WO2009149493A1

WO2009149493A1 - Novel receptor hetero-dimers/-oligomers

Info

Publication number: WO2009149493A1
Application number: PCT/AU2009/000720
Authority: WO
Inventors: Kevin Pfleger; Heng Boon See; Ruth Marie Seeber; Matthew Blake Dalrymple; James Williams; Elizabeth Mccall
Original assignee: Dimerix Bioscience Pty Ltd
Priority date: 2008-06-09
Filing date: 2009-06-09
Publication date: 2009-12-17
Also published as: AU2009257174A1

Abstract

A hetero-dimeric or hetero-oligomeric receptor, comprising at least one vasopressin receptor subunit associated with at least one angiotensin receptor subunit.

Description

NOVEL RECEPTOR HETERO-DIMERS/-OLIGOMERS

Field of the Invention

The present invention relates to a hetero-dimeric or hetero-oligomeric receptor, comprising at least one vasopressin receptor subunit associated with at least one angiotensin receptor subunit.

Background Art

Proteins do not act in isolation in a cell, but in stable or transitory complexes, with protein-protein interactions being key determinants of protein function (Auerbach et al., (2002), Proteomics 2:611-623). Furthermore, proteins and protein complexes interact with other cellular components like DNA, RNA and small molecules. Understanding both the individual proteins involved in these interactions and their interactions are important for a better understanding of biological processes.

The primary physiological function of arginine vasopressin (AVP; vasopressin; also known as antidiuretic hormone) reported by lnoue (Inoue, T., et al., (2001 ) Physiological effects of vasopressin and atrial natriuretic peptide in the collecting duct. Cardiovascular Research 51 :470-480) is that of water regulator, acting upon the kidneys to maintain body fluid homeostasis. In addition, AVP is a potent vasoconstrictor, acting upon smooth muscle to increase blood pressure (Rang, H. P., et al., Pharmacology: 3^rd Edition, 1995, Published by Churchill Livingstone, Edinburgh, UK.).

Due to its involvement in the regulation of blood pressure (BP), the AVP system has been utilised as a target for pharmaceutical intervention in the treatment of deleterious heart conditions. An increase in circulating AVP has been associated with congestive heart failure (CHF), thus numerous vasopressin receptor antagonists have been designed in an effort to ameliorate physiological responses stemming from excessive activation of AVP signalling pathways (Goldsmith, Steven, R., (2006) Vasopressin receptor antagonists: Mechanisms of action and potential side effects, Cleveland Clinic Journal of Medicine 73:S20-S23). The renin-angiotensin system (RAS) plays an important role in the sympathetic nervous system and fluid homeostasis. Renin is a proteolytic enzyme secreted by the kidnies that mediates the formation of angiotensin I (Angl) from a globulin precursor, angiotensinogen (Rang, HP., et al., Pharmacology: 3^rd Edition, 1995, Published by Churchill Livingstone, Edinburgh, UK.). Angl itself appears to have little physiological importance other than providing a substrate for a second enzyme, angiotensin-converting enzyme (ACE), which converts Angl to the highly active angiotensin Il (Angll). However, it should be noted that Angll can be generated by alternative, ACE-independent mechanisms. Angll can in turn be metabolised to Anglll by aminopeptidases.

Angll is an extremely potent vasoconstrictor and as a consequence it has been extensively studied in the context of heart disease and hypertension pathogenesis (Ramasubbu, K. (2007) Anti-angiotensin Therapy: New Perspectives. Cardiology Clinics 25:573-580). In order to counter the deleterious vasoconstrictor effects of Angll in patients with hypertension, therapeutic strategies have been developed that intervene at the level of Angll signalling. In particular, compounds that inhibit the activity of ACE, preventing the conversion of Angl to Angll, and those that specifically block the activation of angiotensin receptors (ATRs), have been employed in the treatment of such conditions (Matchar, D.B. (2008) Systematic Review: Comparative Effectiveness of Angiotensin-Converting Enzyme Inhibitors and Angiotensin Il Receptor Blockers for Treating Essential Hypertension. Annals of Internal Medicine 148: 16-29).

The preceding discussion is intended only to facilitate an understanding of the invention. It should not be construed as in any way limiting the scope or application of the following description of the invention, nor should it be construed as an admission that any of the information discussed was within the common general knowledge of the person skilled in the appropriate art at the priority date. Disclosure of the Invention

The inventors have discovered that the angiotensin receptor and the vasopressin receptor associate. This has important implications regarding therapies for ailments associated with either receptor.

Recent studies have shown that GPCRs may not only act as monomers but also as homo- and hetero-dimers which causes altered ligand binding, signalling and endocytosis (Rios et al. (2000) Pharmacol. Ther. 92:71-87). The effect of drugs acting as agonists or antagonists of a specific receptor may therefore depend on the binding partners of this receptor. It may be desirable to limit the effect of a drug to a cellular response mediated by a specific receptor dimer. As Milligan (Milligan G. (2006), Drug Discovery Today 11 :541-549) observes, while homo- dimerisation and -oligomerisation have limited implications for the drug discovery industry, "differential pharmacology, function and regulation of GPCR hetero- dimers and -oligomers suggest means to selectively target GPCRs in different tissues and hint that the mechanism of function of several pharmacological agents might be different in vivo than anticipated from simple ligand screening programmes that rely on heterologous expression of a single GPCR".

The phrase "vasopressin receptor" or "VR" is to be understood to at least include the G protein-coupled receptor vasopressin receptor 2 (V₂R; V2R) analogous to that activated by arginine vasopressin (AVP) in renal tubules of the kidney that has, among other functions, a major regulatory role in homeostatic fluid maintenance (Zingg, H., (1996) Vasopressin and oxytocin receptors, Baillieres Clinical Endocrinology and Metabolism 10:75-96). The phrase "vasopressin receptor" or "VR" is also to be understood to at least include vasopressin receptor 1a (Vi_aR; V1aR), a second subtype of vasopressin receptor known to be expressed in blood vessels and myocardium, whose function is primarily the control of AVP-mediated vasoconstriction (Zingg, H., (1996) Vasopressin and oxytocin receptors, Baillieres Clinical Endocrinology and Metabolism 10:75-96). The phrase "vasopressin receptor" or "VR" is also to be understood to at least include vasopressin receptor 1 b (V_1bR; V1 bR; also known as V3R or V₃R), a third subtype of vasopressin receptor known to be expressed in the anterior pituitary, whose function is primarily the regulation of ACTH release from corticotropes (Rang, HP., et al., Pharmacology: 3^rd Edition, 1995, Published by Churchill Livingstone, Edinburgh, UK.). The phrase "vasopressin receptor" or "VR" is to be further understood to include newly discovered VR family members.

The phrase "angiotensin receptor" or "ATR" is to be understood to mean either angiotensin Il receptor 1 (AT1 R; ATiR) or angiotensin Il receptor 2 (AT2R; AT₂R), being G protein-coupled receptors analogous to those described by Porello et al. (Porello, E.R., Delbridge, L.M. and Thomas, W.G. (2009) The Angiotensin Il Type 2 (AT2) Receptor: An Enigmatic Seven Transmembrane Receptor. Frontiers in Bioscience 14:958-972), which are activated by angiotensin Il (Angll) and/or angiotensin III (Anglll). "Angiotensin receptor" or "ATR" is to be further understood to include newly discovered angiotensin receptor family members.

In a first aspect of the invention, there is provided a hetero-dimeric or hetero- oligomeric receptor, comprising at least one vasopressin receptor subunit associated with at least one angiotensin receptor subunit.

In a second aspect of the invention, there is provided a method for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of a vasopressin receptor-related compound.

In one embodiment, the vasopressin receptor-related compound is selective for the vasopressin receptor relative to the angiotensin receptor.

In one embodiment, the vasopressin receptor-related compound is coadministered with an angiotensin receptor-related compound.

In a third aspect of the invention, there is provided a method for the treatment of a patient suffering from an arginine vasopressin-related ailment by administering a therapeutically effective amount of an angiotensin receptor-related compound.

In one embodiment, the angiotensin receptor-related compound is selective for the angiotensin receptor relative to the vasopressin receptor.

In one embodiment, the angiotensin receptor-related compound is coadministered with a vasopressin receptor-related compound.

In a fourth aspect of the invention, there is provided a method for the manufacture of a medicament for the treatment of a patient suffering from an angiotensin- related ailment comprising use of a therapeutically effective amount of a vasopressin receptor-related compound.

In one embodiment, the medicament contains an angiotensin receptor-related compound.

In a fifth aspect of the invention, there is provided a method for the manufacture of a medicament for the treatment of a patient suffering from an arginine vasopressin-related ailment comprising use of a therapeutically effective amount of an angiotensin receptor-related compound.

In one embodiment, the medicament contains a vasopressin receptor-related compound.

In a sixth aspect of the invention, there is provided a method for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of an arginine vasopressin-selective binding agent, or fragment thereof.

In a seventh aspect of the invention, there is provided a method for the treatment of a patient suffering from an arginine vasopressin-related ailment by administering a therapeutically effective amount of an angiotensin-selective binding agent, or fragment thereof.

In an eighth aspect of the invention, there is provided a method for the treatment of a patient suffering from an arginine vasopressin-related ailment or an angiotensin-related ailment comprising administering a therapeutically effective amount of a vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/- oligomer, such as a selective allosteric modulator.

In a ninth aspect of the invention, there is provided the use of a therapeutically effective amount of a vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer selective agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero- dimer/-oligomer, such as a selective allosteric modulator, for the manufacture of a medicament for the treatment of a patient suffering from a vasopressin-related ailment or an angiotensin-related ailment.

In a tenth aspect of the invention, there is provided a method for screening a test compound for potential therapeutic activity against an angiotensin-related ailment using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) exposing the test compound to a vasopressin receptor; b) assessing whether and/or the extent to which the activity of the vasopressin receptor is modulated compared to activity of the vasopressin receptor in the absence of the test compound; said modulation being indicative of potential therapeutic activity against the angiotensin-related ailment.

In an eleventh aspect of the invention, there is provided a method for screening a test compound for potential therapeutic activity against an arginine vasopressin- related ailment using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) exposing the test compound to an angiotensin receptor; b) assessing whether and/or the extent to which the activity of the angiotensin receptor is modulated compared to activity of the angiotensin receptor in the absence of the test compound; said modulation being indicative of potential therapeutic activity against the arginine vasopressin-related ailment.

In a twelfth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity using a detector capable of detecting changes in receptor activity, the method comprising the step of: determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor compared to whether, and/or the extent to which the test compound interacts with the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In a thirteenth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer selective activity using a detector capable of detecting changes in receptor activity, the method comprising the step of: determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor compared to whether, and/or the extent to which the test compound interacts with the vasopressin receptor in the absence of the angiotensin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In a fourteenth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer selective antagonism or selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the vasopressin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In a fifteenth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer selective antagonism, selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the angiotensin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b). detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In a sixteenth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer selective inverse agonism using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising a constitutively active vasopressin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally ; c) determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin / angiotensin receptor hetero-dimer/-oligomer.

In a seventeenth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer inverse agonism using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; Ni). a third agent, comprising a constitutively active angiotensin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In an eighteenth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the vasopressin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting an increase in the signal as a determination of whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer and optionally; c) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin / angiotensin receptor hetero-dimer/- oligomer.

In a nineteenth aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the angiotensin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b) detecting an increase in the signal as a determination of whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer and optionally; c) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer.

In the aforementioned methods of the invention, the step of determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor may be performed by way of one or more of the methods described in the applicant's co-pending international patent application "Detection System and Uses Therefor" PCT/AU2007/001722 (published as WO 2008/055313).

In a twentieth aspect of the invention, there are provided selective agonists and/or selective antagonists and/or selective inverse agonists and/or selective allosteric modulators of the vasopressin receptor/angiotensin receptor hetero-dimer/- oligomer.

In a twenty-first aspect of the invention, there is provided a cell, or fraction of a cell, in which both a vasopressin receptor and an angiotensin receptor are over- expressed. In a twenty-second aspect of the invention, there is provided a cell, or fraction of a cell, in which a vasopressin receptor is over-expressed with an endogenously expressed angiotensin receptor.

In a twenty-third aspect of the invention, there is provided a cell, or fraction of a cell, in which an angiotensin receptor is over-expressed with an endogenously expressed vasopressin receptor.

Brief Description of the Drawings

Figures 1 to 3 are illustrative of the technique by which the association of the vasopressin receptor and the angiotensin receptor was detected.

Figure 1 shows the composition of the agents forming the basis of the system for detecting molecular associations: A first agent comprises a first interacting group coupled to a first reporter component; a second agent comprises a second interacting group coupled to a second reporter component; and a third agent comprises a third interacting group.

Figure 2 shows how the administration of the modulator modulates the association of the second interacting group with the third interacting group, preferably by interacting with the third interacting group, either alone, or simultaneously with the first interacting group.

Figure 3 shows that if the first and third interacting groups are associated, modulation of the association of the second and third interacting groups consequently modulates the proximity of the first and second reporter components thereby modulating the signal that is able to be detected by the detector. Therefore monitoring the signal generated by proximity of the first and second reporter components by the detector constitutes monitoring the association of the first and third agents. If the first and third interacting groups are not associated, the first and second reporter components will remain spatially separated and generation of a detectable signal is unlikely.

Figure 4 shows the vasopressin receptor 2 (V2R) as IG1 , Rlucδ as RC1 , beta- arrestin 2 (barr2) as IG2, Venus as RC2 and hemagglutin epitope-tagged AT1 R (HA-AT1 R) as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing V2R/Rluc8 and barr2/Venus with either pcDNA3 or HA-AT1 R following treatment with either 10^"6M arginine vasopressin (AVP) or angiotensin Il (Angll) only or both AVP and Angll combined.

Figure 5 shows the V2R as IG1 , Venus as RC1 , beta-arrestin 2 (barr2) as IG2, Rlucδ as RC2 and AT1 R as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing V2RΛ/enus and barr2/Rluc8 with AT1 R following treatment with various doses of arginine vasopressin (AVP) or angiotensin Il (Angll). In parallel, eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing AT1 RΛ/enus and barr2/Rlucδ following treatment with increasing doses of angiotensin Il (Angll). Data presented as percentage of maximum response.

Figure 6 shows the vasopressin receptor 2 (V2R) as IG1 , Venus as RC1 , beta- arrestin 2 (barr2) as IG2, Rlucδ as RC2 and AT1 R as IG3, in parallel with AT2R as IG1 , Venus as RC1 , beta-arrestin 2 (barr2) as IG2, Rlucδ as RC2 and AT1 R as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently co-expressing V2RΛ/enus and barr2/Rlucδ with AT1 R or AT2R/Venus and barr2/Rlucδ with AT1 R. Cells were treated with 10^"8M Angll (at 0 minutes) followed by AT1 R-specific antagonist Valsartan (10^"6M) or PBS vehicle control (after approximately 20 minutes). Data presented as percentage of maximum BRET signal.

Figure 7 shows the angiotensin receptor 2 (AT2R) as IG1 , Venus as RC1 , beta- arrestin 2 (barr2) as IG2, Rlucδ as RC2 and AT1 R as IG3, in parallel with AT1 R as IG1 , Venus as RC1 , beta-arrestin 2 (barr2) as IG2, Rlucδ as RC2 and in the absence of IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently co-expressing AT2R/Venus and barr2/Rluc8 with AT1 R, or AT1 R/Venus and barr2/Rluc8. Cells were treated with 10^"8M Angll (at 0 minutes) followed by AT1 R-specific antagonist Valsartan (10^"6M) or PBS vehicle control (after approximately 20 minutes).

ABBREVIATIONS

ACE Angiotensin-converting enzyme

Angl Angiotensin I peptide

Angll Angiotensin Il peptide

Anglll Angiotensin III peptide

AT₁R Angiotensin receptor type 1

AT₂R Angiotensin receptor type 2

AVP Arginine vasopressin barr beta-arrestin.

BP Blood pressure

BRET Bioluminescence resonance energy transfer.

CB Cannabinoid receptor.

CSF Cerebrospinal fluid.

DOP Delta opioid. eBRET extended BRET: BRET monitored over extended time periods.

ECFP Enhanced Cyan Fluorescent Protein, which is a variant of the

Aequorea victoria green fluorescent protein gene (GFP).

EGFP Enhanced Green Fluorescent Protein is a red-shifted variant of wild-type GFP.

EYFP Enhanced Yellow Fluorescent Protein.

FRET Fluorescence resonance energy transfer.

GPCRs G-protein coupled receptors.

HA Hemagglutin epitope-tag.

His(6) Histidine tag consisting of 6 consecutive histidine residues.

HPA Hypothalamic-pituitary-adrenal IG lnteracting group.

KOP Kappa opioid.

LPO Lateral preoptic area mRFP1 Monomelic red fluorescent protein.

NPY Neuropeptide Y.

OR Opioid receptor.

PBS Phosphate-buffered saline. pcDNA3 Eukaryotic expression vector.

PVN Paraventricular nucleus.

RC Reporter component.

REM Rapid eye movement.

RET Resonance energy transfer.

Rluc Renilla luciferase.

Rlucδ An improved Renilla luciferase.

SWS Slow wave sleep.

TYFP Topaz Yellow Fluorescent Protein.

Venus An improved Yellow Fluorescent Protein.

VR Vasopressin receptor.

Wt Wild type.

Best Mode(s) for Carrying Out the Invention

General

All publications, including patents and patent applications, cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors that are reported in the publications and which may be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional molecular biology, chemistry and fluorescence techniques, within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, eg., Coligan, Dunn, Ploegh, Speicher and Wingfield "Current protocols in Protein Science" (1999) Volume I and Il (John Wiley & Sons Inc.); and Bailey, J. E. and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986; Lakowicz, J. R. Principles of Fluorescence Spectroscopy, New York : Plenum Press (1983) for fluorescence techniques.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" includes a plurality of such proteins, and a reference to "an analyte" is a reference to one or more analytes, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

The invention described herein may include one or more ranges of values (e.g. size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

Throughout this specification, unless the context requires otherwise, the word "comprise" or variations, such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer, or group of integers, but not the exclusion of any other integers or group of integers. Specific

As is apparent from the preceding summary of the invention, the invention relates, inter alia, to hetero-dimeric or hetero-oligomeric receptor, comprising at least one vasopressin receptor subunit associated with at least one angiotensin receptor subunit.

The terms "hetero-dimer" and "hetero-oligomer", and variations such as "hetero- dimeric" and "hetero-oligomeric", as used herein, refer to an entity within which at least one vasopressin receptor is associated with at least one angiotensin receptor.

The phrase "associated with", as used herein, refers to combination via any known direct or indirect stabilising atomic or molecular level interaction or any combination thereof, where the interactions include, without limitation, bonding interactions such as covalent bonding, ionic bonding, hydrogen bonding, coordinate bonding, or any other molecular bonding interaction, electrostatic interactions, polar or hydrophobic interactions, or any other classical or quantum mechanical stabilising atomic or molecular interaction.

Instances of different tissues having different repertoires of hetero-dimers have been reported. For example, 6'guanidinoaltrindole, an analogue of a well-known KOP receptor ligand, has been identified as a DOP-KOP hetero-dimer selective agonist, with efficacy as a spinally selective analgesic, leading to the conclusion that DOP-KOP heterodimers are expressed in the spinal cord, but not in the brain (Waldhoer, M. et al. (2005) A hetero-dimer selective agonist shows in vivo relevance of G-protein coupled receptor dimers. Proc. Natl. Acad. Sci. USA 102:9050-9055). Accordingly, the hetero-dimeric or hetero-oligomeric receptor, comprising at least one vasopressin receptor subunit associated with at least one angiotensin receptor subunit represents a novel drug target. As is the case with 6'guanidinoaltrindole, known ligands may exhibit differing abilities to trigger a hetero-dimeric receptor, which may uncover new applications for pre-existing molecules:

- Hilairet S. et al. 2003 (J. Biol. Chem. 278:23731-23737) have recently shown that CB1 antagonists suppress appetite by acting through a

CB1/OxR1 hetero-dimer pair.

- It has been shown that somatostatin SSTR5 receptor will hetero- dimerise with a dopamine D2 receptor (Rocheville M. et al. (2000) Science 288:154-157). - An angiotensin AT1 receptor/bradykinin B2 receptor hetero-dimer is believed to be responsible for pre-eclampsia in pregnant women. Evidence suggests that the hetero-dimer is more sensitive to Angiotensin Il (AbdAlla S. etal. (2001) Nat. Med. 7:1003-1009).

As will be apparent from the following examples, the inventors herein have identified and characterised the molecular association of the vasopressin receptor with the angiotensin receptor.

It will be apparent to a person skilled in the art that association of the vasopressin receptor with the angiotensin receptor enables the use of compounds related to one receptor, including and without limitation, ligands of one receptor (be they agonists, inverse agonists or antagonists) in the treatment of ailments related to the other receptor.

Thus, the present invention encompasses a method for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of a vasopressin receptor-related compound.

The phrase "vasopressin receptor-related compound" is to be understood to mean a compound that interacts with the vasopressin receptor; a compound that binds to a compound that interacts with the vasopressin receptor, including but not limited to vasopressin; or a compound that modulates the production of a compound that interacts with the vasopressin receptor, including but not limited to vasopressin.

In one form of the invention, the vasopressin receptor-related compound is a vasopressin receptor agonist, inverse agonist or antagonist.

In one embodiment, the vasopressin receptor-related compound is an allosteric modulator of the vasopressin receptor.

In one embodiment, the vasopressin receptor-related compound modulates the production of vasopressin.

In one embodiment, the vasopressin receptor-related compound is a vasopressin binding agent, or a vasopressin binding fragment thereof.

In one embodiment, the vasopressin binding agent is an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-id iotypic antibody.

In one embodiment, the vasopressin receptor-related compound is selective for the vasopressin receptor relative to the angiotensin receptor. In one form of the invention, the vasopressin receptor-related compound is selective for the vasopressin receptor relative to the angiotensin receptor by a factor of at least 10. In one form of the invention, the vasopressin receptor-related compound is selective for the vasopressin receptor relative to the angiotensin receptor by a factor of at least 100. In one form of the invention, the vasopressin receptor- related compound is selective for the vasopressin receptor relative to the angiotensin receptor by a factor of at least 1000.

In the context of vasopressin receptor-related compounds that modulate the production of a compound that interacts with the vasopressin receptor, the phrase "selective for the vasopressin receptor relative to the angiotensin receptor" is to be understood to mean that the compound modulates the production of a compound that interacts with the vasopressin receptor to a greater extent than it modulates the production of angiotensin.

In the context of vasopressin receptor-related compounds that are vasopressin binding agents, or vasopressin binding fragments thereof, the phrase "selective for the vasopressin receptor relative to the angiotensin receptor" is to be understood to mean that the vasopressin binding agent, or the vasopressin binding fragment thereof, binds vasopressin selectively relative to angiotensin.

In one embodiment, the vasopressin receptor-related compound is co- administered with an angiotensin receptor-related compound.

The phrase "angiotensin receptor-related compound" is to be understood to mean a compound that interacts with the angiotensin receptor; a compound that binds to a compound that interacts with the angiotensin receptor, including but not limited to angiotensin; or a compound that modulates the production of a compound that interacts with the angiotensin receptor, including but not limited to angiotensin.

In one embodiment, the angiotensin receptor-related compound is an agonist, inverse agonist or antagonist of the angiotensin receptor.

In one embodiment, the angiotensin receptor-related compound is an an allosteric modulator of the angiotensin receptor.

In one embodiment, the angiotensin receptor-related compound modulates the production of angiotensin.

In one embodiment, the angiotensin receptor-related compound is an angiotensin binding agent, or an angiotensin binding fragment thereof.

In one embodiment, the angiotensin binding agent is an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody. The present invention further encompasses a method for the treatment of a patient suffering from an arginine vasopressin-related ailment by administering a therapeutically effective amount of an angiotensin receptor-related compound.

In one embodiment, the angiotensin receptor-related compound is an allosteric modulator of the angiotensin receptor.

In one form of the invention, the angiotensin receptor-related compound is a compound that modulates the production of angiotensin.

In one form of the invention, the angiotensin receptor-related compound is an angiotensin binding agent, or an angiotensin binding fragment thereof.

In one embodiment, the angiotensin binding agent is an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-id iotypic antibody.

In one form of the invention, the angiotensin receptor-related compound is selective for the angiotensin receptor relative to the vasopressin receptor by a factor of at least 10. In one form of the invention, the angiotensin receptor-related compound is selective for the angiotensin receptor relative to the vasopressin receptor by a factor of at least 100. In one form of the invention, the angiotensin receptor-related compound is selective for the angiotensin receptor relative to the vasopressin receptor by a factor of at least 1000.

In the context of angiotensin receptor-related compounds that modulate the production of a compound that interacts with the angiotensin receptor, the phrase selective for the angiotensin receptor relative to the vasopressin receptor is to be understood to mean that the compound modulates the production of a compound that interacts with the angiotensin receptor to a greater extent than it modulates the production of vasopressin.

In the context of angiotensin receptor-related compounds that are angiotensin binding agents, or angiotensin binding fragments thereof, the phrase selective for the angiotensin receptor relative to the vasopressin receptor is to be understood to mean that the angiotensin binding agent, or the angiotensin binding fragment thereof, binds angiotensin selectively relative to vasopressin.

In one form of the invention, the vasopressin receptor-related compound is an agonist, inverse agonist or antagonist of the vasopressin receptor.

In one embodiment, the vasopressin binding agent is an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-idiotypic antibody.

The present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of a vasopressin receptor-related compound.

In one form of the invention, the angiotensin receptor-related compound is an agonist, inverse agonist or antagonist of the angiotensin receptor.

The present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an arginine vasopressin- related ailment by administering a therapeutically effective amount of an angiotensin receptor-related compound.

Thus, the present invention encompasses a method for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of an arginine vasopressin-selective binding agent, or fragment thereof.

The arginine vasopressin selective binding agent may be an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-id iotypic antibody. The present invention further encompasses a method for the treatment of a patient suffering from an arginine vasopressin-related ailment by administering a therapeutically effective amount of an angiotensin-selective binding agent, or fragment thereof.

The angiotensin-selective binding agent may be an antibody, including a humanised antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody and/or an anti-id iotypic antibody.

The present invention further encompasses a method for the treatment of a patient suffering from an arginine vasopressin-related ailment or an angiotensin- related ailment by administering a therapeutically effective amount of a vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/oligomer, such as a selective allosteric modulator.

The present invention further encompasses the use of a therapeutically effective amount of a vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/- oligomer, such as a selective allosteric modulator, for the manufacture of a medicament for the treatment of a patient suffering from an arginine vasopressin- related ailment or an angiotensin-related ailment.

In one form of the invention, the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective agonist, inverse agonist, partial agonist, antagonist or other molecule that interacts with the hetero-dimer/-oligomer, such as an allosteric modulator, is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer by a factor of at least 10. In one form of the invention, the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective agonist, inverse agonist, partial agonist, antagonist or other molecule that interacts with the hetero-dimer/-oligomer, such as an allosteric modulator, is selective for the vasopressin / angiotensin receptor hetero-dimer/-oligomer by a factor of at least 100. In one form of the invention, the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective agonist, inverse agonist, partial agonist, antagonist or other molecule that interacts with the hetero-dimer/- oligomer, such as an allosteric modulator, is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer by a factor of at least 1000.

The present invention further encompasses a method for the treatment of a patient suffering from an arginine vasopressin-related ailment by administering a therapeutically effective amount of a selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimerA-oligomer, such as a selective allosteric modulator.

In one embodiment, the selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/-oligomer, such as a selective allosteric modulator is co- administered with a vasopressin receptor-related compound.

In one embodiment, the vasopressin receptor-related compound is a vasopressin receptor agonist, inverse agonist or antagonist.

In one embodiment, the selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/-oligomer, such as a selective allosteric modulator is coadministered with an angiotensin receptor-related compound.

In one embodiment, the angiotensin receptor-related compound is an angiotensin receptor agonist, inverse agonist or antagonist.

The present invention further encompasses a method for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of a selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/-oligomer, such as a selective allosteric modulator.

In one embodiment, the vasopressin receptor-related compound is a vasopressin receptor agonist, inverse agonist or antagonist. In one embodiment, the selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/-oligomer, such as a selective allosteric modulator is coadministered with an angiotensin receptor-related compound.

In one embodiment, the angiotensin receptor-related compound is an angiotensin agonist, inverse agonist or antagonist.

The present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an arginine vasopressin- related ailment comprising use of a therapeutically effective amount of a selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, inverse agonist or antagonist.

The present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an arginine vasopressin- related ailment comprising use of a therapeutically effective amount of a selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/-oligomer, such as a selective allosteric modulator.

In one embodiment, the medicament contains an angiotensin receptor-related compound. In one embodiment, the angiotensin receptor-related compound is an angiotensin agonist, inverse agonist or antagonist.

In one embodiment, the medicament contains a vasopressin receptor-related compound. In one embodiment, the vasopressin receptor-related compound is a vasopressin receptor agonist, inverse agonist or antagonist.

The present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an angiotensin-related ailment comprising use of a therapeutically effective amount of a selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, inverse agonist or antagonist.

The present invention further encompasses a method for the manufacture of a medicament for the treatment of a patient suffering from an angiotensin-related ailment comprising use of a therapeutically effective amount of a selective angiotensin receptor / vasopressin receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimer/-oligomer, such as a selective allosteric modulator.

In one embodiment, the medicament contains an angiotensin receptor-related compound. In one embodiment, the angiotensin-receptor-related compound is an angiotensin receptor agonist, inverse agonist or antagonist.

Arginine vasopressin-related ailments include ailments that are related to increased or decreased production of arginine vasopressin, and/or increased or decreased responsiveness of cells to arginine vasopressin. The following list (AIi, F. et al., (2007) Therapeutic potential of vasopressin receptor antagonists. Drugs 67(6):847-858) provides some examples of arginine vasopressin-related ailments: - Diabetes insipidus;

Chronic heart failure;

Hyponatraemia;

Fluid overload;

Polycystic kidney disease; - Enuresis

However, it should be understood that the phrase arginine vasopressin-related ailment is not limited thereto.

Known vasopressin receptor-related compounds include; arginine vasopressin (AVP; vasopressin; antidiuretic hormone), lypressin (Lys⁸-vasopressin), desmopressin (1-deamino-D-Arg⁸-vasopressin), terlipressin (triglyceryl-lysine vasopressin), felypressin (Phe²-Lys⁸-vasopressin), conivaptan (vaprisol; a dual V_1aR and V₂R antagonist), tolvaptan (a V₂R antagonist; AIi, F. et al., (2007) Therapeutic potential of vasopressin receptor antagonists. Drugs 67(6):847-858), lixivaptan (a V₂R antagonist; AIi, F. et al., (2007) Therapeutic potential of vasopressin receptor antagonists. Drugs 67(6):847-858), satavaptan (SR-121463; a V₂R antagonist; AIi, F. et al., (2007) Therapeutic potential of vasopressin receptor antagonists. Drugs 67(6):847-858) and SSR149415 (a Vi_bR-selective antagonist; Griebel, G. et. al. (2005) Non-peptide vasopressin V1 b receptor antagonists as potential drugs for the treatment of stress-related disorders. Current Pharmaceutical Design 11 (12):1549-1559).

Angiotensin-related ailments include aliments that are related to increased or decreased production of angiotensin, and/or increased or decreased responsiveness of cells to angiotensin. Listed below is a number of conditions that have either been proposed to stem from a dysregulated angiotensin system, or, could potentially be treated using angiotensin-based interventions: - Chronic heart failure;

- Atherosclerosis/ ischemia;

- Hypertension; - Hyperkalemia;

- Preeclampsia;

- Diabetes mellitus;

- Diabetic retinopathy;

- Sarcoidosis; - Alzheimer's Disease

However, it should be understood that the phrase angiotensin-related ailment is not limited thereto.

Known angiotensin receptor-related compounds include angiotensin Il (Angll) and angiotensin III (Anglll). Known antagonists for ATR include: CGP-42112A (AT₂R antagonist; Sigma #C-160), Eprosartan (ATiR; market name Teveten®, Abbott Laboratories USA), Losartan (ATiR; market name Cozaar®, Merk & Co), Valsartan (AT₁R; market name Diovan®, Novartis), Telmisartan (ATiR, market name Micardis®, Boehringer Ingelheim), lrbesartan (AT-iR, market name Avapro®, SanofiAventis), Olemsartan (AT₁R, market name Benicar®, Daiichi Sankyo Inc), PD123319 (AT₂R, Tocris), ZD-7115 (AT₁R), Saralasin ((Sar¹- Ala⁸)Angll), Sarthran

and DuP753 (AT₁R).

There are three different vasopressin receptor subtypes that have been identified thus far, all sharing a high degree of homology both amongst themselves and the closely related oxytocin receptor (OTR). The \Λ_A vasopressin receptor subtype (V_1AR) is primarily involved in vasoconstriction and is expressed on vascular smooth muscle and cardiomyocytes, whereas the VIB subtype (V₁BR; also known as V₃R) is expressed within the anterior pituitary where it regulates the release of adrenocorticotropic hormone (ACTH; also known as corticotropin). The third vasopressin receptor subtype, V₂R, is found predominantly in the renal ducts of the kidneys and is involved in water retention. In the presence of high circulating levels of AVP, V₂R is also capable of inducing vasodilation (Goldsmith, S. R. (2006) Vasopressin receptor antagonists: Mechanisms of action and potential side effects. Cleveland Clinic Journal of Medicine 73:S20-S23).

The discovery of the novel hetero-dimers of the present invention provides a context for certain experimental observations.

A study of rat aortic smooth muscle revealed the presence of both Angll and AVP binding sites in these cells, which exhibited contractions following addition of either peptide (Penit, J. et ai, (1983) Vasopressin and angiotensin Il receptors in rat aortic smooth muscle cells in culture. American Journal of Physiology 244:E72-82). It is well established that Angll plays an important role in regulating the release of AVP from the posterior pituitary and early research clearly demonstrated the ability of Angll to promote water intake in rats (Epstein A. (1970) Drinking induced by injection of angiotensin into the brain of the rat. Journal of Physiology (London) 210:457-474). Subsequent research has predominantly focused upon the interplay between these systems in 2 related contexts; hypertension and fluid homeostasis.

An investigation of acute hypertension in rats found that increased blood pressure (BP) following artificial constriction of the aorta could be delayed and/or inhibited by pretreatment with either a vasopressin or angiotensin receptor antagonist (Salgado H. and Salgado M. (1989) Acute aortic coarctation hypertension: role of vasopressin and angiotensin II. American Journal of Physiology 257: H 1480- 1484). The researchers suggested a temporal difference in these effects with Angll apparently mediating the short-term, rapid rise in BP while AVP contributes to the maintenance of elevated BP. Another report concerning Angll-dependent AVP release and BP found that addition of either an ATiR or AT₂R antagonist was capable of blocking Angll-mediated AVP release (Hogarty D. et ai, (1992) The role of angiotensin, ATi and AT₂ receptors in the pressor, drinking and vasopressin responses to central angiotensin. Brain Research 586:289-294). Only the ATiR antagonist (Losartan) was capable of inhibiting elevations in BP following Angll administration, indicating this receptor subtype is responsible for the pressor effects of Angll.

Using a 2 kidney, 1 clip (2K, 1C) model of renovascular hypertension, researchers demonstrated that the expression of ATiR and Vi₃R mRNA were altered within particular regions of the brain under experimental conditions (Jackiewicz, E. et al., (2004) Altered expression of angiotensin AT1a and vasopressin Via receptors and nitric oxide synthase mRNA in the brain of rats with renovascular hypertension. Journal Physiology and Pharmacology 55:725-737). Significant differences in the expression profiles of \Λ_aR and ATiR between control animals and mice with a genetic predisposition to excessive water consumption (polydipsia) have been observed (Tribollet, E. et al., (2002) Up-regulation of vasopressin and angiotensin Il receptors in the thalamus and brainstem of inbred polydipsic mice. Neuroendocrinology 75:113-123). Of note, binding to Vi₃R in the thalamic paraventricular nucleus (PVN) was increased almost 10-fold in polydipsic animals, whereas in the hypothalamic PVN Vi₃R binding was detected in controls but was completely absent in polydipsic mice.

Another study looking at the regulation of thirst and sodium balance administered a variety of angiotensin and vasopressin receptor antagonists into the lateral preoptic area (LPO) of the brain (Saad, W. et al., (2005) Interaction between arginine vasopressin and angiotensin Il receptors in the central regulation of sodium balance. Regulatory Peptides 132:53-58). Both ViR and V₂R antagonists, alone or in combination, were able to reduce the salt intake elicited by AVP and completely blocked this effect if administered prior to AVP treatment. Interestingly, both ATiR (Losartan) and AT₂R antagonists could also inhibit the actions of AVP upon salt intake. The authors concluded, "these results suggest that LPO are implicated in sodium balance that is mediated by Vi, V₂, ATi and AT₂ receptors", however receptor interactions at a molecular level (heterodimers) was not proposed as a potential mechanism.

In one embodiment, the present invention provides a method for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of a vasopressin receptor-related compound selected from the group: vasopressin (arginine vasopressin; AVP; antidiuretic hormone), lypressin (Lys⁸-vasopressin), desmopressin (1-deamino-DArg⁸- vasopressin), terlipressin (triglyceryl-lysine vasopressin), felypressin (Phe²-Lys⁸- vasopressin), conivaptan (also known as vaprisol; a dual Vi₃R and V₂R antagonist), tolvaptan (a V₂R antagonist; AIi, F. et al., (2007) Therapeutic potential of vasopressin receptor antagonists. Drugs. 67:847-858), lixivaptan (a V₂R antagonist; AIi, F. et al., (2007) Therapeutic potential of vasopressin receptor antagonists. Drugs. 67:847-858), satavaptan (also known as SR-121463; a V₂R antagonist; AIi, F. et al., (2007) Therapeutic potential of vasopressin receptor antagonists. Drugs. 67:847-858) and SSR149415 (a V-i_bR-selective antagonist; Griebel, G. et. al., (2005) Non-peptide vasopressin V1b receptor antagonists as potential drugs for the treatment of stress-related disorders. Current Pharmaceutical Design. 11 :1549-1559).

In one form of the invention, the vasopressin-receptor related compound is a vasopressin receptor agonist, inverse agonist or antagonist selected from the foregoing group.

In one embodiment, the present invention provides a method for the treatment of a patient suffering from an arginine vasopressin-related ailment by administering a therapeutically effective amount of an angiotensin receptor-related compound selected from the group: angiotensin Il (Angll), angiotensin III (Anglll), CGP- 42112A (AT₂R; Sigma #C-160), Eprosartan (ATiR; market name Teveten®, Abbott Laboratories USA), Losartan (ATiR; market name Cozaar®, Merk & Co), Valsartan (AT-|R; market name Diovan®, Novartis), Telmisartan (AT₁R, market name Micardis®, Boehringer Ingelheim), lrbesartan (ATiR, market name Avapro®, SanofiAventis), Olemsartan (ATiR, market name Benicar®, Daiichi Sankyo Inc), Candesartan (market name Atacand®, AstraZeneca) PD123319 (AT₂R, Tocris), ZD-7115 (AT₁R), Saralasin ((Sar¹-Ala⁸)Angll), Sarthran ((Sar¹- Th^Angll) and DuP753 (AT₁R).

In one form of the invention, the angiotensin receptor related compound is an angiotensin receptor agonist, inverse agonist or antagonist selected from the foregoing group.

The present invention also includes a method for screening a test compound for potential therapeutic activity against an angiotensin-related ailment using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) exposing the test compound to a vasopressin receptor; b) assessing whether and/or the extent to which the activity of the vasopressin receptor is modulated compared to activity of the vasopressin receptor in the absence of the test compound; said modulation being indicative of potential therapeutic activity against the angiotensin-related ailment.

The present invention also includes a method for screening a test compound for potential therapeutic activity against an angiotensin-related ailment, the method comprising the steps of: a) exposing the test compound to a vasopressin receptor; b) assessing the extent to which the activity of the vasopressin receptor is modulated; said modulation being indicative of potential therapeutic activity against the angiotensin-related ailment.

In one embodiment, the method for screening a test compound for potential therapeutic activity against an angiotensin-related ailment using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one embodiment, the method for screening a test compound for potential therapeutic activity against an angiotensin-related ailment using a detector capable of detecting changes in receptor activity comprises the steps of: contacting a cell expressing vasopressin receptor with said test compound; detecting a change in vasopressin receptor activity.

Methods for assessing the extent to which the activity of a vasopressin receptor is modulated and detectors capable of detecting changes in receptor activity are well-known to those skilled in the art.

The present invention also includes a method for screening a test compound for potential therapeutic activity against an arginine vasopressin-related ailment using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) exposing the test compound to an angiotensin receptor; b) assessing whether and/or the extent to which the activity of the angiotensin receptor is modulated compared to activity of the angiotensin receptor in the absence of the test compound; said modulation being indicative of potential therapeutic activity against the arginine vasopressin-related ailment.

The present invention also includes a method for screening a test compound for potential therapeutic activity against an arginine vasopressin-related ailment, the method comprising the steps of: a) exposing the test compound to an angiotensin receptor; b) assessing the extent to which the activity of the angiotensin receptor is modulated; said modulation being indicative of potential therapeutic activity against the arginine vasopressin-related ailment.

In one embodiment, the method for screening a test compound for potential therapeutic activity against an arginine vasopressin-related ailment using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one embodiment, the method for screening a test compound for potential therapeutic activity against an arginine vasopressin-related ailment using a detector capable of detecting changes in receptor activity comprises the steps of: contacting a cell expressing angiotensin receptor with said test compound; detecting a change in angiotensin receptor activity.

Methods for assessing the extent to which the activity of an angiotensin receptor is modulated and detectors capable of detecting changes in receptor activity are well-known to those skilled in the art.

The present invention comprises a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor; and b) determining whether, and/or the extent to which the test compound interacts with the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

The present invention comprises a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor; and b) if the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor, determining whether, or the extent to which the test compound interacts with the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity using a detector capable of detecting changes in receptor activity comprises the steps of: contacting a cell expressing angiotensin receptor and vasopressin receptor with said test compound; detecting a change in angiotensin receptor or vasopressin receptor activity.

The present invention comprises a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor; and b) if the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor, determining whether, or the extent to which the test compound interacts with the vasopressin receptor in the absence of the angiotensin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer. The present invention further provides a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity using a detector capable of detecting changes in receptor activity, the method comprising the step of: determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor compared to whether, and/or the extent to which the test compound interacts with the vasopressin receptor in the absence of the angiotensin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/oligomer selective activity using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective antagonism or partial agonism, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist or partial agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the vasopressin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist or partial agonist of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer; c) if the test compound is an antagonist or partial agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an antagonist or partial agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic or partial agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective antagonism or selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the vasopressin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an antagonist, partial agonist, or negative allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective antagonism, selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective antagonism, selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity comprises the steps of: contacting a cell expressing angiotensin receptor and vasopressin receptor with said test compound; detecting a change in angiotensin receptor or vasopressin receptor activity.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective antagonism or partial agonism, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist or partial agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the angiotensin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b). detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist or partial agonist of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer; c) if the test compound is an antagonist or partial agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/oligomer, determining whether, or the extent to which the test compound is an antagonist or partial agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic or partial agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/oligomer is selective for the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective antagonism, selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the angiotensin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b). detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/oligomer selective antagonism, selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/oligomer selective inverse agonism, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising a constitutively active vasopressin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; c) if the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin / angiotensin receptor hetero-dimer/-oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective inverse agonism using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising a constitutively active vasopressin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally ; c) determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective inverse agonism using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective inverse agonism using a detector capable of detecting changes in receptor activity comprises the steps of: contacting a cell expressing angiotensin receptor and vasopressin receptor with said test compound; detecting a change in angiotensin receptor or vasopressin receptor activity.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer inverse agonism, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising a constitutively active angiotensin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; c) if the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer inverse agonism using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising a constitutively active angiotensin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

The present invention further provides a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the vasopressin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting an increase in the signal as a determination of whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer and optionally; c) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin / angiotensin receptor hetero-dimer/- oligomer.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity comprises the step of: administering said test compound to an animal.

In one embodiment, the method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity comprises the steps of: contacting a cell expressing angiotensin receptor and vasopressin receptor with said test compound; detecting a change in angiotensin receptor or vasopressin receptor activity.

The present invention further provides a method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) defermining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the angiotensin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b) detecting an increase in the signal as a determination of whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer and optionally; c) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer.

In a preferred embodiment of the invention, the step of determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor are performed by way of the methods described in the applicant's co-pending international patent application "Detection System and Uses Therefor", PCT/AU2007/001722 (published as WO 2008/055313). However, for the sake of clarity, it should be understood that the methods of the present invention are not restricted to methods where the step of determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor are performed by way of the methods described in the applicant's co-pending international patent application "Detection System and Uses Therefor", PCT/AU2007/001722 (published as WO 2008/055313).

Alternate methods of determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor include assays observing a change in coupling to signalling pathways such as a change in G-protein utilisation, ligand binding assays, signalling assays such as those monitoring changes in Ca²⁺, inositol phosphate, cyclic adenosine monophosphate (cAMP), extracellular-signal regulated kinase (ERK) and/or mitogen-activated protein kinase (MAPK), receptor trafficking assays, beta- arrestin translocation assays, enzyme-linked immunosorbent assays (ELISAs) and any other assay that can detect a change in receptor function as a result of receptor heterodimerization.

The present invention includes selective agonists and/or antagonists and/or inverse agonists of the vasopressin receptor/angiotensin receptor hetero-dimer/- oligomer.

The present invention includes selective agonists and/or selective antagonists and/or selective inverse agonists and/or selective allosteric modulators of the vasopressin receptor/angiotensin receptor hetero-dimer/-oligomer. The present invention comprises a cell, or fraction of a cell, in which both a vasopressin receptor and an angiotensin receptor are over-expressed.

The present invention comprises a cell, or fraction of a cell, in which a vasopressin receptor is over-expressed with an endogenously expressed angiotensin receptor.

The present invention comprises a cell, or fraction of a cell, in which an angiotensin receptor is over-expressed with an endogenously expressed vasopressin receptor.

Throughout this specification, unless the context requires otherwise, the phrase "fraction of a cell¹ includes, without limitation, cell membrane preparations. As would be understood by a person skilled in the art, cell membrane preparations are useful in binding assays, or as antigens against which antibodies, including antibody therapeutics, may be raised.

The present invention comprises a cell in which both a vasopressin receptor and an angiotensin receptor are over-expressed.

The present invention comprises a cell in which a vasopressin receptor is over- expressed with an endogenously expressed angiotensin receptor.

The present invention comprises a cell in which an angiotensin receptor is over- expressed with an endogenously expressed vasopressin receptor.

The phrase "over-expressed", as used herein in the context of receptors, refers to an abnormal level of expression of the receptor within the cell relative to the natural level of expression. This may include a level of expression considered to be within the physiological range, but expressed in cells not normally expressing the receptor. This may also include a level of expression considered to be within the physiological range, but in cells not normally expressing the receptors modified in any way, such as by fusion to other proteins or by the addition of immunolabels. Cells in which a receptor is over-expressed may be identified by standard assay techniques well known in the art.

As used herein the term "patient" refers to any animal that may be suffering from one or more of angiotensin- or arginine vasopressin-related ailments. Most preferably the animal is a mammal. The term will be understood to include for example human, farm animals (i.e., cattle, horses, goats, sheep and pigs), household pets (i.e., cats and dogs) and the like.

The phrase "therapeutically effective amount" as used herein refers to an amount sufficient to modulate a biological activity associated with the interaction of angiotensin receptor agonist, inverse agonist, antagonist or allosteric modulator with the angiotensin receptor or vasopressin receptor agonist, inverse agonst, antagonist or allosteric modulator with the vasopressin receptor or of angiotensin receptor/ vasopressin receptor hetero-dimer/oligomer-specific agonist, inverse agonist, antagonist or allosteric modulator with an angiotensin receptor/ vasopressin receptor hetero-dimer/oligomer.

In the context of aspects of the invention where both a vasopressin receptor- related compound, such as and without limitation a vasopressin receptor agonist, inverse agonist or antagonist, and an angiotensin receptor-related compound, such as and without limitation an angiotensin receptor agonist, inverse agonist or antagonist, are administered in combination, a therapeutically effective amount of a vasopressin receptor-related compound or a therapeutically effective amount of an angiotensin receptor-related compound in combination may be lower than therapeutically effective amounts of vasopressin receptor-related compound or angiotensin receptor-related compound when administered alone. That is, the administration of a vasopressin receptor-related compound and an angiotensin receptor-related compound in combination may generate a therapeutic effect at what would otherwise be sub-therapeutic doses of either.

Medicaments of the invention are, in various aspects, administered by injection, or prepared for oral, pulmonary, nasal or for any other form of administration. Preferably the medicaments are administered, for example, intravenously, subcutaneously, intramuscularly, intraorbital^, ophthalmically, intraventricularly, intracranially, intracapsularly, intraspinally, intracisternally, intraperitoneally, buccal, rectally, vaginally, intranasally or by aerosol administration.

The mode of administration is in one aspect at least suitable for the form in which the medicament has been prepared. The mode of administration for the most effective response is in one aspect determined empirically and the means of administration described below are given as examples, and do not limit the method of delivery of the composition of the present invention in any way. All the above formulations are commonly used in the pharmaceutical industry and are commonly known to suitably qualified practitioners.

The medicaments of the invention in certain aspects include pharmaceutically acceptable nontoxic excipients and carriers and administered by any parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections. In addition the formulations optionally contain one or more adjuvants.

The pharmaceutical forms suitable for injectable use optionally include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Alternatively, the compounds of the invention are, in certain aspects, encapsulated in liposomes and delivered in injectable solutions to assist their transport across cell membrane. Alternatively or in addition such preparations contain constituents of self-assembling pore structures to facilitate transport across the cellular membrane. The carrier, in various aspects, isa solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity is maintained, for example and without limitation, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the injectable compositions is in certain aspects, brought about by the use in the compositions of agents delaying absorption. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation in certain aspects include without limitation vacuum drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

Contemplated for use herein are oral solid dosage forms, which are described generally in Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990 Mack Publishing Co. Easton PA 18042) at Chapter 89, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatised with various polymers (E.g., U.S. Patent No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10, Banker and Rhodes ed., (1979), herein incorporated by reference. In general, the formulation will include the compounds described as part of the invention (or a chemically modified form thereof), and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

For the vasopressin receptor-related compounds or angiotensin receptor-related compounds of the invention the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations that will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. In one aspect, the release will avoid the deleterious effects of the stomach environment, either by protection of the composition or by release of the compounds beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance, a coating impermeable to at least pH 5.0 is used. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This coating includes, without limitation, sugar coatings, or coatings that make the tablet easier to swallow. Exemplary capsules consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used. The shell material of cachets in certain aspects is thick starch or other edible paper. For pills, lozenges, moulded tablets or tablet triturates, moist massing techniques are also contemplated, without limitation.

In certain aspects, the therapeutic is included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1mm. The formulation of the material for capsule administration is, in certain aspects, a powder, lightly compressed plugs or even as tablets. In one aspect, the therapeutic could be prepared by compression.

Colourants and flavouring agents are optionally included. For example, compounds may be formulated (such as, and without limitation, by liposome or microsphere encapsulation) and then further contained within an edible product, such as for example and without limitation a refrigerated beverage containing colorants and flavouring agents.

The volume of the therapeutic is in one aspect, diluted or increased with an inert material. These diluents could include for example and without limitation, carbohydrates, especially mannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts are also optionally used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

In other embodiments, disintegrants are included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite are also contemplated. Another form of the disintegrants is the insoluble cationic exchange resins. Powdered gums are also optionally used as disintegrants and as binders and these include, without limitation, powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders are contemplated to hold the therapeutic compounds together to form a hard tablet and include, without limitation, materials from natural products such as acacia, tragacanth, starch and gelatin. Other binders include, also without limitation, methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) are contemplated for use in alcoholic solutions to granulate the therapeutic.

Antifrictional agents are optionally included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants are optionally used as a layer between the therapeutic and the die wall, and these include but are not limited to: stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Exemplary soluble lubricants include sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, and Carbowax 4000 and 6000. Glidants that improve the flow properties of the compound during formulation and to aid rearrangement during compression are optionally added. The glidants include without limitation starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment, a surfactant is added in certain embodiments as a wetting agent. Surfactants include, for example and without limitation, anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents are optionally used and include, without limitation, benzalkonium chloride or benzethomium chloride. The list of potential nonionic detergents that are contemplated in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. When used, these surfactants are present in the formulation of the compounds either alone or as a mixture in different ratios.

Additives that potentially enhance uptake of the compounds are for instance and without limitation the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulations are also contemplated. In certain aspects, the compounds are incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms i.e., gums. In some aspects, slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings have a delayed release effect.

In other aspects, a mix of materials is used to provide the optimum film coating. Film coating is carried out, for example and without limitation, in a pan coater or in a fluidized bed or by compression coating. Also contemplated herein is pulmonary delivery of the compounds. In these aspects, he compounds are delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered-dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of this invention are, for example and without limitation, the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn Il nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.

All such devices require the use of formulations suitable for the dispensing of the compounds. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, optionally comprise the compounds suspended in water. The formulation also includes, in one aspect, a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). In one embodiment, the nebulizer formulation also contains a surfactant, to reduce or prevent surface induced aggregation of the compounds caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device comprise, in one aspect a finely divided powder containing the compounds suspended in a propellant with the aid of a surfactant. The propellant is any conventional material employed for this purpose, such as and without limitation, a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1 ,1 ,1 ,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include, without limitation sorbitan trioleate and soya lecithin. Oleic acid is also contemplated as a surfactant in certain aspects.

Formulations for dispensing from a powder inhaler device comprise a finely divided dry powder containing the compound and optionally include a bulking agent, such as and without limitation lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. In certain embodiments, the compound(s) is/are prepared in particulate form with an average particle size of less than 10 microns, most preferably 0.5 to 5 microns, for most effective delivery to the distal lung.

Nasal delivery of the compounds is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with, for example and without limitation, dextran or cyclodextran.

It will be appreciated that in certain aspects, the medicaments of the invention are given as a single dose schedule, or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of delivery for example with 1 to 10 separate doses, is optionally followed by other doses given at subsequent time intervals required to maintain or reinforce the treatment. The dosage regimen is, at least in part, determined by the need of the individual and the judgement of the practitioner.

The invention will now be further described by way of reference only to the following non-limiting examples. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES

General methods

Briefly, referring to Figures 1 to 3, the IGs are provided in the form of the two receptors (VR and ATR). One of the two is attached to an RC (IG1-RC1 , IG3). A second IG (IG2-RC2) is derived from a molecule that interacts with the receptors upon ligand binding (e.g. beta-arrestin, or a mutant thereof). The detection system not only detects the formation of the VR-ATR heterodimer but can distinguish whether a ligand or drug acts as an agonist, partial agonist, antagonist, inverse agonist, partial inverse agonist or allosteric modulator at the receptor hetero-dimer.

HEK293FT cells were seeded in 6-well plates at a density of approximately 630,000 cells/well and maintained at 37 ⁰C, 5% CO₂ in Complete Media (DMEM containing 0.3 mg/ml glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco)) supplemented with 10% fetal calf serum (FCS; Gibco). Transient transfections were carried out 24 h after seeding using GeneJuice (Novagen) according to manufacturer instructions. 24 h post-transfection, cells were washed with PBS, detached using 0.05% trypsin/0.53 mM EDTA, resuspended in HEPES- buffered phenol red free Complete Media containing 5% FCS and added to a poly-L-lysine-coated white 96-well microplate (Nunc). 48 h post-transfection, eBRET assays were carried out following pre-incubation of cells with EnduRen™ (Promega) at a final concentration of 30 μM, at 37 ⁰C, 5% CO₂ for 2 h. eBRET measurements were taken at 37 °C using the Victor Light plate reader with Wallac 1420 software (Perkin-Elmer). Filtered light emissions were sequentially measured for 2-3 s in each of the 'donor wavelength window' (400-475 nm) and 'acceptor wavelength window' (520-540 nm).

The BRET signal observed between interacting proteins is normalized by subtracting the background BRET ratio. This can be done in one of two ways (see Pfleger et al. (2006) Cell Signal 18:1664-1670; Pfleger et al. (2006) Nat Protoc 1 :336-344): 1 ) the ratio of the 520-540 nm emission over the 400-475 nm emission for a cell sample containing only the donor construct is subtracted from the same ratio for a sample containing the interacting acceptor and donor fusion proteins; 2) the ratio of the 520-540 nm emission over the 400-475 nm emission for a cell sample treated with vehicle is subtracted from the same ratio for a second aliquot of the same cell sample treated with ligand. In the following examples, the second calculation will be used and the signal is described as the 'ligand-induced BRET ratio'.

EXAMPLE 1 MEASUREMENT OF DETECTABLE SIGNALS INDICATIVE OF THE MOLECULAR ASSOCIATION OF THE VASOPRESSIN RECEPTOR WITH THE ANGIOTENSIN RECEPTOR

Referring now to Figure 4, eBRET signals were measured from cells transiently expressing V2R/Rluc8 and barr2/Venus with either pcDNA3 or hemagglutin epitope-tagged AT1 R (HA-AT1 R) following treatment with either 10^"6M arginine vasopressin (AVP) or angiotensin Il (Angll) only or both AVP and Angll combined.

Prior to ligand treatment (added at 0 minutes), a baseline eBRET signal was recorded for each of the combinations. Angll treatment of cells co-expressing V2R/Rluc8 and barr2Λ/enus with pcDNA3 did not result in a ligand-induced BRET signal. AVP treatment of cells co-expressing V2R/Rluc8 and barr2/Venus with HA- AT1 R resulted in the eBRET signal reaching a peak of about 0.05. A signal was also observed following Angll treatment of cells co-expressing V2R/Rluc8, barr2/Venus and HA-AT1 R and this signal also reached approximately 0.05. Treatment of cells co-expressing V2R/Rluc8, barr2/Venus and HA-AT1 R with both Angll and AVP resulted in a peak signal of about 0.13, substantially greater than that observed following addition of Angll or AVP alone.

This example demonstrates that a signal resulting from the proximity of RC1 and RC2 is detected specifically for the combination where the vasopressin receptor 2 (V2R) as IG1 , Rlucβ as RC1 , beta-arrestin 2 (barr2) as IG2, Venus as RC2 and hemagglutin epitope-tagged AT1 R (HA-AT1 R) as IG3, and when the modulator, in this case Angll, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.

This example demonstrates that the inventors have identified the molecular association of the vasopressin receptor with the angiotensin receptor.

This example also demonstrates the greater than additive effect of combined treatment with IG1 ligand (AVP) and IG3 ligand (Angll; modulator). This provides further and distinct evidence for the molecular association of the vasopressin receptor with the angiotensin receptor, as this greater than additive effect is indicative of RC1 and RC2 proximity as a result of IG1-IG2 association in addition to IG2-IG3-IG1 association. This provides evidence against signals originating from non-specific IG1-IG2 association in the absence of an IG1-specific ligand. Without wishing to be bound by theory, this greater than additive effect may also be partly due to IG1 ligand acting as a modulator to modulate the association of IG2 and IG3 via allosteric effects on IG3. Furthermore, this more than additive effect may also be partly due to an active IG conformation (one that is bound to agonist) being more favourable for signal generation, perhaps enabling increased proximity of RC1 and RC2, or more favourable relative orientation of RC1 and RC2.

This example also demonstrates that IG3 can be tagged, such as by the addition of a hemagglutin (HA) epitope-tag, however, this tag does not constitute a reporter component and does not interfere with and/or contribute to the signal generated by the proximity of RC1 and RC2. Such tagging enables additional information to be ascertained, such as the relative expression level of IG3.

EXAMPLE 2 MEASUREMENT OF A DETECTABLE DOSE-DEPENDENT SIGNAL INDICATIVE OF THE MOLECULAR ASSOCIATION OF THE VASOPRESSIN RECEPTOR WITH THE ANGIOTENSIN RECEPTOR RESULTING IN A DOSE-RESPONSE CURVE WITH A HILL SLOPE GREATER THAN 1 Referring now to Figure 5, eBRET signals were measured from cells transiently co-expressing V2RΛ/enus and barr2/Rluc8 with AT1 R following treatment with various doses of arginine vasopressin (AVP) or angiotensin Il (Angll). In parallel, eBRET signals were measured from cells transiently co-expressing AT1 R/Venus and barr2/Rluc8 following treatment with increasing doses of angiotensin Il (Angll). Data presented as percentage of maximum response.

These observations enabled generation of dose-response curves with distinct EC₅₀ values and Hill slopes. The EC₅₀ value for Angll treatment of V2RΛ/enus, barr2/Rluc8 and AT1R (approximately 3nM) was similar to the EC_5O value for Angll treatment of barr2/Rluc8 and AT1 RΛ/enus (approximately 5nM) and substantially different from the EC₅₀ value for AVP treatment of V2RΛ/enus, barr2/Rluc8 and AT1R (approximately 50OnM). Furthermore, the Hill slope generated for data with Angll treatment of V2RΛ/enus, barr2/Rluc8 and AT1 R was substantially greater than 1 , in contrast to that generated for data with Angll treatment of barr2/Rluc8 and AT1 R/Venus and for data with AVP treatment of V2RΛ/enus, barr2/Rluc8 and AT1 R.

This example demonstrates that a signal resulting from the proximity of RC1 and RC2 is detected specifically for the combination where V2R is IG1 , Venus is RC1 , beta-arrestin 2 (barr2) is IG2, Rlucδ is RC2 and HA-AT1 R is IG3, and when the modulator, in this case Angll, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.

This example also demonstrates that the molecular association of the vasopressin receptor with the angiotensin receptor is detected in a dose-dependent manner.

This example also demonstrates that the molecular association of the vasopressin receptor with the angiotensin receptor results in a dose-response curve with a Hill Slope greater than 1.

EXAMPLE 3 EVIDENCE FOR A DIFFERENTIAL EFFECT OF AN AT1 R ANTAGONIST ON THE MOLECULAR ASSOCIATION OF THE VASOPRESSIN RECEPTOR WITH THE ANGIOTENSIN 1 RECEPTOR (AT1 R) IN CONTRAST TO THE MOLECULAR ASSOCIATION OF AT1 R AND AT2R

Referring now to Figures 6 and 7, eBRET signals were measured from cells transiently co-expressing V2R/Venus and barr2/Rluc8 with AT1 R (Figure 6), or

AT2R/Venus and barr2/Rluc8 with AT1 R (Figures 6 and 7), or AT1 R/Venus and barr2/Rluc8 (Figure 7). Cells were treated with 10^"8M Angll (at 0 minutes) followed by AT1 R-specific antagonist Valsartan (10^"6M) or PBS vehicle control (after approximately 20 minutes). Data presented as percentage of maximum BRET signal (Figure 6) or ligand-induced BRET ratio (Figure 7).

Treatment with Angll resulted in an increase in eBRET signal for both heterodimer combinations, although the rate of increase appeared to be greater for the AT2RΛ/enus, barr2/Rluc8 and AT1 R combination (Figure 6). Treatment with Valsartan resulted in a rapid reduction in the Angll-induced eBRET signal with the AT2R/Venus, barr2/Rluc8 and AT1 R combination, however, such a reduction was not observed for the V2RΛ/enus, barr2/Rluc8 and AT1 R combination (Figure 6). The BRET signal observed for the AT1 R/Venus and barr2/Rluc8 combination was greater than that observed for the AT2RΛ/enus, barr2/Rluc8 and AT1R combination, however, in both cases, treatment with Valsartan resulted in a rapid reduction in the Angll-induced eBRET signal (Figure 7), unlike with the V2RΛ/enus, barr2/Rluc8 and AT1 R combination (Figure 6).

This example provides evidence indicating that the molecular association of the vasopressin receptor and AT1 R can influence the ability of an AT1 R-specific antagonist to inhibit the effect of an ATR agonist in a manner different to that observed as a result of the molecular association of AT1 R and AT2R, and different to that observed following treatment of AT1 R by the same AT1 R-specific antagonist in the absence of other overexpressed receptors.

Claims

The Claims Defining the Invention are as Follows:

1. A hetero-dimeric or hetero-oligomeric receptor, comprising at least one vasopressin receptor subunit associated with at least one angiotensin receptor subunit.

2. A method for the treatment of a patient suffering from an angiotensin-related ailment by administering a therapeutically effective amount of a vasopressin receptor-related compound.

3. A method according to claim 2 characterised in that the vasopressin receptor- related compound is selective for the vasopressin receptor relative to the angiotensin receptor.

4. A method for the treatment of a patient suffering from an arginine vasopressin-related ailment by administering a therapeutically effective amount of an angiotensin receptor-related compound.

5. A method according to claim 4 characterised in that the angiotensin receptor- related compound is selective for the angiotensin receptor relative to the vasopressin receptor.

6. A method for screening a test compound for potential therapeutic activity against an angiotensin-related ailment using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) exposing the test compound to a vasopressin receptor; b) assessing whether and/or the extent to which the activity of the vasopressin receptor is modulated compared to activity of the vasopressin receptor in the absence of the test compound; said modulation being indicative of potential therapeutic activity against the angiotensin-related ailment.

7. A method for screening a test compound for potential therapeutic activity against an arginine vasopressin-related ailment using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) exposing the test compound to an angiotensin receptor; b) assessing whether and/or the extent to which the activity of the angiotensin receptor is modulated compared to activity of the angiotensin receptor in the absence of the test compound; said modulation being indicative of potential therapeutic activity against the arginine vasopressin-related ailment.

8. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective activity using a detector capable of detecting changes in receptor activity, the method comprising the step of: determining whether, and/or the extent to which, the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor compared to whether, and/or the extent to which the test compound interacts with the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the angiotensin receptor while the angiotensin receptor is associated with the vasopressin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

9. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/oligomer selective activity using a detector capable of detecting changes in receptor activity, the method comprising the step of: determining whether, and/or the extent to which, the test compound interacts with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor compared to whether, and/or the extent to which the test compound interacts with the vasopressin receptor in the absence of the angiotensin receptor; such that a test compound that exhibits greater affinity and/or potency and/or efficacy when interacting with the vasopressin receptor while the vasopressin receptor is associated with the angiotensin receptor is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

10. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/oligomer selective antagonism or selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the vasopressin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

11. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective antagonism, selective partial agonism or selective negative allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the angiotensin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b). detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

12. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective inverse agonism using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising a constitutively active vasopressin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally ; c) determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin / angiotensin receptor hetero-dimer/-oligomer.

13. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer inverse agonism using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether, and/or the extent to which, the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising a constitutively active angiotensin receptor; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b) detecting a decrease in the signal as a determination of whether and/or the extent to which the test compound is an inverse agonist of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, or the extent to which the test compound is an inverse agonist of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer.

14. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the angiotensin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the vasopressin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the vasopressin receptor; b) detecting an increase in the signal as a determination of whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer and optionally; c) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin / angiotensin receptor hetero-dimer/- oligomer.

15. A method for screening a test compound for vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer selective positive allosteric modulation using a detector capable of detecting changes in receptor activity, the method comprising the steps of: a) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the vasopressin receptor coupled to a first reporter component; ii). a second agent, comprising an interacting group coupled to a second reporter component; iii). a third agent, comprising the angiotensin receptor; iv). an agonist of the angiotensin receptor, the vasopressin receptor and/or the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer; wherein proximity of the first and second reporter components generates a signal; and wherein the modulator modulates the association of the interacting group with the angiotensin receptor; b) detecting an increase in the signal as a determination of whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer and optionally; c) determining whether and/or the extent to which the test compound is a positive allosteric modulator of the vasopressin receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the vasopressin receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the vasopressin receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the vasopressin receptor / angiotensin receptor hetero- dimer/-oligomer.

16. Selective agonists and/or selective antagonists and/or selective inverse agonists and/or selective allosteric modulators of the " vasopressin receptor/angiotensin receptor hetero-dimer/oligomer.

17. A cell, or fraction of a cell, in which both a vasopressin receptor and an angiotensin receptor are over-expressed.

18. A cell, or fraction of a cell, in which a vasopressin receptor is over-expressed with an endogenously expressed angiotensin receptor.

19. A cell, or fraction of a cell, in which an angiotensin receptor is over-expressed with an endogenously expressed vasopressin receptor.