CN1285357A - Snake venom enzyme nerve growth factor - Google Patents

Abstract

The present invention relates to a preparation method of nerve growth factor by using venin. It is characterized by that colbravenom undergoes three-step chromatographic process of CM-sepnadex C50, CM-sphatose FF and Sepndex G75 under the condition of below 10 deg.C to prepare semi-product, to it are added a stabilizing ageng albumin and excipient dextran for freeze-dried product, and It is filtered, filled and freeze-dried so as to obtain the invented product.

Description

Snake venom enzyme nerve growth factor

What the present invention relates to is the processing method for preparing nerve growth factor with snake venom.

Nerve growth factor is a chemical name, Chinese name: neural live plain (the neural factor of giving birth to), and English name: NERVE GROWTH FACTOR, be called for short: NGF.

Nerve growth factor (NGF) is the cell growth regulator of finding the earliest, also is one of most important bioactive molecules of neural system.From finding NGF 40 years so far, brand-new field of neuroscience has been opened up in its discovery and research, has promoted the research and the progress of development biology and cytobiology.For this reason, Nobel judging panels in 1986 have spoken highly of the theory significance and the using value of this work, and should year Nobel Medical Biology award and give two scientist Levi-Montalcini (Italy) and the Cohen (U.S.) that makes outstanding contribution in NGF research.

According to reported literature NGF from many biomaterials separation and purification obtain, these materials comprise: snake venom, mouse submandibular gland, the mature placenta of people, cavy prostate gland and ox seminal vesicle etc., but content the abundantest be still male mice submaxillary gland and multiple snake venom.

Because NGF plays an important role in neural growth and damage, therefore more external drugmakers all drop into a large amount of man power and materials NGF are developed and studies.What foreign study and exploitation were in a leading position at present is Grentech company, and mainly with NGF treatment peripheral nerve injury and some nervus deformability diseases, it is clinical to have entered the second phase at present, but does not form commodity as yet.And the domestic present NGF commodity that are given the ratification that do not have as yet.External like product price of producing is too expensive.It is reported only 0.01mg of NGF that U.S. Sigma company produces, promptly up to 118 dollars, real non-general compatriots can bear.Consider China's snake venom resource than horn of plenty, cheap, if therefrom separate purification NGF very big pharmaceutical use and economic worth will be arranged.

The research of neurotization is the problem that domestic scholars is devoted for years to research always.A large amount of experimental studies results show: nerve growth factor has neuronal development and differentiation such as promoting sympathetic neuron, Sensory neurone and forebrain cholinergic neuron, and keeps their normal function, promotes the reparation after their damages.So it is clinical that nerve growth factor is applied to, remove patient's misery, be to work as forward swing in the previous important problem of our face.Therefore we at first study the nerve growth factor purifying process, and we have set up the technology of a Chinese style scale production nerve growth factor at present, and pharmacology and the clinical application of furtheing investigate nerve growth factor for us lay the foundation.

Purpose of the present invention just provides a kind of processing method for preparing nerve growth factor with snake venom.

The objective of the invention is to take following technical scheme to realize: on the lot of experiments basis, to determine the optimum manufacturing process route of nerve growth factor.From the base-material of the preferred cobra venom of technical parameters such as productive rate, specific activity, through three step chromatographies, the nerve growth factor of the single component of purifying as separation and purification of nerve growth factor.

The nerve growth factor that adopts above-mentioned technical scheme to prepare, animal experiment through preclinical pharmacology shows, nerve growth factor be a kind of to central nervous system and peripheral nervous system growth, grow and the necessary nerve growth active substance of surviving: confirm to have following effects after deliberation: (1) promotes neuronic growth and differentiation.(2) the sophisticated neurone of nutrition is kept its survival and function.(3) have the effector neuron that protection damages, promote the nerve fiber regeneration reparation on this basis.(4) nerve growth factor is the motor neuron chemotropic factor of damaged or generation.

Preparation technology:

All operations is all finished below 10 ℃.

1. CM-Sephadex C50 chromatography

Get cobra venom 10g, be dissolved among 0.01M acid sodium damping fluid, (FH5.8) 100ml, after the dissolving, centrifugal 10000 change, 10 minutes, get supernatant,, change damping fluid 3 times above-mentioned damping fluid 5000ml dialysis, sample is splined on and uses the good chromatography column of 0.01M acetate buffer balance (in 5.5 * 100cm) then, use the 0-0.7MNaCL linear gradient elution, collection has the active protein peak of nerve growth factor, and is concentrated freeze-dried.

2. CM-SpharoseFF chromatography

With above-mentioned spissated sample, be dissolved in the 20ml 0.01M sodium acetate buffer (FH5.8), be splined on and use the good chromatography column of 0.01M sodium acetate buffer (PH5.8) balance (in 2.6 * 50cm), use the 0-1MNaCL linear gradient elution, collection has the active protein peak of nerve growth factor, and is concentrated freeze-dried.

3. SephdexG75 chromatography

With the sample under the above-mentioned CM-SpharoseFF chromatography, be dissolved in 10ml 0.01M sodium acetate buffer, PH5.8 includes among the 0.15MNaCL, and centrifugal 10000 change, get supernatant, last sephdexG75 (2.6 * 100cm) posts (used the 0.01M sodium acetate buffer, PH5.8, it is good to include the 0.15M/L balance), collection contains the active symmetrical peak of NGF, after electrophoresis and high performance liquid chromatography prove simple spike, the dialysis desalination, freeze-drying promptly gets work in-process (about 3mg).

4. preparation prescription:

Raw material: the nerve growth factor work in-process, by the half-finished activity of mensuration of quality standard, every specification 1000 units feed intake by 135%-145%.Add 1% dextran (molecular weight 10,000), and contain the 1.5mg human serum albumin in every medicine, filter packing, freeze-drying makes.

Indication: be used for the treatment of the peripheral nerve injury that a variety of causes causes;

1. peripheral nerve injury due to tension injury, incision, the contusion etc.;

2. metabolic disease: diabetes, uremia etc. cause peripheral neuropathy;

3. protrusion of intervertebral disc, cervical spondylosis causes DPN;

4. the DPN that causes such as antitumor drug taxol, infectious peripheral nerve unit etc.

Usage and consumption and route of administration:

1. each 1000-2000 unit, intramuscular injection, once a day, 20 days is a course of treatment.

2. in the four limbs trauma surgery, can be at administration of art localized pulverization or injured nerve place local injection, each 1-2 props up.

Precaution:

1. this product is the protein preparation, the careful usefulness of allergic constitution.If anaphylaxis occurs, handle by anaphylaxis.

Need vibration repeatedly to dissolve when 2. dissolving medicine.

Validity period and holding conditions:

Preserve tentative 2 years below 10 ℃.

Claims (1)

1, snake venom enzyme nerve growth factor is characterized in that: he has a scientific preparation process:

All operations is all finished below 10 ℃.

1. CM-Sephadex C50 chromatography

Get cobra venom 10g, be dissolved among 0.01M sodium acetate buffer, (FH5.8) 100ml, after the dissolving, centrifugal 10000 change, 10 minutes, get supernatant,, change damping fluid 3 times above-mentioned damping fluid 5000ml dialysis, sample is splined on and uses the good chromatography column of 0.01M acetate buffer balance (in 5.5 * 100cm) then, use the 0-0.7MNaCL linear gradient elution, collection has the active protein peak of nerve growth factor, and is concentrated freeze-dried.

2. CM-SpharoseFF chromatography

With above-mentioned spissated sample; Be dissolved in the 20ml 0.01M sodium acetate buffer (FH5.8), be splined on and use the good chromatography column of 0.01M sodium acetate buffer (PH5.8) balance (in 2.6 * 50cm), use the 0-1MNaCL linear gradient elution, collection has the active protein peak of nerve growth factor, and is concentrated freeze-dried.

3. SephdexG75 chromatography

With the sample under the above-mentioned CM-SpharoseFF chromatography, be dissolved in 10ml 0.01M sodium acetate buffer, PH5.8 includes among the 0.15MNaCL, and centrifugal 10000 change, get supernatant, last sephdexG75 (2.6 * 100cm) posts (used the 0.01M sodium acetate buffer, PH5.8, it is good to include the 0.15M/L balance), collection contains the active symmetrical peak of NGF, after electrophoresis and high performance liquid chromatography prove simple spike, the dialysis desalination, freeze-drying promptly gets work in-process (about 3mg).

4. preparation prescription:

Raw material: the nerve growth factor work in-process, by the half-finished activity of mensuration of quality standard, every specification 1000 units feed intake by 135%-145%.Add 1% dextran (molecular weight 10,000), and contain the 1.5mg human serum albumin in every medicine, filter packing, freeze-drying makes.