5 Diuretic and Uricosuric Properties of Tienilic Acid (Ticrynafen) in Mice, Rats, and Anesthetized Beagle Dogs Diuretic Agents Downloaded from pubs.acs.org by EAST CAROLINA UNIV on 11/13/16. For personal use only.
Antihypertensive Activity in SH Rats and Structure—Activity Relationship P. BESSIN, J. BONNET, M. F. MALIN, C. JACQUEMIN, N. D E BREZE, I. PELAS, L . DESGROUX, B. AGIER, and M. DUTARTE
Albert Rolland Center for Research and Pharmacology (CREPHAR), 4, Rue de la Division Leclerc, 91380 Chilly-Mazarin, France
In the last twenty years, two series of compounds have been discovered which exhibit marked diuretic and saluretic a c t i v i t y . These are (a) the phenoxyacetic acids, the most prominent member being ethacrynic acid (1,2, Figure 1) and (b) the sulfamoylbenzoic acids, the most potent being furosemide (3) and bumetanide (4,5). The latter compound differs from furosemide in several respects, the most obvious being that the 5-chloro substituent is replaced by phenoxy. These very potent natriuretic agents are sufficiently effective to satisfy the physician for use in the treatment of hypertension and cardiac diseases; however, they possess unwanted side effects which include potassium depletion, a diabetogenic propensity (6,7,8,9) and hyperuricemia (10-23). To solve the problem of potassium depletion, a search for new diuretic agents led to the discovery of compounds which block the tubular sodium-potassium exchange either directly, i.e., triamterene (24) and amiloride (25), or by blocking the action of aldosterone, i.e., spironolactone. In regard to diuretic-induced hyperuricemia, the discovery in 1967 of t i e n i l i c acid was a major contribution to diuretic therapy. This compound, (2% 3-dichloro-4-(2-thienylcarbonyl)phenoxy7acetic acid, is an aryloxyacetic acid analog of ethacrynic acid which exhibits diuretic and uricosuric activity in mice, rats and dogs (28), as well as in man (29,30,31). More recently, another aryloxyacetic acid, an indanone (32,33,3^,35), has been reported to possess potent diuretic and uricosuric activity in chimpanzees and i n man. The purpose of the studies which we w i l l describe was to evaluate the diuretic and uricosuric effects of t i e n i l i c acid in mice, rats and dogs and the antihypertensive properties in SH rats preliminary to c l i n i c a l t r i a l s . METHODS Diuretic and Uricosuric Activity in Mice (36) - I n i t i a l screening 0-8412-0464-0/78/47-083-056$07.25/0 © American Chemical Society
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Tienilic Acid in Mice, Rats, and Beagles
ETHACRYNIC ACID
BENZIODARONE
Figure 1.
Compound structures
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of d i u r e t i c agents were performed using groups o f 6 male Swiss mice, weighing 22 + 1 g, randomly s e l e c t e d and d i s t r i b u t e d i n p a i r s i n t o metabolism cages f o r 2 o r 4 hours. Food and water were withheld f o r 2 hours p r i o r to the experiments, u r i n e was c o l l e c t e d at 2 and 4 hours and the urine volumes and e l e c t r o l y t e s measured. A l l mice were administered 1 ml o f 0 . 9 Î s a l i n e o r a l l y and e i t h e r simultaneously t r e a t e d o r not ( c o n t r o l group) with d i u r e t i c agents. Test compounds ( t i e n i l i c a c i d , e t h a c r y n i c a c i d and furosemide) were d i s s o l v e d o r suspended i n the same 0 . 9 Ï s a l i n e and administered by gavage. Four groups r e c e i v e d t i e n i l i c a c i d o r a l l y a t doses o f 20, 50, 100 and 200 mg/kg and were compared to furosemide a t 20 mg/kg, e t h a c r y n i c a c i d a t 20 mg/kg and benziodarone (a potent n o n - d i u r e t i c u r i c o s u r i c agent) a t 100 mg/kg by the same route. F o r the g e n e r a l s c r e e n i n g o f d i u r e t i c and u r i c o s u r i c drugs, such as t i e n i l i c a c i d and i t s congeners, the compounds were administered a t doses o f 100 mg/kg o r a l l y i n comparison t o f u r o s e m i d e o r e t h a c r y n i c a c i d a t 20 mg/kg. Na and Κ analyses were determined u s i n g the flame photometer (Eppendorf), c h l o r i d e s by m i c r o a n a l y s i s (37) and u r i c a c i d by the enzymatic spectrophotometric technique (3SJ or by a colôrimetric method (.29). S t a t i s t i c a l a n a l y s i s was made by a p p l i c a t i o n o f Student's t t e s t w i t h simultaneous c a l c u l a t i o n o f means (M) and standard d e v i a t i o n o f the mean (S/VrT). +
T i e n i l i c Acid-Benziodarone and Furosemide-Benziodarone R e l a t i o n s h i p s i n Mice - Groups o f 6 mice weighing 22 + 1 g were admini s t e r e d simultaneously e i t h e r 100 mg/kg o r 200 mg/kg o f t i e n i l i c a c i d , o r 200 mg/kg o f benziodarone, o r 20 mg/kg o f furosemide o r a combination o f the same doses o f t i e n i l i c a c i d + benziodarone o r furosemide + benziodarone. A f t e r dosing, a l l animals were d i s t r i b u t e d by p a i r s i n t o metabolism cages f o r 2 hours. Urine samples were c o l l e c t e d f o r the determination o f e l e c t r o l y t e and u r i c a c i d e x c r e t i o n using the same a n a l y t i c a l methods as p r e v i ously d e s c r i b e d . The s t a t i s t i c a l s i g n i f i c a n c e was based on a comparison o f t i e n i l i c a c i d + benziodarone or furosemide + benziodarone versus t i e n i l i c a c i d alone, furosemide alone o r benziodarone alone (Student's t t e s t ) . D i u r e t i c A c t i v i t y i n Rats - Groups o f 3 animals weighing 400 + 5 g, deprived o f food and water o v e r n i g h t , were given 20 ml/kg o f water by i n t u b a t i o n on the day preceding the experiment. A f t e r dosing, the animals were placed i n metabolism cages f o r the c o l l e c t i o n o f a 5 hour sample o f u r i n e . D i u r e t i c drugs were administered as i n the previous experiment i n a volume o f 20 ml/kg of 0.9% s a l i n e a t the beginning o f the experiment, while a c o n t r o l group was administered only the s a l i n e s o l u t i o n . Statistical s i g n i f i c a n c e r e f e r s to a comparison between t r e a t e d and c o n t r o l r a t s (Student's t t e s t ) . Dose-response r e l a t i o n s h i p s are i l l u s t r a t e d i n F i g u r e 5 u s i n g the r e g r e s s i o n l i n e procedure.
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Phenol-Red Retention Test i n Rats (40,41) - Groups o f 5 randomized male r a t s , weighing 180 + 2 0 g, were given the t e s t compounds o r a l l y 30 minutes before the intravenous i n j e c t i o n o f 75 mg/kg o f phenol-red as a 1.5% s o l u t i o n i n 0.9% sodium c h l o r i d e . Heparini z e d blood samples were taken from the r e t r o - o r b i t a l plexus a t 15, 30, 45 and 60 minutes; c o l o r was developed by the a d d i t i o n o f 0.05 ml o f 0.1 M sodium hydroxide and read a t 540 my on the s p e c t r o c o l o r i m e t e r (Eppendorf). R e s u l t s were c a l c u l a t e d as percentage o f v a r i a t i o n i n r e l a t i o n to the c o n t r o l value. S t a t i s t i c a l s i g n i f icance was c a l c u l a t e d by comparison with t r e a t e d and c o n t r o l r a t s (Student's t t e s t ) . A n t i h y p e r t e n s i v e A c t i v i t y i n Spontaneous Hypertensive Rats (Wistar-Okamoto S t r a i n ) - The s t u d i e s were performed i n Wistar hypertensive male r a t s which were approximately 25 weeks o l d and randomly placed i n groups o f s i x animals. The s y s t o l i c blood pressure was determined by the t a i l c u f f technique (Physiograph Narco System) before treatment and a t 2, 5 and 24 hours a f t e r each d a i l y a d m i n i s t r a t i o n o f the drug o r the v e h i c l e , i n acute (1 day) and i n subacute experiments (8 days). In c h r o n i c s t u d i e s , the blood pressure was measured i n d i r e c t l y before and 1.5, 3·5, 6, 12 and 18 months a f t e r the beginning o f the experiment. In the l a s t study, t i e n i l i c a c i d was mixed with the d a i l y food a t a l e v e l c a l c u l a t e d to provide an o r a l intake o f 100 and 200 mg/kg. In the acute o r a l experiments, the r a t s (N = 6) were given 100, 200 and 400 mg/kg o f t i e n i l i c a c i d ; i n the subacute e x p e r i ments, the d a i l y o r a l dose was 200 mg/kg. S t a t i s t i c a l a n a l y s i s was determined by Tukey's m u l t i p l e comparisons procedure which permits the s t a t i s t i c a l c l a s s i f i c a t i o n o f means between themselves or compared to c o n t r o l data. In F i g u r e 17, s t a t i s t i c a l s i g n i f i cance i s i l l u s t r a t e d by the a d d i t i o n of a broken l i n e to the main s o l i d l i n e . Each p o i n t represents a mean value, and the v e r t i c a l bars i n d i c a t e the standard e r r o r o f the mean. In the chronic study, s t a t i s t i c a l s i g n i f i c a n c e was c a l c u l a t e d by Student's t p a i r e d t e s t . In a l l the experiments, the blood pressure was measured i n d i r e c t l y i n a c o n t r o l group o f SH r a t s under the same c o n d i t i o n s ^ I n a d d i t i o n , i n the c h r o n i c study, the t r e a t e d SH r a t s were compared to c o n t r o l Wistar normotensive r a t s . T r i g l y c e r i d e - L o w e r i n g E f f e c t i n Obese Rats ( F a t t y S t r a i n ) Measurement o f the t r i g l y c e r i d e - l o w e r i n g e f f e c t was c a r r i e d out i n groups o f 5 to 7 obese male r a t s ( F a t t y s t r a i n ) and compared to normal heterozygous r a t s as a f i r s t c o n t r o l group and to normal Wistar r a t s as a second c o n t r o l group. Obese r a t s were randomly d i s t r i b u t e d i n t o two groups o f 5 to 7 animals which were given e i t h e r 200 mg/kg o f t i e n i l i c a c i d as an o r a l d a i l y dose f o r 7 days or the v e h i c l e alone. The other c o n t r o l groups (heterozygous r a t s and normal Wistar r a t s ) were given only the f l u i d v e h i c l e . Blood samples were taken a t the end o f the
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experiment from the r e t r o - o r b i t a l plexus i n order to determine plasma glucose, t o t a l l i p i d s , c h o l e s t e r o l and t r i g l y c e r i d e l e v e l s a f t e r d a i l y treatment with t i e n i l i c a c i d . Statistical signifi cance was c a l c u l a t e d i n comparison with t r e a t e d and c o n t r o l r a t s (Student's t t e s t ) . D i u r e t i c , u r i c o s u r i c and Clearance S t u d i e s i n Anesthetized Beagle Dogs under Hydropenic C o n d i t i o n s - The d i u r e t i c , u r i c o s u r i c and clearance s t u d i e s were performed i n beagle dogs weighing 10 t o 13 kg which had been f a s t e d overnight and then a n e s t h e t i z e d by i . v . i n j e c t i o n o f mebubarbital, 30 mg/kg. During the experiment, the dogs were i n t r a v e n o u s l y i n f u s e d a t a r a t e o f 2 ml/minute w i t h 0.6% s a l i n e , 0.08% PAH, 0.4% c r e a t i n i n e and mebubarbital, 5 mg/kg/hour u s i n g a constant r a t e i n f u s i o n pump. A f t e r m e s i a l laparotomy and c a t h e t e r i z a t i o n o f the u r e t e r s , each study s t a r t e d with a c o n t r o l p e r i o d o f two hours. Blood and u r i n e samples were c o l l e c t e d a t 15 minute i n t e r v a l s . Then, the l y s i n e s a l t o f t i e n i l i c a c i d was i n j e c t e d i n t r a v e n o u s l y a t 5, 10 and 20 mg/kg. A group o f 4 t o 6 beagle dogs was used f o r each dose and f o r the c o n t r o l (placebo) e v a l u a t i o n . Sodium and potassium determinations were made u s i n g the Eppendorf Flame Photometer, c h l o r i d e by the c o l o r i m e t r i c micromethod (37) and urate by the enzymatic procedure (38). Other measurements and c a l c u l a t i o n s which were made are as f o l l o w s : PAH (42), c r e a t i n i n e (43), urea (44), pH, blood pressure, osm ( o s molar c l e a r a n c e ) a f t e r c c y o s c o p i c determination (using the f r e e z i n g p o i n t d e p r e s s i o n ) , T-HpO ( f r e e water reabsorption) and Τ H 0 = osm - g (ml/min). The osm - Τ H O r e l a t i o n s h i p was studied u s i n g Τ HpO c o r r e c t e d f o r osmolar clearance, and c r e a t i n i n e c l e a r ance as p r e v i o u s l y described (45): Τ Η 0 / osm. A l l the c l e a r 2
ρ
creat. ance s t u d i e s were determined using standard procedures. S t a t i s t i c a l s i g n i f i c a n c e was c a l c u l a t e d as p r e v i o u s l y described by the a p p l i c a t i o n o f Tukey's t e s t (Section 5 ) . Data are i l l u s t r a t e d i n v a r i o u s f i g u r e s as the mean values and the standard e r r o r o f the mean ( v e r t i c a l bars) o r as percentage o f c o n t r o l values. RESULTS D i u r e t i c A c t i v i t y i n Mice - As shown i n Figure 2, the o r a l admin i s t r a t i o n o f t i e n i l i c a c i d (20, 50, 100, 200 mg/kg p.o.) gave a s i g n i f i c a n t i n c r e a s e i n u r i n e volume and e l e c t r o l y t e e x c r e t i o n . However, i n comparison with furosemide o r e t h a c r y n i c a c i d , t i e n i l i c a c i d i s about 1/5 to 1/10 as potent. Nevertheless, contrary to other d i u r e t i c and n a t r i u r e t i c agents, t i e n i l i c a c i d i n c r e a s e s urate e x c r e t i o n . The u r i c o s u r i c a c t i v i t y o f t i e n i l i c a c i d i n mice i s doser e l a t e d , as shown i n F i g u r e 3, i n comparison with that o f f u r o s e mide and e t h a c r y n i c a c i d i n e f f e c t i v e d i u r e t i c doses. I n the same experiment, benziodarone, which possesses s i g n i f i c a n t u r i c o s u r i c
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