CN110755426B

CN110755426B - Application of rapamycin and structural analogs thereof in preparing medicines for treating diseases caused by ectopic overexpression of Msi1 gene

Info

Publication number: CN110755426B
Application number: CN201810837686.8A
Authority: CN
Inventors: 于政权; 宋永利
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-07-26
Filing date: 2018-07-26
Publication date: 2022-09-30
Anticipated expiration: 2038-07-26
Also published as: CN110755426A

Abstract

The invention discloses application of rapamycin in preparing a medicament for treating diseases caused by ectopic overexpression of an Msi1 gene. The invention uses rapamycin which is an inhibitor of an m-TOR pathway to treat skin abnormal Paget-like diseases caused by the ectopic overexpression of an Msi1 gene for the first time. The rapamycin medicament is applied to a mouse model, can well slow down the phenotype of diseases, and achieves the effect of relieving the diseases. And the developed medicine is applied to EMPD patients, and has achieved better clinical effect. The invention provides a new medicine for treating the external mammary gland Paget disease of mice and human.

Description

Application of rapamycin and structural analogs thereof in preparing medicines for treating diseases caused by ectopic overexpression of Msi1 gene

Technical Field

The invention relates to the field of biological medicine, in particular to application of rapamycin and structural analogs thereof in preparing a medicine for treating diseases caused by ectopic overexpression of an Msi1 gene.

Background

The target protein of Rapamycin (Rapamycin) is a classical serine/threonine protein kinase, which was extracted from S.hygroscopicus fermentation broth in 1975. Originally used as antifungal drugs. Previous studies reported that many genetic diseases in humans, such as tumors, diabetes, etc., are associated with abnormal activation of the m-TOR pathway, and that the m-TOR protein, with a molecular weight of 289KD, is essential for growth and development in mammals (Inoki K,2005, Nat Genet,37(1): 19-24). It has been found that m-TOR and Rictor can bind to each other to form a protein complex which phosphorylates the amino acids of the AKT protein and activates AKT, and if molecules preventing the binding of m-TOR and Rictor can be found, the activation of AKT can be prevented, and the activation of AKT is often closely related to the development of tumors, and the activation of AKT is prevented, so that the probability of tumorigenesis can be reduced (Sargasov D,2005, Science,307(5712): 1098-.

Rapamycin is a macrolide antibiotic with antioxidant and anti-aging effects, and can block PI3K-AKT-mTOR signaling pathway by blocking m-TOR, affecting cell cycle and cell growth, and using Rapamycin in cells, which can form a complex with FKBP12, and further combine with m-TOR to form a complex. In addition, it can also inhibit IL-2-mediated T cell differentiation, and can effectively prevent the occurrence of immune rejection (Andoh T F,1996, Kidney int,50(4): 1110-. In addition, Rapamycin may have a very good ameliorating effect on Th 1-and Th 2-mediated colonic inflammation (Heller F,2002, Immunity,17(5): 629-638).

Rapamycin was discovered in 1976 and although many physiological actions were reported, it was not approved as a therapeutic drug for immune rejection until 1999, many physiological actions of m-TOR have not been discovered so far, and further studies on physiological actions in vivo have been carried out (Knoop, C,2006, Semin Respir Crit Care Med). Numerous studies have shown that in malignant tumors, abnormal expression or activity of molecules in The PI3K/Akt/mTOR pathway leads to cells escaping normal senescence, can proliferate rapidly without limit, and cause normal cells to become malignant, leading to tumorigenesis (Bjorntti, M.A.2004, The mTOR pathway: a target for Cancer therapy, Nat Rev Cancer and). In glioma cells, the PI3K/Akt/mTOR signaling pathway is in an overactivated state (Nature,2008,455(7216: 1061-8)). Therefore, inhibition of the m-TOR pathway can block activation of the PI3K pathway, and can inhibit generation of partial tumors and partial gliomas.

Musashi1 belongs to a member of the RNA-binding protein family, is highly conserved evolutionarily (Gunter, K.M,2011, IUBMB LIFE63,678-685), is first discovered in experiments by the famous neurobiologist Montell in the 90 th century, and when studying the developmental process of drosophila sensory organs, important results are found, wherein wild-type sensory organ precursor cells (SOPs) form a non-neural progenitor cell IIa and a non-neural progenitor cell IIb when asymmetrically dividing, and when Musashi1 is mutated, the division mode of the SOP cells is greatly changed, two identical IIa cells are formed after division, and as a result, the phenotype of drosophila shows double seta. Due to its phenotypic characteristics, it is thought that Miyamato Musashi, the Japanese famous warrior, was held by the same warrior as a twin sword, and in order to commemorate the warrior, this gene was named Musashi (Fox, RG,2015, Annu Rev Cell Dev Biol 31, 249-267).

Musashi1 is mainly expressed in stem cells and progenitor cells, while expression in differentiated cells is relatively reduced (Sakakibara, S,2001, J.Neurosci.21,8091-8107), which when Musashi1 is knocked out in a nematode, causes a disturbance in male mating behavior, suggesting that Msi1 may regulate the activity of differentiated neural cells (Yoda, A.2000, Genecls 5, 885-895). Musashi1 can bind to (G/A) U _n AGU (n-1-3) motif, which is located in the 3' UTR region of the mRNA of the target gene (Ohy)ama, T,2012, Nucleic Acids Res.40, 3218-3231). In vivo, few Msi1 target genes were identified, such as m-numb (Imai, T,2001, Mol, cell. biol), CDKN1A (Battelli, S,2006, Nature395, 124-125). In addition, 64 Msi1 target genes were identified in 293T cells by RIP-ChIP method (de Sousa Abreuet al, 2009) compared to the control group, the mRNA of these 64 target genes was enriched to Msi 1-related subpopulations, which mainly include two classes, one, cell cycle, cell proliferation, cell differentiation and apoptosis, depending on their function for tumor formation; second, protein modification, including ubiquitination, ubiquitin cycle (Raquel de Sousa Abreu,2009, The joural of biological chemistry,284,18, 12125-.

At present, no good treatment method exists at home and abroad for treating the extramammary Paget disease, and once diagnosis is confirmed, a surgical excision method is required for treatment.

Disclosure of Invention

The invention aims to provide application of Rapamycin and structural analogues thereof in preparing a medicament for treating diseases caused by ectopic overexpression of an Msi1 gene.

In order to achieve the object, the invention provides the application of Rapamycin and structural analogues thereof in preparing medicines for treating diseases caused by ectopic overexpression of the Msi1 gene. Wherein the structural analog of Rapamycin is everolimus (RAD001), KU-0063794, AZD8055, Temsirolimus (Temsirolimus), INK128, Ridaforolimus (Ridaforolimus) or a combination thereof.

Preferably, the ectopic overexpression refers to overexpression of the Msi1 gene in the basal layer of the skin.

More preferably, the disease is Paget's disease caused by ectopic overexpression of the Msi1 gene.

Most preferably, the disease is an extramammary Paget disease due to ectopic overexpression of the Msi1 gene.

In a second aspect, the invention provides a medicament or a composition for treating a disease caused by ectopic overexpression of the Msi1 gene, wherein the effective component is Rapamycin and/or a structural analogue thereof.

In a third aspect, the invention provides a topical cream for treating Paget disease, in particular external mammary Paget disease, caused by overexpression of the Msi1 gene, the cream formulation being as follows: per 100g of distilled water, 0.5g of Rapamycin, 6g of white vaseline, 0.17g of ethylparaben, 2g of glyceryl monostearate, 2g of stearic acid, 802.8 g of tween-80, 13g of octadecanol, 33g of glycerol and 0.04g of sodium dodecyl sulfate.

When in use, the Rapamycin cream is smeared on the surface of the skin of an affected part of an EMPD patient, and the effective dose is 12.5mg cream/m ² Skin.

In a fourth aspect, the invention provides a drug for inhibiting expression of an Msi1 gene, the active ingredient of the drug is Rapamycin and/or a structural analogue thereof, and the drug can be used for treating Paget diseases caused by high expression of an Msi1 gene, in particular extramammary Paget diseases.

In the present invention, human and mouse Msi1 genes have accession numbers NM _002442.3 and NM _008629.1 in GenBank, respectively.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention uses m-TOR pathway inhibitor Rapamycin and structural analogues thereof for the first time to treat skin abnormal Paget-like diseases caused by ectopic overexpression of the Msi1 gene. The Rapamycin medicament is applied to a mouse model, can well slow down the phenotype of the disease, and achieves the effect of relieving the disease. And the developed medicine is applied to EMPD (external mammary gland Paget disease) patients, and has already achieved better clinical effect. The invention provides a new method for treating the external mammary Paget disease of mice and human.

Drawings

FIG. 1 is a schematic diagram of the construction process of the mouse model for the extramammary paget disease in example 1 of the present invention. A mice (K14rtta transgenic mice) were crossed with B mice (TRE-Msi1 transgenic mice).

FIG. 2 shows the results of the identification of the mouse model of the extramammary paget disease in example 1 of the present invention. K14rttA, 500 bp; b, Tre-msi1, 300bp and 500 bp.

FIG. 3 shows the results of genotyping mice overexpressing the Musashi1 gene of example 1. A: comparison; b: msi1 overexpressing mice (DTG mice).

FIG. 4 shows PAS staining results of DTG mice in example 1 of the present invention. The arrow indicates positive cells.

FIG. 5 shows the immunohistochemical staining results of the paget-like disease markers in example 1 of the present invention.

FIG. 6 shows HE staining results of DTG mice in example 1 of the present invention after Rapamycin was used.

FIG. 7 shows that the expression of pS6 is high and the m-TOR pathway is over-activated in the DTG mouse in example 1 of the present invention.

FIG. 8 shows that the human EMPD sample pS6 is highly expressed and the m-TOR pathway is over-activated in example 1 of the present invention.

FIG. 9 shows that the expression of pS6 was decreased and the m-TOR pathway was inhibited after Rapamycin was administered to mice in example 1 of the present invention.

FIG. 10 is a graph showing that the expression of pS6 was decreased and the m-TOR pathway was inhibited in the EMPD patients treated with Rapamycin in example 2 of the present invention.

FIG. 11 shows the decrease of PAS positive cells after the administration of Rapamycin to mice in example 1 of the present invention.

FIG. 12 shows the decrease of PAS positive cells after Rapamycin administration to EMPD patients in example 2 of the present invention.

FIG. 13 shows the reduction of CAM5.2 expression in DTG mice of example 1 after using Rapamycin.

FIG. 14 is a graph showing the decrease in CAM5.2 expression following Rapamycin administration in EMPD patients of example 2 of the invention.

FIG. 15 is a graph showing the reduction of symptoms in EMPD patients treated with Rapamycin cream in example 2 of the present invention.

FIG. 16 shows the CT examination results of the EMPD patients of example 2 of the present invention who returned to a normal phenotype after using the Rapamycin cream.

FIG. 17 shows the preparation of Rapamycin cream in example 2 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The technical process of the invention is as follows:

1. construction of mammary gland paget disease transgenic mouse model

A transgenic mouse model with expression of Musashi1 controlled by Doxycine (Dox). Dox is added into drinking water to induce the overexpression of an exogenous gene Musashi 1.

2. Abnormal activation of m-TOR (messenger-responsive receptor) channel of Paget-like transgenic mouse model is detected

Musashi1 shows typical symptoms of the extramammary paget disease after the specific overexpression of mouse skin, and shows paget-like cells, and the abnormal activation of the m-TOR channel is found in Musashi1 overexpression mice through detection. Musashi1 was found to be highly expressed by human samples and the m-TOR pathway was also aberrantly activated.

3. Use of specific drugs

In DTG mice, the mice were injected with Rapamycin at a dose of 4mg/kg, and after injection of the drug, the mice had a reduced skin paget-like disorder. Since abnormal activation of the m-TOR pathway was detected in DTG mice, the m-TOR pathway inhibitor Rapamycin was used to verify whether it had a disease-modifying effect.

4. After the drug is administered, the results of the drug effect are analyzed

The effect of phenotypic change and recovery was observed by HE staining. The mechanism of drug effect and phenotype recovery was further examined from molecular mechanisms by immunostaining.

Example 1 use of Rapamycin in the treatment of extramammary Paget disease due to overexpression of the Msi1 Gene

Preparation of an extramammary mouse paget disease model (inducible mouse model)

This example prepared a transgenic mouse model (DTG mouse) with expression of Musashi1 controlled by doxycyline (dox). Overexpression of the foreign gene Musashi1 was induced by feeding Dox to mice. After Musashi1 is overexpressed for 48h (i.e., 48h after Dox administration), symptoms of the primary extramammary paget disease can develop.

The specific preparation method of the DTG mice comprises the following steps:

the K14rtta transgenic mouse and a TRE-Msi1 transgenic mouse are mated (the construction method of the two transgenic mice is shown in Wang S, Li N, Yousefi M, Nakauka-Ddamba A, Li F, Parada K, et al (2015) Transformation of the endogenous polypeptide by the MSI2 RNA-binding protein. nat Commun 6:6517), a progeny mouse is generated, when the progeny mouse grows to 6 weeks, tetracycline with the final concentration of 0.2g/L is added into drinking water of the mouse, the mouse is allowed to freely drink water, the drinking water is ensured to be sufficient, and the gene Musashi1 is induced to be overexpressed. After being fed with tetracycline for 48 hours, the mice with the symptoms like the external mammary gland paget disease can appear, namely an external mammary gland mouse paget disease model (DTG mice).

Identification of the genotype of the DTG mice:

1. extracting mouse genome DNA as a template, performing PCR amplification (the PCR target is K14 rta gene) by taking F1 and R1 as primers, and detecting an amplification product to generate a characteristic band of 500 bp. And (3) PCR reaction system: mix 6. mu.L (Kangwei century, cat # 01037/30252), F10.6. mu.L, R10.6. mu.L, genomic DNA template 1. mu.L, ddH ₂ O3.8. mu.L. Reaction procedure: 5min at 95 ℃; 30s at 95 ℃,30 s at 61 ℃,30 s at 72 ℃ and 35 cycles; 72 ℃ for 2 min.

2. Extracting mouse genome DNA as a template, performing PCR amplification (the PCR target is TRE-Msi1 gene) by taking F2 and R2 as primers, detecting the amplification product, and generating two characteristic bands of 300bp and 500 bp. And (3) PCR reaction system: mix 6. mu.L (Kangwei century, cat # 01037/30252), F20.6. mu.L, R20.6. mu.L, G0.6. mu.L, genomic DNA template 1. mu.L, ddH ₂ O3.2. mu.L. Reaction procedure: 5min at 95 ℃; 30s at 95 ℃,30 s at 56 ℃,30 s at 72 ℃ and 35 cycles; 72 ℃ for 2 min.

Mice identified as mammary paget disease that meet both 1 and 2 above. If other genotypes are present, they are littermate control mice.

The primer sequences used were as follows (SEQ ID NOS: 1-5):

F1:5′-CACGATACACCTGACTAGCTGGGTG-3′

R1:5′-CACGATACACCTGACTAGCTGGGTG-3′

F2:5′-CCCTCCATGTGTGACCAAGG-3′

R2:5′-GCACAGCATTGCGGACATGC-3′

G:5′-GCAGAAGCGCGGCCGTCTGG-3′

(II) DTG mouse drug treatment

When DTG mice grow for 6 weeks, injecting the DTG mice, injecting a Rapamycin solution into a part of the DTG mice to serve as an experimental group, and injecting physiological saline into the other part of the DTG mice to serve as a control group (the preparation method of the Rapamycin solution is that the Rapamycin mother solution is prepared by using absolute ethyl alcohol, the concentration of Rapamycin in the mother solution is 10mg/mL, the concentration of Tween 805% and the concentration of PEG-4005%, and then the mother solution is diluted by using the physiological saline to the final concentration of Rapamycin of 1mg/mL to obtain the Rapamycin solution), wherein the daily dosage of each mouse is 0.8mg of Rapamycin/kg of body weight, and the continuous injection is carried out for 5 days. The total dose of Rapamycin administered per mouse was 4mg Rapamycin/kg body weight. On day 5, the injection was completed, and sampling was started for subsequent experiments.

(III) sampling

The control group was a DTG mouse born in the same litter and injected with physiological saline. DTG mice injected with Rapamycin solution served as experimental groups. The mice were sacrificed by dislocation of the neck, the hair on the back was removed by electric clippers, a small rectangular back skin was taken with scissors, fixed with 4% paraformaldehyde for 24 hours, washed with PBS 3 times for 20min each time, and trimmed to a tissue size of about 1cm × 0.75cm using a razor blade. The hair is cut at the angle (in the direction of the hair) and a small piece is cut off in preparation for dehydration.

(IV) dehydration

The dehydration procedure and procedure are as follows

The temperature of the last 3 steps is set to 60 ℃ to dissolve the wax, and the rest is normal temperature.

(V) embedding

Embedding in paraffin, vertically embedding, cutting into 5 μm size, spreading in 39 deg.C water bath, observing under solid microscope whether the film is completely spread, taking out the spread film with adhesive glass slide, and air drying.

(VI) phenotypic analysis of mice (HE staining)

Hematoxylin-eosin staining (HE staining) is the most common method used in cytology, histology, embryology and pathology, and can visually observe abnormal changes of tissue cells.

1. Baking slices: 63 ℃ and 1 h.

2. Dewaxing and hydrating.

3. Xylene I:15 min.

4. Xylene II 15min

5. Gradient ethanol treatment:

100% ethanol I, II: each for 5min

95% ethanol I, II: each for 5min

80% ethanol for 5min

70% ethanol for 5min

Distilled water: 5min

And (3) hematoxylin: for 10min

Tap water: 5min

95% ethanol I, II: each for 3min

Eosin: 10s

95% ethanol I, II: each for 3min

100% ethanol I, II: each for 3min

Xylene I:10min

Xylene II:10min

And sealing and taking a picture.

(VII) immunohistochemistry

1. Baking slices: 63 ℃ and 1 h.

2. Dewaxing and hydrating.

3. Xylene I:15 min.

4. Xylene II:15 min.

5. Gradient ethanol treatment:

100% ethanol I, II: each for 5min

95% ethanol I, II: each for 5min

80% ethanol for 5min

70% ethanol for 5min

Distilled water: and 5 min.

6. Antigen retrieval: boiling sodium citrate with pH of 6.0 for 20 min.

7. And (3) natural cooling: about 1 h.

PBS treatment 3 times, 5 min/time.

9.H ₂ O ₂ Light shielding for 20 min.

PBS treatment 3 times, 5 min/time.

11. And (3) sealing: the confining liquid acts for at least 1 h.

12. A first antibody: the prepared primary antibody is added according to the proper proportion and amount and stays overnight at 4 ℃.

13. Rewarming: standing at room temperature for half an hour.

PBS treatment 3 times, 5 min/time.

Solution B: room temperature for 30 min.

PBS treatment 3 times, 5 min/time.

Solution C: room temperature for 30 min.

PBS treatment 3 times, 5 min/time.

19. Distilled water: the treatment is carried out for 5 min.

DAB, stopping color development under the condition of proper color development time according to the characteristics of the antibody, and putting the antibody into distilled water.

21. And (3) hematoxylin: counterstaining for 5 min.

22. Tap water: the treatment is carried out for 5 min.

23. Gradient ethanol treatment:

70% ethanol for 3min

80% ethanol for 3min

90% ethanol for 3min

3min with 100% ethanol

Xylene I:5min

Xylene II:5min

And sealing and taking a picture.

Wherein, the B liquid and the C liquid are from a China fir Jinqiao kit, and the catalog number is as follows: sp9001, sp 9002.

(eighth) Western blot

1. Extraction of proteins

(1) Cut the skin with proper size and cut into pieces with scissors.

(2) To the above sample was added 400. mu.L of protein lysate (PMSF: IP volume ratio 1:100), and the homogenized tissue was dispersed by a homogenizer and lysed on ice for 30 min.

(3) Centrifuging at 12000rpm for 10min at 4 deg.C

(4) And (3) sucking the supernatant, taking a part of samples to detect the concentration, using the samples in a Western blot experiment, and storing the rest samples at-80 ℃.

2. Detection of protein concentration

Protein concentration was measured using Bycyanus kit BCA, 50. mu.g of each lane.

3. Electrophoresis

Concentrating the glue: 60V, separation gel: 80V.

4. Rotary film

And (5) wet-rotating, and rotating the film for 1h at 330 mA.

5. Sealing of

Blocking with 5% skimmed milk powder for 1 h.

6. A primary antibody

According to the molecular weight of protein, the membrane with proper size is cut, sealed and added with proper amount of primary antibody. 4 ℃ overnight.

7. Rewarming

Shake on a shaker for 30 min. Recovering the primary antibody.

8. Washing film

TBST 3X10 min/time

9. Second antibody

Secondary antibodies from pelagic days were incubated for 1h at room temperature.

10. Washing membrane

TBST treatment was carried out 3 times at a rate of 10 min.

11. Color development

Primary antibodies for immunohistochemistry: pS6(cell signaling technology, 1; 400, # 4858). CAM5.2(1: 50; ZSGB-BIO, ZM-0316 histochemical secondary antibody and DAB color development kit (China fir gold bridge kit).

Primary antibody for Western blot: pS6(cell signaling technology, dilution ratio 1; 1000). Beta-tubulin (Shanghai assist saint Biotech Co., Ltd., dilution ratio 1; 4000,30101ES50), secondary antibody for Western blot (Biyunnan, dilution ratio 1: 10000, cat # A0208 (rabbit source), A0216 (mouse source).

PAS staining kit: purchased from Regen Biotechnology Ltd, Beijing (cat # DG 0005).

Upon HE staining, the DTG transgenic mice were observed to develop paget-like cells. Through immunohistochemistry and western blot detection, pS6 expression is found to be increased, and PAS staining results show that a large number of PAS positive cells are generated in DTG transgenic mice, which indicates that the paget-like disease is induced in the mice and mTOR pathway is activated. After Rapamycin administration, pS6 expression decreased, cam5.2 expression decreased, and PAS positive cells decreased, indicating that after administration, the mTOR pathway was inhibited and the disease was inhibited.

FIG. 1 is a schematic diagram of the construction process of a mammary paget disease mouse model. A mice (K14rtta transgenic mice) were crossed with B mice (TRE-Msi1 transgenic mice).

FIG. 2 shows the results of the identification of a mammary paget disease mouse model. A is K14rttA, 500 bp; b, Tre-msi1, 300bp and 500 bp.

FIG. 3 shows the results of genotyping the mice overexpressing the gene Musashi 1. A: comparison; b: msi1 overexpressing mice (DTG mice).

FIG. 4 shows PAS staining results of DTG mice. The arrow indicates positive cells.

FIG. 5 is the results of immunohistochemical staining of paget-like disease markers.

FIG. 6 shows HE staining results of DTG mice with Rapamycin.

FIG. 7 shows that DTG mouse pS6 is highly expressed and the m-TOR pathway is over-activated.

FIG. 8 shows that the human EMPD sample pS6 is highly expressed and the m-TOR pathway is over-activated.

FIG. 9 shows that pS6 expression is decreased and the m-TOR pathway is inhibited after mice are administered Rapamycin.

FIG. 11 shows the decrease of PAS positive cells after mice use of Rapamycin.

FIG. 13 shows that the expression of CAM5.2 is reduced in DTG mice when Rapamycin is administered.

Example 2 preparation of Rapamycin cream and its use in the treatment of extramammary Paget disease caused by Msi1 gene overexpression

Preparation method of Rapamycin cream

1. The formula of the Rapamycin cream comprises the following components:

per 100g of distilled water, 0.5g of Rapamycin, 6g of white vaseline, 0.17g of ethylparaben, 2g of glyceryl monostearate, 2g of stearic acid, 802.8 g of tween-80, 13g of octadecanol, 33g of glycerol and 0.04g of sodium dodecyl sulfate.

2. The preparation method comprises the following steps:

the Rapamycin cream is of the O/W type (oil-in-water).

(1) Oil phase: adding the substances (white vaseline: 6g, ethylparaben: 0.17g, glyceryl monostearate 2g, stearic acid 2g, and tween-802.8 g) into an oil pan, heating and melting with a heating device, adjusting temperature to 80 deg.C, and keeping the temperature.

(2) Water phase: the substances (octadecanol 13g, glycerol 33g, distilled water added), heating to melt by a heating device, adjusting the temperature to 80 ℃, and preserving the heat.

(3) Emulsification forming: and opening a vacuum device, slowly pumping the water phase, the Rapamycin and the oil phase into a homogenizing pot in sequence, starting the homogenizing device, and stopping homogenizing after homogenizing for half an hour. Continuously stirring until the mixture is condensed to obtain the product.

FIG. 17 shows the preparation of a finished Rapamycin cream.

Therapeutic example of Rapamycin cream

The patient diagnosed as EMPD is determined as a volunteer by recruitment, and the ointment is applied to the affected part (with erythema) near the scrotum and also applied to the healthy tissue part once in the morning and at night, and each cm ² The cream is applied to the skin in an amount of about 12.5mg (preferably, the amount of the cream is adjusted according to the actual erythema area of the affected part so as to cover the erythema) and the cream is extended to the healthy tissue part. After the medicine is continuously applied for 2 weeks, the symptoms of the EMPD patients are relieved and even recovered after the medicine is applied by comparing the symptoms before and after the medicine is applied.

Significant disease remission was observed after application of Rapamycin cream in human EMPD cases.

FIG. 10 shows that pS6 expression is decreased and the m-TOR pathway is inhibited in EMPD patients following Rapamycin.

FIG. 12 shows the decrease of PAS positive cells after the administration of Rapamycin to EMPD patients.

FIG. 14 shows that CAM5.2 expression is reduced in EMPD patients following Rapamycin.

FIG. 15 shows that EMPD patients have reduced symptoms after using the Rapamycin cream.

FIG. 16 shows the results of CT examination of EMPD patients with Rapamycin cream, which returned to a substantially normal phenotype.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of agriculture in China

Application of rapamycin and structural analogues thereof in preparing medicines for treating diseases caused by ectopic overexpression of Msi1 gene

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Claims

1. The application of the rapamycin and the structural analogue thereof in preparing the medicine for treating the extramammary Paget disease is disclosed, wherein the structural analogue of the rapamycin is everolimus, KU-0063794, AZD8055, temsirolimus, INK128, despholimus or the combination thereof.

2. The use according to claim 1, wherein the medicament is a topical cream formulated as follows: per 100g of distilled water, 0.5g of rapamycin, 6g of white vaseline, 0.17g of ethylparaben, 2g of glyceryl monostearate, 2g of stearic acid, 802.8 g of tween-802.8 g, 13g of octadecanol, 33g of glycerol and 0.04g of sodium dodecyl sulfate.