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Pharmacologic Profiles of GA0113, a Novel Quinoline Derivative Angiotensin II AT1-Receptor Antagonist

Ebisu, Hajime; Nishikawa, Masakuni; Tanaka, Mikio; Okazoe, Takashi*; Morizawa, Yoshitomi*; Shinyama, Hiroshi; Nakamura, Norifumi

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Journal of Cardiovascular Pharmacology: October 1999 - Volume 34 - Issue 4 - p 526-532
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Most angiotensin II (Ang II) AT1-receptor antagonists, including losartan (DuP 753) and candesartan cilexetil (TCV-116; Fig. 1), possess a biphenyltetrazole moiety within their chemical structure, and antagonists without this moiety are generally not active (1-3). However, some agents with a biphenyltetrazole moiety display relatively low oral bioavailability (BA) (4,5), which may result in variability of action. Therefore our target was to find a novel biphenyltetrazole replacement. A benzene ring of biphenyl with no tetrazole moiety was replaced with heterocycles (i.e., quinoline, indazoles). The tetrazole moiety was substituted with acidic groups (i.e., sulfonamides) in line with the a finding that the replacement of the tetrazole group by more lipophilic acidic groups improves oral BA (6,7). After synthesizing a large number of compounds by using this strategy, we finally obtained GA0113, N-{2-{6-[(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-yl) methyl] quinolin-2-yl} phenyl} trifluoromethanesulfonamide (Fig. 1) for trial as a novel Ang II-receptor antagonist. In this study, we describe the pharmacologic profile of the new angiotensin II antagonist GA0113 in a number of in vitro and in vivo experiments.

FIG. 1
FIG. 1:
Chemical structures of GA0113 and TCV-116.


Ang II-receptor binding assay

A binding assay for AT1 and AT2 receptors was performed by using commercially available membrane fractions expressing either human AT1 or AT2 (CRM036 or CRM048; NEN, Boston, MA, U.S.A.). For the binding assays, 20 μl of GA0113 (10−11-10−5M), CV-11974, an AT1 antagonist and a metabolite of TCV-116 (10−11-10−5M), PD123319, a selective AT2 antagonist (10−10-10−5M), Ang II (10−5M, for nonspecific binding) or assay buffer [50 mM Tris-HCl buffer (pH 7.4) containing 50 mM MgCl2 and 0.25% bovine serum albumin (BSA), for total binding] and 30 ml of [125I] Sar1, Ile8-Ang II (1.7 × 10−9M) were added to the wells of the assay plate. Assay buffer (150 μl) containing membrane fractions was added to the assay plate to initiate the binding reaction. Incubation was performed for 2 (AT1) or 3 h (AT2) at 37°C. After incubation, to separate bound and free radioactivity, we immediately filtered the reaction mixture under pressure through glass fiber GF/C (AT1) or GF/A (AT2) filters (Whatman International Ltd., Maidstone, Kent, England) presoaked with 0.5% polyethylenimine solution, and each plate well and filter was rinsed 3 times with phosphate-buffered saline (PBS). The radioactivity trapped on the filters was counted by a τ-counter (COBRA II 5003; Packard Japan, Tokyo, Japan).

For saturation binding studies, AT1 receptors were incubated in GA0113 (1.1 × 10−8M) with increasing concentrations of [125I] Sar1, Ile8-Ang (0.11-3.4 × 10−9M).

The inhibitory concentration of test compound that caused 50% inhibition of the specific binding of [125I]Sar1, Ile8-Ang II (IC50) was determined by displacement curves. The dissociation constant (Kd) was obtained from a Scatchard plot (8).

Ang II-induced contraction in isolated rabbit aortae

Male New Zealand White rabbits weighing 1.5-2.3 kg (Keari, Osaka, Japan) were anesthetized with sodium pentobarbital (20 mg/kg, i.v.) and killed by bleeding. The thoracic aorta was rapidly isolated and cut into helical sections. The vascular endothelium was removed by gently rubbing the intimal surface with a cotton ball. Aortic strips were suspended in an organ bath containing Krebs-Henseleit solution (in mM: NaCl, 118.4; KCl, 4.7; CaCl2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25.0; and glucose, 10.0) kept at 37°C and aerated with 95% O2/5% CO2. The resting tension was adjusted to 1.0 g. The developed tensions were measured isometrically with a force-displacement transducer (TB-611T, TB-612T; Nihon Kohden, Tokyo, Japan) connected to a carrier amplifier (AP-601G; Nihon Kohden).

The concentration-contractile response to Ang II (10−10-3 × 10−6M) was obtained first, and the strips were then washed several times with fresh media. To determine antagonism against Ang II-induced contraction, the strips were treated with GA0113 (10−10-3 × 10−6M), CV-11974 (10−10-3 × 10−6M), or each vehicle for 15 min, and then the contractile response for Ang II was obtained again.

The pD′2 values were determined according to the method of Ariens and van Rossum (9).

Antihypertensive effects in renal artery ligated hypertensive rats (RALHR)

Male Wistar rats (303-377 g; Keari) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), and subjected to a midline abdominal incision. The left renal artery was exposed and completely ligated with 3-0 silk suture. On day 5 after surgery, rats with a systolic blood pressure (SBP) of 150-214 mm Hg were chosen for the following study. These animals received GA0113 (0.01-1 mg/kg, p.o.) or a vehicle at a volume of 10 ml/kg, and their SBP and heart rate (HR) were measured by the tail-cuff method (BP-98; Softron, Tokyo, Japan) immediately before administration and 1-144 h after administration.

ED25 values (the dose that reduced blood pressure by 25 mm Hg) was estimated by using linear-regression analysis.

Antihypertensive effects in spontaneously hypertensive rats (SHRs)

Male 13-week-old SHR/Izm (Disease Model Cooperative Research Association, Kyoto, Japan) with SBP 181-200 mm Hg at the start were used and were not different among the groups. They received GA0113 (0.03 and 0.1 mg/kg), TCV-116 (candesartan cilexetil), an AT1-selective antagonist (0.3 and 1 mg/kg), or a vehicle once a day (between 09:00 and 10:00 a.m.) for 15 days. SBPs and HRs were measured on days 1-15, as described earlier in RALHRs.

Taking the SBP data into consideration, we express peak value as the maximum absolute difference between the curve of vehicle and the curve of drugs at 2-11 h after dosing. Trough is expressed as the difference between both curves at 24 h after dosing. The trough per peak (T/P) expresses the resulting ratio between these differences. The values of trough and peak were obtained directly from the curve of averaged results of each group.

Pharmacokinetics in rats

To evaluate absorption, GA0113 (0.1 mg/kg) was administered p.o. or i.v. to 20-h fasted Sprague-Dawley rats (6-7 weeks old; Keari). Blood was collected by repeated jugular venipuncture at 72 h. Plasma was harvested after centrifugation and stored frozen at −20°C until analyzed. Plasma levels of GA0113 were measured by reverse-phase, high-performance liquid chromatography (Lc Module I; Waters Millipore Corp., Milford MA, U.S.A.) with an Inertsil ODS-2 column (4.6 × 250 mm, S-5 μm, GL Science, Tokyo, Japan) by using a 60:40 (vol/vol) acetonitrile-20 mM KH2PO4 solution (pH 5.0) at 1.2 ml/min and detected at 285 nm.

Plasma terminal half-lives (T1/2) were calculated by linear regression of the log plasma concentration-time profile for each animal. The areas under the concentration-time curves (AUC0-∞) were calculated by linear trapezoidal approximation to the last time point and extrapolated to infinity by using T1/2. The systemic plasma clearance (CL) was calculated for each animal by the relation dose/AUC0-∞. The volume of distribution (Vdss) was calculated by using statistical moment analysis. The oral BA (%) was calculated by AUC0-∞ p.o./AUC0-∞ i.v. × 100.


GA0113, TCV-116, and CV-11974 were supplied by the Research Center, Asahi Glass Co. Ltd. For the in vivo study, GA0113 was prepared by dissolving >3 mg in a mixture of Na2CO3 (2 M, 0.1 ml) and EtOH (0.1 ml) and then diluted with saline (i.v.) or distilled water (p.o.). TCV-116 was suspended in 5% gum arabic. For the in vitro study, GA0113 and CV-11974 were dissolved with dimethyl sulfoxide (DMSO) and then diluted with an assay buffer (pH 7.4). Ang II (Peptide Institute, Osaka, Japan), [125I]-Sar1, Ile8-Ang II (NEN), and PD123319 (RBI, Natick, MA, U.S.A.) were purchased from the sources indicated.

Statistical analysis

Except for the pharmacokinetics data (mean ± SD), the results are expressed as mean ± SEM. The values for the different groups were compared by using analysis of variance and Dunnett's test; p values of <0.05 were considered to be significant.


Radioligand-binding assays

GA0113 and CV-11974 displaced the specific binding of [125I]-Sar1, Ile8-Ang II to binding sites in AT1 in a concentration-dependent manner (Fig. 2A), and the IC50 values were 1.1 × 10−8 and 1.3 × 10−7M, respectively. In contrast, specific binding of [125I]-Sar1, Ile8-Ang II to binding sites in AT2 was displaced by PD123319 but not by GA0113 (Fig. 2B). The binding of various concentrations [125I]-Sar1, Ile8-Ang II in AT1 was determined with or without GA0113 and analyzed by the Scatchard method. GA0113 increased the Kd value of [125I]-Sar1, Ile8-Ang II binding from 2.6 × 10−9 to 7.1 × 10−9M without affecting Bmax (data not shown).

FIG. 2
FIG. 2:
Effects of GA0113, CV-11974, and PD123319 on specific binding of [125I]-Sar1, Ile8-angiotensin II to human AT1 receptors (A) and AT2 receptors (B). All values represent mean ± SEM (n = 3).

Ang II-induced contraction in isolated rabbit aortae

GA0113 (10−6-10−11M) inhibited the Ang II-induced contraction in the rabbit aorta in a concentration-dependent manner (Fig. 3). At the tested concentration, GA0113 had no effect on resting tension, indicating absence of AT1 antagonistic activity. The antagonism of Ang II produced by GA0113 and CV-11974, a metabolite of TCV-116, was characterized by a rightward shift in the concentration-response curve of Ang II and by a significant reduction in the maximal contractile response, indicating insurmountable antagonism. The pD′2 values of the GA0113 and CV-11974 were 8.82 and 10.08, respectively.

FIG. 3
FIG. 3:
Effects of GA0113(A) and CV-11974 (B) on concentration-contractile response curve for angiotensin II in rabbit isolated aortic rings. All values represent mean ± SEM (n = 7).

Antihypertensive effects in RALHRs

Administration of GA0113 (0.01-1 mg/kg) reduced SBP in a dose-related manner with ED25 of 0.015 mg/kg, and at dose 0.1 mg/kg or more, SBP was reduced for >24 h (Fig. 4A). GA0113 did not affect HR significantly (Fig. 4B).

FIG. 4
FIG. 4:
Effects of oral administration of GA0113 on systolic blood pressure(A) and heart rate (B) in renal artery-ligated hypertensive rats. The initial values of systolic blood pressure (mm Hg) and heart rate (beats/min) were not different among the groups (one-way analysis of variance): 162 ± 4 and 455 ± 31 (vehicle), 176 ± 8 and 476 ± 14 (0.01 mg/kg), 182 ± 9 and 472 ± 35 (0.1 mg/kg), 170 ± 11 and 455 ± 20 (1 mg/kg). All values represent mean ± SEM (n = 4-5). Significantly different from vehicle group, *p < 0.05; **p < 0.01 (repeated-measures analysis of variance followed by Dunnett's test).

Antihypertensive effects in SHRs

The decrease in SBP was potentiated day by day and reached a plateau by day 4 (Fig. 5A). After termination of treatment, SBP gradually returned to the initial level, and no subsequent increase in SBP (rebound phenomenon) was observed. Likewise, TCV-116 showed gradually potentiated reduction in SBP (Fig. 6A). GA0113 at 0.03 and 0.1 mg/kg showed a T/P ratio >0.5 on and after day 3 and day 1, respectively. TCV-116 showed a T/P ratio >0.5 on and after day 4, with only a dose of 1 mg/kg (Table 1). Neither GA0113 nor TCV-116 affected HR significantly (Figs. 5B and 6B).

FIG. 5
FIG. 5:
Effects of repeated oral administration of GA0113 on systolic blood pressure(A) and heart rate (B) in spontaneously hypertensive rats. The initial values of systolic blood pressure (mm Hg) and heart rate (beats/min) were not different among the groups (one-way analysis of variance): 189 ± 2 and 370 ± 9 (vehicle), 191 ± 2 and 400 ± 15 (0.03 mg/kg), 189 ± 2 and 398 ± 8 (0.1 mg/kg). All values represent mean ± SEM (n = 7). Significantly different from vehicle group, *p < 0.05; **p < 0.01 (repeated-measures analysis of variance followed by Dunnett's test).
FIG. 6
FIG. 6:
Effects of repeated oral administration of TCV-116 on systolic blood pressure(A) and heart rate (B) in spontaneously hypertensive rats. The initial values of systolic blood pressure (mm Hg) and heart rate were (beats/min) not different among the groups (one-way analysis of variance): 202 ± 1 and 380 ± 10 (vehicle), 206 ± 2 and 375 ± 14 (0.3 mg/kg), 205 ± 2 and 399 ± 9 (1 mg/kg). All values represent mean ± SEM (n = 7). Significantly different from vehicle group, *p < 0.05; **p < 0.01 (repeated-measures analysis of variance followed by Dunnett's test).
Effects of GA0113 and TCV-116 repeated p.o. administration on trough/peak ratio in SHR

Pharmacokinetics in normotensive rats

The mean observed pharmacokinetic parameters are summarized in Table 2. After i.v. administration, CL and Vdss were 99.0 ± 13.3 ml/h/kg and 1.42 ± 0.30 L/kg, respectively. After p.o. administration, the maximal plasma level (Cmax), T1/2, and the time to Cmax (Tmax) were 43.0 ± 2.4 ng/ml, 12.2 ± 2.2 h, and 3.2 ± 1.8 h, respectively. The AUC0-∞ after p.o. administration was 961.4 ± 156.8 ng · h/ml, equivalent to 94% (BA) of the AUC0-∞ after i.v. administration (1,022.7 ± 121.4).

Pharmacokinetic parameters of GA0113 in normotensive rats given single p.o. or i.v. doses


Although GA0113 has a quinoline ring as a key structure, unlike TCV-116 and other AT1 antagonists with biphenyltetrazole, its pharmacologic profile regarding blocking activity and specificity to the AT1 receptor in vitro was similar to that of TCV-116 (CV-11974): GA0113 competitively bound to the AT1 receptor in membrane fractions, but blocked Ang II-induced vascular contraction in an insurmountable manner. The precise mechanism underlying the difference is unclear, but the insurmountable antagonism of TCV-116 has been explained by an extremely slow dissociation of the agent from the receptor (10), although the plasma Ang II level was not measured in this study. Treatment with Ang II-receptor antagonists is accompanied by a marked increase in plasma Ang II levels, as a result of a blockage of the negative-feedback control of renin release (11-14). Therefore it is possible that the AT1-blocking action of AT1-receptor antagonists is attenuated by an increase in plasma Ang II level, especially for antagonists with competitive inhibition. As to mode of inhibition, GA0113 may have an advantage over other competitive antagonists by rigidly blocking the AT1 receptor even where the plasma Ang II level is markedly elevated.

In RALHRs and SHRs, GA0113 produced more potent and long-lasting antihypertensive action than did other AT1 antagonists (15-18). Despite the decrease in blood pressure of >60 mm Hg, no reflex tachycardia was observed in RALHRs. When it was administered repeatedly to SHRs, the antihypertensive action of GA0113 as well as TCV-116 was gradually potentiated and reached a plateau within 4 days. The potency of GA0113 anti-hypertensive activity in SHRs was −10 times greater than that in TCV-116. In addition, GA0113 showed an ideal T/P ratio of >0.5 during the treatment period, superior to that of TCV-116. Pharmacokinetic examination revealed that GA0113 has longer T1/2 (12.2 h) and much better bioavailability (94%) than TCV-116, with a large Vdss, whereas the T1/2 and BA of TCV-116 have been reported to be 3.8 h and 19-28%, respectively (19). It has been proposed that a T/P ratio >0.5 is necessary for optimal 24-h control of blood pressure in a once-daily regimen (20,21), because the intermittent reduction of blood pressure causes a baroreflex-mediated increase in sympathetic tone, which may result in cardiovascular complications, such as left ventricle hypertrophy (22). More recently, some active metabolites of GA0113 were found in rat plasma after oral administration of GA0113. The in vitro and in vivo potency of the main metabolite was found to be similar to that of GA0113, and the Cmax is reached ∼24 h after GA0113 dosing (data not shown). Therefore the metabolite may contribute partially to the prolonged cardiovascular action of GA0113. Taken together, the pharmacokinetic advantages, such as longer half-life and excellent BA in GA0113, show promise for well-controlled antihypertensive effect through once-daily dosing in hypertensive patients, or less, depending on patient variability.

In conclusion, although GA0113 is not structurally related to TCV-116 and other AT1-receptor antagonists having a biphenyltetrazole moiety, it showed insurmountable AT1 antagonism and a well-controlled antihypertensive effect with good pharmacokinetic characteristics. These properties suggest that GA0113 may become a potent agent for the treatment of hypertension and other Ang II-related cardiovascular diseases.


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Angiotensin II; Antihypertensive agent; AT1-receptor antagonist; GA0113; Hypertension; Rat

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