EA026837B1 - RADIOLIGAND METHOD FOR QUANTITATION OF RECEPTOR ACTIVITY OF β-ADRENORECEPTORS ON THE SURGACE OF HUMAN T-LYMPHOCYTES - Google Patents

Abstract

The invention relates to medicine. In accordance with the invention, a modified radioligand method for quantitative assessment of receptor activity of β-adrenoreceptors on the surface of human lymphocytes is proposed that can reliably detect specific binding to β-adrenoreceptors and/or β-adrenoreceptors at the level of 0.5 fmol per million cells, based thereon a clinical analysis procedure may be established.

Description

The invention relates to the field of medicine. In accordance with the invention, a modified radioligand method for quantitatively evaluating the receptor activity of β-adrenergic receptors on the surface of human leukocytes, which allows to reliably detect specific binding to βι-adrenergic receptors and / or ₂ -adrenergic receptors at a level of 0.5 fmol / million cells, on the basis of which an analysis can be made for clinical research.

State of the art

In the structure of non-infectious pathology, cardiovascular and broncho-pulmonary pathologies are of the greatest importance. Diseases of the cardiovascular system are in first place among the causes of morbidity and mortality, both in Russia and in other countries of the world [Chazova I.E., Chuchalin A.G., Zykov K.A., Ratova L.G. / / Systemic hypertension. 2013.10 (1). from. 5-35].

Among broncho-pulmonary pathologies, bronchial obstructive diseases require special attention. [С1оЬа1 ΙηίιίαΙίνο ίοτ AkShta (ΟΙΝΑ) (irba! Sat 2014); С1оаа1 51та1сду Изοτ Изе Фадио818, шападстсш апб ргсуепйоп οί стошс оЬагисОуе р1тоиагу Фксакс (SORI). IrbaSh 2014.] Many patients with pathological data require the appointment of drugs that affect β-adrenergic receptors.

Adrenoreceptors, by their structure, belong to the group of receptors connected to guanine nucleotide-binding proteins (C-protein). Anatomically, β-adrenergic receptors are divided into β ₁ -, β ₂ - and β ₃ -adrenoreceptors, among which the most significant for the diagnosis and treatment of cardiovascular and bronchopulmonary diseases are:

1) βι-adrenergic receptors, the maximum number of which are found in the right atrium,

2) β ₂ -adrenergic receptors, which are mainly present in the lungs [§ioio M., UokShtaka T., Uadita T., HuatatoU I. Mge 8s1. 1993. ν.52 (12). P.1063-70].

For lung diseases, β-adrenergic agonists are often the basis of therapy, and for cardiovascular diseases, β-adrenergic blockers, which are ligands for the corresponding receptors. Changes in the expression and affinity of receptors that occur under the influence of appropriate drugs often lead to a decrease in the effectiveness of the prescribed therapy and, in some cases, to the development of serious side effects, such as an increase in the heart rate, prolongation of the CT interval, a decrease in the level of potassium, bronchospasm, etc.

The interaction of the ligand-receptor depends on the type of ligand (agonist or inverse agonist) and is manifested in a change in cellular activity. Agonists, binding to the receptor, activate C-protein and increase the intracellular content of cyclic adenosine monophosphate (cAMP). These changes in the cell simultaneously with the onset of the clinical effect also cause a change in receptor activity. Often, the appointment of agonists leads to a decrease in sensitivity to the ligand and desensitization of the receptors [Muksu 1., Ta1s K., BeGkoM ^ K. 1. 1. VU1: Svet. 1975. No. 250. R. 5727-5729; Rstkshk 1. R., \ Sa1bo S. L., Nagbey T.K. Rebetaboi Prgos. 1982. V. 41. P. 1327], which, in some cases, causes an adverse change in the clinical course of the disease, in the form of worsening bronchial obstruction and the lack of response to the therapy.

The use of antagonists, on the contrary, leads to an increase in the density of β-adrenergic receptors [Parfenova EF, Krasnikova TL, Aripova NA et al. Ger. Archive. 1993.65 (4). from. 49-52] on the surface of mammalian cells and may affect their affinity.

Changes in receptor activity are noted not only under the influence of pharmacological preparations. It was noted that in diseases of the cardiovascular and bronchopulmonary systems, there is already an initially altered background of receptor activity. In patients with ventricular extrasystole, the number of β ₂ -adrenoreceptors on peripheral blood lymphocytes is statistically higher than in the normal group [T. Krasnikova, V.B. Yurkova, M.M. Kuzmina et al. Cardiology. 1989. No. 7. S.25-29].

In patients with arterial hypertension, a statistically significantly increased expression of β ₂ -adrenoreceptors is also observed in relation to the norm, which, in addition, correlates with the severity of the disease. Moreover, the presence of heart failure in the latter leads to a decrease in the rate of maximum binding of β ₂ ligands. [Tsshd R., Kantai 8.Ts., Kobsk S.S. c1 a1. At 1. Kekr1g. SGF Sage Meb. 1997.155 (3). R. 1130-1134].

When examining patients with obstructive pulmonary pathology, conflicting data are noted: some sources report an initially low density of β ₂ receptors on the surface of blood cells, while others do not note a connection between changes in the density of receptors on the surface of blood cells and the presence of bronchial asthma [§1ai1su R. S., b. Oig1x11. § b. Iibsteoob, 8. A11gsb, R.A. 1i8s1. 1. C1. I and νсδΐ. 1980.V. 65 (3). P.577-585].

In recent decades, a number of both direct and indirect methods have been developed to determine receptor activity: the assessment of changes in affinity, expression of receptors, as well as their activity over

- 1,026,837 due to changes in secondary messengers.

One way to assess the state of the adrenergic receptor apparatus of lymphocytes is to assess the severity of adreno-dependent intracellular biochemical processes after the action of an agonist on a homogeneous population of lymphocytes [B.Ya. Zonis, V.V. Karpov, M.G. Lukashevich. Pulmonology. 1997, No. 4, p. 73-77.]. The study of secondary messengers, such as, for example, cyclo-AMP (cAMP) and protein kinase A (PKA), allows you to indirectly evaluate the expression of β-adrenergic receptors [Alabat O., Zljekp Ipdeta R.K. Paganto 1. Thy. 2011.24 (1). R. 174-81]. However, this approach, firstly, provides only indirect information about the receptor activity of lymphocytes, and secondly, does not allow to assess changes in receptor activity on the cell surface.

Real-time polymerase chain reaction (PCR) and Western blot analysis with antibodies against β-adrenergic receptors demonstrate higher sensitivity. The amount of material required for such analyzes is small. However, these methods provide information only on the level of expression of β-adrenoreceptor genes in the studied cells: RT PCR - on the level of transcription, and Western blot - on the level of translation of Schi, Wai L., Wapd 1. e! a1. PbO8 Opera. 2012.7 (12). R. e52911.]. Thus, the receptor activity of β-adrenergic receptors per se, i.e. the binding of specific ligands, these methods cannot be measured.

Another significant drawback of the solutions of the aforementioned prior art is that the known methods for determining the expression of adrenoreceptor and intracellular activity allow us to evaluate the changes occurring on the cell surface only indirectly.

The methods of binding to radioactive ligands make it possible to assess the receptor parameters of interest both initially and against the background of the use of drugs that affect β-adrenergic receptors, and also allow their affinity to be determined [B1apke81eup M., Otaaita δ.Τ, Nes1ot8 M..P. Υ. e! a1. Eig.

1. Cn. Paganto 1. 1992.42 (6). R. 613-8]. The density of surface β-adrenoreceptors is determined by specific binding using a specific ligand labeled with the radioactive isotope ¹²⁵ 1 or ³ N. Non-specific binding is determined by adding a significant excess of unlabeled ligand. The specific binding of the ligand to the receptor is calculated by the difference between the total and nonspecific binding, the density of the receptors and the dissociation constant are determined in Scatchard coordinates using the linear regression coefficient. From the displacement curves of the labeled ligand, the receptor-ligand dissociation constant is calculated according to the Cheng-Prussov formula [Sbat. T., Znbegt, K., LeGk ^ b / K. 1. 1. Cbn. 1way 81. 1976, ν. 57 (1). R. 149-55; Krasnikova T.L., Korichneva I.L., Radyukhin V.A. Biochemistry. 1989.54 (2), p. 235-243].

However, the described traditional radioligand method for determining the amount and affinity of β-adrenergic receptors is laborious, expensive and requires the collection of a large amount of blood, especially when studying the effect of drugs. The latter is most significant in assessing the dynamics of the effect of drugs acting on β-adrenergic receptors, which makes this method of little use in real clinical practice.

Thus, there is a need to develop improved methods for radioligand analysis, which are essentially devoid of the disadvantages of the solutions known from the prior art.

The invention provides a method for the selective quantitative assessment of the relative receptor activity of βι-adrenergic receptors on the surface of human leukocytes by specific binding of [ ¹²⁵ 1] cyanopindolol to cells, in which, in order to determine the specific receptor activity of cellular βι-adrenergic receptors, the binding difference [ ¹²⁵ 1] cyanopindolol cells in the presence of 1.5-2.0 uM nonradioactive and cyanopindolol binding of ^[125 1] -tsianopindolola cells in the presence of 0,20-0,28 M βι-specific l ganga COP-20712 (chemical name- [dihydrochloride 2 - ((3-carbamoyl-4-hydroxy) phenoxy) ethylamino] -3- [4- (1-methyl-4trifluoromethyl-2-imidazolyl) phenoxy] -2-propanol) for which the reaction mixture containing an aqueous solution of [ ¹²⁵ 1] -cyanopindolol, phosphate buffer solution (PB§), a solution of unlabeled ligand (cyanopindolol or COP-20712) and a cell suspension are incubated for 45-60 minutes at 37 ° C with careful stirring, the process is stopped by the addition of ice water (cold water with ice), the cells are centrifuged at 1800-2000d for 10-12 minutes, the supernatant is discarded The precipitate is carefully suspended in cold (+ 4 ° С) РВ§ and centrifuged again under the same conditions, the supernatant is discarded and the precipitate is carefully suspended in cold РВ§ and centrifuged at 10000-12000 d for 2-3 minutes, the supernatant is discarded, and The precipitate is calculated on a γ-counter, measuring the amount of radioactive material in each sample, after which the relative receptor activity of β ^ adrenoreceptors is calculated by the difference in the total binding and beta-specific binding of cyanopindolol per 1 million cells.

A method for the quantitative selective assessment of the relative receptor activity of β ₂ -adrenoreceptors on the surface of human leukocytes by specific binding of [ ¹²⁵ 1] cyanopindolol to cells is proposed, in which, in order to determine the specific receptor activity of cellular βι-adrenoreceptors, the binding difference of [ ¹²⁵ 1] cyanopindolol by cells is determined in the presence of 1.5-2.0 μM non-radioactive cyanopindolol and binding of [ ¹²⁵ 1] cyanopindolol to cells in

- 2 026837 the presence of 0.14-0.18 μM in _2- specific ligand 1C1 118551 (chemical name is (±) -erythrochloride (8 *, 8 *) - 1- [2,3- (dihydro-7-methyl -1H-inden-4-yl) oxy] -3 - [(1-methylethyl) amino] -2-butanol), for which the reaction mixture containing an aqueous solution of [ ¹²⁵ !] - cyanopindolol, phosphate buffer solution (ΡΒδ), a solution of unlabeled ligand (cyanopindolol or 1C1 118551) and a cell suspension are incubated for 45-60 minutes at 37 ° C with gentle stirring, the process is stopped by the addition of ice water (cold water with ice), the cells are centrifuged at 1800-2000 d for 10-12 min, the supernatant is discarded, the precipitate is carefully suspended in cold (+ 4 ° С) ΡΒδ and centrifuged again under the same conditions, the supernatant is discarded and the precipitate is carefully suspended in cold ΡΒδ and centrifuged at 10000-12000d for 2-3 min , the supernatant discarded, and the pellet to calculate a γ-counter, measuring the amount of radioactive material in each sample, then calculating a relative receptor activity by a ₂ -adrenoceptor difference total binding and of specific binding of beta-cyanopindolol attributing and 1 million cells.

The technical result is an improved method of analysis, practicable in clinical practice, characterized by greater sensitivity, expressness and precision, as well as significantly less (up to 5 times) the volume of blood taken for examination.

List of drawings

In FIG. Figure 1 shows the dependences of the binding efficiency of [ ¹²⁵ 1] -cyanopindolol by lymphocytes in the presence of various concentrations of beta-active ligands: βι-ligand СОР-20712 (1) and β ₂ ligand 1С1 118551 (2).

In FIG. 2 shows the total specific binding of [ ¹²⁵ 1] -cyanopindolol on T-lymphocytes of 18 healthy volunteers.

FIG. 3 characterizes (T-specific binding of [ ¹²⁵ 1] -cyanopindolol on T-lymphocytes of 18 healthy volunteers.

In FIG. 4 shows ( _2- specific binding of [ ¹²⁵ 1] -cyanopindolol on T-lymphocytes of 2 healthy volunteers initially, 30 minutes and 2 hours after inhalation ( ₂ -action short-acting agonists (salbutamol).

In FIG. Figure 5 shows the nature of the (specific binding of [ ¹²⁵ 1] -cyanopindolol on Tlymphocytes of 2 patients with bronchial asthma (BA) and arterial hypertension (AH) initially, 30 minutes and 2 hours after inhalation ( ₂ -action short-acting agonists (salbutamol).

In FIG. Figure 6 shows the βι-specific binding of [ ¹²⁵ 1] cyanopindolol on the lymphocytes of 2 healthy volunteers.

In FIG. 7 shows βι-specific binding of [ ¹²⁵ 1] cyanopindolol on the lymphocytes of 2 patients with cardiac arrhythmias (LDCs).

SUMMARY OF THE INVENTION

To determine the number and affinity of β-adrenergic receptors by radioligand analysis, [ ¹²⁵ 1] -cyanopindolol is usually used, and for the selective analysis of β-adrenergic receptors, ³ HCOP12177 (specific ligand for β-adrenergic receptors) are used. The use of ligands labeled with tritium ( ³ N) significantly reduces the sensitivity, which is about 100 times lower than when using [ ¹²⁵ 1] cyanopindolol. Thus, the specific activity of [ ¹²⁵ 1] -cyanopindolol is about 2000 Ci / mmol, while the specific activity of ³ H-COP12177 usually does not exceed 3050 Ci / mmol.

As a result of extensive research, the authors were able to develop a method of using [ ¹²⁵ 1] cyanopindolol as a radioactive ligand, which allows to overcome the disadvantages of the prior art and to analyze with sensitivity up to 0.2-0.4 fmol (0.2-0.4-10 ^-15 mol) in the sample. To achieve this, a number of problems were solved, in particular, the determination of specific binding to cells, binding to specific subtypes of β-adrenergic receptors, and reproducibility of analysis results over time.

The authors found that specific binding to cells can be estimated as the difference between the binding of [ ¹²⁵ 1] -cyanopindolol in the presence of 1.7 μM non-radioactive (cold) cyanopindolol and in its absence. The total specific binding of [ ¹²⁵ 1] -cyanopindolol is at the level of 1 fmol / million cells (about 4000 cpm / million cells). It was also found that the determination can be carried out for 20 hours without changing specific binding.

The main condition is that the cells are not subjected to freezing, since double freezing-thawing leads to an almost complete loss of specific binding. Several series of experiments with total lymphocytes showed that the specific binding of [ ¹²⁵ 1] cyanopindolol is a fairly objective criterion for cell viability and coincides with cytological criteria.

Clinical significance may be given only for individual types of receptors. However, the specific binding, as described above, is determined immediately by several types of receptors. Therefore, the authors established the concentrations of cold non-radioactive ligands specific for β! - 3 026837 and in _2- adrenoreceptors, which can competitively displace [ ¹²⁵ 1] -cyanopindolol. SSR-20712 (dihydrochloride 1- [2 - ((3-carbamoyl-4hydroxy) phenoxy) ethylamino] -3- [4- (1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy was proposed as a specific βι ligand ] -2-propanol) as well as β ₂ ligand successfully applied 1C1 118551 (hydrochloride (±) -eritro- (* 8, 8 *) - 1- [2,3- (dihydro-7metil-1H-indene 4-yl) oxy] -3 - [(1-methylethyl) amino] -2-butanol).

An experiment on the displacement of [ ¹²⁵ 1] -cyanopindolol from lymphocyte receptors by various concentrations of 1C1 118551 shows that there is a threshold concentration above which more complete displacement of the labeled ligand no longer occurs (Fig. 1), and the amount of [ ¹²⁵ 1] cyanopindolol remaining is about 30 % In FIG. Figure 1 shows the binding of [ ¹²⁵ 1] -cyanopindolol to lymphocytes in the presence of beta-active ligands of the βι-ligand SSR-20712 (1), and the β2 ligand 1C1 118551 (2). This effect is understandable, because [ ¹²⁵ 1] -cyanopindolol is not a strictly specific ligand for β-adrenergic receptors and is quite effective in binding to other receptors, for example, serotonin [V.,, V. c. ., A11ep V.S., Neyey T.E.// SagFouaks Ke8. 2006. D.71 (1). P.69-78]. Thus, a decrease in the binding of [ ¹²⁵ 1] -cyanopindolol in the presence of a specific ligand 1C1 118551 is a characteristic of precisely β2-adrenergic receptor activity of lymphocytes.

In a similar experiment with mouse hybridoma cells with β ₁ -adrenoreceptors exposed on the surface (1KS-6), the concentration of SSR-20712 was determined, which provides specific displacement of [ ¹²⁵ 1] cyanopindolol from βι-adrenergic receptors.

The invention allows to analyze the relative receptor activity of human blood cells and changes in the receptor activity of cells in response to exposure to the body of various substances, especially drugs. In the context of the present invention, relative cell receptor activity is defined as determining the availability of a receptor on the cell surface for a specific ligand under given specific conditions. Relative receptor activity is a relative value that depends on: the number of receptors on the surface of the cells, the affinity of these receptors and the availability of these receptors for a specific ligand.

The relative receptor activity of cellular β-adrenergic receptors is defined as (total binding - β-specific binding) / 1 million cells.

The values of the receptor activity of T-lymphocytes of healthy volunteers for β ₂ -adrenoreceptors are shown in FIG. 2 and 3. Under the general specific activity of receptors (OS) is meant the difference in the amount of [ ¹²⁵ 1] -cyanopindolol bound to cells in the presence of 1.7 μM non-radioactive cyanopindolol and in its absence. The specific activity of β ₂ -adrenoreceptors (BS) of T-lymphocytes is defined as the difference in the amount of [ ¹²⁵ 1] cyanopindolol bound to cells in the presence of 0.16 μM 1C1 118551 and in its absence.

The data presented show that the value of the activity of β ₂ -adrenoreceptors, which the authors assess by the ability to specifically bind [ ¹²⁵ 1] -cyanopindolol, varies greatly among different people, and the application of the concepts normal or average does not seem to be correct. Moreover, fluctuations in the values of the total receptor activity in different people (Fig. 2) do not correlate with the fluctuations in their specific β ₂ receptor activity (Fig. 3).

As a result, the most significant are not the absolute values of the binding of the labeled ligand by the cells, but the dynamics of changes in these values under the influence of external factors, i.e. during a functional test.

The test is carried out using a β ₂ short-acting agonist (the active substance is salbutamol) using two healthy volunteers as an example. Changes noted 30 minutes after inhalation of the β ₂ agonist show that β ₂ receptor binding is reduced. Since the measurement error in triplet parallel samples does not exceed 10%, changes in receptor activity of more than 20% can be considered reliable. A study of the relative receptor activity 2 hours after inhalation of the β ₂ agonist shows a restoration of receptor activity to almost the initial level (Fig. 4). Despite the significant difference in the absolute value of [ ¹²⁵ 1] cyanopindolol binding by T-lymphocytes in different donors, a similar dynamics of changes is observed for β ₂ -adrenoreceptor activity (Fig. 4).

When assessing the relative receptor activity in patients with cardiorespiratory pathology (two patients with bronchial asthma and arterial hypertension), the dynamics of β ₂ receptor activity is observed, which is opposite to that observed in healthy volunteers. 30 minutes after inhalation of the β ₂ agonist, an increase in β ₂ receptor binding and restoration of relative receptor activity was observed after 2 hours almost to the initial level (Fig. 5).

In a preferred embodiment of the invention, to determine the specific binding of [ ¹²⁵ 1] cyanopindolol by βι-adrenergic receptors, the authors found that the concentration of CCP20712 should be 0.24 μM.

In 10 healthy volunteers, the inventors found that due to the small number of βι-4,026,837 adrenergic receptors on the cell surface (Fig. 6, using two volunteers as an example), it is very difficult to reliably determine the number of βι-adrenoreceptors. Nevertheless, patients with cardiac arrhythmias (frequent ventricular extrasystole) exhibit higher βι-adrenergic receptor activity, which can be successfully diagnosed using the proposed method. Total and βι receptor binding are shown in FIG. 7.

In general, this approach, of course, does not provide information on the exact number of β-adrenergic receptors on the surface of lymphocytes, because the amount of cell-bound ligand, in this case [ ¹²⁵ Σ] cyanopindolol, depends not only on the number of receptors exposed on the outside of the cells, but also on the affinity of the receptors and their availability for interaction with the ligand from the solution.

Therefore, not limited to a specific theory, the authors believe that in this case it is more correct to talk about receptor activity and its dynamics, which, quite possibly, will be more significant for clinical practice than determining the expression and affinity of receptors.

The proposed method can significantly increase the sensitivity compared with the definitions of ligands labeled with ³ N. In addition, with the same sensitivity of the traditional method using ¹²⁵ Σ, the proposed method allows you to selectively evaluate the receptor activity of both βι-adrenergic receptors and β ₂ -adrenoreceptors.

An important advantage is that at the same time a three to five-fold decrease in the volume of the collected material (blood) is achieved - 20 ml instead of 100-150 ml, which is required in the methods of the prior art. This is of particular importance if this study is performed in patients in real clinical practice, selectively determining binding to β ₁ - and β ₂ -adrenoreceptors, which is especially important in patients with comorbid pathology receiving treatment with drugs that affect β-adrenergic receptors. This makes it possible to use this method not only for scientific research, but also in real clinical practice.

It is also obvious to a specialist in the field of (bio) analytical chemistry that this significantly reduces the time spent on each analysis, and the measured values of the radioactivity of the samples are shifted to a region of significantly higher values, which leads to a decrease in the relative error of determination.

Information confirming the possibility of carrying out the invention Materials

Specific ligands are used for analysis: СОР-20712 (β-adrenergic blocker), 1C1 118551 (β ₂ adrenergic blocker), and cyanopindolol obtained from §1§sh (USA). Bovine serum albumin is also obtained from §1§ (USA). The drug salbutamol (Ventolin, a short-acting β ₂ agonist) is a commercial preparation of the company O1axo8bLk1che Pagtaseijsac (Poland) and is available as a metered-dose aerosol for inhalation (100 μg / dose). Radioactive Na ¹²⁵ 1 in a solution of IAOH with a concentration of 1000 MBq / ml was obtained from the B / O Isotope (Russia). Kit for the isolation of T-lymphocytes: Rap T Ce11 1vo1ayoi Κίΐ II Litap 1x1 t1, 1x2 t1 of the company MSheput Vu! Es.

Patient Characterization

All included volunteers sign an informed consent before inclusion in the study in accordance with all the rules of ethics of the Helsinki Declaration and the National Standard of the Russian Federation Good Clinical Practice GOST R 52379-2005.

The analysis included 18 healthy volunteers without cardiovascular and bronchial obstruction; 2 patients with moderate asthma of a controlled course and arterial hypertension; 2 patients with cardiac arrhythmias (frequent ventricular extrasystole).

All volunteers are over 18 years old and do not take drugs that affect β-adrenergic receptors. Participants undergo a medical examination, examination using standard methods for assessing the state of the cardiovascular and broncho-pulmonary systems before blood sampling. Patients with asthma within 48 hours do not take long-acting β ₂ -agonists and molinolytic drugs; short-acting - within 12 hours before the study.

The study of βι-adrenergic receptors is carried out in 10 healthy volunteers and 2 patients with cardiac arrhythmias. The determination of β ₂ -adrenoreceptor activity is carried out in 18 healthy volunteers and 2 patients with bronchial asthma and arterial hypertension.

Blood collection and cell isolation

Peripheral blood sampling for studies on T-lymphocytes is carried out three times for each volunteer: once - control (initially), second time - after 30 minutes and a third time - 2 hours after salbutamol inhalation 400 μg. In the study of mononuclear lymphocytes, blood sampling is carried out once.

Mononuclear lymphocytes are isolated according to the method described in the publication [Vouit A. I. SNP.

Bb. 1ue51. 1968. 21. R. 77-89]. From the obtained total lymphocyte fraction, T-lymphocytes are isolated using the Rap T. Ce11 1b1a1uyu II итitap 1x1t1, 1x2t1 kit of the Miuepuyuuyu company according to the instructions attached to the kit.

- 5,026,837

Preparation of [ ¹²⁵ 1] -cyanopindolol

The introduction of the radioactive isotope ¹²⁵ 1 into the cyanopindolol molecule is carried out by a modified method described in the article Oghettoob R. S, Niyet V.M. Teerterabot, Amylobacterium, Amyloidis arthritis. It is. ί., 1963, 89 (1). R. 114-123.

The radioiodination reaction mixture contains 1 μg cyanopindolol, 0.2 M potassium phosphate buffer (pH 7.0), 1 mCi Na ¹²⁵ 1 in a volume of 50 μl. The reaction is initiated by adding 10 ml of a solution of chloramine T (10 mg / ml). The incubation duration at room temperature is 1 min. The reaction was stopped by adding 10 μl of sodium thiosulfate (20 mg / ml) and after 5 min of incubation at room temperature, 1 μl of a solution of cold sodium iodide (6 mg / ml) was added.

The target product is isolated by HPLC (chromatograph manufactured by OSHOP, France) on a ShSkoSh 100 C-18 column (5 μm, 4x150 mm) in ion-pair mode. Buffer A - 10% acetic acid, buffer B 100% acetonitrile. Elution with a gradient of acetonitrile concentration from 0 to 100% in 20 min with a flow rate of 0.5 ml / min. Detection of the eluate by UV absorption at 280 nm and by a flow detector of radioactivity. Fractions containing [ ¹²⁵ 1] -cyanopindolol are combined, evaporated to dryness, dissolved in 70% ethanol and stored at -20 ° C.

Total binding of [ ¹²⁵ 1] -cyanopindolol to cells

All reactions and measurements are carried out in three parallels (triplets). The total specific binding of [ ¹²⁵ 1] -cyanopindolol to cells is determined by incubating the cells for 1 h at 37 ° C with gentle stirring on a shaker at a speed of 100 rpm in test tubes with a capacity of 1.5 ml (Eppendorf). The composition of the reaction mixture: 100 μl of an aqueous solution of [ ¹²⁵ 1] -cyanopindolol in phosphate buffer solution (PB§) with a concentration of 1000 counts / μl, 10 μl of a solution of unlabeled cyanopindolol of different concentrations (or 10 μl of water) and 100 μl of a cell suspension with a concentration 5-10 million / ml.

After incubation, the process is stopped by adding 400 μl of ice water (cold water with ice) to each sample. Cells are centrifuged on a Mugo8rsh12 Vu8ai (Latvia) centrifuge at 2000d for 10 min, the supernatant is discarded, the pellet is carefully suspended in 200 μl of cold (+ 4 ° С) РВ§ and centrifuged again under the same conditions. The supernatant is discarded and the pellet is carefully suspended in 200 μl of cold (+ 4 ° C) PB§ and centrifuged at 10000 d for 2 minutes. The supernatant is discarded, and the pellet in the tubes is counted on a γ-counter \ ν; · ι11; κ Χνί / agb 1470 (RetktpE1teg), measuring the amount of radioactive material in each sample. Account efficiency is about 60%.

Specific β-binding of [ ¹²⁵ 1] cyanopindolol to cells

All reactions and measurements are carried out in three parallels (triplets). The specific binding of [ ¹²⁵ 1] cyanopindolol to cells is determined by incubating the cells for 1 h at 37 ° C with gentle stirring on a shaker at a speed of 100 rpm in test tubes with a capacity of 1.5 ml (Eppendorf). Composition of the reaction mixture: 100 μl of an aqueous solution of [ ¹²⁵ 1] -cyanopindolol in PB§ with a concentration of 1000 counts / μl, 10 μl of a solution of unlabeled ligand (СОР-20712, 1С1 118551 or cyanopindolol) of different concentrations (or 10 μl of water) and 100 μl μl of a suspension of cells with a concentration of 10 million / ml Further procedures are carried out as in the determination of total binding (see above).

Claims (2)

CLAIM 1. A method for the selective quantitative assessment of the relative receptor activity of βι-adrenergic receptors on the surface of human leukocytes by specific binding of [ ¹²⁵ 1] cyanopindolol with cells, characterized in that in order to determine the specific receptor activity of cellular βι-adrenoreceptors, the difference in binding of [ ¹²⁵ 1] -cyanopindolol by cells in presence of 1.5-2.0 uM nonradioactive and cyanopindolol binding of ^[125 1] cyanopindolol cells in the presence of 0,20-0,28 M βι-specific ligand GRA-20712 (chemical on - [dihydrochloride 2 - ((3-carbamoyl-4-hydroxy) phenoxy) ethylamino] -3- [4- (1-methyl-4trifluoromethyl-2-imidazolyl) phenoxy] -2-propanol), for which the reaction mixture, containing an aqueous solution of [ ¹²⁵ 1] -cyanopindolol, phosphate buffer solution (PB§), a solution of unlabeled ligand (cyanopindolol or СОР-20712) and a cell suspension are incubated for 45-60 minutes at 37 ° C with gentle stirring, the process is stopped by adding ice water (cold water with ice), the cells are centrifuged at 1800-2000d for 10-12 minutes, the supernatant is discarded, the sediment is carefully suspended end up in a cold (+ 4 ° С) РВ§ and centrifuged again under the same conditions, the supernatant is discarded and the precipitate is carefully suspended in cold РВ§ and centrifuged at 10000-12000d for 2-3 minutes, the supernatant is discarded, and the precipitate is calculated on γ counter, measuring the amount of radioactive material in each sample, and then calculate the relative receptor activity of βι-adrenergic receptors according to the difference in total binding and beta-specific binding of cyanopindolol per 1 million cells. 2. A method for the quantitative selective assessment of the relative receptor activity of β2-adrenergic receptors on the surface of human leukocytes by specific binding of [ ¹²⁵ 1] cyanopindolol to cells, characterized in that in order to determine the specific receptor - 6,026,837 cell activity in ₁ -adrenoreceptors determine the difference in the binding of [ ¹²⁵ 1] -cyanopindolol by cells in the presence of 1.5-2.0 μM non-radioactive cyanopindolol and the binding of [ ¹²⁵ 1] cyanopindolol by cells in the presence of 0.14-0.18 μM B2 -specific ligand 1C1 118551 (chemical name hydrochloride (±) -erythro (8 *, 8 *) - 1- [2,3- (dihydro-7-methyl-1 H-inden-4-yl) oxy] -3 - [(1 methyl ethyl) amino] -2-butanol), for which a reaction mixture containing an aqueous solution of [ ¹²⁵ 1] cyanopindolol, phosphate buffer solution (PB8), a solution of unlabeled ligand (cyanopindolol or 1C 1 118551) and a suspension of cells, incubated for 45-60 minutes at 37 ° C with gentle stirring, the process is stopped by the addition of ice water (cold water with ice), the cells are centrifuged at 1800-2000d for 10-12 minutes, the supernatant is discarded, the precipitate is carefully suspended in cold (+ 4 ° C) PB8 and centrifuged again under the same conditions, the supernatant is discarded and the precipitate is carefully suspended in cold PB8 and centrifuged at 10000-12000 d for 2-3 minutes, the supernatant is discarded, and the precipitate is calculated on γ counter measuring the amount of radioactive th material in each sample, then calculating a relative receptor activity by difference β2adrenoretseptorov total binding, and specific binding of beta-cyanopindolol, referred to 1 million cells.