Na+,2Cl−,K+ cotransport system as a marker of antihypertensive activity of new torasemide derivatives

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European Journal of Pharrnacolog% 219 (1992) 385-394 ~3 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2~)99/92/$05.00

385

EJP 52617

Na+,2CI-,K + cotransport system as a marker of antihypertensive activity of new torasemide derivatives Bernard

Mascreel

", P a t r i z i a F e r r a r i b, M a r r a F e r r a n d i ", B e r n a r d P i r o t t c ~, M a r c S c h y n t s ~, Paolo Parenti b and Jacques Delarge a

a Department of Medicinal Chemistry, Uni~'ersity of Liege, 3 Rue Fusch, B-4000 I.i~ge, Belgium and t, Istituto di Ricerche, Farmitalia Carlo Erha. Nerriano, Italy Received 25 November 1991. revised MS received 7 April 1992, accepted 9 June 1992

A series of compounds related to torascmide, a loop diuretic, were synthesized and examined for their diuretic potency and inhibitory activity on the erythrocyte and renal medullary thick ascending limb vesicle Na ~,2CI-,K + cotransport in Milan hypertensive (MHS) and normotensivc (MNS) rat strains, where previous studies had demonstrated an alteration of thc cotransport system genetically related to hypertension. From the results of the screening, structure-activity relationships wcrc drawn and two compounds, JDL 961 and C 2921 were selected. Their IC50 on renal vesicle cotransport wcrc similar in thc two strains (JDL 961: MHS = 1.8 p,M; MNS = 1.2 p,M; C 2921: MHS = 4 ~M; MNS = 3.8 /zM), and wcrc 4 - 8 times lower than those of torasemidc (MHS = 13 ~M: MNS = 31 /zM, P < 0.01) and 50-60 times lower than those of bumctanidc (MHS = 145 /.~M; MNS = 206/,tM, P < 0.05) taken as reference compounds. Their ability to reducc the devclopmcnt rate of hypcrtcnsion was tested both in MHS and in Okamoto spontaneously hypertensive rats (SHR) strain, in which cotransport alterations arc opposite to those of MHS. Both torasemide derivatives (7.5 m g - k g - ' os per day) prevented development of hypertension in thc two strains. The time course of this hypotcnsive activity was faster and the percentage of blood pressure fall greater in MHS (20-25%) than in SHR rats (12-15%), even though the absolute value of blood prcssure fall was similar in MHS (JDL 961 = - 17 mm Hg; C 2921 = - 30 mm Hg) and SHR (JDL 961 = - 25 mm Hg; C 2921 = - 20 mm Hg). A supcrimposablc effcct of bumetanidc was obscrved in the two strains, but at 8 times highcr daily dose (60 m g ' kg- l). These rcsults suggest that new loop diuretics can be selected for their antihypcrtcnsivc activity on the basis of their in vitro potency in inhibiting the Na +.2CI ,K+. Torascmide; Bumetanide; Cotransport; Red blood cells; Hypertension; Diuretics (loops)

1. Introduction T o r a s e m i d e a n d its derivatives r e p r e s e n t a new class o f l o o p d i u r e t i c s able to inhibit NaCI r e a b s o r p t i o n in the thick a s c e n d i n g limb o f H e n l c ' s loop ( D e l a r g e , 1978; D e l a r g e et al., 1980; W i t t n e r et al., 1987). Previous studies, c o n d u c t e d in i s o l a t e d p e r f u s e d r a b b i t cortical thick a s c e n d i n g limb tubules, s h o w e d that t o r a s e m i d e a n d s o m e o f its derivatives are a b l c to inhibit, in a d o s e - d e p e n d e n t way, the short circuit c u r r e n t , c o r r e s p o n d i n g to the rate o f c h l o r i d e r e a b s o r p t i o n , at lower c o n c e n t r a t i o n s t h a n the r e f e r e n c c c o m p o u n d , f u r o s e m i d e ( W i t t n e r et al., 1987). This activity is mainly d u e to a s t r o n g inhibition o f the

Correspondence to: B. Masereel, Department of Medicinal Chemistry. University of Liege, 3 Rue Fusch. 40(X) Liege, Belgium. Fax 32.41.221 855.

N a + , 2 C I - , K + c o t r a n s p o r t t h a n to an inhibition of the b a s o l a t e r a l C I - c h a n n e l s ( W a n g e m a n n et al., 1986). T h e possibility to study the m e c h a n i s m o f action o f loop d i u r e t i c s on a simple cell m o d e l such as the e r y t h r o c y t e has b e e n d e m o n s t r a t e d by m a n y a u t h o r s (Palfrey et al., 1980; Ellory et al., 1982; G a r a y ct al., 1990). E r y t h r o c y t e s posses a C l - - d e p e n d e n t N a + , K + c o t r a n s p o r t which is i n h i b i t e d by f u r o s e m i d e , bum e t a n i d c , p i r e t a n i d e and e t h a c r i n i c acid in the s a m e rank o f p o t e n c y as in the isolated cortical thick a s c e n d ing limb o f H e n l e ' s loop (Ellory et al., 1982). It s c e m s t h e r e f o r e i n t e r e s t i n g to e v a l u a t e the inhibitory p o t e n c y on N a + , 2 C I - . K * c o t r a n s p o r t o f a new series of t o r a s e m i d e derivatives b o t h on e r y t h r o c y t e s a n d vesicle p r e p a r a t i o n s o f m e d u l l a r y thick a s c e n d i n g limb o f H e n l e ' s loop from rat. F u r t h e r m o r e , since loop d i u r e t ics are c o n s i d e r e d as the first-line t r e a t m e n t in mild to m o d e r a t e essential h y p e r t e n s i o n , we have c o n s i d e r e d the possibility to select, on the basis of the in vitro inhibitory activity on the N a + , 2 C I - , K + c o t r a n s p o r t ,

386

new torasemide derivatives able to lower blood pressure more effcctively in those forms of essential and genetic hypertension where alterations of Na +,2C1-.K cotransport activity have bccn described (Garay et al., 1980; Bianchi et al., 1985; Canessa, 1989; Cusi et al., 1991). To approach this problcm we have uscd both for the in vitro cell studies and for the in vivo treatments an animal model of genetic hypertension, the Milan rat strain (MHS), charactcrized by a spontaneous devclopment of hypertension due to a primary increased rate of the Na + reabsorption at renal tubular level (Boberg and Person, 1986; Salvati ct al., 1987). "/'his renal defect is underlied by a faster Na+,2CI ,K + cotransport, as detected both in vesicle preparations of thick ascending limb (Ferrandi et al., 1990) and in intact erythrocytes (Fcrrari et al., 1987) of MHS when compared with their normotensive controls MNS. The cotransport alteration has been demonstrated to be primarily determined and genetically associated to hypertension (Bianchi et al., 1985). Kinetic studies performed on erythrocytes revealed that the faster cotransport rate is due to a higher affinity for the internal N a ' site (Ferrari et al., 1991). A first screening of several torasemide derivatives, including also torasemide, furosemide and bumetanide as reference compounds, was conducted by measuring both the in vivo diuretic activity on S p r a g u e - D a w l e y rats and the in vitro IC50 on the erythrocytic N a + , 2 C I - , K + cotransport of both MHS and MNS rats. On the basis of these tests and of a structure-activity relationship analysis we selected, among the others, two diuretic molecules with the lowest ICs~ for the MHS than the MNS cotransport system, that also showed specificity and selectivity in inhibiting the N a + , 2 C I ,K" cotransport of medullary thick ascending limb vesicles of both strains. The antihypertensive activity of these two molecules was tested both in MHS and in another rat strain of genetic hypertension, the spontaneously hypertensive rats (SHR), where the N a + , 2 C I - , K + crythrocytic cotransport activity has opposite characteristics as compared to MHS, that is: decreased rate and lower affinity for the internal Na ~ site when S H R were compared with the respective control strain W i s t a r - K y o t o rats (WKY) (De Mendoca et al., 1982; Rosati et al., 1988). These two hypertensive rat models, with opposite defect of cotransport, have been chosen to verify wether a specific and sensitive in vitro inhibitor of the Na ~,2CI-,K* cotransport is more effective in reducing the blood pressure increase in those forms of hypertension sustained by a hyperactivity of the cotransport system. The two torasemide derivatives selected on the basis of the in vitro screening were able to prevent the development of hypertension in both MHS and S H R at doses 8 times lower than the reference compound

bumetanide. In MHS the antihypertensive activity appeared earlier than in S H R and accounted for a 2025% reduction of systolic blood pressure as compared to a 12-15% in S H R rats.

2. Materials and methods

2.1. Chemistry All compounds were synthesized from 4-chloropyrid-3-yl sulphonamide according to general synthetic pathways previously reported (Thunus et al., 1979; Delarge et al., 1980). Their elemental analyses for C, H, N and S were within (I.4% of the theorical values. N M R and I.R. spectra were in accordance with chemical structures.

2.2. Lipophilicity 2.2.1. Shake-flask method Lipophilicity of compounds at pH 7.4 (log P ' ) was expressed as the logarithm of their partition coefficient in the n - o c t a n o l / w a t e r system by the shake-flask method (Cloux et al., 1988). 2.2.2. Recersed phase high performance liquid chromatography (RP-HPLC) A R P - H P L C system was used for the determination of the log P' of all drugs. A reversed phase column (RP-18) was brought to equilibrium with n - p r o p a n o l / phosphate buffer pH 7.4 (30:70). Compounds were dissolved and eluted with the same solution. A series of standards with a wide range of lipophilicity determined by the shake-flask method was run and a calibration curve was established for each session. KNO.~ was injected to determine the void volume. Log k', determined for each sample, is defined as log (t r L~)/to where tr is the retention time of the drug and t 0 the retention time of KNO.~. The calibration curve was calculated from log P' and log k' of standards: log P' = (2.522 x log k ' ) + 0.5806; n = 6; r = 0.995. Log P' of other compounds was interpolated. 2.3. In t'itro tests 2.3.1. Erythrocyte ouabain-insensiti t'e S°Rb uptake Fresh blood (10 ml) from both MHS and MNS rats was collected in heparinized tubes, centrifuged, and the plasma removed. The red cell pellet was washed 4 times with an ice-cold K÷-free physiological solution. The washed hematocrit of thc erythrocyte pellet was approximately 90%. Aliquot (1 ml) of this pellet was preincubated for 30 min at 37°C in the presence of 1 mM ouabain and different concentrations of the tested compound from 10 ~ to 10 -4 M at a final hematocrit

387

23.2. Ouabain-insensitice 86Rb uptake in medullary thick

of 15%. After the preincubation period, 3 # C i / m l 86Rb and 5 mM KCI (final concentration) were added to start flux measurements. Aliquots of cells were taken in duplicate, at 10 and 45 min, and separated from the medium by layering the samples on phtalate oil and spinning them for 10 s in a microfugc. The tips of the tubes were sliced off, mixed with water to suspend the pellet, and counted for 86Rb content. The S~'Rb uptake, at different drug concentrations, was expressed as the difference between the counts at 45 and 10 rain, each of them corrected for their hemoglobin content (cpm ~"Rb/Hb). Control samples were performed with and without (1.8 mM bumetanide, a saturating concentration. IC5o of the derivatives was calculated according to the non-linear least squares program and expressed in /xM.

ascending limb cesicles Vesicles from medullary thick ascending limb of Henle's loop were prepared from 2-month-old rats (MHS and MNS) of 200-220 g body weight according to the method previously described (Burnham et al., 1985; Burnham et al., 1986; Ferrandi et al., 1990). For all drugs, the dose-dependent inhibitory potency of ouabain insensitive X~'Rb fluxes was determined using the method reported (Cusi et al., 1981). These experiments included a control sample, both for MHS and MNS vesicles, in absence (total ouabain-insensitivc ~¢'Rb uptake) and in presence of 0.8 mM bumetanide, a concentration that has been demonstrated to fully inhibit the Rb uptake into renal vesicles of both strains (Fcrrandi et al., 1990).

TABLE 1 Lipophilicity (log P'), minimal efficient diuretic dose (mg.kg-~) and concentration of Rl-substituted compounds required for half maximal inhibition of the ouabain insensitive e~'Rb uptake (IC5o, # M ) o n MNS and MHS erythrocytes. Mcans+S.E.

No.

Internal No.

RI

Log P'

Min. diuretic

ICso (#M)

dose

n

MNS

+ 0.45

1.0

14.11/+ 1.4

[/- 1] /V-.x

+ 1.13

3.2

28.80

28.40

['/

+ 0.28

3.9

17.9(I

18.10

+ (I.79

2.9

8.50

11.20

+ 0.84

4.5

17.20

11.90

- 0.28

3.2

13.0tl

32.00

+0.09

1.8

6.3(I

5.30

+0.68

1.3

3.3(I

2.10

+ 1.(14

1.1

1.20

0.94

+0.61

1.5

3.30 +_0.7

1.70_+1/.19

MIlS

R1

\

NH

O

Ctl 3

S O , N I t - - C - - NI I - - C H - - C H I

1

~

Torasemide

"

. ~

7.80 ± l.(I a

CH 3 2

JDL 414 CF3

3

JDL 466

]l

F 4

JDL413

5

JDL 468

('1 -2 ~ v ~ If

]1

Br 6

JDL 882 k

7

JDL 861

~"'~

8

JDL 888

(

9

JDL 941

(

10

C 2921

~']

;' P

<

0.01.

~.~

"~,..

388

2.4. In t,it:o tests 2.4.1. Diuresis Male S p r a g u e - Dawley rats (140-160 g) were housed in plastic metabolism cages (three r a t s / c a g e ) and urine collected for 4 h. Logarithmically spaced doses wcrc administered orally in a dose volume of 10 m l - k g (salinc solution containing 0.1% of sodium carboxymcthylcellulose). Simultaneously 30 ml. kg -~ of saline solution were injected s.c. For each compound, regression analysis of urinary volume and administered doscs led to establish the minimal cfficient diuretic dose, which corrcsponds to diuresis induced by 1 m g . k g - ~ of torasemidc. 2.4.2. In cico antihypertensice treatment Chronic treatment was conducted for 5 weeks in malc rats of Milan (MHS) and O k a m o t o ( S H R ) strains. At thc beginning of treatment the rats were 4 weeks old. At that age, both strains can bc considered in a prehypertensive phase. Four groups of nine animals were formcd for cach strain and subdivided into groups of three rats for each cage. Bumetanidc (60 mg • kgper day), J D L 961 (7.5 m g . k g - t per day) and C 2921 (7.5 m g . kg- ~ per day) were given in tap water. These doses were chosen because they are the lowest doses producing the maximal natriuresis and diuresis in S p r a g u e - D a w l e y rats, as verified in preliminary experiments (data not shown). Two days before starting treatment and every two days after, the 24-h water consumption of each cage was measured in order to calcu-

late the right amount of compounds to be dissolved in tap water to achieve the required daily doses for each rat. The control received only tap water. Body weight was recorded every two days. Systolic blood pressure and heart rate were recorded by the tail cuff method before starting daily treatment (basal values). Systolic blood pressure and heart rate were then recorded once a week at 10 a.m. for 5 weeks during chronic treatment and 1 week after ending treatment (recovery) in order to cvaluatc the active duration period of the drug possible inhibitory effect on hypertension development. Results wcrc cxprcsscd as means + S.E. Differences between treated and control rats were evaluated by means of a two-way analysis of variancc followed by Dunnett's tcst.

3. Results 3.1. Diuresis

As shown in tables 1 and 2, the minimal efficient diuretic dose of all Rl-cycloalkyl substituted compounds (6-14), decreases with the RI C-atom number and is positively correlated with the IC5~~ on erythrocyte ouabain-insensitive Rb uptakc of MNS (P < 0.001) and MHS (P = 0.005) (fig. 1). For meta-toluyl substitutcd derivatives listed in tables 1 (1-5) and 3 (15-22), no correlation was found bctwccn crythrocytic ICs0 and diuretic activities. This could bc duc to differences in absorption a n d / o r drug metabolism.

TABLE 2 Lipophilicity (log P'). minimal efficient diuretic dose (mg.kg i) and activity on MNS and MHS erythrocytes of Rl- and R2-substituted compounds related to torasemide. The 1Cs. values represent drug concentration (~M) for half maximal inhibition of the ouabain insensitive S~'Rb uptake. Means + S.E. No.

Internal No.

RI

Lx~gP'

Min. diuretic dose

IC~. (/,tM)

n

MNS

MIlS

RI

\

NH

0 R2

11

JDL 960

~

- 1.3(I

4.9

16.00

26.(XI

12

JDL 959

~

-(I.22

1.1

7.7(I

8.70

13

JDI. 1035

~

+ 0.41

0.5

3.76 _+1.5

3.00 + 0.04

14

JDL 961

~

* 11.69

< 0.1

1.30 _+0.2

1.611 _+0.3

389 2

b. o

1

14

0

13~l.i

6

-1

[]

-2

'

7

'5

-6

log

-'4

-

IC50

9

MNS

[M]

8

b.

ol o

0

13 ~

61_1 11

-1

[]

-2

log

Ic50

MHS

[MI

Fig. 1. Rl-cycloalkyl substituted compounds. Correlation between lipophilicity (log P ' ) and crythrocytic cotransport inhibitory potency (1Cs.) of normotensive (MNS) and hypertensive (MHS) rats ( P < 0.001 ).

3.2. In citro study Tables 1-4 show the ICs0 of the torasemide derivatives and the reference compounds, bumetanide and furosemide, on the erythrocyte ouabain-insensitive Rb uptake of MHS and MNS rats. Most of the synthesized compounds were more active than furosemide but less active than bumetanide (table 4). A significantly lower ICso for torasemidc (P < 0.01) (table 1) and furosemide (P < 0.05) (table 4) was found in MHS than in MNS erythrocytes, but not for bumetanidc (table 4). No significant selectivity of other torasemide derivatives was observed between MHS and MNS ouabain-insensitive Rb uptake. Since the cotransport inhibitory potency of bumetanidc, furosemide and torasemide is reported to be quite different depending on the type of tissue and species considered (Haas and McManus, 1982; Jonhson et al., 1982; Haas and McManus, 1983; Schlatter et al., 1983; Wittner et al., 19871, the cotransport activity, expressed as ouabain-inscnsitive, bumetanide-sensitive Rb uptake, was also determined in vesicles from medullary thick ascending limb of Henle's loop of the two strains. As previously reported (Ferrandi et al., 199(I), we confirmed that the bumetanide-sensitive Nat,2CI-,K* cotransport activity was higher in MHS than MNS renal vesicles (MHS: 491 + 35 vs. MNS:

339 _+48 pmol Rb/min per mg; P < 0.05). Moreover, both torasemide (P < 0.01) and bumetanide (P < 0.05), but not furosemide, were more active in inhibiting MHS than MNS renal vesicles cotransport (table 5). In particular, the affinity of bumctanide for the renal vesicle cotransport was 10(KI times lower than for erythrocytes, whilst the affinities for torasemide were comparable. One possible explanation of this difference can be the quite artificial intravesicular ionic composition compared to the physiological condition of erythrocytes which might affect the binding affinities of the cotransport inhibitors. Two torasemide derivatives, C 2921 and JDL 961, selected for their diuretic potency and activity on the erythrocyte cotransport, were also tested on medullary thick ascending limb vesicles (table 5), showing comparable affinities as those observed on erythrocytcs. Moreover, C 2921 and JDL 961 showed at least 4-8 times lower 1Csn than torasemide and about 50-60 times lower ICso than bumetanide and furosemide for the renal cotransport of both strains. No selectivity between MHS and MNS cotransportcr was detected, while bumetanide and torasemide showed an approximatively 2 times higher IC50 value for MNS than for MHS.

"r" E 60 E

40' e,i

o

o

20-

u o

o

lltl

._= E e,t

$ or"

07

=

-r

- 40

I 2

Treatment I 3

i 4

I 5

I 6

WEEKS

Fig. 2. C h r o n i c treatment of male hypertensive rats (MHS). Effect on systolic blood pressure and heart rate. Means +S.E.; ** P < 0.001: * P < 0.05; nine rats for each group. (C3) Control (tap water); ( a ) bumetanide (60 m g . k g i per day); ( l l ) J D L 961 (7.5 m g . k g i per day); ( o ) (72921. (7.5 m g . k g i per day).

3911 TABLE 3 R2-substituted compounds, lipophilicity (log P'), minimal efficient diuretic dose (mg.kg ' l ) and biological activity on MNS and MHS erythrocytes. The IC50 values represent drug concentration (~M) for half maximal inhibition of the ouabain insensitive ~'Rb uptake. Means+ S.E. No.

Internal No.

R2

Log P'

Min. diuretic dose

IC5o (IzM) MNS

n MHS

R1

ICH3" ~ N I I

15

JDL 494

0

C! 13-- NH - -

71.1.011

42.311

1

- 11.21

7.6

+ 0.06

2.7

> 101)

> 101)

2

+ 1.12

3.3

> 100

> 100

2

( ) •----N

+ 0.45

1.8

8.80

6.20

2

+ 0.88

1.2

2.711

1.50

2

+ 1.21

4.6

2.70

3.30

2

+ 1.39

4.3

1.80

4.98

2

- 0.13

1.9

5.90

7.5O

2

Ct 13\ 16

C 2785

N--

/ CH3

C2H5\ 17

C 2973

N-C2H5 /

18

C 2744

\

19

C 2776

F

211

C 2777

(

21

C 2979

CH 3- - C N

22

C 2770

3.3.

],

~

~I

,~,

N

Structure-activity relationships

Taking into account the results on diuresis and

erythrocyte Rb fluxes, the structurc-activity study in-

volved the comparison of torasemide derivatives with the main substitution in R1 a n d / o r R2 position. In R1 position (table 1), replacing - C H 3 with - C F 3 or a halide generates either equally or less potent com-

TABLE 4 Lipophilicity (log P') and activity of bumetanide and furosemide on MNS and MHS erythrocytes. The IC5o values represent drug concentration (tzM) for half maximal inhibition of the ouabain insensitive 86Rb uptake. Mean _+S.E. No.

Log P'

IC5o (p.M) MNS

n MHS

C4 H g - - NH ~ , , , C O O H 23

Bumetanide

- 0.30

0.30 ± 0.11

0.14 + 0.3

5

12.40 +_ 1.3 a

4

SO2NIt 2

24

Furosemide

~ _ , , / / C I t 2~ N H ~---() ~ C O O H

~LJ

CI" y

SO2NH2 ~' P < 0.05.

- (I.76

29.70 + 5.3

391

pounds: - C H 3 = - E l > - B r > - F > - C F 3. The trend observed throughout the Rl-cycloalkyl substituted compounds listed in tables 1 and 2 is that increasing the C-atom number enhances the inhibitory potency. Optimum affinity was obtained for a 8-membered ring in this position: C8 > C7 > C6 > C5. Indeed JDL 941 and J D L 961 with a cyclooctyl Rl-moicty arc 10 times more active than torascmide. For R2-substitutcd compounds, aminoethyl (table 2) or aminoisopropyl (table 1) seems to bc the best short aminoalkyl residues. The aminomethyl mocity (JDL 494) strongly decreases the biological response. The blending of the distal sulfonylurca nitrogen with a 6- or 7-membcrcd ring (table 3) increases the activity (C 2776, C 2777, C 2779, C277(1) whereas disubstitution of this nitrogen with two short alkyl residues (C 2785, C 2973) deletes the crythrocytc eotransport affinity in spite of a diuretic activity. Moreover, a correlation was found between the hydrophobicity of all active torasemide derivatives and their potency to inhibit the MNS (P = 0.038) or MHS (P = 0.(1241 erythrocytie cotransporter. These correlations become excellent (fig. 1) if Rl-cycloalkyl substituted compounds only arc considered (P < 0.001). 3.4. In ciz'o study The effect of C 2921 and J D L 961 on blood pressure in two strains of spontaneously hypertensive rats (MHS and SHR) was performed by treating the rats for 5 weeks at the daily oral dose of 7.5 m g - k g - ' . Bumetanide was chosen as reference compound and orally tested at 60 mg. kg- ~. These doses were chosen because of their ability to induce maximal diuresis and natriuresis in preliminary experiments. 3.4.1. Effect of long-term treatment on MItS rats Four-week-old MHS, at a prehypertensive stage, started to develop hypertension at the beginning of the experiment. Figure 2 shows the increases of systolic blood pressure and heart rate (expressed as ..4 values

TABLE 5 Concentration ( # M ) required for a 5(}% inhibition of the ouabain-insensitive X"Rb uptake in medullary thick ascending limb vesicles of MNS and MHS rats. No.

1 Torasemide 23 Bumetanide 24 Furosemide 10 C 2921 14 JDL 961 " P < 0.01; h p < 0.05.

IC5~, (p.M)

n

MNS

MHS

31.1 + 2.9 206 _+21 155 _+47 3.8 1.2

13.6+ 2.2 " 145 + 18 h 151 __+42 4.0 1.8

3 8 6 2 2

290 250 " /

210 170 0 ¢1

130 " 90-

8

50-

40

~

30 ~

c¢ -Z.a

z8

s~ ss

s

-¢c~ 2 0

o~

~0 Treatment 1 B

1 1

l 2

I 3

I 4

I 5

I 6

WEEKS

Fig. 3. Chronic treatment of male MIlS rats. Effect on body weight and water intake. Means_+ S.E.; ** P < 0.(101; * P < 0.05; nine rats for each group. ([3) Control (tap water); (z~) bumetanide ((31) mgkg- i per day); ( • ) JDL 961 (7.5 rag- k g i per day): (2~) (.'2921 (7.5 mg.kg I perday).

from the pretreatment basal values) of the four groups of MHS, treated as previously described. Systolic blood pressure of control rats increased during the 5 weeks of observation from 124.4 _+ 2.7 (mm Hg) to 162.2 _+ 3.5 (mm Hg). Daily treatment with bumetanide, J D L 961 and C 2921 effectively reduced the increase in systolic blood pressure, especially in the third and fourth weeks of observation while in the fifth week this effect was no longer statistically significant. After the recovery period the systolic blood pressure returned to the control values. The reduction in systolic blood pressure caused by JDL 961 and C 2921 was comparable to that of bumetanide. Throughout the treatment, heart rate showed no statistically significant difference among the four groups. Figure 3 shows the ponderal curves and the amounts of 24-h water intake per 100 g body weight for the four groups of MHS. It can be seen that the animals treated with the diuretics showed a statistically significant reduction of body weight compared to the controls, throughout the observation period. Moreover, the 24-h water consumption significantly increased in the treated animals, especially at the beginning of the experiment and returned to the control values after the recovery period.

392

3.4.2. Lffect of long-term treatment on S t t R rats Four-week-old S H R were still dcvcloping hypertension. Thc cffccts of bumetanide, J D L 961 and C 2921 on systolic blood pressure and heart rate are shown in fig. 4. Systolic blood pressure of control rats progressively increased throughout the observation period (from 136.1 + 2.9 to 191.7 + 3.7 mm Hg). Daily trcatment with the three diurctics became effectively lowered systolic blood prcssurc only during the fourth and fifth weeks of obscrvation. Then, during the recovcry period, systolic blood pressure returned to thc control values. The hypotcnsivc effect of the three compounds was comparable. Heart rate decreased with age in control and C 2921 trcatcd groups whereas it did not change throughout the observation period in the bumetanidc and J D L 961 treated groups and was higher than in the control rats. As shown in fig. 5, the pondcral curvcs of the S H R rats treated with J D L 961 and C 2921 can superimpose those of the controls while the bumetanide treated group started with a higher body weight in basal condition and maintained this difference for all the observation period. Figure 5 also indicates the 24-h water consumption. It can be seen that both J D L 961 and C

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