CN117756920A - Albumin drug conjugate for delivering tacrolimus to podocyte in targeted manner and preparation method thereof - Google Patents
Albumin drug conjugate for delivering tacrolimus to podocyte in targeted manner and preparation method thereof Download PDFInfo
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- 229960001967 tacrolimus Drugs 0.000 title claims abstract description 74
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 title claims abstract description 74
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Abstract
The invention belongs to the technical field of pharmaceutical preparations, and provides a preparation method of an albumin drug conjugate for delivering tacrolimus to podocytes in a targeted manner, which comprises the following steps: the method comprises the following steps: firstly, coupling tacrolimus and albumin by glutaraldehyde to obtain a tacrolimus-albumin conjugate; then, the mixture is stirred and reacted with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) to activate the free hydroxyl groups on the albumin; then adding RLD to react to obtain an RLD-tacrolimus-albumin conjugate; the RLD-tacrolimus-albumin conjugate of the invention does not release tacrolimus when entering blood because of blood dilution; the particle size of the RLD-tacrolimus-albumin conjugate is smaller, so that the medicine can pass through glomerular basement membrane more easily; targeting of RLD increases glomerular podocyte uptake of tacrolimus and decreases pancreatic, thymus and lymph node uptake of tacrolimus; thereby enhancing the therapeutic effect of tacrolimus and reducing the risk of raising blood sugar and infection.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an albumin drug conjugate for delivering tacrolimus to podocytes in a targeting way, and a preparation method and application thereof.
Background
Tacrolimus (TAC) is a drug recommended by the kdago guidelines for treating immune-related kidney diseases such as membranous nephropathy, igA nephropathy, lupus nephritis, and the like, significantly improving podocyte injury and kidney prognosis in patients. However, the tacrolimus Mo Sishui is poorly soluble and has low oral bioavailability, and is mainly combined with erythrocyte and plasma proteins after intravenous injection, and the concentration is rapidly reduced. Clinically, intravenous or oral administration of tacrolimus often causes elevated blood glucose and increases the risk of infection. Therefore, if the tacrolimus can be subjected to dosage form modification, the water solubility is improved, and meanwhile, the tacrolimus is delivered in a targeting manner, so that the curative effect can be greatly improved, and the side effect can be reduced.
Albumin is a commonly used nano carrier and has the characteristics of wide sources, good biocompatibility, no immunogenicity, easy modification and the like. Albumin supplementation also increases plasma osmotic pressure and improves fluid distribution, and is clinically used for treating nephrotic syndrome hypoproteinemia and edema. The existing clinical albumin nanometer preparation methods comprise a desolvation method, a ultrasonic emulsification method, a nab technology and the like, wherein the methods are to physically wrap the tacrolimus, and after the tacrolimus is diluted by blood, part of the tacrolimus can be released to the blood in advance and the purpose of targeted cell delivery cannot be achieved; it is reported that the nano-particle size of the tacks Mo Sibai proteins is more than 100nm, which limits their passage through glomerular filtration barriers. At the same time, podocytes have no specificity for albumin uptake. Thus, these disorders require higher doses of tacrolimus Mo Sibai protein in nanometers to achieve the same therapeutic effect.
Integrin αvβ3 expressed on the basal surface of podocytes anchors podocytes to glomerular basement membrane. The polypeptide arginine-leucine-aspartic acid (RLD) is the shortest polypeptide that specifically binds to integrin αvβ3, and studies have shown that RLD binding does not activate the Rac1 signaling pathway to induce podocyte injury. Human Protein Altas data shows that αvβ3 protein expression levels in pancreas, thymus and lymph nodes are significantly lower than in kidneys. Thus, the RLD modified tacrolimus-albumin conjugate can be used to achieve podocyte targeted drug delivery.
Disclosure of Invention
In order to solve the problems, the invention discloses a preparation method of an albumin drug conjugate for delivering tacrolimus to podocytes in a targeting way, so as to solve the problems that the existing tacrolimus preparation worsens blood sugar and increases infection risk.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a preparation method of an albumin drug conjugate for delivering tacrolimus to podocytes in a targeting way, which comprises the following steps:
s01: dissolving tacrolimus in a first solvent, dissolving albumin in a second solvent, and coupling the tacrolimus and the albumin by using glutaraldehyde to obtain a tacrolimus-albumin conjugate;
s02: dispersing the tacrolimus-albumin conjugate into MES buffer, and activating the carboxyl group of the tacrolimus-albumin conjugate by using N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide;
s03: the activated tacrolimus-albumin conjugate and polypeptide arginine-leucine-aspartic acid (RLD) are subjected to temperature control and stirring reaction, unreacted small molecules and catalyst are removed from the obtained product by using a dialysis method, and then the product is subjected to freeze-drying, so that the RLD-tacrolimus-albumin conjugate is obtained.
In S01, the first solvent is one or more of acetone, methanol, acetonitrile, dimethyl sulfoxide and N, N-dimethylformamide; the mass volume ratio of tacrolimus to the first solvent is 2-10:1; preferably, the mass to volume ratio of tacrolimus to the first solvent is 5:1.
The second solvent is one or more of deionized water and PBS, and the mass volume ratio of albumin to the second solvent is 20-100:1; preferably, the mass to volume ratio of albumin to the second solvent is 40:1.
Further, in the step S01, the mass ratio of tacrolimus to albumin is 1:5-15, more preferably 1:8, and the mass-volume ratio of tacrolimus to glutaraldehyde is 4:0.5 to 2.0, more preferably a ratio of 4:1.
Further, in step S01, the coupling temperature is 5 to 20 ℃, more preferably 10 ℃, and the coupling time is 12 to 36 hours, more preferably 24 hours.
Further, in step S02, the mass ratio of tacrolimus-albumin conjugate, N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 1: 0.5-3: 0.5 to 3, more preferably 1:1:1.
further, in step S02, the method further includes the steps of: after adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the tacrolimus-albumin conjugate, stirring for 5-30 min, more preferably 10min, adding N-hydroxysuccinimide, and stirring at 20-25 ℃ for reacting for 0.5-12 h, more preferably 2h to obtain the activated tacrolimus-albumin conjugate.
Further, in step S03, the mass ratio of tacrolimus-albumin conjugate to RLD added in step (2) is 1:0.5 to 5, more preferably 1:1.
further, in step S03, the method further includes the steps of: after RLD is added to the activated tacrolimus-albumin conjugate, the pH is adjusted to 7.0-7.5, and the mixture is stirred and reacted for 6-24 hours at 20-25 ℃, more preferably 12 hours.
The invention also provides an application of the albumin drug conjugate for delivering tacrolimus to the target podocyte, which is prepared by the preparation method, in a drug for treating kidney diseases.
The beneficial effects of the invention are as follows:
the RLD-tacrolimus-albumin conjugate obtained by covalent bond coupling in the invention can not release tacrolimus because of blood dilution when entering blood; the solubility of tacrolimus in the aqueous solution reaches 20 mug/ml; the particle size of the RLD-tacrolimus-albumin conjugate is 20-40 nm, so that the medicine can pass through glomerular basement membrane more easily; the targeted effect of RLD increases the uptake of tacrolimus by glomerular podocytes and reduces the uptake of tacrolimus by pancreas, thereby enhancing the therapeutic effect of tacrolimus and reducing the risk of raising blood glucose.
Drawings
FIG. 1 is a TEM image of the RLD-tacrolimus-albumin conjugate prepared in example 1;
FIG. 2 is a MALDI-TOF chart of the RLD-tacrolimus-albumin conjugate prepared in example 1;
FIG. 3 is a Fourier transform infrared spectrum of the RLD-tacrolimus-albumin conjugate prepared in example 1;
FIG. 4 shows the serum release profile of the RLD-tacrolimus-albumin conjugate prepared in example 1.
Detailed Description
The present invention is further illustrated in the following drawings and detailed description, which are to be understood as being merely illustrative of the invention and not limiting the scope of the invention.
Example 1
Preparation of RLD modified tacrolimus-albumin conjugate
100mg of tacrolimus was dissolved in 20ml of acetone, 800mg of human albumin was dissolved in 20ml of deionized water, and the tacrolimus acetone solution was added dropwise to the albumin solution while stirring. After that, 50. Mu.L of 50% glutaraldehyde solution was added dropwise, and the mixture was left to stir at 10℃for 24 hours. And (3) fully dialyzing for 24 hours by using a dialysis bag with a molecular cutoff of 3kDa and deionized water, removing unreacted components, and replacing the dialysate every 4 hours. After the dialysis is finished, the white tacrolimus-albumin conjugate is obtained by freeze drying.
50mg of tacrolimus-albumin conjugate was dispersed into 5mL MES buffer (50 mM, pH 6.0). 50mg of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) is added and stirred, after 10 minutes, 50mg of N-hydroxysuccinimide is added and stirring is continued for 2 hours at room temperature of 20-25 ℃. Then 50mg of RLD is added, the pH of the system is adjusted to 7.5 by 0.1M NaOH, and stirring is continued for 12 hours at room temperature of 20-25 ℃. The obtained product is fully dialyzed for 24 hours by using a dialysis bag with molecular retention of 3kDa and deionized water, unreacted components are removed, and the obtained product is freeze-dried, and the measured recovery rate is 80 percent and the purity is 97 percent.
Example 2
RLD-tacrolimus-albumin conjugate transmission electron microscope characterization
0.5mg of RLD-tacrolimus-albumin conjugate was dissolved in 0.5ml of deionized water, one drop was taken onto a 300 mesh copper mesh, and dried and then negatively dyed with 1% phosphotungstic acid for 5min. And then observed under a transmission electron microscope. As shown in FIG. 1, the particle size of the RLD-tacrolimus-albumin conjugate is 20-40 nm.
Example 3
Characterization of RLD-Tacrolimus-Albumin conjugate MALDI-TOF
The RLD-tacrolimus-albumin conjugate sample was purified using a desalting column followed by freeze-drying. The RLD-tacrolimus-albumin conjugate was dissolved in ultrapure water and diluted to 100mmol/L. Protein standards were mixed with the samples. The sample solution and the saturated matrix solution (saturated 2, 5-dihydroxybenzoic acid) were mixed uniformly in a 1:1 ratio. Mu.l of the mixed solution was applied to the sample target and air-dried. And (5) sampling and measuring to obtain a mass spectrogram and the molecular weight of the protein. As shown in fig. 2, the RLD-tacrolimus-albumin conjugate has a molecular weight of 68.524KDa, and an average of 2 tacrolimus molecules per albumin can be conjugated, calculated from the albumin and tacrolimus molecular weights.
Example 4
Fourier transform infrared spectrum characterization of RLD-tacrolimus-albumin conjugate
1mg of human albumin, tacrolimus, RLD, and RLD-tacrolimus-albumin conjugate samples were ground into fine powder in an agate mortar, the obtained powder was uniformly mixed with dried potassium bromide, placed into a mold, and compressed into tablets on a tablet press. And placing the sample on a detection window of a Fourier transform infrared spectrometer for infrared scanning. Test resolution of 4cm -1 The test range is 400-4000cm -1 . As shown in fig. 3, the RLD-tacrolimus-albumin conjugate has characteristic absorption peaks for all individual material components.
Example 5
Solubility of RLD-tacrolimus-albumin conjugate:
preparing RLD-tacrolimus-albumin conjugate aqueous solutions with different concentrations, adding acetonitrile with equal volume, oscillating for 1min on a vortex oscillator, and then performing ultrasonic crushing for 5min in an ice water bath. Zinc sulfate-methanol solution is added to extract tacrolimus. Then redissolved in 10mM ammonium acetate-0.5% formic acid in water. Chromatographic conditions: a Waters TDMC chromatographic column (5 μm,10 mM. Times.2.1 mM) was used, the column temperature was 55deg.C, mobile phase A was 10mM ammonium acetate-0.5% formic acid aqueous solution, mobile phase B was 0.5% formic acid methanol solution, and rapid gradient elution was performed at a flow rate of 0.25ml/min and a sample injection amount of 20. Mu.l. Mass spectrometry conditions: electrospray ionization source (ESI), electrospray voltage 3.0kV, cone hole voltage 20V, extraction voltage 5V, cone hole temperature 105 ℃, drying gas temperature 400 ℃, nitrogen flow 400L/h, purge gas flow 50L/h, collision air pressure 0.325Pa, monitoring mode of MRM mode, TAC m/z 821.30-768.20. Standard curves were established using TAC standards of 20, 10, 8, 4, 2, 1, 0.1ng/ml, and TAC concentrations were calculated from the standard curves. The calculated RLD-tacrolimus-albumin conjugate was about 20ug/ml water soluble.
Example 6
Stability of RLD-tacrolimus-albumin conjugate:
2mg of the RLD-tacrolimus-albumin conjugate sample was dissolved in 2ml of PBS and placed in a dialysis bag with a molecular cut-off of 3kDa, and the two ends were fastened. PBS release medium containing 0.1% Tween-80 was prepared, placed in a constant temperature shaker, continuously shaken at 250rpm/min, and 1ml release medium was withdrawn at 0h, 6h, 12h, 24h, 48h, 72h, 96h and 120h, respectively, while 1ml fresh release medium was replenished. The TAC content was measured as in example 5, and a release curve was plotted. As shown in fig. 4, RLD-tacrolimus-albumin conjugate was released slowly, only about 5.3% after 12 hours.
Example 7
The only difference from example 1 is that: in step S01, the mass ratio of tacrolimus to albumin is set to 1:5, and the other conditions are the same. The RLD-tacrolimus-albumin conjugate was tested for water solubility of about 21.7ug/ml as per example 5.
Example 8
The only difference from example 1 is that: in step S01, the mass ratio of tacrolimus to albumin was set to 1:15, and the other conditions were the same. The RLD-tacrolimus-albumin conjugate was tested for water solubility of approximately 5.4ug/ml as in example 5.
Comparative example 1
The only difference from example 1 is that: in the step S01, the mass ratio of tacrolimus to albumin is 1:4, and the mass volume ratio of tacrolimus to glutaraldehyde is 1:1, the remaining steps and conditions were the same as in example 1. The solubility of tacrolimus in the RLD-tacrolimus-albumin conjugate is 4ug/ml, the TEM detection particle size is 96.4-183.6 nm, and the release rate of tacrolimus in the RLD-tacrolimus-albumin conjugate is 37.4% after 12h by dialysis, which suggests that too high tacrolimus and glutaraldehyde may cause a large amount of albumin self-crosslinking.
Performance test 1
RLD-tacrolimus-albumin conjugate reduces diabetic nephropathy mouse proteinuria:
RLD-tacrolimus-albumin conjugates were synthesized according to example 1, and mice were randomized as designed into 4 groups of normal control, diabetic nephropathy + free tacrolimus treatment, diabetic nephropathy + RLD-tacrolimus-albumin conjugate treatment, each group of 10. Free tacrolimus group and RLD-tacrolimus-albumin conjugate group mice were given tail intravenous infusion of an equivalent amount of tacrolimus of 0.5mg/kg/d for a continuous treatment of 28d. After the treatment is finished, urine albumin/urine creatinine is measured by taking random urine, and fasting blood glucose is measured. The results are shown in table 1, and it can be seen that RLD-tacrolimus-albumin conjugate significantly reduced urinary albumin/urinary creatinine levels in diabetic nephropathy mice without significant impact on abdominal blood glucose.
Table 1 groups of mice urine albumin/urine creatinine (mg/g) and fasting blood glucose (mmol/L)
| Grouping | Urinary albumin/urinary creatinine | Fasting blood sugar |
| Normal control group (n=10) | 16.7±7.4 | 5.3±1.2 |
| Diabetic nephropathy group (n=10) | 347.9±95.2 | 20.7±4.3 |
| Free tacrolimus treatment group (n=10) | 205.7±84.6 | 26.3±8.7 |
| RLD-tacrolimus-albumin conjugate treatment group (n=10) | 96.0±32.5 | 21.3±79 |
Performance test 2
RLD-tacrolimus-albumin conjugate improves tacrolimus tissue distribution:
RLD-tacrolimus-albumin conjugates were synthesized according to example 1, and mice were randomly divided into 2 groups of free tacrolimus, diabetic nephropathy + RLD-tacrolimus-albumin conjugate groups of 6 each, as designed. Each mouse was given an equivalent amount of tacrolimus of 0.5mg/kg/d by tail vein and treated continuously for 3d. After the treatment, the renal cortex and pancreas were taken and weights (mg) were recorded. The tissue tacrolimus was then ground, extracted, TAC content was measured as in example 5 and corrected for body weight. The results are shown in table 2, and it can be seen that RLD-tacrolimus-albumin conjugate significantly increased renal uptake of tacrolimus and decreased pancreatic uptake of tacrolimus.
TABLE 2 Tacrolimus content (ng/mg) in different organs of mice of each group
| Organ | Free tacrolimus | RLD-tacrolimus-albumin conjugates |
| Renal cortex (n=6) | 5.8±1.1 | 13.6±3.0 |
| Pancreas (n=6) | 4.4±1.2 | 2.7±0.8 |
It should be noted that the foregoing merely illustrates the technical idea of the present invention and is not intended to limit the scope of the present invention, and that a person skilled in the art may make several improvements and modifications without departing from the principles of the present invention, which fall within the scope of the claims of the present invention.
Claims (9)
1. A method for preparing an albumin drug conjugate for targeted delivery of tacrolimus to podocytes, comprising the steps of:
s01: dissolving tacrolimus in a first solvent, dissolving albumin in a second solvent, and coupling the tacrolimus and the albumin by using glutaraldehyde to obtain a tacrolimus-albumin conjugate;
s02: dispersing the tacrolimus-albumin conjugate into MES buffer solution, and activating carboxyl of the tacrolimus-albumin conjugate by using N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to obtain an activated tacrolimus-albumin conjugate;
s03: the activated tacrolimus-albumin conjugate reacts with polypeptide arginine-leucine-aspartic acid to obtain the RLD-tacrolimus-albumin conjugate.
2. The method of claim 1, wherein in step S01, the first solvent is one or more of acetone, methanol, acetonitrile, dimethyl sulfoxide, and N, N-dimethylformamide; the second solvent is one or more of deionized water and PBS.
3. The method for preparing the albumin drug conjugate for delivering tacrolimus to foot cells in a targeted manner according to claim 1, wherein in the step S01, the mass ratio of tacrolimus to albumin is 1:5-15, and the mass volume ratio of tacrolimus to glutaraldehyde is 4:0.5 to 2.0.
4. The method for preparing the albumin drug conjugate for delivering tacrolimus to podocytes in a targeted manner according to claim 1, wherein in the step S01, the coupling temperature is 5-20 ℃, and the coupling time is 12-36 h.
5. The method for preparing an albumin drug conjugate for targeting podocyte delivery of tacrolimus according to claim 1, wherein in step S02, the mass ratio of tacrolimus-albumin conjugate, N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 1: 0.5-3: 0.5 to 3.
6. The method of claim 1, wherein in step S02, the method further comprises the steps of: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the tacrolimus-albumin conjugate, stirring for 5-30 min, adding N-hydroxysuccinimide, and stirring at 20-25 ℃ for reacting for 1-12 h to obtain the activated tacrolimus-albumin conjugate.
7. The method for preparing an albumin drug conjugate for targeting podocyte delivery of tacrolimus according to claim 1, wherein in step S03, the mass ratio of tacrolimus-albumin conjugate to polypeptide arginine-leucine-aspartic acid added in step (2) is 1:0.5 to 5.
8. The method of claim 1, wherein in step S03, the method further comprises the steps of: adding polypeptide arginine-leucine-aspartic acid into the activated tacrolimus-albumin conjugate, adjusting the pH to 7.0-7.5, and stirring at 20-25 ℃ for reaction for 6-24 hours; and (5) purifying and freeze-drying, and measuring the particle size to be 20-40 nm.
9. Use of an albumin drug conjugate for targeted delivery of tacrolimus to podocytes prepared by the preparation method of any one of claims 1-8 in a medicament for treating kidney diseases.
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