Application of absolute ethyl alcohol in preparation of medicine for preventing and treating echinococcosis

Abstract

The invention discloses application of absolute ethyl alcohol in preparation of medicine for preventing and treating echinococcosis, and belongs to the technical field of biological medicine. The invention provides application of absolute ethyl alcohol in preparation of a medicine for preventing and treating echinococcosis. The echinococcosis is hepatic vesicular echinococcosis. The invention also discloses a pharmaceutical composition for preventing and treating the hepatic vesicular echinococcosis. The pharmaceutical composition comprises more than one absolute ethyl alcohol. The invention aims to provide the application of the absolute ethyl alcohol in preparation of the medicine for preventing and treating echinococcosis, the absolute ethyl alcohol has strong inhibitory activity on the echinococcosis, has great application potential, and provides a novel, safe and sustainable medicine for preventing and treating the echinococcosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of anhydrous ethanol in the preparation of a medicine for preventing and treating echinococcosis. Background Art

[0002] Alveolar echinococcosis is a serious parasitic disease caused by Echinococcus multilocularis, with pathological features of chronic infiltrative growth, which often leads to extensive liver damage. Currently, the main treatment for alveolar echinococcosis is radical liver resection, but due to the large surgical trauma and high requirements on the patient's physiological conditions, many elderly patients or patients with underlying diseases cannot bear it. In addition, for patients who cannot undergo radical resection, interventional external drainage is often used to relieve symptoms and prolong survival time.

[0003] In the treatment of hepatic cystic echinococcosis, anhydrous ethanol has been used for sclerotherapy and has shown good results. However, since the structure of hepatic alveolar echinococcosis is different from that of hepatic cystic echinococcosis, the application of anhydrous ethanol in hepatic alveolar echinococcosis has not been fully studied and reported. Anhydrous ethanol has the ability to destroy the cell membrane structure and can theoretically be used to inactivate the pathogen of hepatic alveolar echinococcosis. However, its effectiveness and safety in clinical application still need to be further verified to ensure its safety and effectiveness during treatment. Summary of the invention

[0004] The present invention aims to provide the use of anhydrous ethanol in the preparation of a drug for preventing and treating echinococcosis, thereby providing a novel, safe and sustainable drug for the prevention and treatment of alveolar echinococcosis.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] Application of anhydrous ethanol in the preparation of drugs for the prevention and treatment of echinococcosis.

[0007] Preferably, the echinococcosis is alveolar echinococcosis.

[0008] The present invention also provides a pharmaceutical composition for preventing and treating alveolar echinococcosis, comprising one or more of the anhydrous ethanol.

[0009] Preferably, the anhydrous ethanol is used as an active ingredient and a pharmaceutically acceptable carrier.

[0010] The carrier used in the pharmaceutical composition of the present invention is a common carrier available in the pharmaceutical field, including: preservatives, solubilizers, stabilizers, etc. for injectable preparations. The pharmaceutical preparation can be administered parenterally, such as intraperitoneally or topically, using an insulin needle for direct injection of anhydrous ethanol.

[0011] The present invention also provides a pharmaceutical preparation comprising a therapeutically effective amount of the anhydrous ethanol and a pharmaceutically acceptable excipient.

[0012] The pharmaceutical preparation is in the following dosage form: an injectable preparation, including an injectable solution or suspension.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects:

[0014] (1) Highly efficient inactivation of pathogens: Anhydrous ethanol can effectively inactivate the pathogens of alveolar echinococcosis.

[0015] (2) An insulin needle is used to inject anhydrous ethanol under direct vision, ensuring the accuracy and minimal invasiveness of the injection and reducing damage to surrounding healthy tissues.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The HE staining results of the control group and the experimental group in Example 1 of the present invention are shown in FIG. Figure 1 A in the figure is the HE staining result of the control group. Figure 1 B in the figure is the HE staining result of the experimental group;

[0018] Figure 2 The PAS staining results of the control group and the experimental group in Example 1 of the present invention, wherein: Figure 1 A in the figure is the PAS staining result of the control group. Figure 1 B in the figure shows the PAS staining results of the experimental group. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0020] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0021] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0022] Example 1

[0023] The experimental animals were 8-week-old SD rats weighing 200±20g. The rats were raised in an SPF environment. The experiment was carried out after adapting to the environment for one week without abnormalities. The breeding environment was SPF grade, the temperature was 24℃, and the 12-hour light and dark alternation was strictly observed. During the experiment, the animals were fed with sterile ordinary feed and sterile water was used throughout the feeding process.

[0024] Establishment of hepatic alveolar echinococcosis model and treatment plan: 4 months after modeling, the abdominal cavity of rats was opened, and rats with hepatic alveolar cyst lesions with a diameter of 0.75±0.25cm were randomly selected. Six rats were injected with normal saline and 12 rats were injected with anhydrous ethanol. The group injected with normal saline was the control group, and the group injected with anhydrous ethanol was the experimental group. Insulin needles were inserted into the center and around the liver HAE lesions. The volume of the injected liquid was equal to the volume size. The volume was referenced to [(long * short 2 ) / 2], multi-point injection was used in the injection range, especially around the lesion, to ensure that the solution could fully infiltrate, and the injection time was 1-2min. After the injection, the needle should be slowly rotated and withdrawn to avoid liquid outflow. Serum, liver, and spleen tissues of rats in each group were taken for use one month after the treatment of the two groups of animals. No rats died in the normal saline group, with a total of 6 rats. One rat died in the anhydrous ethanol group, 2 rats did not meet the standards after treatment, and 9 rats met the standards.

[0025] The effect of the above embodiment 1 was verified by the following test:

[0026] The efficacy and safety were evaluated by HE staining, glycogen staining PAS, IL-2, IL-10 and liver function. The test plan is as follows:

[0027] Hematoxylin-eosin (HE) staining

[0028] 1) Paraffin slicer cuts tissue wax block into 5μm slices, places in warm water of pathology tissue baking machine, adheres slides to take tissue slices to the middle and upper 1 / 3, avoid any possible folding or damage. Transfer the slices to 60℃ pathology tissue baking machine for drying for 30-60 minutes. Place the dried slices in a slice box for further processing;

[0029] 2) Place the slide on a metal grill and use a pathology tissue baking machine at 60-70°C to moderately soften the wax for 15 to 30 minutes;

[0030] 3) Dewaxing: Dewaxing in xylene I, II, and III for 5 min each;

[0031] 4) Dehydration: Place the sections in anhydrous ethanol, 90%, 80%, and 70% ethanol aqueous solutions for five minutes each;

[0032] 5) Rinse the sections three times with running water, 5 min each time;

[0033] 6) Use paper to remove floating impurities on the surface of the hematoxylin stain solution, and immerse the tissue sections in the hematoxylin stain solution for 3-5 minutes to fully stain the cell nuclei. Wash the sections thoroughly to remove unreacted hematoxylin residues until the water in the water tank is transparent and clear;

[0034] 7) Immerse the slices in 1% hydrochloric acid and ethanol aqueous solution for 2-3 seconds. Immediately rinse the slices with running water to remove the residual hydrochloric acid and ethanol, and soak the slices in clean water for 10 minutes to turn blue;

[0035] 8) Immerse tissue sections in 0.5% eosin aqueous solution for 5 minutes, and wash the sections with running water to remove excess eosin stain until the water becomes clear;

[0036] 9) Dehydration: Place tissue sections in 70% ethanol solution for 1 min, 80% ethanol solution for 1 min, 90% ethanol solution for 1 min, and anhydrous ethanol for 2 min;

[0037] 10) Transparent: transfer the sections to xylene I and II for 5 min each;

[0038] 11) Sealing: Place the slices in a fume hood, wait for the residual xylene to evaporate and dry completely, then drop neutral resin solution in the center of the tissue slices, gently place a cover glass and carefully eliminate bubbles. Place the slides in a well-ventilated environment, wait for the neutral resin to cure naturally, and then observe the staining results. Figure 1 .

[0039] Depend on Figure 1 It can be seen that the normal structure of the liver tissue of the rats in the control group (A) disappeared through HE staining, and irregular alveolar echinococcosis cysts were seen inside, with a large number of lymphocytes and monocytes infiltrating at the edge, and fibrous tissue proliferation around them. In the experimental group (B), broken cysts were seen, and coagulative necrosis tissue was seen around the cysts.

[0040] Periodic acid-Schiff (PAS) staining

[0041] 1) Place the slide on a metal grill and use a pathology tissue baking machine at 60-70°C to moderately soften the wax for 15 to 30 minutes;

[0042] 2) Place the sections in xylene I, II, and III for 5 minutes each;

[0043] 3) Immerse tissue sections in anhydrous ethanol, 90%, 80%, and 70% ethanol solutions for 5 minutes each;

[0044] 4) Wash the slices three times with running water, 5 minutes each time;

[0045] 5) The periodic acid solution was equilibrated to room temperature, and about 100 μL of periodic acid solution was added to each slice. The chemical reaction was carried out in a wet box for 10 minutes. The residual periodic acid solution on the slice was carefully removed, and the slice was placed in distilled water and thoroughly washed on a shaker for 5 minutes;

[0046] 6) Add 100 μL Schiff reagent to each slide, place in a humidified box, and incubate in a dark oven at 37°C for 30-60 minutes. Carefully remove the staining solution and wash the slides in distilled water. Place the slides on a shaker and wash for 5 minutes. The ideal staining time for different types of tissues may vary, so it is recommended to adjust the staining duration accordingly based on the actual staining results in each experiment;

[0047] 7) Add 100 μL of hematoxylin staining solution to each slide, ensuring that the staining treatment time is within 30 seconds. Then completely remove the staining solution and rinse the sample with distilled water at least twice, 3 seconds each time, until the floating color on the slide is completely removed;

[0048] 8) Differentiation: Add hydrochloric acid ethanol differentiation solution to treat the slide for half a minute. Remove the differentiation solution and rinse twice with tap water for 3 seconds each time until the floating color is removed, and finally soak in tap water to turn blue for 5 minutes;

[0049] 9) Dehydration and transparency: Soak the tissue sections in 70%, 80%, 90% and anhydrous ethanol for 2 minutes each. Transfer the sections to xylene I for 5 minutes and xylene II for 5 minutes;

[0050] 10) Sealing: Add a drop of neutral resin solution to the center of the tissue slice and gently place a cover glass to eliminate bubbles. Place the slide in a fume hood and wait for the neutral resin to cure naturally before observing the staining results. Figure 2 .

[0051] Depend on Figure 2 It can be seen that the stratum corneum and germinal layer of the alveolar larvae of the rats in the control group (A) are regular and have a regular shape through glycogen staining (PAS). The stratum corneum and germinal layer of the alveolar larvae of the rats in the experimental group (B) are broken and have an irregular shape.

[0052] Serum ELISA for ALT / AST

[0053] 1) Serum collection: The anesthetized rats were fixed in supine position on an animal platform, the hair in the chest area was cut off, and the skin was disinfected;

[0054] Hold a No. 4-5 needle syringe in your right hand, insert the needle into the left 3rd-4th intercostal space at the point where the heart beats strongest (usually 0.5cm below the line connecting the two upper limbs and slightly to the left of the midline), pierce the heart, slowly draw back the syringe, wait for the blood to automatically enter the syringe, and then transfer it to the blood collection tube.

[0055] 2) Reagent preparation: ALT / AST matrix solution preheated to 37°C; 2,4-dinitrophenylhydrazine solution; 0.4 mol / L sodium hydroxide solution (diluted at a ratio of 4 mol / L sodium hydroxide solution: double distilled water = 1:9 before use, prepared as needed);

[0056] 3) Take a 96-well plate, set up a measurement well and a control well for each sample, and add 20 μL of matrix solution to each well. Add 5 μL of the sample to be tested to the measurement well. Gently shake the well plate to mix, and react at 37°C for 30 minutes;

[0057] 4) Add 20 μL of 2,4-dinitrophenylhydrazine solution to the measurement well and control well respectively, and add 5 μL of the sample to be tested to the control well. Gently shake the plate to mix well and react at 37°C for 20 min.

[0058] 5) Add 200 μL of 0.4 mol / L sodium hydroxide solution to the measurement well and control well respectively, shake the 96-well plate gently to mix, and place at room temperature for 15 minutes. Use a microplate reader to measure the OD value of each well at a wavelength of 505 nm (ALT) / 510 nm (AST). Use the absolute OD value (OD value of the measurement well minus the OD value of the control well) to check the standard curve and obtain the corresponding ALT / AST activity value. ALT / AST activity (U / L) = Substitute the standard curve to obtain ALT / AST activity (Kamen's unit) × 0.482 × N. (0.482: Conversion of Karmen's unit to U / L; N: dilution multiple before sample testing).

[0059] Serum Elisa detection of IL-2, IL-10, VEGF, HIF1-a

[0060] 1) Take out the ELISA kit from the 4°C refrigerator 10 minutes before the experiment, allow the liquid inside to return to room temperature, and prepare the reagents required for the experiment according to the instructions.

[0061] 2) Preparation of standard gradient working solution: Add 1 mL of universal diluent to the standard, mix well by pipetting, and pipette gently to avoid splashing. After all the white powder is dissolved, a 300 pg / mL standard is obtained, and then dilute it in multiples according to Table 1 below.

[0062] Table 1 Preparation scheme of standard products with different concentrations

[0063]

[0064]

[0065] Take out the strips needed for the experiment from the aluminum foil bag, seal the remaining strips in a sealed bag, and store them in a refrigerator at 4°C.

[0066] Sample addition: Add the standard into the reaction wells in descending order of concentration, add 100 μL of universal diluent into the last well, add 100 μL of the sample to be tested into the corresponding reaction wells, cover with the sealing film and incubate at 37°C for 1h (to reduce errors, the sample to be tested should be diluted at least 1 times with universal diluent before adding into the ELISA well, and all the sample wells to be tested should be set up with at least 3 replicates).

[0067] 3) Adding antibodies: After the incubation, take out the ELISA plate, pour out the liquid in the plate vertically, add 100 μL of the pre-prepared antibody to each well without washing, cover with a sealing film and incubate at 37°C for 1 hour.

[0068] 4) Washing: After incubation, remove the ELISA plate, pour out the liquid in the plate vertically, add 300 μL of 1× washing solution to each well and gently shake the ELISA plate to wash away the residual antibodies in the plate, discard the washing solution and pat dry on absorbent paper, and repeat washing 3 times.

[0069] 5) Add enzyme binding working solution: Add 100 μL of the enzyme binding working solution prepared in advance to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0070] Wash the plate: Wash the plate 5 times as described above.

[0071] 6) Add substrate: Add 90 μL of substrate working solution to each well, cover with sealing film and incubate at 37°C in the dark for 15 min.

[0072] 7) Termination: After the incubation, remove the ELISA plate, add 50 μL of stop solution to each well, use an ELISA reader set to a wavelength of 450 nm, and detect the OD value of each well.

[0073] 8) Use Excel to calculate the standard curve, and use the standard curve to calculate the content of the target cytokine in the serum of each well.

[0074] The results are as follows Figure 1-2 and Table 2-4.

[0075] Depend on Figure 1 and Figure 2 It can be seen that the stratum corneum and germinal layer were intact and clear in the HAE lesions of the control group rats. The vesicle structure in the central area of ​​the experimental group was incomplete, the germinal layer and stratum corneum were poor, and the liver tissue structure in the treatment edge zone was disordered, showing coagulative necrosis.

[0076] Table 2 Liver function test results of rats in each group

[0077]

[0078] Note: Comparison of serum AST and ALT levels of rats before operation with those on days 1, 3 and 7 *P<0.05; comparison of serum AST and ALT levels of rats before operation with those on day 14 P>0.05.

[0079] As shown in Table 2, after the anhydrous ethanol treatment, the liver function of rats reached the highest value on the first day, decreased on the third and seventh days, and returned to normal after the fourteenth day.

[0080] Table 3 Expression of VEGF and HIF1a in serum of HAE rats

[0081]

[0082]

[0083] Note: P<0.05 is statistically significant

[0084] As shown in Table 3, according to the OD value of the samples, the serum VEGF concentration of the experimental group rats was 507.10±57.45ng / ml, and the VEGF concentration of the control group was 742.20±79.95ng / ml. After anhydrous ethanol treatment, the IL-2 content was lower than that of the control group (t=7.166, P<0.05). The HIF-1a content of the experimental group was 936.60±142.00ng / L, and the HIF-1a content of the control group was 1189.00±49.85ng / L. After anhydrous ethanol treatment, the IL-10 content was lower than that of the control group (t=3.448, P<0.05). There is no blood vessel supply in the hepatic alveolar cysts, and the nutrition supply during the growth period mainly comes from the blood vessels of the marginal zone. After anhydrous ethanol injection treatment of hepatic alveolar cysts, VEGF and HIF1-a were lower than those of the control group, with statistical significance.

[0085] This indicates that after the alveolar larvae are treated with anhydrous ethanol, the blood vessels supplying the marginal zone of the alveolar larvae can be effectively destroyed, thereby inhibiting the growth of the alveolar larvae.

[0086] Table 4 Expression levels of IL-2 and IL-10 in serum of HAE rats

[0087] Cytokines IL-2 IL-10 OD content in serum of rats in the experimental group (ng / L) 134.70±16.87 132.8±6.35 OD content in serum of rats in the control group (ng / L) 77.75±10.38 167.3±6.25 t 5.75 4.91 P <0.05 <0.05

[0088] Note: P<0.05 is statistically significant

[0089] As shown in Table 4, according to the OD value of the sample, the serum IL-2 concentration of the experimental group rats was 134.70±16.87ng / ml, and the IL-2 concentration of the control group was 77.75±10.38ng / ml. After anhydrous ethanol treatment, the IL-2 content was higher than that of the control group (t=5.75, P<0.05). The IL-10 content of the experimental group was 132.8±6.35ng / L, and the IL-10 content of the control group was 167.3±6.25ng / L. After anhydrous ethanol treatment, the IL-10 content was lower than that of the control group (t=4.91, P<0.05).

[0090] Th1 cells mainly secrete factors such as IL-2, IFN-γ, and TNF, while Th2 cells mainly secrete factors such as IL-4, IL-5, and IL-10. IL-2 has a certain inhibitory effect on the growth of hydatids, while overexpression of IL-10 can enable Echinococcus tapeworms to escape the host's immune attack. Therefore, increased IL-2 and decreased IL-10 represent effective treatment.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of anhydrous ethanol in the preparation of drugs for the prevention and treatment of echinococcosis.

2. The application according to claim 1, characterized in that: The echinococcosis is alveolar echinococcosis.

3. A pharmaceutical composition for preventing and treating alveolar echinococcosis, comprising one or more anhydrous ethanol as described in claim 1.

4. A pharmaceutical composition according to claim 3, characterized in that: The invention comprises the anhydrous ethanol as claimed in claim 1 as an active ingredient and a pharmaceutically acceptable carrier.

5. A pharmaceutical preparation comprising a therapeutically effective amount of anhydrous ethanol as claimed in claim 1, and a pharmaceutically acceptable excipient.

Images