WO1998016219A1

WO1998016219A1 - The medicine containing triptolide for preventing and/or treating acute graft rejection

Info

Publication number: WO1998016219A1
Application number: PCT/CN1997/000100
Authority: WO
Inventors: Leishi Li
Original assignee: Nanjing General Hospital Of Nanjing Command Pla
Priority date: 1996-10-15
Filing date: 1997-10-10
Publication date: 1998-04-23
Also published as: CN1179306A

Abstract

The invention relates to the medicine containing triptolide for prevention and/or treatment of acute graft rejection. The medicine significantly prolongs the survival of the graft at the dose of 120-180 νg/kg/day. To obtain better effect, triptolide can be administered with cyclosporin A, or with cyclosporin A, azathioprine and corticoid(s).

Description

Medicine for preventing and treating acute rejection of transplantation by triptolide

The invention relates to a medicament for treating acute graft rejection of an epoxy diterpene hydrazone compound. BACKGROUND-Tripterygium wilfordii has been widely used in folk medicine for a long time, and has also been used to treat arthritis and skin diseases. A large number of clinical and experimental studies since the 1970s have shown that Tripterygium wilfordii has anti-inflammatory, immunosuppressive and anti-fertility pharmacological effects.

To prevent acute rejection of grafts, triple anti-rejection drugs based on cyclosporine A, azathioprine, and corticosteroids are currently used. Ming Ming's public

The object of the present invention is to use triptolide as a medicine for preventing and treating acute rejection of a graft. Triptolide is also known as triptolide. It is a colorless needle-like crystal with a melting point of 227 -228 ° C. It has the following structure:

According to the inventor's research, triptolide has a significant effect on prolonging the survival time of allografts, and can be used as a drug for preventing and treating acute rejection of grafts.

According to the research of the inventor, Balb / c (H-2b) mice were used as donors, C57BL / 6 (H-2d) mice were used as recipients, and allograft skin transplantation was performed under sterile conditions. Triptolide at 200 μm / kg / day has obvious anti-rejection effect, and the survival time of skin grafts is similar to that of cyclosporine A at 20 mg / kg / day (see Example 1).

Balb / c (H-2b) mice were used as donors, C57BL (H-2d) mice were used as recipients, and allogeneic mouse heterotopic heart transplantation was performed under aseptic conditions. The experiment proved that the dose was 200 m / kg / day Glaunolactone also has significant anti-rejection effects. (See Example 2).

Inbred Lou rats were used as donors, and inbred Wistar-Fister rats were used as recipients. Allogeneic rat heterotopic kidney transplantation was performed under aseptic conditions. The experiment proved that the dose was 200 im / kg / day Esterase also has a marked anti-rejection effect (see Example 3).

Triptolide can be treated synergistically with cyclosporine A. Small doses of triptolide plus J and cyclosporine A have significant therapeutic effects. The survival time of skin and plants has been more than doubled compared to the control group (see Embodiments 1, 3).

According to the inventor's clinical research, compared with the triple anti-rejection therapy currently used at home and abroad, mainly cyclosporine A, azathioprine and corticosteroids, the application of triptolide can significantly reduce allogeneic The incidence of acute cell rejection after cadaveric kidney transplantation, postponing the time of postoperative acute rejection, significantly improves the one-year survival rate after allogeneic kidney transplantation (see Example 4).

According to the inventor's research, the medication on the day of the operation had almost no preventive effect, and the survival time of the graft prolonged accordingly with the advance of the medication time. The preventive and preventive effect of the drug administration on the 14th day before the operation was significantly greater than that on the 3rd and 7th days before the operation.

Using triptolide-containing enzymes as a drug for acute rejection of grafts has obvious curative effects. In particular, the anti-rejection effect of triptolide is completely different from that of cyclosporine A, because when the two drug doses When there is no obvious anti-rejection effect, the combined use can produce significant effects through the mutual synergy between the two. Nie Jia way of implementing the present invention

Example 1

Experiments have verified that triptolide has the effect of prolonging the survival time of allografts. Animals: Balb / c (H-2b) mice were used as donors; C57BL / 6 (H-2d) mice were used as recipients. Male and female. Allograft skin transplantation under aseptic conditions

Experimental drug Source Concentration Diluent Triptolide A Institute of Dermatology, Chinese Academy of Medical Sciences 0. 03mg / ml 1% Tween 80

CsA Sandoz Ltd 3mg / ml 1% Tween 80 Control solution 1% Tween 80 Administration method: Oral administration 0.5ml / 10g / time

1.1 The dose-effect relationship of the effect of triptolide on acute rejection of allograft skin grafts:

Balb / c mice were divided into 3 groups, namely the experimental control group (n-8); the triptolide treatment group (n = 18) and the CsA treatment group (n = 17). Each treatment group was administered 14 days before skin transplantation and divided into three treatment groups according to different dosages: high, medium and low. The triptolide dosage was 200, 100, 50〃g / kg / day, CsA is 20, 10, 5 mg / kg / day. The experimental results are shown in Table 1. The average skin graft survival time of Balb / c mice in the control group was 9.8 ± 0.4. The mean survival time of the grafted skin grafts in the triptolide group was 10.6 ± 0.5, 13.8 ± 0.4, and 17.2 ± 0.4 days from low to high. The mean survival time for the CsA treatment group was 11.7 ± 0.53, 13.7 ± 0.5, and 20.5 ± 0.5 days.

Table 1. The dose-effect relationship of triptolide in the prevention and treatment of acute rejection of allogeneic skin grafts

Group Number of cases Survival time Average survival time

Tian Tian Control group 6 9, 10, 10, 10, 10, 10 9.8 ± 0.4

Triptolide

50 g / kg / day 5 10, 10, 11, 11, 11 10.6 ± 0.5

100 // g / kg / day 6 13, 14, 14, 14, 14, 14 13.8 ± 0.4

20 (^ g / kg / day 5 17, 17, 17, 17, 18 17.2 ± 0.4

CsA treatment group

5mg / kg / day 6 11, 11, 12, 12, 12, 12 11.7 ± 0.5

10mg / kg / day 6 13, 13, 14, 14, 14, 14 13.7 ± 0.5

20mg / kg / day 6 20, 20, 21, 21, 21, 21 20.5 ± 0.5

Therefore, triptolide has an anti-acute rejection effect on grafts. The dose of the drug is closely related. Triptolide at 200 μg / kg / day has obvious anti-rejection effect, and the survival time of skin grafts is similar to that of CsA at 20 mg / kg / day.

1.2 Time-effect relationship of the effect of triptolide on acute rejection of allogeneic skin grafts: The treatment group was administered on the 28th, 14th, 7th, 3rd days before the skin transplantation and the day of the surgery, the dose was 200 ig / kg / day. The experimental results are shown in Table 2. On the day of surgery, there was almost no prevention and treatment of the drug, and the effect of the transplantation time prolonged with the advancement of the medication time. The effect of treatment on the 14th day before the operation was significantly better than that of the animals on the 3rd and 7th days before the operation. Although administration of 28 days before surgery can further improve the survival time of the graft, there is no obvious advantage compared with 14 days before surgery. Table 2. Time-response effects of triptolide on acute rejection of allograft skin grafts

Group Number of days in advance of medication Number of cases Survival time Mean survival time

Day day day control group 6 9, 10, 10, 10, 10, 10 9. 8 ± 0.4

Triptolide treatment group

0 6 10, 10, 10, 11, 11, 11 10. 6 ± 0.5

-3 6 12, 12, 12, 12, 13 '13 12. 8 ± 0. 8

-7 6 13, 14, 14, 14, 14, 14, 14 13. 8 ± 0.4

-14 5 17, 17, 17, 17, 17, 18 17. 2 ± 0. 4

-28 6 17, 17, 18, 18, 19, 19 18. 0 ± 0.5

Synergistic effect of triptolide and CsA:

Balb / c mice were divided into experimental control group (n = 8); low-dose triptolide treatment group (n = 6); low-dose CsA treatment group (ii = 6) and drug synergy treatment group (n = 6) . All the treatment groups were given triptolide 50g / kg / day; or CsA5mg / kg / day; or triptolide 50xg / kg / day and CsA5mg / kg / day before skin transplantation. The experimental results are shown in Table 3. It can be seen from the table that the drug synergistic treatment group, that is, the low-dose triptolide and the small-dose CsA treatment group, the treatment group showed a significant therapeutic effect, and the survival time of the graft was more than doubled compared with the control group. Table 3. Synergistic comparison of triptolide and CsA

Day day day control group 6 9, 10, 10, 10, 10, 10 9. 8 soil 0.4 low-dose triptolide treatment group

50 g / kg / day -14 5 10, 10, 11, 11, 11 10. 6 soil 0.5 low-dose CsA treatment group

5mg / kg / day -14 6 11, 11, 12, 12, 12, 12 11. 7 ± 0.5

CsA + Triptolide -14 6 19, 21, 21, 25, 25, 26 22. 9 soil 2. 9

0 6 13, 13, 16, 16, 16, 17 15. 2 ± 1. 7

Therefore, this study suggests that the pathway of rejection between Tripterygium wilfordii and CsA is not exactly the same. Therefore, when two drugs with no obvious anti-rejection effect are used in combination, a significant effect can be produced through the mutual synergy between the two drugs.

Example 2. The effect of triptolide on the prevention and treatment of acute rejection of allogeneic heart transplantation. Animals: The donors were Balb / c (H-2b) mice and the recipients were C57BL / 6H-2d) mice. Allogeneic mouse heterotopic heart transplantation was performed under sterile conditions.

Experimental grouping: Animals were divided into triptolide treatment group, cyclomycin A treatment and experimental control. The triptolide treatment group received 50 g / kg / day (n-6) and 200 g / kg / day (n = 6); the cyclosporine A treatment group received 5 mg / kg / day ( n = 6) and 20 mg / kg / day (ιι = 6). The control group (n = 6) was given only the same amount of 1% Tween 80 solution. Table 4. Triptolide to prolong survival time of allograft heart. Group number of survival time mean survival time Day triptolide

200 / z g / kg / day 5 11, 11, 14, 14, 14 12. 8 ± 1. 6 cyclosporine A treatment group

20mg / kg / day 4 15, 17, 18, 19, 19 17. 6 ± 1. 7 Transplanted heart control group

6 5, 6, 7, 7, 8, 8 6. 8 ± 1.2

Example 3. The effect of triptolide on the prevention and treatment of acute rejection of allogeneic transplantation was experimentally determined. Animals: Inbred Lou rats were used as donors, and Wistar-Fister rats were used as recipients. Allogeneic rat heterotopic kidney transplantation was performed under aseptic conditions.

Experimental grouping: Animals were divided into triptolide treatment group, cyclosporine A treatment group, combined treatment group, experimental control group and sham operation group. Among them, triptolide treatment group gave triptolide 50 μ / kg / day (n = 12), 100〃g / kg / day (n = 10), and 200〃g / kg / day (n = 12). ); The cyclosporine A treatment group was given cyclosporine A 5 mg / kg / day (n = 10), 10 mg / kg / day (n = 11), and 20 mg / kg / day (n = 12). The combination treatment group received 5 mg / kg / day of cyclosporine A and 50 μg / kg / triptolide (n = 12). The control group (n = 12) and the sham operation group (n = 6) were given only the same amount of 1% Tween 80 solution. The experimental results are shown in Tables 5, 6, and 7. Table 5. Survival rates of transplanted kidneys in rats in the triptolide treatment group. Rats in the treatment group. Time (days) Pingyu Yujian (days).

Triptolide

100 μ g / kg / day 3 12, 13, 15 13. 3

Cyclosporine A treatment group

20mg / kg / day 3 24, 26, 27 25. 6 Table 6. Changes in the long and short diameters of the transplanted kidney at 7 and 14 days after the triptolide treatment group (cm)

7 days 14 days

Long diameter Short diameter Long diameter Short diameter Sham group 0.67 ± 0.23 0.45 ± 0.17 0.89 ± 0.34 0.54 ± 0.23 n = 3 η = = 3

Transplantation control group 9.03 ± 0.78 6.33 ± 0 · 54 12 · 67 ± 1.12 5.72 ± 1.01 n == 4 η == 3

Triptolide

50 g / kg / day 2.63 ± 0.62 2.05 ± 0.38 3.41 ± 0.29 3.11 ± 0.67 n == 4 η = 3

100 μ g / kg / day 2.83 ± 0 · 39 2.33 ± 0.57 4.00 ± 0.52 2.93 ± 0.47 η = = 4 η = = 5

200 μ g / kg / day 3.72 ± 0.61 3.01 ± 0.36 5.17 ± 0.85 3.49 ± 0 · 45 η = = 3 η = = 4

Cyclosporine A treatment group

5mg / kg / day 2.84 ± 0.57 2.11 ± 0.41 3.63 ± 0.54 2.96 ± 0.60 η = 4 η = 4

10mg / kg / day 3.17 ± 0.29 2.83 ± 0.34 5.28 ± 0.59 3.42 ± 0.35 η == 5 η = 3

20mg / kg / day 5.33 ± 0.74 3.87 ± 0.67 5.15 ± 0.45 4.65 ± 0.26 η == 4 η == 5

Pathological examination results: A large number of inflammatory cell infiltration, interstitial edema, and tubular inflammation in the control group can be seen on the 7th day after kidney transplantation. Pathological changes of acute cellular differentiation reactions such as interstitial edema and tubulitis; 7 and 14 days after surgery In the 200 ig / kg / day triptolide treatment group, the transplanted kidney tissue had only mild inflammatory cell infiltration and no tubulitis disease; in the 100 μg / kg / day triptolide treatment group, the transplanted kidney tissue Moderate inflammatory cell infiltration and tubulitis; 50 g / kg / day triptolide treatment Pathological changes in transplanted kidney tissue in the treatment group were similar to those in the control group. Only mild inflammatory cell infiltration was seen in the transplanted kidney tissue of the 20 mg / kg / day cyclosporine A treatment group, and moderate inflammatory cells were seen in the transplanted kidney tissue of the 10 mg / kg / day cyclosporine A treatment group. Pathological changes of acute cellular rejection such as infiltration, interstitial edema, and tubulitis; 5 mg / kg / day cyclosporine A and pathological changes of transplanted kidney tissue were similar to the control group. Table 7. Changes in long and short diameters (cm) of transplanted xylitol and cyclosporine A in 7 and 14 days after surgery

7 days 14 days

Long diameter Short diameter Long diameter Short diameter Sham group 0.67 ± 0.23 0.45 ± 0.17 0.89 ± 0.34 0.54 ± 0.23 n = 3 n = 3

Kidney transplantation control group 9.03 ± 0.78 6.33 ± 0.54 12.67 ± 1.12 5.72 ± 1.01 n = 4 n = 3

Combination therapy group:

Cyclosporine A 5mg / kg / day + triptolide 50 μg / kg / day

3.67 ± 0.46 2.83 ± 0.29 4.78 ± 0.41 3.13 ± 0.75 n = 5 n = 4

Cyclosporine A 5mg / kg / day + triptolide 100 μg / kg / day

2.67 ± 0.27 2.17 ± 0.46 3.48 ± 0.31 2.18 ± 0.29 n = 4 n = 3

Pathological examination results: Compared with the control group, although the rats in the 5 mg / kg / cyclosporine A + 50 μg / kg / triptolide combination treatment group had moderate inflammatory cell infiltration in the transplanted kidney tissue And a small amount of tubulitis lesions; 5mg / kg / cyclosporine A + 100Lig / kg / triptolide in rats in the combined treatment group, transplanted kidney tissue with mild inflammatory cell infiltration and tubulitis lesions; but The pathological changes in the transplanted tissues were much lighter than those in the control group. Example 4.

Eighty-three patients who underwent kidney transplantation were selected in our institute, including 71 males and 12 females. The patients were divided into two groups according to whether they received triptolide treatment. The first group received cyclosporine A and thiazole. Purine and corticosteroid-based triple anti-rejection therapy totaled 22; in the second group, 61 patients were treated with triptolide before surgery. The doses were: cyclosporine A 8 mg / kg / day, azathioprine 50 mg / kg / day, methylprednisolone 40 mg / kg / day, triptolide A 120 mg / kg / day. The comparison of the incidence of postoperative acute rejection in the two groups is shown in the following table: Group 1 Group 2

Cases Occurrence Cases Occurrence Rate%% Number% 30 days after transplantation:

Graft function is normal 13 59.1 56 91.8 Graft super rejection 1 4.6 0 0 Critical changes in the graft kidney 2 9.1 5 8.2 Acute graft rejection 6 27.3 0 0

Subtotal 22 61 120 days after kidney transplant:

Graft function is normal 15 75 37 91.2 Critical graft change 2 10 4 9.8 Acute renal transplant rejection 3 15 0 0

Subtotal 20 41 1 to ²⁰ days after kidney transplantation to 1 year after surgery:

Graft function is normal 14 70 26 92.9 Critical graft change 1 5 1 3.5 Acute renal transplant rejection 5 25 1 3.5

Subtotal 20 28 Industrial applicability

The use of this drug has a significant effect on prolonging the survival time of the graft. It can be used in conjunction with sporin A, or in combination with cyclosporine A, azathioprine, and corticosteroids for better efficacy.

Claims

Rights request

1. A drug for treating acute rejection of grafts,

It is characterized by containing triptolide.

2. A drug for preventing and treating acute rejection of a graft according to claim 1,

It is characterized in that the administered dose is 120-180 g / kg / day.

3. A drug for acute rejection of a chronic graft according to claim 1,

It is characterized in that triptolide is used in conjunction with cyclosporin A.

4. A drug for preventing and treating acute rejection of a graft according to claim 1,

It is characterized in that triptolide is used in combination with cyclosporine A, azathioprine and corticosteroids.

5. A drug for acute rejection of a chronic graft according to claim 1, 3 or 4,

It is characterized in that the medication is started before the operation, and the preferred time is 10-16 days before the operation.