CN115607566A - Enema, preparation method and application thereof, and colorectal cancer treatment method - Google Patents

Abstract

The invention relates to an enema, a preparation method, application and a colorectal cancer treatment method. The enema can realize controllable release in a tumor slightly acidic environment, so that the enema can be attached to a tumor envelope in a targeted manner and wraps a tumor tissue, a tumor mass is reduced and even falls off, and intestinal obstruction symptoms are effectively relieved; the enema has low toxic and side effects and good biological safety, and can not affect normal intestinal wall tissues; no operation is needed, the anus can be reserved, the sequelae are few, and the life of the patient is not affected.

Description

Enema, preparation method and application thereof, and colorectal cancer treatment method

Technical Field

The invention relates to the technical field of biological materials, and particularly relates to an enema, a preparation method and application thereof, and a colorectal cancer treatment method.

Background

Colorectal cancer is one of the most common malignancies worldwide, with over 120 million patients diagnosed with colorectal cancer each year, over 60 million patients dying from colorectal cancer, the third and fourth leading causes of cancer death worldwide. In China, the incidence rate of colorectal cancer is second to that of lung cancer and gastric cancer, the incidence rate is the third place, the mortality rate is the second place, and the incidence rate is gradually increased in recent years.

Colorectal cancer is characterized by a high proportion of rectal cancer, about 60%, and the mid-low rectal cancer (the distance between the distal margin of a tumor and the anal margin is less than 15 cm) is the most common rectal cancer, and accounts for about 70% -80%. Surgical resection and postoperative chemotherapy are the primary methods of treating low-to-mid rectal cancer.

However, some patients have to adopt a permanent external colostomy due to the difficulty of surgically preserving the anus as the tumor penetrates into the pelvic cavity and is close to the dentate line, which leads to difficult excretion for the patients with prognosis, and 3.4-31% of patients also have incision infection. In addition, the middle-low rectal cancer patients are the middle-aged and the old, and the survival and the life quality of the middle-low rectal cancer patients are seriously threatened by the sequelae of the surgical operation.

Therefore, it has long been a challenge to treat low-and mid-grade rectal cancer to keep the anus and the function thereof as far as possible on the basis of ensuring radical treatment of tumors. Besides deepening research on conventional therapies such as surgery, radiotherapy and chemotherapy, the development of a new treatment strategy which is milder, safer and promising is urgent.

At present, no effective solution is provided aiming at the problems of serious surgical operation sequelae, incapability of retaining anus and the like in the related technology.

Therefore, there is a need in the art to develop a drug effective in treating colorectal cancer, which reduces the suffering of patients.

Disclosure of Invention

The invention aims to provide an enema, a preparation method, application and a colorectal cancer treatment method aiming at the defects in the prior art, so as to solve the problems of serious surgical sequelae, incapability of retaining anus and the like in the related technology.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect of the invention there is provided an enema comprising an LDH/EDTA layered double hydroxide material.

In some of these embodiments, the enema is a formulation administered by enema.

In some of these embodiments, the enema further comprises a mixed solution.

In some of these embodiments, the enema is used to treat colorectal cancer.

In some of these embodiments, the enema is in a liquid formulation.

In some of these embodiments, the enema is a suspension.

In some of these embodiments, the LDH/EDTA layered double hydroxide has a mass of 0.1g to 0.5g.

In some of these embodiments, the LDH/EDTA layered double hydroxide material is present at a concentration of 0.005g/mL to 0.01g/mL.

In some of these embodiments, the volume of the mixed solution is 10mL to 100mL.

In some of these embodiments, the mixed solution is sterile phosphate buffer or physiological saline.

In a second aspect of the present invention, there is provided a method of preparing an enema according to the first aspect, wherein the method comprises:

mixing the LDH/EDTA layered double hydroxide material with sterile phosphate buffer solution or normal saline to obtain the enema.

In a third aspect of the invention, there is provided a use of the enema preparation of the first aspect in the manufacture of a medicament for the treatment of colorectal cancer.

In some of these embodiments, the LDH/EDTA layered double hydroxide material is used in an amount of 0.5 g/time.

In some of these embodiments, the dosing cycle is once a week for six consecutive weeks.

In a fourth aspect of the invention, there is provided a method of treatment of colorectal cancer, the method comprising:

the enema according to the first aspect is infused into the colorectal of a patient, thereby treating colorectal cancer.

In some embodiments, the patient is administered a regimen comprising one or more characteristics selected from the group consisting of:

the patient keeps the head and feet low for at least 0.5h; and/or

The inclination angle of the head-low and foot-high positions is 20-40 degrees.

In some of these embodiments, the head low and foot high positions are inclined at an angle of 30 °.

In some of these embodiments, the patient maintains a head-low-foot high position for 1h.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the enema provided by the invention can effectively treat colorectal cancer through simple enema, can effectively perform targeted treatment on the colorectal cancer by directly performing enema at a straight position, reduces side effects on the whole body, is good in biological safety, does not need to perform an operation, can keep the anus, has few sequelae, and reduces pain of a patient.

Drawings

FIG. 1 is a transmission electron microscope image of LDH/EDTA.

FIG. 2 shows the Zeta potential of LDH/EDTA.

FIG. 3 shows the release of the drug at different pH levels of LDH/EDTA.

FIG. 4 is an image of intestinal wall tissue, wherein FIG. 4a is an image of normal intestinal wall tissue of example 2 of the present invention; fig. 4b is an image of cancerous bowel wall tissue in accordance with example 2 of the present invention.

FIG. 5 is an enteroscopy view, wherein FIG. 5a is an enteroscopy view before perfusion for example 2 of the present invention; fig. 5b is a retroperfused enteroscope of example 2 of the present invention.

Figure 6 is a graphical representation of the intensity of EpCAM before and after treatment according to example 2 of the present invention;

FIG. 7 is a view on an enteroscope, wherein FIG. 7a is a view on an enteroscope before being perfused according to example 3 of the present invention; FIG. 7b is a retroperfused enteroscope of example 3 of the present invention.

Figure 8 is a graphical representation of the intensity of EpCAM before and after treatment according to example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments provided in the present application without any inventive step are within the scope of protection of the present application.

The invention will be further illustrated with reference to the following specific examples. It should be understood that the following specific examples are given by way of illustration of the present invention, and the detailed implementation and specific operation procedures are not intended to limit the scope of the present invention.

In the invention, the LDH material composition selects zinc-aluminum ions which are relatively safe to organisms. In order to ensure that the LDH is continuously adhered to the surface of the tumor cell so as to break the cell connection and avoid the LDH from being taken into the cell by the cell to play a role of chelation, the invention prepares the LDH with positive electricity and the particle size of which is about 0.8 mu m. In fact, the particle size of the resulting LDH/EDTA layered double hydroxide material is also around 0.8 μm, which is strongly electropositive.

In the invention, the LDH material has stronger pH sensitivity and can responsively release EDTA in a tumor slightly acidic environment.

In some of these examples, the process is simpler and more stable using a homogeneous alkaline liquor method for preparing LDH materials. Specifically, a mixed solution in which a zinc source (zinc metal salt) and an aluminum source (aluminum metal salt), hexamethylenetetramine (HMT) and sodium chloride are dissolved is placed in a 120 ℃ oil bath pot, and after the oil bath is refluxed for 2 hours under the argon protection atmosphere, ethylenediamine tetraacetic acid disodium salt is added to replace interlayer anions, and the EDTA-loaded LDH is prepared and is marked as LDH/EDTA. Wherein, the temperature of the oil bath pan can be 80 ℃, and the time of refluxing the oil bath can be 22 hours. Among them, EDTA is largely intercalated between LDH layers by anion exchange, and subsequently released in a slightly acidic body environment (pH 6.5).

In some of these embodiments, the mass ratio of ethylenediaminetetraacetic acid disodium salt to the zinc source may be 1.2g:0.594g. The zinc source is preferably selected from zinc nitrate and the like. The aluminum source is preferably selected from aluminum nitrate and the like. Wherein, the molar ratio of the zinc source to the aluminum source can be 2:1. the mass ratio of the sodium nitrate to the zinc source may be 594. The mass ratio of the hexamethylene tetramine to the zinc source can be 99.

In the synthesis reaction of the LDH/EDTA, the synthesis reaction is carried out in the protective atmosphere of argon, helium and the like, so that carbon dioxide in the air is prevented from entering a system, and carbonate ions are formed, inserted into LDH layers and difficult to replace.

In the invention, an LDH/EDTA material system is filtered and cleaned by water for three times after being prepared, the powder is stored in a refrigerator at 4 ℃ after being cooled and dried, and the powder is dispersed into sterile phosphate buffer solution or physiological saline according to the concentration in the current preparation. The LDH/EDTA material system has no obvious acute or chronic pathological toxicity and abnormality on main tissues and blood indexes of an organism and has better system in-vivo safety.

In the invention, the LDH/EDTA material system can not enter cells due to large size and has electropositivity on the surface, and can be continuously adsorbed on the surface of electronegative cell membranes, EDTA is released in a tumor metaacidic microenvironment to chelate calcium ions, and the expression of related proteins depending on the calcium ions is inhibited, so that the cell connection is damaged, and adherent cells fall off greatly. And importantly, these exfoliated dispersed cells remain tightly encapsulated by the material system, inhibiting their ability to migrate to the wall.

EXAMPLE 1 enema

(I) Experimental method

The preparation process of the LDH/EDTA layered double hydroxide material is as follows:

weighing a certain amount of Zn (NO 3) 2.6H 2O (0.594 g), al (NO 3) 3.9H 2O (0.375 g), naNO3 (0.085 g) and hexamethylene tetramine (HMT) (0.702 g) and dissolving in 150mL deionized water, magnetically stirring for 20min under the protection of a high-purity argon atmosphere to discharge air, then moving the solution to a 120-DEG C oil bath pot for stirring, refluxing under the condition of high-purity argon as a protective gas, slowly adding Na2H2EDTA solution (1.2 mg and dissolving in 50mL deionized water) after 100min, continuing to react for 22H at 100 ℃, finally filtering and collecting precipitates, fully cleaning with deionized water to obtain LDH/EDTA, and finally freeze-drying to obtain powder which is stored in a refrigerator at 4 ℃.

The preparation process of the enema comprises the following steps:

adding 0.5g of LDH/EDTA layered double hydroxide material into 10-100 mL of sterile PBS buffer solution, and ultrasonically mixing uniformly to form white suspension.

(II) results of experiment

FIG. 1 is a transmission electron microscope image of LDH/EDTA, and FIG. 1 shows that the prepared LDH/EDTA is in a monodisperse two-dimensional lamellar morphology, and the particle size is 0.8-1.2 μm, so that phagocytosis by cells is avoided.

FIG. 2 shows the Zeta potential of LDH/EDTA, and it can be seen from FIG. 2 that the prepared LDH/EDTA is strongly electropositive, thereby ensuring rapid attachment to the cell membrane surface.

FIG. 3 shows the release of drug at different pH values of LDH/EDTA, and the analysis in FIG. 3 shows that the material system has more sensitive pH responsiveness, and at pH 6.5, EDTA is rapidly released from LDH/EDTA, and the release concentration is as high as 0.225mM and 0.908mM within 0.5h and 24h respectively. However, at pH 7.4, the final release concentration of EDTA was as low as 0.209mM over 24 h. This demonstrates that the material system protects normal tissue and vasculature while destroying tumor cell junctions.

Example 2

Examination of the Effect of the enema preparation of example 1 on the treatment of intestinal tumor

Experimental methods

(one) patient screening criteria

The patient inclusion criteria were as follows:

(1) Advanced colorectal cancer, tumor growth in the cavity, loss of operative opportunities

(2) Local recurrence and metastasis of colorectal cancer, blocking the intestinal lumen;

(3) The chemotherapy and immunotherapy of colorectal cancer have poor effect, and local tumors are definite;

(4) CT, MRI scoring, endoscopy diagnosis and pathological diagnosis are definitely CRC;

(5) Age 18 years old or older;

(6) The distance between the tumor part of the colorectal cancer and the anus is less than 15cm;

(7) Important viscera diseases such as non-strict center of gravity, liver, kidney and the like are not seen;

(8) The patient compliance is good, and the patient is willing to cooperate with CRC patients who are in clinical trials.

Exclusion criteria for patients were as follows:

(1) The patient has severe complications such as malnutrition, dyscrasia, ascites and other tumor terminal stage;

(2) Patients have a blood disease basis such as blood coagulation factor disorder;

(3) Patients have had bowel perforations or a greater risk of potential perforation due to colorectal cancer;

(4) The evaluation shows that the radiotherapy and chemotherapy and the immunotherapy are still available;

(5) The tumor growth cavity is not obvious in protrusion, and the invasive growth is taken as the main part;

(6) There has been extensive systemic metastasis;

(7) Pregnant or lactating women;

(8) Patients who have multiple organ tumors or have severe uncontrolled medical illness or acute infection at the same time;

(9) Patients were unable to understand the purpose of the study or did not agree to the requirements of the study;

(10) Lack of legal capability or restricted legal capability;

(11) Researchers judge that compliance is not good and cannot cooperate with sample collection;

(12) Any history, at the discretion of the investigator, may interfere with the outcome of the trial or increase patient risk.

(II) embodiment

Under the guidance of an endoscope, the enema is perfused and administered, the administration dose is 0.5 g/time, the administration system is 50 mL-100 mL/time, and the administration period is once per week and six weeks continuously;

prior to perfusion, the LDH/EDTA layered double hydroxide material is diluted in a sterile phosphate buffered saline solution to form an enema;

during perfusion, the patient maintains a head-foot-high (30 ° tilt) position for 1h.

(III) clinical evaluation criteria

(1) Performing fecal occult blood test to assess whether treatment causes bleeding;

(2) Blood routine, thromboelastogram, liver and kidney functions, electrolytes, heart and lung functions, tumor markers and blood metal index detection prompt whether treatment is safe or not;

(3) Collecting the intestinal exfoliated tissues and secretions of a patient after treatment, evaluating the types and the number of the exfoliated cells treated by LDH/EDTA, and judging whether the treatment causes wound or bleeding or not through the secretions;

(4) Contrast B ultrasonic abdominal imaging examination tumour occupy-place condition before and after treatment, detect the longest path of tumour through the enteroscope, carry out abdominal or rectum reinforcing CT and MRI inspection to patient before and after the treatment simultaneously, aassessment patient's lump size after the treatment.

Results of the experiment

Placing the LDH/EDTA layered double hydroxide material in a buffer solution on a sterile operation table for ultrasonic treatment so as to make the enema be in suspension; a patient lies on an operating table and keeps a head-down and feet-up (30-degree inclined) position; the operator pours the enema into the colon and rectum of the patient under the endoscope; after perfusion, the patient remained in a low head (30 ° tilt) position for 1h.

Figure 4 is an endoscopic view of perfusion. As shown in fig. 4a, the normal intestinal wall tissue was not enriched with LDH/EDTA layered double hydroxide material, i.e. indicating that LDH/EDTA layered double hydroxide material did not affect the normal intestinal wall tissue. As shown in fig. 4b, LDH/EDTA layered double hydroxide material coats tumor tissue like a "spider web". Therefore, the LDH/EDTA layered double hydroxide material is released in a large amount in a tumor microacid environment and is attached to the tumor surface in a targeted manner.

FIG. 5 is an enteroscopy of the patient before and after perfusion. As shown in FIG. 5a, prior to perfusion, the patient has multiple masses attached to the intestinal wall. As shown in FIG. 5b, after a single infusion treatment, the lump in the area indicated by the arrow in FIG. 5a did not drop out in FIG. 5b, and the patient had significantly relieved symptoms of ileus.

Patient feces were collected before and after perfusion treatment, and the intensity of tumor cell marker EpCAM expressed by fecal exfoliated cells before and after treatment was compared based on flow cytometry. As shown in figure 6, the intensity of surface expression of EpCAM on exfoliated cells in feces after treatment was significantly enhanced.

From the above, local perfusion treatment under endoscope guidance can effectively relieve intestinal obstruction symptoms, and a large amount of tumor cell shedding can be seen based on fecal tissue analysis after treatment of patients.

Example 3

Examination of the Effect of the enema preparation of example 1 on the treatment of intestinal tumor

Experimental methods

The experimental method of this example is substantially the same as example 2.

Results of the experiment

Placing the LDH/EDTA layered double hydroxide material in a buffer solution on a sterile operation table for ultrasonic treatment so as to make the enema be in suspension; a patient lies on an operating table and keeps a head-down and feet-up (30-degree inclined) position; the operator pours the enema into the colorectal of the patient under the endoscope; after perfusion, the patient remained in a low head (30 ° tilt) position for 1h.

FIG. 7 is an enteroscope before and after perfusion for the patient. As shown in fig. 7a, prior to perfusion, the patient had multiple masses attached to the intestinal wall. As shown in FIG. 7b, after a single infusion treatment, the mass in the area indicated by the arrow in FIG. 7a is significantly reduced in FIG. 7b, and the patient's ileus symptoms are significantly alleviated.

Patient feces were collected before and after perfusion treatment, and the intensity of tumor cell marker EpCAM expressed by fecal exfoliated cells before and after treatment was compared based on flow cytometry. As shown in figure 8, the intensity of surface expression of EpCAM on exfoliated cells in feces after treatment was significantly enhanced.

From the above, local perfusion treatment under endoscope guidance can effectively relieve intestinal obstruction symptoms, and a large amount of tumor cell shedding can be seen based on fecal tissue analysis after treatment of patients.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims (10)

1. An enema characterized by comprising LDH/EDTA layered double hydroxide material.

2. Enema according to claim 1, characterized in that the mass of the LDH/EDTA layered double hydroxide is between 0.1g and 0.5g; and/or

The concentration of the LDH/EDTA layered double hydroxide material is 0.005 g/mL-0.01 g/mL.

3. The enema according to claim 1 or 2, wherein the enema is a preparation administered by enema; and/or

The enema is used for treating colorectal cancer; and/or

The enema is a liquid preparation; and/or

The enema is a suspension.

4. An enema according to any one of claims 1 to 3, further comprising a mixed solution.

5. An enema according to claim 4, wherein said mixed solution is a sterile phosphate buffer or physiological saline; and/or

The volume of the mixed solution is 10 mL-100 mL.

6. A method for preparing an enema preparation according to any one of claims 1 to 5, comprising:

mixing the LDH/EDTA layered double hydroxide material with a sterile phosphate buffer solution or physiological saline to obtain the enema.

7. Use of an enema as claimed in any one of claims 1 to 5 in the manufacture of a medicament for the treatment of colorectal cancer.

8. Use according to claim 7, characterized in that the LDH/EDTA layered double hydroxide material is used in an amount of 0.5 g/time.

9. The use according to claim 7, wherein the administration cycle is once a week for six consecutive weeks.

10. A method of treating colorectal cancer, comprising:

infusing an enema preparation as claimed in any one of claims 1 to 4 into the colorectal of a patient;

the patient maintains the low head and high foot position for at least 0.5h; and/or

The inclination angle of the head-low and foot-high positions is 20-40 degrees.

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