JPH03170498A - Peptide and drug having interleukin-1 like activity and containing the peptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a peptide and a drug containing this peptide that has interleukin-1 (hereinafter referred to as IL-1)-like activity. [Prior Art] IL-1 is a glycoprotein with a molecular weight of 14,000 to 18,000 that is produced by monocytes, macrophages, B cells, vascular endothelial cells, etc. by stimulating these cells. This IL-1 is present in a very small amount of less than 10''lOmol, and is contained in concanavalin A (hereinafter referred to as .Con A).
Proliferative effect on thymocytes in coexistence, differentiation of thymocytes into T cells, amplification of T cell division, enhancement of antibody production in B cells, increase in polynuclear leukocytes, promotion of fibroblast division, in hepatocytes It exhibits various physiologically active effects such as suppressing albumin production, suppressing the proliferation of specific cancer cells, protecting against radiation, and restoring infection resistance, and is deeply involved in the homeostasis maintenance mechanism of the body. For this reason, various research and development efforts are underway to effectively utilize IL-1 as a pharmaceutical product, etc., and a method for mass production using genetic recombination technology has been established. However, as mentioned above, IL-1 has a variety of physiologically active actions, and because its physiologically active actions are expressed in extremely small amounts of 10" a+ol or less, it is not used in large amounts as a therapeutic agent for specific diseases. If IL-1 is administered in large doses, there is a risk that the bioactive effects of !L-1, other than those necessary for the treatment of the disease, will be increased by administering a large amount of IL-1, making it impossible to maintain homeostasis in the body. Therefore, as a way to avoid the above-mentioned dangers,
Various IL-1-like activating substances that have some of the physiological activities of IL-1 have been obtained, and research and development is progressing to use these IL-1-like activating substances as drugs depending on the type of disease. ing. As such an IL-1-like activator, for example, a glycopeptide with a molecular weight of 9,100 and consisting of 58 amino acids contained in salivary gland hormones is known [Japan Society of Immunology General Meeting, Academic Conference Record Vol. 18, p. 568 (1988),
Special Publication No. 60-4200. This IL-1-like activator is an extract made from salivary gland hormone derived from the parotid gland of non-human dairy animals (mainly cows), and has physiologically active effects such as increasing white blood cells and lowering serum calcium. have [Problems to be Solved by the Invention] However, there are only a few types of IL-1-like active substances known to date, and the types of diseases to which they can be applied are limited. It is desired to develop drugs using this active substance. Furthermore, although IL-1-like activators made from polypeptides derived from animals other than humans are useful for humans, they also have the problem of causing side effects such as fever and antigenicity. One way to suppress the occurrence of such side effects is to use ILs with as short an amino acid sequence as possible.
It is desired to obtain a -1-like active substance and a drug using this active substance. Therefore, the object of the present invention is to provide a protein that has a short amino acid sequence and has some of the physiologically active effects that IL-1 has, namely, the specific antibody production enhancement effect and the proliferative effect on thymocytes in the coexistence of Con A. have, I
The object of the present invention is to provide a peptide that can be used as an L-1-like active substance and a drug containing this peptide and having IL-1-like activity. [Means for Solving the Problems] The present invention has been made to achieve the above object, and the peptide of the present invention has the following formula Thr-^sp-As
9-Thr-^1 a- 1 1 e-Va I -L
It is characterized by having an amino acid sequence represented by eu-Leu-Lys. Furthermore, the drug having IL-1-like activity of the present invention is characterized by containing a peptide having the amino acid sequence represented by the above formula. In the above formula, Thr represents threonine, Asp represents aspartic acid, Ala represents alanine, He represents isoleucine, Vat represents valine, Leu represents leucine, and Lys represents lysine. Furthermore, all of these amino acids are L-type. The present invention will be explained in detail below. First, the peptide of the present invention having the amino acid sequence represented by the above formula will be explained. This peptide has the physicochemical properties shown below. ■Properties: White powder ■Molecular weight: 1088 ■Solubility: Soluble in water (physiological saline), insoluble in alcohol ■pi:. 7.8 The method for producing the peptide of the present invention is not particularly limited, and may be a chemical synthesis method such as a solid phase method or a liquid phase method, except that the L-type amino acids are arranged as described above. , Gosei D
It can be obtained by biochemical methods such as incorporating NA into microorganisms such as Escherichia coli and allowing the microorganisms to produce it, and methods such as extraction from salivary gland hormones or subunits thereof derived from the parotid glands of warm-blooded animals including humans. The peptide of the present invention obtained in this way is IL-1
It has a part of the physiologically active action that it has, that is, the action of enhancing specific antibody production and the action of proliferating thymocytes in the coexistence of Con A.Its amino acid sequence has a total of 10 amino acids. Next, the drug having IL-1-like activity of the present invention is explained. This drug contains the above-mentioned peptide of the present invention. IL-1 of the present invention Drugs with similar activity can be prepared into various dosage forms for treatment and treatment of humans and other warm-blooded animals, such as oral preparations (tablets, granules, powders, capsules, etc.), injections, etc. (solvent, lyophilized agent, etc.), transdermal agent (patch, ointment), transmucosal agent, implant, eye drops, etc.For formulation, sodium chloride, glycine, lactose, mannitol, etc. , sorbitol, sucrose, starch, dextran, gelatin, etc. can be used as adjuvants. Other therapeutically useful agents can also be included. The content of the peptide of the present invention in a drug varies depending on its dosage form, but is generally 5 to 100% by weight for solid and semisolid forms, and 0.1 to 10% by weight for liquid forms. The dosage of the drug having IL-1-like activity of the present invention depends on the type of warm-blooded animal including humans, the type of disease, severity of symptoms, dosage form, doctor's diagnosis, etc. Although it can vary more widely, the amount of the peptide of the invention generally ranges from 5 μg to 10 μg/kg, preferably from 10 μg to 4 μg/kg per day.
kg. However, as mentioned above, depending on the severity of the patient's symptoms, the doctor's diagnosis, etc., it is of course possible to administer an amount smaller than the lower limit or larger than the upper limit of the above range. Furthermore, the drug having IL-1-like activity of the present invention can be administered once a day or in several divided doses so as to achieve the above-mentioned dosage. [Examples] Examples of the present invention will be described below. Example of Preparation of Peptides of the Invention Chloromethylated 2% divinylbenzene cross-linked styrene polymer resin (CJ content 1.16 mmoleq/g
)lg in Bethucel and Boc-Lys (z) −
OH- (Boc represents a protective group, t-butyloxycarbonyl group, and 2 represents a protective group, carbobenzoxy group) and triethylamine in the above resin.
! Add 3 equivalents of B to the resin and shake in 10 ml of dimethylformamide (DMF) at 35°C for 30 hours.
oc-Lys(z)-0- was attached. After this, Bo
The c-Lys-0- bound resin was thoroughly washed with DMF and then ethanol, and then dried under reduced pressure. In order to quantify the amount of Boa-Lys(z)-0- bound to the resin, a portion (10 µ) of the dried resin bound to Boc-Lys(z)-0- was taken and diluted with 6N hydrochloric acid/ Hydrolysis was performed at 110°C for 24 hours using a dioxane solution (12N hydrochloric acid diluted twice with dioxane), and the solution from which the resin was removed was subjected to amino acid analysis. LyS(Z)10- is 0.
It was confirmed that they were bound at a rate of 23 mmol eq/g. Thereafter, using the above-described resin to which Boc-Lys (z)-0- was bound, amino acids having protecting groups were sequentially bound by peptide bonding according to the procedure shown below. 1) The above-mentioned resin to which Boc-Lys(z)-0- is bound is washed with 10 ml of dichloromethane by shaking for 10 minutes. Repeat this operation two more times. 2> The resin obtained in 1) above was reacted for 1 minute using 10 ml of 50% trifluoroacetic acid (TFA)/dichloromethane solution, and then reacted for 5 minutes using the same solution to remove the protecting group Boc. to leave. 3) Perform the same cleaning operation as in 1) above. 4) Using 10 ml of DMF, shake and wash for 10 minutes. Repeat this operation two more times. 5) Shake for 1 minute with 10 ml of 10% triethylamine/DMF solution, then shake for 5 minutes with the same solution to neutralize. 6) Wash the resin obtained in 5> above by shaking with 10 ml of DMF for 10 minutes. Repeat this operation two more times. 7) Wash the resin obtained in 6> above with 10 ml of dichloromethane by shaking for 10 minutes. Repeat this operation two more times. 8) Boa-Lys (z
) 3 equivalents of Boa-Leu-OH and Boc
3 equivalents of dicyclohexylcarbodiimide (DCC) is added to -Lys(z)-, and the mixture is reacted at room temperature for 3 hours. 9) Among the operations shown in 1) to 8> above, Boc of 8)
-Leu-OR, sequentially Boc-Leu-Of
{,Boc-Val-OHSBBoc-Ile-OHSB
oc-Ala-011, Boc-Thr(Bzl)10
8 (Bzl represents a benzyl group which is a protecting group),
Boa-Asp(OBzl)-0H, Boa-Asp(
OBzl)-0H, and Boc-Thr(Bzl)
Repeat the operations shown in 1) to 8) above except for using -011. However, Boa-1 1e-OH and Val-
In the reaction with Leu-Leu-Lys(z)-resin, the reaction solution is washed away and then the reaction is repeated again. That is, after the above operation 8), the above operations 7> and 8) are performed again. 10) After this, the peptide-bound resin is washed with ethanol and dried under reduced pressure. By the above procedure, Lys (z), Leu, Le
u SMal, lie SAla SThr(Bzl)
, Asp(OBzl) , Asp(OBzl), Boa
A resin in which -Thr(Bzl) was sequentially bonded was obtained. The yield at this time was 1.34 g. 1 g of the resin thus obtained was treated with hydrogen fluoride in the presence of anisole at 0°C for 1 hour to cleave all the protecting groups and release the peptide from the resin. It was distilled off under reduced pressure. After washing the resin with ether, IM acetic acid was added to extract the target substance.
The extract was subjected to column chromatography (2.5 cm) using Sephadex G-25 (manufactured by Pharmacia).
The target substance was eluted using IM acetic acid (φ×100 cm), and the fraction containing the target substance was lyophilized. The yield at this time was 130 mg. After this, the target peptide 42 is purified using a preparative HPLC (high performance liquid chromatography) column (product name: MMC-DODS-5, manufactured by YMC).
I got ■. The analysis results of the obtained peptide are as follows. - Amino acid analysis value (hydrolyzed with 6N hydrochloric acid at 110°C for 24 hours. Values represent relative values based on the Ala value as the reference value, and numbers in brackets represent theoretical values.) Lys: 0. 94 (1), Asp:
2. 06 (2) Thr: 1. 88 (
2), Ala: IVat: 0. 53
(1), Ile: 0. 53 (1) Leu
: 2. 07 (2)・Specific optical rotation [α]20 -14. 3@D (c-0.13, using 0.1M ammonia water as a solvent) - Infrared absorption spectrum (IR) 1138 Formulation example Injection containing 500 μg of the peptide of the present invention and 10 μg of lactose (freeze-dried agent) ) was prepared in the following manner. First, 500 g of the peptide of the present invention and 10 g of lactose were added to distilled water for injection 11, and dissolved while adjusting the pH to 9.0. Next, the obtained solution was sterile-filtered using a Millipore filter (GS) (manufactured by Millipore), and the filtrate was aliquoted into ampoules in 1 ml portions. After that, the peptide 500 of the present invention was obtained by freeze-drying the separated filtrate.
An injection (lyophilized product) containing 10 μg of lactose and 10 μg of lactose was obtained. When using this injection (lyophilized agent), add distilled water for injection to bring the total volume to 1 ml. Test for evaluating the proliferation effect on thymocytes in the presence of Con A. C3H/HeJ mouse thymocytes were cultured in RPMI1640 culture medium (trade name, manufactured by Nissui Seiyaku ■) containing 5% fetal bovine serum, and the cells in the culture medium were After adjusting the number to 5 x 105 cells/ml, a 96-well microplate was used, 4 holes were made into 1 group, and the 4 groups were prepared according to the present invention in the coexistence of Con A as follows. The proliferative effects of the peptides on thymocytes were evaluated. a The peptide of the present invention was added to one well of the microplate containing the mouse thymocytes at a rate of 10 μg/ml.
Also Con A at 0.5μg/m! Added at 5% co
After culturing at 37° C. for 48 hours in the presence of 2, 3H-thymidine (0.8 μCI/well) was added and the cells were further cultured for 12 hours. Thereafter, the cells were collected using a cell harvester, and the degree of proliferation of the thymocytes was evaluated by measuring radiation from 3H-thymidine newly incorporated into DNA by replication using a liquid scintillation counter. b The same procedure as above a was carried out except that the addition ratio of the peptide of the present invention was 100 μg/ml. C The same procedure as above a was carried out except that the addition ratio of the peptide of the present invention was 250 μg/ml. d The same procedure as above a was carried out except that the addition ratio of the peptide of the present invention was 500 μg/ml. In addition, as a comparison of the evaluations a to d above, the degree of proliferation of thymocytes was evaluated in the following manner using the other four groups. Only the peptide of the present invention was added at 250 ml without adding eConA.
The procedure was the same as in a above except that it was added at a ratio of μz/ml. 500% of the peptide of the present invention alone without adding fConA.
The procedure was the same as in a above, except that it was added at a ratio of μg/ml. g Con A alone without adding the peptide of the present invention
．． The procedure was the same as in a above except that the mixture was added at a rate of 5 μg/ml. h The same procedure as above a was carried out except that the peptide of the present invention and Con A were not added. The radiation measurement results (scintillation counting results) for a to h above are shown in Table 1. (Left below) *: Values are [average count value (el)l') ]±

[Standard error] is shown, and the numbers in brackets represent relative values based on the average count value of h. As is clear from Table 1, the peptide of the present invention and Co
When coexisting with Con A, the peptide of the present invention and Con A were approximately 18 to 46 times lower than when Con A was not added.
The measured value increases approximately 13 to 15 times when only on A is added. Further, when only the peptide of the present invention was added, no increase in the measured value was observed. An increase in the measured value means that the amount of 3H-thymidine newly incorporated into DNA due to replication is large, which means that the number of proliferating thymocytes is large. It was confirmed that it has IL-1-like activity, which shows a strong proliferative effect on thymocytes in the coexistence of . Enhancement of Specific Antibody Production Three groups of 6-week-old female inbred C 5 7 B L/6 mice with 8 mice per group were prepared, and 10 6 sheep red blood cells were administered to each mouse through the tail vein. One of the three groups was used as a comparison group, and the spleen of each individual was removed on the fifth day after the administration of sheep red blood cells, and the number of anti-sheep red blood cell antibody-producing cells in the spleen was determined using the PFC method (plaque test). The samples were counted and the average value for the group was determined. The remaining two groups were used as test groups, and the number of anti-sheep red blood cell antibody-producing cells was counted in the following manner, and the average value for the groups was determined. B. To each individual in one of the two groups, 100 μg of the peptide of the present invention was administered at the same time as the sheep red blood cells were administered, and the other conditions were the same as in the comparative example group, and the average value as a group was determined. (b) To each individual in the other group, 500 μg of the peptide of the present invention was administered at the same time as the sheep red blood cells were administered, and the other conditions were the same as in the comparative example group, and the average value as a group was determined. These results are shown in Table-2. Table-2 *: 0. in t-test. There is a significant difference at a risk rate of 1% or less. As is clear from Table 2, the peptide of the present invention was
In the test group administered μg, the number of anti-sheep erythrocyte antibody-producing cells increased approximately 12 times compared to the comparative example group, confirming that the peptide of the present invention has a strong adjuvant effect (immune support effect). It was done. That is, it was confirmed that the peptide of the present invention has an IL-1-like activity of enhancing specific antibody production. [Effects of the Invention] As explained above, the peptide of the present invention has a short amino acid sequence and exhibits some of the physiologically active effects of IL-1, namely, the enhancement of specific antibody production and the effect on thymocytes in the presence of Con A. It has a physiologically active effect similar to a proliferative effect. Therefore, the peptide of the present invention can be used as an IL-1-like active substance with a low risk of side effects. Furthermore, since the drug containing the peptide having IL-1-like activity of the present invention has a low risk of side effects when administered, it can enhance vaccine efficacy, increase host defense ability, and can be used during chemotherapy or radiotherapy for cancer patients. It becomes possible to improve the immunocompromised state of patients.

Claims (2)

[Claims] (1) The following formula Thr-Asp-Asp-Thr-Ala-Ile-V
A peptide characterized by having an amino acid sequence represented by al-Leu-Leu-Lys. (2) A drug having interleukin-1-like activity, characterized by containing the peptide according to claim (1).