CN100546656C - A kidney-targeted drug carrier and its prodrug, preparation method and application - Google Patents

Abstract

The invention discloses a new kidney-targeting drug carrier, a prodrug formed as a drug carrier, a preparation method and an application thereof. Low-molecular-weight chitosan or its derivatives are obtained by degrading and derivatizing natural chitosan, which has good kidney-targeting properties. connected to obtain the prodrug. The drug molecule is connected with the carrier through chemical bonds, and involves its preparation method and application. In vitro and in vivo experimental studies indicated that the prodrug could reach the kidney in vivo and release the active drug. The preparation method of the invention is simple, the process is mature, the performance is stable, and it is suitable for large-scale continuous production. Its applications include immunosuppressants for treating kidney diseases, anti-rejection drugs for nephritis, drugs for nephritis complicated by kidney infection and preparations thereof.

Description

A kidney-targeted drug carrier and its prodrug, preparation method and application

technical field

The invention belongs to the field of pharmacy, and particularly relates to low-molecular-weight chitosan or its derivatives, a drug carrier with good kidney-targeting efficiency, and a novel kidney-targeted prodrug formed as a drug carrier, as well as a preparation method and application thereof.

Background technique

The kidney is one of the vital organs of the human body. If the physiological function of the kidney deviates, it often leads to serious diseases. Kidney-related diseases mainly include various infection inflammations, diabetes, high blood pressure, tumors, etc., such as acute and chronic nephritis, acute and chronic renal failure, urinary tract infection, kidney cancer , Wilms tumor, renal pelvis tumors, etc. In recent years, the incidence and prevalence of end-stage renal disease are increasing year by year. According to statistics, in 1989, there were 187,000 patients with end-stage renal disease in the United States, and in 1998, nearly 400,000 people became ill. These patients have to rely on dialysis or kidney transplantation to maintain their lives, but the average annual mortality rate is still as high as 20%, which is comparable to the mortality rate of some malignant tumors. In addition, about 10 times as many people suffer from chronic renal failure due to diseases of the cardiovascular system. Among them, diabetes and high blood pressure are the main causes of kidney disease, and about 30% of patients with advanced diabetes will suffer from renal failure. Because diabetes can lead to glomerular sclerosis, renal arteriosclerosis and pyelonephritis and other diseases, it can lead to renal failure in the advanced stage.

Kidney disease requires long-term medication. However, all the drugs used have different degrees of extrarenal effects, and the targeting is not strong. In addition, many drugs will be degraded by other organs (such as liver, etc.) before reaching the kidney; even if the drug can reach the kidney, it may not be able to act on the target cells; transmission will also be affected. Therefore, renal targeting of drugs is necessary to increase drug safety and efficacy. In order to realize the kidney-targeted drug delivery, people have conducted in-depth research on the kidney-targeted drug delivery system so far. Drugs, antibodies, and gene therapy are used to achieve the goal of kidney-targeted drug delivery.

Among the various kidney-targeting carriers that have been studied, LMWP is ideal. LMWPs with a molecular weight below 30,000 Daltons are freely filtered by the glomerulus and rapidly absorbed through the proximal tubule. After LMWP is endocytosed by proximal tubule cells, it migrates to lysosomes via endosomes. In lysosomes, LMWPs are broken down into small peptides and individual amino acids. Studies have shown that about 80% of the injected dose of LMWP (such as lysozyme, cytochrome c, aprotinin, etc.) is finally absorbed by the kidney. There are many patents around this aspect, such as: US6562371, US2003211142, JP2001055343. Suzuki et al. investigated the potential of alkyl glycosides as kidney-targeting vehicles. They used different sugar molecules as probes, combined different sugar molecules with model drugs (arginine vasopressin, AVP) through alkyl spacers of different lengths, and studied the effect of AVP-glycosyl complexes on rats after intravenous injection. It was found that the tissue distribution characteristics were largely dependent on the sugar molecules in the conjugate: glucose (Glc), mannose (Man) and 2d-Glc-C8-AVP all showed good kidney-specific distribution. Glc-S-C8-derivatives have obvious kidney targeting, suggesting that Glc-S-C8- is a suitable carrier for kidney targeting. The related patent is: EP0953357. According to Journal of Controlled Release.95 (2004): 229-237 report: Yamamoto Y et al. prepared vinylpyrrolidone/dimethylmaleic acid copolymer (PVD) by free radical polymerization, and investigated the molecular weight and charge pair PVD in Effects of distribution in mice. PVD with a molecular weight of 6000-8000 Daltons has good renal targeting, and about 80% of the administered dose of PVD is taken up by the kidneys. Other studies have shown that because a large number of γ-glutamyl transpeptidase can be specifically concentrated in the brush border of the renal proximal tubule, this enzyme can convert glutamyl derivatives of amino acid polypeptides into γ-glutamine γ-glutamyl amino acid derivatives are then hydrolyzed to glutamic acid, so the kidneys are highly active in the uptake and metabolism of glutamyl derivatives of amino acids and peptides, so γ-glutamyl amino acid derivatives can be used as kidney-targeted prodrugs .

It can be seen that in the field of research on kidney-targeted drug carriers, domestic research is still at a relatively low stage, and no related patents have been found; international research is also mainly focused on active targeting, and it is also hoped to find More kidney-targeting drug carriers with excellent effects can better treat human kidney diseases and serve human beings.

At present, there are many related patents about the polymer of chitosan: the chitosan drug carrier disclosed in the patent CN1698899A uses polymer chitosan as a drug carrier to make the drug reach the effect of controlled release; the patent CN1554267A discloses A method for preparing water-soluble chitosan; patent CN1743008A discloses that chitosan is used to wrap medicine, form nanoparticles, and passively target to the liver, etc. However, there is no suggestion that low molecular weight chitosan can be used as a good kidney-targeted drug carrier and the preparation method and application of prodrugs.

Contents of the invention

The present invention is based on the above researches, after screening a large number of drug carriers, the obtained low-molecular-weight chitosan or its derivatives all have good kidney-targeting properties. Chitosan is derived from the shell of shrimp and crab. Its structure is similar to that of cellulose, and it is a polysaccharide that is extremely abundant in nature. Chitosan is cheap, easy to obtain, has excellent biodegradability and biocompatibility, so the prepared low molecular weight chitosan can be used as an ideal drug carrier for kidney targeting. By using low-molecular-weight chitosan as a drug carrier for anti-tumor drugs, or glucocorticoids or immunosuppressants, these drugs can be specifically transported to the kidney, with little distribution in other tissues, under the action of kidney enzymes , to release drugs, play a role of local anti-tumor or local immunosuppression, reduce the toxic and side effects caused by anti-tumor drugs or glucocorticoids, and enhance the curative effect.

The purpose of the present invention is to disclose a new drug carrier with good kidney targeting efficiency, which is connected with antineoplastic drugs, glucocorticoids or immunosuppressants through chemical bonds to form prodrugs and preparations thereof, so that the drugs can be Targeting the kidney, reducing the concentration and side effects of the drug in other parts of the body, reducing the dose of the drug, increasing the efficacy and producing a local immunosuppressive effect.

Low-molecular-weight chitosan (LMWC) with a molecular weight of 1,000-100,000 Daltons has good kidney targeting. After intravenous injection into mice, it quickly accumulates in the kidney within 15 minutes, and only a small amount of other tissues LMWC, after injection, LMWC is gradually degraded by enzymes in the body and excreted from the body.

LMWC is a suitable kidney-targeting carrier: ①The carrier has functional groups for drug linking; ②LMWC specifically accumulates in the kidney; ③The physical and chemical properties of LMWC determine the physical and chemical properties of the drug to be linked; ④Drug-LMWC conjugates in the kidney Stable in the circulation, but when it reaches the kidneys, the active drug is released under the action of enzymes. ⑤ LMWC is cheap, easy to obtain, has excellent biocompatibility, biodegradability, and will not cause the accumulation of drug carriers in the body. LMWC is one of the kidney-targeted drug carriers.

LMWCs can be linked to drugs in a variety of ways. Drugs can be directly linked to LMWC through chemical bonds formed by the amino groups and hydroxyl groups on the sugar units of the carrier. The parent drug is released from the drug-spacer-LMWC or drug-LMWC conjugate through enzymatic and chemical hydrolysis.

Technical scheme of the present invention is:

According to one aspect of the present invention, a kidney-targeting drug carrier is disclosed: low-molecular-weight chitosan or its derivatives, as a drug carrier, can target covalently bound drugs to the kidney, which is a new renal Targeted drug carrier; Figure 1 shows its structural formula.

In the formula:

R can be a hydrogen atom (-H), or an alkyl group (-CH ₃ , -C ₂ H ₅ , etc.), or a carboxymethyl group (-CH ₂ COOH);

m and n are positive integers

The molecular weight of low molecular weight chitosan or its derivatives is between 1,000-100,000.

In a preferred embodiment of the present invention, the low molecular weight chitosan or its derivative is a chitosan with a deacetylation degree of 40%-60% and good water solubility. The degree of deacetylation refers to the content of free-NH2 on the sugar unit in the chitosan molecule, expressed by the percentage of the number of deacetylated sugar units in the total number of sugar units in the chitosan molecule. Its preparation method is as follows:

1) Dissolve chitosan in 0.3-3% (w/v) hydrochloric acid aqueous solution, filter, and prepare 2-10% (w/v) chitosan hydrochloric acid solution, after completely dissolving, add hydrogen peroxide , so that the concentration is 0.1-1.5% (w/v), stirred and reacted at 40-70° C. for 2 hours. Adjust the pH value to 13-14 with sodium hydroxide, centrifuge at 4000 rpm to obtain a low molecular weight chitosan precipitate, wash with water until neutral, and dry in vacuum.

2) Dissolve the dry product in 0.5-10% (v/v) acetic acid solution to prepare 0.1-0.9% (w/v) chitosan acetic acid solution. After completely dissolving, add acetic anhydride. It is 8-12 times of the molar number of amino groups in chitosan, after reacting at normal temperature for 3-8 hours, adjust the pH value to 13-14 with sodium hydroxide, add acetone precipitation product equivalent to 1.5-5 times the volume of the reaction solution, After centrifugation at 4000 rpm, a precipitate was obtained. The precipitate was treated with 1 mol/L potassium hydroxide methanol solution for more than 5 hours, and washed with methanol until neutral.

3) The above precipitate was dissolved in water and further purified by gel permeation chromatography (GPC). The purified product is freeze-dried to obtain water-soluble chitosan with a deacetylation degree between 40% and 60%.

The prodrug formed by the low molecular weight chitosan of general formula (I) (seeing Fig. 1) or derivative thereof and antineoplastic drug or glucocorticoid or immunosuppressant:

in:

LMWC is low molecular weight chitosan or its derivatives, the molecular weight is between 1,000-100,000.

n is an integer, and the maximum does not exceed the number of active functional groups on each sugar unit on the LMWC;

L is a linking group, selected from the group consisting of ester group, carbonate group, amide group, amide ester group, ether group, amine group, amino acid ester group and amino acid amide;

D is an antineoplastic drug, or a glucocorticoid, or an immunosuppressant; it is characterized in that the drug is covalently linked to the amino group and hydroxyl group in the sugar unit of low molecular weight chitosan or its derivatives through chemical bonds.

In a preferred embodiment of the present invention, in the prodrug, the drug combined with the drug carrier is composed of antineoplastic drugs or glucocorticoids, or immunosuppressants. Among them, the antineoplastic drugs are selected from the group consisting of fluorouracil, chlorambucil, cyclophosphamide, melphalan, sarcoidin and their derivatives, but not limited to these listed drugs; the glucocorticoids are selected from hydrogenated Cortisone, cortisone, dexamethasone, prednisone, prednisolone, dexamethasone, betamethasone, and the group consisting of their derivatives, but not limited to these listed drugs; immunosuppressants Selected from the group consisting of triptolide, cyclosporin A, tacrolimus, rapamycin, mycophenolate mofetil and their derivatives, but not limited to these listed drugs. The drug combined with the drug carrier in the prodrug is preferably triptolide, chlorambucil or prednisolone succinate monoester.

In a preferred embodiment of the present invention, the prodrug obtained is: triptolide succinate monoester-chitosan structural formula (II) or chlorambucil-chitosan structural formula (III) or prednisone Prednisolone succinate monoester-chitosan structural formula (IV) (or called: prednisolone succinate half ester-chitosan) structure is as follows:

in:

L stands for succinic acid linking group

LMWC stands for low molecular weight chitosan or its derivatives, the deacetylation degree is 40%-60%, and the molecular weight is between 1,000-100,000.

n is an integer, and the maximum does not exceed the number of active functional groups on each sugar unit on the LMWC.

Succinic acid is used as a spacer to link drug molecules with low molecular weight chitosan carriers. For drugs with carboxyl groups: chlorambucil, the prodrug can be obtained directly by dehydration with a condensing agent; for drugs without carboxyl groups: triptolide, it needs to be derivatized to generate derivatives with carboxyl groups; for existing succinic acid Drugs of monoester derivatives: prednisolone succinate monoester, directly adopt its succinate monoester derivatives. The prodrug preparation method is briefly described as follows:

(1) The drug is connected to the linking group by one selected from an ester bond, a carbonate bond, an amide bond, an amide ester bond, an ether bond, an amine bond, an amino acid ester bond, and an amino acid amide bond; preferably the drug triptolide Alcohol needs to react with succinic acid to obtain triptolide succinate monoester; the preferred drugs prednisolone succinate monoester and chlorambucil can be directly linked to the carrier without derivatization.

(2) medicine or the medicine derivative that makes, N-hydroxysuccinimide is dissolved in ethanol; Low molecular weight chitosan or derivative are dissolved in distilled water or pH value at 6.0-8.0MOPS damping fluid (if low molecular weight Chitosan or derivatives are insoluble in water, just dissolve with 0.5%-10% HCl solution solution, then adjust near neutrality with MOPS damping fluid), after being completely dissolved, slowly add ethanol mixture dropwise to The water phase is added according to the calculation of water: alcohol = 1:9-9:1 (v/v), and finally water-soluble carbodiimide is added as a condensation agent, reacted overnight at 4°C, and 1.5-5 times the volume of the reaction solution is added The product was precipitated with acetone, washed several times, and dried to obtain the prodrug.

(3) The preparation method of triptolide succinate monoester: triptolide and succinic anhydride are dissolved in the organic solvent dichloromethane, chloroform or pyridine in the ratio of 1:1-1:100, add catalyst Dimethylaminopyridine and triethylamine reacted completely at room temperature and purified by silica gel column chromatography. Triptolide succinate monoester was obtained.

Condensing agents may include: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl), N-cyclohexyl-N'-trimethylaminopropylcarbodiimide Aminoiodide (EDCI), N-cyclohexyl-N'-morpholineethylcarbodiimide methyl p-toluenesulfonate.

According to one aspect of the present invention, there is provided a pharmaceutically acceptable formulation of the above prodrug. Including tablets, suppositories, soft or hard gelatin capsules, solutions, suspensions or aerosols, lyophilized powder injections, preferably lyophilized powder injections. Wherein, the prodrug preparation can be administered through oral, intranasal, rectal, transdermal or injection, preferably the preparation administered by injection. Its preparation methods are well known to those skilled in the art.

Another object of the present invention is to provide the pharmaceutical evidence that the low molecular weight chitosan and the prodrug using it as a drug carrier can specifically accumulate in the kidney and release the original drug in the kidney. Using FITC-labeled low molecular weight chitosan and prednisolone succinate-chitosan prodrugs to carry out in vitro and in vivo tests in order to achieve the following purposes: 1. Determine that low molecular weight chitosan has good kidney function Targeting efficiency; 2. Ensure that the prodrug will not degrade in the body before reaching the kidney; 3. After the prodrug reaches the kidney, it can be hydrolyzed to release the drug.

1. In vivo distribution of fluorescently labeled low molecular weight chitosan:

After injecting FITC-labeled low-molecular-weight chitosan through the tail vein of mice, the mice were sacrificed at 15 minutes, 30 minutes, 60 minutes and 120 minutes, and the hearts, livers, spleens, lungs, and kidneys were taken and weighed to prepare After homogenizing and precipitating protein, measure the content with a fluorescence spectrophotometer, calculate the average amount of chitosan in each tissue of each mouse as a percentage of the total injection, and the results are shown in Figure 2. It shows that low molecular weight chitosan has good kidney targeting efficiency.

2. In vitro release of prednisolone succinate-chitosan prodrug:

The in vitro stability test confirmed that the conjugate was mixed with rat plasma at 37°C, shaken, and the amount of free drug in the plasma was measured regularly by high performance liquid chromatography (HPLC). The results confirm that the prodrug is relatively stable, and the amount of free drug released in 2 hours is less than 10% of the bound drug. Considering that the prodrug can be rapidly absorbed by the kidney in the blood (<15 minutes), it is necessary to avoid the prodrug from reaching the kidney. before releasing the drug. However, the prodrug can release the drug rapidly in the homogenate of rat kidney tissue, and the results are shown in Figure 3. The curve (◆) in the figure represents the cumulative release of prodrug in rat plasma; the curve (■) represents the cumulative release of prodrug in rat kidney tissue homogenate; the curve (▲) represents the precursor Cumulative drug release in the blank control solution.

3. In vivo distribution of prednisolone succinate-chitosan prodrug:

A common way to evaluate the targeting of a drug or preparation is to measure the relative concentration of the drug in each organ at a certain time point, that is, the targeting efficiency. Inject the prodrug and prednisolone injection through the tail vein of the mice, kill them regularly, measure by HPLC, calculate the drug concentration in each gram of organs, and find that in the group injected with the prodrug, the drug concentration in the kidney is much higher than that in the kidney. In the control group, the distribution of prednisolone in various organs and tissues was relatively balanced, and the drug concentration in the kidney tissue was far lower than that in the prodrug group. The results are shown in Figure 4. The results prove that: prednisolone succinate-chitosan prodrug has good targeting efficiency.

In summary, low-molecular-weight chitosan is a new drug carrier with good kidney targeting efficiency. The model drug and its prodrug can specifically deliver the drug to the kidney, activate and release the original drug. Produce local immunosuppressive effect, effectively reduce its toxic and side effects, and enhance its curative effect.

Description of drawings:

Figure 1. Structural formula of low molecular weight chitosan or its derivatives

Figure 2. Distribution of fluorescently labeled chitosan in various organs and tissues

Figure 3. The release curve of prednisolone succinate-chitosan prodrug in rat plasma, kidney tissue homogenate and blank control solution

Figure 4. Distribution of prednisolone succinate-chitosan prodrug and prednisolone reference substance in various organs and tissues of mice

Figure 5. The 400 MH NMR spectrum of water-soluble chitosan with a molecular weight of 20,000 Daltons

Figure 6. Distribution of prednisolone succinate-chitosan prodrug (Mw=10000) in various organs and tissues of mice

Figure 7. Distribution of prednisolone succinate-chitosan prodrug (Mw=40000) in various organs and tissues of mice

specific embodiment

The drug carrier, prodrug and preparation method thereof of the present invention will be further described in conjunction with the implementation below, which does not limit the present invention, and the scope of the present invention is defined by the claims.

Example 1:

Preparation of low molecular weight water-soluble chitosan

Dissolve 3g chitosan in 150ml 0.45% (w/v) hydrochloric acid aqueous solution, after treating to dissolve completely, filter, add 1.88ml hydrogen peroxide, stir reaction at 40-70 ℃ for 2 hours. Adjust the pH value to 13 with sodium hydroxide, precipitate out, centrifuge at 4000 r/c to obtain a low molecular weight chitosan precipitate, wash with water until neutral, and then vacuum dry.

Take 1g of the dry product and dissolve it in 170ml of 3% acetic acid solution. After completely dissolving, add 6ml of acetic anhydride, stir and react at room temperature for 4 hours, adjust the pH value to 13 with sodium hydroxide, add 500ml of acetone, and centrifuge to obtain a precipitate , the precipitate was treated with 1 mol/L potassium hydroxide in methanol for 5 hours, washed with methanol to neutrality, dissolved in water, and further purified by gel permeation chromatography (GPC). After freeze-drying, water-soluble chitosan is obtained. Take 5 mg and dissolve it in 0.5 ml of heavy water (D ₂ O), and measure it by 400 M NMR, see Figure 5, the degree of deacetylation is 56%.

Example 2:

Preparation and in vivo distribution of fluorescently labeled chitosan

Accurately weigh 300mg of chitosan (Mw=20000, deacetylation degree is about 50%, water-soluble) and dissolve in 10ml of 1mol/L HCl solution, then adjust the pH value to 6.9 with NaOH. Add 21 mg FITC (fluorescein isothiocyanate), mix well, react at room temperature for 24 hours, and dialyze. The dialyzed sample was freeze-dried (chitosan itself is a good scaffold base, so there is no need to add other scaffold bases). The fluorescein content was determined to be 6.56% (w/w). Take 20 Kunming mice, male, weighing between 25-30 grams, each group of 5 mice, a total of 4 groups. Calculated according to the injection dose of 40mg/kg, inject FITC-chitosan into the tail vein, kill the mice at 15min, 30min, 60min, and 120min respectively, take the heart, liver, spleen, lung, and kidney, weigh them, and prepare a tissue homogenate. After the protein was precipitated, the content was measured with a fluorescence spectrophotometer, and the average amount of chitosan in each tissue of each mouse was calculated as a percentage of the total injection amount. The results are shown in Figure 2. The results proved that low molecular weight chitosan is a drug carrier with good kidney targeting.

Example 3:

Preparation of prednisolone succinate-chitosan prodrug

Precisely weighed 115mg of prednisolone succinate monoester, 28.8mg of N-hydroxysuccinimide, dissolved in 20ml ethanol; accurately weighed 100mg of chitosan (Mw=10000 Dalton, deacetylation degree is 50% About, water-soluble) dissolved in 30ml distilled water, after completely dissolving, slowly add the ethanol mixture dropwise to the water phase under stirring condition, finally add 120mg 1-(3-dimethylaminopropyl)-3-ethylcarbodi Imine hydrochloride was used as a condensation agent, reacted overnight at 4°C, precipitated the conjugate with 150ml of acetone, washed the precipitate several times, and dried to obtain the prodrug. Sealed and stored at low temperature.

Accurately weighed 115mg of prednisolone succinate, 28.8mg of N-hydroxysuccinimide, dissolved in 20ml of ethanol; accurately weighed 100mg of chitosan (Mw=20000 Daltons, water insoluble), added 10ml After 0.5% HCl is dissolved, add 20ml of MOPS buffer solution with pH=7.2, slowly add the ethanol mixture to the water phase, and finally add 120mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt Acetate as the catalyst, reacted overnight at 4°C, precipitated the conjugate with 150ml of acetone, washed the precipitate several times, and dried to obtain the prodrug. Sealed and stored at low temperature.

Example 4:

In Vitro Drug Release of Prednisolone Succinate-Chitosan Prodrug

An ideal targeted prodrug should be stable before reaching the kidney, and release the active parent drug after reaching the target organ or target cell to take effect. The present invention studies the drug release behavior of the prodrug in rat plasma, rat kidney tissue homogenate and control solution in detail. Studies have shown that the prodrug is relatively stable in rat plasma, and the drug released within 15 minutes is less than 10% of the total drug, while the drug release rate in rat kidney homogenate is relatively fast. This ensures that the prodrug can reach the kidney and release the parent drug.

Take 5ml of heparin-anticoagulated fresh plasma from SD rats, preheat at 37°C, add prodrug to make the concentration 200μg/mL, shake in a water bath at 37°C, take 0.2ml of regular samples, add methanol to precipitate protein, and vortex shake for 5 Minutes, centrifugal (14000 rpm, 15 minutes), high-performance liquid chromatographic determination of free drug concentration in the supernatant.

Take 5ml of 33% kidney tissue homogenate from SD rats, preheat at 37°C, add prodrug to a concentration of 200μg/mL, shake in a water bath at 37°C, take 0.2ml of regular samples, add methanol to precipitate protein, and vortex shake for 5 minutes , centrifugal (14000 rev/min, 15 minutes), high performance liquid chromatographic determination supernatant free drug concentration. It was found that the prodrug was relatively stable in rat plasma, but could rapidly release the parent drug in kidney tissue homogenate, the results are shown in Figure 3. The curve (◆) in the figure represents the cumulative release of prodrug in rat plasma; the curve (■) represents the cumulative release of prodrug in rat kidney tissue homogenate; the curve (▲) represents the precursor Cumulative drug release in the blank control solution.

Example 5:

Three different molecular weights, about 50% degree of deacetylation of prednisolone succinate-chitosan prodrug in vivo tissue distribution study:

Take 20 Kunming mice, male, weighing between 25-30 grams, and randomly make a group of 5 mice, a total of 4 groups. Calculated according to the injection dose of 28mg/kg, prednisolone was injected into the tail vein. The mice were killed at 15min, 30min, 60min, and 120min respectively, and the heart, liver, spleen, lung, kidney, and plasma were collected and prepared into 33% tissue homogenate and 50% plasma, adding methanol and 10% trichloroacetic acid to precipitate proteins , vortex shaking for 5 minutes, centrifugation (14000 rpm, 15 minutes), high performance liquid chromatography was used to measure the free drug concentration in the supernatant, and the average concentration of prednisolone per gram of organ tissue (or per ml of blood plasma) was calculated. content. The results are shown in Figure 4.

Another 20 Kunming mice, male, weighing between 25-30 grams, were randomly divided into groups of 5 each, a total of 4 groups. Tail vein injection of prednisolone succinate-chitosan prodrug, the Mw of chitosan is 20000 Daltons, the injection dose is calculated according to the amount of prednisolone in the prodrug, which is equivalent to prednisolone Songlong 28mg/kg. The mice were sacrificed at 15min, 30min, 60min, and 120min respectively, and the heart, liver, spleen, lung, kidney, and plasma were collected and prepared into 33% tissue homogenate and 50% plasma, adding NaOH to hydrolyze the prodrug, and then adding methanol and 10% trichloroacetic acid precipitation protein, vortex shaking 5 minutes, centrifugation (14000 rpm, 15 minutes), high performance liquid chromatography measures the free drug concentration of the supernatant, calculates the average organ tissue per gram (or per ml Plasma), the content of prednisolone. The results are shown in Figure 4.

Another 20 Kunming mice, male, weighing between 25-30 grams, were randomly divided into groups of 5 each, a total of 4 groups. Prednisolone succinate-chitosan prodrug was injected into the tail vein, and the Mw of chitosan was 10000 Daltons. The injection dose is calculated according to the amount of prednisolone in the prodrug, which is equivalent to 28 mg/kg of prednisolone. The biological sample processing method is the same as above, and the results are shown in Figure 6.

Another 20 Kunming mice, male, weighing between 25-30 grams, were randomly divided into groups of 5 each, a total of 4 groups. Tail vein injection of prednisolone succinate-chitosan prodrug, the Mw of chitosan is 40000 Daltons. The injection dose is calculated according to the amount of prednisolone in the prodrug, which is equivalent to 28 mg/kg of prednisolone. The biological sample processing method is the same as above, and the results are shown in Figure 7.

The results all proved that prednisolone succinate-chitosan prodrug has good renal targeting efficiency.

The invention discloses a new drug carrier with good kidney targeting efficiency, and prepares a prodrug through a chemical bond with the drug, so that the drug can be targeted to the kidney, reduce the concentration and side effects of the drug in other parts of the body, and reduce the The dose of the drug increases the efficacy.

Any one of the above prodrugs and its clinically available formulations can be used in the prevention and control of acute and chronic nephritis, renal tumors, anti-rejection after renal transplantation and other renal diseases.

The preparation method of the invention is simple, the process is mature, the performance is stable, and it is suitable for large-scale production.

Claims (6)

1, a kind of prodrug of kidney-targeted medicine vector formation is characterized in that described prodrug is to combine formation with low-molecular weight chitoglycan with glucocorticoid, has structure shown in the general formula (I):

Wherein:

LMWC is a low-molecular weight chitoglycan;

N is a positive integer, and maximum is no more than the active function groups number on each sugar unit on the LMWC;

L is a linking group, is selected from ester group, carbonate group, amide groups, amide ester group, ether, amido, aminoacid ester group, amino acid amide;

D is a glucocorticoid;

It is characterized in that medicine is covalently bound by the amino in the sugar unit of chemical bond and low-molecular weight chitoglycan, hydroxyl;

The molecular weight of described low-molecular weight chitoglycan is 1, and 000-100 is between 000; Be the chitosan with good aqueous solubility of deacetylation between 40%-60%.

2, the prodrug that forms according to the kidney-targeted medicine vector of claim 1 is characterized in that described glucocorticoid is selected from hydrocortisone, cortisone, dexamethasone, prednisone, prednisolone, dexamethasone, betamethasone.

3, the prodrug that forms according to the kidney-targeted medicine vector of claim 2 is characterized in that described prodrug is prednisolone succinate monoester-chitosan, has structural formula (IV):

Wherein:

L represents the succinic acid linking group

LMWC represents low-molecular weight chitoglycan, and deacetylation is at 40%-60%, and molecular weight is 1, and 000-100 is between 000.

N is a positive integer, and maximum is no more than the active function groups number on each sugar unit on the LMWC.

4, the prodrug that forms according to each described kidney-targeted medicine vector of claim 1-3, the acceptable preparation of materia medica that it is characterized in that described prodrug comprise tablet, suppository, soft or hard-gelatin capsules, solution, suspensoid or aerosol, freeze-dried powder.

5, the method for the prodrug that forms of a kind of kidney-targeted medicine vector for preparing claim 1 is characterized in that:

(1) medicine is connected with linking group by being selected from a kind of in ester group, carbonate group, amide groups, amide ester group, ether, amido, aminoacid ester group, the amino acid amide base;

(2) medicaments derivative, the N-hydroxy-succinamide that makes is dissolved in ethanol; Low-molecular weight chitoglycan is dissolved in distilled water or pH value at the 6.0-8.0MOPS buffer; If low-molecular weight chitoglycan is water insoluble, just, regulate near neutral then with the MOPS buffer with the HCl solution dissolving of 0.5%-10%; After treating to dissolve fully, under stirring condition, alcohol mixture slowly is added drop-wise to water, press water: alcohol=1: 9-9: 1 (v/v) calculates adding, adding water-soluble carbodiimide at last is condensing agent, 4 ℃ of reactions are spent the night, add the acetone precipitation product that is equivalent to reactant liquor 1.5-5 times volume, and after the washing precipitation for several times, the dry prodrug that gets;

Wherein condensing agent is selected from: N-(3-dimethylamino-propyl)-N '-ethyl-carbodiimide hydrochloride, N-cyclohexyl-N '-trimethylamine propyl group carbodiimide iodized salt, N-cyclohexyl-N '-morphine quinoline ethyl carbodiimide methyl tosylate.

6, according to the application in each the medicine of the anti-rejection of prodrug after preparation prevention and control acute and chronic nephritis, tumor of kidney, renal transplantation of claim 1-3.

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