once a week for 5 weeks. At week 4 of the experiment, the FDP group was perfused with gastric FDP (400 mg/kg) for two consecutive weeks. At 6-8 weeks of experiment, all female mice model mice and normal male mice (10 weeks old) were mated 1:1 with cages, at the last 2 weeks of experiment, the mated mice were kept separately, after 10 weeks, all female mice were weighed and the cervical pulp dislocation was sacrificed, the oviduct was dissected, and the red beaded embryo was positive pregnant.
The results are shown in Table 3 and FIG. 4, the conception rate of female mice is reduced after the cyclophosphamide molding, and FDP can significantly improve the conception rate, and has certain reference significance for treating infertility. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 compared to the model set.
TABLE 3 Table 3
| Group of | Pregnant rate(％) |
| Sham | 80.55±4.81 |
| Vehicle | 38.88±4.81 ### |
| FDP(400mg/kg) | 61.11±9.62 * |
Example 11 effects of FDP (400 mg/kg) and NMN combined gastric lavage on mice primary dysmenorrhea model
Experimental materials: female ICR mice, weighing 20-22g, were purchased from the university of Yangzhou comparative medical center. FDP, NMN, estradiol benzoate, oxytocin were purchased from Sigma, USA.
Experimental grouping: female ICR mice, 56 in each group, were evenly divided into normal, model, FDP (400 mg/kg), different doses of NMN (800 mg/kg and 1200 mg/kg), FDP (400 mg/kg) +NMN (800 mg/kg) and FDP (400 mg/kg) +NMN (1200 mg/kg). Each of the other groups except the normal group (normal saline subcutaneous injection) was subcutaneously injected with estradiol benzoate (10 mg/kg) for 10 consecutive days. The model group, FDP group, NMN group and FDP and NMN combination groups were given by gavage on day 4 with equal amounts of solvent, FDP (400 mg/kg), NMN (800 mg/kg and 1200 mg/kg), FDP (400 mg/kg) +NMN (800 mg/kg) and FDP (400 mg/kg) +NMN (1200 mg/kg), respectively, for 7 days (1 time per day). The mice were observed for the number of writhing reactions and the time of first writhing reactions within 30 minutes by intraperitoneal injection of oxytocin (10U/kg) 10min after the last administration of day 10.
As shown in table 4 and fig. 5, the combined administration of FDP (400 mg/kg) and NMN (1200 mg/kg) significantly reduced the increase in the number of torsion responses caused by the primary dysmenorrhea model, significantly increased the time to first torsion response, and had significant advantages over FDP or NMN alone. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum ** P<0.01 in comparison with the model set, & P<0.05 sum && P<0.01 compared with FDP (400 mg/kg) +NMN (1200 mg/kg) group, $$$ P<0.001 compared to FDP (400 mg/kg) +NMN (800 mg/kg).
TABLE 4 Table 4
Example 12 effects of co-administration of FDP (400 mg/kg) and NMN on uterine tissue COX-2, PGF2α, PGE2, PGF2α/PGE2 gene expression.
Animal modelling and administration methods were the same as in example 5. Taking uterine tissue, weighing about 30mg on an electronic balance, adding Trizol for cleavage, extracting total RNA of the uterine tissue, performing reverse transcription to obtain cDNA, preparing a reaction system, and amplifying to detect the change of COX-2, PGF2α and PGE2 gene expression of the uterine tissue.
Results as shown in table 5 and fig. 6, mRNA expression levels of COX2 and pgf2α were elevated in mice following primary dysmenorrhea model, and co-administration of FDP (400 mg/kg) and NMN (1200 mg/kg) reduced mRNA expression levels of COX2 and pgf2α in uterine tissue, with significant advantages over administration of FDP (400 mg/kg) or NMN (1200 mg/kg) alone; the mRNA expression level of PGE2 in uterine tissue of mice after the primary dysmenorrhea model is reduced, the ratio of pgf2α/PGE2 is increased, the combined administration of FDP (400 mg/kg) and NMN (1200 mg/kg) can increase the mRNA expression level of PGE2 in uterine tissue, reduce the ratio of pgf2α/PGE2, and has significant advantages compared with the single administration of FDP (400 mg/kg) or NMN (1200 mg/kg). These results suggest that the pgf2α/PGE2 ratio of uterine tissue can be reduced more significantly after co-administration of FDP and NMN than when FDP or NMN is administered alone, and that the therapeutic effect on primary dysmenorrhea is more pronounced. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum ** P<0.01 in comparison with the model set, & P<0.05 sum && P<0.01 compared with FDP (400 mg/kg) +NMN (1200 mg/kg) group, $ P<0.05 sum $$$ P<0.001 compared to FDP (400 mg/kg) +NMN (800 mg/kg).
TABLE 5
Example 13 effects of combined administration of FDP (400 mg/kg) and NMN on pathological forms of uterine tissue.
Animal modelling and administration methods were the same as in example 5. Uterine tissue was taken and fixed with 4% paraformaldehyde. The fixed tissue was dehydrated with fractionated ethanol and then embedded in paraffin. The uterine tissue was then cut into 5 μm thick sections, morphologically assessed by hematoxylin and eosin (H & E) staining, followed by observation of the stained sections under an optical microscope (Olympus BX 53) and analysis using a pre-installed software program.
The results are shown in FIG. 7, and compared with the blank group, the model group mice have increased secretion of the lamina propria gland, with increased inflammatory cell number and edema, thickened smooth muscle of uterus, hypertrophy of cells, disorder of arrangement and uterine edema; the combined administration of FDP (400 mg/kg) and NMN (1200 mg/kg) reduced the pathological changes in uterine tissue edema and inflammation in the mice of the model group.
Example 14 effect of co-administration of FDP (400 mg/kg) and NMN on conception rate in female mice.
The implementation materials are as follows: female ICR mice, weighing 20-22g, were purchased from the university of Yangzhou comparative medical center. FDP, NMN and cyclophosphamide were purchased from Sigma, USA.
Grouping animals: female ICR mice were divided into 7 groups of 10 animals each, each group being divided into a physiological saline group, a model group, FDP (400 mg/kg), NMN groups of different doses (800 mg/kg and 1200 mg/kg), FDP (400 mg/kg) +NMN (800 mg/kg) and FDP (400 mg/kg) +NMN (1200 mg/kg). Saline was injected into the saline group, and the remaining 6 groups were intraperitoneally injected with cyclophosphamide (20 mg/kg) once a week for 5 weeks. At week 4 of the experiment, the corresponding doses of compound were administered intragastrically for two consecutive weeks in FDP (400 mg/kg), different doses of NMN (800 mg/kg and 1200 mg/kg), FDP (400 mg/kg) +NMN (800 mg/kg) and FDP (400 mg/kg) +NMN (1200 mg/kg) groups. At 6-8 weeks of experiment, all female mice were mated 1:1 with normal male ICR mice (10 weeks old) and at the last 2 weeks of experiment, the mated mice were kept separately, after 10 weeks, all female mice were weighed and the cervical pulp dislocation was sacrificed, the oviduct was dissected, and the red beaded embryo was a positive pregnancy.
As shown in Table 6 and FIG. 8, the conception rate of female mice after the molding of cyclophosphamide was decreased, and FDP (400 mg/kg) and NMN (1200 mg/kg) were administered in combinationThe drug can obviously improve the conception rate after the drug is taken, and has obvious significance compared with the single drug administration of FDP (400 mg/kg) or NMN (1200 mg/kg). Thus, FDP and NMN have significant advantages for treating infertility after co-administration. ### P<0.001 sum #### P<0.0001 in comparison to the blank group, * P<0.05 sum ** P<0.01 in comparison with the model set, & P<0.05 And && P<0.01 compared with FDP (400 mg/kg) +NMN (1200 mg/kg) group, $$ P<0.01 compared to FDP (400 mg/kg) +NMN (800 mg/kg).
TABLE 6
| Group of | Pregnant rate(％) |
| Sham | 80.55±4.81 |
| Vehicle | 38.88±4.81 ### |
| FDP(400mg/kg) | 61.11±9.62 **& |
| FDP(400mg/kg)+NMN(800mg/kg) | 65.56±5.09 *** |
| FDP(400mg/kg)+NMN(1200mg/kg) | 80 *** |
| NMN(800mg/kg) | 43.9±6.97 $$ |
| NMN(1200mg/kg) | 62.23±3.86 **&& |
In summary, the invention adopts the continuous subcutaneous injection of the estradiol benzoate and the oxytocin for intraperitoneal injection to manufacture a primary dysmenorrhea model, and examines the treatment effect of the combined administration of FDP and NMN on the primary dysmenorrhea model. The results show that the combined administration of FDP (400 mg/kg) and NMN (1200 mg/kg) can remarkably reduce the torsion reaction times of mice, increase the time for the first torsion reaction, reduce the expression of the COX-2 gene of uterine tissue inflammatory factor, reduce the expression of PGF2α gene, increase the expression of PGE2 gene, reduce the ratio of PGF2/PGE2, and have remarkable advantages compared with the single administration of FDP (400 mg/kg) or NMN (1200 mg/kg). Through H & E staining, FDP (400 mg/kg) and NMN (1200 mg/kg) can be combined to reduce infiltration of inflammatory cells, increase tight connection between tissues and reduce the degree of inflammatory pathological injury. In addition, the combined administration of FDP (400 mg/kg) and NMN (1200 mg/kg) can also increase the conception rate of female ICR mice, and has certain reference significance for treating infertility. These results all indicate that the combined administration of FDP (400 mg/kg) and NMN (1200 mg/kg) has certain pharmacological effects in the treatment of primary dysmenorrhea and infertility. FDP and NMN are used as active substances and are combined with other carriers or are prepared into a compound for use with other compounds, and various medicament forms for treating primary dysmenorrhea and infertility are prepared according to a conventional preparation method, or are prepared into a compound preparation with other medicaments for treating primary dysmenorrhea and infertility, so that the medicament effect can be further increased, adverse reactions can be reduced, and an economic, stable, safe and reliable solution is provided for treating primary dysmenorrhea and infertility.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will understand that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A pharmaceutical composition, characterized in that the pharmaceutical composition consists of fructose-1, 6-diphosphate or a pharmaceutically acceptable salt thereof and nicotinamide mononucleotide NMN or a pharmaceutically acceptable salt thereof; the molar ratio of the 1, 6-fructose diphosphate or a pharmaceutically acceptable salt thereof to NMN or a pharmaceutically acceptable salt thereof is 1:3 to 1:5, wherein the 1, 6-fructose diphosphate or a pharmaceutically acceptable salt thereof is calculated as 1, 6-fructose diphosphate and the NMN or a pharmaceutically acceptable salt thereof is calculated as NMN.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable salt of fructose-1, 6-bisphosphate is the trisodium salt of fructose-1, 6-bisphosphate.
3. Use of a pharmaceutical composition according to any one of claims 1-2 for the preparation of a medicament for the prevention and/or treatment of primary dysmenorrhea, infertility.
4. The use according to claim 3, wherein the pharmaceutical composition is capable of reducing the number of torsion reactions after dysmenorrhea and increasing the time for the first torsion reaction; the pharmaceutical composition has the effects of treating primary dysmenorrhea by reducing the expression of COX-2 genes of uterine tissues, reducing the expression of PGF2 alpha, increasing the expression of PGE2 and obviously reducing the ratio of PGF2/PGE 2.
5. Use according to claim 3, characterized in that said pharmaceutical composition is capable of reducing hypertrophy of uterine tissue, reducing inflammatory cell infiltration and reducing the pathological extent of uterine tissue.
6. The use according to claim 3, wherein said pharmaceutical composition is capable of increasing the conception rate of a female individual.
7. A pharmaceutical formulation comprising a therapeutically effective amount of the pharmaceutical composition of any one of claims 1-2 and a pharmaceutically acceptable carrier, adjuvant or vehicle.
8. The pharmaceutical formulation of claim 7, wherein the pharmaceutical formulation is in the form of a gastrointestinal administration, an injection, a vaginal gel administration, or a skin administration, including a capsule, a powder, a tablet, a granule, a pill, an injection, a syrup, an oral liquid, an inhalant, a cream, an ointment, a suppository, or a patch.
9. Use of a pharmaceutical formulation according to claim 7 or 8 for the preparation of a medicament for the prevention and/or treatment of primary dysmenorrhea, infertility.
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