CN100430417C - Preparation method of transferrin (Tf)-nerve growth factor (NGF) conjugate - Google Patents

Abstract

The present invention relates to a conjugate capable of fully performing the function of controlling and treating NGF clinical diseases. The preparation technology is characterized in that firstly, Tf and NGF are extracted and purified from mammal blood plasma and cobra poison by a biochemical method; then, the Tf and the NGF are coupled to form a Tf-NGF conjugate by a protein couplant; the couplant and the uncombined Tf and NGF are removed by a chromatography method, and then a pure Tf-NGF conjugate is prepared. The conjugate has the Tf receptor combination and the NGF biological activity, the Tf can be used as a transport factor to be combined with receptors on vascular endothelial cells to introduce macromolecule medicines NGF to enter the brain tissues and perform the functions on controlling and treating the NGF clinical diseases, and the conjugate is mainly used for preventing and treating brain neural injury diseases of parkinsons disease, Alzheimer's Disease, etc.

Description

Preparation method of transferrin (Tf)-nerve growth factor (NGF) conjugate

technical field

The invention belongs to the field of biological medicine, especially the preparation method of transferrin (Tf)-nerve growth factor (NGF) conjugate.

Background technique

Nerve Growth Factor (NGF, hereinafter referred to as NGF) is a project of the Nobel Prize in Medicine in 1986. A large number of experiments have proved that this substance has a regulatory effect on the development, differentiation, survival and functional repair of nerve cells. It has also been found that the brain and Spinal cord injury diseases, especially diseases such as Parkinson's disease and Alzheimer's disease, are closely related to the degeneration and apoptosis of nerve cells and the changes in the levels of neurotrophic factors such as NGF. Relevant reports have confirmed the preventive and therapeutic effects of NGF on nerve injury diseases. However, since NGF is a non-lipophilic biomacromolecule, it is difficult to enter cells, especially through the blood-brain barrier (Blood Brain Barrier BBB) of the endothelium of the brain capillary wall, which brings difficulties to the prevention and treatment of these diseases with NGF. At present, the cell body translocation function mediated by transferrin Tf (hereinafter referred to as Tf) and its receptor (Transferrin Receptor TfR) has been confirmed. In recent years, extensive research has been carried out on the method of coupling drugs with Tf or its receptor antibody as a carrier for targeted therapy, and some results have been achieved. However, there has been no report on a relatively mature preparation process.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method of a medicine that can effectively exert the clinical disease prevention and treatment effect of NGF for diseases such as Parkinson's whose etiology is closely related to the level of NGF.

The present invention solves the above technical problems with the following technical solutions:

The preparation method of Tf-NGF conjugate is:

Step 1: Extract and purify Tf and NGF from mammalian plasma and cobra venom respectively by biochemical methods.

The second step: use protein coupling agent to couple Tf and NGF to form Tf-NGF conjugate, use chromatography to remove Tf and NGF in the coupling agent and non-binding part, and obtain the pure product of Tf-NGF conjugate, which is filtered Bacteria, NGF activity detection, and qualified Tf-NGF conjugates can be used for drug efficacy experiments in animal models of diseases.

The Tf-NGF conjugate has the biological activity of binding to the Tf receptor and NGF, and can use Tf as a transport factor to bind to the receptor on the vascular endothelial cells to mediate the macromolecular drug NGF to cross the blood-brain barrier and enter the brain tissue, effectively It is mainly used to prevent and treat brain neuron damage diseases, such as Parkinson's disease, Alzheimer's disease and other brain diseases.

Detailed ways

By using the preparation method of the transferrin (Tf)-nerve growth factor (NGF) conjugate of the present invention, the Tf-NGF conjugate that can effectively play the role of NGF in preventing and treating clinical diseases can be produced.

The preparation process steps of coupling Tf and NGF to form Tf-NGF conjugates are:

a. Biochemically extract and purify Tf and NGF from mammal plasma and cobra venom respectively.

b. Use a protein coupling agent to couple Tf with NGF to form a Tf-NGF conjugate, and label part of the NGF with the isotope iodine ¹²⁵ and then couple it with Tf to form a Tf-NGF-I ¹²⁵ conjugate. Use chromatography to remove Tf, NGF, and I ¹²⁵ in the coupler and non-binding parts, respectively. The pure products of Tf-NGF conjugates and Tf-NGF-I ¹²⁵ conjugates are obtained, and the qualified Tf-NGF conjugates can be used for drug efficacy experiments in animal models of diseases after bacterial filtration and testing of NGF activity.

When using the produced Tf-NGF conjugates to carry out the mouse blood-brain barrier penetration test, use NGF-I ¹²⁵ and Tf-NGF-I ¹²⁵ conjugates to perfuse the carotid arteries of mice, and then use normal saline for 10 minutes. The animals were perfused, and the residual NGF-I ¹²⁵ and Tf-NGF-I ¹²⁵ in the cerebral blood vessels were washed away, and the brain tissue was taken out to determine the isotope content. The results are as follows:

The content of I ¹²⁵ in Tf-NGF-I ¹²⁵ group in brain tissue was about 6.9 times higher than that in NGF-I ¹²⁵ group.

When producing the animal model of rat Parkinson's disease, the test rats were injected with the neurotropic damage agent methyl-phenyl-tetrahydropyridine (MPTP). Then use the above-mentioned Tf-NGF conjugates to protect the experimental mice in the model treatment group. By means of immunohistochemistry, electron microscopy and biochemical analysis, it can be observed that the brain tissue, substantia nigra cholinergic nerve Cells etc. are well protected. For example, the number of TH positive cells in the substantia nigra showed 797.0±121.4 in the MPTP group, 2330.0±260.3 in the Tf-NGF conjugate group, and 2381.0±158.0 in the normal control group. The experiments showed that the number of cell survival in the Tf-NGF conjugate group was significantly higher than that in MPTP group, and there was no significant difference in the number of cell survival between the Tf-NGF conjugate group and the normal control group.

The following are examples of preparation of Tf-NGF conjugates:

Example 1:

1. Tf extraction and purification:

1. Salting out: Take 200ml of rat plasma, add 54ml of 0.1mol/L NaHCO ₃ , mix well, add 5.4ml of triaminoacetic acid-Cl ₃ dropwise, stir at 25°C for 5 hours, then add 63g of ammonium sulfate, and place at 25°C for 3 hours Hour. Then centrifuge at 7000rpm for 30 minutes, discard the supernatant, take the precipitate and dissolve it with 30ml PH7.80.02mol/L Tris-HCl buffer solution, put it into a dialysis bag, dialyze the above buffer solution for 12 hours, and change the solution three times in between.

2. Column chromatography: take the above-mentioned salting-out extract and put it on the QAE-Sephadex A-25 column, equilibrate elution with pH7. , 0.175mol/L NaCl phosphate buffer for step gradient elution, collect the third component, the third component is Tf. The third component was dialyzed against distilled water, freeze-dried and stored.

2. NGF extraction and purification:

1. Gel filtration chromatography: Take 2 grams of cobra venom, dissolve it with 25 ml of 1% glacial acetic acid (V/V), put it on a Sephadex G-50 column, and elute with 1% glacial acetic acid for equilibrium. Fraction 2 was collected, which contained NGF. Use 2mol/L NaOH to adjust the pH value to 6.0 for later use.

2. Ion-exchange chromatography: take the second fraction collected by the above-mentioned gel filtration chromatography and put it on SP-sephadex C-25 column, equilibrate elution with pH7.40.05mol/L phosphate buffer, and then use L NaCl, 0.125mol/L NaCl, 0.175mol/L NaCl phosphate buffer for step gradient elution, collect the third component, the third component is NGF. The third component was dialyzed against distilled water, freeze-dried and stored.

Three. Tf-NGF conjugate preparation process:

1. NGF activation reaction: Take 400mg of NGF and dissolve it in 0.16mol/L boric acid buffer solution containing 0.001mol/L EDTA (ethylenediaminetetraacetic acid), and add dropwise 2-iminohydrosulfur chloride dissolved in 7ml of the same buffer solution Alcohol 20mg. The reaction was stirred for 1.5 hours and terminated by adding 37 mg of glycine. The reaction solution was put on a sephadex G-25 column, and eluted with 0.001mol/L EDTA pH7.40.01mol/L phosphate buffer for equilibrium elution, and the first fraction was collected.

2. Tf activation reaction: Take 400mg of Tf and dissolve it in pH 7.40.01mol/L phosphate buffer, add dropwise 45mg of N-hydroxysuccinimide dissolved in 3ml of dimethyl sulfoxide, stir for 1.5 hours, and add sephadex G- 25 column, balanced elution with pH7.40.01mol/L phosphate buffer, and collected the first fraction.

3. Tf, NGF combination reaction: mix the Tf and NGF components after the above activation reaction, and stir for 3 hours. The reaction solution was concentrated and applied to a sephadex G-200 column, and eluted with PH7.40.01mol/L phosphate buffer for equilibrium, and the first component was Tf-NGF conjugate. Further bacteria-filtered, freeze-dried and preserved.

Example 2:

1. The extraction and purification of Tf is the same as in Example 1.

2. NGF extraction and purification:

1. Gel filtration chromatography is the same as in Example 1;

2. Ion exchange chromatography: take the second fraction collected by gel filtration chromatography and put it on the CM-sephacell column, elute with pH 5.8 0.05mol/L acetate buffer, and then use 0.05～0.5mol/L NaCl Acetic acid buffer was used for linear gradient elution, and the third component was collected, which was NGF. The third component was dialyzed against distilled water, freeze-dried and stored.

Three. Preparation of Tf-NGF conjugates:

1. NGF activation reaction: Take 400mg of NGF and dissolve it in 0.16mol/L boric acid buffer solution containing 0.001mol/L EDTA (ethylenediaminetetraacetic acid), and add dropwise 2-iminohydrosulfur chloride dissolved in 7ml of the same buffer solution Alcohol 30mg. All the other are the same as Example 1. 2.Tf activation reaction: get Tf400mg and dissolve with PH 7.40.01mol/L phosphate buffer, add dropwise the 30mg N-hydroxysuccinimide that dissolves with 3ml dimethyl sulfoxide, all the other are with embodiment 1 .

3. The binding reaction of Tf and NGF is the same as in Example 1.

Example 3:

1. The extraction and purification of Tf is the same as in Example 1.

2. NGF extraction and purification:

1. Gel filtration chromatography is the same as in Example 1;

2. Ion exchange chromatography: take the second fraction collected by gel filtration chromatography and put it on the SP-sephadex C-25 column, elute with pH 7.4 0.05mol/L phosphate buffer in balance, and then use 0.05～0.5mol /LNaCl phosphate buffer for linear gradient elution, collect the third component, the third component is NGF. The third component was dialyzed against distilled water, freeze-dried and stored.

Three. Preparation of Tf-NGF conjugates:

1. NGF activation reaction: Take 400mg of NGF and dissolve it in 0.16mol/L boric acid buffer solution containing 0.001mol/L EDTA (ethylenediaminetetraacetic acid), and add dropwise 2-iminohydrosulfur chloride dissolved in 7ml of the same buffer solution Alcohol 43mg. All the other are the same as Example 1. 2.Tf activation reaction: get Tf 400mg and dissolve it with PH 7.40.01mol/L phosphate buffer, add dropwise 20mg N-hydroxysuccinimide dissolved with 3ml dimethyl sulfoxide, and all the other are the same as the examples 1.

3. The binding reaction of Tf and NGF is the same as in Example 1.

The following are application examples of Tf-NGF conjugates:

The pharmacological effect of the Tf-NGF conjugate of the present invention on the mouse Parkinson's disease model:

Select 40-day-old mice (14-16 grams) to be randomly divided into 3 groups: 1, MPTP experimental group (making Parkinson's model); 2, Tf-NGF treatment group (for Parkinson's model treatment); 3, control group (injection physiological saline). Experimental results: After 30 days of the experiment, the experimental mice were anesthetized with pentobarbital sodium, followed by arterial perfusion with normal saline and 4% paraformaldehyde, and the brain tissue was taken for experimental observation. HE staining light microscope analysis and electron microscope analysis respectively showed that the nucleus of neurons in the MPTP experimental group was condensed, vacuolar degeneration appeared, Nissl bodies dissolved, and a large number of obvious myelin sheaths fell off. In the normal control group and the Tf-NGF treatment group, no above-mentioned pathological changes were observed. Immunohistochemical analysis: the count of dopamine cells in the substantia nigra of the brain showed that the survival number of cells in the Tf-NGF treatment group was significantly higher than that in the MPTP experimental group (P<0.05), but there was no significant difference from the normal control group (P>0.05). The experimental results show that the Tf-NGF conjugate can pass through the blood-brain barrier, and can produce good pharmacological effects on the mouse model of Parkinson's disease.

Claims (1)

1. a transferrin Tf and NGF are coupled to form a preparation method for Tf-NGF conjugate, which is characterized in that the processing steps are: 1. Tf extraction and purification: 1. Salting out: Take 200ml of rat plasma, add 54ml of 0.1mol/L NaHCO ₃ , mix well, add 5.4ml of triaminoacetic acid-Cl ₃ dropwise, stir at 25°C for 5 hours, then add 63g of ammonium sulfate, and place at 25°C for 3 hours Then centrifuge at 7000rpm for 30 minutes, discard the supernatant, take the precipitate and dissolve it with 30ml PH7.8 0.02mol/L Tris-HCl buffer solution, put it into a dialysis bag, dialyze the above buffer solution for 12 hours, and change the solution three times in the middle; 2. Column chromatography: take the above-mentioned salting-out extract and put it on the QAE-Sephadex A-25 column, equilibrate with PH7.4 0.05mol/L phosphate buffer for elution, and then use 0.075mol/L NaCl, 0.125mol/L NaCl, 0.175mol/L NaCl phosphate buffer for step gradient elution, collect the third component, the third component is Tf; dialyze the third component against distilled water, freeze-dry and save; 2. NGF extraction and purification: 1. Gel filtration chromatography: take 2 grams of cobra venom, dissolve it with 25 ml of 1% glacial acetic acid (V/V), put it on a Sephadex G-50 column, and elute with 1% glacial acetic acid balance; collect the second component, the first Component 2 contains NGF; use 2mol/L NaOH to adjust the pH value to 6.0 for later use; 2. Ion-exchange chromatography: take the second fraction collected by the above-mentioned gel filtration chromatography and put it on SP-sephadex or CM-sephacell column, equilibrate elution with phosphoric acid or acetate buffer, and then use phosphoric acid or acetate buffer as gradient respectively Elution, collect the third component, the third component is NGF; dialyze the third component against distilled water, freeze-dry and save; 3. Preparation of Tf-NGF conjugates: 1. NGF activation reaction: Dissolve 400mg of NGF in 0.16mol/L boric acid buffer solution containing 0.001mol/L EDTA, add dropwise 20-43mg of 2-imino hydrochloride mercaptan dissolved in 7ml of the same buffer solution; stir the reaction After 1.5 hours, 37 mg of glycine was added to terminate the reaction; the reaction solution was put on a sephadex G-25 column, and eluted with PH7.4 0.01 mol/L phosphate buffer solution of 0.001 mol/L EDTA in equilibrium, and the first component was collected; 2. Tf activation reaction: Take 400mg of Tf and dissolve it in pH 7.4 0.01mol/L phosphate buffer, add dropwise 20-45mg of N-hydroxysuccinimide dissolved in 3ml of dimethyl sulfoxide, and stir for 1.5 hours. Sephadex G-25 column, equilibrated with pH7.4 0.01mol/L phosphate buffer for elution, and collected the first fraction; 3. Tf, NGF combination reaction: mix the Tf and NGF components after the above activation reaction, stir and react for 3 hours; the reaction solution is concentrated on a sephadex G-200 column, and balanced with pH7.4 0.01mol/L phosphate buffer Elution, the first component is Tf-NGF conjugate; further bacterial filtration, freeze-dried storage.