JPH02157229A - Immunosuppression agent - Google Patents

Abstract

PURPOSE:To obtain immunosuppression agent having peptidergic protease- inhibiting activity with small side effect by containing peptide having specific amino acid sequence or salt of said peptide as active ingredient. CONSTITUTION:A peptide having amino acid sequence expressed by formula l corresponding to a part of amino acid sequence in coat protein (gp 120) of HIV or pharmacologically allowable salt of said peptide is contained in the aimed immunosuppression agent as active ingredient. A peptide related to coat protein of HIV such as a peptide having amino acid sequence expressed by formula II, adventitia protein of HIV or modified compound of said compound, etc., is shown as above-mentioned peptide. Said immunosuppression agent is used to suppression of rejection in transplantation of organ or bone marrow, or prevention and curing of autoimmunity disease such as articular rheumatism, myasthenia gravis, Crohn's disease, aplastic anemia or allergy of mammal.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an immunosuppressant used for suppressing rejection reactions in organ transplants such as kidney transplants.

[Prior Art/Problems to be Solved by the Invention] Conventionally, cyclophosphamide, azathioprine, etc. are known as immunosuppressants, but these immunosuppressants have side effects such as bone marrow suppression, hair loss, liver damage, Because it is associated with teratogenicity, etc.
There is a long-awaited need for an immunosuppressant that has fewer side effects and can be administered over a long period of time.

An object of the present invention is to provide a novel immunosuppressant having peptide protease inhibitory activity and fewer side effects.

[Means to solve the problem]

The present inventors have discovered that the pathogenic virus of acquired immunodeficiency syndrome (
As a result of various studies on HIV outer membrane protein-related peptides, such as peptides consisting of the amino acid sequence of coat protein ff (gp120) of HIV (hereinafter referred to as HIV), we have found that at least It has been found that a peptide having this sequence and its salt have excellent immunosuppressive effects.

That is, the amino acid sequence (Proc) is already known as an antigenic determinant in inducing neutralizing antibodies against HIV.

Natl, 8cad, Sci, LISA,
88. 3198-3202 (198B)
) contains the amino acid sequences -Arg-[1e- and -Lys5et-, which are the reaction sites of Dice's Tribun inhibitor and Pohrman-Birk inhibitor, which are widely known as proteinaceous trypsin inhibitors, as well as mammalian serum. It was discovered that the synthetic peptide containing this part has an amino acid sequence that is highly homologous to the amino acid sequence of the reaction site of inter-α-trypsin inhibitor, which is a type of protease inhibitor present in That's what I came to think. Therefore, we synthesized a new peptide containing this part and examined the pharmacological effects of HIVO outer membrane protein-related peptides, including the synthetic peptide, and found that at least peptides having the amino acid sequence shown in (1) below and salts thereof. was found to have excellent immunosuppressive effects.

The present invention has been completed based on such new findings, and includes at least a peptide having the amino acid sequence of formula (1) below [hereinafter, the peptide having the amino acid sequence represented by formula (1) is referred to as peptide (1). , peptide (1) and other peptides having the amino acid sequence of formula (1) are collectively referred to as peptide (1) derivatives] or their pharmacologically acceptable salts (hereinafter also simply referred to as salts). It is a protease inhibitor as a component.

Asn-^sn-Thr-Arg-Lys-3er-I
Ie-Arg-11e-GlnArg-Gly-Pro
-Gly-Arg-8Ia-Phe-Val-Thr4
1e-(I)Gly-Lys-11e-Gly- In this specification, when amino acids, peptides, protecting groups, etc. are indicated by abbreviations, they are IUPACIUB.
Comm1ssion on Biological
These are based on nomenclature abbreviations or common abbreviations in the field, examples of which are given below.

Cys Nijistin Thr: Threonine Ar
g: Arginine Pro Nibroline Asn:
Asparagine Lys: Lysine Ser: Serine
IIe: Isoleucine Gln: Glutamine
Gly nigericin Ala: Alanine P
he: phenylalanine Val: valine t
lis: histidine t-Boc: t-butyloxycarbonyl In the present specification, pharmacologically acceptable salts are not particularly limited as long as they have low toxicity, and include hydrochloride, acetate, phosphoric acid salt, etc. Salts, percussion salts such as citrate and succinate, and inorganic acid salts such as alkali metal salts (sodium salt, potassium salt, etc.) are mentioned.

In the present invention, the amino acid sequence represented by the formula (+) is HI
This corresponds to a part of the amino acid sequence in VO coat protein M (gp 120). However, the peptide (
+) The inducer is not particularly limited as long as it is a peptide having at least the amino acid sequence represented by the above formula (1),
Peptides with one or more amino acids additionally bonded to the amino acid sequence, peptides modified with polyethylene glycol or its derivatives (polyethylene glycol, monomethoxypolyethylene glycol, etc.) to reduce its antigenicity, etc. More specifically, the above peptide (I), a peptide having an amino acid sequence represented by the following formula (hereinafter referred to as peptide (II)), an outer membrane protein of H[V, and modified compounds thereof Examples include peptides related to the HIV envelope fluorophore.

The peptide (1) derivative in the present invention is produced by decomposition of gp120, a peptide synthesis method used in addition to conventional peptide chemistry, a modification method, and the like.

The peptide synthesis method may be either a solid phase method or a liquid phase method. The solid phase method is performed, for example, as follows. That is, first, a C-terminal amino acid with a protected amino group is bonded to an insoluble carrier via a carboxyl group. Any known insoluble carrier may be used. Next, after removing the amino protecting group, the amino acids with protected amine groups are sequentially peptide-bonded according to the amino acid sequence, reacted step by step to synthesize the entire amino acid sequence, and then the insoluble carrier is removed. At this time, it is preferable that the reactive functional group is protected by a protective group known in peptide synthesis.

The peptide thus produced can be purified to any degree of purity by purification methods commonly used in peptide chemistry, such as using ion exchange resins, partition chromatography, gel chromatography, reversed phase chromatography, and the like.

The peptide produced as described above can be converted into a salt by a method known per se, and a free peptide can be produced by hydrolyzing the salt by a method known per se.

[Action/Effect]

Peptide (1) derivatives including Peptide (1) and Peptide (II) of the present invention have excellent immunosuppressive activity and are effective against supplementary mammals such as humans, dogs, cows, horses, mice, and rats. For example, it can be used to suppress rejection reactions during organ or bone marrow transplantation, and as a prophylactic and therapeutic agent for autoimmune diseases such as rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, Crohn's disease, aplastic anemia, and allergies. Can be done.

Peptide (1) derivatives and salts thereof are formulated as ordinary freeze-dried products and administered orally or parenterally, and the dosage form varies depending on the route of administration and the like.

Preferred dosage forms include capsules, tablets, injections, etc., and such preparations can be prepared by mixing with carriers, excipients, patches, embossing agents, creams, ointments, etc. by methods known per se. be done.

For example, when the peptide (1) mfi form or its salt is used as a therapeutic agent for chronic rheumatoid arthritis, the dosage is, for example, in the case of an injection, 1 to 50 mg/day of the peptide <I> sg derivative or its salt. The body weight is generally measured and the dose is divided into one to several doses per day.

Experimental Example 1 lXl fractionated from saliva of healthy people by density gradient centrifugation
1 μg of 0' lymphocytes, PHA (phytohemagglutinin) and peptide (1), peptide (■), or a peptide consisting of 15 amino acid sequences different from peptide (1) (i.e., GlnGly-Pro-Lys-Gl).
u-Pro-Phe-^rg-Asp-Tyr-Val
-AspArg-Phe-Tyr, peptide 15) was placed in a flat-bottomed microplate and cultured for 72 hours at 37°C in a 5% carbon dioxide incubator. Add 'H-thymidine to 5 μCi. After the culture was completed, cells were collected using a cell harvester, and a stimulation index was calculated and evaluated using the following formula from the radioactivity taken into the cells using a liquid scintillation counter.

Stimulation index - (A-C) / (B-C) A: Radioactivity of cells added with PIIA B: Radioactivity of cells added to PH8 C: Radioactivity of background The inhibitory effect on bulb proliferation is shown in FIG.

As is clear from these results, it is clear that the peptide (1) derivative and its salt, which are the active ingredients of the present invention, suppress the proliferation of lymphocytes.

Experimental example 2 LD of peptide (1) and peptide (II).

When examined, all of them were 1 g or more.

〔Example〕

The present invention will be explained in more detail with reference to synthesis examples and formulation examples below, but these are not intended to limit the present invention in any way.

Note that all amino acids used in the following examples are L-units unless otherwise specified.

Synthesis example 1 Peptide (1) was synthesized.

Peptide synthesis was performed by a solid-phase method using Peptide Synthesizer-43OA manufactured by Applied Biosystems. That is, 0.5 mmol of Shi-Boc-L-glycine-bonded phenylacetamidomethyl resin (styrene-
1% divinylhensen copolymer), and added t-Bo to the N-terminus.
2 mmol of t-Boc amino acid protected with C, N,N
- Condensation was carried out by the symmetrical amino acid anhydride method using 1 mmol N,N-dicyclohexylcarbodiimide in fluoromethylformamide, followed by the sequential extension method in which the peptide bonds were extended one by one. However, the condensation of Asn and GinArg was carried out twice by an active ester method using 1 mmol of hydroxybenzotriazole in N,N-dimethylformamide.

The side chain protecting group of t-Boc amino acid is 2.
4.6-) Limethylbenzenesulfonyl group, Nα-butoxycarbonyl-Nε-2-chlorobenzyloxycarbonyl group was used for Lys, and benzyl group was used for Ser and Thr, respectively.

The standard synthesis program for peptides is 60% deprotection.
trifluoroacetic acid/dichloromethane for 1 min or 15 min, 3 x 1 min with dichloromethane as a wash, 2 x 1 min with 10% N, N-diisopropylethylamine as a neutralization, and 1 x 1 min with N, N-dimethylformamide as a wash. Symmetrical amino acid anhydride method using NN-dicyclohexycarbodiimide in N,N-dimethylformamide as condensation for 6 times for 20 min, N,N as washing.
- twice for 1 minute with dimethylformamide and four times with dichloromethane. In these standard synthesis programs, the reaction temperature, reaction time, etc. for each amino acid are controlled in detail by a microcomputer.

The peptide resin obtained by the automatic peptide synthesizer is
The support resin and the protecting groups on the amino acid side chains were cleaved with trifluoromethanesulfonic acid.

That is, 1 g of synthetic peptide resin and 1 g of thioanisole
ml of ethanedithiol 50 IP was stirred at room temperature for 10 minutes, and while cooling the flask with ice water, 10 ml of trifluoroacetic acid was added. Add and stir for 10 minutes.

Next, 1d of trifluoromethanesulfonic acid is added, and the flask is removed from the ice water and stirred at room temperature for 30 minutes. Add cold diethyl ether to the flask until no more peptide precipitates appear, stir for 1 minute, filter the crystals with a Teflon filter, further dissolve this in a small amount of trifluoroacetic acid, transfer to cold diethyl ether, and pass through the Teflon filter again. Deli! This was dissolved in 2N acetic acid to obtain a crudely purified peptide.

The crudely purified peptide was purified using a reversed-phase column (diameter 2 cm, length 25
High performance liquid chromatography (column:
Purification was performed using YMCR-005-5). Elution was performed using 0.1% trifluoroacetic acid as the mobile phase and a linear concentration gradient of 90% acetonitrile from 5% to 60% (flow rate 5 affi/ll1 in). The chromatogram is as shown in FIG. 2 (detected by ultraviolet absorption at 220 nm).

One nanomole of the purified peptide was analyzed using a gas phase sequencer. As a result, the amino acid residues detected in analysis cycles 1 to 24 were, in order, 8sn, Asn,
Thr+ Arg+ Lys, Ser,
Ile, Arg+ l1eGin, Arg,
GIy+ Pro+ GIy+ Arg+^Ia+ P
He, Val.

Thr, lie, Gly, Lys, lle,
It was confirmed that the target peptide (1) was obtained.

The yield of peptide after purification was 85 μ.

Synthesis example 2 Peptide (U) was synthesized.

Peptide synthesis was performed in the same manner as in Synthesis Example 1. In addition, as protecting groups for the t,-Boc amino acid side chain, a dinitrophenyl group was used for His, and a 4-methoxyhensyl group was used for Cys.

Since the synthesized peptide resin contains His and Met, the following pretreatment was performed before the cleavage with trifluoromethanesulfonic acid as in Synthesis Example 1. 24 mK of N,N-dimethylformamide and 1 m of thiophenol were added to the peptide resin, and after stirring at room temperature for 1 hour, suction filtration was performed using a Teflon filter. Washed three times with dichloromethane and dried. Furthermore, while maintaining this resin at O'C, it was reacted for 3 hours in m-cresol 1 ml, dimethyl sulfide 3 d, trifluoroacetic acid 5 ml, and difluoromethanesulfonic acid ld, and washed with cold diethyl ether on a Teflon filter. After drying, it was cut in the same manner as in the synthesis example.

It was purified by high performance liquid chromatography in the same manner as in Synthesis Example 1. The chromatogram is shown in Figure 3 (22
(detected by ultraviolet absorption at 0 nm).

Results of analysis using the same method as Synthesis Example 1 Analysis cycle 1~
The amino acid residues detected in 36 are CysThr,
Arg, Pro, ^sn, Asn, A
sn, Thr, 8rg, LysSer,
rle, Arg, Ile, Gin,
Arg, Gly, Pro, Gly.

Arg, Ala, Phe, Val, Thr,
Ile, Gly, Lys, Ile.

Gly, Asn, Met, Arg, Gin,
It was confirmed that the target peptide (■) was obtained.

Formulation Example 1 After the peptide (1) was purified, it was injected into ampoules, freeze-dried and sealed to obtain an injection preparation of 100 μ/ampule.

Formulation Example 2 Macrogol ointment (8th edition Japanese Pharmacopoeia) (mixture of 50g of Macrogol 4000 and 50g of Macrogol 40050) was mixed with 1g of lyophilized peptide (I) and kneaded thoroughly to obtain a 1% ointment preparation. .

Formulation Example 3 In Formulation Example 1, peptide (1) was replaced with peptide (
II) to obtain an injectable preparation.

Formulation Example 4 In Formulation Example 2, peptide (1) was replaced with peptide (
II) to obtain an ointment.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the proliferation response of lymphocytes based on antigen stimulation by the immunosuppressant of the present invention. FIG. 2 is a high performance liquid chromatography chart of peptide (n). FIG. 3 is a high performance liquid chromatography chart of peptide (1). Figure 2 Figure 3

Claims (1)

[Scope of Claims] An immunosuppressant containing as an active ingredient a peptide having at least the following amino acid sequence or a pharmacologically acceptable salt thereof. [There is a gene sequence]