Introduction

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The regulation of body fluid volume is important in almost every area of medicine. AVP is well known to play a major role in water metabolism by inducing water reabsorption at the renal collecting duct through stimulation via V₂ receptors. In various water retaining conditions, such as advanced cardiac failure (Cas et al., 1995; Goldsmith et al., 1983; Szatalowicz et al., 1981), cirrhosis (Bichet et al., 1982), nephrotic syndrome and syndrome of inappropriate ADH secretion (Barter and Schwartz, 1967; Zerbe et al., 1980), the kidneys fail to excrete the amount of water ingested, causing water retention and leading to an excessive water state. Thus, water diuretics (aquaretics) have exciting therapeutic implications in the management of patients with water excess and consequent dilutional hyponatremia (Schrier and Niederberger, 1993; Verbalis, 1993).

Although several potent peptide vasopressin antagonists are currently available (Laszlo et al., 1991; Manning and Sawyer, 1989), none has emerged as a clinically useful antidiuretic antagonist because of their low oral bioavailability, species differences and especially, antidiuretic (agonistic) effects when tested in humans (Albrightson-Winslow et al., 1989; Allison et al., 1988; Brooks et al., 1988; Mah and Hofbauer, 1988). Because two nonpeptide, orally effective AVP antagonists, OPC-21268 and OPC-31260, were respectively reported in 1991 and 1992 (Yamamura et al.), there has been great interest in AVP research and clinical use, encouraging the development of various nonpeptide AVP antagonists (Freidinger and Pettibone, 1997; Serradeil-Le et al., 1993, 1996; Tahara et al., 1997) and some of them have been clinically developed in patient.

The AVP receptor subtypes originally proposed by Michell et al. (1979) were based on primary intracellular signaling mechanisms: cyclic AMP-independent (V₁) and cyclic AMP-dependent (V₂) pathways. Recently, cDNAs encoding the AVP receptors have been cloned and their primary structure identified. AVP receptors were further classified into at least three subtypes: V_1a- (Morel et al., 1992; Thibonnier et al., 1994), V_1b- (De Keyzer et al., 1994; Sugimoto et al., 1994) and V₂-receptors (Birnbaumer et al., 1992; Lolait et al., 1992). Species differences between humans and rats have been reported with peptide AVP antagonists to V₂-receptors and with nonpeptide antagonists to V_1a-receptors (Liu et al., 1994; Hirasawa et al., 1994; Pettibone et al., 1992). In these previous reports, crude membrane preparations or V_1a-receptors expressed only transiently in COS-7 cells were used to determine the species differences. We designed the stable expression of three cloned human AVP receptors in HeLa cells and attempted to develop more potent nonpeptide antagonists to human AVP V₂-receptors. OPC-41061, 7-chloro-5-hydroxy-1-[2-methyl-4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-1-benzazepine, was selected as the most potent human V₂-receptor antagonist through a series of structural conversions of OPC-31260 (fig. 1). In this study, we determined the pharmacological profile of OPC-41061 and its antagonistic action on human AVP receptors using intact HeLa cells stably expressing each subtype of human AVP receptors and on rat AVP receptors using plasma membrane preparations.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Chemical structures of OPC-41061 and OPC-31260.

Furthermore, aquaretics may be well-suited for chronic therapy devoid of the well-known complications of conventional diuretics (saluretics) (e.g., hyponatremia or hypokalemia), and aquaretics may provide the optimum diuretic therapy in water-retaining states. However, peptide V₂-receptor antagonists have failed to show continuous aquaresis by chronic treatment in rats (Hofbauer et al., 1986; Mah et al., 1988) and there have been no reports examining chronic aquaretic treatment in detail. Therefore, in this study we focused on the aquaretic effect by multiple dosing as compared with a single dosing, and on the changes that occur by chronic aquaresis in conscious male rats.

	Materials and Methods

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Materials. OPC-41061 and OPC-31260 were synthesized by the Tokushima 2nd Factory of Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan). AVP was purchased from Peptide Institute Inc., Japan, lipofectamine and fetal calf serum from GIBCO BRL., penicillin, streptomycin and BSA from Sigma, DMEM from Nissui Pharmaceutical Co., [³H]-AVP from New England Nuclear, AVP RIA kit from Mitsubishi Petrochemical Co., and G418 (Geneticin disulfate) and other chemicals from Wako Pure Chemicals, Japan.

Animals. Male Sprague-Dawley rats (SD rats) were purchased from Charles River Japan, Inc., at 6 or 7 wk of age and housed during the experiment in an air-conditioned (temperature-, humidity- and light-controlled) animal room with free access to food and water. All experiments were performed under the regulations of the Guideline for Animal Experimentation in Otsuka Pharmaceutical Co., Ltd.

Preparation of HeLa cells expressing human AVP receptors. The cDNAs for human vasopressin V_1a-receptor was cloned in our laboratory (Hirasawa et al., 1994). For V_1b- and V₂-receptors, cDNA synthesized from human pituitary and kidney RNA was amplified the full length of coding region by using primers for V_1b- (Sugimoto et al., 1994) and V₂- (Birnbaumer et al., 1992) receptors. Cloned cDNAs and PCR products were subcloned into pBluescript KS II (+) (Stratagene, La Jolla, CA). Nucleotide sequence analysis was performed by the ABI 373A DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) for both complete strands. We confirmed that the clones obtained were identical to the previously reported human V_1b- and V₂-receptor cDNA by sequencing, respectively. These cDNA fragments were ligated in SR promoter based mammalian expression vector pME18S and resulting constructs were used for transfection.

Preparation of rat liver and kidney plasma membranes. Liver and kidney plasma membranes were prepared from SD rats (300-400 g, Charles River, Yokohama, Japan). Details of the methods used have been published previously (Yamamura et al., 1991, 1992). Protein concentration was measured by the method of Bradford using BSA as a standard.

Radioligand binding assay to HeLa cells. After reaching confluence in 12-well (V_1a-, V_1b-HeLa) or 24-well (V₂-HeLa) dishes, the cells were washed twice with ice-cold PBS and incubated for 2 hr with [³H]-AVP at 4°C in DMEM medium adjusted to pH 7.4 using 10 mM HEPES-NaOH containing 0.3% BSA. For the competition experiment, OPC-41061 and OPC-31260 were dissolved in dimethyl sulfoxide, diluted with DMEM medium and added into the wells at several appropriate concentrations. After 2 hr of incubation, the cells were rinsed twice with ice-cold phosphate-buffered saline, lysed in 500 (12-well) or 250 (24-well) µl of 0.1 N NaOH containing 0.1% sodium dodecyl sulfate, transferred into scintillation vials, combined with 5 ml of Aquazol II (NEN, Boston, MA) and detected using a liquid scintillation counter (LSC-1050, Aloka, Tokyo, Japan). The protein content of the lysed cells was determined by the method of Bradford.

Radioligand binding assay to rat kidney and liver plasma membranes. Binding assay to rat kidney and liver plasma membranes was performed as described previously (Yamamura et al., 1991, 1992).

cAMP production in V₂-HeLa by AVP. After reaching confluence in 24-well dishes, the V₂-HeLa cells were washed twice with ice-cold PBS and incubated for 10 min with AVP at 1 nM in the presence or absence of OPC-41061 at 37°C in DMEM medium adjusted to pH 7.4 using 10 mM HEPES-NaOH containing 1 mM 3-isobutyl-1-methylxantine and 0.3% BSA. For the competition experiment, OPC-41061 and OPC-31260 were dissolved in DMSO, diluted with DMEM medium and added into the wells at several appropriate concentrations. After 10 min of incubation, the medium was evacuated and the cells were rinsed once with ice-cold PBS. cAMP was extracted from the cells using 250 µl of 0.1 N HCl solution and stored at 20°C until determination using a radioimmunoassay kit (Yamasa, Tokyo, Japan).

Single-dosing experiments in rats. OPC-41061 suspended in 1% hydroxymethyl cellulose or the solvent was administered orally at doses of 0.3 to 10 mg/kg to rats at 8 wk of age (body weight: 280-340 g) by a gastric tube. Seven animals in each dosing group were then placed individually in metabolic cages and spontaneously voided urine was collected for the periods of 0 to 2, 2 to 4, 4 to 6, 6 to 8 and 8 to 24 hr. Another five animals in each group were decapitated at 4 hr postdosing and trunk blood was collected. After centrifugation at 3000 rpm for 10 min, the serum was obtained and frozen until use. Osmolality was determined by freezing point depression using a Fiske osmometer (Model 3400, Boston, MA). Electrolytes were measured by the ion-electrode method (CX-3, Beckman Instruments, Fullerton, CA), and creatinine and urea nitrogen were measured using a COBAS autoanalyzer (COBAS FARAII, Roche, Basel, Switzerland). AVP was measured by radioimmunoassay using a AVP-RIA kit (Mitsubishi Petrochemical Co. Ltd., Tokyo, Japan).

Multiple-dosing experiments in rats. Male SD rats at 7 wk of age (body weight: about 260 g) were divided into two groups. One group (group A) were periodically examined for aquaretic effect and the other (group B) were decapitated for determination of AVP-receptors and AVP content. Six animals were used in each group. OPC-41061 was suspended in 1% hydroxymethyl cellulose solution and orally administered at doses of 1 and 10 mg/kg once daily at around 9:30 A.M. The rats in group A were placed individually in metabolic cages and spontaneously voided urine was periodically (day 1, 1, 3, 7, 10, 14, 21 and 28) collected for the periods of 0 to 4 and 4 to 24 hr. The rats in group B were decapitated at 24 hr postdosing on days 7, 14 and 21 and those in group A were decapitated at 24 hr postdosing on day 28. In the control group, rats were decapitated at predosing (day 1) to obtain basal values. The kidney and liver (for analysis of AVP receptors) and the pituitary (for determination of AVP content) were quickly removed and immediately cooled with ice-cold saline. The removed pituitary was weighed and then stored frozen at 80°C until extraction.

Binding data analysis. The dissociation constant (K_d) and the number of binding sites (Bmax) were determined by Scatchard analysis of saturation binding of [³H]-AVP. The IC₅₀ values of test compounds were determined by displacement experiments. The inhibition constant (K_i) was calculated from the IC₅₀ values using the Cheng and Prusoff equation (1973).

In vivo data analysis. Data from the single-dosing experiment were analyzed by one-way analysis of variance (ANOVA) followed by two-tailed Dunnett's test at a significance level of P < .05. Data from the repeated-dosing experiment were analyzed by ANOVA based on repeated measurements. When ANOVA indicated statistical significance, differences were analyzed by two-tailed Dunnett's multiple-comparison test at each measurement day. Differences were considered significant at P < .05.

	Results

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Antagonistic affinities of OPC-41061 for AVP receptors. As shown in table 1, HeLa cells transfected with each subtype of human AVP receptors constantly expressed a sufficient number of receptors for [³H]-AVP binding assay through repeated passaging. In V₂-HeLa cells, the expressed V₂-receptors acted functionally and stimulated adenylate cyclase after stimulation by AVP (fig. 2). AVP, even at the minimum concentration of 10¹² M, increased the production of cAMP (281 ± 98% from the basal value), with the maximum increase achieved at 10⁸ M. 

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of AVP on cyclic AMP production (A) and inhibitory effect of OPC-41061 on cAMP production induced by 1 nM AVP (B) in V2-HeLa cells. A, Values are expressed as the percent increase of cAMP production from the baseline for 10 min. Values represent the mean ± S.E.M. of three separate determinations performed in triplicate. B, Values are expressed as the mean ± S.D. of triplicate assays. Statistical analysis was not performed because inhibitory effect of OPC-41061 was clearly shown and these data were from one typical experiment of the three performed.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of OPC-41061 and OPC-31260 on [3H]-AVP binding to V2-HeLa cells. Values are expressed as the mean ± S.E.M. of at least five separate determinations performed in duplicate.

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Aquaretic effect by single-dosing in conscious rats. A single oral administration of OPC-41061 increased urine volume and decreased urine osmolality in a dose-dependent manner at doses of 0.3 to 10 mg/kg in normally hydrated conscious rats (fig. 4). The maximum urine output for 2 hr postdosing was 18.0 ± 2.6 ml, which was 12 times higher than the control, and urine osmolality reached a minimum of 175 ± 15 mOsm/kg (vs. 714 ± 136 mOsm/kg for the control). No significant increase in urine output was seen after 4 hr postdosing (data not shown). Urinary Na excretion during the 4 hr postdosing increased dose-dependently. However, the magnitude of the increase was considerably smaller than that by natriuretic agents such as furosemide [data shown previously (Yamamura et al., 1992)].

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Aquaretic effect of a single oral administration of OPC-41061 in normally hydrated conscious rats. OPC-41061 was administered orally in 1% hydroxymethyl cellulose solution. Values are expressed as the mean ± S.E.M. (n = 7). Changes in urine volume and osmolality were analyzed by linear regression analysis and significance level was confirmed. Differences vs. the control group were then analyzed by t test using a closed testing procedure (Marcus et al., 1976; Hothorn and Lehmacher, 1991). * P < .05, ** P < .01.

Aquaretic effect by multiple-dosing in conscious rats. OPC-41061 was orally administered once daily at 1 and 10 mg/kg/day for 28 days. No differences in body weight gain were seen between the OPC-41061 groups and the control group, indicating that OPC-41061 did not affect body weight gain (fig. 5). Figure 6 and table 4 show the effects on urine excretion and serum parameters in rats after multiple dosing of OPC-41061. Throughout the experiment, OPC-41061 at doses of 1 and 10 mg/kg significantly increased urine volume collected during 0 to 4 hr postdosing and decreased urine osmolality. At 1 mg/kg urine volume remained constant throughout the study, but at 10 mg/kg it gradually decreased. However, urine osmolality remained constant at both doses and at each level showed significant differences compared with the control, suggesting that 4-wk repeated administration of OPC-41061 did not alter the compound's aquaretic effect. The excretion of Na and urea nitrogen in the urine collected during 0 to 4 hr postdosing were significantly increased in the OPC-41061 groups, but there were no differences in the net excretion collected during 0 to 24 hr postdosing. There were no differences in the net urinary excretion of creatinine between the OPC-41061 groups and the control group (10 mg/kg: 41.8 ± 1.1 mg/kg/24 hr, 1 mg/kg: 39.4 ± 1.7 mg/kg/24 hr, control: 42.2 ± 1.4 mg/kg/24 hr at day 28), indicating that OPC-41061 did not affect glomerular filtration rate. Urinary AVP excretion was significantly increased in the urine collected during 0-4 hr and 0-24 hr postdosing and remained almost constant throughout the study. It is important to note that, in addition to the constant change in urine osmolality, the constant AVP excretion during the study period further support the conclusion that repeated administration did not alter the aquaretic effect of OPC-41061.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effects of multiple dosing of OPC-41061 on body weight in male SD rats. Values are expressed as the mean ± S.E.M. (n = 6). Differences between the OPC-41061 groups and the control group were analyzed by ANOVA based on repeated measurements, but no statistical significance was seen.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effects of multiple dosing of OPC-41061 on urine collected during 0 to 4 hr postdosing in male SD rats. Values are expressed as the mean ± S.E.M. (n = 6). Differences between the OPC-41061 groups and the control group were analyzed by ANOVA based on repeated measurements (## P < .01) and any significant differences were then analyzed by two-tailed Dunnett's multiple-comparison test at each measurement day (* P < .05, ** P < .01 vs. the control).

	Discussion

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Aquaretics have exciting therapeutic implications in the management of patients with water excess and consequent dilutional hyponatremia, as in patients with congestive heart failure and cirrhosis, or in patients with euvolemic hyponatremia, as in patients with syndrome of inappropriate ADH secretion. There is an obvious need for a potent V₂-receptor antagonist that can be safely administered orally over the long term in a clinical setting. Previous reports of chronic blockade of vasopressin receptors by a peptide V₂-antagonist did not show persistent aquaresis (Hofbauer et al., 1986; Mah et al., 1988). A peptide V₂-antagonist, d-(CH₂)₅-D-Tyr(Et)VAVP, increased urine volume and decreased urine osmolality after i.v. or s.c. administration in normally hydrated or dehydrated Sprague-Dawley rats in acute experiments. However, by chronic administration by i.v. infusion or repeated s.c. dosing, water excretion and intake increased markedly on the first day but then subsequently reverted to normal, although the marked initial water loss was fully compensated for by an increased water intake. The lack of a chronic effect with peptide antagonists is thought to be due to the activation of endogenous compensatory mechanisms such as hormones, prostaglandins and renal and systemic hemodynamics. Alternatively, the lack of effect may be due to an intrinsic agonism which might have contributed to the normalization of water balance by limiting the maximum antiantidiuretic effects of renal tubular AVP receptors.

OPC-41061 did not increase cyclic-AMP production in V₂-HeLa cells and did not show any antidiuretic effect in water-loaded, alcohol-anesthetized rats (data not shown), indicating that OPC-41061 possesses no agonistic activity for V₂-receptors. Therefore, OPC-41061 is expected to produce a chronic aquaretic effect during multiple dosing. In this report, we examined changes not only in the aquaretic effect of OPC-41061 but also in the circulating AVP system and targeted organ function by chronic oral administration of OPC-41061 for 28 days in rats. Urine volume did not change throughout the study period in the 1-mg/kg group, but it decreased slightly in the 10-mg/kg group. The slight decrease in urine volume was considered to be the result of activated compensatory mechanisms. There have been similar reports that the natriuresis by chronic hydrochlorothiazide or furosemide administration markedly abated and sodium excretion was returned to the control level in a so-called "braking effect" (Kahn et al., 1983; Walter and Shirley, 1986). However, in our study, urine volume was decreased only slightly in the first few days and did not return to the control level as was seen in the experiments with peptide AVP antagonists and with hydrochlorothiazide and furosemide. Furthermore, urine osmolality and urinary AVP excretion showed constant change throughout the study period, indicating that the aquaretic effect of OPC-41061 was unchanged during the 28 days of repeated administration. Although OPC-41061 increased the excretion of Na and urea nitrogen in the urine collected during 0 to 4 hr postdosing, serum Na concentration was increased and serum urea nitrogen was decreased significantly at 4 hr postdosing. This discrepancy seems attributable to the fact that hemoconcentration by the increased free water clearance exceeds the Na excretion but not the urea excretion. These changes then returned to the control levels at 24 hr postdosing, because there were no differences in the urine collected during 0 to 24 hr postdosing between the treated and control groups. The excretion of AVP was increased during 0 to 4 hr postdosing and remained almost the same throughout the study period. Serum AVP concentration was increased at 4 hr postdosing and subsequently returned to the control values. There were also no differences in serum AVP concentration, pituitary AVP level, or the number and dissociation constant of AVP receptors in the liver and kidney between the treated and control groups. Although oral administration of OPC-41061 increased AVP secretion from the pituitary and increased circulating AVP for several hours after dosing, both parameters recovered to the basal level by 24 hr postdosing. Because the marked aquaresis by OPC-41061 did not last past 4 hr postdosing, and the initial water loss was fully compensated for by an increasing water intake, there seemed to be no significant changes in the AVP producing and secreting organ and the AVP target receptors. In other circulating hormone systems, serum renin activity tended to be decreased and aldosterone was significantly decreased following by chronic administration. The suppression of renin and aldosterone secretion might be caused by a direct action of the increased AVP on the juxtaglomerular apparatus (Reid et al., 1983) or secondary to an apparent increases in serum sodium after OPC-41061 dosing.

Congestive heart failure is a complex clinical syndrome characterized by a number of neuroendocrine responses. Recent work has established the importance of the renin-angiotensin systems and angiotensin-converting enzyme inhibitors have emerged as distinctly useful drugs in CHF. Therefore, it may be desirable for drugs used in the treatment of CHF to not activate the renin-angiotensin-aldosterone system. Diuretic therapy is a mainstay in the treatment of edematous conditions in CHF. The conventional diuretics (natriuretics) now in use are effective for the treatment of edematous states, but they are associated with activation of the renin-angiotensin-aldosterone systems. Furthermore, natriuretics may cause electrolyte imbalance, such as hyponatremia or hypokalemia. Serum sodium concentration is one of the most powerful predictors of cardiovascular mortality, with hyponatremia patients showing substantially shorter survival than patients with a normal serum sodium concentration (Lee and Packer, 1986). It is not known whether neurohormonal activation and decreased serum Na level are the results of a more advanced degree of heart failure or whether they contribute directly to the progression of mortality. As a result of the aquaresis demonstrated in this report, aquaretics have a potential medical benefit for the treatment of edematous conditions in CHF by removing excess water from the body without activating the renin-angiotensin-aldosterone system or causing serum electrolytes imbalances.

In conclusion, OPC-41061 was characterized as a highly potent nonpeptide human AVP V₂-receptor antagonist with no agonistic properties. The compound's aquaretic effects by single administration were confirmed by an increase in urine volume and a decrease in urine osmolality followed by an increase in serum sodium concentration. Chronic administration for 28 days also confirmed a sustained aquaretic effect with no change in pituitary AVP content or target AVP receptors. Thus, these results support the contention that OPC-41061 may be therapeutically useful for the treatment of various diseases involving body fluid retention. It is also expected that aquaretics will be well-suited for chronic therapy without the well-known side effects of conventional diuretics (saliuretics) on the balance of electrolytes, such as hyponatremia or hypokalemia, and that aquaretics may make possible optimum diuretic therapy in water-retaining states.