CH658133A5 - METHOD FOR PRODUCING A STABLE RECEPTOR PREPARATION. - Google Patents

Description

The invention relates to a method for producing a stable receptor preparation for determining the amount of substances binding to brain receptors (primarily benzodiazepines, β-adrenergic blocking agents and Y-aminobutyric acid), the receptor preparation and a method for determining substances binding to brain receptors.

In therapy, it is often necessary for diagnostic and / or therapeutic purposes to determine the amount of substances present in the body fluids that bind to brain receptors. So you can e.g. draw conclusions for individual brain diseases from the y-aminobutyric acid content of the spinal fluid.

It is also known that the body fluids usually contain the substance to be determined in a very low concentration (from 10 ~ 8 - 10-12 moles), so measuring their amount requires special, sensitive methods.

Mass spectrometric, high-performance liquid chromatographic and ion-exchange fluorometric methods suitable for determining the amount of y-aminobutyric acid are described in the following publications: J.

Chromate 118, 395 (1976), Brain Res. 167, 297 (1979) and Anal. Biochemistry 101: 349 (1980). Although the listed methods are sufficiently sensitive, their common disadvantage is that the biological sample to be examined cannot be used directly when measuring, but only after laborious sample preparation (cleaning consisting of several steps, preparation of derivatives, etc.).

To carry out the newly developed radioreceptor test for determining the amount of y-aminobutyric acid [J. Neurochem. 28, 1121 (1977), Brain Res. 182, 99 (1980), Clinica Chimica Acta 109, 77 (1981)], it is not necessary to prepare the body fluid to be examined. However, it is disadvantageous that the receptor suspension required to carry out the examination must be freshly prepared from the brain of experimental animals using the known methods. This requires the maintenance of an animal house and a special biochemical laboratory, which hospitals and clinics generally do not have. Such a receptor preparation is therefore necessary which can be kept indefinitely in the dry state and can be safely used to determine the amount of substances which bind to brain receptors.

The preparation of an easy-to-use, indefinitely dry receptor preparation is described in German Offenlegungsschrift No. 2 902 071. According to the known method, the receptor preparation is produced by homogenizing animal brain tissue with the aqueous solution of a neutral, water-soluble substance - expediently sucrose - and freezing the homogenate.

The disadvantage of the known receptor preparation, however, is that it can only be used to determine the amount of benzodiazepines and is also not suitable for measuring the different types of benzodiazepines separately. Determination of the amount of other brain receptor binding agents, e.g. y-aminobutyric acid and ß-adrenergic blockers are not possible with the known receptor preparation.

In the course of our investigations it was observed that this deficiency can be attributed to the fact that the known receptor preparation contains the majority of the brain receptors in a form inactivated by endogenous substances. By completely removing the inactivating endogenous substances, the brain receptor sites can be released and the preparation can thus determine any substance that can bind to brain receptors.

The invention thus relates to a method for producing a stable receptor preparation which is suitable for determining the amount of substances which bind to brain receptors, according to claim 1.

Preferred embodiments of the invention form the subject of claims 4 to 6.

With this method, a solid, stable, indefinitely long-lasting receptor preparation is obtained, which contains all brain receptor sites in an active state and is generally suitable in the radioreceptor test for determining the amount of all substances which can bind to the brain receptors.

An animal (rat, cattle, chicken, etc.) brain is expediently used as the starting material, of which a brain homogenate is produced in the manner described in German Offenlegungsschrift No. 2 902 071. Sucrose is advantageously added to the aqueous medium as a water-soluble inert substance. With the first centrifugation carried out at an acceleration of 800-1100 g, the coarse precipitate is separated from the brain or partial brain homogenate; the supernatant contains the basic substance of the receptor preparation. The second centrifuge, carried out at an acceleration of 18,000 - 22,000 g,

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The base material of the receptor preparation is separated in a state contaminated with cell nuclei and mythochondrium. In this step, the basic substance of the receptor preparation contains the vast majority of the active receptor sites still in a state inactivated by endogenous substances. This solid substance is then homogenized again in distilled water. Then the cell membranes spring open and the endogenous substances blocking the active receptor sites enter the aqueous medium. The homogenization is again carried out in cold (4-10 ° C) distilled water. At least 15 parts by weight of distilled water are expediently used to homogenize 1 part by weight of solid material again. In general, the more homogenized the more distilled water, the more effectively the blocking endogenous substances can be removed from the active receptor sites. The upper limit of the amount of distilled water is essentially determined by economic considerations. 10-20 parts by weight of distilled water are generally used to homogenize 1 part by weight of solid material again.

To promote the cracking of the cell membranes, the homogenate containing distilled water can, if desired, be frozen and thawed.

With the first centrifugation carried out at an acceleration of 7000-9000 g, the cell nuclei and mythochondrium, which contain no active receptor sites and whose presence would adversely affect the properties (e.g. filterability) of the receptor preparation, are removed from the homogenized product containing distilled water. The supernatant separates the base material of the preparation by the following centrifugation, which is carried out with an acceleration of 35,000 - 45,000 g, which is then carried out with an aqueous buffer solution with a pH between 6 and 8

- expediently with a tris-citrate buffer solution (pH = 7.1)

- is washed at least once. The efficiency of cleaning can be increased if the suspension consisting of the washing liquid and the solid substance is frozen and thawed at least once during washing. If washing is carried out only once, the washing liquid is removed and the solid obtained is suspended in an aqueous buffer solution with a known amount and a known salt content. This suspension is freeze-dried. If the solid is washed in several steps, the suspension formed directly with the last washing liquid is lyophilized; the amount and the salt content of the washing liquid (buffer solution) must of course also be known in this case.

The stable, solid receptor preparation produced in this way can be used to determine the amount of substances that bind to brain receptors as follows:

The aqueous solution which contains the substance to be investigated, which is marked as radioactive (usually with tritium), is added to the suspension of the receptor preparation prepared with aqueous buffer solution with a pH between 6 and 8 or distilled water. The mixture is incubated, then the solid is filtered off, washed with buffer solution and the radioactivity of the solid is determined. Then a known amount of non-radioactive substance to be examined is added to the receptor preparation carrying the radioactively labeled substance to be examined, the mixture is incubated, the solid substance is filtered off, washed with buffer solution and the radioactivity is measured again. The latter operation is repeated several times with different, known amounts of a non-radioactive substance to be examined. Based on the measurement results, a concentration / radioactivity calibration curve is recorded (the term “concentration” stands for the concentration of the non-radioactive substance to be investigated).

The amount of the substance to be examined in the body fluid (e.g. in the backbone fluid) is determined with the aid of the above calibration line so that a known amount of body fluid is added to the receptor preparation bearing the radioactive substance to be examined, the mixture incubated, the solid is filtered off, washed and its radioactivity measured and the concentration belonging to the measured value is read from the calibration line.

The invention is illustrated in more detail by the following examples, without restricting the scope thereof.

example 1

i5 Production of a receptor preparation

Male rats from the CFY strain were decapitated. Her brain was immediately removed and washed with ice-cold physiological saline. The brain part called the bridge and the occipital brain were removed and a homogenate was formed from the residue 20 with 0.32 molar aqueous sucrose solution, the volume of which was 15 times. The homogenate is centrifuged for 10 minutes with an acceleration of 1000 g, then the supernatant is separated off and centrifuged for 10 minutes with an acceleration of 20 000 g. The separated solid substance is homogenized again in cold (+ 4 ° C) distilled water, which has a 15-fold volume, then the homogenate is centrifuged at an acceleration of 8,000 g for 10 minutes. The supernatant is separated off and centrifuged for 30 minutes at an acceleration of 40,000 g for 30 minutes. The solid obtained is homogenized in 50 times the volume of a 50 millimolar tris-citrate buffer solution (pH = 7.1) and the suspension is centrifuged again at an acceleration of 40,000 g. The latter washing step is repeated. The solid is then frozen frozen in a tenfold buffer solution and held at -20 ° C for 10 hours. The suspension is thawed, diluted five times with distilled water and centrifuged again at an acceleration of 40,000 g. This freeze-thaw-centrifuge cycle is repeated three times -40. After the last thawing, the suspension is divided into parts, frozen and freeze-dried. A powdered receptor preparation is obtained, which is mixed with buffer solution or distilled water before use (measurement). The powdered receptor preparation can be stored for years without modification.

Example 2

Recording the calibration curve for the radioreceptor test see above. Solutions or suspensions of the following composition are used to record the calibration curve:

Solution I.a: 0.7435 g NaCl, 0.0186 g KCl, 0.0144 g CaCl2, 0.012 g MgSO4 dissolved in 100 ml twice-distilled water

Solution I.b: 1 g of bovine serum albumin dissolved in 10 ml of solution La

Solution II.a: 1 mg of y-aminobutyric acid dissolved in 1000 ml of solution V 60

Solution ILb: Mixture of 10 jj.1 solution Il.a and 10 ml solution V

Solution II.c: mixture of 100 p.1 solution ILb and 900 | il Lö-

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solution V

Solution Il.d: mixture of 750 (il solution II.c and 250 fil solution V

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Solution ILe: Mixture of 500 ml solution II.c and 500 ml solution V

Solution Il.f: Mixture of 250 p.1 solution II.c and 750 nl solution V

Solution Il.g: mixture of 100 jxl solution II.c and 900 ji.1 solution V

Suspension III: 0.2 g of the receptor preparation prepared according to Example 1 is suspended in 3 ml of twice-distilled water, then the suspension is diluted with 17 ml of solution V.

Solution IV: 3H-Y-aminobutyric acid solution with an activity of 40 nCi / 500 jj.1 (solvent: aqueous hydrochloric acid solution with a pH value of 4)

Solution V: 50 millimolar tris citrate buffer solution (pH = 7.1)

(a) Determination of the Complete Binding Capacity (Cc Value)

The following solutions or suspensions are mixed in succession:

500 yy.1 solution IV,

10 | il solution I.b,

10 (il solution V and 500 yy.1 suspension III.

The mixture is incubated at 4 ° C for 20 minutes, then the solid is filtered on a Whatman GF / C micropore glass fiber filter. The solid is stirred with 10 ml of liquid scintillation measuring solution with a known composition, the suspension is left to stand in the refrigerator for 1 hour, then shaken and the radioactivity of the suspension is determined. The radioactivity value thus obtained is marked by Cc; this is the total amount of y-aminobutyric acid that can be bound by the preparation.

(b) depressing the radioactively marked y-aminobutyric acid by non-radioactive y-aminobutyric acid (determination of the C ^ r values)

The procedure is as in paragraph (a), except that instead of 10 jil solution V, 10 jil solution II.c, Il.d, ILe, Il.f and Il.g are used in succession. The measured radioactivity values are identified by Cx.

(c) Determination of the amount of non-specifically bound y-aminobutyric acid (Cb value)

The procedure is as in paragraph (a), but instead of 10 (j.1 solution V 10 jj.1 solution Il.a are used. In this case, the entire amount of specifically bound 3H-y-aminobutyric acid is replaced by the non-radioactive y The measurement of the radioactivity of the sample gives the correction factor which characterizes the amount of the non-specific binding ^ -y-aminobutyric acid. This correction value (Cb value) is obtained when the calibration line is recorded subtracted from the Cc and Cx values.

The measurement results are shown in Table 1.

Table 1

Solution sign

Concentration of the inactive y-amino-butyric acid nM

Counts / minute

Cc-Cb Cx "Cb

Il.a II.c o o

160 2049

2.15

Table 1 (continued)

Solution sign

Concentration of the inactive y-aminobutyric acid nM

Counts / minute

Cc - Cb Cx - Cb

Il.d

7.5

2367

OO

""H

ILe

5.0

2780

1.55

Il.f

2.5

3284

1.30

Il-g

1.0

3754

1.13

V.

-

4221

1.00

If the Cc-Cb / Cx-Cb values are shown as a function of the concentration of the inactive y-aminobutyric acid, the calibration line is obtained.

When determining the concentration of y-aminobutyric acid in the body fluid, the procedure is such that 10 ml of body fluid are added to the mixture instead of 10 fxl buffer solution (solution V) or inactive y-aminobutyric acid solution of known concentration, then the radioactivity of the preparation is measured and measured by the Calibration line that reads the y-aminobutyric acid concentration belonging to the measured radioactivity.

Example 3

Determination of the amount of other substances specifically binding to brain receptors

3.1 Determination of 3H-diazepam

5 mg of the lyophilized receptor preparation prepared according to Example 1 are suspended in a mixture of 0.9 ml of aqueous tris-citrate buffer solution (pH = 7.1) and 0.1 ml of twice-distilled water. 2nM 3H-diazepam is added to the suspension, incubated at 4 ° C for 30 minutes, then the solid is filtered on a Whatman GF / C micropore glass fiber filter and washed eight times with 2.5 ml of ice-cold tris-citrate buffer solution . The radioactivity is measured as in Example 2. In this way the total binding capacity (Cc) of the preparation is determined. The calibration line is recorded using the depressing method described in Example 2 and the amount of non-specifically bound 3H-diazepam (correction factor, Cb) is determined as in Example 2.

3.2 Determination of the 3H-diazepam dependent on y-aminobutyric acid:

The procedure is as in Example 3.1, but 2 × 10-5 mol of y-aminobutyric acid are also added to the incubation mixture.

3.3 Determination of the Ca2 + -dependent 3H-y-aminobutyric acid:

5 mg of the lyophilized receptor preparation prepared according to Example 1 are suspended in a mixture of 0.9 ml tris-HCl buffer solution (pH = 7.4) and 0.1 ml twice-distilled water. 250 nM calcium chloride, 40 μM isoguväcin and 5.4 μM 3H-y-aminobutyric acid are added to the suspension. The mixture is incubated for 30 minutes at room temperature, then the solid is filtered and washed at room temperature with tris-HCl buffer solution (pH = 7.4). The radioactivity is measured as in Example 2; the correction factor (Cb) is determined using the method described in Example 2.

3.4 Determination of 3H-dihydroalprenolol

5 mg of the lyophilized receptor preparation prepared according to Example 1 are dissolved in 1 ml tris-HCl buffer solution (pH = 8.0)

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suspended. 2 nM 3H-dihydroalprenol are added to the suspension, incubated for 30 minutes at room temperature, then the solid is filtered and washed at room temperature with tris-HCl buffer solution (pH = 8.0). The radioactivity is measured as in Example 2; the correction factor (Cb) is determined using the method described in Example 2.

The amount (Cc-Cb) of the specifically bound 3H ligand is given in Table 2.

Table 2

Example of specifically bound 3H ligand number fmol / ml

3.1

111

3.2

231

3.3

22

3.4

19th

v

Claims (9)

658 133 1. A process for the preparation of a stable receptor preparation which is suitable for determining the amount of substances binding to brain receptors, wherein a brain or partial brain homogenate prepared with the aqueous solution of a water-soluble inert substance with an acceleration of 800 to 1100 g for 8 to 20 minutes is centrifuged and the substance of the preparation is separated from the supernatant, characterized in that the supernatant is centrifuged at an acceleration of 18,000 to 22,000 g for 10 to 20 minutes, the solid substance thus obtained is homogenized again in distilled water, the homogenate is then Centrifuged at an acceleration of 7,000 to 9,000 g for 5 to 15 minutes, the supernatant separated, centrifuged at an acceleration of 35,000 to 45,000 g for 20 to 30 minutes, the solid obtained with an aqueous buffer solution with a pH Value between 6 and 8 washed at least once and that of the solid and the washing liquid existing suspension is freeze-dried. 2. The method according to claim 1, characterized in that the second homogenate is frozen and thawed before centrifuging. 2nd PATENT CLAIMS 3. The method according to claim 1 or 2, characterized in that the suspension formed during washing from the solid substance and the washing liquid is frozen and thawed at least once. 4. The method according to any one of claims 1 to 3, characterized in that the renewed homogenization takes place in distilled water at a temperature of 4 to 10 ° C. 5. The method according to any one of claims 1 to 4, characterized in that 10 to 20 parts by volume of distilled water are used for the renewed homogenization based on 1 part by volume of solid substance. 6. The method according to any one of claims 1 to 3, characterized in that tris-citrate buffer solution is used for washing. 7. Receptor preparation prepared according to one of claims 1 to 6. 8. A method for determining the amount of substances binding to brain receptors by means of a radioreceptor test, characterized in that a receptor preparation according to claim 7 is used for the test. 9. The method according to claim 8, characterized in that the amount of y-aminobutyric acid present in the backbone liquid is determined.