JPH06327497A - Method for measuring activity of actylcholine esterase at local site of brain - Google Patents

Abstract

PURPOSE:To measure the AchE activity at a local site of a brain by measuring the concentration of radioactivity at the local site of the brain with a radio tracer, measuring the volume of blood flow rate at the local site, and subsequently calculating the AchE activity from the relation of the measured results with the AchE activity. CONSTITUTION:The concentration of radioactivity at a local site of a brain is measured with a radio tracer, and an AchE activity is calculated from the relation of the AchE activity with both the measured concentration of the radioactivity at the local site of the brain and the measured blood flow rate at the local site. The AchE activity can be measured also in vivo. The radio tracer is characterized in that (a) it has an ester group approximately specifically hydrolyzed with the acetyl choline esterase in the molecule; (b) it has high liposolubility and is incorporated into brains in dependence on the blood flow; (c) it is hydrolyzed with the acetylchloline esterase of the brain, and the produced alcohol is accumulated in the brain; and (d) an alcohol produced outside the brain is not transferred into the brain.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a brain local acetylcholinesterase (hereinafter abbreviated as AchE) useful for diagnosing dementia, evaluating the efficacy of drugs for improving brain function, and elucidating the mechanism of aging.
It relates to a method for measuring activity.

[0002]

2. Description of the Related Art Acetylcholinergic nerves in the brain are deeply involved in memory,
It is considered that this disorder of the nervous system is a cause of the memory disorder such as Alzheimer's dementia. Since the AchE activity decreases as the function of the acetylcholine-acting nerve decreases, the activity measurement greatly contributes to the diagnosis of dementia and aging, the evaluation of treatment and the elucidation of pathological conditions.

Conventionally, as a method for measuring local AchE activity in the brain, an in vitro method using a part of the brain after death was used.
In addition to the measurement with the method described above, a method of staining a brain slice histochemically and measuring it by image processing (Biegon and Wol)
ff, J. Neurosci. Meth. 16: 39-
45, 1986) is known.

However, all of these methods were intended for the post-mortem brain, and could not be applied to living humans or animals. Therefore, in vivo
There has been a demand for a method capable of measuring AchE activity even in the case of o.

[0005]

Under the circumstances, as a result of intensive studies by the present inventors, radioactivity in the brain was measured using a radiotracer having a specific property, and blood in the brain was also measured. The present invention has been completed by finding that the local AchE activity in the brain can be determined by measuring the flow rate and calculating the relationship between these values and the AchE activity.

That is, the present invention provides (A) the following properties (a) to (d): (a) an ester group which is almost specifically hydrolyzed by acetylcholinesterase in the molecule, and (b) an ester. The body is highly lipophilic and is taken up by the brain depending on the blood flow. (C) The alcoholic body produced by being hydrolyzed by acetylcholinesterase in the brain has a low permeability through the blood-brain barrier and accumulates in the brain (d). The alcohol concentration generated outside the brain does not transfer to the brain. The radioactivity in the brain is measured using a radiotracer, (B) the blood flow in the brain is measured, and (C) (A), ( The present invention provides a method for measuring local acetylcholinesterase activity in the brain, which comprises calculating the acetylcholinesterase activity from the relationship between B) and the acetylcholinesterase activity.

In the present invention, first, the above-mentioned property (a)
Radioactivity in the brain is measured using a radiotracer having (d). These radiotracers are taken into the brain depending on the blood flow, and are accumulated in the brain when hydrolyzed by AchE in the brain. For example, the radiolabeled N-methylhydroxypiperidine acyl derivative shown below is used. Etc. can be used.

[0008]

[Chemical 1]

To label these compounds, AchE
Atoms that remain when hydrolyzed to an alcohol form are labeled with a radioactive isotope by a conventional method. For example, in the case of an acyl derivative of N-methylhydroxypiperidine, hydrogen of the corresponding amide group of piperidine may be replaced with a radioactive methyl group by ¹⁴ CH ₃ I, ¹¹ CH ₃ I or the like according to a conventional method. The radioactivity concentration in the brain was measured by administering these radiotracers, and after a lapse of a predetermined time, an autoradiography method using a brain section, a positron emission tomography method (PET), a single photon emission computed tomography method (SPECT). It can be measured with.

On the other hand, the blood flow in the brain is, for example, ¹²³ I-labeled N-isopropyl-p-iodoamphetamine (IM
P) and the reference sample method (L
ear et. al. J. Cereb. Blood
Flow Metabol. 2: 179-185,18
82; Kuhl et. al. J. Nuc. Med.
23: 196-203, 1982), and it is also possible to measure by other known methods.

In order to calculate the AchE activity in the brain region using the thus-measured local radioactivity concentration and blood flow volume, a Kinetic model incorporating a blood flow-dependent brain distribution and metabolic processes was constructed. . That is, the uptake of radiotracer (hereinafter simply referred to as "tracer") into the brain depends on the blood flow rate, and the tracer taken up into the brain is hydrolyzed by AchE competitively with acetylcholine to become an alcohol. Also, the alcoholic body generated in the blood does not transfer to the brain. This is expressed as in Expression (1) using a differential equation.

[0012]

[Equation 1]

Here, C _b is the concentration of the unchanged tracer in the brain, C _p is the concentration of the unchanged tracer in the blood, F is the local blood flow in the brain, and λ is the cerebral blood distribution coefficient in the equilibrium state of the tracer. Yes, F = λK holds. K is the blood-brain barrier (BBB) permeation rate constant of the tracer. The theoretical formula of this blood flow-dependent uptake into the brain is Iodoantipyri
This is the same as the theoretical formula for measuring the regional blood flow in the brain using ne (Sakurada et. al., Am. J. Phy.
siol. 234: H59-66, 1978). There are various theoretical formulas for the blood flow-dependent uptake into the brain, and an appropriate formula is used according to the tracer used.
Further, V _m and K _m are the maximum rate of hydrolysis by AchE of the tracer and the Michaelis constant, C _a is the concentration of free acetylcholine in the brain, K _ma is the Michaelis constant of hydrolysis of acetylcholine, and C _m is the tracer metabolite in the brain. Is the alcohol body concentration, k _el is the rate of elimination of alcohol bodies from the brain. Acetylcholine in the brain is usually accumulated in synapses and is released during neurotransmission, so the free acetylcholine concentration C _a may be regarded as almost zero. The tracer used in the measurement from a tracer level, C _b is negligible much smaller than the K _m.
In addition, alcohol may be eliminated from the brain very slowly, so k _el may be considered to be zero. Therefore, the above simultaneous differential equation (1) can be rewritten as follows.

[0014]

[Equation 2]

Here, k _m (= V _m / K _m ) is Ac in the brain
It will show the hydrolytic activity of the tracer by hE. Equation (3) is obtained by solving the change in the unchanged blood concentration after rapid intravenous injection of the tracer as C _p = ΣC _oi exp (−k _e i t).

[0016]

[Equation 3]

If the time t is sufficient, the exponent term can be regarded as 0. In other words, the concentration of unchanged drug in blood and brain is 0.
Becomes At this time, only the alcohol metabolite exists in the brain, and its concentration is represented by the formula (4).

[0018]

[Equation 4]

Therefore, when a tracer labeled with a radioisotope is administered intravenously and the concentration of unchanged drug in blood and brain becomes 0, the radioactivity concentration in the brain is the same as the blood concentration of unchanged drug. AUC (area under the blood concentration curve), tracer cerebral blood partition coefficient, cerebral regional blood flow and cerebral regional Ac
It will depend on hE activity. This expression is a basic kinetic analysis of the tracer, in order to determine the k _m by using this, must measure absolute values of AUC and cerebral local blood flow of the unchanged drug concentration in blood. However, since the tracer is rapidly decomposed by esterase in blood, it takes time to measure the concentration of unchanged drug. Therefore, a method of performing measurement using a striatum with extremely high AchE activity as an internal standard was examined. When the subscript of the striatum is s and the subscript of the region of interest in the brain is o, and the ratio of the in-brain radioactivity concentrations is calculated, equation (5) is obtained.

[0020]

[Equation 5]

The AUCs cancel each other out. Here, if C _o / C _s is Z,

[0022]

[Equation 6]

When this is rewritten, equation (7) is obtained.

[0024]

[Equation 7]

Here, λ _o K _o / λ _s K _s is the ratio of the blood flow volume between the striatum and the region of interest, which is defined as F, and k _mo is the tracer hydrolysis of the region of interest to be obtained. It is active, and if this is Y,

[0026]

[Equation 8]

When this is solved for 1 / Y, equation (9) is obtained.

[0028]

[Equation 9]

[0029] The Y has exhibits hydrolytic activity of the tracer, the ratio phi = k of the metabolic activity k _mo metabolic activity k _ma and tracer acetylcholine in order to determine the metabolic activity y of acetylcholine by AchE _mo / k _ma And the following equation (10) is used.

[0030]

[Equation 10]

Two parameters A = φ (1 / k_ms+1
/ K_s) (Λ_o/ Λ_s) And B = φ (1 / K_s) (Λ_o/
λ_s) Is unknown, but AchE activity in animals
y and the striatal ratio Z of the radioactivity concentration in the brain are determined by punch out
It can be actually measured using the obtained brain tissue piece. Well
Also, as a tracer for measuring local blood flow in the brain
〔^{one two Three}I] When using IMP, the blood flow is C_b/ AUC
(C_bIs local radioactivity concentration, AUC is blood IMP
The striatal ratio F of the local blood flow in the brain is
^{one two Three}It becomes equal to the striatal ratio of I concentration. Therefore, this is also a
Can be measured from a brain tissue piece obtained by punch-out
It Therefore, tracers in various parts of the brain and striatum
Distribution coefficient ratio λ ₀/ Λ_sDoes not change much depending on the part
Assuming that, linearly using the measured y, Z and F
Determine unknown parameters A and B by least squares method
You can In addition, by measuring the concentration of unchanged drug in blood,
Actual measurement, measurement of absolute regional blood flow, and punch-out
K using the obtained brain tissue_mFrom the actual measurement of
Use to determine all parameters such as λ and K
Is possible. Ac in vitro in animals
Since hE activity can be measured, it is difficult to determine the parameters.
Various methods can be applied, and studies using pathological animals are also possible
Is. In the case of human,
Find the average value and use it. Because of this an accidental death
Which case using autopsy brain, k_mAnd φ are measured and healthy people
The average value of is calculated. In addition, throw a tracer on a healthy person
Giving and C by a method such as PET_mIs measured and then AU
Find C. These values and the brain blood flow in healthy people
From equation (4), λ and K are calculated. More than
Determine the average value of parameters A and B of healthy subjects from the results
You can Patterns determined in male white rats
The parameters are shown in Table 1 as an example.

[0032]

[Table 1]

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, in the autoradiography method using a brain slice, a highly accurate result in which the influence of blood flow is corrected by the double tracer method is obtained, and AchE activity measurement in a minute site in the brain of an animal is measured. And imaging are possible. In addition, using the ¹¹ C-labeled body, PET can be used to measure local AchE activity in the brain of living humans, animals, etc., which enables early diagnosis of dementia, determination of the efficacy of therapeutic agents, and elucidation of the pathology of dementia and aging. Useful for.

[0034]

EXAMPLES Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

Example 1 Ibotenic acid was injected into the Meinert nucleus on the left side of the cerebrum of male white rats to destroy the Meinert nucleus. On the right side, only needle insertion was performed for 1 minute. This treatment reduces the activity of the cholinergic nervous system on one side of the brain, since the Meynert nucleus is bundled with cholinergic nerve bundles. Two weeks after that, [ ¹⁴ C] MP3A, [ ¹⁴ C] MP4A and [ ¹⁴ C] MP4P were respectively administered to 5 rats in a total of about 10 μCi / 0.3 ml, and 28 minutes later [ ¹²³ I]
N-isopropyl-p-iodoamphetamine (IM
P) about 300 μCi / 0.3 ml, bolus from tail vein
Was administered. 2 minutes after administration of IMP (30 minutes after administration of MP3A, MP4A or MP4P), decapitated the rat,
The brain was immediately removed, frozen for 10 minutes with dry ice, embedded in a compound, and a frozen block was prepared in dry ice-acetone. Set this on a cryostat at -20 ° C and set the thickness to 2 for autoradiography.
A 0 μm section was collected from the target site on a cover glass. Separately, thickness 3 for tissue punch-out
Several 0 .mu.m sections were collected on the aluminum foil from the target site by several sheets.

The sections for autoradiography were exposed to hydrochloric acid gas for several seconds to fix N-methylpiperidine as a hydrochloride, and then dried on a hot plate at 55 ° C. for 1 minute, and a Lumirror film (thickness 4 μm). And immediately exposed to an imaging plate (IP, made by Fuji Photo Film). Then, after the exposure for 4 hours, the IP was read by a bio image analyzer BA-100 (manufactured by Fuji Photo Film Co., Ltd.) to obtain an image of ¹²³ I + ¹⁴ C. This section was left for 1 week in a cool and dark place, and after ¹²³ I was sufficiently attenuated (half-life was 13 hr, it was attenuated to about 1/8000). The second exposure to IP was carried out for 24 hours, and ¹⁴ I got the image of C. After that, the same exposure as the first exposure was performed for 4 hours, and the BA's decec was obtained between the obtained image and the initially obtained ¹²³ I + ¹⁴ C image.
Tor Response was subtracted to obtain an image of ¹²³ I alone.

¹⁴ obtained by autoradiography
The C and ¹²³ I image data can be processed by the image processing device NEXUS.
Detector R by 600 (Nexus)
After esponce background processing and registration of the two images, they were converted into images showing the striatal ratio of each radioactivity concentration. Brain local AchE activity was calculated using these images and the parameters shown in Table 1. The cerebral cortex and striatum were cut out from the punch-out section, homogenized, incubated with acetylcholine and DTNB, and the color development was measured with an absorptiometer to measure AchE activity. The wet tissue weight was calculated by measuring the protein concentration in the homogenate by the Lowry method and using 10%, which is the ratio of the normal brain tissue protein amount to the wet weight.

When the AchE activity value obtained by the present invention was compared with the AchE activity actually measured from the excised brain tissue homogenate, it showed a good agreement as shown in FIG. 1, particularly 4-propionyl-N-methylpiperidine ( M
A high correlation was observed in P4P).

[Brief description of drawings]

FIG. 1 is a diagram showing a correlation between cerebral cortex AchE activity measured according to the present invention using MP3A, MP4A or MP4P and AchE activity measured using excised brain tissue homogenate. In the figure, ● indicates the value obtained from the cerebral cortex on the normal side where the Meynert nucleus was not destroyed, and ○ indicates the value obtained from the cerebral cortex on the destroyed side.

Front page continuation (72) Inventor Toru Yokoshima ▲ Hiki ▼ 2117 Tokai-mura Muramatsu, Naka-gun, Ibaraki Prefecture Daiichi Pure Chemicals Co., Ltd. Tokai Research Institute (72) Inventor Shin-ichiro Nagatsuka 2117 Tokai-mura Muramatsu, Naka-gun, Ibaraki Prefecture Tokai Research Institute, Ltd.

Claims (1)

[Claims] 1. (A) The following properties (a) to (d): (a)
It has an ester group that is almost specifically hydrolyzed by acetylcholinesterase in the molecule, and (b) the ester is highly lipophilic and is taken up by the brain depending on the blood flow, (c)
Using a radiotracer having an alcoholic body produced by hydrolysis by acetylcholinesterase in the brain, which has low permeability through the blood-brain barrier and accumulates in the brain, and (d) an alcoholic body produced outside the brain does not transfer to the brain. The radioactivity concentration in the brain is measured, (B) the blood flow in the brain is measured, and the acetylcholinesterase activity is calculated from the relationship between (C), (A), (B) and the acetylcholinesterase activity. Method for measuring local acetylcholinesterase activity in the brain.