CN1107507C

CN1107507C - Immunosuppressive medicine

Info

Publication number: CN1107507C
Application number: CN99106183A
Authority: CN
Inventors: 肖培根; 冯璞; 曹丽; 卞庆亚; 罗崇念
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 1999-05-10
Filing date: 1999-05-10
Publication date: 2003-05-07
Anticipated expiration: 2019-05-10
Also published as: CN1236617A

Abstract

The present invention relates to a new immunosuppressive medicine which is prepared from triptergium polyglycoside and white peony chief glycoside according to a certain proportion. The experiments in bodies and out of bodies prove that the immunosuppression effect of the present invention is obviously better than independent triptergium polyglycoside and independent white peony chief glycoside. Besides, the poisonous effect and the side effect are also obviously lessened. The present invention has obvious preventive and therapeutic action on autoimmune diseases and immunological rejection after organ transplantation.

Description

A kind of immunosuppressive drug

Technical field

The present invention relates to a kind of medicine that is mainly used in autoimmune disease and organ transplant rejection that is combined into by Tripterygium glycosides and white peony root's total glycoside.

Background technology

The immunosuppressant of at present clinical plant extract commonly used is Tripterygium glycosides (GTW), and the rejection of GTW after to autoimmune disease, anaphylactic disease and organ transplantation all has significant curative effect.But the major defect that GTW exists is that digestive system, genitourinary system, nervous system and medulla hematopoietic system are all had in various degree toxic and side effects, and lethal report is arranged.The toxic and side effects of GTW and therapeutic effect all strengthen with the increase of taking dose.And how to keep or strengthen the curative effect of Radix Tripterygii Wilfordii, and to reduce its using dosage and reduce toxic and side effects be a unsolved always difficult problem.The effect that white peony root's total glycoside (TGP) has the two-ways regulation immunologic function, when high dose, has immunosuppressive action, and when low dosage, mainly show as immunological enhancement, have antiinflammatory and immunoregulatory function clinically, heavy dose produces effect to rheumatoid arthritis, and safety and low toxicity, the general situation that can correct patient in the same fashion of improving disease symptoms.Thereby TGP all has very big-difference with GTW to the effect characteristics of immunne response and the situation of clinical practice, but exists the complementarity of effect.And the site of action of the two immunne response, effect link and character also respectively have characteristics.The compositions identical with the present invention had not yet to see report.

Summary of the invention

Main purpose of the present invention is, TGP and GTW are made up, and provides a kind of simple GTW therapeutical effect strong, and the little immunosuppressive drug of toxic and side effects.

When technical scheme of the present invention comprises the steps: in the body experiment, Glucosidorum Tripterygll Totorum ground be powder, be dissolved in 1% carboxymethyl cellulose and become suspension; White peony root's total glycoside (TGP) is extracted by the Chinese medicine Radix Paeoniae Alba, and wherein content of paeoniflorin is not less than 350 μ g/mg, and TGP directly prepares with normal saline; Merging group medicine before animal level medicine by 1: 5-10 fully mixes and gets final product.The administration volume is 0.2m/kg.Matched group gavages the normal saline of equivalent.During experiment in vitro, the preparation of tripterygium glycosides is with a little distilled water disintegrate tablet, add ethyl acetate extraction then three times, remove ethyl acetate, dilute with complete RPMI-1640 after adding a little Tween-80 hydrotropy during use,-20 ℃ of refrigerators are preserved behind the filtering with microporous membrane, dilute with the RPMI-1640 that contains 10%FCS during use.Directly with complete 1640 liquid preparation ,-20 ℃ of preservations are standby for TGP.

The specific embodiment:

Experimentation proves that the immunosuppressive action of Tripterygium glycosides and white peony root's total glycoside conjugate is obviously strong than the effect of its separate constituent, and promptly the two has the collaborative effect that suppresses immune response, and the synergism of the aspect that has no side effect.Main The pharmacological results comprises:

1, use in conjunction has the collaborative effect that suppresses whole immunne response in the GTW body of TGP and low dose, the effect when its action effect is used separately above them.Concrete experiment is:

1.1GTW the two share the inhibitory action to mice delayed hypersensitivity (DTH) with TGP

DTH is the tissue injury that is caused by the reaction of the lymphocyte of sensitization and antigen-specific, its reaction principle is that the delayed hypersensitivity cell combines with antigen, cause corresponding specific cell activation, discharge multiple lymphokine, causing the local organization monocyte infiltration is main inflammatory reaction.2,4-dinitrochlorobenzene (DNCB) is a kind of hapten, can be combined into complete antigen inducing specific cellullar immunologic response with the protein carrier of body, produces the DTH reaction of skin.Oral seven days of TGP and low dose of GTW all have certain inhibitory action to normal mouse DTH, but usefulness a little less than.And the effect that low dose of GTW and TGP share obviously strengthens, and surpasses the action intensity of heavy dose of GTW.The results are shown in Table 1.

Table 1GTW, TGP and the two are share the influence to BALB/c mouse DTH

Heavy (mg) suppression ratio (%) of group dosage (mg/kg) ear

Contrast-9.20 ± 1.33

GTW 10 7.87±1.65 ^* 14.5

GTW 30 7.38±1.07 ^* 19.7

TGP 50 7.92±1.19 ^*，a 13.9

GTW+TGP 10+50 6.32±0.83 ^* 31.2

The result is represented by means standard deviation, and compare with matched group sample number=10, ^*P＜0.05, ^*A:P＜0.05 is compared with GTW (10mg/kg) group in P＜0.01.

1.2 GTW and TGP and the two are share the influence to mouse spleen antibody forming cell (PFC)

The results are shown in Table 2, the GTW of doses and TGP produce cell to the mice specific antibody all inhibitory action, and low dose of GTW and TGP share then can obviously suppress PFC, consistent with the GTW effect of heavy dose.

Table 2GTW, TGP and share influence to spleen cell PFC quantity

Group dosage (mg/kg) PFC number/spleen suppression ratio (%)

Contrast 0 89410 ± 1540-

GTW 10 69121±1213 ^** 22.69

GTW 30 41263±1012 ^** 53.88

TGP 50 72129±1354 ^* 19.25

GTW+TGP 10+50 39746±921 ^** 55.52

The result is represented by means standard deviation, and sample number=10 are compared with matched group, ^*P＜0.05, ^*P＜0.01.

1.3GTW and TGP and the two are share the influence to the allogeneic skin graft rejection

BALB/c mouse is divided into 5 groups, and promptly matched group, GTW small dose group (10mg/kg), the heavy dose of group of GTW (30mg/kg), TGP organize (50mg/kg) and medicine Synergistic treatment group (GTW10mg/kg+TGP50mg/kg).In moving preceding 5 days of skin beginning oral administration, once a day, until the skin death of being ostracised.Carried out skin graft operation on the 6th day.The result shows that GTW30mg dosage group skin survival period prolongs 62.96%, and the skin survival period and the matched group of low dose of GTW group and TGP group do not have significant difference (P＞0.05).But can obviously suppress graft-rejection after the two combination, the skin survival period of Synergistic treatment group prolongs 56.79%, basic effect near heavy dose of GTW group, but all can not prevent the rejection of graft fully, and make its long-term surviving, the results are shown in Table 3.Experimental result shows: the effect of the inhibition graft-rejection of GTW and TGP is limited separately, by uniting different medicines such as using TGP, can improve the inhibitory action of GTW to transplant rejection.

Table 3 GTW and TGP and the two are share the mice allogeneic skin graft

The influence of C57BL/6 → BALB/c rejection

Group dosage (mg/kg) time-to-live (my god) the mean value variance (my god)

Contrast 07,7,8,8,8,8,8,9,9,9 8.1 ± 0.7

GTW 10 7，8，8，8，8，9，9，9，9，10 8.5±0.8

GTW 30 11，12，12，13，13，13，14，14，15，15， 13.2±1.24 ^**

TGP 50 7，7，8，8，8，8，8，9，9，10 8.36±0.98

GTW+TGP 10+50 11，11，12，12，13，13，13，13，14，15 12.71±1.18 ^**

Compare with matched group, ^*P＜0.01.

1.4 GTW and TGP and the two are share the influence to the mouse spleen lymphocyte breeder reaction

The BALB/c mouse oral administration, once a day, continuous 7 days.Got spleen prepared cell suspension and measures the breeder reaction that it stimulates mitogen ConA and LPS respectively in the 8th day.Found that GTW and TGP all have than the obvious suppression effect the lymphopoiesis of ConA and LPS stimulation, the work of GTW is more remarkable in order to the effect of heavy dose group, it is close with heavy dose of GTW that low dose of GTW (10mg/kg) and TGP share its inhibitory action of (50mg/kg) back, sees Table 4.

Table 4 GTW, TGP and combination thereof are in vivo to the influence of lymphproliferation response

Group dosage (mg/kg) optical density value

ConA LPS

Contrast-0.884 ± 0.152 0.563 ± 0.134

TGP 50 0.740±0.131 ^* 0.408±0.129 ^*

GTW 10 0.695±0.119 ^* 0.291±0.104 ^*

GTW 30 0.528±0.142 ^** 0.257±0.121 ^**

GTW+TGP 10+50 0.531±0.139a 0.201±0.069a

The result is represented by means standard deviation, and compare with the GTW group sample number=10, a:P＜0.05, ^*Compare with matched group, ^*P＜0.05, ^*P＜0.01.

1.5 GTW, TGP and the two combination thereof are to the effect of mixed lymphocyte culture reaction (MLR)

MLR is that the T lymphocyte reacts the antigenic specific immune response of the main tissue intersolubility (MHC) of homogeneous variant cell, is equivalent to the external model of graft-rejection.The present invention measures two-way and unidirectional MLR with the spleen cell of BALB/c (H-2d) mice and C57BL/6 (H-2b) mice.Find that GTW can obviously suppress MLR, its effect is dose dependent: low dose of TGP does not have obvious effect to MLR, but the TGP of 25-50 μ g/ml has certain inhibitory action to MLR.GTW and TGP (the 25 μ g/ml) inhibitory action to MLR when share significantly strengthens, and the results are shown in Table 5.Show that GTW and TGP share MLR is had coordinate repression.

Table 5 GTW, TGP and combination thereof are in vivo to the inhibitory action of mixed lymphocyte culture reaction

Group dosage (μ g/ml) optical density value (OD570nm)

Two-way unidirectional

Contrast-0.638 ± 0.029 0.437 ± 0.048

0.31 0.508±0.049 ^* 0.327±0.057 ^*

GTW 0.62 0.430±0.065 ^** 0.281±0.044 ^**

1.25 0.318±0.011 ^** 0.250±0.005 ^**

2.5 0.237±0.036 ^** 0.208±0.006 ^**

6.25 0.669±0.067 0.415±0.090

12.5 0.660±0.171 0.374±0.020

TGP

25 0.529±0.158 ^* 0.315±0.012 ^*

50 0.418±0.450 ^* 0.287±0.020 ^**

25+0.31 0.422±0.070 ^a 0.275±0.004 ^a

25+0.62 0.303±0.112 ^a 0.181±0.024 ^b

TGP+GTW

25+1.25 0.267±0.003 ^b 0.168±0.015 ^a

25+2.5 0.185±0.015 ^a 0.120±0.001 ^b

N=3, compared with the control, ^*P＜0.05, ^*P＜0.01; Compare a:P＜0.05, b:P＜0.01 with corresponding GTW dosage.

2.GTW TGP and the two are share the influence to hemopoietic function of bone marrow

GTW has certain toxic action to medulla hematopoietic system, thereby in research GTW and TGP immunity pharmacology, observed their effects, whether the toxic action of hemopoietic system has also been had corresponding enhancing trend collaborative when suppressing the immunne response function to understand them to hemopoietic system.

GTW, TGP and the two are combined in and use the effect that spleen colony CFU-S is formed in the body

Spleen colony CFU-S is the index that detects the myeloid stem cell number in the body, and CFU-S can further be divided into erythrocyte, granulocyte and megalokaryocyte.The LACA mice is used 850Rad's ⁶⁰The Co radiation gamma, within 4hr through the medullary cell 5 * 10 of tail venoclysis with the strain mice ⁴/ Mus, random packet administration, continuous 7 days.Experimental result shows: in the dosage range of this research, low dose of GTW and TGP and the two combination group there is no the CFU-S number is had a significant effect, but GTW has minimizing effect (P＜0.05) to CFU-S when heavy dose.TGP and low dose of and heavy dose of GTW result who share and the result that independent GTW organizes do not have significant change, and prompting TGP does not have obvious influence to the effect that suppresses the hemopoietic system myeloid stem cell in the GTW body.

Table 6 GTW and TGP and the two are share the influence to mouse spleen CFU-S

Group dosage (mg/kg) CFU-S/ spleen

Contrast-11.77 ± 1.80

GTW 10 11.50±1.71

GTW 30 8.33±1.10 ^*

TGP 50 11.98±3.02

GTW+TGP 10+50 11.67±2.28

GTW+TGP 30+50 8.49±1.51 ^*

The result is represented by means standard deviation, and sample number=5 are compared with matched group, ^*P＜0.05.

3.GTW and TGP share the preventive and therapeutic effect to experimental autoimmune encephalomyelitis

Experimental change autoimmunity encephalomyelitis (Experimental Autoimmune Encephalomyelitis, EAE) be a kind of a kind of autoimmune disease model that is brought out in laboratory animal with Fu Shi Freund's complete adjuvant subcutaneous injection by central nervous system's myelin autoantigen basic protein (MBP), the essence of its disease is by CD ₄ ⁺The delayed allergy that the autoreactivity lymphocyte produces central nervous system (CNS), owing to show in symptom, aspect such as pathological characters and pathogenesis and mankind itself's immune disease-multiple sclerosis (Multiple Sclerosis, MS) very similar, and being acknowledged as the highly desirable experimental animal model of MS, the present invention uses this model and has observed GTW and TGP and share prevention and therapeutical effect to EAE.Main result is as follows:

3.1GTW, TGP and share preventive effect to rat EAE morbidity

Induce EAE with CFA-GPSCH immunity Wistar rat, animal is divided into 5 groups at random, and 9 every group, began oral administration the same day in inducing, every day 1 time, continuous 7 days, regularly observe animal every day, the record result.Experimental result shows GTW15mg/kg, and 30mg/kg, TGP50mg/kg, GTW+TGP (15mg/kg+50mg/kg) all have the effect of prevention EAE morbidity, and the morbidity number of animals reduces, and disease time postpones, and the state of an illness alleviates.Wherein the effect of share with heavy dose of GTW group and low dose of GTW and TGP is best, and the action effect of the two is basic identical.The results are shown in Table 7.

Table 7GTW, TGP and share preventive effect to rat EAE

Group dosage (mg/kg) EAE sickness rate clinical score EAE time of occurrence (my god)

Contrast-88.9 (8/9) 3.45 ± 1.23 9.6 ± 1.8

GTW 15 66.7(6/9) 2.50±0.50 ^* 11.8±2.4

GTW 30 33.3(3/9) 2.01±0.42 ^** 13.3±2.2 ^**

TGP 50 55.6(5/9) 3.00±0.62 14.5±3.1 ^**

GTW+TGP 15+50 22.2(2/9) 2.25±0.35 ^** 14.7±2.6 ^**

Compare with matched group, ^*P＜0.05, ^*P＜0.01, sample number=9

3.2GTW, TGP and share therapeutical effect to rat EAE

After the rat of immune induction animal EAE began symptom to occur, with all animal random packet, gastric infusion was treated, continuous 7 days, and every day the observed and recorded animal state of an illness, until transference cure, the results are shown in Table 8.GTW and TGP can make the EAE symptom of rat obviously alleviate, and the course of disease also obviously shortens, and the effect of heavy dose of GTW is the most obvious, and TGP is basic identical with heavy dose of GTW with the effect that low dose of GTW share.

Table 8 GTW, TGP and share therapeutical effect to the Wistar rat

Group dosage (mg/kg) Mus count the clinical score EAE persistent period (my god)

Contrast 9 3.22 ± 0.92 8.22 ± 1.47

GTW 15 9 1.88±0.73 ^** 6.33±1.66 ^**

GTW 30 9 1.67±0.81 ^** 5.67±1.33 ^**

TGP 50 9 2.11±0.99 ^** 7.30±1.61 ^*

GTW+TGP 15+50 9 1.77±0.78 ^** 6.00±1.00 ^**

Compare with matched group, ^*P＜0.05, ^*P＜0.01.

3.3GTW, TGP and share effect to EAE rat specific immune response

Because EAE is by CD ₄ ⁺The organ specific autoimmune disease of a kind of CNS that Thl is cell-mediated, autoreactive T cell to CNS myelin basic protein (MBP) antigenic specificity plays an important role in the generation evolution of EAE disease, it is the main cytology basis that EAE takes place, thereby, the research medicine has important value to the lymphocytic effect of autoreactivity T to the mechanism of inquiring into the Drug therapy autoimmune disease.The present invention has studied GTW, and TGP and the two combination vivo medicine-feeding thereof are to the mass action of the EAE Rats Spleen lymphproliferation response of MBP stimulation.Animal grouping and administering mode are with 3.1.Behind immune induction EAE, put to death animal on the 20th day, get spleen lymphocyte and measure it, the results are shown in Table 9 the breeder reaction that MBP stimulates.

Table 9GTW, TGP and share the EAE rat that vivo medicine-feeding stimulates MBP

The influence of spleen lymphocyte breeder reaction

Group dosage (mg/kg) optical density value suppression ratio (%)

Normal control-0.197 ± 0.021-

EAE contrast-0.432 ± 0.033 ^*-

GTW 15 0.320±0.017 ^** 25.92

GTW 30 0.224±0.017 ^** 48.14

TGP 50 0.239±0.021 ^** 44.67

TGW+TGP 10+50 0.187±0.019 ^** 56.71

Compare with the normal control group, ^*P＜0.01 is compared with the EAE matched group, ^*P＜0.01, sample number=9.

Advantage of the present invention is that effect is affirmed, can obviously reduce the toxic and side effect of thunder godvine, but not reduce its curative effect, and part solves The difficult problem that in clinical application, mainly faces of thunder godvine, medicament sources is convenient, be easy to get, methods for the treatment of is easy, is in middle medical knowledge Opinion instructs lower, uses the modern medicine and pharmacology ways and means creatively to study a comparatively desirable immunodepressant that obtains.

Claims

1, a kind of immunosuppressive drug, it is characterized in that said immunosuppressive drug by Tripterygium glycosides GTW and white peony root's total glycoside TGP by weight 1: the suspension that is used as autoimmune disease and organ transplant rejection medicine that 5-10 forms.

2, the preparation method of immunosuppressive drug as claimed in claim 1, when it is characterized in that being used for treated in vitro, with GTW ethyl acetate extraction three times, remove ethyl acetate, add a small amount of Tween-80 dissolving, use the RPMI-1640 dilute filtration, TGP directly with the RPMI-1640 preparation, all preserves down at-20 ℃.

3, the preparation method of immunosuppressive drug as claimed in claim 1 when it is characterized in that being used for vivo medicine-feeding, is dissolved in GTW in 1% carboxymethyl cellulose and becomes suspension, and TGP directly prepares with normal saline.

4, the application of immunosuppressive drug as claimed in claim 1 in preparation autoimmune disease and organ transplant rejection therapeutic agent.