JPH08157364A - Immunorejection suppressing agent - Google Patents

Abstract

PURPOSE: To obtain an immunorejection suppressing agent capable of suppressing immunorejection after organ transplantation, etc., and having little side effect by compounding amrinone as an active component. CONSTITUTION: This immunorejection suppressing agent contains amlinon, which is called by a chemical name of 5-amino-(3,4'-bipyridine)-6(1H)-one and has cardiotonic effect, as an active component. The agent is formulated into an oral preparation, an injection, etc., by a conventional method. In the case of oral administration, this is administered at a daily dose of 1-20mg/kg in one or several divided doses. In the case of parenteral administration, preferably the agent is administered at 0.5-10mg/kg/day by intravenous injection and at 0.05-33mg/kg/hr by intravenous drip infusion. Especially, in the case of acute cardiac insufficiency, it is administered at a single dose of 1mg/kg by intravenous injection and at 0.3-0.9mg/kg/hr by intravenous drip infusion. After the transplantation of heart, kidney, bone marrow, liver, pancreas, etc., immunorejection is suppressed by administering the amurinone. This is especially effective after heart transplantation. This immunorejection suppressing agent has a completely different structure from those of agents such as ciclosporin and corticosteroid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immune rejection inhibitor for suppressing immune rejection after organ transplantation and the like.

[0002]

BACKGROUND OF THE INVENTION Amrinone is a compound having cardiotonic action, and its chemical name is 5-amino- (3,4'-bipyridin) -6 (1H) -one. The cardiotonic action of amrinone is described, for example, in Japanese Examined Patent Publication No. 60-32630. A therapeutic agent for acute heart failure containing amrinone as an active ingredient has already been manufactured and sold. Examples of the therapeutic agent for acute heart failure include "Kartonic", a registered trademark of Yamanouchi Pharmaceutical Co., Ltd. It is considered that the mechanism of the cardiac action of amrinone is related to the accumulation of intracellular cAMP, which is an intracellular signal transmitter. More specifically, amrinone is proposed to exhibit cardiotonic action by suppressing type III phosphodiesterase and accumulating intracellular cAMP. Here, phosphodiesterase (hereinafter referred to as PDE) is an intracellular adenosine cyclic 3 ', 5'-monophosphate (cAMP).
And is classified into four different types I to IV.

Further, amrinone suppresses the production of tumor necrosis factor in endotoxin shock, which suggests that it has an action of suppressing inflammation (Circulatory Shock 36, 200-207 (199).
2)). Furthermore, it has been reported that amrinone may be related to thrombin-induced platelet-derived growth factor (PDGF) -like protein (Shaddy RE, Paisley JE, Hansen.
JC, Diatric Research 30, 4, 351-354 (1991)).

[0004]

However, it has not been known so far that amrinone suppresses immune rejection after transplantation. In addition, since the mechanism of action of suppressing immune rejection is generally considered to be different from the mechanism of action such as cardiotonic action and inflammation suppressing action, it could not be predicted that amrinone suppresses immune rejection.

[0005] In a normal transplantation, some mismatch of histocompatibility antigens is unavoidable, and suppression of immune rejection is essential for successful transplantation. Known immunorejection suppressants include cyclosporine and corticosteroid drugs. Cyclosporine suppresses the immune response by inhibiting the activation of T cells, and corticosteroid drugs reduce the number of T cells and inhibit the nucleic acid metabolism of lymphocytes to suppress their function. , Suppresses the migration and metabolism of macrophages and suppresses the immune response. However, since these drugs have side effects such as nephrotoxicity and hypertension, it has been desired to develop an immunorejection suppressant having fewer side effects.

[0006]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide an immune rejection inhibitor which has a completely different structure from drugs such as cyclosporin and corticosteroid drugs. That is, according to the present invention, there is provided an immune rejection inhibitor after transplantation, which comprises amrinone as an active ingredient. In addition, this transplant is preferably an organ transplant, more preferably a heart transplant.

[0007]

[Function] Transplant includes organ transplant and skin transplant.
This is because immune rejection generally occurs during these transplants. However, corneal transplantation is not included. Because there are no blood vessels in the cornea, it is difficult to induce immune rejection. The transplant is preferably an organ transplant, and the organ transplant includes a heart transplant, a kidney transplant, a bone marrow transplant, a liver transplant, a pancreatic transplant and the like. Of the organ transplants, heart transplant is more preferable.

The immunorejection inhibitor is applied orally or parenterally. When applied orally, divided into 1 to several times, 1.0 to 20.0 mg / kg / day, preferably 2.0 to 5.0 mg / kg / day
Is administered. When applied parenterally, it is preferably administered intravenously. For intravenous injection, divide 0.5 into 1 to several times
Administer ~ 10.0mg / kg / day. In the case of intravenous infusion, 0.05 to 3.0 mg / kg / hr should be administered. Or single IV 0.5
~ 10.0 mg / kg, followed by intravenous drip infusion of 0.05 to 3.0 mg / kg / hr, preferably single intravenous infusion of 1.0 to 1.5 mg / kg followed by drip infusion of 0.3 to 1.2 mg / kg / You may go hr. For reference, the administration method and dose of amrinone currently clinically used in acute heart failure are shown as 1.
0 mg / kg IV and 0.3-0.9 (preferably 0.6) mg / kg / hr
Intravenous drip infusion.

The above-mentioned preparation is easily manufactured by a conventional method.
For example, in the case of oral administration, any of tablets, pills, capsules, granules, powders, solutions and syrups may be used. The carrier and excipient used in the preparation may be any one ordinarily used in oral preparations, and may be solid or liquid. Examples thereof include lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cocoa butter, ethylene glycol and the like, and other commonly used ones. Further, a moisturizing agent, a suspending agent, a sweetening agent, a flavoring agent, a flavoring agent, a preservative and the like may be contained. In the case of an injection, either an injection in ampule or a lyophilized preparation may be used, and a carrier and an excipient used in the preparation may be any as long as they are usually used for an injection. For example, propylene glycol, polyethylene glycol, olive oil, ethyl oleate, and other commonly used substances can be used. It may also contain moisturizers, suspending agents, preservatives and the like.

Examples of preferable injection formulations are shown below. Amlinone as lactate, 50mg (as free base)
And 2.5 mg of sodium pyrosulfite and 315 mg of D-sorbitol were prepared in 10 ml of water for injection to prepare an ampoule injection. The pH was appropriately adjusted to 3.5 to 4.5 with lactic acid or sodium hydroxide.

[0011]

INDUSTRIAL APPLICABILITY The immunorejection suppressant of the present invention can suppress the immune rejection reaction that occurs after transplantation and has few side effects.

[0012]

EXAMPLES The present invention will be described in detail below with reference to examples of heart transplantation using mice. However, the present invention is not limited to the following examples. [Animal] In the following examples, DBA2 mice (H-2
d), C57BL / 6 mice (H-2b) and C3H /
He mice (H-2k) were used, and male and 7-9 week old mice were selected as these mice.

[Transplantation] C57BL / 6 mice (H-2b) using DBA2 mice (H-2d) as heart donors
Was used as a heart recipient. Heterotopic heart transplant is C
The method of Orry et al. was improved. Ischemic time is usually 4
After 5 to 60 minutes, the survival rate was about 80%. Cases dropped out within 72 hours due to problems in experimental procedures were excluded from the experiment. Survival of the transplanted heart was confirmed by electrocardiography by palpation of the posterior region every day. The date of refusal was the day of suspension of mind. Amrinone was dissolved in 0.5N lactic acid and then diluted with distilled water to a final concentration of 5 mg / l. The drug is 10 mg / kg once a day
Alternatively, 40 mg / kg was orally administered. Dosing began on the day of heart transplantation and continued for 60 days while the graft was beating.

[Histopathological Examination] Five days after the transplantation, the mice were killed. Grafts were cut laterally to maximize the perimeter of the ventricles and fixed with 10% formalin.
The transplanted tissue was embedded in paraffin and then stained with hematoxyline and eosin. The histopathological observation was performed by scoring by an observer who did not know the background data. Cellular wetting, necrosis and myocardial inflammation were scored on a scale of 0 (none), 1 (minor), 2 (minor), 3 (normal), 4 (severe).

[Measurement of cytotoxic T lymphocyte (CTL) activity] Culture and measurement of cytotoxic T lymphocytes were carried out according to the methods described in the literature (Kruisbeek AM Isolati).
on and Fractionation of Mononuclear Cell Populatio
ns. In: Coligan JE, Druisbeek AM, Margulies D.
H., Shevachi EM Strober W. (eds) Current Protoco
ls in Immunology. New York: Green Publishing Asso
ciates and Wiley-Interscience; 1991: 3.1. 1-5 and
Wunderlich J., Shearer G., Induction and Measure
ment of Cytotoxic T Lymphocyte Activity. In Coliga
n JE, Druisbeek AM, Margulies DH, Shevachi
EM Strober W. (eds) Current Protocols in Immunolo
gy. New York: Green Publishing Associates and Wile
y-Interscience; 1991: 3.11. 1-7). The spleen of the mouse was removed and a single cell suspension was prepared. Red blood cells are A
CK lysis buffer (0.15M NH ₄ Cl, 1.0
mM KHCO ₃ , 0.1 mM Na ₂ EDTA; pH =
7.4), after dissolving in Lympholyte-M high specific gravity solution, baking soda and centrifugation (25 ° C., 20 minutes, 1
500 gf) was removed from the lymphocyte fraction. Cytotoxic cells were 7 × 10 ⁶ C57BL / 6 splenocytes (recipient system) treated with mitomycin C (25 μg / ml, Sigma, St. Louis, MO, USA) and 5 × 10 ⁶ DBA2-stimulated cells ( Donor system)
It was induced by culturing for 5 days. The cytotoxic activity is ^51A- labeled DBA2-targeted splenocytes (donor) or C3H / He-targeted cells (third party group) in which cytotoxic cells are labeled.
In addition, it was evaluated by measuring the cell solubility. In addition, both of these cells were previously subjected to lipopolysaccharide (lip
Opolysaccharide) had been stimulated for 2 days. The results when added to DBA2 target splenocytes (donor) are shown in FIG. 4 (A), while the results when added to C3H / He target cells (third party group) are shown in FIG. 4 (B). . Target cells (1.7 × 10 ⁴ ) and C57BL / 6 so that the ratio of target cells (T) to C57BL / 6 effector cells (E) are 100: 1, 50: 1, 25: 1.
Effector cells were cultured in triplicate in round bottom 96-well tissue culture plates at 200 μl / well. 5
After culturing in humid air containing% CO ₂ for 4 hours at 37 ° C., 100 μl was sampled from each well and the radioactivity was measured by an automatic gamma scintillation counter. The spontaneously released radioactivity was 30% or less of the total uptake. The specific cytotoxic activity was calculated as follows.

[0016]

Here, cpm is a count per minute measured by an automatic gamma scintillation counter. The maximum cpm was 0.1 for target cells labeled with ⁵¹ Cr.
Obtained by treatment with% Triton. In the in vitro experiment, amrinone was dissolved in 0.5N lactic acid solution and diluted with distilled water to prepare a 0.025N lactic acid solution having a final concentration of 1 mg / ml. The drug was added to the co-culture at a concentration of 5 μg / ml. The solvent was similarly added to the control. For comparison, non-sensitized cells that did not induce cytotoxicity were used as controls.

[Measurement of Cytokine] Mixed culture was carried out as described above with a ratio of reacting cells: stimulating cells of 7: 5. The total lymphocyte count was adjusted to 1 × 10 ⁷ / well. The mouse IL-2 and IFN-γ concentrations in the culture supernatant were measured with an ELISA kit (Endogen, Boston, Massachusetts, USA). The detection sensitivity of the kit is 3 pg / ml or less for IL-2,
For IFN-γ, it was 15 pg / ml or less. Supernatants were collected 12 and 24 hours after culture.

The experimental results will be described below. [Effect of amrinone on graft survival] Fig. 1 shows the survival rate of the control group and the graft treated with amrinone. Untreated C5
7BL / 6 mouse recipients (n = 6) averaged 10.
He rejected the transplantation of DBA2 in 5 days. Amrinone 10
At mg / kg / day and 40 mg / kg / day, the engraftment period was extended to 12 and 22 days, respectively.
When the dose of amrinone is 40 mg / kg / day,
The extension of engraftment period was significant (p <0.01). On the other hand, when the dose of amrinone was 10 mg / kg / day, the engraftment period was slightly extended, but it was not significant.

[Histological Examination] The histological examination is C57B.
It was performed on the transplanted DBA heart 5 days after the transplantation to L / 6. Grafts treated with amrinone 40 mg / kg / day were compared to the control group. A photograph observed with an optical microscope is shown in FIG. Both FIG. 2A and FIG. 2B are magnified 100 times. FIG. 2 (a) is a photograph of a transplanted heart not treated with amrinone. Extensive infiltration of mononuclear cells is observed. On the other hand, FIG. 2 (b) is a photograph of a transplanted heart not treated with amrinone. Comparing with Fig. 2 (a), the inflammation of the amrinone-treated graft was clearly mild. FIG. 3 shows the histological results of the grafts engrafted for 5 days. The degree of cell infiltration was significantly (p <0.05) milder in the amrinone-treated group than in the untreated group. Therefore, it can be seen that amrinone suppressed the inflammatory reaction, which is a type of immune rejection reaction. The bar indicates the mean ± standard deviation.

[Action of amrinone on in vitro CTL activity] In order to clarify the action mechanism of suppressing graft rejection, the action of amrinone on CTL was examined. D of C57BL / 6 splenocytes (recipient)
A specific lytic effect on BA2 splenocytes (donor) was measured, and the result is shown in FIG. 4 (A) as a specific cytotoxic activity. On the other hand, the C3H / He splenocytes (third party) of C57BL / 6 splenocytes (recipient) were similarly measured, and the results are shown in FIG. 4 (B). In FIG. 4 (A), as compared with non-sensitized C57BL / 6 cells, control effector cells treated with the drug, that is, cells corresponding to the so-called control treated with the solvent instead of amrinone, were treated with M for 5 days.
LC showed increased lytic activity against DBA2 target cells.
On the other hand, the CTL activity of the effector cells treated with 5 μg / ml of amrinone was E: T ratio 25: 1, 50: 1, 1
It was significantly decreased at 00: 1. However, 2.5
The treatment with μg / ml did not show the inhibitory effect on the lytic activity.

Next, in FIG. 4 (B), C57BL / 6 effector cells were used for the third cell line (C
When 3H / He) was used as a target cell, it showed almost no cytolytic activity. This suggests that the MLC-induced cytolytic activity is specific to DBA2. That is, the cytotoxic activity (CTL activity) specific to donor cells was significantly reduced by the administration of 5 μg / ml of amrinone. This suggests that amrinone can suppress the production of cytotoxic T lymphocytes in vivo as well. The therapeutic concentration of amrinone in human plasma is considered to be 0.6-6.4 μg / ml.
In FIG. 4, ★ indicates that p <0.05 is significant, and ★★ indicates that p <0.01 is significant.

[Effect of amrinone on the production of interleukin (IL) -2 and interferon (IFN) -γ] I in the supernatant of different one-way reactive MLC
The concentrations of L-2 and IFN-γ were measured 12 hours and 24 hours after culturing. Amrinone was added to the culture supernatant at a concentration of 5 μg / ml. As shown in FIGS. 5A and 5B, the levels of IL-2 and IFN-γ were significantly suppressed by amrinone after 12 hours and 24 hours.

That is, administration of 5 μg / ml of amrinone significantly suppressed the concentrations of IL-2 and IFN-γ produced in the immune response. From this, it was revealed that amrinone suppressed the immune rejection reaction. Furthermore, the IFN-γ level of the amrinone-treated group was below the detection limit of the kit. From this, it was revealed that the suppressive effect of amrinone was more remarkable for IFN-γ than for IL-2. In FIG. 5, * indicates that p <0.05 is significant, and ★★ indicates that p <0.01 is significant.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of amrinone on graft survival (%).

FIG. 2 is a photograph of the morphology of the organism, the heart cross-section of a mouse that has undergone heart transplantation. (A) When untreated with amrinone.
(B) When treated with amrinone.

FIG. 3 is a graph showing a histological score of heart transplantation.

FIG. 4 is a graph showing the specific cytotoxic activity of amrinone.

FIG. 5 is a graph showing the suppression of interleukin-2 and interferon-γ production by amrinone.

Claims (3)

[Claims] 1. An agent for suppressing immune rejection after transplantation, which comprises amrinone as an active ingredient. 2. The immune rejection inhibitor according to claim 1, wherein the transplant is an organ transplant. 3. The immune rejection inhibitor according to claim 2, wherein the organ transplant is heart transplant.