CN101336933A - Preparation method of medicine for treating vitiligo and clinical use thereof - Google Patents

Abstract

The invention relates to a preparation method of a medicament for treating vitiligo and an application thereof in clinics. The precursor of the medicament is a basic medium containing 15% human AB-type serum, and the basic medium is made from 15 mL human AB-type serum, 85 mL low-sugar DMEM culture medium, 0.24 g Hepes, 0.2 g sodium hydrogen carbonate and 0.03 g L-glutamine. The medicament is prepared by the steps of: culturing a first passage, a second passage and a third passage porcine dermal papilla cells with the basic medium, collecting culture supernatant of the first passage, the second passage and the third passage porcine dermal papilla cells, and making into lyophilized powder. The inventive medicament has distinct therapeutic effect on vitiligo; and the used cells and culture serum have no antigenicity in human body and may not cause any allergic reactions after applying in human body. In addition, the inventive medicament is applied by subcutaneous injection, so as to remarkably improve the effective availability.

Description

A kind ofly treat the preparation method of vitiligo medicine and the application in clinical thereof

Technical field

The present invention relates to a kind of preparation method of medicine, particularly relate to a kind of the treat preparation method of vitiligo medicine and the application in clinical thereof.

Technical background

Vitiligo is clinical common pigment obstacle dermatoses, does not also have a kind of effective Therapeutic Method at present.

The vitiligo basic pathology is changed to the intraepidermal melanocyte in skin lesion district and disappears, and clinical manifestation is skin symmetry or asymmetric depigmentation patch.Its definite cause of disease and pathogenesis are not clear, may be unusual with autoimmune, multiple factors such as inherited genetic factors, melanocytic fault of construction or desmoenzyme Developmental and Metabolic Disorder are relevant.Many scholars' the white macula mesocuticle melanocyte that studies confirm that lacks rather than functionally inactive, can only external source provide so treat convalescent melanocyte, and its source and Restoration Mechanism it be unclear that, and now thinks to derive from the hair follicle external root sheath more.

Melanocyte can be present in around stratum basale, corium, the blood vessel and hair follicle in the normal skin, all can become melanocytic source place.Clinical observation finds that the recovery of pigment and hair follicle have substantial connection behind the therapy of vitiligo, shows as follicular orifice pigment island on every side, increases gradually, enlarges and merges, and at the position of no hair follicles such as the palm sole of the foot and mucosa, vitiligo is difficult to recovery.Further studies show that, have " melanocyte depots " in the hair follicle, the melanocyte of dormancy is not got involved when vitiligo in the hair follicle, can reactivate under therapeutical effect, division growth, by no function state-transition is functional status, melanocytic source when becoming the therapy of vitiligo recovery.

It is generally acknowledged that (dermal papilla cells is DPC) in the growth of hair follicle and periodically play an important role in the adjusting and controlling growth for human hair papilla cell.Hair follicle is melanocytic to be reactivated and division growth mechanism to regulating to illustrate human hair papilla cell, must set about from the excretory biological activity of human hair papilla cell self, and this also is one of important content of vitiligo research from now on.The present invention is setting up on the human hair papilla cell in-vitro culture model basis, the culture supernatant of application 1,2,3 generation human hair papilla cell is made conditioned medium, observe human dermal papilla cells conditioned medium (dermal papilla cell conditional mediumDPCCM) to leukodermic therapeutical effect, and human dermal papilla cells conditioned medium made lyophilized powder, Preliminary Applications finds that in patients with vitiligo it has definite therapeutic effect to vitiligo.

Summary of the invention

In view of above-mentioned, the object of the present invention is to provide the leukodermic medicine of a kind of treatment.

The purposes of the lyophilized powder that another object of the present invention is to prepare in treatment vitiligo medicine.

The objective of the invention is to be achieved through the following technical solutions:

A kind of with base culture base 1,2,3 generation human hair papilla cell culture supernatant preparation treatment vitiligo medicine, it is characterized in that described basal medium is by 15ml people AB type serum and 85ml low sugar DMEM culture medium, 0.24gHepes, 0.2g sodium bicarbonate, the proportioning of 0.03g L-glutaminate constitutes.

The beneficial effect of advantage of the present invention and generation is:

Human hair papilla cell is the secreted cell, when In vitro culture, can secrete numerous protein, these protein can promote the g and D of hair follicle, proved that at experiment in vitro it has definite biologic activity, but it is applied to the clinical treatment vitiligo and yet there are no report, the present invention is on the basis of original cultivation human hair papilla cell, improve its culture medium, personnel selection AB type blood serum substituting calf serum is successfully turned out human hair papilla cell, collects and cultivates 1,2,3 generation human hair papilla cell basal medium, be used for the clinical treatment vitiligo, observe its therapeutic effect, finding has definite therapeutical effect, has obtained good economic benefit and social benefit.

The medicine of using the present invention's preparation has tangible curative effect in the treatment vitiligo, used cell of this medicine and cultivation serum are all to the equal no antigen of human body, be used for human body and do not have any anaphylaxis, the present invention's base culture base human hair papilla cell that contains people AB type serum, cultivate human hair papilla cell with calf serum than before and obtained breakthrough technically, for this medicinal application is laid a good foundation in clinical, this also is the core place of key technology of the present invention.In addition, the used medicine of the present invention is a subcutaneous injection, and its active drug availability improves greatly, and its preparation technology is very fastidious, and is practical, can be the extensive patients service.

Description of drawings

Fig. 1 treats sketch map before the vitiligo for the present invention, is the boundary by the saggital midline of vitiligo skin lesion, and the left side skin lesion of figure is a therapentic part, and the right side skin lesion of figure is the blank position

Fig. 2 treats vitiligo sketch map after January for the present invention

The specific embodiment

Below by embodiment the present invention is further described again:

The preparation method of medicine and clinical practice thereof are to realize by following steps:

(1). reagent and material

Low sugar DMEM culture medium: available from Sigma company.Hepes: available from Boehringer Mannheim company.Healthy people AB type serum: southwest hospital of Third Military Medical University Blood Transfusion Department provides.

(2). the method for medication preparation

2.1 the preparation of basal medium:

2.1.1 the preparation of people AB type serum

A) gather healthy blood donor (getting rid of HIV, HBV, HCV, infection by Treponema pallidum) whole blood 200ml and go into not have the blood bag of anticoagulant through looking into;

B) will place 1 hour in 37 ℃ of water tanks of whole blood blood bag;

C) 2000rpm is centrifugal 30 minutes;

D) with serum separator separation of human AB type serum, isolating people AB type serum can be put into-20 ℃;

E) people AB type serum bag is taken out from-20 ℃ of household freezers, put into 4 ℃ of cold closetes 12 hours;

F) people AB type serum room temperature was placed 2 hours;

G) people AB type serum was placed 1 hour in 37 ℃ of water tanks;

H) people AB type serum is placed 1 hour removal complement in 56 ℃ of water tanks, its upper and lower temperature difference should be less than 0.5 ℃;

I) on super-clean bench with 2% iodine tincture sterilization serum bag drain pipe 5 minutes, empty doing;

J) use the shears that has disinfected to cut off and seal medicated cap, serum is poured in the 100ml bottle that has disinfected;

K) be packed as the 15ml/ bottle with suction pipe ,-20 ℃ frozen standby.

The step of above-mentioned people AB type serum preparation also can be:

A) gather the blood bag that healthy blood donor whole blood 200ml goes into not have anticoagulant;

B) will place 1 hour in 37 ℃ of water tanks of whole blood blood bag;

C) 2000rpm is centrifugal 30 minutes;

D) with serum separator separation of human AB type serum, it is standby that isolating AB type serum can be put into-20 ℃ of household freezers;

E) people AB type serum bag is taken out from-20 ℃ of household freezers, put into 4 ℃ of cold closetes 12 hours;

F) people AB type serum room temperature was placed 2 hours;

G) people AB type serum was placed 1 hour in 37 ℃ of water tanks;

H) people AB type serum is placed 1 hour removal complement in 56 ℃ of water tanks, its upper and lower temperature difference should be less than 0.5 ℃;

I) on super-clean bench with 2% iodine tincture sterilization serum bag drain pipe 5 minutes, empty doing;

J) use the shears that has disinfected to cut off and seal medicated cap, serum is poured in the 100ml bottle that has disinfected;

K) be packed as the 15ml/ bottle with suction pipe ,-20 ℃ frozen standby.

2.1.2 the preparation of basal medium: with 15ml people AB type serum and 85ml low sugar DMEM culture medium, 0.24gHepes the 0.2g sodium bicarbonate is behind the 0.03g L-glutaminate mixing, with the filtering with microporous membrane degerming of 0.22 micron pore size, directly use or-20 ℃ frozen standby.

2.2 the collection of human dermal papilla cells conditioned medium

(1) get a palm concentric reducer skin behind normal health youth's (getting rid of HIV, HBV, HCV, infection by Treponema pallidum) pillow of unexpected death through looking into, with two step enzyme method separation of human hair papilla cells (referring to the clock white jade, Yang Xichuan, Yang soldier, etc.Two step enzyme digestion separation of human scalp hair papilla cells [J].Chinese journal of dermatology, 2003; 36 (7): 406.)；

In the 50ml culture bottle, add the 5ml basal medium when (2) cultivating, add the 7ml basal medium in the 100ml culture bottle, changed one time basal medium in 3 days;

(3) open super-clean bench and also use disinfection by ultraviolet light 30 minutes, contagion gown during operation, wear disposable breathing mask and medicated cap, with behind the 75% alcohol disinfecting both hands on super-clean bench with 1,2,3 generation human hair papilla cell culture supernatant aseptic pouring in the aseptic centrifuge tube of 50ml from culture bottle, cover centrifuge tube and tighten with revolving a flap, 3000rpm is centrifugal 10 minutes in IK16 type centrifuge (U.S. SIGMA company);

(4) on super-clean bench, open centrifuge tube,, move in the aseptic glass container with the supernatant in the aseptic straw sucking-off centrifuge tube (carefully can not stir bottom precipitation), add aseptic rubber closure after, perform labelling ,-20 ℃ frozen standby.

2.3 the preparation of human dermal papilla cells conditioned medium lyophilized powder

(1) cryodesiccation chamber's disinfection by ultraviolet light is 4 hours;

Contagion gown when (2) operating, wear disposable breathing mask and medicated cap,, cover bottle stopper and reserve the lyophilizing hole with on super-clean bench, human dermal papilla cells conditioned medium being divided into 3ml/ lyophilizing bottle behind the 75% alcohol disinfecting both hands with aseptic straw;

(3) the lyophilizing bottle is put into freeze dryer;

(4) freeze-drying process:

I-40 ℃ 4 hours

II-20 ℃ 3 hours (beginning to be evacuated to end)

III-5 ℃ 10 hours

5 ℃ of IV 4 hours

15 ℃ of V 3 hours

25 ℃ of VI 5 hours

(5) under vacuum environment, vacuum package lyophilizing bottle;

(6) open freeze dryer cryodesiccation chamber door, take out the lyophilizing bottle, add bottle cap and pressurize the package bottle stopper;

(7) labelled, indicate sign and date, 4 ℃ of preservations are standby.

The physicochemical property of treatment vitiligo medicine is:

Color: white;

Form: Powdered;

PH value: 7.35;

Dissolubility: every bottle can dissolve in 3 seconds fully with the 2ml normal saline, and dissolving back liquid is orange red.

Carry out the preparation of medicine according to the method for said medicine preparation before using: every bottle of lyophilized powder 3ml physiological saline solution (original concentration before the recovery lyophilizing).

(3), the biological safety embodiment of medicine

1.1 acute toxicity test in mice: we select 7～9 ages in week, body weight for use is the Kunming mouse of 18～22g.Set up 5 dosage groups such as 50.0ml/kg, 40.0ml/kg, 32.0ml/kg, 25.6ml/kg, 20.5ml/kg by trial test common ratio 0.8, every group of 10 mices, male and female half and half.Toxic reaction, death condition and the time of animal are observed, write down to single intraperitoneal injection immediately behind the medicine, observed continuously 14 days.

1.2 rabbit pyrogen testing: selecting 3 body weight is the Japanese white big ear rabbit of 1.8～2.4kg.Press 10ml/kg ear vein drug administration by injection.Measure rabbit anus body temperature, the degree of depth is 6 centimetres, later on every 1 hour take temperature 1 time, and totally 3 times.

1.3 antibacterial culturing: randomly draw 5 lyophilizing bottles, the physiological saline solution dissolving, 4 road methods of scoring are inoculated on the common blood plate, cultivate observed result after 48 hours.

1.4 fungal culture: randomly draw 5 lyophilizing bottles, the physiological saline solution dissolving is inoculated on the husky Bao Shi culture medium, cultivates observed result after 10 days.

1.5 result: after acute toxicity test in mice was presented at lumbar injection various dose human dermal papilla cells conditioned medium sample solution, each treated animal was not found tangible poisoning symptom, whole test no animal dead or other unusual sign in the time limit.After the rabbit pyrogen testing was presented at ear vein injection human dermal papilla cells conditioned medium sample solution, the body temperature of each animal changed the requirement that all meets pyrogen testing.Antibacterial and fungal culture there is no antibacterial and conk.

(4), the clinical practice of medicine

(1) physicochemical property of treatment vitiligo medicine is:

Color: white;

Form: Powdered;

PH value: 7.35;

Carry out the preparation of medicine according to the method for said medicine preparation before using: every bottle of lyophilized powder 2ml physiological saline solution (original concentration before the recovery lyophilizing).Drug solubility: every bottle can dissolve in 3 seconds fully with the 2ml normal saline, and dissolving back liquid is orange red.

(2) therapeutic scheme: selecting for 50 example ages is subjects at 12～46 years old patients with vitiligo, subcutaneous multi-point injection behind the routine disinfection pars affecta skin, 1 time weekly, the each 2ml at most of each affected part (diameter is less than 3 centimetres), totally 4 times, after finishing, treatment observed 6 months.

Human dermal papilla cells conditioned medium is applied to affected part under the situation of patient's informed consent after identifying by bio-safety.In the patients with vitiligo of 50 routine human dermal papilla cells conditioned medium treatments, all the other all have certain curative effect except that 6 examples are invalid.

Claims (2)

1, a kind of with base culture base 1,2,3 generation human hair papilla cell culture supernatant preparation treatment vitiligo medicine, it is characterized in that described basal medium is by 15ml people AB type serum and 85ml low sugar DMEM culture medium, 0.24gHepes, 0.2g sodium bicarbonate, the proportioning of 0.03g L-glutaminate constitutes.

2, the lyophilized powder by the described culture supernatant preparation of claim 1 is preparing the purposes for the treatment of in the vitiligo medicine.

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