Chapter 15 Nonpeptide vasopressin receptor antagonists: development of selective and orally active V1a, V2 and V1b receptor ligands

D. Poulain, S. Oliet and D. Theodosis(Eds.) Progress in Brain Research, Vol. 139

© 2002 ElsevierScienceB.V. All rightsreserved CHAPTER 15

Nonpeptide vasopressin receptor antagonists: development of selective and orally active Via, V2 and Vlb receptor ligands C. Serradeil-Le Gal 1,,, j. Wagnon 2, G. Valette 1, G. Garcia J.P. Maffrand 1 and G. Le Fur 3

2, M.

Pascal l,

1 Exploratory Research Department, Sanofi-Synth(labo Recherche, 195 Route d'Espagne 31036 Toulouse C~dex, France 2 Exploratory Research Department, Sanofi-Synth~labo Recherche, 371 Rue du Professeur J. Blayac 34184 Montpellier C~dex 04, France 3 Sanofi-Synthdlabo Recherche, 174 Avenue de France, 75635 Paris C~dex 13, France

Abstract: The involvement of vasopressin (AVP) in several pathological states has been reported recently and the selective blockade of the different AVP receptors could offer new clinical perspectives. During the past few years, various selective, orally active AVP Via (OPC-21268, SR49059 (Relcovaptan)), V2 (OPC-31260, OPC-41061 (Tolvaptan), VPA-985 (Lixivaptan), SR121463, VP-343, FR-161282) and mixed VIa/V 2 (YM-087 (Conivaptan), JTV-605, CL-385004) receptor antagonists have been intensively studied in various animal models and have reached, Phase Ilb clinical trials lbr some of them. For many years now, our laboratory has focused on the identification of nonpeptide vasopressin antagonists with suitable oral bioavailability. Using random screening on small molecule libraries, followed by rational SAR and modelization, we identified a chemical series of l-phenylsulfonylindolines which first yielded SR49059, a Via receptor antagonist prototype. This compound displayed high affinity for animal and human Via receptors and antagonized various Via AVP-induced effects in vitro and in vivo (intracellular [Ca2+] increase, platelet aggregation, vascular smooth muscle cell proliferation, hypertension and coronary vasospasm). We and others have used this compound to study the role of AVP in various animal models. Recent findings from clinical trials show a potential interest for SR49059 in the treatment of dysmenorrhea and in Raynaud's disease. Structural modifications and simplifications performed in the SR49059 chemical series yielded highly specific V2 receptor antagonists (N-arylsulfonyl-oxindoles), amongst them SR121463 which possesses powerful oral aquaretic properties in various animal species and in man. SR121463 is well-tolerated and dose-dependently increases urine output and decreases urine osmolality. It induces free water-excretion without affecting electrolyte balance in contrast to classical diuretics (e.g. furosemide and hydrochlorothiazide). Notably, in cirrhotic rats with ascites and impaired renal function, a 10-day oral treatment with SR121463 (0.5 mg/kg) totally corrected hyponatremia and restored normal urine excretion. This compound also displayed interesting new properties in a rabbit model of ocular hypertension, decreasing intraocular pressure after single or repeated instillation. Thus, V2 receptor blockade could be of interest in several water-retaining diseases such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), liver cirrhosis and congestive heart failure and deserves to be widely explored. Finally, further chemical developments in the oxindole family have led to the first specific and orally active Vlb receptor antagonists (with SSR149415 as a representative), an awaited class of drugs with expected therapeutic interest mainly in ACTH-secreting tumors and various emotional diseases such as stress-related disorders, anxiety and depression. However, from the recently described tissue locali-

* Correspondence to: C. Serradeil-Le Gal, Exploratory Research Department, Sanofi-Synthrlabo Recherche, 195 Route d'Espagne 31036 Toulouse Cfdex, France. Tel.: +33-5-6116-2384; Fax: +33-5-6116-2586; E-mail: claudine.serradeil@ sanofi-synthelabo.com

198 zation for this receptor, we could also speculate on other unexpected uses. In conclusion, the development of AVP receptor antagonists is a field of intensive pharmacological and clinical investigation. Selective and orally active compounds are now available to give new insight into the pathophysiological role of AVP and to provide promising drugs.

Keywords: Vasopressin; Vla receptor; Vlb receptor; V2 receptor; Nonpeptide antagonist; SSR149415; SR49059; SR121463; Aquaretics; ACTH

Introduction

AVP, functions and receptors Vasopressin (AVP) is a cyclic nonapeptide which exerts a variety of biological effects in mammals. The primary role of AVP involves the regulation of water and solute excretion by the kidney. However, this hormone is also actively involved in a number of other physiological functions including blood pressure control, platelet aggregation, liver glycogenolysis and neoglucogenesis, uterus contraction, cell proliferation, adrenocorticotropin (ACTH) release by the adenohypophysis, aldosterone secretion by the adrenals and clotting factor release (Barberis et al., 1999). Together with oxytocin (OT), another structurally related nonapeptide, AVP is also implicated in intemeuronal communication in the central nervous system (CNS) and modulates several behavioral functions such as feeding, memory, thermoregulation and the control of adaptative, social and sexual processes (Dreifuss et al., 1991). These central and peripheral effects of AVP are based upon a local or systemic release pattern into the organism and occur via interaction with specific seven transmembrane G-protein-coupled receptors. Three AVP receptors (Via, Vlb (or V3) and V2) and one type of OT receptor have been cloned in animal species and in man and have been clearly identified by their primary structure, gene localization, mRNA distribution, pharmacology and functions (Lolait et al., 1995; Thibonnier et al., 1998). Briefly, the AVP V1 (Via and V~b) receptors mediate phospholipase C activation and intracellular calcium mobilization. AVP Via receptors have a ubiquitous central and peripheral localization (liver, platelet, uterus, brain etc). The AVP Vlb receptors are involved mainly in the stimulating effect of AVP on ACTH secretion in the pituitary but, as recently demonstrated, Vlb receptors have a wide distribution in the rat brain and also an endocrine role

in other organs such as the pancreas and the adrenals (Lee et al., 1995; Grazzini et al., 1996). Finally, to complete the review of this field, two putative receptors for AVP (or related peptides) have been reported. First, the AVP-activated Ca 2+ mobilizing (VACM-1) receptors, unlike other AVP receptors, appear to possess only one transmembrane domain and reportedly bind AVP activating a Ca 2+ second messenger pathway. VACM-1 also modulates the cAMP response of V2 receptors when expressed in CHO cells. The cDNA for VACM-1 has been cloned from rabbit, rat and human tissues and mRNA is present in numerous, if not all, tissues, including brain, kidney and endothelial cells. Recently, it has been shown that the VACM-1 protein is identical to cullin-5, which belongs to a newly discovered family of proteins implicated in the cell cycle, clearly different from the AVP/OT receptor family (Burnatowska-Hledin et al., 1995, 2000). Secondly, a novel dual angiotensin II (Ang II)/AVP receptor with an AT1/V2 receptor profile has revealed specific binding for both Ang II and AVP with positive coupling to adenylyl cyclase in response to both hormones. It has been cloned in the rat and in man. This receptor localized in the renal thick ascending limb tubules and collecting ducts, may be involved in renal tubular Na + and fluid reabsorption. Recent data have also shown a wide distribution in the rat CNS (Ruiz-Opazo et al., 1995; Hurbin et al., 2000). However, clear characterization of this 7-TM receptor using reference peptide and nonpeptide AVP/OT ligands is needed to further explore this entity. At present, the biological role of these atypical proteins, VACM-1 and the mixed AVP/Ang II receptor, remains to be defined.

Design of AVP receptor antagonists In the 1980s, a number of cyclic and linear peptide receptor antagonists and agonists derived from

199 the natural hormone and exhibiting various selectivity profiles for AVP and OT receptors were designed by M. Manning in collaboration with W. Sawyer. Amongst them, d(CHz)sTyr(Me)AVP, a selective AVP Via receptor antagonist still used as a V~a reference; d(CH2)5[Tyr (Et) 2, Val 4, D-ArgS]Vp, a potent V2/V ~a antagonist with aquaretic properties in the rat; d(CHz)5[D-Ile 2, Ile4]AVP, the first selective V2 peptide ligand and Aaa-D-Tyr(Et)-PheVal-Asn-Abu-Pro-Arg-Arg(NH2), the first linear Vz/VI~ antagonist, represented key steps in the story of peptide AVP receptor antagonists (Manning and Sawyer, 1989, 1991; Jard, 1998). They offered the first valuable pharmacological tools for the classification of AVP/OT receptors and for the acute characterization of their role. They also represented the first generation of radiolabeled ligands for mapping the distribution of AVP and OT receptors. Of note, the team from SmithKline and Beecham Laboratories was particularly active in developing peptide V2 receptor antagonists for clinical purposes. Unfortunately, due to profound species differences well-known in the field of AVP, molecules which were potent V2 receptor antagonists in several animal models turned out to be V2 receptor agonists in humans (Allison et al., 1988). More recently, in the 1990s, besides the molecular cloning of the 4 AVP/OT receptor subtypes, the first nonpeptide AVP Vla/V2 receptor antagonists appeared and allowed, due to their oral bioavailability, clinical evaluation of their therapeutic uses. Chemical structures selective for Via receptors or V2 receptors or exhibiting a mixed V1a/V2 antagonist profile became available and were intensively studied in various animal models and evaluated in clinical trials (Albright and Chan, 1997; Thibonnier et al., 2001). Finally, very recently in the new millennium, the first selective Vlb ligands were discovered (Derick et al., 2000; Serradeil-Le Gal et al., 2002). In the present chapterz we will first review the different orally active, nonpeptide AVP receptor antagonists reported up to now and their potential therapeutic indications. Since our laboratory has focused for many years on the identification of nonpeptide AVP antagonists with suitable oral bioavailability, we will present some recent pharmacological and clinical data supporting their main therapeutic indications. For more details of the development of

orally active Via, V2 AVP receptor antagonists in general, the reader is referred to the many excellent and accurate reports that have been published recently (Albright and Chan, 1997; Freidringer and Pettibone, 1997; Mayinger and Hensen, 1999; Thibonnier, 1999; Paranjape and Thibonnier, 2001; Thibonnier et al., 2001). The second part of this chapter focuses on the pharmacological characterization of SSR149415, the first selective V~b receptor antagonist described so far (Serradeil-Le Gal et al., 2002). This molecule constitutes a unique probe for exploring the poorly known V~b receptor and the interest of Vlb blockade in several pathological states.

Nonpeptide AVP Vla and/or V2 receptor antagonists and their potential clinical indications Resulting from high throughput screening (HTS) of thousands of small nonpeptide molecules (molecular weight ~ 500) belonging to a variety of chemical libraries, the first orally active AVP receptor antagonists were reported in the 1990s and today, an impressive number of patents covers the field of nonpeptide AVP ligands. Only some of them, with different selectivity profiles (Via, V2, Via and V2) are undergoing clinical trials (Phase IIb for the most advanced) and the results are eagerly awaited to evaluate both the benefit and the safety of this class of drugs. The term 'Vaptan' has been coined to officially name all the members of this new class of drugs (e.g. Relcovaptan (SR49059), Tolvaptan (OPC-41061), Lixivaptan (VPA 985) and Conivaptan (YM-087)).

Nonpeptide Via receptor antagonists The first Via receptor antagonists, OPC-21268 and SR49059 (Relcoptan) were described in 1991 and 1993, respectively, and have been involved in various clinical trials (Yamamura et al., 1991; Serradeil-Le Gal et al., 1993). They are still the major players identified in this field. These molecules, belonging to different chemical series (quinolinone and indoline series, respectively), were obtained following chemical optimization and SAR of a lead compound found by random screening. It is important to underline that the OPC-quinolinone series was the starting point for the design of many AVP antago-

200 TABLE 1 Orally active, nonpeptide AVP Via receptor antagonists reported in 2001 and their chemical structures

( 1) (2) (3) (4) (5) (6)

~o

Name

Company

Chemical series

Development status

OPC-21268 SR49059 (Relcovaptan) None None None None

Otsuka Sanofi-Synthflabo Eli Lilly Yamanouchi Fujisawa Yamanouchi

Quinolinone derivative N-Sulfonyl-indoline derivative Azetidinone derivatives Benzazepine derivatives Benzazepine derivatives Triazole derivatives

Phase II Japan; stopped US/Europe Phase II/stopped Preclinical Preclinical Preclinical Preclinical

(4) o

OPC-21268

oyo

~"~ (3)

o~c"~,

(1) ~OCH3 SR49059 OCH3

\

(6)

nists reported by several competitors (Tables 1-3). Of note, structure-activity relationships among analogues of OPC-21268 yielded also derivatives with marked affinity for the human OT receptor further developed by Merck as selective OT receptor antagonists for the treatment of preterm labor (Freidringer and Pettibone, 1997). In addition, rational modifications of these selective Via structures (OPC-21268 and SR49059) further yielded potent selective V2, mixed V1 and V2, and more recently, pure Vlb receptor antagonists (in the case of SR49059) with oral bioavailability, as illustrated in Tables 2, 3 and 5. More recently, two other chemical series of Via receptor ligands described in patents by Eli Lilly as azetidine derivatives and by Yamamouchi as triazole derivatives, have emerged (see illustration with compounds 3 and 6, respectively, in Table 1). However, up to now, no specific lead compounds have been identified and no pharmacological studies are reported with these new derivatives.

N--.

Clinical indications for nonpeptide Via receptor antagonists Due to the ubiquitous localization of Via (brain, vessels, platelet, uterus, adrenals... ) and to their various central and peripheral biological effects (see Section 1), Via blockade could be of interest in a large number of diseases. AVP may be increased in several pathological situations such as hypertension, congestive heart failure, dysmenorrhea, brain edema, small cell lung cancers and various CNS disorders (depression, anxiety... ) (Table 4). Due to profound interspecies variability in AVP/ OT receptors (Allison et al., 1988; Pettibone et al., 1992), the affinity of OPC-21268 for the human Via receptor was very weak and this compound failed to prevent AVP-induced contractions in various human vascular preparations in vitro (Serradeil-Le Gal et al., 1993; Burrell et al., 1994). If some clinical trials have been reported with this compound, its loss of affinity for the human Via receptors has severely precluded clinical developments. At variance with OPC-21268, SR49059 exhibited a constant high affinity for animal and human Via receptors and

201 TABLE 2 Orally active, non-peptideAVP V2 receptor antagonists reported in 2001 and their chemical structures

( 1) (2) (3) (4) (5) (6) (7) (8) (9)

Name

Company

Chemical series

Developmentstatus

OPC-31260 OPC-41061(Tolvaptan) SR121463 VPA-985 (Lixivaptan) WAY-140288 VP-343 and VP-339 VP-365 FR161282 None

Otsuka Otsuka Sanofi-Synthrlabo Wyeth-Ayerst Wyeth-Ayerst Wakamoto Wakamoto Fujisawa Ortho-McNeil

Benzazepine derivative Benzazepine derivative N-Arylsulfonyl-oxindolederivative Benzodiazepine derivative Benzodiazepine derivative Quinoxaline derivative Benzodiazepine derivative Benzodiazepine derivative Benzothiazepine derivatives

Phase II Phase II Phase II Phase II Preclinical Preclinical Preclinical Preclinical Preclinical

H3C~N, CH3

H•O•N'•I""i 0

CH3CH20~~O O~N OPC.31260 °

H

HNOC/~OCH3 I

SR121463

OH

O C I ~ NH CH3 OPC-41061

.3c-~,.-~

H3C~N H3C

. VPA-985

O

~

~H3

WAY140288~

F

has been intensively studied in pharmacological and clinical trials as a prototype drug (Serradeil-Le Gal et al., 1993). From several proof of concept studies performed with SR49059 - - highly speculative for some of them - - a number of clinical indications for AVP Via receptor antagonists have clearly emerged. Since AVP exerts a powerful local vasoconstriction effect in several crucial vascular beds (renal, gastric, c o r o n a r y . . . ), SR49059 effects were assessed in patients suffering from Raynaud's disease. In a double-blind, placebo-controlled randomized cross-over study, at 300 mg p.o. once daily, SR49059 significantly antagonized (15%) cold-induced decrease in finger systolic blood pressure following a cold immersion test and accelerated temperature recovery (Hayoz et al., 2000). These preliminary results suggested an involvement of AVP in the abnormal vasoactive response of Raynaud's patients

o

°

O~]~ NH

O~

NH

(9) ~

together with a potential interest of V~a blockade in this pathology. Interesting results were also obtained with SR49059 in the treatment of dysmenorrhea. AVP plasma levels are thought to be increased in primary dysmenorrhea and this hormone is clearly involved in the development of pain by stimulating both vascular and uterine Via receptors (Bossmar et al., 1997). In a double-blind, randomized cross-over Phase lib study, SR49059 (100 and 300 mg) induced a dose-related positive effect in reducing pelvic pain during the first 24 h in primary dysmenorrhea, with a simultaneous decrease in the need for analgesic rescue (Brouard et al., 2000). Up to now, no convincing results are available with Via receptor antagonists in hypertension, either in various animals models or in human, even if AVP seems to be involved in some forms of essential hypertension, in particular, in African-American

202 TABLE 3 Orally active, non-peptide AVP Va/V 2 receptor antagonists reported in 2001 and their chemical structures

(1) (2) (3) (4) (5)

Name

Company

Chemical series

Development status

YM-087 (Conivaptan) YM-471 JVT-605 CL-385004 None

Yamanouchi Yamanouchi Japan Tobacco Wyeth-Ayerst Fujisawa

Benzazepine derivative Benzazepine derivative Thiazepine derivative Benzodiazepine derivative Benzazepine derivatives

Phase II Preclinical Preclinical Preclinical Preclinical

YM-471

~

c.,

@,

H3

0 ~ / O ~

O ~ ] , , ~ NH

O~L.NH

INNH CH3

O /~ /CH3 O(CH2)3C--N~.~'~ N\CH CL-38500 NH

JTV-605

YM-O87

" - u N H

(5)

o

TABLE 4 Main potential clinical indications for AVP Via, V2, dual Vla/V 2 and Vlb receptor antagonists Via antagonists

Dysmenorrhea, preterm labor Raynaud's disease Hypertension Congestive failure Brain edema Motion sickness Oncology (SCLC) CNS disorders

V2 antagonists

Mixed Vla/V 2 antagonists

Vlb antagonists

Congestive heart failure SIADH Liver cirrhosis with ascites and water retention Hyponatremia Nephrotic syndrome Brain edema Glaucoma Hypertension Diabetic nephropathy Meniere's disease

Congestive heart failure Hypertension Brain edema

Stress-related disorders, anxiety, depression ACTH-secreting tumors, Cushing's syndrome HPA axis disorders

patients with elevated AVP p l a s m a levels. A n exploratory study with SR49059 (300 mg, single dose) in a situation o f osmotic release o f AVP (induced by a 5% hypertonic saline infusion) in black hypertensive patients failed to demonstrate a sustained blood pressure reduction (Thibonnier et al., 1999). It is

generally assumed that blockade of both Vla and V 2 receptors needs to be achieved to reach a significant improvement in blood pressure alterations. A number of other clinical indications remain to be investigated, in particular CNS disorders with compounds able to cross the b l o o d - b r a i n barrier (Table 4).

203

Nonpeptide V2 receptor antagonists While only two AVP Via receptor antagonists have been described, numerous selective, orally active AVP V2 receptor antagonists have been reported by several pharmaceutical companies. Ten lead compounds can be identified at various stages of investigation, with four molecules currently involved in clinical developments: OPC-31206, OPC-41061 (Tolvaptan), SR121463 and VPA-985 (Lixivaptan) (Table 2). By modifying the dihydroquinolinone chemical structure of their V~a receptor antagonist, OPC-21268, Otsuka reported in 1992 an orally active Vz compound, OPC-31260, and more recently a back-up/follow-up molecule, OPC-41061 (Tolvaptan) in the benzazepine chemical series (Yamamura et al., 1992, 1998). As shown in Table 2, the other V2 receptor antagonists reported by various pharmaceutical firms are all benzazepine derivatives, except SR121463, an oxindole derivative resulting from optimization and simplification of our Via receptor antagonist, SR49059. In order to simplify the 1-phenylsufonyl indoline structure of SR49059 bearing three asymmetric carbons, we designed Narylsufonyl-oxindoles yielding highly specific V2 ligands among which SR121463 was chosen for further preclinical and clinical development (SerradeilLe Gal et al., 1996; Serradeil-Le Gal, 2001).

Clinical indications for V2 receptor antagonists The generation of receptor-specific AVP V 2 antagonists, so-called 'aquaretics', able to block the antidiuretic action of AVP in the collecting duct cells, and thus to specifically promote water excretion by preventing the insertion of AVP-specific water channels (aquaporin, AQP-2) into the luminal membrane, could be of high therapeutic value for the treatment of several water-retaining disorders such as SIADH, liver cirrhosis, certain stages of congestive heart failure and hypertension, nephrotic syndrome, renal failure... (Table 4). In most of these diseases an abnormal increase of circulating AVP plasma level, activating renal V2 receptors, seems to be the key event in water retention and subsequent hypotonic hyponatremia (Goldsmith et al., 1989; Gavras, 1991; Sorensen et al., 1995). Thus, for these pathologies, there is great clinical interest in the development

of potent Va receptor antagonists to provide specific water diuretic/aquaretic compounds devoid of the well-known side effects of classical diuretic or saliuretic agents on the urine Na + and/or K + loss. According to the above rationale, several clinical trials are reported with OPC compounds and VPA-985 in CHF, cirrhosis with ascites, SIADH with subsequent hyponatremia and in congestive heart failure (Thibonnier, 1999; Paranjape and Thibonnier, 2001). Whatever the compound used, urine volume was increased, urine osmolality decreased and a normalization of serum Na + observed. Results of repeated chronic treatments in these pathologies are eagerly awaited, but are available in some animal models. Of note, SR121463 demonstrated benefit in a model of cirrhosis (CC14-induced) in rats with ascites, water retention and impaired renal function after chronic treatment. Ten-day repeated oral administration of SR121463 (0.5 mg/kg) normalized serum Na + and totally corrected hyponatremia. SR121463 restored normal urine excretion, urine osmolality and renal function since after a water overload, cirrhotic rats excreted similar urine volume as control noncirrhotic rats (Jimenez et al., 2000). In addition, due to the extrarenal localization of AVP V2 receptors (brain, endothelial, lung... ), other therapeutic areas deserve to be explored. For exampie, V2 receptor blockade could be of interest in brain edema or more surprisingly in glaucoma. As recently shown, in a rabbit model of ocular hypertension (c~-chymotrypsin-induced), SR121463, after single or repeated (10 days at 1%) instillation, markedly decreased intraocular pressure with similar efficacy to the currently used el (clonidine) or [3-adrenergic (timolol) treatments, demonstrating the potential benefits of V2 receptor antagonists in decreasing intraocular pressure via a mechanism of action that remains to be elucidated. Moreover, it suggested the presence of ocular V2 receptors (Lacheretz et al., 2000).

Dual nonpeptide VI~, V2 receptor antagonists The class of dual Vla and V 2 receptor antagonists has been extended and includes now several lead compounds reported in Table 3, but only one molecule YM-087 (Conivaptan) has been tested in humans; clinical trials are reported mainly in CHF (Norman et al., 2000; Udelson et al., 2000). Of note, all these

204 compounds are benzazepine derivatives obtained by modifying the chemical structure of the first AVP receptor antagonists (OPC-2128, OPC-31260). According to the compound, the affinity and activity ratio at Via and V2 receptors is highly variable, generating various pharmacological profiles for these drugs. Interestingly, this Via/V2 ratio seems a key factor when considering therapeutic purposes. Dual Via and V2 receptor blockade is expected to modify both systemic hemodynamic and renal parameters. This strategy could be of interest in developing antihypertensive agents. Pure V~a receptor antagonists are reported to be inactive per se in various animal and human models of hypertension (even with increased AVP plasma levels). It is assumed that blockade of both Via and V2 receptors will achieve a decrease in blood pressure by modifying both peripheral resistances and circulating blood volume. Similarly, dual Vla/V2 blockade could improve hemodynamic and fluid status in CHE Finally, treatment of brain edema with this class of drugs needs also to be explored, based on the rationale that pure Via and pure V2 receptor antagonists have both shown a benefit in the development of this pathology by decreasing brain water content and restoring brain Na + content with a decrease in neurogenic inflammation. An involvement of peripheral and probably direct effect on brain vessel permeability and choroid plexus could be speculated even if the intrinsic mechanism of action is not known (Bemana et al., 1997; Laszlo et al., 1999).

Vlb receptor antagonists Vlb receptor ligands The recently cloned Vlb receptor, mainly found in the adenohypophysis, is involved in the stimulating effect of AVP on ACTH secretion (De Keyzer et al., 1994; Sugimoto et al., 1994). AVP is a direct ACTH secretagogue but also synergizes corticotropin-releasing factor (CRF)-induced ACTH release in the pituitary (Gillies et al., 1982). This receptor has a wide distribution in various tissues such as the brain, adrenals, kidney and pancreas. To date, due to the lack of selective Vlb receptor ligands (agonists/antagonists) and to the absence of orally active Vlb receptor antagonists, the Vlb receptor is still poorly characterized and the precise role of AVP

via central and peripheral Vlb receptors remains to be elucidated. Interestingly, to explore the functions of this receptor, a knockout mouse has recently been generated (Lolait et al., 2000). In contrast to the numerous potent and selective, peptide and nonpeptide Via and V2 receptor ligands, only a few non-selective Vlb peptides are available: [D-3(pyridyl)Ala2]AVP, a specific Vlb agonist in rats turned out to be a Via, V1b ligand at human AVP receptors; dDAVP displays both agonist V2/Vlb properties and a recent series of dDAVP analogues modified at position 2 yielded full Vlb/partial Via agonists (Derick et al., 2000). Finally, the reference peptide antagonist, [deaminopenicillamine-O-Me-Tyr, Arg]AVP, (dPen), is a dual Vlb/Vla ligand. Recently, we have developed the first selective, nonpeptide and orally active Vlb receptor antagonist to be described, SSR149415 (Serradeit-Le Gal et al., 2002 (Table 5)). It results from chemical optimization in the field of the indoline/oxoindole chemical series which has previously yielded nonpeptide molecules highly selective for the Via (SR49059, Tables 1 and 6) and the for the V2 (SR121463, Tables 2 and 6) receptors.

Pharmacological profile of SSR149415 As shown in Table 6, SSR149415 displays nanomolar affinity for human Vlb receptors. It exhibits a TABLE 5 Orally active nonpeptide AVP Vlb receptor antagonists reported in 2002 Name

Company

Chemical series

Development status

SSR149415

SanofiSynth61abo

N-Arylsulfonyloxindole derivative

Preclinical

~

OH

OCH3 -

I SOa /~OCH3 CHaO

0

205 TABLE 6 Comparative affinities of the nonpeptide compounds, SR49059, SR121463 and SSRl49415 for human AVP (Via, Vlb, V2) and OT receptors

Ki (nM) SR49059 SR121463 SSR149415 AVP OT

h-Via

h-Vib

h-V2

h-OT

6.34-0.6 4604-120 91 4-23 1.7 64

220:t:30 >10,000 1.54-0.8 1.1 1782

275-t-50 4.1-t-0.8 14124-314 1.1 167

320-t-168 12134-383 174-t-35 16 0.9

Affinities of the natural hormones, AVP and OT, as references. Inhibition constants (Ki) were determined from competition experiments calculated according to the Cheng and Prussoff equation. Values are the mean + SEM of at least three determinations.

highly selective profile versus Via, V2 and OT human receptors and has no measurable affinity for a number of other receptors (n = 100). As illustrated in Fig. 1, SSR149415 dose-dependently antagonized [3H]AVP binding to human Vm receptors with an affinity for human glb receptors close to that of the natural hormone, AVP ( K i values of 1.54 4- 0.82 and 0.80 :k 0.25 nM, respectively). SSR149415 exhibited much higher affinity than the nonselective reference

100 1 7.

80

,'v

60

~,

40 20

I m

• 0

10"1°

-

- ,---i

10 "9

- -,---

10 .8

10 7

10 .6

CONCENTRATION [MI Fig. 1. Inhibition of [3H]AVP specific binding to human Vlb receptors by SSR149415 (v) and reference peptide compounds: AVP (o); dDAVP, [desamino-[D-Arg]vasopressinl (I); ripen, [desamino-[o-Arg]vasopressin] (o) and dPal, [(deamino-Cys, D3-(Pyridyl)-Ala2-ArgS)-vasopressin] (V). Binding assays were performed for 45 min at 20°C in the presence of 30 gg/assay of CHO membranes expressing the human Vlb receptors. Results represent data from a typical experiment performed in duplicate, which was repeated three times without noticeable differences.

agonist (dDAVP, [D-3(pyridyl)Ala2]AVP) and antagonist (dPen) Vlb peptides (Ki values of 20-4-8, 12 + 5 and 21 4- 6 nM, respectively). Ki values obtained for these peptides are consistent with affinities previously reported for the human Vlb receptor. In saturation binding experiments followed by Scatchard analysis, SSR149415 inhibited [3H]AVP binding in a competitive manner with a Ki value of 2.51 4- 0.45 nM (Serradeil-Le Gal et al., 2002). Earlier cellular events upstream of ACTH release, provoked by occupancy of corticotroph V i b receptors by AVP, include the activation of phospholipase C, protein kinase C and the mobilization of intracellular free Ca e+ mainly via Gq/ll G-protein recruitment. In CHO cells transfected with the human Vlb receptor other intracellular pathways have also been described (e.g. cAMP production, stimulation of DNA synthesis and cell proliferation), clearly depending on the level of V1b receptor expression (Thibonnier et al., 1997). In CHO cells expressing the human Vlb receptors AVP-induced cell proliferation with an ECs0 value of 0.144-0.12 nM (n = 4) as measured by a colorimetric method with the tetrazolium compound (MTS) according to (Thibonnier et al., 1998). In this latter model, SSR149415 (Fig. 2) dose-dependently antagonized stimulation of cell proliferation by AVP (3 nM) with a Ki value of 0.43 4- 0.41 nM consistent with the SSR149415 affinity found in binding studies using the same cellular preparation. In vivo, pharmacology performed measuring ACTH secretion induced by various stimulants, such as hormones and physical stress, confirmed the full antagonist profile of SSR149415. In all these situations, SSR149415 antagonized ACTH secretion, which constitutes a critical response of the organism to stress in emotional situations (Aguilera and Rabadan-Diehl, 2000). It is important to note that SSR149415 demonstrated high potency by the oral route on the potentiation of the effect on CRF by AVP, a mechanism described as typically Vlbmediated. Significant inhibition of ACTH secretion was observed from a dose of 3 mg/kg p.o., total blockade occurred at 10 mg/kg p.o., an effect that lasted for more than 4 h (Fig. 3). Conversely, the selective, orally active Via receptor antagonist, SR49059, was unable to inhibit AVP plus CRFinduced ACTH secretion, demonstrating a specific Vtb-mediated effect. Various physical stresses are

206

(J o~

80

"~

60

z

40

_o

:!11

(A)

=o 100

100 • ................................................................... ~ ' - ' ,,

/

O - e ,uIn,,~.

,,,u+

"' :Z >

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SSR149415 [M} Fig. 2. Inhibition by SSR149415 of AVP-inducedproliferation of CHO cells transfected with the human Vlb receptor. CHO cells were grown for 48 h in 96-well plates (5000 cells/well). Cell proliferation was measured using the CellTiter 96 cell proliferation assay from Promega (Madison, WI). Cells were washed with 200 Ixl PBS and treated with AVP (3 riM) and increasing concentrations of SSR149415. After 18 h of incubation (37°C, 5% CO2, 80% humidity), 20 Ixl dye solution was added to each well. The plate was incubated for 4 h and absorbance was recorded at 492 nm wavelength. Results represent data from a typical experiment which was repeated four times without noticeable change.

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. able to induce A C T H secretion. The stress-induced release of A C T H is believed to involve the activation of several humoral and neural pathways, including that mediated by AVP (Linton et al., 1985). As shown in Fig. 4, in rats submitted to an immobilization period of 15 min, there was a significant increase (more than 5-fold) in plasma A C T H levels. Pretreatment with SSR149415 ( 3 - 1 0 m g / k g i.p.) 30 rain before the restraint stress period, caused dosedependent inhibition of plasma A C T H elevation in comparison with stressed animals treated with the corresponding vehicle. The regulation of A C T H secretion and consequently of the HPA axis are largely mediated by AVP and V lb receptors, and S SR 149415 offers a new tool for controlling emotional or physical stress. Indeed, several neuroendocrine studies strongly suggest that dysregulation of the HPA system plays a causal role in the development and the course of diseases such as generalized anxiety, depression and addiction (Holsboer, 1999). In conclusion, SSR149415 is a potent, selective and orally active Vlb receptor antagonist. Whatever the model, SSR149415 is devoid of any V]b agonist

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Fig. 3. Effect of oral SSR149415 on the potentiation of exogenous CRF by AVP on ACTH secretion in conscious rats. Dose-effect (A) and time-course (B) studies. (A) SSR149415 (1-10 mg/kg) was administered by gavage 2 h prior to CRF (0.1 txg/kg i.v.) and AVP (0.03 Ixg/kg i.v.) injection. Inset: effect of a selective orally active Via receptor antagonist, SR49059 (1-10 mg/kg) in this model. (B) The time-course effect of SSR149415 was studied at 10 mg/kg p.o. on plasma ACTH secretion (n = 5-10). Values are expressed as the percentage of ACTH secretion versus the CRF plus AVP control. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett's test or using the nonparametric Kruskal-Wallis test. (* P < 0.05; ** P < 0.01 versus control).

effect when tested alone. It is a unique tool for exploring the localization and the role of V]b receptors. This class of drugs exhibits a promising therapeutic profile for the treatment of stress-related disorders, anxiety and depression. However, due to the ubiquitous localization of the Vlb receptor, several other

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Fig. 4. Effect of SSR149415 on restraint stress-induced ACTH secretion in conscious rats. Rats (8-10 per group) received intraperitoneal injection of the vehicle (2 ml/kg 5% DMSO, 5% cremophor, 90% saline) or SSR149415 (3 and 10 mg/kg). Thirty minutes after the injection, rats were placed in plexiglass restrainers for 15 min. At the end of the immobilization period, they were sacrificed by decapitation and plasma ACTH levels were measured by RIA. Data are mean 4- SEM. Statistical significance was assessed by an ANOVA on transformed logarithmic data followed by Dunett's test (** P < 0.01 vs. control - unrestraint animals; ~ P < 0.01 vs. stress control - restraint animals).

therapeutic indications need to be investigated (Table 4). Conclusion

AVP mediates a wide number of biological effects and may be involved in several pathological states. Selective blockade of the different AVP receptors, therefore, could offer new clinical perspectives for treating several diseases (Table 4). In 2001, four classes of orally active AVP receptor nonpeptide antagonists (Via, V2, Vla + V2, and V~b) were available, constituting the 'Vaptan family'. In the past decade, various, selective, nonpeptide and orally active AVP Via (OPC-21268, SR49059 (Retcovaptan)), V2 (OPC-31260, OPC-41061 (Tolvaptan), VPA985 (Lixivaptan), SR121463, VP-343, FR-161282, WAY-140288) and mixed V]a/V2 (YM-087 (Conivaptan), JTV-605, CL-385004) receptor antagonists have been intensively studied in various animal models and have reached, for some of them, Phase IIb clinical trials. It should be noted, however, that no compounds are on the market, many clinical trials and proof of concept studies are still ongoing and the results of long-term treatments are eagerly awaited to evaluate the benefit and the safety of this new class of drugs.

A wide variety of therapeutic indications could be targeted with these compounds: SR49059, a potent and selective Via receptor antagonist may be of interest in the treatment of dysmenorrhea and in Raynaud's disease while V2 receptor antagonists were of benefit in several water-retaining diseases (CHF, SIADH and hepatic cirrhosis) by inducing powerful aquaretic effects without modification of Na+ and/or K + excretion, at variance with classical diuretics. They improved urine excretion, urine osmolality and renal function, and subsequently, normalized serum Na+ (partially or totally) and corrected hyponatremia. Extrarenal localization of V2 receptors has also been evidenced. Other therapeutic domains with V2 receptor antagonists remain to be explored, notably in glaucoma where SR121463, a selective V2 compound, decreased intraocular pressure by local instillations, as do the reference drugs currently used (timolol and clonidine). Finally, we have developed the first selective, orally active Vtb receptor antagonist. This compound constitutes an invaluable tool for exploring this poorly characterized receptor and the precise role of AVP via central and peripheral V]b receptors. SSR149415, a representative member of this class, has potential for the treatment of anxiety, depression and stressrelated disorders. In addition, in view of the recently

208

described tissue localization of the Vlb receptor protein and mRNA (brain, pancreas, adrenals... ), still other potential uses may be possible.

Abbreviations ACTH Ang II AVP CHF CRF dDAVP dPen

adrenocorticotropin angiotensin II arginine vasopressin congestive heart failure corticotropin releasing factor desamino-[D-Arg]vasopressin [deaminopenicillamine-O-MeTyr,Arg]vasopressin high throughput screening HTS OT oxytocin SIADH syndrome of inappropriate antidiuretic hormone secretion VACM-1 receptor vasopressin-activated Ca2+ mobilizing receptor

Acknowledgements The authors would like to acknowledge the teams of Sanofi-Synth61abo Recherche from the Discovery, Preclinical, Clinical and Chemical Development Departments for their work on the Vasopressin program. Our thanks are extended to external contributors for their work and their interest in our compounds. We are thankful to Dr R. Pruss and A.J. Patacchini for helpful comments on the manuscript and M. Laborde for her skilful secretarial assistance. C.S.L.G. thanks J.L., Y. and M. Le Gal for their invaluable support and their encouragement.

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