COMPOSITION COMPRISING BOTULINUM TOXIN OR SALT THEREOF FOR INCREASING ENDOMETRIAL BLOOD FLOW RATE

Abstract

A composition may increase a rate of endometrial blood flow and a composition for increasing implantation potential, including botulinum toxin or a salt thereof, and a pharmaceutical composition may preventing or treating subfertility or infertility, including botulinum toxin or a pharmaceutically acceptable salt thereof. Such compositions can increase a rate of endometrial blood flow and improve embryonic implantation potential just by being applied to the endometrium, and thus may be helpful to patients with subfertility or infertility, especially those who have repetitive failures in embryonic implantation. Since the compositions include botulinum toxin which is a substance that has been widely applied to the human body for cosmetic purposes, studies on human toxicity may have a shortened period, and thus the disclosure is very useful in economical and industrial aspects.

Claims

1. A composition suitable for increasing a rate of endometrial blood flow, the composition comprising: botulinum toxin or a salt thereof.

2. The composition of claim 1, wherein the botulinum toxin is botulinum toxin A.

3. The composition of claim 1, wherein a concentration of the botulinum toxin or a salt thereof is in a range of from 0.1 to 1 unit.

4. The composition of claim 1, which is suitable to increase expression of Ccl7, Cyr61, Itgb3, Foxc1, Clec14a, Hif3a, Gpx1, Cd34, Lif, Itgb3, Stc2, Dll1, Cd160, Calca, Cd31, or a combination of these.

5. The composition of claim 1, which is suitable to decrease expression of Ccbe1, Tgfbi, Stc1, Adra2b, Tbx4, or a combination of these.

6. The composition of claim 1, which is suitable to increase angiogenesis in an endometrium to increase endometrial blood flow rate.

7. A composition suitable for increasing implantation potential, the composition comprising: botulinum toxin or a salt thereof.

8. The composition of claim 7, wherein the botulinum toxin is botulinum toxin A.

9. The composition of claim 7, wherein a concentration of the botulinum toxin or a salt thereof is in a range of from 0.1 to 1 unit.

10. A pharmaceutical composition for prevention or treatment of subfertility or infertility, the pharmaceutical composition comprising: botulinum toxin or a pharmaceutically acceptable salt thereof.

11. The pharmaceutical composition of claim 10, wherein the botulinum toxin is botulinum toxin A.

12. The pharmaceutical composition of claim 10, wherein a concentration of the botulinum toxin or a pharmaceutically acceptable salt thereof is in a range of from 0.1 to 1 unit.

13. The pharmaceutical composition of claim 10, wherein the composition is suitable for application to an endometrium of a subject.

14. The pharmaceutical composition of claim 10, wherein the composition is suitable to increase angiogenesis in an endometrium to increase a rate of endometrial blood flow and to improve embryonic implantation potential.

15. The pharmaceutical composition of claim 10, wherein the subfertility or infertility is due to damaged endometrium, hypofunction of the uterus, recurrent pregnancy loss, recurrent failure of implantation of unknown cause, failure of implantation due to a thin endometrium, or a combination of these.

16. A method of preventing or treating subfertility or infertility, the method comprising: administering the pharmaceutical composition of claim 10 a subject with subfertility or infertility.

17. The composition of claim 1, comprising the salt of the botulinum toxin.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0055] FIGS. 1 and 2 show results of RNA sequencing (RNA-seq) of the RNAs extracted from both sides of the mice uterine tissues after 8 days of treating one side of the uterus with botulinum toxin A (BoTA) and treating the other side of the uterus with saline as a control group. Among the genes changed after a BoTA treatment are genes related to angiogenesis (FIG. 1) and genes related to embryo implantation (FIG. 2) (downregulation (blue) and upregulation (red)).

[0056] FIGS. 3 and 4 confirm similar patterns exhibited when the gene expression patterns of mice showing endometrium receptivity among the RNA-seq results performed by the same processes as in FIGS. 1 and 2 are compared with those of humans (reference: Identification of gene expression changes associated with uterine receptivity in mice, Jia-Peng He et al. Frontiers in Physiology, 2019), and show a heatmap of differentially expressed genes (DEG) based on the downregulation (green) and upregulation (red) list. Euclidian distance measurement and an average linkage clustering algorithm were used.

[0057] FIG. 5 is a diagram comparing the expression level of CD31 protein (red) which is an angiogenesis marker on the surface of the endometrium, in which extracted uterine tissues were sectioned (Area #1, #2, #3) after 8 days of the same drug treatment as in FIGS. 1 and 2, wherein CD31 protein expression is significantly increased in the BoTA-treated group.

[0058] FIGS. 6 and 7 are diagrams comparing numbers of implanted embryos after treating one side of the mice uterus with BoTA and treating the other side of the uterus with saline, injecting superovulation-inducing hormone after 7 days, and mating with a fertile male after 10 days, and the number of implantations on the side treated with BoTA was shown to be significantly increased.

MODE OF DISCLOSURE

[0059] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited thereto.

Example

[0060] 1. Experimental Method

[0061] In order to solve the problem of implantation and pregnancy failure due to an endometrial factor, botulinum toxin A (BoTA) was injected into the uterine cavity to identify an increase of endometrial angiogenesis.

[0062] BoTA was treated by applying on the endometrium and after 8 days of BoTA treatment, expression levels of the genes related to angiogenesis and endometrial receptivity were observed. To this end, dorsal parts of the mice were incised to expose upper parts of both sides of the mice uterus, and BoTA was injected to one side by using a 1 cc syringe and the same amount of saline was injected to the other side as a control group. After the drug injection, 8 days after the incised back of the mice was stitched and recovered, both sides of uterine tissues were extracted to use in additional experiments. At this time, the botulinum toxin A used in all experiments was botulinum toxin type A provided by Hugel for free, and the unit concentration shown in the drug composition was identically applied to the experiment. Based on the application of 1 unit of botox per 1 cc of saline when applied to the human skin, a BoTA concentration used in the experiment was 0.5 unit, 1 unit, and 2 units per 1 cc of saline, and an amount of 30 μg was injected into the mice uterine cavity.

[0063] In order to identify an efficacy of BoTA on embryo implantation, pregnant mare serum gonadotropin (PMSG) was injected 7 days after the injection of BoTA/saline, hCG (human chorionic gonadotropin) was injected after 9 days, the mouse was mated with a fertile male mouse after 10 days, the uterus was extracted on the 12th day, and the numbers of implanted embryos were compared.

[0064] 2. Experiment Result

[0065] (1) Change of Expression of Genes Related to Angiogenesis and Embryo Implantation after Treatment of BoTA in Uterine Cavity.

[0066] In order to identify genes related to angiogenesis and embryo transplant that are differentially expressed, 573 genes differentially expressed in the BoTA-treated group in comparison to the control group were classified according to the genetic ontology. Groups related to angiogenesis (GO: 0001525, Table 1) and embryo transplant (GO: 0007566, Table 2) were classified.

TABLE-US-00001 TABLE 1 Filter: Fold change P-value 10 BotoxDay 8/ BotoxDay 8/ Gene Control Control symbol Day 3 Day 3 Description Ccl7 7.979 0.029 chemokine (C-C motif) ligand 7 Cyr61 4.683 0.004 cysteine rich protein 61 Itgb3 2.894 0.002 integrin beta 3 Foxc1 2.803 0.001 forkhead box C1 Clec14a 2.779 0.034 C-type lectin domain family 14, member a Hif3a 2.153 0.049 hypoxia inducible factor 3, alpha subUnit Gpx1 2.125 0.026 glutathione peroxidase 1 Cd34 2.047 0.038 CD34 antigen Ccbe1 0.448 0.018 collagen and calcium binding EGF domains 1 Tgfbi 0.326 0.015 transforming growth factor, beta induced

TABLE-US-00002 TABLE 2 Filter: 4. Fold change P-value Gene BotoxDay 8/ BotoxDay 8/ symbol ControlDay 3 ControlDay 3 Description Lif 4.021 0.008 leukemia inhibitory factor Itgb3 2.894 0.002 integrin beta 3 Stc2 2.522 0.033 stanniocalcin 2 Stc1 0.447 0.018 stanniocalcin 1

[0067] As shown in FIGS. 1 and 2 (Tables 1 and 2), 8 days after BoTA treatment, 10 significantly differentially expressed genes were classified into angiogenesis gene oncology (GO) term. Genes classified to be upregulated were Ccl7, Cyr61, Itgb3, Foxc1, Clec14a, Hif3a, Gpx1 and Cd34, and genes classified to be downregulated were Ccbe1 and Tgfbi. Ccl7 is known to participate in macrophage recruitment which is essential for vascular remodeling through interaction with Hif-1a. Cyr61 and Itgb3 regulate angiogenesis by increasing adhesive strength of endothelial cells through interaction with each other. Foxc1 expressed by pericytes of the brain is required for endothelial proliferation and vascular remodeling in the brain. Hypoxia-inducible factors are one of the most important genes in angiogenesis that induces initiation of transcription of hypoxia-reactivity factors. The lack of glutathione peroxidase-1 is related to functional disorders of endothelial progenitor cells and causes angiogenic regulation disorders. Cd34 is known as a hematopoietic stem cell marker and is essential for inflammation and angiogenesis. Tgfbi downregulated in the BoTA-treated uterus is known to have an anti-angiogenesis and anti-cancer effect through interaction with FAS1 domain. Four significant genetic lists were classified to embryo transplant GO term: Lif, Itgb3, Stc2, and Stc1 which are a number of the most important factors in embryo transplant through activation of JAK/STAT and MAPK signal transduction and increased angiogenesis. It has been reported that during pregnancy, RNA levels of both Stc1 and Stc2 are increased in the endometrium, and Stc1 and Stc2 induction increase in the embryo transplant site. However, in decidualization, Stc1 is consistently expressed in the entire decidualization process, while Stc2 has a decreased expression at the end of the decidualization process. According to this report, Stc1 is involved in the entire decidualization process, while Stc2 may be mainly involved in the first decidualization process.

[0068] A recently reported study (reference: Identification of Gene Expression Changes Associated with Uterine Receptivity in Mice, Jia-Peng He et al. Frontiers in Physiology, 2019) identified changes of gene expression related to uterine receptivity in both humans and mice. A total of 115 differentially expressed genes were simultaneously identified in both humans and mice, and among them, 50 genes were upregulated in a receptive uterus compared to a non-receptive uterus, and 25 genes were downregulated. In order to find a correlation between our differentially expressed genes (DEG) 8 days after BoTA treatment and 115 DEGs reported in the reference, gene expression patterns of the two studies were compared. Among the 25 downregulated genes in receptive uteri of both humans and mice, 7 genes were also downregulated in our data, and 2 genes showed the opposite pattern (FIGS. 3 and 4). On the other hand, among the 50 upregulated genes in receptive uteri of humans and mice, 9 genes were upregulated in BoTA treated group compared to a control group, and 2 genes were downregulated (FIGS. 3 and 4). According to these data, it may be inferred that BoTA treatment on the uterus has a positive effect on angiogenesis, embryo transplant, and uterine receptivity.

[0069] (2) Identification of Increased Angiogenesis by BoTA Treatment in the Uterine Cavity

[0070] Angiogenesis is strongly related to an increase of endometrial receptivity and regeneration of endometrial tissues.

[0071] As shown in FIG. 5, expression levels of CD31 protein, which is a surrogate marker for angiogenesis in immunofluorescence staining, were compared at a BoTA-treated paraffin-embedded endometrium site and a control site, and the expression level of CD31 protein in the BoTA-treated endometrial epithelial layer was significantly increased compared to the control group.

[0072] (2) Identification of Increased Embryonic Implantation Potential by BoTA Treatment in the Uterine Cavity.

[0073] As shown in FIGS. 6 and 7, a significantly increased number of embryos implanted with clearly increased angiogenesis were identified in the BoTA-treated uterus compared to the saline-treated control group.