CN115721614B - alpha-KG sustained release preparation and application thereof - Google Patents
alpha-KG sustained release preparation and application thereof Download PDFInfo
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- CN115721614B CN115721614B CN202111001331.3A CN202111001331A CN115721614B CN 115721614 B CN115721614 B CN 115721614B CN 202111001331 A CN202111001331 A CN 202111001331A CN 115721614 B CN115721614 B CN 115721614B
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- ketoglutarate
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Abstract
The invention provides an alpha-ketoglutarate (alpha-KG) slow release preparation, a preparation method and application thereof, which are mainly prepared from alpha-ketoglutarate sodium salt and hypromellose, and have the advantages of simple components, safety, effectiveness, lasting effect, relatively simple preparation process and suitability for mass production; after the alpha-KG slow release preparation is orally taken, the alpha-KG can be slowly released, the stimulation to the gastrointestinal tract is avoided, and meanwhile, the alpha-KG in the human body is continuously supplemented, so that the alpha-KG slow release preparation is maintained at a required steady state level of the human body, and is particularly suitable for the aged with reduced alpha-KG level, thereby improving the health state of the human body and reducing various diseases related to the reduction of the alpha-KG such as osteoporosis and the like; recent studies have found that continuous low dose supplementation of α -KG in animals has significant anti-cancer effects. The alpha-KG sustained release preparation provided by the invention has important application value in the aspects of improving health condition, resisting tumor, prolonging life and the like.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to the field of pharmaceutical preparations, and particularly relates to an alpha-KG sustained-release preparation and a preparation method and application thereof.
Background
Alpha-Ketoglutarate (Alpha-KG) is a key metabolic intermediate product of tricarboxylic acid cycle of organisms, and has important effects on stem cell development, anti-aging, protein synthesis promotion, energy metabolism regulation and the like. alpha-KG contributes to various metabolic processes of nutrients (e.g., amino acids, glucose, fatty acids), provides energy (ATP) for vital activities of cells and material elements (e.g., biofilm lipids) required for cell proliferation. The research result of Randall M.Chin in 2014 in the journal of Nature shows that alpha-KG can delay senility and prolong the service life of adult nematodes by 50%. The research results of Ana P.Gomes in the journal of Nature in 2020 show that the content of the metabolite alpha-KG in the serum of the elderly (more than or equal to 60 years) is significantly reduced compared with the young; azar AsadiShahmirzadi et al report in Cell metaolism that the addition of α -KG to the diet of middle aged mice results in longer mice life and improved overall health. Recently, in one study published by Yuan Wang et al, nature Communications, alpha-KG was found to alleviate age-related osteoporosis and promote bone regeneration. At present, with the acceleration of population aging, the requirements of the aged population on life health and quality of life are improved, the alpha-KG of the aged is safely and continuously supplemented, the health state of the aged is improved, the occurrence of various senile diseases such as osteoporosis and the like is reduced, the service life is prolonged, and the method has important social significance.
Tumors are one of serious diseases seriously endangering human life health, and are expressed by abnormal cell hyperproliferation and differentiation, and invasion and metastasis of malignant tumor cells seriously affect the structure and functions of multiple organs of the organism, thus forming serious threat to human health and life. With the continued development of biomedicine, the continued development of chemotherapeutic drugs and targeted therapies has resulted in a continual increase in survival rates of cancer patients. However, the existing anticancer drugs often have toxic and side effects such as leucocyte and platelet reduction caused by immunosuppression, inappetence of digestive system, nausea and vomiting and the like, so that it is important to find an effective treatment mode with small toxic and side effects. Recent studies indicate that low doses of continuous supplementation with α -KG have significant anti-tumor effects (see figures 10-13) and no adverse side effects.
Aiming at the fact that the in-vivo level of the alpha-KG is reduced along with the increase of the age, and the effects of improving the health and the service life, improving the osteoporosis and resisting the tumor are achieved based on the alpha-KG, the preparation which is suitable for middle-aged and elderly people to supplement the alpha-KG is required to be developed, so that the alpha-KG of the crowd is maintained at the normal level, diseases related to the reduction of the alpha-KG are prevented, tumors are prevented, good health conditions are kept, and important social benefits are achieved.
Because the alpha-KG has strong acidity and high solubility, a large amount of acidic substances are dissolved in the gastrointestinal tract in a short time after being directly taken orally, and certain irritation is generated to the gastrointestinal tract; on the other hand, the alpha-KG has small molecular weight, is easy to be absorbed by organisms, has high metabolism speed, and is difficult for common preparations to maintain the concentration of the alpha-KG in the bodies at a long-term steady-state level.
Based on the physicochemical properties of α -KG and the rapid absorption and metabolism thereof in vivo, it is necessary to develop an oral dosage form capable of slowly releasing α -KG in the digestive tract, which is helpful for maintaining the concentration of α -KG in vivo within a physiological concentration range, and avoiding adverse effects caused by short-time impact release of α -KG in the digestive tract and rapid rise and fall of blood concentration.
The oral sustained release preparation refers to a preparation which can release the drug continuously for a long time after administration. Because the administration times are reduced, the fluctuation of the difference between peaks and valleys of the blood concentration is small, and meanwhile, the irritation to the gastrointestinal tract is reduced, the curative effect is more durable and safer, and the medicine is widely selected. Therefore, the research and development of the alpha-KG oral sustained-release preparation can help to overcome the weakness of the alpha-KG, and is expected to become an ideal product for supplementing the alpha-KG and maintaining the stable physiological concentration of the alpha-KG in the body.
Currently, some related products of alpha-KG, such as arginine-alpha-ketoglutarate (AAKG), are used as a health-care beverage for metabolism or a dietary supplement for directly supplementing the alpha-KG, such as a tablet of Kirkman in the United states, and related products for supplementing the alpha-KG are not reported in China, particularly, related reports of a slow-release preparation product of the alpha-KG are not seen.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an alpha-KG sustained-release preparation which mainly comprises alpha-ketoglutarate sodium salt and hydrophilic polymer skeleton gel material, can slowly release the alpha-KG after being orally taken, avoid the stimulation to gastrointestinal tract, and continuously supplement the alpha-KG in vivo to maintain the alpha-KG at the required steady state level of the organism, thereby improving the health state of human bodies and preventing various diseases related to the reduction of the alpha-KG. Meanwhile, the sustained low-dose supplementation of the alpha-KG in the animal body has remarkable anti-tumor effect, so that the alpha-KG sustained release preparation can be used for anti-tumor treatment. The slow release preparation is especially suitable for the elderly with reduced alpha-KG level, and has important application value in the aspects of preventing osteoporosis, preventing and treating tumors, improving health condition, prolonging service life and the like.
In addition, when the common drug sustained release agent is detected by a liquid phase mass spectrum combined technology, the polymer in the sustained release agent is easy to cause the residue of a detection pipeline, so that the technical difficulties of product detection and quality control analysis are caused. Therefore, how to prepare the alpha-KG sustained release agent meeting the application purpose by optimizing the preparation process, and the detection instrument pipeline is not blocked during the liquid phase mass spectrum combined detection, thereby facilitating the detection and ensuring the quality monitoring, and being the technical problem to be solved at present.
In view of these problems, the present invention provides a mixture, formulation or pharmaceutical formulation that is relatively simple in formulation, has a fixed content of ingredients, and is convenient to formulate and quantitatively analyze, to solve one or more of the problems currently existing in the art, including technical problems and problems in application.
Therefore, in one aspect, the invention provides an alpha-KG sustained release preparation and a hydrophilic gel skeleton, and the preparation mainly contains active ingredients and hydrophilic gel skeleton materials, and the two materials are mixed according to a certain proportion, and the product can realize the sustained release effect of the active ingredients under physiological conditions through the following preparation process. The combination can lead the alpha-KG to exist stably, is beneficial to long-term storage and does not influence the slow release effect.
In some embodiments, an alpha-ketoglutarate or an alpha-ketoglutarate salt, or an analog having similar activity thereto, is included. By "analog" is meant that, based on the structure of the active substance α -ketoglutarate or α -ketoglutarate, certain groups are attached which do not affect or substantially affect the physiological activity of the α -ketoglutarate or α -ketoglutarate.
The "hydrophilic gel skeleton" described in the present invention refers to some hydrophilic polymers which contain a large amount of hydrophilic groups, such as-OH groups, and which are capable of binding active substances and hydrophilic substances together after wetting, and are hydrated to form a gel after contact with an aqueous solution, and the active substances are contained in the gel and can be slowly released in an aqueous physiological environment, thereby achieving the purposes of slow release in the digestive tract, prolonged absorption time and reduced adverse irritation.
In some embodiments, the hydrophilic gel skeleton is any one selected from hypromellose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, and chitosan.
In some embodiments, the alpha-ketoglutarate is alpha-ketoglutarate sodium salt or alpha-ketoglutarate potassium salt.
In some embodiments, the α -ketoglutarate is disodium α -ketoglutarate; the hydrophilic gel skeleton is hydroxypropyl methylcellulose.
In addition, the research shows that most of polymer materials used for preparing the sustained-release preparation have the defects of low stability, premature decomposition and other stability of the prepared sustained-release preparation due to the influence of low pH, so that the polymer materials are not suitable for preparing the a-ketoglutarate sustained-release preparation with obvious acidity. Through repeated experiments, the hydrophilic polymer material such as hypromellose can bear the influence of low pH of alpha-KG, has unaffected stability, and is suitable for preparing alpha-KG sustained release preparations. The alpha-KG sustained release agent prepared by the hydrophilic gel skeleton has stable property and is beneficial to preservation; in addition, the hydroxypropyl methylcellulose used by the sustained-release preparation can resist the acidic environment in the stomach, so that the alpha-KG is ensured to enter the intestinal tract in a gel form for slow release.
The alpha-KG has a simple structure and no ultraviolet absorption, so that the liquid phase mass spectrum combination technology is required to be used for measuring the content of the alpha-KG, and when common polymer materials such as sodium carboxymethylcellulose (a salt-containing substance) and the like are adopted for preparing the alpha-KG sustained-release preparation, the prepared alpha-KG sustained-release preparation often causes residues of detected substances in an instrument pipeline and a chromatographic column when active components are released by mass spectrum detection and analysis, and the detection and Quality Control (QC) analysis of products are seriously influenced, so that the technical problem to be solved is needed.
Aiming at the problems, a large number of experiments show that the alpha-KG sustained release preparation prepared by adopting hydrophilic polymer materials such as hypromellose and the like has no residual problem of an instrument system pipeline when the active ingredient is released by mass spectrum detection and analysis. And the sustained release preparation prepared from the hypromellose has relatively simple process and easy preparation, and is the best choice for preparing the alpha-KG sustained release preparation. It should be noted that while hypromellose is described herein as the best choice for preparing an α -KG sustained release formulation, other hydrophilic bone polymer materials are not excluded as the same desirable choice.
In some embodiments, the composition ratio of the alpha-ketoglutarate or the alpha-ketoglutarate to the hydrophilic gel skeleton or the hypromellose is 1:0.1-0.4. In some embodiments, the ratio of the hydrophilic gel matrix to the active ingredient of the present invention can be obtained effectively by experiment.
In some aspects, a lubricant and a wetting agent are also included; the lubricant is any one selected from magnesium stearate, talcum powder, microcrystalline cellulose and compressible starch; the wetting agent is 50% -90% ethanol solution.
In some aspects, the lubricant is magnesium stearate; the wetting agent is 70% ethanol solution.
In some embodiments, the sustained release formulation is in the form of a tablet or capsule; the content of alpha-KG in each tablet or capsule is 100 mg-1000 mg.
In a second aspect of the present invention, the present invention provides a method for preparing an α -KG sustained release preparation, which mainly comprises the following steps:
1) Mixing alpha-ketoglutaric acid or alpha-ketoglutaric acid salt with hydrophilic gel skeleton material, sieving, grinding, and mixing;
2) Adding wetting agent to obtain soft material, extruding, granulating, and drying;
3) Making into capsule, or adding lubricant, and making into tablet.
In some embodiments, the hydrophilic gel skeleton is any one selected from hypromellose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, and chitosan.
In some embodiments, the alpha-ketoglutarate is alpha-ketoglutarate sodium salt or alpha-ketoglutarate potassium salt.
In some embodiments, the α -ketoglutarate is disodium α -ketoglutarate; the hydrophilic gel skeleton is hydroxypropyl methylcellulose.
In some embodiments, the composition ratio of the alpha-ketoglutarate or the alpha-ketoglutarate to the hypromellose is 1:0.1 to 0.4.
In some embodiments, the wetting agent is a 50% -90% ethanol solution; the lubricant is any one selected from magnesium stearate, talcum powder, microcrystalline cellulose and compressible starch.
In some embodiments, the wetting agent is a 70% ethanol solution; the lubricant is magnesium stearate.
In a third aspect of the present invention, there is provided an application of the α -KG sustained release preparation as described above in preparing a medicament in the field of antitumor or the α -KG sustained release preparation prepared by the method as described above.
In some embodiments, the anti-tumor field includes one or more of lung cancer, breast cancer, liver cancer, acute leukemia and malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, nephroblastoma, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, gastric cancer, medullary thyroid cancer.
In some embodiments, the anti-tumor domain is a liver cancer domain.
In a fourth aspect, the invention provides a pharmaceutical combination or combination formulation comprising α -KG, and doxorubicin or/and cisplatin.
In some embodiments, the α -KG is in the form of a sustained release formulation, although α -KG may also be a non-sustained release formulation. When the alpha-KG is in the form of a sustained release formulation, it may comprise any of the sustained release formulations as described above and different dosage forms, as well as different proportional relationships between the drugs.
In some embodiments, the anti-tumor includes one or more of lung cancer, breast cancer, liver cancer, acute leukemia and malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, nephroblastoma, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, gastric cancer, medullary thyroid cancer.
In some embodiments, the anti-tumor field is a breast cancer field.
Further, the anti-tumor field is lung cancer field.
The "drug combination" of the present invention is generally a combination of two different drugs for the treatment of a disease, and may include the administration of the two drugs separately in a certain order, for example, the α -KG sustained release formulation of the present invention is administered first, followed by administration of doxorubicin and/or cisplatin at intervals, and it is understood that such order may also be administered after administration of doxorubicin and/or cisplatin at intervals, using the α -KG sustained release formulation. Of course, in other embodiments, both drugs are present in a mixed state and administered simultaneously or nearly simultaneously. In addition, the combination is not limited to the combination of two drugs of the present invention, and may be combined with other drugs, so that three drugs can be administered simultaneously or sequentially, or three or more drugs can be administered simultaneously or sequentially.
The invention has the beneficial effects that:
(1) The alpha-KG oral sustained release preparation has simple components, only comprises active components and the sustained release agent, but does not comprise other components, is safe and effective, and has lasting effect; after oral administration, the alpha-KG is slowly released, so that the stimulation to the gastrointestinal tract is avoided, the alpha-KG in the human body is continuously supplemented, the steady-state level of the alpha-KG required by the human body is maintained for a long time, various diseases related to the reduction of the alpha-KG are prevented, the health state of the human body is improved, and the service life is prolonged;
(2) The preparation process is simple, the operation is easy, and the method is suitable for expanding production;
(3) Provides the application of the alpha-KG oral slow release preparation in anti-tumor treatment;
(4) The medicine can avoid adverse effects caused by short-time impact release of alpha-KG in the alimentary canal and rapid rise and fall of blood concentration, has good clinical compliance, provides an ideal dosage form for stably supplementing the alpha-KG for people with reduced alpha-KG, especially middle-aged and elderly people, meets physiological requirements, and is beneficial to promoting health; simultaneously, a new treatment mode and a pharmaceutical preparation are provided for tumor patients.
(5) Provides the pharmaceutical composition combination of alpha-KG and doxorubicin and the application thereof in anti-tumor treatment, and has synergistic effect;
(6) Provides a combined preparation of alpha-KG and cisplatin and the application thereof in anti-tumor treatment, and can also achieve the synergistic effect.
Drawings
FIG. 1 is a schematic view showing the α -KG slow-release granule produced in example 1;
FIG. 2 is a sustained release capsule of α -KG prepared in example 1;
FIG. 3 is a sustained release tablet of α -KG prepared in example 2;
FIG. 4 is a schematic diagram showing the content of alpha-KG in the alpha-KG slow-release capsules and the alpha-KG slow-release tablets prepared in example 1 and example 2;
FIG. 5 is a chart of the measurement of α -KG by LC-MS in example 3;
FIG. 6 is a standard curve of the determination of α -KG using LC-MS in example 3;
FIG. 7 is a graph showing the release profile of the α -KG sustained release capsules and α -KG stock from the capsules in example 3;
FIG. 8 is a graph showing the release profile of the sustained release of the α -KG tablets and the α -KG drug substance from the capsules in example 3;
FIG. 9 is a graph showing comparison of alpha-KG plasma levels of the impact dose (after 1h of gastric lavage) and the mode of administration of the sustained release dosage form (drinking water containing alpha-KG) of the mice of example 5;
FIG. 10 shows the therapeutic effect of α -KG on nude mice model of liver cancer transplantation in example 6;
FIG. 11 shows the results of a test of a-KG alone or in combination with cisplatin as a clinical chemotherapeutic in example 6 on lung cancer transplants (nude mouse model), wherein (A) is a tumor growth curve; and (B) is a weight change curve of the mice.
FIG. 12 is a graph showing the therapeutic effect of α -KG alone or in combination with doxorubicin as a clinical chemotherapeutic in example 6 on breast cancer transplants (nude mouse model), wherein (A) tumor growth curves; (B) Mice body weight change curves (x) represent statistically very significant differences).
FIG. 13 shows the results of inhibition of tumor angiogenesis by α -KG in example 6.
Detailed Description
The invention will now be described in further detail with reference to the following examples, which are intended to facilitate an understanding of and how the invention may be practiced, but which are merely limited examples of how the invention may be practiced and do not constitute any limitation to the invention and its applications. The specific scope of the invention is presented in the claims of the invention. The reagents used in this example are all known products and are obtained by purchasing commercially available products.
Example 1: preparation of alpha-KG slow-release granule and capsule
Precisely weighing 50.0g of alpha-ketoglutarate disodium salt and 20.0g of HPMC (hydroxypropyl methylcellulose), sieving with a 100-mesh sieve, grinding, mixing uniformly, wetting with ethanol solution, preparing soft materials, extruding, rounding, granulating, placing in a vacuum drying oven at 50 ℃, drying for 6h, and sieving with a 20-mesh sieve to obtain alpha-KG slow-release granules; weighing 280mg of slow release particles, and filling the slow release particles into HPMC hollow capsules, namely alpha-KG slow release capsules, respectively dissolving three slow release capsules into 500ml of water, and performing mass spectrometry to obtain the result that the average content of alpha-KG in the slow release particles is 185.80mg; the slow release granule is shown in figure 1, and the slow release capsule and the alpha-KG content result are shown in figures 2 and 4.
Example 2: preparation of alpha-KG sustained release tablet
Precisely weighing 100.0g of alpha-ketoglutarate disodium salt and 40.0g of HPMC, sieving with a 100-mesh sieve, grinding and mixing uniformly, wetting with ethanol solution, preparing soft materials, extruding, rounding and granulating, placing in a 50 ℃ vacuum drying oven, drying for 6 hours, sieving with a 20-mesh sieve, adding 1% of magnesium stearate, tabletting with a full-automatic tablet press to prepare alpha-KG sustained-release tablets, wherein each tablet weighs 290-300mg, and the average hardness of the tablets is 65.66N according to the tablet requirement by a tablet hardness tester. Three sustained release tablets are randomly taken and dissolved in 500ml of water, and the mass spectrum measurement result shows that the average content of alpha-KG in the sustained release tablets is 190.16mg, and the sustained release tablets and the content of alpha-KG are shown in figures 3 and 4.
Example 3: determination of biological release degree of alpha-KG slow-release capsule and slow-release tablet
Respectively taking an alpha-KG slow release tablet (embodiment 2), a slow release capsule (embodiment 1) and an alpha-KG raw material capsule (containing an equivalent amount of alpha-ketoglutarate disodium salt raw material powder and containing no slow release reagent of the invention), placing the alpha-KG raw material capsule and the slow release capsule in 37 ℃ and 500mL of water, adopting a dissolution paddle method, stirring at 100rpm continuously, respectively taking 100 mu L of clear solution at 5min, 15min, 30min, 1h, 4h and 6h, filtering by a 0.45 mu m filter membrane, taking 10 mu L of sample solution, adding 90 mu L of acetonitrile, centrifuging at 12000rpm for 5min, taking supernatant, diluting 50% acetonitrile water for 50 times, adopting LC-MS to measure the alpha-KG content, wherein the alpha-ketoglutarate disodium salt standard substance is prepared into corresponding standard curve samples, the concentrations are respectively 50, 100, 200, 400, 800 and 1000ppb, further calculating the biological release degree, wherein the detection patterns of the LC-MS are respectively shown in figure 5, the standard curves are shown in figure 6, the alpha-KG content at different times is measured, the alpha-KG content of the alpha-KG is calculated, the slow release curve of the alpha-KG capsule is the alpha-KG capsule, the alpha-KG release tablet is the slow release tablet, and the alpha-KG content of the alpha-KG capsule is the slow release tablet.
As can be seen from fig. 7 and 8, the alpha-ketoglutarate disodium salt raw material capsule is completely dissolved and released in 10 minutes; the alpha-KG slow release capsule is gradually released after 1 hour, and the alpha-KG slow release tablet is gradually released in 6 hours, so that the slow release effect is very obvious.
Meanwhile, the invention also discusses a slow release preparation prepared from alpha-ketoglutarate and salts thereof and other hydrophilic gel skeleton materials, such as sodium carboxymethyl cellulose, hydroxyethyl cellulose and chitosan, and has similar slow release effect to that of the embodiment 3, thereby proving that the hydrophilic gel skeleton materials can also be used as materials aiming at the alpha-ketoglutarate and salts thereof without other additional components, and the formula is simple and the preparation process is simple.
Example 4: influence of different polymer frameworks on measurement mode for preparing alpha-KG sustained-release preparation
The method provided in example 2 is adopted to prepare the alpha-KG sustained-release tablets, wherein the hydrophilic gel skeleton is respectively hydroxypropyl methylcellulose, sodium carboxymethylcellulose, hydroxyethyl cellulose or chitosan, different alpha-KG sustained-release tablets are prepared, the stability of each alpha-KG sustained-release tablet is detected, the influence on an LC-MS pipeline in the detection process is detected, and a biological release curve is detected, wherein the stability detection method comprises the following steps: the prepared alpha-KG slow-release tablet was placed in an oven at 40℃for 15 days, the appearance was examined and the content of alpha-KG therein was examined.
As can be seen from table 1, the stability of the prepared α -KG sustained release tablet and the LC-MS detection process are greatly affected by using different matrix materials, and when hydroxyethyl cellulose or chitosan is used as the matrix material, the prepared sustained release preparation has low stability and premature decomposition; in addition, when sodium carboxymethyl cellulose, hydroxyethyl cellulose or chitosan is used as a framework material, the prepared alpha-KG sustained-release preparation can often cause residues of detection objects in an instrument pipeline and a chromatographic column, and seriously influence the sensitivity and the detection efficiency of alpha-KG detection. In summary, the alpha-KG sustained-release tablet with optimal performance can be prepared only when hydroxypropyl methylcellulose is used as a framework material, and is convenient to adopt LC-MS detection and analysis, and quantitative detection and quality control analysis are ensured.
Example 5: comparing the blood concentration of mice alpha-KG intragastric administration (simulating oral common preparation, non-slow release alpha-KG original drug released in short time impact) with free drinking administration of alpha-KG drinking water in equal dose (simulating low dose long time continuous intake of alpha-KG slow release preparation)
The 18 mice were divided into 3 groups of 6: control (control group: clear water), α -KG sodium gastric lavage group, α -KG sodium Na drinking group (simulated slow release reagent). Another 3 mice served as blank (blank) as standard blank plasma.
(1) The mice in the control group drink clean water freely without other treatment; (2) The dosage of the stomach-lavage group is 300mg/KG, namely, each mouse is given 200ul of alpha-KG solution with the concentration of 30mg/ml every day, the oral administration of the common alpha-KG sodium bulk drug is simulated, and the total dosage of one day is taken in a short time; (3) drinking water containing alpha-KG sodium administration group: an equivalent dose (300 mg/KG) of water was calculated per day for each mouse (weight about 20 g), and the alpha-KG sodium content was 1mg/mL, 200mg of alpha-KG sodium was weighed and dissolved in 200mL of drinking water (concentration 1 mg/mL), and the mice were free to drink, simulating the long-term continuous ingestion of the low dose of the alpha-KG sustained-release preparation.
The total daily dosage of alpha-KG-Na taken by animals in the stomach-filling group and the drinking group is consistent (the dosage of the alpha-KG-Na active ingredient is the same). 1 hour after the third day of the stomach irrigation or drinking water administration, the orbit is sampled, heparin sodium is used for sampling blood, shaking is carried out, and the supernatant is taken after centrifugation for 10min at 4 ℃. 40. Mu.L of plasma sample was added to 160. Mu.L of pre-chilled methanol (1:4) at minus 20℃and shaken for 3min, centrifuged (12000 rpm,10 min) at 4℃and 100. Mu.L of supernatant was detected by mass spectrometry. Taking 4 mu L of alpha-KG-Na standard mother solution, respectively adding the alpha-KG-Na standard mother solution into 40 mu L of blank serum, preparing a 1/10 concentration solution in the same blood taking process, uniformly mixing, respectively adding 4 times of precooled methanol (156 mu L) to precipitate protein, centrifuging at 4 ℃ (12000 rpm,10 min), and taking supernatant and sampling. The final concentration of standard yeast is: 1000. 800, 500, 200, 100, 50, 25, 10, 0ppb, mass spectrometry detection.
The results showed that both short-time high-dose impact release and long-time low-dose slow release of α -KG produced an elevated α -KG level in the plasma of mice, with similar blood levels at this time point (one hour after gastric lavage) (fig. 9). This demonstrates that the alpha-KG sustained release dosage form can continuously supplement the alpha-KG of the organism, and the blood concentration is basically the same as that of one hour after the stomach irrigation. Because the sustained release dosage form of the medicine is released continuously, adverse effects caused by short-time impact release of alpha-KG in the digestive tract and rapid rise and fall-back of blood concentration can be avoided. The slow release reagent has obvious advantages.
Example 6: nude mouse tumor transplantation tumor animal model test
The research shows that the alpha-KG has inhibition effect on various tumors, including lung cancer, breast cancer, liver cancer, acute leukemia, malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, nephroblastoma, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, gastric cancer, medullary thyroid cancer and the like, and the embodiment only selects lung cancer, breast cancer and liver cancer for experiments.
The nude mice are used for respectively constructing (1) SK-HEP-1 liver cancer cell transplantation tumor, (2) A549 human lung cancer cell transplantation tumor and (3) MDA-MB-231 human breast cancer transplantation tumor, and the nude mice are used for researching the anti-tumor effect of alpha-KG in animal bodies under the condition of slow release administration mode, and the specific operation is as follows:
1. SK-HEP-1, A549, MDA-MB-231, which had good growth status, were collected, cells were resuspended in medium and counted, and placed on ice.
2. After the cell suspension is uniformly mixed, 2X 10 is injected into the armpit of a BALB/c female nude mouse with the weight of 18-20g 6 100. Mu.L of cells, in which MDA-MB-231 cells were first mixed with Matrigel and inoculated subcutaneously.
3. When nude mice grow 5X 5mm (about 2 weeks) subcutaneously, they are randomly grouped and dosing is started, 6-7 animals per group.
4. The SK-HEP-1 liver cancer model implementation animal groups are respectively as follows: 1) A vehicle (water) group; 2) And the drinking water group contains alpha-KG-Na.
The animal group of the A549 lung cancer model is as follows: 1) A vehicle (water) group; 2) A drinking water group containing alpha-KG-Na; 3) Standard chemotherapeutic cisplatin control group (CDDP, 4mg/kg/w, i.p.); 4) alpha-KG in combination with CDDP.
The MDA-MB-231 model implementation groups are respectively as follows: 1) A vehicle group; 2) An alpha-KG-Na drinking water group; 3) A chemotherapeutic drug Doxorubicin (DOX) treatment group (DOX, 1mg/kg/w, i.p.); 4) alpha-KG-Na was used in combination with DOX.
5. Tumor size was measured using vernier calipers, 2 times per week. The length (L) and width (W) of the tumor were recorded, and the tumor size was calculated as tumor volume= (l×w) 2 )/2。
6. Tumor growth curves were drawn according to tumor sizes of nude mice, and immunohistochemical determination of angiogenic factor CD31 expression was performed on tumor tissues.
The results show that in the liver cancer model, the drinking water containing the alpha-KG single medicine has good anti-tumor effect (figure 10) and can obviously inhibit the growth of tumor cells. The results of the anti-tumor mechanism study of the alpha-KG are shown in figure 13, and the results of the anti-tumor mechanism study of the alpha-KG are shown in figure 13, wherein the alpha-KG can inhibit tumor angiogenesis and obviously inhibit tumor growth. The medicine adopting the slow release reagent can obviously inhibit the growth of tumor cells, and has potential therapeutic application value.
In the lung cancer model, the drinking water containing alpha-KG single medicine (600 mg/KG/day) has an anti-tumor effect, the curative effect is slightly better than that of the standard chemotherapeutic medicine Cisplatin (CDDP) (4 mg/KG/week) (figure 11A), no obvious adverse toxic and side effects are seen, and the body weight is normal (figure 11B). Whereas the mice in the standard chemotherapeutic CDDP treatment group had significantly reduced body weight (FIG. 11B), the efficacy of α -KG was superior to Cisplatin (CDDP) for the lung cancer model.
In the breast cancer model, the alpha-KG single drug (1200 mg/KG/day) has an anti-tumor effect, the curative effect of the alpha-KG single drug is slightly superior to that of the clinical chemotherapeutic drug Doxorubicin (DOX) (1 mg/KG/week) (figure 12A), no obvious adverse toxic and side effects are seen, and the body weight is normal (figure 12B); the combination of the two drugs of alpha-KG (1200 mg/KG/day) and doxorubicin (1 mg/KG/week) showed good synergistic antitumor effect, which has significant statistical significance compared with the effect of the two individual drugs (FIG. 12).
It is understood herein that the combination of α -KG and doxorubicin has a synergistic effect. The α -KG may be a sustained release agent of the present invention, or may be a non-sustained release agent, or may be in the form of a drug substance. It will be readily appreciated that the interaction of the drugs is an interaction between the active ingredients and is not necessarily related to the particular dosage form, i.e. the combination has a significant anti-cancer effect, whether it is a sustained release agent or a non-sustained release agent.
Although the present invention is disclosed above, the present invention is not limited thereto. Since various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, the scope of the invention shall be defined by the following claims.
Claims (7)
1. An alpha-KG slow release preparation is characterized in that the slow release preparation is mainly prepared from active ingredient alpha-ketoglutarate or alpha-ketoglutarate, hydrophilic gel skeleton material and wetting agent;
the alpha-ketoglutarate is alpha-ketoglutarate sodium salt or alpha-ketoglutarate potassium salt;
the hydrophilic gel skeleton material is selected from any one of hypromellose and sodium carboxymethyl cellulose;
the wetting agent is 50% -90% ethanol solution;
the composition ratio of the alpha-ketoglutarate or the alpha-ketoglutarate to the hydrophilic gel skeleton is 1:0.1-0.4.
2. The α -KG sustained release formulation of claim 1 wherein the α -ketoglutarate is disodium α -ketoglutarate.
3. An α -KG sustained release formulation as claimed in any one of claims 1 to 2 further comprising a lubricant.
4. The α -KG sustained release formulation of claim 3 wherein the wetting agent is a 70% ethanol solution; the lubricant is magnesium stearate.
5. The α -KG sustained release formulation according to any one of claims 1 to 4 wherein the formulation of the sustained release formulation is a tablet or capsule; the content of alpha-KG in each tablet or capsule is 100 mg-1000 mg.
6. A method for preparing the alpha-KG sustained release preparation of claim 1, comprising the steps of:
1) Taking active ingredients as slow release reagents: alpha-ketoglutaric acid or alpha-ketoglutarate, and hydrophilic gel skeleton material which plays a role in slow release of the active ingredient, sieving, grinding and mixing uniformly;
2) Adding wetting agent to obtain soft material, extruding, granulating, and drying;
3) Making into capsule, or adding lubricant and making into tablet.
7. Use of an α -KG sustained release formulation as defined in any one of claims 1 to 5 or prepared by the method of claim 6 for the preparation of an antitumor drug, wherein the tumor is selected from lung cancer, breast cancer, liver cancer.
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