JP2003513053A - Use of dopamine D3 receptor agonists in the treatment of salt-dependent hypertension - Google Patents

Abstract

(57) Abstract: The use of dopamine D ₃ receptor agonist for the production of a medicament for the diagnosis and treatment of salt-dependent hypertension.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the use of dopamine D ₃ receptor agonist for the manufacture of a medicament in the diagnosis and treatment of salt-dependent hypertension.

[0002]

[Prior art]

A relationship between renal dopamine receptor function and decreased renal sodium excretion in certain essential hypertension has been postulated, although the exact cause of the renal defect is unknown (T. Hussein et al. Hypertension, 1998, 32: 187-197).
. Salt-dependent hypertension is present in about 10-15% of cases in humans. The diagnosis is made by requiring the patient to maintain a low salt diet for several weeks, resulting in a decrease in blood pressure in the case of salt-dependent hypertension. However, the problem that patients often do not maintain such a diet permanently makes it difficult to make a reliable diagnosis. Even when diagnosed, it is less certain that patients will continue on a low salt diet forever.

[0003]

[Problems to be Solved by the Invention]

An object of the present invention is to find a drug that enables simple diagnosis and treatment of salt-dependent hypertension.

[0004]

[Means for Solving the Problems]

We have found that this task is achieved by using dopamine D ₃ receptor agonists for the manufacture of a medicament for the diagnosis and treatment of salt-dependent hypertension.

Suitable dopamine D ₃ receptor agonists in principle refer to all agonists which are selective for this receptor subtype. One of such agonists
One example is R (+)-7-hydroxy-2-dipropylaminotetralin (7OH-DPAT). Particularly preferred agonists are those that exhibit high concentrations of peripheral plasma. It was possible to prove its effect using various animal experiments.

Dahlrat (see Dahl et al., Nature 1962, 194: 480-482) represents an animal model of salt-dependent hypertension. Hypertension can be induced by the high sodium diet in so-called salt-sensitive Dahl rats, even when they are young.
However, this effect does not occur in so-called salt-resistant Dahl rats. 7OH-DPAT, a dopamine D ₃ receptor agonist, in salt-resistant Dahl rats
Administration increased renal glomerular filtration rate, natriuresis, and excretion capacity, but had no effect on renal function in salt-sensitive Dahl rats.

Dopamine D ₃ Receptor m in Salt-Resistant and Salt-Sensitive Dahl Rats
RNA measurements revealed that mRNA expression was 60% lower in salt-sensitive Dahl rats than in salt-resistant Dahl rats. Similarly, radiolabelled selective dopamine D ₃ ligands to membrane preparations were obtained from kidney salt-sensitive Dahl rats, ^{[3 H]} binding capacity -7-OH-DPAT is compatible kidney salt resistant Dahl rats It was about 50% lower than the prepared preparation.

[0008] In salt-resistant rats, which naturally do not develop hypertension when a high-salt diet is taken, the selective dopamine D ₃ receptor antagonist 5-amino-
3- (4- (4- (2-t-butyl-6-trifluoromethyl) pyrimidine-4
Sub-chronic administration of -yl) piperazin-1-yl) -2-methyl-but-2-en-1-ylmercapto) -4-methyl-1,2,4 (4H) -triazole had a significant effect on arterial blood pressure. It was found to show a significant increase. These findings, it can be inferred that critically involved in the development of the dopamine D ₃ receptor precisely salt-dependent hypertension.

Dopamine D ₃ receptor agonists can be used to diagnose salt-sensitive hypertension. This is the oral or subcutaneous administration of the D ₃ agonist,
It is performed by measuring urine volume and sodium excretion under standardized conditions. Increased urine output and sodium excretion indicate a normal response. Without this reaction, salt-sensitive hypertension would be indicated. Such a distinction is important in subsequent therapeutic decisions.

In order to manufacture a drug, a D ₃ receptor agonist is added to a conventional drug dosage form,
For example, it can be converted into an oral, parenteral, subcutaneous, intraperitoneal or topical dosage form. Suitable forms include, for example, tablets, capsules, infusions or injectable solutions,
There is a liquid or spray suitable for drinking. To suit the desired mode of administration, the novel pharmaceutical formulations contain conventional carriers and diluents in addition to the active ingredient. For topical external administration, such as ethanol, isopropanol, ethoxylated castor oil, ethoxylated hydrogenated castor oil, polyacrylic acid, polyethylene glycol, polyethylene glycol stearate, ethoxylated fatty alcohol, liquid paraffin, mineral oil and wool fat. Pharmaceutical excipients can be used. Suitable for internal administration are eg lactose, propylene glycol, ethanol, starch, talc and polyvinylpyrrolidone.

Antioxidants such as tocopherol and butylated hydroxyanisole and butylated hydroxytoluene, aroma improving additives, stabilizers, emulsifiers and lubricants may also be present. The substances present in the formulation together with the active ingredient and the substances used for the production of pharmaceutical formulations are toxicologically acceptable and compatible with the particular active ingredient. The pharmaceutical formulation is
For example, it is prepared by a conventional method of mixing the active ingredient with a conventional carrier and diluent.

[0012]

【Example】

The present study was performed in normotensive Sprague-Dawley rats and two animal models of arterial hypertension, the Okamoto species spontaneously hypertensive rats (SHR) and salt-sensitive Dahl rats (DS). Male SHR rats aged 8-10 weeks and body weight 180-260 g (n = 8) and its control group, male Wistar Kyoto rats (WKY) (n = 8).
) Was fed a normal rat diet with free access to raw water. Male DS rats aged 6 to 7 weeks and body weights 100 to 150 g (n = 10) and their normotensive control, salt-resistant Dahl rats (DR, n = 10), 2 before clearance test.
The rat diet supplemented with 4% by weight sodium chloride was given for 2 to 25 days with free drinking of raw water.

Clearance test Sodium thiopental (80 mg / Kg body weight, Trapana
1 (R), Byk Gulden, Konstanz, DE) was anesthetized by intraperitoneal injection and placed on a heated bed to maintain a rectal temperature of 37.1 ° C. A tracheotomy was performed to facilitate spontaneous breathing. Two catheters were inserted into the right jugular vein. The first catheter, equal to 0.8% of body weight Ringer saline (111 mmol NaCl, 30 mmol NaHCO 3, 4.7 mmol KCl) the speed 1.5-3.
It was injected at 0 ml / hour. During this experiment, [ ³ H] -insulin (1.2 μCi / ml) dissolved in Ringer's solution was infused through a second catheter at a rate of 0.6 mg / hr to measure glomerular filtration rate.

Blood samples were removed and a cannula was inserted into the left carotid artery for continuous measurement of systemic blood pressure. Urine samples were removed from the bladder catheter. After releasing the rat from this procedure for 80-100 minutes, two consecutive clearance periods were examined every 20 minutes to establish a baseline. Ringer's saline infusion was then given in 2 consecutive doses, 0.
The injection was replaced with a solution of the D ₃ receptor agonist, R (+)-7-hydroxy-2-dipropylaminotetralin, dissolved in Ringer's solution at 01-0.1 μg / kg body weight / min. The Ringer's solution was then reinjected and 10 minutes later the clearance period was measured. A 180 μl blood sample was taken 10 minutes after each 20 minute clearance period.

Glomerular filtration rate (GFR) was measured using renal [ ³ H] -insulin clearance. Mean arterial blood pressure (MAP) and heart rate (HR) were measured continuously during this test. After the clearance survey was completed, the kidney was removed, the blood was exsanguinated, and the weight was measured. Urine volume was weighed. Blood samples were centrifuged and hematocrit values were measured. Urine and plasma sodium concentrations were measured by flame photometry using ELEX® 6361 provided by Eppendorf, Hamburg. ³ H-insulin radioactivity was measured by liquid phase scintillation counting.

The results showed that a uniform and dose-dependent response to the administration of selective D ₃ receptor agonists occurred in spontaneously hypertensive rats, normotensive WKY rats and salt-resistant Dahl rats. Increased natriuresis, diuresis and glomerular filtration rate in these three groups were clearly in contrast to the results in salt-sensitive Dahl rats. In this type of animal, no change in renal function was observed upon dopamine D ₃ receptor stimulation.

Binding studies of [ ³ H] -7-OH-DPAT with dopamine D ₃ mRNA in kidney tissue Kidney tissue was obtained by first anesthetizing the rat. The kidneys were exposed, removed, weighed, and the tunic removed using sterile engineered material.

RT-PCR (RT-PCR: Reverse Transcriptase-Polymerase Chain Reaction) for measuring dopamine D ₃ mRNA in renal tissue. Both Darle species (DS and DR) and kidneys obtained from SHR and WKY rats. Were incised in an arrow shape using a sterile knife under HEPES at 4 ° C. (Terada et al., 1993). The kidneys were laterally divided into 3 equal-sized pieces, first shock-frozen in 1.5 ml Eppendorf tubes in liquid nitrogen and then stored at −80 ° C. until further RNA preparation. RNA was isolated by the method of Chomjinski and Sakki (1987). This requires homogenization of the tissue in the presence of acidic phenol / chloroform mixture and centrifugation. The upper aqueous phase is R
Including NA. It was purified from residual phenol using isopropanol and precipitated using ethanol. This is then recovered in _{DEPC-H 2} O, after quantification, it was used for RT-PCR. All steps were performed under sterile conditions to prevent RNase contamination.

500 ng of total RNA was transcribed into complementary DNA (cDNA) in a 15 μl mixture. Primers for subsequent RT-PCR were selected based on appropriate literature or using published sequences in the gene database and synthesized by MWG-Biotech (Ebersburg). D ₃ receptors relative quantitation of mRNA expression, Wong et al (1994) was performed by describing the primer dropping method. In the first step, the target sequence is amplified in sequence for a certain number of times, and in the second step,
In this case, a primer pair for amplification of the β-actin gene can be added and PCR can be continued for additional times.

Comparison of signal intensities after PCR of various samples showed significant differences between the expression of DS rats and their control, DR rats. DS rats showed a significant reduction in expression of about 60%. 3 RTs in each rat
-The PCR reaction was investigated in groups of 3 rats (n = 3/9). In fact, the direct contrast of WKY to SHR showed a slight increase in expression. However, this did not reach significant levels. Comparison with WKY and SHR rats and DS rats, _however, in the expression of D 3-mRNA, Note showed greater significant difference.

7-OH-DPAT Binding Kidneys from both Dahl rat species (DS and DR) after feeding normal and high salt diet were buffered (TRIS 25 mM / HEPES 40 mM (pH 4.7), sucrose 320 mM, EDTA 0.5 mM
) And was homogenized immediately and centrifuged at 1000 × g for 15 minutes (4 ° C.). After discarding the supernatant, the pellet was resuspended in buffer and recentrifuged. The second supernatant was centrifuged at 100,000 xg for 30 minutes (4 ° C). The final pellet was resuspended in buffer without sucrose and EDTA and stored at -80 ° C until binding experiments were performed. Binding studies were performed with 100 mg of membrane protein. Saturation experiments were performed using 10 μM unlabeled 7-OH-DPAT with 0
． Performed with 5-50 nM [ ³ H] -7-OH-DPAT to measure non-specific binding. Specific binding was calculated by subtracting non-specific binding from total binding.

The dissociation constant (K _D ), which describes the affinity of the ligand for the receptor, showed no significant difference and was about 10 nM for both Dahl species. On the other hand, specific binding (bmax) appeared significantly lower in DS than in DR. This difference was even more pronounced when the rats were already on a high salt diet. In this case, the specific binding to DS rat is D
Only 50% of the value for R animals.

[0023] Dopamine D_ThreeSalt-dependent arterial hypertension in salt-resistant Dahl rats by receptor inhibition
Induction of tenderness   Salt-resistant Dahl rats (DR) fed a normal diet show dopamine D in drinking water. _Three Antagonist, 5-amino-3- (4- (4- (2-t-butyl-6-tri
Fluoromethyl) pyrimidin-4-yl) piperazin-1-yl) -2-methyl
But-2-en-1-ylmercapto) -4-methyl-1,2,4 (4H) -to
Riazole was treated at a dose of 40 mg / kg per day for 1 week. Arterial blood pressure
Was measured by tail plethysmometry before and during the weekend. Then
Animals were fed ad libitum on a high salt diet (4 wt% NaCl in diet). Experimental opening of arterial blood pressure
It was measured every week from the beginning to 4 weeks. Dopamine D with normal salt diet_ThreeAnn
Treatment of animals with tagonists did not cause changes in blood pressure (approximately 100 mmH
value of g). However, the high salt diet showed an increased occurrence in arterial pressure.
This averaged a maximum of 148 mmHg on day 29, so far the plateau phase
Was not shown.

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Claims (2)

[Claims] 1. Use of a dopamine D ₃ receptor agonist for the manufacture of a medicament in the diagnosis and treatment of salt-dependent hypertension. 2. The use according to claim 1, wherein R (+)-7-hydroxy-2-dipropylaminotetralin is used as the dopamine D ₃ receptor agonist.