CA2389643A1 - Use of dopamine-d3 receptor agonists for the therapy of salt-dependent hypertension - Google Patents

Abstract

The invention relates to the use of dopamine-D3 receptor agonists in order t o produce a medicament for the diagnosis and therapy of salt-dependent hypertension.

Description

THERAPY OF SALT-DEPENDENT HYPERTENSION

The present invention relates,to the use of dopamine D3 receptor agonists for producing a drug product for the diagnosis and treatment of salt-dependent hypertension.
A connection between:renal dopamine receptor functian and diminished renal sodium excretion in some types of essential hypertension has been suggested, although the exact cause of the renal defect is unclear (cf. T. Hussain et al., Hypertension, 1998 , 32:187-197).
Salt-dependent hypertension is present in about 10 to 15% of cases in humans. The diagnosis has to date been made by requiring the patients to maintain a low-salt diet for several weeks, which results in a reduction in the hypertension in cases of salt-dependent hypertension. However, one difficulty is that the patients frequently do not persistently maintain such a diet, which makes it difficult to make a reliable diagnosis. Even when the diagnosis has been made it is often not ensured that the patient will maintain a low-salt diet permanently.
It is an object of the present invention to find drug prodcuts which make simple diagnosis and therapy of salt-dependent hypertension possible.
We have found that this object is achieved by employing dopamine D3 receptor agonists for producing a drug product for the diagnosis and treatment of salt-dependent hypertension.
Suitable dopamine D3 receptor agonists are in principle all the agonists which are selective for this receptor subtype. One example of such an agonist is R(+)-7-hydroxy-2-dipropylaminotetralin (7 OH-DPAT). Particularly suitable agonists are those which display high peripheral plasma levels.
It has been possible to demonstrate the effect by means of various animal experiments.
The Dahl rat (cf. Dahl et al., Nature 1962, 194:480-482) represents an animal model of salt-dependent hypertension.
Hypertension can be induced in so-called salt-sensitive Dahl rats by a high-sodium diet even when they are juveniles, whereas this effect does not occur in so-called salt-resistant Dahl rats.
Whereas in salt-resistant Dahl rats administration of the dopamine D3 receptor agonist 7 OH-DPAT increased the glomerular filtration rate, natriuresis and volume excretion, administration had no effect on renal functions in salt-sensitive Dahl rats.
Determination of the dopamine D3 receptor mRNA of salt-resistant Dahl rats and salt-sensitive Dahl rats revealed that expression of the mRNA was 60% less in the salt-sensitive Dah7. rats than in the salt-resistant Dahl rats.
There was likewise a reduction of about 50% in the ability to bind the radiolabeled selective dopamine D3 ligand [3H]-7-OH-DPAT
in membrane preparations from kidneys of salt-sensitive Dahl rats compared with corresponding preparations from kidneys of salt-resistant Dahl rats.
In salt-resistant rats, which by definition do not develop hypertension when they receive a high-salt diet, there was found to be a significant increase in arterial blood pressure when the animals received subchronic administration of the selective dopamine D3 receptor antagonist 5-amino-3-(4-(4-(2-t-butyl-6-trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)-2-methyl-but-2-en-1-ylmercapto)-4-methyl-1,2,4(4H)-triazole while receiving a high-salt diet.
It can be inferred from these findings that it is the dopamine D3 receptors which are crucially involved in the development of salt-dependent hypertension.
Dopamin D3 receptor agonists can be used to diagnose salt-sensitive forms of hypertension. This is done by administering a D3 agonist orally or parenterally, and measuring the urine volume and the sodium excretion under standardized conditions. An increase in the urine volume and the sodium excretion would indicate a normal reaction; the absence of this reaction would point to a salt-sensitive form of hypertension.
Such a distinction is important for subsequent therapeutic decisions.
To produce a drug product the D3 receptor agonists can be converted into conventional pharmaceutical dosage forms, for example for oral, parenteral, subcutaneous, intraperitoneal or topical administration. Suitable forms are, for example, tablets, i capsules, solutions for infusion and injection, drinkable forms or sprays.
Appropriate for the desired mode of administration, the novel pharmaceutical preparations contain conventional carriers and diluents in addition to the active ingredient. For local external administration it is possible to use pharmaceutical excipients such as ethanol, isopropanol, ethoxylated castor oil, ethoxylated hydrogenated castor oil, polyacrylic acid, polyethylene glycol, polyethylene glycol stearate, ethoxylated fatty alcohols, liquid paraffin, petrolatum and wool fat. Suitable for internal administration are, for example, lactose, propylene glycol, ethanol, starch, talc and polyvinylpyrrolidone.
It is also possible for antioxidants such as tocopherol and butylated hydroxyanisole and butylated hydroxytoluene, flavor-improving additives, stabilizers, emulsifiers and lubricants to be present.
The substances present together with the active ingredient in the preparation, and the substances used in the production of the pharmaceutical preparation, are toxicologically acceptable and compatible with the particular active ingredient. The pharmaceutical preparations are produced in a conventional way, for example by mixing the active ingredient with conventional carriers and diluents.
Experiments The investigations were carried out on normotensive Sprague-Dawley rats and on two animal models of arterial hypertension, the spontaneously hypertensive rats of the Okamoto strain (SHR) and the salt-sensitive Dahl rats (DS).
Male SHR rats (n=8) and their control group, male Wistar-Kyoto rats (WKY), (n=8), with an age of 8 to 10 weeks and a body weight of 180 to 260 g, were fed with normal rat feed, with free access to tap water. Male DS rats (n=10) and their normotensive controls, salt-resistant Dahl rats (DR, n=10), with an age of 6 to 7 weeks and a body weight of 100 to 150 g, received, for a period of 22 to 25 days before the clearance tests, a rat feed supplemented with 4~ by weight sodium chloride, likewise with free access to tap water.

Clearance tests The rats were anaesthetized by an intraperitoneal injection of sodium thiopental (80 mg/kg body weight; Trapanal~ from Byk Gulden, Konstanz, DE) and placed on a heated stage in order to maintain a rectal temperature of 37.1°C. A tracheostomy was performed to facilitate spontaneous breathing. Two catheters were introduced into the right jugular vein. Ringer's saline (111 mmol NaCl, 30 mmol NaHC03, 4.7 mmol KC1) was infused at a rate of 1.5 to 3.0 ml/h, equivalent to 0.8% of the body weight, through the first catheter. Throughout the experiment, [3H]-inulin (1.2 ~Ci/ml) dissolved in Ringer's solution was in infused at a rate of 0.6 ml/h through the second catheter, in order to determine the glomerular filtration rate.
A cannula was inserted into the left carotid artery to remove blood samples and for continuous measurement of the systemic blood pressure. Urine samples were removed through a bladder catheter. After the rats had recovered from this operation for a period of 80 to 100 min, two consecutive clearance periods of 20 min each were investigated to establish the base line. Then infusion of Ringer's saline was replaced by that of. a solution of the D3 receptor agonist R(+)-7-hydroxy-2-dipropylaminotetralin, dissolved in Ringer's solution, in two consecutive doses of 0.01 to 0.1 ~g/kg body weight/min. Then Ringer's solutian was reinfused and, after 10 min, a clearance period was carried out.
After 10 min of each 20-minute clearance period a 180 ~1 blood sample was taken.
The glomerular filtration rate (GFR) was determined by means of the renal [3H]-inulin clearance. The mean arterial blood pressure (MAP) and the heart rate (HR) were determined continuously during the tests. After completion of the clearance investigations, the kidneys were removed, exsanguinated and weighed. The urine volume was determined by gravimetry. The blood samples were centrifuged and the hematocrit was determined. The sodium concentrations in urine and plasma were determined by flame photometi:y using an ELEx~ 6361 apparatus supplied by Eppendorf, Hamburg. The 3H-inulin radioactivity was determined by liquid phase scintimetry.
The result showed that a uniform and dose-dependent reaction to the administration of the selective D3 receptor agonist occurred in spontaneously hypertensive rats, normotensive WKY rats and salt-resistant Dahl rats. The natriuresis, diuresis and increase in the glomerular filtration rate in these three groups clearly contrasted with the results for the salt-sensitive Dahl rats. In animals of this strain there was no observable change in renal function due to dopamine D3 receptor stimulation Investigation of the dopamine D3 mRNA and [3H]-7-OH-DPAT binding 5 in kidney tissue Kidney tissue was obtained by initially anesthetizing the rats.
The kidneys were exposed, removed, weighed and freed from the capsule using sterile operating materials.
RT-PCR to determine dopamine D3. mRNA in kidney tissue (RT-PCR: Reverse transcriptase-polymerase chain reaction) The kidneys from both Dahl strains (DS and DR), and SHR and WKY
rats, were divided sagittally with a sterile scalpel under HEPES
at 4°C (Terada et al., 1993). The kidney was divided into 3 transverse pieces of equal size and initially shock-frozen in 1.5 ml Eppendorf tubes in liquid nitrogen and then stored at -80 °C until further preparation of the RNA. The RNA was isolated by the method of Chomczynski and Sacchi (1987). This entailed the tissue being homogenized in the presence of an acidic phenol/chloroform mixture and centrifuged. The upper aqueous phase contains the RNA. This is purified from residual phenol with isopropanol and precipitated with ethanol. This can then be taken up in DEPC-H20 and, after quantification, employed in the RT-PCR. All the steps were carried out under sterile conditions, and contamination with RNases was precluded.
500 ng of total RNA were transcribed into complementary DNA
(cDNA) in 15 ~1 mixtures. The primers for the subsequent RT-PCR
were selected either on the basis of appropriate literature or with the aid of published sequences in gene databases and were synthesized by MWG-Biotech (Ebersberg). The relative quantification of the D3 receptor mRNA expression was carried out by the primer dropping method as described by Wong et al. (1994).
In the first stage, the target sequence is amplified in a.
particular number of cycles in order, in the second stage, to add the primer pair for amplification of the housekeeping gene, in this case beta-actin, and to allow the PCR to continue for further cycles.
Comparison of the signal intensities after the PCR of the various samples showed a significant difference between expression in the DS rats and that in their controls, the DR rats. DS rats showed a significant reduction of about 60% in expression. The investigations were carried out with groups of three rats with three RT-PCR reactions for each rat (n=3/9). Direct comparison of the WKY with the SHR in fact revealed a slight increase in expression. However, this did not reach the significance level.
Comparison of DS rats with the WKY and SHR rats however showed an even larger significant difference in the expression of D3-mRNA.

7-OH-DPAT binding Kidneys from animals of both Dahl strains (DS and DR) after normal and high-salt diets were homogenized immediately after removal using a potter in buffer solution (TRIS 25 mM / HEPES
40 mM (pH 4.7), sucrose 320 mM, EDTA 0.5 mM) and centrifuged at 1000 x g for 15 minutes (4°C). After the supernatant had been discarded, the pellet was resuspended in buffer and recentrifuged. The second supernatant was centrifuged at 100,000 x g for 30 minutes (4°C). The final pellet was resuspended in buffer without sucrose and EDTA and stored at -80°C until the binding experiments were carried out. Binding studies were carried out with 100 mg of membrane protein. Saturation experiments were carried out with 0.5 to 50 nM [3H]-7-OH-DPAT, employing 10 ~M unlabeled 7-OH-DPAT to measure the nonspecific binding. The specific binding was calculated by subtracting the nonspecific binding from the total binding.
The dissociation constant (KD) expressing the affinity of the ligand for its receptor showed no significant differences and was around 10 nM for both Dahl strains. On the other hand, the specific binding (bmax) emerged as significantly lawer for DS
than for DR. The difference was even clearer when the rats had previously received a high-salt diet. In this case, the specific binding for DS rats was only 50$ of the value for DR animals.
Induction of salt-dependent arterial hypertension in salt-resistant Dahl rats by inhibition of dopamine D3 receptors Salt-resistant Dahl rats (DR) on a normal diet were treated for one week with the dopamine D3 antagonist 5-amino-3-(4-(4-(2-t-butyl-6-trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)-2-methylbut-2-en-1-ylmercapto)-4-methyl-1,2,4(4H)-triazole in a dose of 40 mg/kg per day in the drinking water. The arterial blood pressure was measured by tail plethysmometry before and at the end of this week. The animals were then exposed to a high-salt diet (4~ by weight NaCl in the feed) ad libitum. The arterial blood pressure was determined each week for up to 4 weeks after the start of the experiment.

It emerged that treatment of the animals with the dopamine D3 antagonist with a normal salt diet caused no change in the blood pressure (values around 100 mmHg). However, with the high-salt diet there was an increasing rise in the arterial pressure, which on day 29 did not as yet show a plateau phase, with an average maximum of 148 mmHg.

Claims (2)

We claim:

1. The use of dopamine D3 receptor agonists for producing a drug product for the diagnosis and therapy of salt-dependent hypertension.

2. The use as claimed in claim 1, where R(+)-7-hydroxy-2-dipropylaminotetralin is used as dopamine D3 receptor agonist.