JP2001081030A - Side-effect protective agent in immunosuppresant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a therapeutic agent that can draw out the immunosuppresant effect and the inhibitory effect of coronary artery sclerosis in maximum with the side-effect controlled by using a diterpenoid having the inhibitory action against production of platelet-derived growth factor(PDGF) and an immunosuppresant in combination. SOLUTION: A compound of formula I (X1-X3 are each OH or H) or a compound of formula II is given in combination with an immunosuppresant as a main drug in such a sufficient dose that can inhibit at least the onset of the arteriosclerosis that is brought about by the immunosuppresant. The compound of formula I or II may be extracted from naturally occurring substance, for example, a variety of Multiglycosidororum tripterygii or the synthesized product may be employed. The compound is, for example, triptolide, tripdiolide, triptonide or the like. As the irnmunosuppresant, are cited cyclosporin, tacrolimus and the like. The dose of the immunosuppresant is about 2/3-1/4 of the single dose and the daily dose of the compound of formula I and the formula II is generally about 10-100 μg/kg.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a means for preventing and treating side effects of an immunosuppressant.

[0002]

BACKGROUND OF THE INVENTION Coronary atherosclerosis (G) in grafts after heart transplantation
CA) has become a major clinical challenge in the long-term survival of transplant patients (TransplantProc. 1).
987, 19: 19-25) (Br. Heart J. et al.
1993, 69: 469-470). GCA is recognized to be involved in chronic rejection, but appears in the majority of heart transplant recipients within a few years after heart transplantation (T
ranplant Proc. 1990, 22:11
9-212) (Nature Med. 1997, 3:
1-4). The incidence rate of GCA is about 5 years after transplantation, 50%
(Br. Heart J. 1993, 69:46).
9-470). Pathological assays for such diseases are distinguished from age-related arteriosclerosis and include tubular and intensive myointimal prolifera.
), often a complete elastic layer
plastic lamina) and full length (ent
It is characterized by containing ile length blood vessels (Am. Heart J. 1995, 129: 791).
799) (Transplant Proc. 199).
3, 25: 2077-2079). In such patients, the infiltration of the cells of the proliferating lesions is composed of smooth muscle cells, macrophages, and T lymphocytes (Transplant Proc. 1993, 25:
2077-2079). Recently, accumulation of PDGF (platelet-derived growth factor) has been found in the intimal layer of lesions (Transplant Proc. 1997, 29:
1045-1046).

[0003] The inventor of the present invention has reported that GCA is
glycosidorrum tripterygi
i: may be abbreviated as MT hereinafter) (JP-A-11-92393). Koto Raiden is a rheumatoid art
hritis), systemic lupus erythematosus (system)
mic lupus erythematosus),
And it is a Chinese medicine that has been confirmed to be effective in treating other autoimmune diseases. There are diterpenoids triptolide, triprobediolide, tryptonide, alkanoid serrasinin, celabendin, serafrin, willfordein, willforin, willforgin, willforgin, willfortrin, willofin, and hippolid.
Celastrol, _dulcitol, C _{6 H 13~14} O
_6, it is known to contain glucose, tannins and the like.

[0004] Recently, significant expression of PDGF has been observed in heart transplant patients, and PDGF
Was highly estimated (Am. J. Cardio).
l. 1996, 77: 1210-1215). The present inventor has previously described that the diterpenoid compound in the lightning Koto component is PD
They discovered that they inhibited GF production, and found that they inhibited GCA, and filed a patent application (Japanese Patent Application No. 10-10602).
1). It is known that triptolide, a diterpenoid compound in the lightning Koto component, has an immunosuppressive effect (Int. J. Immunopharmac. 199).
2, 14: 963-969). However, in these inventions, the treatment method that inhibits GCA and maximizes the immunosuppressive effect was not known.

[0005]

The problem to be solved is to use an immunosuppressant in combination with a diterpenoid compound having a PDGF production inhibitory activity, and to maximize the effects of the immunosuppressive effect and the GCA inhibitory effect. Is provided.

[0006]

According to the present invention, a compound of the general formula I or II is used in combination with an immunosuppressive agent as a main agent to suppress the occurrence of arteriosclerosis caused by the immunosuppressant as a main agent. To a protective or therapeutic agent for side effects of an immunosuppressant, which comprises administering a compound of the general formula I or II to the compound of the general formula I or II. The present invention relates to an agent for preventing or treating side effects of an immunosuppressant, which is reduced by an immunosuppressive effect.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The general formula I or general formula II of the present invention
Examples of the compound are as follows.

Embedded image Wherein X ₁ is H or OH; X ₂ and X ₃ are each independently O
H or H. The compound of the general formula I or the general formula II can be subjected to a desired derivatization in consideration of solubilization, absorption, safety and stability. As the method, for example, as disclosed in US Pat. No. 5,663,335, it is also possible to mutate to a pharmaceutically acceptable salt or ester form. Further, the OH group may contain a usual protecting group. Similar effects can be expected for isomers, and solvates and hydrates are also available.

The compound of the general formula I or II may be an extract from a natural product or, of course, a synthetic product. As the extraction raw material, various types of lightning Koto can be suitably used, but of course, are not limited thereto. Examples of the compound name include triptolide, tripdiolide, tryptonide, tripchlorolide, triptonolide and the like.

The present invention relates to the use of the present invention in combination with an immunosuppressant,
It is effective. As the immunosuppressant, a widely known immunosuppressant can be used, and all the compounds confirmed to induce PGDF by administration of the immunosuppressant by confirming the action thereof can be applied to the present invention. As immunosuppressants, cyclosporine,
A general-purpose drug such as tacrolimus may be used, and an older type of azathioprine may be used.
prine), prednisolone (prednisolo)
Ne) may be used in combination. The dose of the immunosuppressant is about 2/3 to 1/4 of the effective dose of the drug alone, and simply about half the dose is sufficient. The most common usage is about 10 mg / kg / day, half of the normal usage of cyclosporin.

The dose of the compound of the general formula I or the general formula II is generally about 10 to 100 μg / kg per day, but depending on the desired condition, administration of about 10 to 100 times this amount is possible. It is. In addition, the compound of the general formula I or the general formula II did not show any particular side effect or death in an acute toxicity test using about 100 times the amount of these compounds in rats.

It is preferable that the administration be started before and / or immediately after transplantation, and that the administration be continued continuously. After the immunosuppressive effect enters a certain stable period, the compound of the general formula I or the general formula II may be used alone, but as long as another immunosuppressant is used, the compound of the general formula I Alternatively, it is preferred to continue to administer the compound of general formula II.

The dosage form of the compound of the general formula I or II of the present invention can be changed depending on the properties of the compound, but it can be prepared as an oral preparation or a parenteral preparation. In one embodiment, in the case of a succinate salt, the succinate salt can be used as an oral preparation such as an injectable injection preparation or a capsule which is soluble. Fat emulsion formulations are well-established as techniques for formulating intravenous administration of physiologically active substances, and can be suitably used as a means for formulating the compound of the general formula I or II of the present invention.

[0013]

Reference Example 1 Production Example 1500 After extracting 1500 tablets of Koto Raijin, it was extracted with acetone, and the obtained extract was concentrated under reduced pressure.
Distilled water was added for further concentration, and acetone was distilled off. The concentrated solution was sequentially extracted with hexane and ether, and the obtained extracts were concentrated and dried under reduced pressure, respectively.

(1) 4.91 g of the ether-extracted fraction was fractionated by silica gel column chromatography (hexane: ethyl acetate volume ratio = 1: 1). At TLC,
Kedde reagent (5% by weight of 3,5-dinitrobenzoic acid)
An ethanol solution and a 2N aqueous solution of sodium hydroxide were mixed at the time of use) to collect a fraction colored red to purple-red, and a reverse-phase preparative HPLC (ODS system, manufactured by Waters, solvent: 30% by weight) Acetonitrile / water mixed solvent, 5
17.7 mg of a single substance
I got This has an Rf value, ¹ H-NMR, LC / M
From the result of S analysis (m / z 360), it was identified as triptolide (C ₂₀ H ₂₄ O ₆ , molecular weight 360).

(2) 850 mg of the hexane-extracted fraction was fractionated by silica gel column chromatography (hexane: ethyl acetate volume ratio = 1: 1), and a Kedde reagent-positive fraction was recovered. HPLC (ODS
(Waters Co., Ltd., solvent: mixed solvent of 60% by weight of acetonitrile / water, 8.5 ml / min) to obtain 13.7 mg of a single substance. This is Rf
Value, ¹ H-NMR and LC / MS analysis (m / z 312), it was found that tryptophenolide (C ₂₀ H ₂₄ O ₃ ,
(Molecular weight 312) (J. Pharmaco
l. Exp. Ther. 1995, 272: 1305
１３1312) Reference Example 2 Preparation Example 1 Powder Powder 10 mg of triptolide and 100 g of crystalline cellulose were uniformly mixed and packaged in 1 g portions to prepare a powder. Reference Example 3 Formulation Example 2 Capsule Triptolide 10 mg Lactose 90 g Magnesium stearate 10 g was uniformly mixed, and the mixed powder was filled into hard gelatin capsules 200 mg each to prepare capsules. Reference Example 4 Formulation Example 3 Powder Powder 10 mg of tryptophenolide and 100 g of crystalline cellulose were uniformly mixed and packaged in 1 g portions to prepare a powder. Reference Example 5 Preparation Example 4 Capsule Tryptophenolide 10 mg Lactose 90 g Magnesium stearate 10 g was uniformly mixed, and the mixed powder was filled into hard gelatin capsules 200 mg each to prepare a capsule.

Experimental Example Effect of Triptolide on Coronary Artery Thickening and PDGF Increase in Transplanted Hearts (Experimental animals and treatment group) Adult male WKAH rats (RT-1 ^k ) and Lewis rats (RT-1 ^l ) weighing 200-240 g ) Were used as donor and recipient, respectively. Ono and Lin for intraperitoneal ectopic heart transplantation
dsey's method (J. Thorac. Cardiova).
sc. Surg. 1969, 57: 225-229). The function of the transplanted heart was evaluated daily by palpation. The transplanted rats were randomly divided into 4 groups (n = 7
/ Group), and cyclosporine was administered after transplantation. In the control group, only cyclosporine was administered and 5 mg
The group administered / kg / day was group A, and the group administered 10 mg / kg / day was group B. In addition, triptolide 35 μg /
kg / day administered to rats corresponding to group A
Those administered to rats corresponding to the group and group B were designated as group D. The pure triptolide was dissolved in a DMSO solution to a concentration of 3.54 μg / μl.
Stored at -20 ° C for no more than days. All recipient rats were given an immunosuppressant daily and 6 days after transplantation.
The animals were sacrificed on day 0.

(Isolation of triptolide from MT) Triptolide was isolated by the method described by Gu and Tao et al. (Int. J. Immuno. Pharmacol. 1).
995, 17: 351-356) (J. Pharmac.
ol. EXPERIMENTAL THERAPEUT
ics 1996, 272: 1305-1312). The aqueous extract of MT was concentrated under reduced pressure and HPLC (h
high performance liquid ch
(Romatography). Fractions showing immunosuppressive activity were mixed with mixed lymphocyte reaction (mixed ly
identified by Mphocytes reaction (MLR) and a nuclear magnetic resonance apparatus (nuclear magn).
etic resonance spectrosco
py) and a mass spectrometer
(Etry). Then, triptolide was isolated from the active fraction by ethyl acetate extraction according to the above Production Example.

(Sample processing and analysis) The transplanted heart was excised and then bisected along the central part of both ventricles and 10%
Fixed with formalin. Specimens were embedded in paraffin, hematoxylin-eosin staining was used to evaluate the degree of rejection, and Elastica Masson staining was used to evaluate intimal thickening of coronary arteries. The left and right coronary arteries were sectioned at 1 mm intervals and processed for light microscopic observation. The incidence of coronary artery disease and the degree of intimal hyperplasia were evaluated for at least 10 sections for each heart. In addition, a cross section of the heart just below the atrial valve was prepared as an example. Microscope (Olympus AX
80T) and a color image analyzer (SP500)
Using F), a double-blind study was performed on all slides separately by two observers. Vessel diameter 50μm
All of the above coronary artery sections were obtained from 206 blood vessels in the control group, 203 blood vessels in the C group, and 207 blood vessels in the D group.
The blood vessels of the book were observed. Oblique sections were excluded from the analysis. The degree of rejection is from 0 (none) to 3 (heavy),
Intimal thickening of the coronary arteries is graded from 0 (normal) to 4 (occlusion),
Lurie et al. (Transplantatio
n 1981, 31: 41-47):
Grade 0: Normal artery with perfect elastic layer, Grade 1: Less than 10% vascular lumen stenosis, Grade 2: 50% vascular stenosis
Below, grade 3; vascular lumen stenosis is 50% or more, grade 4; 1
00% vascular occlusion. For all vessels, the expression of coronary arteriosclerosis (GCA) in the grafts was determined by the method of Nagamine et al. (J. Heart Lung Transplant.
1994, 13: 895-898).
Furthermore, for myocardial tissue obtained from the heart graft, the expression of PDGF-A chain mRNA was determined by Northern blot analysis.
The technique described in Saiki, Oana et al. (Science
1988, 239: 487-491) (Eur. JC).
ell Biol. 1995, 61: 42-49).

(RNA Isolation) Transplanted hearts were excised, snap frozen in liquid nitrogen and stored at -80 ° C until RNA was isolated. RNA is RNAzol (Biotex Labo)
rats, Houston, TX) according to the attached protocol.

(PCR and cDNA probes) PDG
To detect FA mRNA, an 826 base cD
The NA fragment was converted from rat heart graft total RNA to rat PD.
Prepared using two oligonucleotide primers based on the sequence of GF-A. Primer 1 (forward primer sequence): 5'-G
ACCGCGGCCTCGCCTCCCTGCCGAG
CTTCC-3 'primer 2 (reverse primer sequence): 5'-GA
CCGCGGTCTCGAGGTGCTACAGACT
Using 10 μg of GC-3 ′ total RNA, double-stranded cDNA was synthesized using a cDNA synthesis kit from Amersham. PCR amplification of the cDNA is performed using a PCR reaction solution (10
mmol / L Tris · HCl, pH 8.3, 50m
mol / L KCl, 1.5 mmol / L MgC
l _2, 0.2mmol / L dNTP, was carried out in 2UTaq polymerase) in. The PCR reaction is DNA The
Using a rmalCycler, 45 ° C. at 94 ° C. (denaturation), 1 minute at 55 ° C. (annealing), 90 ° C. at 72 ° C.
35 cycles were performed with one second (extension) as one cycle.
The nucleotide sequence of the cDNA probe is Genetics Ge
ne Analysis System (Genetic
It was analyzed and confirmed using the software of s).

(Northern blot analysis) 10 μg of total RNA
Is denatured at 65 ° C. for 2 minutes and contains 1%
Fractionation was performed by electrophoresis in agarose gel, and a nylon membrane (Zeta-Probe Blotting M) was used.
transcribed by blotting on E. embranes, Bio-rad). Hybridization was performed at 65 ° C. with 0.5% SDS, 5 × Denhardts solution, 10% dextran sulfate, 5 × SSC and 50 μg.
/ ML salmon sperm DNA (Salmon spam D
NA), followed by repeated washings and finally at 65 ° C. in 0.1 × SSC / 0.1% SD
Washed with S. The probe was [A- ³² P] -d
Nick transl with CTP (Amersham)
ation labelingkit (Boehrin
ger Mannheim). The intensity of the hybridization signal was measured by fluorescence excited photochemically using an image analyzer (BAS 2000, Fuji).

(Statistical Analysis) Analysis of variance was performed by ANOVA using the Stat View ^™ SE program (Aba).
cus Concept). All p-values were two-sided and p <0.05 was set as significant. The results are expressed as mean ± SD.

(Results) The survival rates of heart grafts after transplantation are shown in Table 1.
As shown in the figure, it was about 60% in the cyclosporin 5 mg / kg / day administration group (Group A), but was almost 100% in the other groups. The reduction of heart graft survival with low doses of cyclosporine was improved by the administration of triptolide. No apparent toxicity was observed in the triptolide administration group. Serum creatinine, GOT and GTP were within normal limits at the time of sacrifice 60 days after transplantation. Histological examination showed that the kidneys, liver, and own hearts of the recipient rats were not damaged at all, either histologically or at the cellular level. That is,
Triptolide administration can reduce the dose of cyclosporine.

Table 1 shows the results of the morphological examination of the engrafted heart grafts. Rejections classified as grades 1 to 3 were seen in three groups. The grade of cardiac rejection is 1.2 for group A
± 0.3, 1.2 ± 0.2 in group B, 1.3 ± in group C
0.5 and 1.2 ± 0.4 for group D. There was no significant difference in the rejection of the engrafted grafts between the groups.

[Table 1]

The incidence of pathological blood vessels was 72% and 68% in the control groups A and B, respectively, but 12% and 10% in the triptolide-administered groups C and D, respectively. %, And the pathological vascular incidence was significantly suppressed by triptolide administration (p <
0.01). Furthermore, the grade of arterial intimal thickening in the tripride-administered group was significantly lower than that in the control group, and the significant difference between the cyclosporine-administered group and the triptolide-administered group was p <0.01, respectively. The maximum grade of intimal hyperplasia was 4 in the control group and 2 in the triptolide group.
Met. That is, in the tryptolite-administered group, intimal hyperplasia was significantly suppressed (p <0.01). FIG. 1 shows marked vascular lumen stenosis due to intimal and myointimal proliferation observed as typical histological changes in the cyclosporine administration group. In the triptolide-administered group, the marked increase in subendothelial proliferation was significantly improved (FIG. 2). That is, the degree of coronary artery lesions in the triptolide-administered group was significantly attenuated. When immunohistological staining was performed using a monoclonal antibody against PDGF, in the cyclosporin group, although a remarkably stained image was seen,
No recognizable staining image was observed in the triptolide-administered group.

[0026] PDGF-A mRNA was significantly positive in all heart grafts. As shown in FIG. 3, PDGF-A mRNA measured by Northern blot analysis
Was significantly lower in the triptolide-administered group than in the control group administered only cyclosporin.
In addition, triptolide was added at 350 μg / kg / day or 3
PDGF-A m when administered at 500 μg / kg / day
The RNA expression level was equivalent to the expression level when 35 μg / kg / day was administered (FIG. 3 (B)). That is, it was confirmed that triptolide exhibited a sufficient effect at a dose of 35 μg / kg / day. From the above results, PDGF-A
It was found that administration of triptolide also reduced the amount of cyclosporine in mRNA expression.

[0027]

The compound of the general formula I or II of the present invention is
An immunosuppressant, which is used in combination with an immunosuppressant as a main agent, and administers a compound of the general formula I or II in an amount sufficient to suppress the occurrence of arteriosclerosis caused by the immunosuppressant as the main agent. The side effects protection / therapeutic agent is
Significantly reduce the dose of conventional immunosuppressive drugs, and then expect a sufficient and sufficient immunosuppressive effect.As a result, significantly suppress the development of arteriosclerosis and achieve long-term normalization of transplanted organs Things.

[Brief description of the drawings]

FIG. 1 is a photograph showing remarkable vascular lumen stenosis due to proliferation of intimal and myointimal grafted coronary arteries in rats to which cyclosporine was administered for 60 days after heart transplantation.

FIG. 2 is a photograph showing improvement in stenosis of a graft coronary artery vascular lumen by administration of triptolide in a rat to which cyclosporine and triptolide were administered for 60 days after heart transplantation.

FIG. 3. PDGF-A measured by Northern blot analysis in rats administered cyclosporine alone or cyclosporine and triptolide for 60 days after heart transplantation
It is a figure which shows the expression level of mRNA.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mitsuhiro Hatta 2-9-17-1 Soshigaya, Setagaya-ku, Tokyo F-term (reference) 4C037 XA03 4C071 AA01 AA08 BB03 BB07 CC14 EE02 FF12 GG03 HH05 LL01 4C086 AA01 AA02 BA17 CA01 MA02 MA04 NA06 NA14 ZA45 ZB08 ZC75

Claims (2)

[Claims] 1. A compound of the general formula I or II which is used in combination with an immunosuppressive agent as a main agent, wherein the compound of the general formula I is sufficient to suppress at least the occurrence of atherosclerosis caused by the immunosuppressant agent as a main agent. Or an agent for preventing or treating side effects of an immunosuppressant, which comprises administering the compound of II. Embedded image In the formula, X ₁ , X ₂ and X ₃ are each independently OH or H. 2. The agent for preventing or treating side effects of an immunosuppressant according to claim 1, wherein the dose of the immunosuppressant as the main agent is reduced by the immunosuppressive effect of the compound of the general formula I or II.