Life Sciences, Vol. Printed in the USA
51, pp. PL 183-187
Pergamon Press
PHARMACOLOGY LETTERS Accelerated
HYPOTENSIVE
Communication
ACTIVITY OF TCV-116, A NEWLY DEVELOPED
RECEPTOR
ANTAGONIST,
IN SPONTANEOUSLY
ANGIOTENSIN
HYPERTENSIVE
II
RATS
Kenji Mizuno, Susumu Niimura, Makio Tani, Ikue Saito, Hironobu Sanada, Michihiko Takahashi, Koichiro Okazaki, Manabu Yamaguchi and Soitsu Fukuchi Third Department of Internal Medicine, Fukushima Medical College, Fukushima 96012, Japan (Submitted July 14, 1992; accepted July 16, 1992; received in final form September ii, 1992)
Abstract: TCV-116, a recently developed angiotensin II (Ang II) receptor antagonist, was administered orally (1 mg/kg per day) to 10-week-old spontaneously hypertensive rats (SHR) for 2 weeks . Blood pressure and plasma components of the renin-angiotensin-aldosterone system were determined in these rats. TCV-116 produced a marked reduction in blood pressure without altering heart rate. Whereas plasma renin concentration (PRC), angiotensin I (Ang I) and angiotensin II (Ang II) all were significantly increased, plasma aldosterone was decreased by approximately 70% compared with control animals. These results not only indicate therapeutic efficacy of this agent in the chronic treatment of human hypertension, but support also the concept that the renin-angiotensin system plays an important role in the control of blood pressure in this animal model of human essential hypertension.
Introduction Although the renin-angiotensin system (RAS) has long been recognized as an important factor in the pathophysiology of several forms of hypertension, the contributory role of this system in the initiation and/or development of hypertension of spontaneously hypertensive rats (SHR) has been mitigated because of normal or even low levels of plasma renin. Progress in drugs which interfere with the RAS at levels of renin and angiotensin converting enzyme (ACE) has indicated the importance of this system in the maintenance of hypertension, inasmuch as these agents actually reduce blood pressure of SHR (1-3). Nonetheless, their mechanism of action has not fully been clarified. They may lower blood pressure by suppressing the generation of angiotensin II (Ang If) in either the circulation or vascular tissues (4-6). Others, however, have suggested that ACE inhibitors lower blood pressure by decreasing sympathetic tone (7), or by elevating the levels of kinins (8) and/or eicosanoids in vascular walls (9). Recently, a new class of highly specific, competitive Ang II receptor antagonists has been described (10,11). Of these compounds, Losartan (known as DuP 753) blocks the vasoconstrictor actions of Ang lI and lacks agonistic or kinin- and prostaglandin-inducing properties (12). More recently, Losartan has also been shown to reduce blood pressure in SHR (12,13), findings suggestive of the contributory role of the RAS in the maintenance of hypertension of this animal model of human essential hypertension. TCV-116, ((-+)-••(cycl•hexy••carb•nyl•xy)ethyl-2-eth•xy-1-[[2'-(•H-tetraz•l•5-y•)bipheny•-4-yl]I H-benzimidazole-7-carboxylate), is a newly synthesized benzimidazole derivative, which possesses potent antagonistic properties highly specific to Ang II receptors in vascular smooth muscle and adrenal glomemlosa cells (14). This agent is a prodrug which is metabolized to an active form (CV-11974) to exert its biological effects (14). However, little is known about the antihypertensive and hormonal activity of this agent in SHR at present. Thus, the purpose of the present study was to examine these pharmacological properties of TCV-116 in SHR and then address the significance of the RAS in pathophysiology of hypertension of SHR.
Correspondence to: K. Mizuno, Third Department of Internal Medicine, Fukushima Medical College, 1 Hikari-ga-oka, Fukushima 96012, Japan. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
PL-184
TCV-II6
in SHR
Vol.
51, No. 20,
1992
Materials and Methods Male SHRs (10-week-old) were maintained on a regular chow diet containing 0.39% sodium and 0.9% potassium and allowed free access to tap water. The rats were divided into two groups and were administered TCV-116 (n=8) or tap water as the control (n=8). TCV-116 was given orally to the rats at a dose of 1 mg/kg/day in a suspension of gum arabic in a volume of 1 ml/kg of water at 4:00 p.m. for 2 weeks. The drug suspension was made up freshly every day. Body weights were determined and systolic blood pressure as well as heart rate was measured with an ultrasound device (UR 5000, Ueda Co., Ltd., Tokyo) before and 1 and 2 weeks after treatment. After 2 weeks of the treatment, rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Blood (6 ml) from the control and TCV-116-treated SHRs was withdrawn by aortic puncture. About 5 ml of the blood were collected into pre-cooled tubes containing Na2-EDTA, o - p h e n a n t h r o l i n e and phenylmethylsulfonyl fluoride (PMSF) at final concentrations of 5 mM, 2 mM and 10 mM, respectively, for determinations of renin concentration, Ang I, Ang II and aldosterone. After centrifugation at 2,500 x g for 30 min at 4°C, the plasma was aspirated and stored at -20°C until assay. The remaining blood was used for determination of serum ACE activity. Plasma immunoreactive Ang 1 and Ang 11 were measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridges (Waters, Milford, MA), as described previously (15,16). In brief, plasma (1 ml) was applied to the cartridges, which had been washed with methanol (3 ml) followed by 0.1 M Tris-acetate buffer, pH 7.4 (10 ml). After washing with distilled water, peptides were eluted with a mixture of methanol/distilled water/trifluoroacetic acid (TFA) (90:9.9:0.1, vol ratio). The extracts were evaporated to dryness in vacuum centrifuge and the residue was redissolved in assay buffer consisting of 0.1 M Tris-acetate containing 2.6 mM Na2EDTA, 1 mM PMSF, and 0.1% bovine serum albumin, pH 7.4, for RIA. Under these assay conditions employed, interassay variation was 9.3% (n=5), and intra-assay variation was 5.6% (n=5). The recovery of Ang II over the measured range (0.05-20 pg/tube), added to the samples and then sent to the Sep-Pak CI 8 cartridge, ranged 82-89%, with an average of 84%. Plasma renin concentration (PRC) was measured by the method previously reported (16). Briefly, plasma samples (50 ul) were incubated with unfractionated plasma (75 ul) from 36 h nephrectomized rats as a substrate for 2 h at 37°C. Generated Ang I was measured by RIA (17). For measurement of plasma aldosterone, a direct RIA was employed with commercially available kits (Aldosterone-RIAKIT 11, Dainabot Co., Ltd., Tokyo). Serum ACE activity was determined by the spectrophotometric method using hippuryl-histidyl-leucine as a substrate (18). Statistical analysis: Results are expressed as means_+SE. Statistical analysis was performed by one way of analysis of variance for repeated measurements and Student's _t-test where appropriate. The null hypothesis was rejected at P
51, pp. PL 183-187
Pergamon Press
PHARMACOLOGY LETTERS Accelerated
HYPOTENSIVE
Communication
ACTIVITY OF TCV-116, A NEWLY DEVELOPED
RECEPTOR
ANTAGONIST,
IN SPONTANEOUSLY
ANGIOTENSIN
HYPERTENSIVE
II
RATS
Kenji Mizuno, Susumu Niimura, Makio Tani, Ikue Saito, Hironobu Sanada, Michihiko Takahashi, Koichiro Okazaki, Manabu Yamaguchi and Soitsu Fukuchi Third Department of Internal Medicine, Fukushima Medical College, Fukushima 96012, Japan (Submitted July 14, 1992; accepted July 16, 1992; received in final form September ii, 1992)
Abstract: TCV-116, a recently developed angiotensin II (Ang II) receptor antagonist, was administered orally (1 mg/kg per day) to 10-week-old spontaneously hypertensive rats (SHR) for 2 weeks . Blood pressure and plasma components of the renin-angiotensin-aldosterone system were determined in these rats. TCV-116 produced a marked reduction in blood pressure without altering heart rate. Whereas plasma renin concentration (PRC), angiotensin I (Ang I) and angiotensin II (Ang II) all were significantly increased, plasma aldosterone was decreased by approximately 70% compared with control animals. These results not only indicate therapeutic efficacy of this agent in the chronic treatment of human hypertension, but support also the concept that the renin-angiotensin system plays an important role in the control of blood pressure in this animal model of human essential hypertension.
Introduction Although the renin-angiotensin system (RAS) has long been recognized as an important factor in the pathophysiology of several forms of hypertension, the contributory role of this system in the initiation and/or development of hypertension of spontaneously hypertensive rats (SHR) has been mitigated because of normal or even low levels of plasma renin. Progress in drugs which interfere with the RAS at levels of renin and angiotensin converting enzyme (ACE) has indicated the importance of this system in the maintenance of hypertension, inasmuch as these agents actually reduce blood pressure of SHR (1-3). Nonetheless, their mechanism of action has not fully been clarified. They may lower blood pressure by suppressing the generation of angiotensin II (Ang If) in either the circulation or vascular tissues (4-6). Others, however, have suggested that ACE inhibitors lower blood pressure by decreasing sympathetic tone (7), or by elevating the levels of kinins (8) and/or eicosanoids in vascular walls (9). Recently, a new class of highly specific, competitive Ang II receptor antagonists has been described (10,11). Of these compounds, Losartan (known as DuP 753) blocks the vasoconstrictor actions of Ang lI and lacks agonistic or kinin- and prostaglandin-inducing properties (12). More recently, Losartan has also been shown to reduce blood pressure in SHR (12,13), findings suggestive of the contributory role of the RAS in the maintenance of hypertension of this animal model of human essential hypertension. TCV-116, ((-+)-••(cycl•hexy••carb•nyl•xy)ethyl-2-eth•xy-1-[[2'-(•H-tetraz•l•5-y•)bipheny•-4-yl]I H-benzimidazole-7-carboxylate), is a newly synthesized benzimidazole derivative, which possesses potent antagonistic properties highly specific to Ang II receptors in vascular smooth muscle and adrenal glomemlosa cells (14). This agent is a prodrug which is metabolized to an active form (CV-11974) to exert its biological effects (14). However, little is known about the antihypertensive and hormonal activity of this agent in SHR at present. Thus, the purpose of the present study was to examine these pharmacological properties of TCV-116 in SHR and then address the significance of the RAS in pathophysiology of hypertension of SHR.
Correspondence to: K. Mizuno, Third Department of Internal Medicine, Fukushima Medical College, 1 Hikari-ga-oka, Fukushima 96012, Japan. 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
PL-184
TCV-II6
in SHR
Vol.
51, No. 20,
1992
Materials and Methods Male SHRs (10-week-old) were maintained on a regular chow diet containing 0.39% sodium and 0.9% potassium and allowed free access to tap water. The rats were divided into two groups and were administered TCV-116 (n=8) or tap water as the control (n=8). TCV-116 was given orally to the rats at a dose of 1 mg/kg/day in a suspension of gum arabic in a volume of 1 ml/kg of water at 4:00 p.m. for 2 weeks. The drug suspension was made up freshly every day. Body weights were determined and systolic blood pressure as well as heart rate was measured with an ultrasound device (UR 5000, Ueda Co., Ltd., Tokyo) before and 1 and 2 weeks after treatment. After 2 weeks of the treatment, rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Blood (6 ml) from the control and TCV-116-treated SHRs was withdrawn by aortic puncture. About 5 ml of the blood were collected into pre-cooled tubes containing Na2-EDTA, o - p h e n a n t h r o l i n e and phenylmethylsulfonyl fluoride (PMSF) at final concentrations of 5 mM, 2 mM and 10 mM, respectively, for determinations of renin concentration, Ang I, Ang II and aldosterone. After centrifugation at 2,500 x g for 30 min at 4°C, the plasma was aspirated and stored at -20°C until assay. The remaining blood was used for determination of serum ACE activity. Plasma immunoreactive Ang 1 and Ang 11 were measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridges (Waters, Milford, MA), as described previously (15,16). In brief, plasma (1 ml) was applied to the cartridges, which had been washed with methanol (3 ml) followed by 0.1 M Tris-acetate buffer, pH 7.4 (10 ml). After washing with distilled water, peptides were eluted with a mixture of methanol/distilled water/trifluoroacetic acid (TFA) (90:9.9:0.1, vol ratio). The extracts were evaporated to dryness in vacuum centrifuge and the residue was redissolved in assay buffer consisting of 0.1 M Tris-acetate containing 2.6 mM Na2EDTA, 1 mM PMSF, and 0.1% bovine serum albumin, pH 7.4, for RIA. Under these assay conditions employed, interassay variation was 9.3% (n=5), and intra-assay variation was 5.6% (n=5). The recovery of Ang II over the measured range (0.05-20 pg/tube), added to the samples and then sent to the Sep-Pak CI 8 cartridge, ranged 82-89%, with an average of 84%. Plasma renin concentration (PRC) was measured by the method previously reported (16). Briefly, plasma samples (50 ul) were incubated with unfractionated plasma (75 ul) from 36 h nephrectomized rats as a substrate for 2 h at 37°C. Generated Ang I was measured by RIA (17). For measurement of plasma aldosterone, a direct RIA was employed with commercially available kits (Aldosterone-RIAKIT 11, Dainabot Co., Ltd., Tokyo). Serum ACE activity was determined by the spectrophotometric method using hippuryl-histidyl-leucine as a substrate (18). Statistical analysis: Results are expressed as means_+SE. Statistical analysis was performed by one way of analysis of variance for repeated measurements and Student's _t-test where appropriate. The null hypothesis was rejected at P
