JP2001194369A - Screening method of medicine acting on central nervous system, and method for evaluating status under stress - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a screening method of a medicine acting on the central nervous system in which a compound to be tested is administered to a mammal abnormal in the immune system, and the concentration of the interleukin 6 in the blood of the mammal, and a method for evaluating the status under stress. SOLUTION: Medicines acting on the central nervous system can be extensively and easily screened, novel medicines acting on the central nervous system can be developed, and the medicines having the central action as the side effect can be screened, and the state under stress of the mammal abnormal in the immune system can be evaluated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for screening a drug acting on the central nervous system and a method for evaluating a stress state of a mammal having an abnormal immune system.

[0002]

2. Description of the Related Art Conventionally, as a method for screening a compound acting on the central nervous system using an animal, for example, a maximum electric shock seizure (ME) for evaluating an antiepileptic drug is used.
S) method and threshold method (PTZ method), forced swimming test for antidepressant evaluation, anti-conflit test for anti-anxiety drug evaluation, anti-hypoxia test for evaluation of brain function improving drugs,
Examples include a method using an electroconvulsive shock-induced amnesia model and a cycloheximide-induced amnesia model for evaluating anti-dementia drugs. Various central nervous drugs have been screened by administering the drug to normal animals or diseased model animals.

Most of such evaluation systems for screening for drugs acting on the central nervous system are based on behavioral observation, and often must be evaluated in combination with double blinds. Could not be evaluated. As described above, although there are many screening methods for drugs acting on the central nervous system depending on the pharmacological action, there is no known method for widely screening drugs acting on the central nervous system irrespective of the pharmacological action. On the other hand, as a stress-related biological substance,
Catecholamines and their metabolites, steroid hormones, etc. are known, and their concentrations are measured,
It can be used as an indicator of a stress state. However, they are not always satisfactory because they are susceptible to coexisting measurement interfering substances.

[0004]

SUMMARY OF THE INVENTION The present invention provides a method for widely and easily screening for a drug acting on the central nervous system and a new index for evaluating the stress state of a mammal having an abnormal immune system. The purpose is to:

[0005]

Under such circumstances, the present inventor has proposed that blood interleukin-6 (hereinafter referred to as "IL-6")
As a result of intensive studies on the correlation between the concentration of) and central nervous system activity, in model animals with abnormal immune system Medium IL-6 concentration changes,
By using this as an index, they have found that a drug acting on the central nervous system can be easily screened, and that a change in blood IL-6 concentration can be an index of a stress state, thereby completing the present invention.

That is, the present invention relates to a method of administering a test compound to a mammal having an abnormal immune system, and administering the test compound to the blood IL-
6. A screening method for a central nervous system acting drug, comprising measuring the concentration of IL-6, and the stress state of a mammal having an abnormality in the immune system by measuring the blood IL-6 concentration in the mammal having an abnormality in the immune system Is provided.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The screening method and the stress state evaluation method of the present invention are characterized by measuring blood IL-6 concentration using a model animal having an abnormal immune system.

[0008] IL-6 is produced from various cells such as monocytes, T cells, B cells, vascular endothelial cells, fibroblasts, and osteoblasts, and induces differentiation of B cells into antibody-producing cells and hepatocytes. Are various physiologically active cytokines, such as induction of protein synthesis in the acute phase, induction of differentiation of brain nervous cells, induction of proliferation and differentiation of hematopoietic cells, and induction of osteoclast differentiation (Experimental Medicine, Vol. 7, No. 1, In recent years, it has been reported that IL-6 induces an acute phase protein, and it is suggested that IL-6 is useful as an inflammatory marker. In addition, studies in open heart surgery cases show that IL-6 in blood can be expressed in the early stage of inflammation. It has been suggested that the initial inflammation may be detected with high sensitivity by using IL-6 as an inflammatory marker (Clinical Pathology, Vol. 42,
11, 1151-1157, 1994). Also,
Recently, it has been reported that laughing lowers blood IL-6 levels in rheumatoid arthritis patients (Journal of Rheumatology).
umatology), Vol. 23, No. 4, pp. 793-794, 1996).

[0009] However, in a mammal in abnormal immune system than ever reports which are discussed in detail IL-6 and central action in general, the blood IL-6 concentration in the mammalian, hypnotic, It is a fact that the central nervous system acting drug having an action such as antipyretic action, analgesic action, cerebral function improving action, etc. could not be expected to significantly decrease.

In the present screening method, a mammal having an abnormal immune system is used. Here, “there is an abnormality in the immune system” refers to a state in which the immune system is enhanced by inflammation or the like, and includes a state in which an autoimmune disease has developed. Autoimmune diseases include chronic inflammatory diseases such as rheumatoid arthritis, and in the present screening method, arthritis is particularly preferred. The autoimmune disease may be one induced by a specific means or a spontaneous autoimmune disease.

Means for inducing an autoimmune disease include a method of inoculating an adjuvant or a chemical substance, use of a transgenic animal, transplantation, and gene knockout. Inoculating an adjuvant to induce immune abnormalities,
As a method of causing arthritis, for example, a method of suspending killed tuberculosis in liquid paraffin and inoculating the suspension into the skin of the ridge of a rat (Arzneim.-Forschung / Drug Research (Arzneim.-Forsch./ Drug Res.), Volume 42, 2
No. pp. 935-944, 1992). In addition, animals having spontaneously occurring immune abnormalities include, for example, MRL
/ Lpr mouse and the like.

By further applying a stress load to a mammal having an abnormal immune system, the blood IL-6 concentration can be effectively increased. As such a stress load, any method such as psychological stress and / or physical stress may be used. For example, forced swimming, irradiation of strong light or sound, restraint, water immersion restraint, overcrowding, electric shock, , Low temperature and / or high temperature.

The above mammals include mammals other than humans, and preferably include rodents such as mice and rats. Preferable model animals used in the screening method of the present invention include mammals other than humans that have developed arthritis, particularly rodents such as mice and rats, and more preferably, they are under stress.

The drugs acting on the central system evaluated by the screening method of the present invention include, for example, an anesthetic action, a hypnotic action, a sedative action, an antipyretic action, an analgesic action, an antidepressant action, a cerebral function improving action or an anti (psychotropic) mind It is suitable for screening a compound having a nerve effect or the like.

In order to measure the concentration of IL-6 in blood, for example, collected blood or plasma or serum separated from blood is used, and IL-6 in whole blood, plasma or serum is used.
The -6 concentration may be measured by a commonly used method such as an RIA method, an EIA method, an ELISA method, and a bioassay method.

Specifically, the screening method of the present invention comprises the steps of (1) administering a non-toxic amount of a compound orally or parenterally to a mammal having an abnormal immune system, and (2) applying no or no stress. After a certain period of time,
(3) It can be carried out by measuring the concentration of IL-6 in blood. In particular, a test compound is administered to a mammal in which inflammation has been induced, and blood is collected after the optimal time of the stress load in which induction of the blood IL-6 is observed by the stress load, and the blood IL-6 concentration is measured. Is preferred. In addition, I in blood
The L-6 concentration may be compared in the same group or in the compound administration group and the non-administration group. The blood IL-6 concentration thus measured is compared before and after administration of the test compound, and when the concentration decreases, it can be determined that the test compound is a drug acting on the central nervous system.

On the other hand, when a stress load is applied to a mammal having an abnormal immune system, the blood IL-6 concentration is increased.
By measuring the -6 concentration, the stress state of a mammal having an abnormal immune system can be measured.

The method for measuring the blood IL-6 concentration for evaluating the stress state of a mammal having an abnormal immune system may be the same as the above-described screening method of the present invention. That is, using the collected blood or plasma or serum separated from the blood, the IL-6 concentration in whole blood, plasma or serum was determined by the RIA method, EIA method, or ELISA.
It may be measured by a commonly used method such as a method and a bioassay method.

The evaluation of the stress state is, specifically,
(1) a non-toxic amount of a central nervous system agonist is orally or parenterally administered to a mammal having an abnormal immune system, (2) blood is collected after a certain period of time, and (3) IL- (4) comparing the measured IL-6 concentration before and after administration of the compound. In particular, a compound having a hypnotic, antipyretic, analgesic or cerebral function improving action, particularly a compound having a hypnotic or cerebral function improving action, is administered to a mammal in which inflammation has been induced, and blood is collected after an optimal time. Then, it is preferable to measure and compare the blood IL-6 concentration.

[0020]

The present invention will be described below in detail with reference to examples.

Example 1 M. tuberculosis H37Ra (manufactured by Difco) was suspended in liquid paraffin (manufactured by Merck) (6 mg / mL) and rat (Lewis strain, 6-7 weeks old, male) 0.1 mL was inoculated intradermally into the ridge of the ridge [Arzneim.-Forsch./Drug Res., 42, No. 2, 93
5-944, 1992]. 18 days later, plethysmometer Ugo Basile 7150
The animals were divided into groups of 5 or 6 animals so that the footpad volumes measured in 1) were equal. The stress-loaded group was placed in a medicinal-type stress cage at the University of Tokyo, and immersed in the aquarium at 23 ° C to the chest, and subjected to stress [Preclinical evaluation method of drug activity, pp. 96-97, Fujimura
Fukuda, Pharmaceutical Daily.] 30 minutes later, under anesthesia with ether, blood is collected and spitz containing serum separating agent (Sepaclean, Eiken Instruments)
To separate the serum. The concentration of IL-6 in each serum was adjusted to MH60
BSF2 cells [European Journal of Immunology (Eur. J. Immuol.), 18, 951-956, 1988
The amount of IL-6 in serum was calculated based on the proliferation activity of a known amount of IL-6. still,
Serum IL-6 was relative (T / C: 100%) with the IL-6 concentration (units / ml) of stressed arthritic rats as 100%.
%)Pointing out toungue. The serum cortisol concentration was measured using an ELISA kit. Table 1 shows the results.

[0022]

[Table 1]

[0023] The above results indicate that by applying water immersion restraint stress to an adjuvant arthritis rat having an abnormal immune state, serum IL-6 increases simultaneously with an increase in serum cortisol known as a stress marker. It indicates that That is, this result indicates that in a mammal having an abnormal immune system, the blood IL-6 concentration is
It indicates that it can be an indicator of a stress state.

Example 2 M. tuberculosis H37Ra (manufactured by Difco) was suspended in liquid paraffin (manufactured by Merck) (6 mg / mL), and rat (Lewis strain, 6-7 weeks old, male) was suspended in liquid paraffin. 0.1 mL was inoculated in the ridge skin [Arzneim.-Forsch./Drug Res., 42, No. 2,
935-944, 1992]. 18 days later, plethysmometer, Ugo Basil 7150 (PLETHYSMO METER, Ugo Basile
The groups were divided into groups of 6 or 7 animals so that the footpad volumes measured in 7150) were equal. Each test compound was administered, 60 minutes later, placed in a medicinal stress-type stress cage at the University of Tokyo, and immersed in a 23 ° C. water tank up to the chest, and subjected to stress [Preclinical evaluation method of drug activity, pp. 96-97, Fujimura Fukuda, Pharmaceutical Daily.] Thirty minutes later, blood was collected under ether anesthesia, and serum was separated using Spitz (Sepaclean, Eiken Instruments) containing a serum separating agent. The concentration of IL-6 in each serum was determined using MH60 / BSF2 cells [European Journal of Immunology (Eur. J. Immuo
l.), 18, 951-956, 1988], and the amount of IL-6 in serum was calculated based on the proliferative activity of a known amount of IL-6. The serum IL-6 was measured at the IL-6 concentration (units / m 2) of the stressed arthritic rat.
l) is shown relative (T / C:%) as 100%. As test compounds, pentobarbital sodium (PE) having a hypnotic effect, aniracetam (AN) having a synaptic transmission efficiency improving effect, cerebral acetylcholine neuromodulating effect and brain energy-metabolism improving effect were used. It was administered immediately before stress loading. Table 2 shows the results.

[0025]

[Table 2]

In the adjuvant arthritis rats having an abnormal immune state, serum IL-6 levels were observed even in the unstressed group.
Was detected, but IL-6 in serum was
Concentrations further increased. In contrast, in the group to which pentobarbital sodium and aniracetam were administered, a decrease in serum IL-6 was observed.

[0027]

According to the screening method of the present invention, a drug acting on the central nervous system can be screened widely and easily, and a new drug acting on the central nervous system can be developed and a drug having a central action as a side effect can be obtained. Screening becomes possible. Further, the evaluation method of the present invention is one of useful means for monitoring the stress state of a mammal having an abnormal immune system.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) A61P 25/04 A61P 25/04 25/20 25/20 25/28 25/28

Claims (5)

[Claims] 1. A test compound is administered to a mammal having an abnormality in the immune system, and interleukin-6 in the blood of the animal is administered.
A method for screening a drug acting on the central nervous system, comprising measuring the concentration. 2. The screening method according to claim 1, wherein the drug acting on the central nervous system has a hypnotic, antipyretic, analgesic or cerebral function improving effect. 3. The screening method according to claim 1, wherein the mammal is further subjected to stress. 4. The mammal according to claim 1, wherein the mammal is a rodent.
4. The screening method according to claim 3. 5. A method for evaluating a stress state of a mammal having an abnormal immune system, comprising measuring a blood interleukin-6 concentration.