Effects of OKY-046, a selective thromboxane synthetase inhibitor, on blood pressure and thromboxane synthesis in spontaneously hypertensive rats

Prostaglandins Leukotrienes and Esent~al Fatty Acids (198% 37. 119-144 Q Lon@nan Group UK Ltd 19X9

Effects of OKY-046, A Selective Thromboxane Synthetase inhibitor, On Blood Pressure and Thromboxane Synthesis in Spontaneously Hypertensive Rats T. GOMI, T. IKEDA, *T. ISHI~ITSU and * Y. UEHARA repayment of ~ep~~ology, Kan~~~Teis~iff ffos~ital, 5-9-22 ~igas~ig~~anda, S~i~agawa, Tokyo ~1, Japan and* The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan

Abstract - The effects of OKY-046, a specific thromboxane (TX) synthetase inhibitor, on blood pressure, urinary TX excretion, TX synthesis in blood platelets, kidney slices and aortic strips, were evaluated in adult spontaneously hypertensive rats (SHR). OKY-046 was dissolved in drinking water at concentrations of 1, IO, 100 mg/dl. The average intakes of OKY-046 were 1.4 + 0.1, 13.0 + 1.1, and 147 + 12 mg/kg/day, in rats who took 1, 10, 100 mg/dl of OKY-046 solutions for drinking water, respectively. The systolic blood pressure was significantly decreased by 34 mmHg only with the high dose of OKY-046 (147 mg/kg/day). OKY-046 suppressed the platelet aggregability to ADP and the release of TX Bz, a stable metabolite of TX AZ, from blood platelets in a dose-dependent fashion. Urinary excretion of TX 62 decreased significantly in both groups treated with moderate (13.0 mg;/kg/day~ .and high doses of OKY-046 (147 mg/kg/day), The release of TX B2 from kidney slices was decreased only by the high dose of OKY-046, while the release of TX B2 from aortic strips was not changed even by the high dose of OKY-046. OKY-046 had no effect on urinary excretion of 6-keto-prostaglandin Flcv, a stable metabolite of prostacyclin, or, on its release from the kidney slices and aortic strips. These results suggest that the effect of OKY-046 on TX synthesis has organ specificity and that the antihypertensive effect of this drug in SHR is related to reduced renal TX synthesis.

Introduction Several lines of evidence suggest that changes in prostaglandin metabolism in kidneys and vascular walls are implicated in the pathogenesis of hypertension (I-7). Urinary excretion of throm-

boxane (TX) BL?.a stable metabolite of TX Al, is elevated in patients with essential hypertension (4). The biosynthesis of TX in the kidney increases as hypertension develops in the Okamoto

139

strain

of spontane~usiy

hypertensive

rats (SHR) (S). The production of TX AZ by incubated thoracic aorta increases in both SHR (6) and Dahl genetic rats susceptible to saltinduced hypertension (7). These findings indicate that changes of TX metabolism in the kidney and vascular walls are related to vascular reactivity and peripheral resistance in certain forms of hypertension. (E)-3-(4-(1-imidazolylmethyl) phenyl)-2propenoic acid hydrochloride (OKY-046, Ono Pharmaceutical Co., Osaka, Japan) is an imidazole derivative with a highly selective inhibitory effect on TX synthetase (8). In animal experiments, OKY-046 shows protective effects against endotoxin shock in rats (9), improves the coronary blood flow in an ischemic canine heart model (lo), and attenuates contractile responses of guinea pig tracheal strips to bronchoactive substances (11). There are two conflicting reports concerning the antihypertensive effect of OKY-046 in SHR. Purkerson et al. (12) have reported that a subcutaneous administration of 40 mg/kg OKY-046 decreased arterial blood pressure and improved renal function in SHR. On the other hand, Tanno et al (13) have observed that an administration of 12 mdkg OKY-046 has no effect on blood pressure in 5week old SHR. In this study, we examined the changes of blood pressure, platelet aggregation, TX synthesis of platelets, kidney and aorta in SHR treated with graded doses of OKY-046 and tried to clarify the relationship between dose, antihypertensive effect and inhibition of TX synthesis in various organs. Method Studies were performed on g-week old female SHR that were bred in the Animal Center of the Kanto Teishin Hospital. Each rat was placed alone into a metabolic cage in a temperaturecontrolled room maintained at 25°C with a light/dark cycle of 12 hours daily. Regular rat chow containing 13 mEq/lOO g of Na and 27 mEq/lOO g of K (Oriental Yeast Co., Tokyo, Japan) was provided on an ad libitum basis. In OKY-046 treated groups, OKY-046 solution with concentrations of 1, 10, and 100 mgidl was provided for drinking water. A group drinking distilled water served as controls. After seven days of oral administration of OKY-046, measurements of biood pressure, and food and water intake were done and 24-hour urine was collected for the measurement of urinary excre-

tion of creatinine, Na, and prostaglandins. The systolic blood pressure was measured by the tail cuff method using an Automated Measurement System for Rat Blood Pressure (Tohiden Co., Tokyo, Japan) (14) without the use of anesthesia. Blood samples for studies of platelet aggregation and TX release from platelets were taken from the abdominal aorta under ether anesthesia. After sampling of blood specimens. the abdominal aorta was cannulated below the renal arteries. The aorta was then clamped above the renal arteries, and the kidneys were perfused to free them from blood with 20 cc of ice-cold Dulbecco’s phosphate-buffered saline (D-PBS, Gibco Laboratories, New York, U.S.A.). The left kidney and thoracic aorta were resected for measuring the release of prostaglandins. A kidney slice, including the hilum, 0.5 mm thick was made by frontal section of each resected kidney with a slicer (Nastume Instrumental Co.. Tokyo. Japan). The aortic strip. 1 cm long, for the measurement of prostaglandin release was prepared after connective tissue around the aortic wall was carefulfy removed. Platelet aggregability was measured by the aggregation threshold method of Sano et al. (15). Blood was mixed with l/l0 volume of 3.8% sodium citrate solution, and platelet rich plasma (PRP) was obtained after centrifugation at 160 g for 10 minutes. Twenty five ~1 PRP was mixed with 25 ~1 of 2-5-2-‘” m$ml of adenosine 5’ - diphosphate (ADP) in the Microtiter Disposable Tray (Tominaga Works LTD. Tokyo, Japan) by Micromixer (Taiyo Bussan Japan) for 15 minutes. The CO., Tokyo, minimum concentration of ADP at which more than 5 aggregated platelets were found under microscopy was determined. The platelet sensitivity to ADP-aggregation was expressed by the absolute value of the exponent (n) of the final minimum ADP concentration, when the endpoint was 2-“mg/ml. The ability of platelets to release TX A1 was determined by the generation of TX B? during spontaneous clotting of whole blood, as reported by Patron0 et al. (16). Two ml of blood was collected in a glass centrifuge tube without any anticoagulant and allowed to clot for 60 minutes at 37°C. Serum was removed from clotted blood following c~ntrifugation at 3000 g for 10 minutes at 4°C. The serum concentration of TX B:! was radioimmunoassayed and the amount of released TX B, was expressed as nanograms per 10’ plate-

141 lets. The measurements of renal and aortic prostaglandin synthesis were performed as described below. The tissue samples of kidney and aorta were separately put into the 3 ml DPBS and incubated in an atmosphere of 95% O1 and 5% CO- in a shaken water bath at 37°C. After 15 minutes preincubation, these tissue samples were put into another 3 ml D-PBS and incubated for 30 minutes under the same conditions as preincubation. The prostaglandin production in the tissue samples was measured from the amounts of prostaglandins released into the incubation medium and expressed as picograms per milligram of the dry weight of each tissue sample. Prostaglandins from serum and urine were extracted by the method of Jaffe et al. (17). A 1 ml sample of urine or serum was mixed with 3 ml petroleum ether to remove neutral lipid. The aqueous layer was exposed to 3 ml of I: I ethyl acetate: isopropanol, and vortexed, and a mixture of 2 ml of ethyl acetate and 3 ml of water was added. After further mixing, the two phases were separated by centrifugation. After the organic phase was evaporated dry in N7 gas at 55°C. it was dissolved in 1 ml of a mixture of benzene. ethyl acetate and methanol (60440:2. v/v). The dissolved material was applied to a silicic acid column and the fractions of &ketoprostaglandin Fr, (6-keto-PG FIN) and TX. B2 were obtained by developing the column serially with an eluent consisting of benzene: ethyl acetate: methanol (60:40:30, v/v for 6-keto-PG F In* 60:40:7, v/v for TX Bl). Prostaglandin concentrations in each fraction extracted from

serum and urine, and in supernatants which were incubated with kidney slices and aortic strips were radioimmunoas~yed by the double-antibody procedure of Morris et a!. (18), utilizing highly specific antisera for 6-keto-PG F1, and TX B2. The antisera for 6-keto-PG FI, and TX Br were purchased from Ono Pharmaceutical Co. (Osaka, Japan). The cross-reactivity of the antiserum for 6-keto-PC F,, was 2.09% with PG E,. 8.4% with PG E:, 4%’ with PG F,,,
The average daily intakes of OKY-046 calculated from water intake were respectively 1.4 I!I 0.1 mg/kg!day, 13.0 + 1.1 mg/kg/day and 147 i 12 mg/kg/day in rats who took 1, 10, 100 mg/dl of OKY-046 solution for drinking water. Systolic blood pressure measured by the tail cuff method decreased by 34 mmHg only in the grou treated with the high dose of OKY-046 kg/day) as compared with the control (147 m Wp

Table 1 Systolic blood pressure, platelet sensitivity to ADP aggregation and thromboxan~ B2 (TX B,) formation in clotting whole blood in control and OKY046 treated groups Variables

Number Body Weight (g) Blood Pressure (mmHg) Food Intake (g/day) Water Intake (ml/day) Urine Volume (ml/day) Platelet Count ( 10’/mm3) Platelet Sensitivity (n) TX Bz (q/l@ of platelet)

OKY-046 Treated Groups

Control Croup

I t&d1

IO m,qdl

100 mg/di

6 231 I!I 6 182 I? 3 18.5 rf: 1.3 31.4 + 2.8 11.5 + 1.1 85.3 t 3.2 10.3 + 0.3 20.0 t 1.3

6 230 1 4 179 + 3 19.1 + 1.2 :!.Y ? 2.7 Il.4 + 1.2 x5.4 * 4.1 8.3 f #.h* 19.9 + 2.1

6 2.17 t: 6 173 rt 3 IX.3 rt 1.0 30.6 + 2.8 11.1 + 0.6 90.1 f 2.0 6.8 f 0.7** 10.0 c 1.9**

6 235 + 4 14x L 2,” lY.0 _+ 1.3 34.6 t 3.x 13.1 * 1.1 89.0 * 3.1 5.1 * 0.2** 2.x f o.s**

II = exponent of the endpoint ADP concentration (2-” mg/ml) Results are expressed as mean i SEM. * :p < 0.05, ** :p < 0.01compared with value in control group

group (p < 11.01, Table 1). There were no differences in body weight. food intake. water intake or urine volume between control and OKY-0% treated groups (Table I). The ADP concentration at which platelet aggregation was induced was higher even in the group treated with the low dose of OKY-046 (1.4 mg/kg/day) than in the control group. OKY046 suppressed the platelet aggregability to ADP in a dose-dependent fashion (Table 1). The mean release of TX B- per 10’ platelets decreased by 60% and 90% in the groups treated with moderate (13.0 m~k~day) and high (147 m~k~day) doses of OKY-046, respectively, as compared with the control group (Table 1). Concerning the urinary excretion of prostaglandins, expressed in relation to mg of creatinine excretion, urinary excretion of TX B2 decreased significantly in both groups treated with moderate and high doses of OKY-046. However, OKY-046 had no effect on urinary excretion of 6-keto-PG F,, (Table 2). Table 3 shows the amounts of prostaglandin released from kidney slices and aortic strips in control and OKY-Oil6 treated groups. The

average release of TX B? from kidney slices decreased by 33% only in the group treated with the high dose of OKY-046 (p < 0.05), while the release of TX B: from the aortic strip was not changed even by the high dose of OKY-0-m used in this study. OKY-046 brought about no change in the release of n-keto-PG F,, from kidney slices or aortic strips. Discussion The aim of the present study was to clarify the factors participating in the pathogenesis of hypertension with respect to platelet function and to vasoconstrictor TX metabolism in the kidney and vascular walls. The chronic oral administration of OKY-046, a specific TX synthetase inhibitor, decreased systolic blood pressure with a concomitant decrease of TX release from kidney slices in X-week old SHR only at a high dose of 147 mg/kg/day. This result suggests that the antihypertensive effect of OKY-046 is closely related to its reduction of renal TX synthesis. There have been c~~n~icting conclusions reported by several investigators

Urinary excretions of sodium (UN, V), potassium (LJ, V). creatinine (U,, F,, (Uh_k~to_P~ Fla V) in V), thromboxane Bz (Urxez V) and 6-keto-prostaglandin control and OKY-046 treated groups

Table 2

Variables

UN,V (mEq/day) U,V U,V

(mEq/day) (mg/dayI

U,xs2V (n~~ay/mg Uh-LcO,-Pf;i- v

OKY-046

Groups 100 v&d1

I mgjdl

1.57 i- 0.17 I 0.32 f 0.91 2.56 + 0.26 2.3x I 11.32

1.68 + 0.18

1.65 F 0.16

I.6

3.32 5.68 I.Yl 2.43

3.29 5.58 I.25 2.62

3.20 + 0.28 5.81 + 0.43 0.67 I!z 0.07* 3.55 t ii.24

3.09 5.34

of Cr)

Treated

Control Grout

10

+ ? + &

0.26 0.73 0.24 0.39

m&i1

rt c rt t

0.16 0.87 0.17* 0.22

t

0.12

la (n~day/mi

of Cr)

Results are expressed

as mean

I

*

SEM,

:p

< 0.01 compared

with value in control

group

Release of thromboxane B2 (TX B3) and 6-keto-prostaglandin F,, (6-ketoPG F,,) from kidney slice and aortic strip in control and OKY-046 treated groups

Table 3

GrOf4p

Control OKY-044 OKY-046 OKY-046

I

mgidt 10 q/d1 100 mddl

Results are expressed

as mean

Kidney Slice TX B2

h-keto-PG

~p~mg~

(pgimg)

21.8 31.3 1x.9 14.Y

_t 1.7 t 2.1 AZ I.6 t 0.7’

321.9 354.6 347.9 343.4

rt SEM,

*

:

+ * * i

F,,

-17.0 77.5 61.2 3i.Y

p < 0.01 compared

Aortic Strip TX B?

h-keto-PC

(iwh9

Whd

42.6 t 42.8 + 39.8 I? 39.1 +

7.4 5.2 2.9 2.7

14709 14213 14805 15452

with value in control

F,,

t 1579 i 1781 + 2236 + 2505 group

concerning the ant~hypertensive effects of TX synthetase ~nhjb~tors in SHR. Purker~~ et al. (12) observed that mean blood pressure in SHR treated with subcutaneous injections of 40 mg&g OKY-046 was tawer by 44.6 mmHg than that of saline injected SHR. Moreover, Uderman et at. (19) reported that the bload pressure decreases by 25 mmHg after 10 days of oral administration of the imidazole derivative UK%,485 at a daily dose of 100 mg/kg in l&week old SHR. In another study by Shibouta et al. (ZO), oral administration of 10 mg/k&lday of pyridine derivative. CV-4151. induced a one week delay in the onset of hypertension in 4-week old SHR during the development phase of hypertensjonbut had no effect on blood pressure in I&week old SHR with established hypertension. On the other hand, Tanno et al. (13) reported that subcutaneous administration of 6 mg/kg OKY046 every 12 hours for 2 weeks had no effect on blood pressure in S-week old SHR. Our results agree with the findings of Purkerson et al. ( 12) and Uderman et al, (19), but differ from those of Shibouta et al. (20) and Tanno et al. (13). The reasons for the differences between their results and ours, although not certain, are likely to be related to differences in the doses of TX synthetase inhibitors and routes of admi~~st~~tjon used in the studies. In this study, an OKY-046 regimen with a high dose of 147 m~k~day induced an antihypertensive effect, while a dose of 13 n~~k~~iay, approximately equivalent to the dose used in the study of Tanno et al. (13) had no effect OII hlooci pressure in SHR. it has been reported that the concentration required for SE; inhibition of TX synthetase activity is 1.1 i( lO_‘”M for OKY-046 and 1.8 x 1c)sM for UK3X,J185 (8. ?i). Therefore, the dose of UK38.485 used by Uderman et ai. (19) is ~~l~pro~~rnate~y~q~~~~~a~ent to the high dose of CKY-046 used in this study. Platelet aggreg~~bi~ity to ADP. which was determined by the platelet aggregation threshold method. was suppressed by treatment with the low dose of OKY-046 and the inhibitory effect bccamc greater with moderate to high dose of OKY-046. TX EQ release from platelets was decreased significantly by treatment with moderate to high doses of OKY-046. OKY-046 inhibited platelet aggregation at about one-tenth the dose which caused inhibition of TX release from platelets. In contrast to our results. Naito et al. (S) showed that OKY-046 ~nhjbit~d TX release from rabbit platelets induced by arachidonic acid

by 95% at a concentration of Wi moi, while it jnh~bited platelet aggregation by 5ft% at the same concentration. Their rest&s indicate that UKY-046 is more potent in inhibiting TX release from platelets than in suppression of platelet aggregabiiity. The reason why our result differs from the observation of Naito et al. (8) is uncertain, but may relate to differences’ in species, in aggregation inducers and in experimental conditions (22, 23). Urinary excretion of TX 32 tended to decrease in the group treated with a low dose of OKY-046 and decreased signj~ca~tly by 5f% and 74% in the groups treated with moderate and high doses of UKY-046, respectivety. UKY-046 caused a decrease of urinary TX Bz excretion in a doscdependent fashion without any change of urinary excretion of 6-keto-PG F,,. The suppressive effect of OKY-046 on urinary excretion of TX seems to be parallel with the effect of this drug on platelet aggregation and TX release from platelets. On the other hand. the signiticant decrease in release of TX 13, from kidney slices was observed onty in the group treated with the high dose of OKY-046. AIthou~h the origin of urinary TX is not well established, our data indicates that TX may mainfy reflect extrarenal TX metab~ljsm of pfatelets. However. Patrrmo et ai. (24) have reported that urinary excretion of TX is not changed by a low dose of aspirin, even though the same dose of aspirin suppresses completely the release of TX from platelets: they have speculated that the urinary excretion of ‘TX may derive from renal produ~t~~~n of TX. A further detailed evaluation is needed to clarify the exact origin of urinary TX and the pathophysiologic roles of renal TX metabolism in the deve~opnle~~t of hyp~rte~si~?n. it has been reported that vascular walls have the ahihty to synthesize prt~stacyc~~n and small but significant amounts of TX AZ (25, 26). The pathophysj~~l~)gi~ role of TX A? in vascular waiis has been challenged by the finding that the amount of vascular TX A? is much smaller than that of vascular prostacy&. However, there are several lines of evidence suggesting that TX As is present in the vascular walls in amounts more than adequate to exert biological effects on both vascular reactivity and on platelet function. ‘The ~rodu~ti~~n of TX AZ hy incubated thoracic aorta is reported to bc increased in both SHR (6) and the Dahl genetic strain of rats susceptible to saftinduced h~~ert~nsi~~~ (7). In this study, WCcould not find any relationship between the antihypcr-

ten&e effect of TX synthetase inhibitor and TX metabolism in the vascular walls, because the release of TX B: from the aortic strips was not changed even by the treatment with highest dose of OKY-046 when there was a significant reduction of blood pressure in SHR. *Another hnding in this study is that there is a discrepancy between the effects of OKY-046 on inhibition of TX formation in platelets, kidney and aorta. OKY-046 inhibited the release of TX from platelets at the middle dose, but the release of TX from the kidney was suppressed only by the high dose. Furthermore, OKY-046 had no effect on TX formation in aortic strips even at the highest dose. These results suggest that the effect of OKY-046 on TX synthesis may be organ specific. Further investigations are required to relate the pharmacological specificity and effectiveness of the drug to its dose-related effects in each organ.

Il.

17.

13.

14.

1.5,

I6

17.

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