Companion preparation, companion preparation suspension as well as preparation method and application of companion preparation suspension

Abstract

The invention provides a partner preparation, a partner preparation suspension as well as a preparation method and application of the partner preparation. The partner preparation comprises a capsule shell, an osmotic pressure regulating solution A and an alkaline regulator, the capsule shell comprises a biocompatible carrier material and a flexible regulator, the osmotic pressure regulating solution A is encapsulated in the capsule shell, and the alkaline regulator is dissolved in the osmotic pressure regulating solution A. When the partner preparation and the weak-base anti-tumor drug are used for treating tumors at the same time, the capsule shell is composed of the biocompatible carrier material and the flexible regulator, so that the partner preparation can penetrate into the deep part of the tumors; after penetrating into the deep part of a tumor, the partner preparation responds to the acidic environment of the tumor to release an alkaline regulator, deeply regulate and control the acidic microenvironment of the tumor and improve the pH value of an extracellular matrix of the tumor, so that a weak-base anti-tumor drug can exist in a non-ionic structure form, the cell permeability of the drug is improved, and the uptake rate of cells to the drug is increased; therefore, the tumor treatment effect of the weak-base anti-tumor medicine is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a companion preparation, a companion preparation suspension, and a preparation method and application thereof. Background Art

[0002] Compared with normal tissues, the pH of tumor extracellular matrix is ​​acidic. Tumor angiogenesis, metastasis, and tumor progression are accompanied by acidification of pH. The acidified tumor microenvironment is associated with overstimulation of several types of ion transporters, such as Na / H ion transporter (NHE1), HCO3 - / Cl - Ion transporters, H / lactate ion transporter (MCT). In addition, high glycolytic metabolism in tumors (Warburg effect) is a major factor in generating an acidic tumor microenvironment.

[0003] At present, the acidic environment of tumors has become an important therapeutic target and has been studied more and more. However, in the related art, the anti-tumor effect of weak alkaline anti-tumor drugs is weakened by the acidic tumor microenvironment, and its effect is reduced. Summary of the invention

[0004] Based on this, it is necessary to provide a companion preparation, a companion preparation suspension, and a preparation method and application thereof to improve the tumor treatment effect of weak alkaline anti-tumor drugs.

[0005] The first aspect of the present invention provides a companion preparation, comprising:

[0006] A capsule shell, including a biocompatible carrier material and a flexibility regulator;

[0007] An osmotic pressure regulating solution A is encapsulated inside the capsule shell; and

[0008] The alkaline regulator is dissolved in the osmotic pressure regulating solution A.

[0009] In some embodiments, the capsule shell has at least one of the following features:

[0010] (1) The mass ratio of the carrier material to the flexibility regulator is (2-6):(0.1-0.5);

[0011] (2) The carrier material includes a lipid material;

[0012] Optionally, the lipid material includes one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine and distearoylphosphatidylethanolamine-polyethylene glycol;

[0013] (3) The flexibility regulator includes one or more of sodium deoxycholate, sodium cholate, bile acid, propylene glycol, oleic acid, Span 80 and Tween 80.

[0014] In some embodiments, the osmotic pressure of the osmotic pressure regulating solution A is the same as the plasma osmotic pressure;

[0015] Optionally, the osmotic pressure regulator contained in the osmotic pressure regulating solution A includes one or more of glucose and sucrose;

[0016] Further optionally, the mass proportion of the osmotic pressure regulator contained in the osmotic pressure regulating solution A is 5-10%.

[0017] In some embodiments, the alkaline regulator satisfies at least one of the following conditions:

[0018] (1) The mass volume ratio of the alkaline regulator and the osmotic pressure regulating solution A is (5-15) g:50 mL;

[0019] (2) The alkaline regulator includes one or more of carbonate and bicarbonate;

[0020] Optionally, the carbonate includes one or more of sodium carbonate, potassium carbonate and ammonium carbonate;

[0021] Optionally, the bicarbonate includes one or more of sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate.

[0022] In some embodiments, the particle size of the companion formulation is 30 nm to 200 nm;

[0023] Optionally, the average particle size of the companion preparation is 50nm to 100nm; further optionally, 50nm to 80nm.

[0024] The second aspect of the present invention provides a companion preparation suspension, which comprises a plurality of companion preparations of the first aspect, wherein the plurality of companion preparations are dispersed in an osmotic pressure adjusting solution B.

[0025] In some embodiments, the osmotic pressure of the osmotic pressure adjusting solution B is the same as the plasma osmotic pressure;

[0026] Optionally, the osmotic pressure regulator contained in the osmotic pressure regulating solution B includes one or more of glucose and sucrose;

[0027] Further optionally, the mass proportion of the osmotic pressure regulator contained in the osmotic pressure regulating solution B is 5-10%.

[0028] In some embodiments, the mass volume ratio of the carrier material included in the plurality of companion formulations to the companion formulation suspension is (2-6) g:100 mL.

[0029] In some embodiments, the zeta potential of the companion formulation suspension is -20 mV to -30 mV.

[0030] The third aspect of the present invention provides a method for preparing a chaperone preparation suspension, comprising the following steps:

[0031] Mixing the carrier material, the flexibility regulator and an organic solvent to prepare a lipid solution;

[0032] Mixing the alkaline regulator and the osmotic pressure regulating solution A to prepare an aqueous solution;

[0033] The lipid solution and the aqueous solution are mixed and then dispersed to prepare colostrum;

[0034] homogenizing the colostrum to obtain an initial suspension;

[0035] The osmotic pressure adjusting solution B is used to replace the external aqueous phase in the initial suspension to prepare the companion preparation suspension.

[0036] In some embodiments, the mass ratio of the carrier material, the flexibility regulator and the alkalinity regulator is (2-6):(0.1-0.5):(5-15).

[0037] In some embodiments, during the dispersion treatment, the rotation speed is 6000 rpm to 8000 rpm, and the dispersion time is 4 min to 8 min;

[0038] Further optionally, the rotation speed of the disperser is 6000 rpm to 7000 rpm; and the dispersion time is 6 min to 8 min.

[0039] In some embodiments, during the homogenization process, the power is 100W to 500W; the homogenization time is 3min to 9min;

[0040] Further optionally, the power of the homogenizer is 300W to 400W; and the homogenization time is 5min to 7min.

[0041] In some embodiments, the step of replacing the external aqueous phase in the initial suspension with the osmotic pressure regulating solution B comprises:

[0042] The initial suspension is concentrated using an ultrafiltration device, and the osmotic pressure regulating solution B is supplemented at the same time.

[0043] In some embodiments, the organic solvent includes one or more of methanol and ethanol.

[0044] The fourth aspect of the present invention provides a comrade preparation freeze-dried powder, which is prepared by freeze-drying the comrade preparation suspension of the second aspect or the comrade preparation suspension prepared by the method of the third aspect.

[0045] The fifth aspect of the present invention provides a use of the partner preparation of the first aspect, the partner preparation suspension of the second aspect, or the partner preparation suspension prepared by the method of the third aspect in the preparation of a pharmaceutical preparation for treating tumors.

[0046] In some embodiments, the tumor comprises a digestive tract tumor;

[0047] Optionally, the digestive tract tumor includes colon cancer.

[0048] The sixth aspect of the present invention provides a pharmaceutical preparation, comprising one or more of the chaperone preparation of the first aspect, the chaperone preparation suspension of the second aspect, and the chaperone preparation suspension prepared by the method of the third aspect, and a drug.

[0049] In some embodiments, the drug includes a weak base drug;

[0050] Optionally, the weak base drugs include one or more of anthracycline drugs, camptothecin drugs and vinca alkaloid drugs;

[0051] Further optionally, the weak base drug includes one or more of doxorubicin, daunorubicin, mitoxantrone, camptothecin, irinotecan, vincristine and vinorelbine; further optionally, the weak base drug includes doxorubicin.

[0052] The above-mentioned companion preparation, companion preparation suspension, and preparation method and application thereof, wherein the companion preparation includes a capsule shell, an osmotic pressure regulating solution A encapsulated in the capsule shell, and an alkaline regulator dissolved in the osmotic pressure regulating solution A, and the capsule shell includes a biocompatible carrier material and a flexible regulator. When the companion preparation and the weak-base anti-tumor drug are used to treat tumors at the same time, since the capsule shell is composed of a biocompatible carrier material and a flexible regulator, the companion preparation can penetrate deep into the tumor; after the companion preparation penetrates deep into the tumor, it releases the alkaline regulator in response to the acidic environment of the tumor, deeply regulates the acidic microenvironment of the tumor, and increases the pH value of the tumor microenvironment, which can make the weak-base anti-tumor drug exist in the form of a non-ionic structure, improve the cell permeability of the drug, and increase the cell uptake rate of the drug, thereby improving the tumor treatment effect of the weak-base anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1This is the particle size distribution diagram of the companion preparation suspension F-Lipo prepared in Example 2;

[0054] Figure 2 This is the potential distribution diagram of the companion preparation suspension F-Lipo prepared in Example 2;

[0055] Figure 3 This is a cryo-electron micrograph of the companion preparation suspension F-Lipo prepared in Example 2;

[0056] Figure 4 The release curve of the drug of the companion preparation suspension F-Lipo prepared in Example 2 under different pH conditions (n=3);

[0057] Figure 5 The results of the study on lactic acid secretion level of CT26 cells after treatment with the companion preparation suspension F-Lipo prepared in Example 2 (n=3);

[0058] Figure 6 The results of the study on lactic acid secretion level of CT26 cells after treatment with the companion preparation suspension F-Lipo prepared in Example 2 (n=3);

[0059] Figure 7 The changes in macrophage phenotype after treatment with the companion preparation suspension F-Lipo prepared in Example 2;

[0060] Figure 8 The change of cytotoxicity after treatment with F-Lipo, the companion preparation suspension prepared in Example 2;

[0061] Fig. 9 The change of cytotoxicity after co-treatment of the companion preparation suspension F-Lipo and DOXIL prepared in Example 2;

[0062] Fig.10 The changes in cell apoptosis after co-treatment of the companion preparation suspension F-Lipo prepared in Example 2 with DOXIL;

[0063] Fig.11 The figure shows the growth inhibition of tumor spheres after co-treatment of the companion preparation suspension F-Lipo prepared in Example 2 with DOXIL.

[0064] Among them, statistical differences are expressed as: *P<0.05, **P<0.01, ***P<0.001; no statistical difference: ns. DETAILED DESCRIPTION

[0065] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0067] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0068] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0069] In this article, when referring to the unit of a data range, if there is a unit only after the right endpoint, it means that the units of the left and right endpoints are the same. For example, 50nm~80nm means that the units of the left endpoint "50" and the right endpoint "80" are both nm (nanometers).

[0070] Only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and likewise any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0071] If there is no special description, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution. If there is no special description, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0072] As used herein, "therapeutically effective amount" refers to the amount of the drug of the present invention that can elicit a biological or medical response in an individual or improve symptoms, slow or delay progression of a disease, or prevent a disease, etc. The "therapeutically effective amount" can be determined by the attending physician or veterinary practitioner and will vary with factors such as the compound, the disease state being treated, the severity of the disease being treated, the age and relevant health conditions of the individual, the route and form of administration, the judgment of the attending physician or veterinary practitioner, etc.

[0073] As used herein, "subject" refers to an animal. Preferably, the animal is a mammal. Subject also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the subject is a human.

[0074] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, the like, and combinations thereof, which are well known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated for use in the therapeutic or pharmaceutical composition.

[0075] There is no particular limitation on the administration of the companion preparation or companion preparation suspension and the antitumor drug of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc., preferably non-oral administration.

[0076] The companion preparation or companion preparation suspension and the anti-tumor drug of the present invention can be administered alone or in combination with other therapeutic drugs (such as other anti-tumor drugs).

[0077] When using a companion preparation, the companion preparation and a therapeutically effective amount of an anti-tumor drug are used in a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of a skilled physician.

[0078] Generally, in this document, "about" is used to adjust a given numerical value above or below that value by 40%, such as 30%, such as 20%, such as 10%.

[0079] The acidic tumor microenvironment weakens the anti-tumor effect of weak-base anti-tumor drugs. The technicians of the present invention have found through research that the weak-base anti-tumor nanomedicines in the tumor microenvironment undergo esterase destruction, phagocytic engulfment and other processes, release the drug from the carrier, and the free drug diffuses into the tumor cells to exert its effect. The cell permeability of weak-base anticancer drugs is related to the pH value. At neutral pH, the drug exists in a non-ionic structure with high permeability, which is conducive to improving the cell uptake rate; at acidic pH, the drug mainly exists in an ionic form with low permeability; therefore, the acidic environment of the tumor cell extracellular matrix will inhibit the uptake of weak-base anti-tumor drugs. Since the diffusion rate of weak-base anti-tumor drugs on the tumor cell membrane is slow, the anti-cancer effect of weak-base anti-tumor drugs in the acidic tumor microenvironment is reduced. In addition, long-term exposure of tumor cell membranes to the acidic tumor microenvironment can activate the multidrug transporter P-glycoprotein (pGP), and P-glycoprotein (pGP) can pump anti-tumor drugs out of cells, leading to drug resistance. Therefore, increasing the pH of tumor extracellular matrix may be one of the key points to improve the anti-tumor effect of weak alkaline drugs.

[0080] In addition, the low permeability of conventional therapeutic drugs in solid tumors seriously impairs the anti-tumor efficacy. In order to achieve significant anti-tumor effects through intravenous injection, drug carriers need to circulate in the blood and leak out from the blood vessels around the tumor, penetrate deeply into the tumor tissue, and finally be taken up by tumor cells. However, the complex biological barriers and tumor microenvironment (TME) in tumor tissue are key factors that hinder the therapeutic effects of nano drug delivery systems. Due to the enhanced permeability and retention (EPR) effect, when nanocarriers pass through the challenges of blood barriers and clearance systems to reach the tumor site, tumor tissue characteristics such as tumor extracellular matrix (ECM), increased interstitial fluid pressure (IFP), tumor vascular system, high heterogeneity of tumors, and tumor-associated fibroblasts (CAFs) will seriously hinder the diffusion and distribution of drug-loaded nanoparticles in the gap, thereby reducing the efficiency of drug delivery in vivo and affecting the anti-tumor therapeutic effect in vivo. Drug carriers can only be enriched around tumor tissues, and most of them are trapped on the surface of solid tumor tissues, resulting in poor anti-tumor effects. Therefore, tumor penetration is considered to be the most important "rate-limiting step" in cancer treatment.

[0081] Based on the above problems, one or more embodiments of the present invention provide a companion preparation, including: a capsule shell, an osmotic pressure regulating solution A and an alkaline regulator; the capsule shell includes a biocompatible carrier material and a flexible regulator, the osmotic pressure regulating solution A is encapsulated inside the capsule shell, and the alkaline regulator is dissolved in the osmotic pressure regulating solution A.

[0082] It should be noted that alkaline regulators refer to alkaline substances that can regulate the acidic microenvironment of tumors. Alkaline regulators can increase the pH value of the extracellular matrix of tumor cells.

[0083] Biocompatible carrier materials refer to carrier materials that do not cause obvious clinical reactions to the organism, such as no thrombosis, no toxicity, no allergic or inflammatory reactions, and no blood cell damage.

[0084] The flexibility regulator refers to a substance that can adjust the flexibility of the carrier material.

[0085] It should be noted that when the chaperone preparation enters the acidic microenvironment of the tumor, some protons will enter the chaperone preparation through the transient pores, combine with the alkaline regulator to produce bubbles, and cause defects in the capsule shell. A large number of protons enter the capsule shell, which can further produce more bubbles, burst the chaperone preparation, and achieve self-feedback release of the chaperone preparation.

[0086] Understandably, when the partner preparation and the weak-base anti-tumor drug are used simultaneously to treat tumors, since the capsule shell is composed of a biocompatible carrier material and a flexible regulator, it can facilitate the penetration of the partner preparation into the deep part of the tumor; after the partner preparation penetrates into the deep part of the tumor, it releases the alkaline regulator in response to the acidic environment of the tumor, deeply regulates the acidic microenvironment of the tumor, and increases the pH value of the tumor extracellular matrix, which can make the weak-base anti-tumor drug exist in the form of a non-ionic structure, improve the drug cell permeability, and increase the cell uptake rate of the drug, thereby enhancing the tumor treatment effect of the weak-base anti-tumor drug.

[0087] This companion preparation targets the acidic microenvironment of tumors and focuses on the uptake characteristics of weak alkaline drugs. It can design targeted treatment strategies in line with actual clinical needs.

[0088] In some embodiments, the mass ratio of the carrier material and the flexibility regulator in the capsule shell is (2-6): (0.1-0.5); for example, it can be (2.5-6): (0.2-0.5), (2.5-5.5): (0.2-0.4), (3-5): (0.3-0.4), (3.5-4.5): (0.2-0.3) or (4-4.5): (0.3-0.5), etc. When the mass ratio of the carrier material and the flexibility regulator in the capsule shell is within the above range, the obtained liposome can achieve good deformation performance. As an example, the mass ratio of the carrier material and the flexibility regulator in the capsule shell can be, but is not limited to, 3.3: 0.3, 4: 0.5, 2: 0.1, 6: 0.5, 5: 0.4, 5.5: 0.3, 6: 0.1, 5: 0.3, 4: 0.4 or 2.5: 0.1, etc.

[0089] In some embodiments, the carrier material comprises a lipid material; optionally, the lipid material comprises one or more of soy lecithin, egg yolk lecithin, hydrogenated soy lecithin, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine and distearoylphosphatidylethanolamine-polyethylene glycol.

[0090] In some embodiments, the flexibility modifier includes one or more of sodium deoxycholate, sodium cholate, cholic acid, propylene glycol, oleic acid, Span 80, and Tween 80.

[0091] In some embodiments, the osmotic pressure of the osmotic pressure adjusting solution A is the same as the plasma osmotic pressure, that is, the osmotic pressure adjusting solution A is an isotonic adjusting solution.

[0092] In a possible embodiment, the osmotic pressure regulator contained in the osmotic pressure regulating solution A includes one or more of glucose and sucrose.

[0093] In some possible embodiments, the mass percentage of the osmotic pressure regulator contained in the osmotic pressure regulating solution A is 5% to 10%; for example, it can be 5.5% to 10%, 5.5% to 9.5%, 6.5% to 8.5%, 7% to 8% or 7.5% to 8%, etc., without specific limitation.

[0094] As an example, the osmotic pressure regulating solution A is a 5 wt % glucose aqueous solution or a 10 wt % sucrose aqueous solution; preferably, it is a 5 wt % glucose aqueous solution.

[0095] As a possible embodiment, the mass volume ratio of the alkaline regulator and the osmotic pressure regulating solution A is (5-15) g:50 mL; for example, it can be (6-15) g:50 mL, (6-14) g:50 mL, (7-13) g:50 mL, (8-12) g:50 mL, (9-11) g:50 mL or (10-115) g:50 mL, etc., without specific limitation. When the mass volume ratio of the alkaline regulator and the osmotic pressure regulating solution A is within the above range, the obtained liposomes can achieve stable encapsulation of the alkaline regulator. As an example, the mass volume ratio of the alkaline regulator and the osmotic pressure regulating solution A can be, but is not limited to, 5g:50 mL, 8g:50 mL, 10g:50 mL, 12g:50 mL or 15g:50 mL, etc.

[0096] In some embodiments, the alkaline regulator includes one or more of carbonate and bicarbonate. Optionally, the carbonate includes one or more of sodium carbonate, potassium carbonate and ammonium carbonate. As an example, the bicarbonate includes one or more of sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate.

[0097] In some embodiments, the particle size of the companion preparation is 30nm to 200nm; for example, it can be 40nm to 200nm, 40nm to 190nm, 50nm to 180nm, 60nm to 170nm, 70nm to 160nm, 80nm to 150nm, 90nm to 140nm, 100nm to 130nm or 110nm to 120nm, etc. When the particle size of the companion preparation is within the above range, based on the penetration retention effect of the tumor, the small particle size can achieve good passive tumor targeting delivery of the companion preparation. As an example, the particle size of the companion preparation can be, but is not limited to, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm or 200nm, etc. Optionally, the average particle size of the companion preparation is 50 nm to 100 nm. Further optionally, the average particle size of the companion preparation is 50 nm to 80 nm.

[0098] One or more embodiments of the present invention further provide a companion preparation suspension, comprising a plurality of the above-mentioned companion preparations, wherein the plurality of companion preparations are dispersed in an osmotic pressure regulating solution B.

[0099] In some embodiments, the osmotic pressure of the osmotic pressure adjusting solution B is the same as the plasma osmotic pressure, that is, the osmotic pressure adjusting solution A is an isotonic adjusting solution.

[0100] As a possible implementation manner, the osmotic pressure regulator contained in the osmotic pressure regulating solution B includes one or more of glucose and sucrose.

[0101] In some possible embodiments, the mass percentage of the osmotic pressure regulator contained in the osmotic pressure regulating solution B is 5% to 10%; for example, it can be 5.5% to 10%, 5.5% to 9.5%, 6.5% to 8.5%, 7% to 8% or 7.5% to 8%, etc., without specific limitation.

[0102] As an example, the osmotic pressure adjusting solution B is a 5 wt % glucose aqueous solution or a 10 wt % sucrose aqueous solution; preferably, it is a 5 wt % glucose aqueous solution.

[0103] In some embodiments, the mass volume ratio of the carrier material included in the plurality of companion preparations to the companion preparation suspension is (2-6) g:100 mL; for example, it may be (2.5-6) ​​g:100 mL, (2.5-5.5) g:100 mL, (3-5) g:100 mL, (3.5-4.5) g:100 mL, (4-4.5) g:100 mL or (3.5-4.5) g:100 mL, etc., without specific limitation. As an example, the mass volume ratio of the carrier material included in the plurality of companion preparations to the companion preparation suspension may be, but is not limited to, 2 g:100 mL, 3.3 g:100 mL, 4 g:100 mL, 5 g:100 mL, 5.5 g:100 mL or 6 g:100 mL, etc.

[0104] In some possible embodiments, the zeta potential of the companion formulation suspension is -20 mV to -30 mV, for example, -21 mV to -30 mV, -21 mV to -29 mV, -22 mV to -28 mV, -23 mV to 27 mV, -24 mV to -26 mV, or -24 mV to -25 mV.

[0105] One or more embodiments of the present invention also provide a method for preparing a companion preparation suspension, comprising the following steps: mixing a carrier material, a flexibility regulator and an organic solvent to prepare a lipid solution; mixing an alkaline regulator and an osmotic pressure regulating solution A to prepare an aqueous solution; mixing the lipid solution and the aqueous solution and dispersing them to prepare colostrum; homogenizing the colostrum to obtain an initial suspension; and replacing the external aqueous phase in the initial suspension with an osmotic pressure regulating solution B to prepare a companion preparation suspension.

[0106] It should be noted that the initial suspension contains two aqueous phases, namely the inner aqueous phase encapsulated in the companion preparation capsule shell and the outer aqueous phase in contact with the outside of the companion preparation capsule shell, and the alkaline regulator that is not completely encapsulated in the capsule shell exists in the outer aqueous phase.

[0107] The osmotic pressure regulating solution A and the osmotic pressure regulating solution B may be the same or different, preferably the osmotic pressure regulating solution A and the osmotic pressure regulating solution B are the same. It should be noted that the osmotic pressure regulating solution A and the osmotic pressure regulating solution B are the same means that the osmotic pressure regulating solution A and the osmotic pressure regulating solution B contain the same type of osmotic pressure regulator, and the mass proportion of the osmotic pressure regulator in the two solutions is the same.

[0108] The preparation method of the companion preparation suspension has a simple process and is suitable for large-scale production by pharmaceutical companies.

[0109] In some possible embodiments, the lipid solution and the aqueous solution are mixed at 60°C to 70°C.

[0110] In some embodiments, the mass ratio of the carrier material, the flexibility regulator and the alkaline regulator is (2-6): (0.1-0.5): (5-15); for example, it can be (2.5-6): (0.2-0.5): (6-15), (2.5-5.5): (0.2-0.4): (6-14), (3-5): (0.3-0.4): (7-13), (3.5-4.5): (0.2-0.3): (8-12), (4-4.5): (0.3-0.5): (9-11) or (3.5-4): (0.2-0.4): (10-11), etc., and is not specifically limited. When the mass ratio of the carrier material, the flexibility regulator and the alkaline regulator is within the above range, the liposome has. As an example, the mass ratio of the support material, the flexibility regulator and the alkaline regulator can be, but is not limited to, 3.3:0.3:10, 4:0.5:15, 2:0.1:5, 6:0.5:8, 5:0.4:5, 4:0.5:12, 5.5:0.3:10, 6:0.1:10, 5:0.3:8, 5:0.3:5 or 6:0.5:15, etc.

[0111] In some embodiments, when the dispersion treatment is performed, the rotation speed is 6000rpm~8000rpm, for example, it can be 6200rpm~8000rpm, 6200rpm~7800rpm, 6400rpm~7600rpm, 6600rpm~7400rpm, 6800rpm~7200rpm or 7000rpm~7200rpm, etc.; the dispersion time is 4min~8min, for example, it can be 4.5min~8min, 4.5min~7.5min, 5min~7min, 5.5min~6.5min or 6min~6.5min, etc. Further optionally, the rotation speed of the disperser is 6000rpm~7000rpm; the dispersion time is 6min~8min. As an example, the mixed lipid solution and aqueous solution are dispersed by a disperser.

[0112] In some embodiments, during homogenization, the power is 100W to 500W; for example, it can be 150W to 500W, 150W to 450W, 200W to 400W, 250W to 350W or 300W to 350W, etc., without specific limitation; the homogenization time is 3min to 9min, for example, it can be 3.5min to 9min, 3.5min to 8.5min, 4min to 8min, 4.5min to 7.5min, 5min to 7min, 5.5min to 6.5min or 6min to 6.5min, etc. Further optionally, the power of the homogenizer is 300W to 400W; the homogenization time is 5min to 7min. As an example, the colostrum is homogenized by a homogenizer.

[0113] In some embodiments, the step of replacing the external aqueous phase in the initial suspension with the osmotic pressure regulating solution B includes: concentrating the initial suspension with an ultrafiltration device, and supplementing the osmotic pressure regulating solution B to achieve replacement of the external aqueous phase in the initial suspension, thereby removing the alkaline regulator contained in the external aqueous phase of the initial suspension, thereby avoiding the alkaline regulator from affecting the body when the companion preparation suspension is used.

[0114] In some embodiments, the organic solvent includes one or more of methanol and ethanol, preferably ethanol.

[0115] One or more embodiments of the present invention further provide a chaperone preparation freeze-dried powder, which is obtained by freeze-drying the above-mentioned chaperone preparation suspension or the chaperone preparation suspension prepared by the above-mentioned method. The chaperone preparation freeze-dried powder obtained by freeze-drying the chaperone preparation suspension can be further conveniently transported and stored, and is convenient for the treatment of diseases.

[0116] One or more embodiments of the present invention further provide a use of the above-mentioned chaperone preparation, the above-mentioned chaperone preparation suspension, or the chaperone preparation suspension prepared by the above-mentioned method in the preparation of a pharmaceutical preparation for treating tumors.

[0117] In some embodiments, the tumor comprises a digestive tract tumor; optionally, the digestive tract tumor comprises colon cancer.

[0118] One or more embodiments of the present invention further provide a pharmaceutical preparation, comprising one or more of the above-mentioned companion preparation, the above-mentioned companion preparation suspension, or the companion preparation suspension prepared by the above-mentioned method, and an anti-tumor drug.

[0119] In some possible embodiments, the anti-tumor drug includes a weak base drug; optionally, the weak base drug includes one or more of anthracycline drugs, camptothecin drugs and vinblastine drugs; further optionally, the weak base drug includes one or more of doxorubicin, daunorubicin, mitoxantrone, camptothecin, irinotecan, vincristine and vinorelbine; further optionally, the weak base drug includes doxorubicin.

[0120] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0121] It should be noted that the reagents and drugs, detection instruments and methods used in the following embodiments, comparative examples and test examples are as follows.

[0122] Reagents and drugs: Soy lecithin, egg yolk lecithin, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine, distearoylphosphatidylethanolamine-polyethylene glycol and hydrogenated soybean lecithin were purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.; methanol and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0123] Detection instruments and methods: laser particle size analyzer (Zetasizer Nano ZS, Malvern, UK); rotary evaporator (RE52CS, Shanghai Yarong Instrument Factory); high performance liquid chromatograph (1260 Infinity, Agilent technologies, USA); inductively coupled plasma (ICP-OES 5100, Agilent Technologies, Santa Clara, CA, USA); inductively coupled plasma-optical emission spectroscopy (ICP-OES, 5100 Agilent). The experimental methods used in the present invention are all conventional methods unless otherwise specified.

[0124] Example 1

[0125] Weigh 3 g of soybean lecithin (as a carrier material), 0.3 g of distearoylphosphatidylethanolamine-polyethylene glycol (as a carrier material), and 0.3 g of sodium cholate (as a flexibility regulator), and fully dissolve them in a mixed solvent of methanol and ethanol in a volume ratio of 2:1 to obtain a lipid solution;

[0126] An aqueous solution was prepared by dissolving 10 g of sodium bicarbonate (as an alkaline regulator) in 50 mL of a 5 wt % aqueous glucose solution (as an osmotic pressure regulating solution A);

[0127] The lipid solution was added to the aqueous solution at 60°C for hydration, and the dispersion was continued at 8000 rpm for 6 minutes using a dispersant to obtain colostrum;

[0128] The colostrum was further homogenized by a homogenizer with a power of 400 W and a homogenization time of 8 min to obtain an initial suspension;

[0129] The external aqueous phase in the initial suspension was replaced by ultrafiltration with a 5 wt % aqueous glucose solution (as osmotic pressure regulating solution B) and the amount was quantified to 100 mL to obtain a companion preparation suspension.

[0130] Example 2-12

[0131] The preparation method of the companion preparation suspension in Examples 2-12 is different from the preparation method of the companion preparation suspension in Example 1 mainly in that at least one of the type / amount of the carrier material, the type / amount of the flexibility regulator, the type / amount of the alkalinity regulator, the type / amount of the osmotic pressure regulating solution A, the type / amount of the osmotic pressure regulating solution B and the organic solvent used in preparing the lipid solution is different; see Table 1 for details.

[0132] Comparative Example 1

[0133] The difference between Comparative Example 1 and Example 1 is that no alkaline regulator is added, and the other aspects are the same, as shown in Table 1 for details.

[0134] Comparative Example 2

[0135] The difference between Comparative Example 2 and Example 1 is that no flexibility regulator is added, and the other aspects are the same, as shown in Table 1 for details.

[0136] The parameter settings in the above embodiments and comparative examples are shown in Table 1.

[0137] Table 1

[0138]

[0139] In Table 1, CS-98 represents soybean lecithin, PC-98T represents egg yolk lecithin, HSPC represents hydrogenated soybean lecithin, DSPC represents distearoylphosphatidylcholine, DSPE represents distearoylphosphatidylethanolamine, and DSPE-PEG2000 represents distearoylphosphatidylethanolamine-polyethylene glycol.

[0140] The companion preparation suspension prepared in Example 1 was recorded as F-Lipo, and the companion preparation suspension prepared in Comparative Example 2 was recorded as T-Lipo, and the following tests were performed.

[0141] Test Example 1 Particle size distribution and zeta potential determination of companion preparation suspension

[0142] Experimental steps: Accurately measure 0.1mL of the companion preparation suspension F-Lipo in a 10mL volumetric flask, add ultrapure water to dilute to the scale, and use a laser particle size analyzer to measure the particle size and Zeta potential of the companion preparation in the companion preparation suspension F-Lipo, and measure it three times in parallel. At the same time, the morphology of the companion preparation in the companion preparation suspension F-Lipo was characterized by cryo-electron microscopy.

[0143] Experimental results: Figure 1-Figure 3 As shown. Figure 1 It can be seen that the particle size of the companion preparation in the companion preparation suspension F-Lipo is concentrated between 50nm-1000nm, and the average particle size (Z average) is about 60nm; Figure 2It can be seen that the Zeta potential of the companion preparation suspension F-Lipo is around -20mV; Figure 3 It can be seen that the chaperone preparation in the chaperone preparation suspension F-Lipo measured under cryo-electron microscopy is round in shape and uniform in size.

[0144] Test Example 2 Determination of alkaline regulator content

[0145] Experimental steps: Take the companion preparation suspension F-Lipo, dilute it 10 times with methanol, centrifuge to obtain NaHCO3 precipitate, and redissolve the precipitate with 1 mL of ultrapure water for detection. Use inductively coupled plasma emission spectrometer to detect the content of sodium bicarbonate in the companion preparation F-Lipo suspension. Use the blank companion preparation without alkaline regulator in Comparative Example 1 as a negative control.

[0146] Experimental results: The sodium intensity was measured at 589 nm and quantified using an inductively coupled plasma emission spectrometer, and the sodium ion content was measured to be 47.7±2.7 mM.

[0147] Test Example 3 Stability Study

[0148] Experimental steps: A laser particle size analyzer was used to measure the change in the hydration radius of the companion preparation suspension F-Lipo within 7 days to investigate the stability of the companion preparation suspension F-Lipo.

[0149] Experimental results: The companion preparation suspension F-Lipo was placed at 4°C for 7 days. The laser particle size analyzer test showed that the particle size of the companion preparation in the companion preparation suspension F-Lipo did not change significantly within 7 days, indicating that the companion preparation suspension F-Lipo has good stability.

[0150] Test Example 4 In vitro release of alkaline regulator in companion preparation suspension

[0151] Experimental steps: Determine the encapsulation efficiency within 48 hours at pH 5.0, 6.5 and 7.4, and investigate the in vitro responsive release ability of the companion preparation suspension F-Lipo. Place the companion preparation suspension F-Lipo in a dialysis bag with a molecular weight cutoff of 8-14kDa, tie the bag tightly, and then suspend it in a 100mL brown wide-mouth bottle containing 50mL of release medium, plug it tightly, and then place the wide-mouth bottle on a 37℃ constant temperature oscillator and oscillate at a rate of 90r / min. Absorb 1mL of the release medium at 1h, 2h, 4h, 6h, 8h, 12h, 24h, and 48h, and add 1mL of the release medium at the same time. Use inductively coupled plasma-optical emission spectroscopy for quantification, calculate the cumulative release percentage of the alkaline regulator at each time point, and draw the cumulative release curve of the alkaline regulator. Use free alkaline regulator as a control.

[0152] Experimental results: Figure 4As shown, under the acidic stimulation condition of pH 5, the release amount of the alkaline regulator in the companion preparation suspension F-Lipo within 48 hours is 70% of the total amount; under the condition of pH 7.4, the release amount of the alkaline regulator is about 30% of the total amount. This shows that the companion preparation suspension F-Lipo can achieve self-feedback drug release in response to the acidic environment of the tumor, and can effectively reduce the premature release of the alkaline regulator in the systemic circulation process, thereby enhancing the enrichment of the alkaline regulator in the tumor location and releasing it in response to the acidic environment in the tumor.

[0153] Test Example 5: Flexibility Study

[0154] Experimental steps: Under the external pressure of 0.1 MPa, the performance of the companion preparation suspension F-Lipo and the companion preparation suspension T-Lipo through the 0.10 μm microporous membrane was investigated. First, the speed V of distilled water passing through the 0.10 μm microporous membrane was measured. 水 Then the speed V of the companion preparation suspension passing through the 0.10 μm microporous filter membrane was measured. 伴侣制剂 , calculate the relative transmittance P = V 伴侣制剂 / V 水 *100%.

[0155] Experimental results: Under the action of 0.1MPa external pressure, the P value of the companion preparation suspension F-Lipo is 78%, and the P value of the companion preparation suspension T-Lipo is 44%. Compared with the companion preparation capsule shell without the flexibility regulator, the companion preparation has better deformability when the companion preparation capsule shell contains the flexibility regulator.

[0156] Experimental Example 6 Study on deep tumor penetration

[0157] Experimental steps: Tumor spheres were prepared using the microporous low-viscosity fluid covering method. Weigh 0.1 g of agarose powder and place it in a conical flask. Add 10 mL of ultrapure water to prepare an agarose solution (1%). Shake gently and heat in a microwave oven for 5 minutes. Take out the conical flask and place it in a biosafety cabinet. While it is hot, add the liquid to the inner circle of the 96-well plate, 60 μL per well, and let it stand for 0.5 h. Then transfer the 96-well plate to a 37°C, 5% CO2 cell culture incubator and continue to stand for 1.5 h. Then take out the 96-well plate and place it in a biosafety cabinet. Digest and collect CT26 tumor cells in the logarithmic growth phase and culture them at a rate of 5×10 3 The cells were inoculated into a 96-well plate at a density of 100 μL per well, and sterile PBS was added to the outer circle of the 96-well plate. The plate was placed in a 37°C, 5% CO2 cell culture incubator and cultured for 7 days, with the medium changed every other day. After 7 days, tumor spheres visible to the naked eye can be formed.

[0158] The companion preparation suspension F-Lipo and the companion preparation suspension T-Lipo were labeled with coumarin 6, and sterilized by filtration with a 0.22μm sterile filter. Fluorescence quantification was performed by fluorescence spectrophotometer, and the quantification was diluted with high-glucose 1640 medium. The tumor sphere culture supernatant was discarded in the biosafety cabinet, and the medium containing T-Lipo / F-Lipo was added, in which the final concentration of coumarin 6 was 100ng / mL, GFP channel / AF488; 3 replicate wells were set for each group and marked, and the cells were placed in a 37℃, 5% CO2 cell culture incubator for further culturing for 6h. Then the culture supernatant was discarded, the tumor spheres were carefully rinsed with PBS 3 times, and the penetration of different fluorescent companion preparations into the tumor spheres in vitro was observed under a laser confocal scanning microscope.

[0159] Experimental results: Figure 5 As shown, the companion preparation suspension F-Lipo has a good ability to penetrate deep into tumors compared with the companion preparation suspension T-Lipo. This indicates that when the companion preparation capsule shell contains a flexibility regulator, the companion preparation has a better ability to penetrate deep into tumors compared with the companion preparation capsule shell that does not contain a flexibility regulator.

[0160] Experimental Example 7 Study on Lactic Acid Metabolism

[0161] Experimental steps: CT26 tumor cells were digested and collected, and 1×10 5 The cells were inoculated at a density of 10 cells / mL in a 12-well plate, 1 mL per well, and administered after 24 hours of culture. pH 6.5 high-glucose 1640 medium, pH 6.5 high-glucose 1640 medium containing NaHCO3 (10mM) and F-Lipo (5, 10, 20mM) were prepared respectively, the original medium of CT26 in the 12-well plate was discarded in a biosafety cabinet, and freshly prepared blank medium or medium containing NaHCO3 (10mM) and F-Lipo (5, 10, 20mM) was quickly added. Three replicate wells were set for each group and marked. The cells were placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, and the cell culture supernatant was collected to determine the lactic acid concentration.

[0162] Quickly mix 1mL enzyme working solution and 0.2mL color developer with 0.02mL cell culture supernatant, and place in a 37℃ constant temperature water bath for accurate reaction for 10min. At the same time, set up a blank control sample (ultrapure water) and a standard control sample (3mmol / L lactic acid) for the same operation. After 10min, add 2mL of stop solution to each tube to terminate the reaction, and immediately measure the OD value of each tube by microplate reader (detection wavelength: 530nm; detection light path: 1cm; ultrapure water zero). The concentration of lactic acid can be calculated according to the following formula: lactic acid concentration (mmol / L) = (experimental sample OD value - blank sample OD value) / (standard sample OD value - blank sample OD value) × 3mmol / L.

[0163] Experimental results: Figure 6 As shown, the companion preparation suspension F-Lipo can penetrate deep into the tumor, self-release drugs in response to the acidic microenvironment of the tumor, and deeply regulate the acidic microenvironment of the tumor.

[0164] Experimental Example 8 Regulation of the immune microenvironment

[0165] Experimental steps: The macrophages (BMDM) cultured in the incubator for 3 days basically adhered to the wall and differentiated into primary macrophages. The cells were scraped off with a cell scraper, resuspended, and cultured at 2×10 5 Cells were seeded on a 12-well plate at a density of 1 cell / mL, and 1 mL of cell suspension was added to each well. After the cells adhered to the wall, the culture medium was replaced to induce macrophage differentiation. IL-4 was diluted to a concentration of 40 ng / mL with serum-containing DMEM, and the M2 induction medium was prepared and cultured for 24 hours, and then added to macrophages to induce M2 macrophages; IFN-γ and LPS were diluted to concentrations of 20 ng / mL and 100 ng / mL with serum-containing DMEM, respectively, and the M1 induction medium was prepared and cultured for 24 hours, and then added to macrophages to induce M1 macrophages. The culture medium containing NaHCO3 (10 mM) and F-Lipo (5, 10, 20 mM) was added to the M1 and M2 macrophages induced for 24 hours, and the serum-containing DMEM culture medium was added to the control group. After 24 hours of administration, the culture medium was removed, the cells were rinsed with PBS, and trypsin was added for 5 minutes. The cells were collected and the phenotype of BMDM was detected by flow cytometry.

[0166] Experimental results: Figure 7 As shown, flow cytometry was used to detect the phenotypic changes of BMDM after drug treatment. The ratios of iNOS / CD206 in the M1, M2, NaHCO3 (10mM), and F-Lipo (5, 10, 20mM) groups were 23.98, 0.31, 1.76, 0.99, 0.88, and 4.7, respectively. From the above results, it can be seen that the companion preparation suspension F-Lipo can significantly reduce the proportion of CD206-positive M2 macrophages, increase the proportion of iNOS-positive M1 macrophages, and alleviate the tumor immunosuppressive microenvironment.

[0167] Test Example 9 Cytotoxicity

[0168] Experimental steps: Digest and collect CT26 cells in the logarithmic growth phase, count them, and use about 5×10 cells per well. 3 The cells were inoculated at a density of 100 μL in a 96-well plate. After culturing for 24 h, one group investigated the safety of F-Lipo loaded with NaHCO3, and the other group investigated the effect of F-Lipo loaded with NaHCO3 on the efficacy of doxorubicin (DOX) in DOXIL liposomes.

[0169] Group 1: 100 μL of blank culture medium was added to each well, and F-Lipo culture medium was added at pH 7.4, where the concentration of NaHCO3 in F-Lipo liposomes was 5 mM, 10 mM, and 20 mM, respectively. The administration time was 6 h, 12 h, 24 h, 36 h, and 48 h.

[0170] The second group: 100 μL of blank culture medium was added to each well, culture medium containing DOXIL at pH 6.5, and culture medium containing DOXIL+F-Lipo were added, where the NaHCO3 concentrations in F-Lipo liposomes were 5 mM, 10 mM, and 20 mM, respectively; the DOX concentrations in DOXIL liposomes were 0.05 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 1.0 μM, and 2.0 μM.

[0171] After administration, the cells were cultured for 24 h. After adding 15 μL of MTT solution (5 mg / mL) to each well, the cells were incubated for another 4 h. The culture supernatant was carefully removed. 150 μL of dimethyl sulfoxide was added to each well. The cells were gently shaken to dissolve the purple crystals. The OD value of each well of the cell culture plate was measured with an ELISA reader (the detection wavelength was 570 nm). The cell survival rate of each drug administration group was calculated according to the formula:

[0172] Cell survival rate (%) = average OD value of experimental group / average OD value of control group × 100%.

[0173] Experimental results: Changes in cytotoxicity after F-Lipo treatment Figure 8 As shown in Figure 2, the companion formulation suspension F-Lipo loaded with NaHCO3 has good safety. The effect of F-Lipo loaded with NaHCO3 on the efficacy of DOX in DOXIL liposomes is shown in Figure 2. Fig. 9 As shown. When the initial culture medium pH value is 6.5, F-Lipo loaded with NaHCO3 can effectively enhance the killing effect of DOX in DOXIL liposomes on tumor cells, and when used in combination with the companion preparation suspension F-Lipo, it has a concentration-dependent relationship. When the concentration of NaHCO3 in F-Lipo liposomes is 20mM, the efficacy of DOX in DOXIL liposomes is the highest.

[0174] Test Example 10 Apoptosis

[0175] Experimental steps: Digest and collect CT26 cells in the logarithmic growth phase, count them, and calculate the number of cells per well to be about 1×10 51 mL of DOX medium and DOX+F-Lipo medium were inoculated at a density of 10 cells per well in a 12-well plate. After culturing for 24 h, 1 mL of DOX medium containing DOX and DOX+F-Lipo medium at pH 6.5 was added to each well. The NaHCO3 concentration in the F-Lipo liposomes was 5 mM, 10 mM, and 20 mM; the DOX concentration in the DOXIL liposomes was 1.0 μM. The cells were stained with the Annexin V-FITC / PI cell apoptosis detection kit: first, Binding buffer (10×) was added to 9 times the volume of sterile ultrapure water to dilute it into 1×Binding buffer working solution; the 12-well plate of CT26 cells was taken out of the incubator, and the CT26 tumor cells were digested and collected into a 1.5 mL EP tube. Note that the blank tube without staining and the single staining tube with only one specific dye should be separated for adjusting the voltage and compensation parameters. Add an appropriate amount of 1×Binding buffer working solution to the cell pellet to make the cell concentration between 1×10 6 About 100 μL of cell suspension, no liquid was added to the blank tube, 5 μL Annexin V-FITC or 5 μL PI was added to each single-staining tube, and 5 μL Annexin VFITC and 5 μL PI were added to each sample tube, mixed and incubated in the dark for 15 minutes. After staining, 400 μL of 1×Binding buffer working solution was added to each tube, and the proportion of Annexin V-FITC or PI-positive cells was detected by flow cytometry to determine the apoptosis of cells. FITC-positive and PI-negative cells reflect that the cells are in the state of early apoptosis, while FITC-positive and PI-positive cells reflect that the cells are in the state of late apoptosis.

[0176] Experimental results: Fig.10 As shown, the efficacy of DOXIL liposomes increased when combined with the companion preparation suspension F-Lipo, and the cell apoptosis rate was the highest and dose-dependent. The companion preparation suspension F-Lipo can effectively enhance the tumor inhibitory effect of DOX in DOXIL liposomes.

[0177] Example 11 Tumor sphere growth inhibition

[0178] Experimental steps: Digest and collect CT26 tumor cells in the logarithmic growth phase, and then culture them at a rate of 5×10 4The cells were inoculated in a 96-well plate at a density of 100 μL per well, and sterile PBS was added to the outer circle of the 96-well plate. The plate was placed in a 37°C, 5% CO2 cell culture incubator and cultured for 7 days, with the medium changed every other day. After 7 days, visible tumor spheres can be formed. In the biosafety cabinet, the supernatant of the tumor sphere culture was discarded, and PBS, medium containing DOXIL, medium containing DOXIL+T-Lipo, and medium containing DOXIL+F-Lipo were added. Among them, the DOX concentration in the DOXIL liposomes was 1.0 μM, and the NaHCO3 concentration was 20 mM. Three replicate wells were set up for each group and marked, and placed in a 37°C, 5% CO2 cell culture incubator. The long and short diameters of the tumor spheres were measured and recorded on the 0th, 2nd, 4th, and 6th days after administration, and the growth of the tumor spheres was observed using an inverted microscope. The volume of the tumor sphere was calculated according to the formula: V=A×B 2 / 2). Wherein, V is the volume of the tumor sphere, A is the long diameter of the tumor sphere, and B is the short diameter of the tumor sphere.

[0179] Experimental results: The results are as follows Fig.11 As shown, both the companion preparation suspension F-Lipo and the companion preparation suspension T-Lipo can effectively improve the tumor inhibition effect of DOX in DOXIL liposomes, and the companion preparation suspension F-Lipo is better than the companion preparation suspension T-Lipo. This shows that the companion preparation suspension F-Lipo has good deep tumor penetration ability, can regulate the deep acidic microenvironment of the tumor, and more effectively improve the uptake and utilization of DOX in DOXIL liposomes, resulting in better tumor inhibition effect.

[0180] Test Example 12 In vivo tumor inhibition activity

[0181] Experimental procedures: subcutaneous injection of 5×10 5 CT26 tumor cell suspension was used to construct a mouse subcutaneous transplant tumor model. When the tumor volume grew to 50-100mm 3 The mice were randomly divided into PBS group (blank control group), F-Lipo group (20 mg / kg), DOXIL group (2 mg / kg), DOXIL group (4 mg / kg), and DOXIL+F-Lipo group (2 mg / kg+20 mg / kg), and sterilized by filtration with a sterile filter before administration. The mice were randomly divided into groups of 6 in each group and administered by tail vein injection once every two days for a total of 5 times. Before each administration, the weight of the tumor-bearing mice was weighed with an electronic scale, and the maximum diameter (denoted as a, unit: mm) and the minimum diameter (denoted as b, unit: mm) of the tumor were measured with a vernier caliper. When the mice reached the end point of the experiment, all tumor-bearing mice were euthanized, and the tumor tissues were dissected and separated. The mass of the tumor in each group was weighed with a balance and recorded, and the tumor inhibition rate was calculated:

[0182] Tumor inhibition rate (%) = (average tumor weight of the control group - average tumor weight of the experimental group) / average tumor weight of the control group.

[0183] Experimental results: as shown in Table 2.

[0184] Table 2

[0185] Group Dosage (mg / kg) Starting number of animals Final number of animals Tumor weight (g) Tumor inhibition rate (%) Blank control group — 6 6 1.28±0.19 — F-Lipo 20 6 6 1.15±0.17 10.16 DOXIL 2 6 6 0.75±0.13 41.41 DOXIL 4 6 6 0.47±0.06 63.28 DOXIL+F-Lipo 2+20 6 6 0.52±0.05 59.38

[0186] Among them, “—” means that the item does not exist.

[0187] As shown in Table 2, the tumor inhibition rate of the F-Lipo (20 mg / kg) group was 10.16%, the tumor inhibition rate of the DOXIL group (2 mg / kg) was 41.41%, the tumor inhibition rate of the DOXIL group (4 mg / kg) was 63.28%, and the tumor inhibition rate of the DOXIL+F-Lipo group (2 mg / kg+20 mg / kg) was 59.38%. Compared with the use of DOXIL alone, the combination with the companion preparation suspension F-Lipo significantly improved the therapeutic effect. This shows that the companion preparation suspension F-Lipo can achieve deep penetration of the drug into the tumor, deeply regulate the acidic microenvironment of the tumor, and exert a strong tumor inhibition effect.

[0188] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0189] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A companion preparation, characterized in that include: A capsule shell, including a biocompatible carrier material and a flexibility regulator; Osmotic pressure regulating solution A, encapsulated inside the capsule shell; and The alkaline regulator is dissolved in the osmotic pressure regulating solution A.

2. The companion formulation according to claim 1, characterized in that The capsule shell has at least one of the following features: (1) The mass ratio of the carrier material to the flexibility regulator is (2-6):(0.1-0.5); (2) The carrier material includes a lipid material; Optionally, the lipid material includes one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine and distearoylphosphatidylethanolamine-polyethylene glycol; (3) The flexibility regulator includes one or more of sodium deoxycholate, sodium cholate, bile acid, propylene glycol, oleic acid, Span 80 and Tween 80.

3. The companion formulation according to claim 1, characterized in that The osmotic pressure of the osmotic pressure regulating solution A is the same as the plasma osmotic pressure; Optionally, the osmotic pressure regulator contained in the osmotic pressure regulating solution A includes one or more of glucose and sucrose; Further optionally, the mass proportion of the osmotic pressure regulator contained in the osmotic pressure regulating solution A is 5% to 10%.

4. The companion formulation according to any one of claims 1 to 3, characterized in that The alkaline regulator satisfies at least one of the following conditions: (1) The mass volume ratio of the alkaline regulator and the osmotic pressure regulating solution A is (5-15) g:50 mL; (2) The alkaline regulator includes one or more of carbonate and bicarbonate; Optionally, the carbonate includes one or more of sodium carbonate, potassium carbonate and ammonium carbonate; Optionally, the bicarbonate includes one or more of sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate.

5. The companion formulation according to any one of claims 1 to 3, characterized in that The particle size of the companion preparation is 30nm to 200nm; Optionally, the average particle size of the companion preparation is 50nm to 100nm; further optionally, 50nm to 80nm.

6. A companion preparation suspension, characterized in that: The method comprises a plurality of the companion preparations according to any one of claims 1 to 5, wherein the plurality of the companion preparations are dispersed in an osmotic pressure regulating solution B.

7. The companion preparation suspension according to claim 6, characterized in that The osmotic pressure of the osmotic pressure regulating solution B is the same as the plasma osmotic pressure; Optionally, the osmotic pressure regulator contained in the osmotic pressure regulating solution B includes one or more of glucose and sucrose; Further optionally, the mass proportion of the osmotic pressure regulator contained in the osmotic pressure regulating solution B is 5-10%.

8. The companion preparation suspension according to claim 6, characterized in that The mass volume ratio of the carrier material included in the plurality of companion preparations to the companion preparation suspension is (2-6) g:100 mL.

9. The companion formulation suspension according to any one of claims 6 to 8, characterized in that The zeta potential of the companion preparation suspension is -20 mV to -30 mV.

10. A method for preparing the chaperone suspension according to any one of claims 6 to 9, characterized in that: The steps include: Mixing the carrier material, the flexibility regulator and an organic solvent to prepare a lipid solution; Mixing the alkaline regulator and the osmotic pressure regulating solution A to prepare an aqueous solution; The lipid solution and the aqueous solution are mixed and then dispersed to prepare colostrum; homogenizing the colostrum to obtain an initial suspension; The osmotic pressure adjusting solution B is used to replace the external aqueous phase in the initial suspension to prepare the companion preparation suspension.

11. The method for preparing the companion formulation suspension according to claim 10, characterized in that: The mass ratio of the carrier material, the flexibility regulator and the alkaline regulator is (2-6):(0.1-0.5):(5-15).

12. The method for preparing the companion formulation suspension according to any one of claims 10 to 11, characterized in that: When dispersing, the rotation speed is 6000rpm~8000rpm, and the dispersing time is 4min~8min; Further optionally, the rotation speed of the disperser is 6000 rpm to 7000 rpm; and the dispersion time is 6 min to 8 min.

13. The method for preparing the companion formulation suspension according to any one of claims 10 to 11, characterized in that: When homogenizing, the power is 100W to 500W; the homogenizing time is 3min to 9min; Further optionally, the power of the homogenizer is 300W to 400W; and the homogenization time is 5min to 7min.

14. The method for preparing the companion formulation suspension according to any one of claims 10 to 11, characterized in that: The step of replacing the external aqueous phase in the initial suspension with the osmotic pressure regulating solution B comprises: The initial suspension is concentrated using an ultrafiltration device, and the osmotic pressure regulating solution B is supplemented at the same time.

15. The method for preparing the companion formulation suspension according to any one of claims 10 to 11, characterized in that: The organic solvent includes one or more of methanol and ethanol.

16. A lyophilized powder of a companion preparation, characterized in that: The chaperone preparation suspension is prepared by freeze-drying the chaperone preparation suspension according to any one of claims 6 to 9 or the chaperone preparation suspension prepared by the method according to any one of claims 10 to 15.

17. Use of the chaperone preparation according to any one of claims 1 to 5, the chaperone preparation suspension according to any one of claims 6 to 9, or the chaperone preparation suspension prepared by the method according to any one of claims 10 to 15 in the preparation of a pharmaceutical preparation for treating tumors.

18. The use according to claim 17, characterized in that The tumors include digestive tract tumors; Optionally, the digestive tract tumor includes colon cancer.

19. A pharmaceutical preparation, characterized in that The invention comprises one or more of the companion preparation according to any one of claims 1 to 5, the companion preparation suspension according to any one of claims 6 to 9, and the companion preparation suspension prepared by the method according to any one of claims 10 to 15, and an anti-tumor drug.

20. The pharmaceutical preparation according to claim 19, characterized in that The anti-tumor drugs include weak base drugs; Optionally, the weak base drugs include one or more of anthracycline drugs, camptothecin drugs and vinca alkaloid drugs; Further optionally, the weak base drug includes one or more of doxorubicin, daunorubicin, mitoxantrone, camptothecin, irinotecan, vincristine and vinorelbine; further optionally, the weak base drug includes doxorubicin.

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