Pharmacological characterization of F-180: a selective human V(1a) vasopressin receptor agonist of high affinity

1. The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F-180 exhibited a high affinity for the human V(1a) receptor subtype (K(i)=11 nM) and was selective for this receptor subtype. 2. Functional studies performed on CHO cells expressing human V(1a) receptors indicate that similarly to AVP, F-180 can stimulate the accumulation of inositol phosphate. The activation constant (K(act)) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V(2) and V(1b) receptor subtypes and an antagonist for the human OT receptor. 3. Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V(1a) receptors, but weak affinity and non selective properties for rat V(1a) receptors. 4. To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. 5. In conclusion, we demonstrate that F-180 is the first selective V(1a) agonist described for human and bovine vasopressin receptors. Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V(1a) receptor subtypes.     

A novel radiolabeled vasopressin antagonist: [3H-Phe]-desGlyd(CH2)5D-Tyr(Et)VAVP, [3H]-SK&F 101926

We report the vasopressin receptor-binding properties of [3H-Phe]-desGlyd(CH2)5D-Tyr(Et)VAVP, [3H]-SK&F 101926, the first radiolabeled vasopressin receptor antagonist. We chose to radiolabel SK&F 101926 because this vasopressin analog is a potent antagonist of vascular V1 and renal V2 vasopressin receptors in all species studied. [3H]-SK&F 101926 bound with a single high affinity to intact vascular smooth muscle cells (A-10; KD = 0.5 nM), and plasma membranes A-10 cells (KD = 0.4 nM) and rat liver (KD = 0.2 nM). In competition experiments with [3H]-SK&F 101926 and [3H]arginine vasopressin ([3H]AVP) using cell and liver membranes, the affinity rank orders of vasopressin analogs were the same and were typical for the V1 receptor subtype. In competition binding experiments with [3H]-SK&F 101926 using cell and liver membranes, guanosine 5'-(beta,gamma-imido)triphosphate did not significantly alter the affinity of the V1 antagonist d(CH2)5Tyr(Me)AVP, but the affinity of AVP was decreased. These data indicate that the V1 receptor can exist in at least two affinity states that are modulated by guanine nucleotides. [3H]-SK&F 101926 also bound specifically and with high affinity to V2 receptors of MDCK cells. We conclude that [3H]-SK&F 101926 binds with high affinity to V1 and V2 vasopressin receptors and is a powerful new tool for the identification of vasopressin receptors and the study of molecular mechanisms involved in the interaction of vasopressin with its receptors.     

Effects of YM471, a nonpeptide AVP V(1A) and V(2) receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells.

YM471, (Z)-4'-[4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl]-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [(3)H]-AVP binding to V(1A) and V(2) receptors with K(i) values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V(1B) and oxytocin receptors with K(i) values of 16.4 microM and 31.6 nM, respectively. In CHO cells expressing V(1A) receptors, YM471 potently inhibited AVP-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, exhibiting an IC(50) value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca(2+)](i) increase (IC(50)=193 nM), and did not affect AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC(50) value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V(1A) and V(2) receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP.     

Selectivity of d[Cha4]AVP and SSR149415 at human vasopressin and oxytocin receptors: evidence that SSR149415 is a mixed vasopressin V1b/oxytocin receptor antagonist

1 A possible role of arginine vasopressin (AVP) V(1b) receptor subtype in stress-related disorders has been recently highlighted by the discovery of the agonist [1-deamino-4-cyclohexylalanine] AVP (d[Cha(4)]AVP) and the antagonist SSR149415. Both compounds have been proposed to target specifically V(1b) receptors, since the reported affinities for the related V(1a), V(2) and oxytocin receptors are in the micromolar or submicromolar range. In the present study, we further investigated the binding affinities of d[Cha(4)]AVP and SSR149415 at recombinant human vasopressin V(1b) (hV(1b)) and oxytocin (hOT) receptors expressed in Chinese hamster ovary (CHO) cells and functional properties of both compounds at hV(1b), hV(1a), hV(2) and hOT receptors. 2 d[Cha(4)]AVP bound to hV(1b) receptors and hOT receptors with pK(i) values of 9.68+/-0.06 and 7.68+/-0.09, respectively. SSR149415 showed pK(i) values of 9.34+/-0.06 at hV(1b) and 8.82+/-0.16 at hOT receptors. 3 d[Cha(4)]AVP stimulated [Ca(2+)](i) increase in hV(1b)-CHO cells with a pEC(50) value of 10.05+/-0.15. It showed pEC(50) values of 6.53+/-0.17 and 5.92+/-0.02 at hV(1a) and hV(2) receptors, respectively, and behaved as a weak antagonist at hOT receptors (pK(B)=6.31+/-0.12). SSR149415 inhibited the agonist-induced [Ca(2+)](i) increase with pK(B) values of 9.19+/-0.07 in hV(1b)-CHO and 8.72+/-0.15 in hOT-CHO cells. A functional pK(i) value of 7.23+/-0.10 was found for SSR1494151 at hV(1a) receptors, whereas it did not inhibit 20 nM AVP response at hV(2) receptors up to 3 microM. 4 Data obtained confirmed the high potency and selectivity of d[Cha(4)]AVP at hV(1b) receptors, but revealed that SSR149415, in addition to the high potency at hV(1b) receptors, displays a significant antagonism at hOT receptors.     

Potent antagonistic action of OPC-31260, a vasopressin V2 receptor antagonist, on [Arg8]vasopressin-induced vasoconstriction in isolated simian femoral arteries.

Using a perfusion technique with isolated vessels, we investigated the effect of OPC-21268 (a selective V1 receptor antagonist) and OPC-31260 (a V2 receptor antagonist) on arginine vasopressin (AVP)- or norepinephrine (NE)-induced vasoconstrictions of isolated simian femoral arteries. The dose-response curve for AVP was shifted to the right in parallel by OPC-31260 but not by OPC-21268. Neither antagonist significantly modified the NE-induced vasoconstriction. On the basis of these results, there are functionally constrictory vasopressin V2 receptors but no V1 receptors in isolated simian femoral arteries.     

Comparison of vasopressin binding sites in human uterine and vascular smooth muscle cells.

Several studies indicate that oxytocin and vasopressin receptors in the human uterus are heterogeneous. We have investigated whether oxytocin and vasopressin bind to separate receptors or one class of receptors in human uterine smooth muscle cells. [3H]d(CH2)5Tyr(Me)AVP, the vasopressin V1A receptor selective radioligand, was used for comparison of vasopressin binding sites in human uterine and vascular smooth muscle cell membranes. Both membrane preparations exhibited one class of high-affinity binding sites with Kd values of 6.44 and 0.47 nM, Bmax values of 166 and 34.8 fmol/mg protein for uterine and vascular smooth muscle cells, respectively. In vascular preparations, the selective vasopressin V1A receptor antagonist, SR 49059 ((2S) 1-[(2R 3S)-(5-chloro-3-(2-chlorophenyl)- -(3.4-dimethoxybenzenesulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2- carbonyl]-pyrrolidine-2-carboxamide), showed high affinity with Ki value of 0.98 nM, confirming that these receptors belong to the vasopressin V1A receptor subtype. On the contrary, in uterine preparations, binding of [3H]d(CH2)5Tyr(Me)AVP was more effectively displaced by oxytocin and the oxytocin receptor selective antagonist, L-371257, (1-[1-[4-[ N-Acetyl-4-piperidinyl)oxy]2-methoxybenzoyl]piperidin-4-yl]- 4H-3,1-benzoxazin-2(1H)-one), than vasopressin and SR 49059, suggesting that binding may be due to cross-reaction with the oxytocin receptors. These results suggest that human uterine smooth muscle cells express only a high density of oxytocin receptors.     

Design and synthesis of the first selective agonists for the rat vasopressin V(1b) receptor: based on modifications of deamino-[Cys1]arginine vasopressin at positions 4 and 8

The neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) mediate a wide variety of peripheral and central physiological and behavioral effects by acting on four different G-protein coupled receptors, termed V1a (vascular), V1b (pituitary), V2 (renal), and OT (uterine). We recently reported that d[Cha4]AVP (A), d[Leu4]AVP (B), d[Orn4]AVP (C), and d[Arg4]AVP (D) have high affinity and are selective agonists for the human V1b receptor. However, peptides A-D were subsequently shown to be potent antidiuretic agonists in the rat and are, thus, not selective V1b agonists in the rat. Peptides A-D served as leads for the studies reported here. They were modified at position 8 by Lys, ornithine (Orn), diaminobutyric acid (Dab), and diaminopropionic acid (Dap) to give d[Cha4,Lys8]VP (1), d[Cha4,Orn8]VP (2), d[Cha4,Dab8]VP (3), d[Cha4,Dap8]VP (4), d[Leu4,Lys8]VP (5), d[Leu4,Orn8]VP (6), d[Leu4,Dab8]VP (7), d[Leu4,Dap8]VP (8), d[Orn4,Lys8]VP (9), d[Orn4,Orn8]VP (10), d[Arg4,Lys8]VP (11), d[Arg4,Orn8]VP (12), and d[Arg4,Dab8]VP (13). All peptides were synthesized by the Merrifield solid-phase method. Their binding and functional properties were evaluated in rat AVP V1a, V1b, and V2 receptors and on the rat OT receptor expressed either in native tissues or in stably transfected cells. They were also examined in rat vasopressor, antidiuretic, and in in vitro (no Mg++) oxytocic assays. Functional studies performed on chinese hamster ovary cells expressing the different AVP/OT receptors confirm that d[Cha4,Lys8]VP (1), d[Cha4,Dab8]VP (3), d[Leu4,Lys8]VP (5), and d[Leu4,Dap8]VP (8) are the first selective agonists for the rat V1b receptor. These selective V1b agonists are promising new tools for studies of the role of the V1b receptor in the rat.     

Pharmacological characterization of RWJ-676070, a dual vasopressin V(1A)/V(2) receptor antagonist

The dysregulation of arginine vasopressin (AVP) release and activation of vasopressin V(1A) and V(2) receptors may play a role in disease. The in vitro and in vivo pharmacology of RWJ-676070, a potent, balanced antagonist of both the V(1A) and V(2) receptors is described. RWJ-676070 binding and intracellular functional antagonist activity was characterized using cells expressing V(1A), V(1B) or V(2) receptors. Its inhibition of V(1A) receptor-mediated contraction of vascular rings and platelet aggregation was determined. V(2) receptor-medated aquaresis was determined in rats, dogs and monkeys. V(1A) receptor-mediated inhibitory activity was assessed in vivo in a vasopressin-induced hypertension model and in normotensive rats and in two hypertensive rat models. RWJ-676070 inhibited AVP binding to human V(1A) and V(2) receptors (Ki=1 and 14 nM, respectively). RWJ-676070 inhibited V(1A) receptor-induced intracellular calcium mobilization and V(2) receptor-induced cAMP accumulation with Ki values of 14 nM and 13 nM, respectively. The compound was slightly less potent against rat V(1A) receptors. RWJ-676070 inhibited V(1A) receptor-mediated vasoconstriction in rat and dog vascular rings and AVP-induced human platelet aggregation. Dose dependent aquaresis was demonstrated in rats, dogs and monkeys following oral administration. RWJ-676070 inhibited AVP-induced hypertension in rats but had no effect on arterial pressure in normotensive and spontaneously hypertensive rats but did decrease arterial pressure in Dahl, salt-sensitive hypertensive rats. RWJ-676070 is a new, potent antagonist of V(1A) and V(2) receptors that may be useful for treatment of diseases benefiting from balanced inhibition of both V(1A) and V(2) receptors.     

Serotonin modulation of cell excitability and of [3H]GABA and [3H]D-aspartate efflux in primary cultures of rat cortical neurons

The effects of 5-hydroxytryptamine (5-HT) on neuronal excitability, evaluated as depolarization-induced firing rate, and on amino acid release, measured as electrically-evoked [(3)H]GABA and [(3)H]d-aspartate efflux, were investigated in rat primary cortical neuronal cultures. 5-HT displayed a concentration-dependent, bimodal effect on neuronal excitability: at 3-10microM it increased excitability through 5-HT(2A) receptors, and was blocked by the selective 5-HT(2A) antagonist MDL 100907, whereas at 30-100microM it reduced excitability through 5-HT(1A) receptors, and was, in turn, blocked by the selective 5-HT(1A) antagonist WAY 100135. The electrically-evoked [(3)H]GABA efflux was concentration-dependently inhibited by 5-HT (pEC(50)=4.74) and such inhibition was prevented by WAY 100135, but not by GR 55562, a selective 5-HT(1D/B) receptor antagonist. Conversely, 5-HT concentration-dependently increased stimulus-evoked [(3)H]d-aspartate efflux (pEC(50)=4.71). The increase was facilitated by methiothepin and was reversed into inhibition by ICS 205930, a selective 5-HT(3) receptor antagonist. In the presence of ICS 205930, the inhibition induced by 5-HT was prevented by the selective 5-HT(1D/B) receptor antagonist GR 55562, but not by WAY 100135. These findings suggest that 5-HT inhibits GABA release through 5-HT(1A) receptors and exerts a dual modulation on glutamate release, mostly facilitatory (through 5-HT(3) receptors) but also inhibitory (through 5-HT(1D/B) receptors), leading to a prevalently positive modulation of the excitatory signal by amino acid neurotransmitter containing neurons.     

Vasopressin V2 (SR121463A) and V1a (SR49059) receptor antagonists both inhibit desmopressin vasorelaxing activity

Although [Arg(8)]vasopressin is a potent vasoconstrictor, it possesses vasorelaxant properties manifested either after vasopressin V1 receptor blockade or directly in some vascular beds. The nature of the receptor involved in the vasorelaxant effect of [deamino-Cys(1) D-Arg(8)]vasopressin (desmopressin), a vasopressin V2 receptor agonist, was studied on rat precontracted aortic rings by the use of highly selective new non-peptide vasopressin receptor antagonists. The present study demonstrates for the first time that desmopressin relaxant effect is antagonized by the vasopressin V2 receptor antagonist SR121463A, but also by the vasopressin V1A receptor antagonist SR49059, suggesting that desmopressin-induced relaxation is mediated by a receptor subtype sharing both V1A and V2 pharmacological profiles.     

KF26777 (2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride), a new potent and selective adenosine A3 receptor antagonist

We investigated the biochemical and pharmacological properties of a new adenosine A(3) receptor antagonist, KF26777 (2-(4-bromophenyl)-7,8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one dihydrochloride). This compound was characterized using N(6)-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide ([125I]AB-MECA) or [35S]guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to membranes from human embryonic kidney 293 (HEK293) cells expressing human adenosine A(3) receptors. KF26777 showed a K(i) value of 0.20+/-0.038 nM for human adenosine A(3) receptors labeled with [125I]AB-MECA and possessed 9000-, 2350- and 3100-fold selectivity vs. human adenosine A(1), A(2A) and A(2B) receptors, respectively. The inhibitory mode of binding was competitive. KF26777 inhibited the binding of [35S]GTPgammaS stimulated by 1 microM 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA). The IC(50) value was 270+/-85 nM; the compound had no effect on basal activity. Dexamethasone treatment for HL-60 cells, human promyelocytic leukemia, up-regulated functional adenosine A(3) receptors expression, and resulted in the enhanced elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) via the adenosine A(3) receptor. KF26777 antagonized this [Ca(2+)](i) mobilization induced by Cl-IB-MECA, with a K(B) value of 0.42+/-0.14 nM. These results indicate that KF26777 is a highly potent and selective antagonist of the human adenosine A(3) receptor.     

Distinct receptor subtypes mediate arginine vasopressin-dependent ACTH release and intracellular calcium mobilization in rat pituitary cells

In the present study, adrenocorticotropic hormone (ACTH) release and intracellular calcium ([Ca(2+)](i)) increase induced by arginine vasopressin (AVP) were characterized in collagenase-dispersed and 3-day cultured rat anterior pituitary cells. AVP and the selective vasopressin V(1b) receptor agonist, [1-deamino-4-cyclohexylalanine]AVP (d[Cha(4)]AVP) induced ACTH release with nanomolar potencies in both cell preparations, and produced a maximal stimulation that was about 1.5 fold greater in the 3-day cultured cells, indicating that the vasopressin V(1b) receptor-ACTH release pathway is enhanced over time in culture. In dispersed cells, AVP, oxytocin and d[Cha(4)]AVP induced [Ca(2+)](i) increases with nanomolar potencies. The selective vasopressin V(1a) receptors antagonist, SR49059 (100 nM), together with the selective oxytocin receptors antagonist (d(CH(2))(5)(1)Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH(2)(9)-vasotocin (100 nM), inhibited the maximal AVP response by ~70%, without affecting the response to d[Cha(4)]AVP, suggesting that the V(1b) receptor was only partially responsible for the AVP-induced [Ca(2+)](i) increase. In contrast, in 3-day cultures, AVP induced an increase in [Ca(2+)](i), while oxytocin and d[Cha(4)]AVP did not. The response to AVP was completely antagonized by SR49059, whereas the vasopressin V(1b) receptor antagonists, SSR149415 and (d(CH(2))(5)(1)Tyr(Me)(2),Thr(4),Orn(8),Tyr-NH(2)(9))-vasotocin had no effect, indicating that the [Ca(2+)](i) increase was mediated exclusively by vasopressin V(1a) receptors. In conclusion, the enhancement of vasopressin V(1b) receptor-mediated ACTH release and the lack of a detectable vasopressin V(1b) receptor coupling to [Ca(2+)](i) increase in cultured cells suggests the activation of a different/additional signaling pathway in the molecular mechanism of ACTH release.     

Characterization of 4-(2-hydroxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-fluorobenzamido]ethyl]piperazine (p-DMPPF) as a new potent 5-HT1A antagonist

BACKGROUND AND PURPOSE: The identification of potent and selective radioligands for the mapping of 5-HT receptors is interesting both for clinical and experimental research. The aim of this study was to compare the potency of a new putative 5-HT(1A) receptor antagonist, p-DMPPF, (4-(2-hydroxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-fluorobenzamido]ethyl]piperazine) with that of the well-known 5-HT(1A) antagonists, WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide) and its fluorobenzoyl analogue, p-MPPF (4-(2-methoxyphenyl)-1-[2'-[N-(2'-pyridinyl)-p-fluorobenzamido]ethyl]piperazine). EXPERIMENTAL APPROACH: Single cell extracellular recordings of dorsal raphe (DR) neurones were performed in rat brain slices. The potency of each compound at antagonizing the effect of the 5-HT(1A) agonist, 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)-tetraline], was quantified using the Schild equation. The pharmacological profile of p-DMPPF was defined using competition binding assays. KEY RESULTS: Consistently with a 5-HT(1A) receptor antagonist profile, incubation of slices with an equimolar (10 nM) concentration of each compound markedly reduced the inhibitory effect of 8-OH-DPAT on the firing rate of DR neurones, causing a significant rightward shift in its concentration-response curve. The rank order of potency of the antagonists was WAY-100635>p-DMPPF>or=p-MPPF. The sensitivity of DR neurones to the inhibitory effect of 8-OH-DPAT was found to be heterogeneous. The binding experiments demonstrated that p-DMPPF is highly selective for 5-HT(1A) receptors, with a K(i) value of 7 nM on these receptors. CONCLUSIONS AND IMPLICATIONS: The potency of the new compound, p-DMPPF, as a 5-HT(1A) antagonist is similar to that of p-MPPF in our electrophysiological assay. Its selectivity towards 5-HT(1A) receptors makes it a good candidate for clinical development.     

SLV313 (1-(2,3-dihydro-benzo[1,4]dioxin-5-yl)-4- [5-(4-fluoro-phenyl)-pyridin-3-ylmethyl]-piperazine monohydrochloride): a novel dopamine D2 receptor antagonist and 5-HT1A receptor agonist potential antipsychotic drug.

Combined dopamine D(2) receptor antagonism and serotonin (5-HT)(1A) receptor agonism may improve efficacy and alleviate some side effects associated with classical antipsychotics. The present study describes the in vitro and in vivo characterization of 1-(2,3-dihydro-benzo[1,4]dioxin-5-yl)-4-[5-(4-fluoro-phenyl)-pyridin-3-ylmethyl]-piperazine monohydrochloride (SLV313), a D(2/3) antagonist and 5-HT(1A) agonist. SLV313 possessed high affinity at human recombinant D(2), D(3), D(4), 5-HT(2B), and 5-HT(1A) receptors, moderate affinity at 5-HT(7) and weak affinity at 5-HT(2A) receptors, with little-no affinity at 5-HT(4), 5-HT(6), alpha(1), and alpha(2) (rat), H(1) (guinea pig), M(1), M(4), 5-HT(3) receptors, and the 5-HT transporter. SLV313 had full agonist activity at cloned h5-HT(1A) receptors (pEC(50)=9.0) and full antagonist activity at hD(2) (pA(2)=9.3) and hD(3) (pA(2)=8.9) receptors. In vivo, SLV313 antagonized apomorphine-induced climbing and induced 5-HT(1A) syndrome behaviors and hypothermia, the latter behaviors being antagonized by the 5-HT(1A) antagonist WAY100635. In a drug discrimination procedure SLV313 induced full generalization to the training drug flesinoxan and was also antagonized by WAY100635. In the nucleus accumbens SLV313 reduced extracellular 5-HT and increased dopamine levels in the same dose range. Acetylcholine and dopamine were elevated in the hippocampus and mPFCx, the latter antagonized by WAY100635, suggesting possible 5-HT(1A)-dependent efficacy for the treatment of cognitive and attentional processes. SLV313 did not possess cataleptogenic potential (up to 60 mg/kg p.o.). The number of spontaneously active dopamine cells in the ventral tegmental area was reduced by SLV313 and clozapine, while no such changes were seen in the substantia nigra zona compacta following chronic administration. These results suggest that SLV313 is a full 5-HT(1A) receptor agonist and full D(2/3) receptor antagonist possessing characteristics of an atypical antipsychotic, representing a potential novel treatment for schizophrenia.     

Development and therapeutic indications of orally-active non-peptide vasopressin receptor antagonists

Vasopressin (AVP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by the stimulation of specific G-protein-coupled receptors (GPCRs) currently classified into V(1)-vascular (V(1)R), V(2)-renal (V(2)R) and V(3)-pituitary (V(3)R) AVP receptors and OT receptors (OTR). The signal transduction pathways coupled to the different subtypes of AVP/OT receptors are reviewed. The recent cloning of the different members of the AVP/OT family of receptors now allows the extensive characterisation of the molecular determinants involved in agonist and antagonist binding, as well as signal transduction coupling. Potential therapeutic uses of AVP receptor antagonists include: the blockade of V(1)-vascular AVP receptors in arterial hypertension, congestive heart failure (CHF) and peripheral vascular diseases; the blockade of V(2)-renal AVP receptors in the syndrome of inappropriate vasopressin secretion, CHF, liver cirrhosis, nephrotic syndrome and any state of excessive retention of free water and subsequent hyponatraemia; the blockade of V(3)-pituitary AVP receptors in adrenocorticotropin (ACTH)-secreting tumours. The pharmacological and clinical profile of orally-active non-peptide AVP receptor antagonists is reviewed.     

Tolvaptan, an orally active vasopressin V(2)-receptor antagonist - pharmacology and clinical trials

Tolvaptan is an orally effective nonpeptide arginine vasopressin (AVP) V(2)-receptor antagonist synthesized by Otsuka Pharmaceutical Co., Ltd. In in vitro receptor-binding studies, tolvaptan blocked the binding of [(3)H]AVP to human V(2) receptors with 29-fold greater selectivity than that for V(1a) receptors, and showed no inhibition of V(1b) receptors. Tolvaptan inhibited not only the binding of [(3)H]AVP but also the AVP-induced production of cyclic AMP in human V(2)-receptor-expressing HeLa cells. In addition, tolvaptan has no intrinsic V(2) receptor agonistic effect. In in vivo studies, tolvaptan showed marked aquaresis in healthy and diseased animals. In rat models with acute and chronic hyponatremia, tolvaptan improved hyponatremia, resulting in the prevention of death, and improved organ water retention. Tolvaptan reduced cardiac preload without unfavorable effects on renal functions, systemic hemodynamics, or circulating neurohormones in dogs with heart failure (HF). Furthermore, in animal models of human polycystic kidney disease (PKD), tolvaptan showed a decrease in kidney weight as well as in cyst and fibrosis volume. In clinical trials including the "ACTIV in CHF" study, tolvaptan in addition to standard therapy increased fluid loss resulting in decreased body weight, and improved edema and serum sodium without affecting blood pressure, heart rate, or renal functions in patients with HF. In patients with hyponatremia, treatment with tolvaptan without fluid restriction appeared to be more effective than fluid restriction alone at correcting hyponatremia without an increase in adverse events. A phase III trial EVEREST is currently being conducted to evaluate the long-term efficacy and safety of tolvaptan in hospitalized patients with severe HF. In conclusion, tolvaptan offers the possibility of a useful therapy in hyponatremia, congestive heart failure, and various other diseases that are associated with volume overload. Furthermore, tolvaptan is also expected to be effective in the treatment of PKD.     

3H] A-369508 ([2-[4-(2-cyanophenyl)-1-piperazinyl]-N-(3-methylphenyl) acetamide): an agonist radioligand selective for the dopamine D4 receptor

Tritiation of the dopamine D(4) receptor selective agonist A-369508 ([2-[4-(2-cyanophenyl)-1-piperazinyl]-N-(3-methylphenyl) acetamide) has provided a radioligand for the characterization of dopamine D(4) receptors. [(3)H] A-369508 binds with high affinity to the major human dopamine D(4) receptor variants D(4.2), D(4.4) and D(4.7) (K(d)=1.7, 4, and 1.2 nM, respectively). It also binds to the rat dopamine D(4) receptor, (K(d)=4.4 nM), implying similar binding affinity across human and rat receptors. A-369508 shows >400-fold selectivity over D(2L), >350-fold selectivity over 5-HT(1A) and >700-1,000-fold selectivity over all other receptors tested. Agonist activity determined by inhibition of forskolin-induced cAMP in Chinese hamster ovary cells transfected with the human dopamine D(4.4) receptor (EC(50)=7.5 nM, intrinsic activity=0.71) indicates that A-369508 is a potent agonist at the human dopamine D(4) receptor. Similar data was observed in other functional assays. [(3)H] A-369508 binds to a single, high affinity site on membranes containing the human dopamine D(4.4) receptor. When compared to the D(2)-like antagonist [(3)H] spiperone, competition binding for agonists like dopamine and apomorphine were 2-10-fold more potent with [(3)H] A-369508, while the antagonists clozapine, haloperidol and L-745870 bind with similar affinity to both ligands. Binding to rat brain regions demonstrated that the most abundant area was cerebral cortex (51.2 fmol/mg protein) followed by hypothalamus, hippocampus, striatum and cerebellum. [(3)H] A-369508 is a useful tool to define the localization and physiological role of dopamine D(4) receptors in central nervous system and can facilitate measuring accurate affinities (K(i)) for structure/activity relationship studies designed to identify dopamine D(4) receptor selective agonists.