CN115721614A - alpha-KG sustained release preparation and application thereof - Google Patents
alpha-KG sustained release preparation and application thereof Download PDFInfo
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- CN115721614A CN115721614A CN202111001331.3A CN202111001331A CN115721614A CN 115721614 A CN115721614 A CN 115721614A CN 202111001331 A CN202111001331 A CN 202111001331A CN 115721614 A CN115721614 A CN 115721614A
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Abstract
The invention provides an alpha-ketoglutaric acid (alpha-KG) sustained release preparation and a preparation method and application thereof, which are mainly prepared from alpha-ketoglutaric acid sodium salt and hydroxypropyl methylcellulose, have simple components, safety, effectiveness, lasting effect and relatively simple preparation process, and are suitable for mass production; after the alpha-KG sustained-release preparation is orally taken, the alpha-KG can be slowly released, the stimulation to gastrointestinal tracts is avoided, the in-vivo alpha-KG is continuously supplemented, and the in-vivo alpha-KG is maintained at a steady-state level required by an organism, so that the alpha-KG sustained-release preparation is particularly suitable for the old with a reduced alpha-KG level, the health state of a human body is improved, and various diseases related to alpha-KG reduction, such as osteoporosis and the like, are reduced; recent studies have found that sustained 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 values in the aspects of improving health conditions, resisting tumors, prolonging service life and the like.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to the field of pharmaceutical preparations, and especially relates to an alpha-KG sustained-release preparation as well as a preparation method and an application thereof.
Background
Alpha-Ketoglutarate (Alpha-KG) is a key metabolic intermediate product of the tricarboxylic acid cycle of an organism and has important effects on stem cell development, aging resistance, protein synthesis promotion, energy metabolism regulation and the like. alpha-KGs contribute to a variety of metabolic processes of nutrients (e.g. amino acids, glucose, fatty acids), provide energy (ATP) for vital activities of cells and material elements (e.g. biofilm lipids) required for cell proliferation. The research results published in journal of Nature of british of Randall m.chi in 2014 show that the alpha-KG can delay senescence and prolong the life of adult nematodes by 50%. The results of studies published by Ana P.Gomes in Nature journal in 2020 show that the content of metabolite alpha-KG in serum of old people (aged 60 or older) is obviously reduced compared with that of young people; azar AsadiShahmirzadi et al, cell Metabolism, reported that addition of α -KG to the diet of middle-aged mice increased the longevity of the mice and improved overall health. Recently, yuan Wang et al, in Nature Communications published, have found that alpha-KG can reduce age-related osteoporosis and promote bone regeneration. At present, with the acceleration of population aging, the requirements of the elderly on life health and quality of life are improved, the alpha-KG of the elderly is safely and continuously supplemented, the health state of the elderly is improved, the occurrence of various senile diseases such as osteoporosis is reduced, the service life of the elderly is prolonged, and the method has important social significance.
Tumors are one of the major diseases seriously harming human life health, and are characterized in that cells are abnormally proliferated and differentiated, and the invasion and metastasis of malignant tumor cells seriously affect the structure and the function of multiple organs of an organism and seriously threaten human health and life. With the continuous development of biomedicine and the continuous development of chemotherapeutic drugs and targeted therapy, the survival rate of cancer patients is continuously improved. However, the existing anticancer drugs often have the toxic and side effects of leucocyte and platelet reduction caused by immunosuppression reduction, inappetence of digestive system, nausea and vomiting, and the like, so that the search for an effective treatment mode with small toxic and side effects is very important. Recent studies have shown that low doses of sustained supplementation with α -KG have significant antitumor effects (see fig. 10-13) without adverse side effects.
Aiming at the problem that the in vivo level of the alpha-KG is reduced along with the increase of the age, and the alpha-KG has the effects of promoting health, prolonging life, improving osteoporosis and resisting tumors, a preparation suitable for supplementing the alpha-KG for middle-aged and elderly people is needed to be developed, so that the alpha-KG of the crowd is maintained at a normal level, diseases related to the reduction of the alpha-KG are prevented, the tumors are prevented and treated, a good health condition is 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 the alpha-KG is directly orally taken, and certain irritation is generated to the digestive tract; on the other hand, the alpha-KG has small molecular weight, easy absorption by organisms and high metabolic rate, and the concentration of the alpha-KG in the bodies is difficult to maintain at a long-term steady-state level by a common preparation.
Based on the physicochemical properties of α -KG and its rapid absorption and metabolism in vivo, it is necessary to develop an oral dosage form capable of slowly releasing α -KG in the digestive tract, which helps to maintain the in vivo α -KG concentration stable within the physiological concentration range, and avoid 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 difference fluctuation between the peak and the valley of the blood concentration is small, the stimulation to the gastrointestinal tract is reduced, the curative effect is more durable and safer, and the medicine is widely selected. Therefore, the development of the alpha-KG oral sustained-release preparation is helpful to overcome the defects 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 vivo.
At present, a plurality of alpha-KG related products, such as arginine-alpha-ketoglutaric acid (AAKG), are used as a body-building beverage for assisting metabolism or dietary supplements for directly supplementing alpha-KG, such as tablets of Kirkman in the United states abroad, but the alpha-KG related products are not applied to supplement of the alpha-KG at home for a while, particularly, the alpha-KG sustained-release preparation products are not reported.
Disclosure of Invention
The invention provides an alpha-KG sustained release preparation, which mainly comprises alpha-ketoglutaric acid sodium salt and hydrophilic polymer skeleton gel material, can slowly release alpha-KG after oral administration, avoids stimulation to gastrointestinal tracts, and continuously supplements the alpha-KG in vivo to maintain the alpha-KG at a steady state level required by an organism, thereby improving the health state of a human body and preventing various diseases related to reduction of the alpha-KG. Meanwhile, the sustained low-dose supplement of the alpha-KG in the animal body has obvious anti-tumor effect, so the alpha-KG sustained-release preparation can be used for anti-tumor treatment. The sustained-release preparation is particularly suitable for the old with reduced alpha-KG level, and has important application values in the aspects of preventing osteoporosis, preventing and treating tumors, improving health conditions, prolonging life and the like.
In addition, when the general drug sustained release preparation is detected by a liquid phase mass spectrometry technology, the high molecular weight in the sustained release preparation easily causes 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 method does not cause the pipeline blockage of a detection instrument during the liquid mass spectrometry detection, is convenient for detection and ensures the quality monitoring, and is a technical problem to be urgently solved at present.
In view of these problems, the present invention provides a mixture, preparation or pharmaceutical preparation having a relatively simple formulation, a fixed content of ingredients and an easy formulation and quantitative analysis, which solves one or more problems, including technical problems and application problems, existing in the art at present.
Therefore, in one aspect, the present invention provides an α -KG sustained release formulation and a hydrophilic gel matrix, and the formulation substantially comprises an active ingredient and a hydrophilic gel matrix material, which are mixed in a certain ratio and processed by the following process to achieve sustained release of the active ingredient in the product under physiological conditions. The combination can allow the alpha-KG to exist stably, is beneficial to long-term storage and does not influence the sustained release effect.
In some embodiments, alpha-ketoglutarate or alpha-ketoglutarate, or analogs with similar activity, are included. By "analogue" is meant that the active substance alpha-ketoglutarate or alpha-ketoglutarate is structurally linked to groups which do not affect or substantially affect the physiological activity of alpha-ketoglutarate or alpha-ketoglutarate.
The term "hydrophilic gel matrix" as used herein refers to a hydrophilic polymer having a plurality of hydrophilic groups, such as-OH groups, which when wetted, bind the active substance to the hydrophilic substance and when contacted with an aqueous solution, hydrate to form a gel, wherein the active substance is contained in the gel and is 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 matrix is any one selected from hypromellose, sodium carboxymethylcellulose, hydroxyethyl cellulose, chitosan.
In some embodiments, the α -ketoglutarate salt is α -ketoglutarate sodium salt or α -ketoglutarate potassium salt.
In some embodiments, the alpha-ketoglutarate is disodium alpha-ketoglutarate; the hydrophilic gel skeleton is hydroxypropyl methylcellulose.
In addition, the research finds that most of high molecular materials used for preparing the sustained-release preparation have the defects of low stability, premature decomposition and the like of the prepared sustained-release preparation due to the influence of low pH, so that the preparation method is not suitable for preparing the obviously acidic a-ketoglutaric acid sustained-release agent. Repeated tests show that hydrophilic high molecular materials such as hydroxypropyl methylcellulose can bear the influence of low pH of alpha-KG, the stability of the hydroxypropyl methylcellulose is not influenced, and the hydroxypropyl methylcellulose is suitable for preparing an alpha-KG sustained-release preparation. The alpha-KG sustained release agent prepared by the hydrophilic gel skeleton has stable property and is beneficial to preservation; in addition, as the hypromellose used in the sustained-release preparation can resist the acidic environment in the stomach, the alpha-KG can be ensured to enter the intestinal tract in a gel form for slow release.
alpha-KG because of its simple structure, no ultraviolet absorption, consequently need select liquid mass spectrometry to determine alpha-KG content, and when adopting common macromolecular material such as sodium carboxymethylcellulose (a salty substance) to prepare alpha-KG sustained-release preparation, the alpha-KG sustained-release preparation who makes often causes the residue of detected thing in instrument pipeline and chromatography post when using mass spectrometry to detect the assay active ingredient release, seriously influences the detection of product and the development of Quality Control (QC) analysis, this is the technical problem who needs to solve urgently.
Aiming at the problems, a large number of experiments find that the alpha-KG sustained-release preparation prepared by adopting hydrophilic high polymer materials such as hydroxypropyl methylcellulose and the like does not have the problem of instrument system pipeline residue when the mass spectrum detection analysis is carried out on the release of active ingredients. And the process for preparing the sustained-release preparation by adopting the hydroxypropyl methylcellulose is relatively simple, is easy to prepare, and is the best choice for preparing the alpha-KG sustained-release preparation. It is to be noted that although hypromellose is described herein as the best choice for the preparation of sustained release formulations of α -KG, other hydrophilic backbone polymeric materials are not excluded as equally desirable choices.
In some embodiments, the composition ratio of α -ketoglutarate or α -ketoglutarate to hydrophilic gel matrix or hypromellose is 1. In some embodiments, the ratio of the hydrophilic gel matrix to the active ingredient of the present invention can be obtained experimentally.
In some embodiments, a lubricant and a wetting agent; the lubricant is any one selected from magnesium stearate, talcum powder, microcrystalline cellulose and compressible starch; the wetting agent is 50% -90% of ethanol solution.
In some embodiments, the lubricant is magnesium stearate; the wetting agent is a 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, the present invention provides a method for preparing an α -KG sustained release formulation, which mainly comprises the following steps:
1) Taking alpha-ketoglutaric acid or alpha-ketoglutaric acid salt, sieving the alpha-ketoglutaric acid or alpha-ketoglutaric acid salt and a hydrophilic gel framework material, and grinding and uniformly mixing the alpha-ketoglutaric acid or alpha-ketoglutaric acid salt and the hydrophilic gel framework material;
2) Adding wetting agent to prepare soft material, extruding, rounding, granulating and drying;
3) Making into capsule, or adding lubricant, and making into tablet.
In some embodiments, the hydrophilic gel matrix is any one selected from hypromellose, sodium carboxymethylcellulose, hydroxyethyl cellulose, chitosan.
In some embodiments, the α -ketoglutarate salt is α -ketoglutarate sodium salt or α -ketoglutarate potassium salt.
In some embodiments, the alpha-ketoglutarate is disodium alpha-ketoglutarate; the hydrophilic gel skeleton is hydroxypropyl methylcellulose.
In some embodiments, the α -ketoglutarate or α -ketoglutarate and hypromellose are present in a ratio of 1.
In some embodiments, the wetting agent is a 50% to 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 a use of the sustained release α -KG formulation as described above for anti-tumor purposes or the sustained release α -KG formulation prepared by the method as described above for the preparation of a medicament in the anti-tumor field.
In some embodiments, the anti-tumor field comprises one or more of the fields of lung cancer, breast cancer, liver cancer, acute leukemia and malignant lymphoma, breast cancer, bronchopulmonary carcinoma, ovarian cancer, nephroblastoma, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, stomach cancer, medullary thyroid cancer.
In some embodiments, the anti-tumor field is a liver cancer field.
In a fourth aspect, the present invention provides a pharmaceutical combination or co-formulation comprising α -KG and doxorubicin or/and cisplatin.
In some embodiments, the α -KG is present in a sustained release formulation, although the α -KG may also be in a non-sustained release formulation. When the α -KG is in the form of a sustained release formulation, it may include any of the sustained release formulations described above as well as different dosage forms and different ratio relationships between the drugs.
In some embodiments, the anti-tumor comprises one or more of the fields of lung cancer, breast cancer, liver cancer, acute leukemia and malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, wilms' tumor, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, stomach cancer, medullary thyroid cancer.
In some embodiments, the anti-tumor field is a breast cancer field.
Further, the anti-tumor field is the field of lung cancer.
The term "combination of drugs" as used herein refers to the combination of two different drugs for the treatment of a disease, and may include the separate and sequential administration of the two drugs, for example, the sustained release α -KG formulation of the present invention may be administered first, followed by a time interval, followed by the administration of doxorubicin or/and cisplatin, and it is understood that the order may also be followed by the administration of doxorubicin or/and cisplatin, followed by a time interval, followed by the administration of the sustained release α -KG formulation. Of course, in other embodiments, the two drugs are present in a mixed state, administered simultaneously or nearly simultaneously. In addition, the so-called combination is not limited to the combination of two drugs of the present invention, and may be combined with other drugs, so that there may be simultaneous or sequential administration of three drugs, or simultaneous or sequential administration of three or more drugs.
The invention has the beneficial effects that:
(1) The alpha-KG oral sustained-release preparation is simple in components, only comprises active components and the sustained-release agent, does not comprise other components, is safe and effective, and has a lasting effect; after being taken orally, the alpha-KG is slowly released, the stimulation to gastrointestinal tracts is avoided, the alpha-KG in vivo is continuously supplemented, the steady state level required by the alpha-KG in the organism is maintained for a long time, and various diseases related to the reduction of the alpha-KG are prevented, so that 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 expanded production;
(3) Provides the application of the alpha-KG oral sustained-release preparation in antitumor treatment;
(4) The adverse effects caused by short-time impact release of alpha-KG in the digestive tract and rapid rise and fall of blood concentration can be avoided, the clinical compliance is good, an ideal dosage form for stably supplementing the alpha-KG is provided for people with reduced alpha-KG, especially for middle-aged and old people, the physiological requirement is met, and the health promotion is facilitated; simultaneously provides a new treatment mode and a new pharmaceutical preparation for tumor patients.
(5) Provides the drug combination of the alpha-KG and the adriamycin and the application thereof in the anti-tumor treatment, and has the synergistic effect;
(6) Provides a combined preparation of alpha-KG and cisplatin and application thereof in antitumor treatment, and can also achieve the synergistic effect.
Drawings
FIG. 1 shows α -KG sustained-release granules prepared in example 1;
FIG. 2 is a view showing the α -KG sustained-release capsule prepared in example 1;
FIG. 3 is a view showing the α -KG sustained-release tablet prepared in example 2;
fig. 4 is a graph showing the α -KG contents of the α -KG sustained-release capsules and the α -KG sustained-release tablets obtained in examples 1 and 2;
FIG. 5 is a detection spectrum for measuring α -KG by LC-MS in example 3;
FIG. 6 is a standard curve for measuring α -KG by LC-MS in example 3;
FIG. 7 is a graph showing the release rate of the α -KG sustained-release capsule and the α -KG raw material from the capsule in example 3;
FIG. 8 is a graph showing the sustained release rate of α -KG sustained release tablets and α -KG drug substance from capsules according to example 3;
FIG. 9 is a graph showing the plasma concentration of α -KG in mice in example 5 comparing the bolus dose of α -KG (after 1h gavage) with the mode of administration of a sustained release formulation (drinking water containing α -KG);
FIG. 10 shows the therapeutic effect of α -KG in example 6 on a nude mouse model of liver cancer transplantable tumor;
FIG. 11 is the results of the test of α -KG in example 6 in combination with cisplatin, a clinical chemotherapeutic agent, alone or in combination with a transplanted tumor of lung cancer (nude mouse model), wherein (A) is the tumor growth curve; and (B) is a mouse body weight change curve.
FIG. 12 is a graph of the therapeutic effect of α -KG in example 6, alone or in combination with the clinical chemotherapeutic agent doxorubicin, on breast cancer transplantable tumors (nude mouse model), wherein (A) the tumor growth curve; (B) Mouse body weight change curves (. X.) represent statistically significant differences.
FIG. 13 shows the results of the inhibition of tumor angiogenesis by α -KG in example 6.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the invention and how it may be carried into effect, and which are merely limited examples of embodiments within the spirit of the invention and are intended to illustrate how the invention may be carried into effect and not to limit the invention or its applications. The specific protection scope of the invention is embodied in the claims of the invention. The reagents used in this example were all known products, and were obtained by purchasing commercially available products.
Example 1: preparation of alpha-KG slow release granule and capsule
Precisely weighing 50.0g of alpha-ketoglutaric acid disodium salt and 20.0g of HPMC (hydroxypropyl methylcellulose), sieving with a 100-mesh sieve, grinding, uniformly mixing, wetting with an ethanol solution, preparing into a soft material, extruding, rounding, granulating, placing in a vacuum drying oven at 50 ℃, drying for 6h, and sieving with a 20-mesh sieve to obtain the alpha-KG sustained-release granules; weighing 280mg of sustained-release granules, filling the granules into HPMC hollow capsules to obtain alpha-KG sustained-release capsules, and respectively dissolving three sustained-release capsules in 500ml of water for mass spectrometry, wherein the result shows that the average content of alpha-KG in the sustained-release granules is 185.80mg; the sustained-release granules are shown in figure 1, and the results of the sustained-release capsules and the contents of alpha-KG are shown in figures 2 and 4.
Example 2: preparation of alpha-KG sustained-release tablet
Accurately weighing 100.0g of alpha-ketoglutaric acid disodium salt and 40.0g of HPMC, sieving with a 100-mesh sieve, grinding and uniformly mixing, wetting with an ethanol solution, preparing into a soft material, extruding, rounding and granulating, placing in a vacuum drying oven at 50 ℃, drying for 6h, sieving with a 20-mesh sieve, adding 1% of magnesium stearate, tabletting with a full-automatic tablet press to prepare the alpha-KG sustained-release tablet, wherein the weight of each tablet is 290-300mg, and the average hardness of the tablet is 65.66N according to the tablet hardness tester, so that the tablet meets the tablet requirements. Three sustained-release tablets were randomly dissolved in 500ml of water, the mass spectrometry results showed that the average content of α -KG in the sustained-release tablets was 190.16mg, and the results of the contents of the sustained-release tablets and α -KG are shown in fig. 3 and 4.
Example 3: determination of biological release degree of alpha-KG sustained-release capsule and sustained-release tablet
Respectively taking an alpha-KG sustained-release tablet (embodiment example 2), a sustained-release capsule (embodiment example 1) and an alpha-KG raw material capsule (containing equal amount of alpha-ketoglutarate disodium salt raw material powder and not containing the sustained-release reagent of the invention), placing the raw material powder in 500mL of water, continuously stirring at 100rpm by using a dissolution instrument paddle method, respectively taking 100 μ L of clear solution in 5min, 15min, 30min, 1h, 4h and 6h, filtering the clear solution by using a 0.45 μm filter membrane, taking 10 μ L of sample solution, adding 90 μ L of acetonitrile, centrifuging at 12000rpm for 5min, taking supernatant, diluting 50 times by 50% acetonitrile, and then measuring the alpha-KG content by using LC-MS, wherein the alpha-KG content is measured by LC-MS, the alpha-KG detection map is shown in figure 5, the alpha-KG content is measured by using a standard curve sample injection, the concentrations are respectively 50, 100, 200, 400, 800 and 1000ppb, the biological release degree is further calculated, wherein the alpha-KG content is measured by LC-MS, the alpha-KG raw material tablet release curves are shown in figure 6, the alpha-KG sustained-KG raw material tablet release curves at different times, and the sustained-KG raw material capsule release curves are calculated by LC-release curves, and the alpha-KG raw material curves are shown in figure 8.
As can be seen from fig. 7 and 8, the α -ketoglutarate disodium salt raw material capsule is completely dissolved and released within 10 minutes; the alpha-KG sustained-release capsule is released gradually after lasting for 1 hour, and the alpha-KG sustained-release tablet is released gradually after lasting for 6 hours, so that the sustained-release effect is very obvious.
Meanwhile, the invention also discusses the sustained release preparation prepared by the alpha-ketoglutaric acid and the salt thereof and other hydrophilic gel framework materials, such as sodium carboxymethylcellulose, hydroxyethyl cellulose and chitosan, and obtains similar effects to those of the embodiment example 3 on the sustained release effect, thereby proving that the hydrophilic gel framework materials can also be used as materials aiming at the alpha-ketoglutaric acid and the salt thereof, and do not contain other additional components, so that the formula is simple, and the preparation process is simple.
Example 4: influence of different polymer skeletons on preparation of alpha-KG sustained-release preparation on measurement mode
In this embodiment, the α -KG sustained-release tablets are prepared by the method provided in embodiment 2, wherein the hydrophilic gel skeleton is made of hypromellose, sodium carboxymethylcellulose, hydroxyethyl cellulose or chitosan, respectively, to obtain different α -KG sustained-release tablets, and the stability of each α -KG sustained-release tablet, the influence on the LC-MS pipeline in the detection process, and the biological release degree curve are detected, wherein the stability detection method is as follows: the prepared alpha-KG sustained-release tablet is placed in an oven at 40 ℃ for 15 days, the appearance of the tablet is inspected, and the content of the alpha-KG in the tablet is detected.
As can be seen from table 1, different framework materials have great influence on the stability of the prepared alpha-KG sustained-release tablet and the LC-MS detection process, and when hydroxyethyl cellulose or chitosan is used as the framework material, the prepared sustained-release preparation has low stability and the defect of premature decomposition; in addition, when sodium carboxymethylcellulose, hydroxyethyl cellulose or chitosan is used as a framework material, the prepared alpha-KG sustained-release preparation often causes residues of a detected object in an instrument pipeline and a chromatographic column, and the sensitivity and the detection efficiency of alpha-KG detection are seriously influenced. In conclusion, the alpha-KG sustained-release tablet with optimal performance can be prepared only when hydroxypropyl methylcellulose is used as a framework material, and LC-MS detection and analysis are conveniently adopted to ensure quantitative detection and quality control analysis.
Example 5: comparing the blood concentration of the mouse alpha-KG intragastrically (simulating oral ordinary preparation, non-sustained release alpha-KG original drug is released in short time by impact) and the drinking water containing alpha-KG with equal dosage for free drinking administration (simulating low dosage of alpha-KG sustained release preparation for long time continuous intake)
18 mice were divided into 3 groups, 6 per group: a control group (a control group: clear water), an alpha-KG sodium intragastric group and an alpha-KG sodium Na drinking group (a simulated slow release reagent). Another 3 mice served as blank controls (blank) and were used as standard blank plasma.
(1) The control group mice had free access to clear water without further treatment; (2) The dose of the gavage group is 300mg/KG once a day, namely each mouse is given 200ul of alpha-KG solution with the concentration of 30mg/ml every day, the oral administration of the alpha-KG solution is simulated, and the total dose of one day is taken in a short time; (3) administration group containing alpha-KG sodium in drinking water: the equivalent dose (300 mg/KG) is calculated according to the drinking of 6mL of water per day by each mouse (the weight is about 20 g), the content of alpha-KG sodium is 1mg/mL, 200mg of alpha-KG sodium is weighed and dissolved in 200mL of drinking water (the concentration is 1 mg/mL), and the mice freely drink the water and simulate the low dose of the alpha-KG sustained-release preparation for long-time continuous intake.
The animals in the gavage group and the drinking group take the alpha-KG-Na daily with the same total dosage (the dosage of the alpha-KG-Na active ingredient is the same). Collecting blood in orbit 1 hr after intragastric administration or drinking water administration on the third day, collecting blood with heparin sodium blood collecting tube, shaking, centrifuging at 4 deg.C for 10min, and collecting supernatant. mu.L of each plasma sample was added to 160. Mu.L of minus 20 ℃ pre-cooled methanol (1. Taking 4 mu L of alpha-KG-Na standard mother liquor, respectively adding 40 mu L of blank serum, preparing a 1/10 concentration solution by the same blood taking process, respectively adding 4 times of precooled methanol (156 mu L) to precipitate protein after uniformly mixing, centrifuging at 4 ℃ (12000rpm, 10min), and taking supernatant for sample injection. The final concentrations of the standard were: 1000. 800, 500, 200, 100, 50, 25, 10, 0ppb, mass spectrometric detection.
The results showed that both short-term large-dose shock release and long-term low-dose slow release of α -KG resulted in elevated α -KG levels in mouse plasma, and that plasma concentrations at this time point (one hour after gavage) were similar (fig. 9). This indicates that the sustained release dosage form of alpha-KG can continuously supplement alpha-KG to the body, and the blood concentration is basically the same as the blood concentration one hour after the intragastric administration. Because the drug of the sustained-release preparation is released continuously, the adverse effects caused by short-time impact release of the alpha-KG in the digestive tract and rapid rise and fall of the blood concentration can be avoided. The sustained release reagent of the invention has obvious advantages.
Example 6: nude mouse tumor transplantation animal model test
Research shows that the alpha-KG has an inhibitory effect on various tumors including lung cancer, breast cancer, liver cancer, acute leukemia, malignant lymphoma, breast cancer, bronchopulmonary carcinoma, ovarian cancer, wilms' tumor, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, stomach cancer, medullary thyroid cancer and the like, and in the embodiment, only lung cancer, breast cancer and liver cancer are selected for experiments.
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 by using a nude mouse, and researching the anti-tumor effect of alpha-KG in an animal body under the condition of a sustained release administration mode, wherein the specific operation is as follows:
1. SK-HEP-1, A549 and MDA-MB-231 in good growth state are collected, the culture medium is resuspended and the cells are counted, and the cells are placed on ice.
2. After the cell suspension is mixed evenly, the axilla of a female BALB/c nude mouse with the weight of 18-20g is injected with 2 x 10 6 100 μ L of cells, wherein MDA-MB-231 cells were first mixed with Matrigel and then inoculated subcutaneously.
3. When tumors grow 5X 5mm subcutaneously (about 2 weeks) in nude mice, the administration is started by randomly grouping, and 6 to 7 mice per group.
4. The SK-HEP-1 liver cancer model implemented animal groups are respectively as follows: 1) Vehicle (water) group; 2) Drinking water containing alpha-KG-Na.
The animal groups implemented by the A549 lung cancer model are respectively: 1) 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) Chemotherapy drug Doxorubicin (DOX) treatment group (DOX, 1mg/kg/w, i.p.); 4) alpha-KG-Na in combination with DOX.
5. Tumor size was measured using a vernier caliper 2 times per week. The length (L) and width (W) of the tumor were recorded and the tumor size could be calculated as tumor volume = (L × W) 2 )/2。
6. And (3) drawing a tumor growth curve according to the tumor size of the nude mice, and performing immunohistochemistry on tumor tissues to determine the expression of the angiogenesis factor CD 31.
The results show that the drinking water containing the alpha-KG single drug has good anti-tumor effect (figure 10) in the liver cancer model, and can obviously inhibit the growth of tumor cells. The results of the research on the anti-tumor mechanism of α -KG are shown in fig. 13, and it can be seen from fig. 13 that α -KG inhibits tumor angiogenesis and significantly inhibits tumor growth. The slow release agent 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 has an anti-tumor effect when being used singly (600 mg/KG/day), the curative effect is slightly better than that of the standard chemotherapeutic drug Cisplatin (CDDP) (4 mg/KG/week) (figure 11A), no obvious adverse side effect is seen, and the weight is normal (figure 11B). However, mice in the CDDP treated group treated with the standard chemotherapeutic drug had significantly reduced body weight (FIG. 11B), and thus the therapeutic effect of α -KG was superior to that of Cisplatin (CDDP) in the lung cancer model.
In a breast cancer model, a single alpha-KG (1200 mg/KG/day) has an anti-tumor effect, the curative effect of the single alpha-KG is slightly better than that of clinical chemotherapeutic adriamycin (DOX) (1 mg/KG/week) (figure 12A), no obvious adverse side effect is seen, and the weight is normal (figure 12B); the combination of the alpha-KG (1200 mg/KG/day) and the adriamycin (1 mg/KG/week) shows good synergistic antitumor effect, and has significant statistical significance compared with the effect of the two drugs which are taken independently (FIG. 12).
It will be appreciated that the combination of α -KG and doxorubicin has a synergistic effect. The α -KG herein may be a sustained-release agent of the present invention, or may be a non-sustained-release agent, and may be in the form of a crude drug. It will be readily appreciated that drug interactions are interactions between active ingredients and are not necessarily related to a particular dosage form, i.e. whether sustained release or not, the combination has a significant anti-cancer effect.
Although the present invention is disclosed above, the present invention is not limited thereto. Since various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention, the invention resides in the claims hereinafter appended.
Claims (24)
1. An alpha-KG sustained release preparation, wherein the sustained release preparation comprises alpha-ketoglutaric acid or alpha-ketoglutarate and a hydrophilic gel matrix material; or mainly comprises alpha-ketoglutaric acid or alpha-ketoglutarate and hydrophilic gel framework material.
2. The sustained release α -KG formulation according to claim 1, wherein said α -ketoglutarate is α -ketoglutarate sodium salt or α -ketoglutarate potassium salt.
3. The sustained release α -KG formulation according to claim 2, wherein said α -ketoglutarate is disodium α -ketoglutarate.
4. The α -KG sustained release formulation according to any one of claims 1 to 3, wherein the α -ketoglutarate or α -ketoglutarate is present in a ratio of 1.
5. The α -KG sustained release formulation according to claim 1, wherein the hydrophilic gel matrix material is selected from one or more of hypromellose, sodium carboxymethylcellulose, hydroxyethyl cellulose and chitosan.
6. The α -KG sustained release formulation according to any one of claims 1 to 5, further comprising a wetting agent and/or a lubricant.
7. The α -KG sustained release formulation according to claim 6, wherein the wetting agent is a 50% to 90% ethanol solution; the lubricant is any one selected from magnesium stearate, talcum powder, microcrystalline cellulose and compressible starch.
8. The α -KG sustained release formulation according to claim 6, wherein the wetting agent is a 70% ethanol solution; the lubricant is magnesium stearate.
9. The α -KG sustained release formulation according to any one of claims 1 to 8, wherein the sustained release formulation is in the form of a tablet or a capsule; the content of alpha-KG in each tablet or capsule is 100 mg-1000 mg.
10. A method for preparing an alpha-KG sustained release formulation, wherein the method comprises the steps of:
1) Taking alpha-ketoglutaric acid or alpha-ketoglutaric acid salt and a hydrophilic gel framework material, sieving, grinding and uniformly mixing;
2) Adding wetting agent to prepare soft material, extruding, rounding, granulating and drying;
3) Making into capsule, or adding lubricant, and making into tablet.
11. The method of claim 10, wherein the α -ketoglutarate salt is α -ketoglutarate sodium salt or α -ketoglutarate potassium salt.
12. The method of claim 11, wherein the alpha-ketoglutarate salt is disodium alpha-ketoglutarate.
13. The method of claim 12, wherein the α -ketoglutarate or α -ketoglutarate salt and the hydrophilic gel matrix material are present in a compositional ratio of 1.
14. The method of claim 10, wherein 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.
15. The method of claim 10, wherein the wetting agent is a 70% ethanol solution; the lubricant is magnesium stearate.
16. Use of the sustained release formulation of α -KG according to any one of claims 1 to 9 or the sustained release formulation of α -KG prepared by the process according to any one of claims 10 to 15 for the preparation of an antitumor medicament.
17. The use of claim 16, wherein the tumor comprises one or more of lung cancer, breast cancer, liver cancer, acute leukemia and malignant lymphoma, breast cancer, bronchopulmonary carcinoma, ovarian cancer, wilms' tumor, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, stomach cancer, medullary thyroid cancer.
18. The application of a drug combination preparation in preparing antitumor drugs, wherein the drug combination preparation comprises alpha-KG and adriamycin or/and cisplatin.
19. The use according to claim 18, wherein the α -KG is in the form of a sustained release formulation.
20. Use according to claim 18, wherein the sustained release formulation comprises a sustained release formulation according to claims 1-10 or a formulation prepared by a method according to any one of claims 11-15.
21. The use of claim 18, wherein the tumor comprises one or more of lung cancer, breast cancer, liver cancer, acute leukemia and malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, wilms' tumor, soft tissue sarcoma, bladder cancer, testicular cancer, prostate cancer, stomach cancer, medullary thyroid cancer.
22. The use of claim 18, wherein the tumor is liver cancer.
23. The use of claim 18, wherein the tumor is breast cancer.
24. The use according to claim 18; wherein the tumor is lung cancer.
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