WO2010003170A1

WO2010003170A1 - Novel receptor hetero-dimers/-oligomers

Info

Publication number: WO2010003170A1
Application number: PCT/AU2009/000774
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: 2008-06-17
Filing date: 2009-06-17
Publication date: 2010-01-14

Abstract

A hetero-dimeric or hetero-oligomeric receptor, comprising at least one alpha-adrenergic 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 alpha-adrenergic 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 a/., (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 catecholamines (CAs; also known as monoamines) that include, but are not limited to, adrenaline (Ad; also known as epinephrine) and noradrenaline (NA; also known as norepinephrine), is the regulation of the sympathetic nervous system which prepares the body for physical activity. In particular, these neurotransmitters and/or hormones are involved in an individual's reaction to stress, which is commonly referred to as the "fight or flight response." Their effects are mediated by numerous G protein- coupled receptors, which include the alpha and beta-adrenergic receptor (αAR and βAR respectively) subtypes. These receptors are sometimes referred to collectively as adrenoreceptors or adrenoceptors.

Adrenergic catecholamines (Ad and NA) fundamentally regulate the vasculature, increasing or decreasing blood pressure (BP) and heart rate (HR) by manipulating the contractile response of smooth muscle. Both Ad and NA are synthesised from the same precursor within the adrenal medulla, although NA is also produced by postganglionic neurons of the sympathetic nervous system (Spence, A.P., et al., Human Anatomy and Physiology: 4^th Edition, 1992, Published by West Publishing Company, Minnesota, USA.)- While NA functions primarily as a neurotransmitter in the regulation of HR and BP, Ad has a broader range of physiological roles that include the mobilisation of carbohydrate and fat stores and the generation of glucose. However, it is the integral role of adrenergic receptors in mediating the sympathetic actions of CAs that has garnered the most attention with respect to pharmacological interventions targeting this system.

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 alpha- adrenergic 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 "alpha-adrenergic receptor" is to be understood to at least include the alpha-adrenergic G protein-coupled receptors (αARs), comprising; the alpha-1 adrenergic receptor subtypes (αiARs) and the alpha-2 adrenergic receptor subtypes (α₂ARs). The phrase "alpha-adrenergic receptor" is to be further understood to include any newly discovered alpha-AR family members.

The phrase "angiotensin receptor" or "ATR" is to be understood to mean either angiotensin receptor 1 (AT1 R; ATiR) or angiotensin 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 alpha-adrenergic 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 an alpha-adrenergic receptor-related compound.

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

In one embodiment, an alpha-adrenergic 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 alpha-adrenergic-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 alpha-adrenergic receptor.

In one embodiment, the angiotensin receptor-related compound is coadministered with an alpha-adrenergic 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 an alpha- adrenergic 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 alpha-adrenergic- related ailment comprising use of a therapeutically effective amount of an angiotensin receptor-related compound.

In one embodiment, the medicament contains an alpha-adrenergic 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 alpha-adrenergic-selective binding agent, or fragment thereof.

In one embodiment, the alpha-adrenergic-selective binding agent is a catecholamine-selective binding agent.

In a seventh aspect of the invention, there is provided a method for the treatment of a patient suffering from an alpha-adrenergic-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 alpha-adrenergic-related ailment or an angiotensin- related ailment comprising administering a therapeutically effective amount of an alpha-adrenergic 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 an alpha-adrenergic 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 alpha-adrenergic-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 an alpha-adrenergic receptor; b) assessing whether and/or the extent to which the activity of the alpha- adrenergic receptor is modulated compared to activity of the alpha- adrenergic 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 alpha-adrenergic- 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 alpha- adrenergic-related ailment.

In a twelfth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor compared to whether, and/or the extent to which the test compound interacts with the angiotensin receptor in the absence of the alpha-adrenergic 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 alpha-adrenergic receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In a thirteenth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer selective activity, the method comprising the step of: determining whether, and/or the extent to which, the test compound interacts with the alpha-adrenergic receptor while the alpha-adrenergic receptor is associated with the angiotensin receptor compared to whether, and/or the extent to which the test compound interacts with the alpha-adrenergic 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 alpha-adrenergic receptor while the alpha- adrenergic receptor is associated with the angiotensin receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In a fourteenth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor; iv). an agonist of the angiotensin receptor, the alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha- adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In a fifteenth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha- adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer and optionally; c) determining whether, and/or the extent to which the test compound is an antagonist, partial agonist or negative allosteric modulator of the alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In a sixteenth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha- ad renergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic / angiotensin receptor hetero-dimer/- oligomer.

In a seventeenth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In an eighteenth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor; iv). an agonist of the angiotensin receptor, the alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic / angiotensin receptor hetero-dimer/-oligomer.

In a nineteenth aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic 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 alpha-adrenergic receptor while the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 an alpha-adrenergic 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 an alpha-adrenergic 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 alpha-adrenergic receptor.

Brief Description of the Drawings

Figures 1 to 3 are illustrative of the technique by which the association of the alpha-adrenergic 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 angiotensin receptor 1 (AT1 R) as IG1 , Venus as RC1 , beta- arrestin 2 (barr2) as IG2, Rlucδ as RC2 and wild-type alpha-adrenergic receptors (ARs) as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing AT1 R/Venus and barr2/Rluc8 with either pcDNA3 or wild- type ARs following treatment with N-phenylpiperazine (NPP). In parallel, eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing barr2/Rluc8 with pcDNA3 following treatment with N-phenylpiperazine (NPP) as a negative control.

Figure 5 shows the angiotensin receptor 2 (AT2R) as IG1 , Venus as RC1 , beta- arrestin 2 (barr2) as IG2, Rlucδ as RC2 and wild-type alpha-adrenergic receptors (ARs) as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing AT2R/Venus and barr2/Rluc8 with either pcDNA3 or wild- type ARs following treatment with N-phenylpiperazine (NPP). In parallel, eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing barr2/Rluc8 with pcDNA3 following treatment with N-phenylpiperazine (NPP) as a negative control.

Figure 6 shows angiotensin receptors (ATRs) as IG1 , Venus as RC1 , beta- arrestin 2 (barr2) as IG2, Rlucδ as RC2 and wild-type alphal b adrenergic receptor (alphalbAR) as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing AT1 RΛ/enus or AT2R/Venus with barr2/Rluc8 and wild-type alphalbAR following treatment with various doses of noradrenaline (NA).

Figure 7 shows the angiotensin receptor 1 (AT1 R) as IG1 , Venus as RC1 , beta- arrestin 2 (barr2) as IG2, Rlucδ as RC2 and wild-type alphal b adrenergic receptor (alphalbAR) as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing AT1 RΛ/enus and barr2/Rluc8 with pcDNA3 or wild-type alphalbAR following treatment with noradrenaline (NA) or angiotensin Il (ATII) alone or in combination (NA + ATII).

Figure 8 shows the angiotensin receptor 2 (AT2R) as IG1 , Venus as RC1 , beta- arrestin 2 (barr2) as IG2, Rlucδ as RC2 and wild-type alphal b adrenergic receptor (alphalbAR) as IG3. eBRET measurements at 37C were carried out on HEK293FT cells transiently expressing AT2R/Venus and barr2/Rluc8 with pcDNA3 or wild-type alphalbAR following treatment with noradrenaline (NA) or angiotensin Il (ATII) alone or in combination (NA + ATII).

ABBREVIATIONS

ACE Angiotensin-converting enzyme

Ad Adrenaline

Angl Angiotensin I peptide

Angll Angiotensin Il peptide

Anglll Angiotensin III peptide

AR Adrenergic receptor

ATM Angiotensin Il peptide

ATiR Angiotensin receptor type 1

AT₂R Angiotensin receptor type 2 barr beta-arrestin.

BP Blood pressure

BRET Bioluminescence resonance energy transfer

CAs Catecholamines

CB Cannabinoid receptor

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 HR Heart rate

IG Interacting group.

KOP Kappa opioid. mRFP1 Monomeric red fluorescent protein.

NA Noradrenaline (A.K.A. Norepinephrine)

NPP N-phenylpiperazine

OR Opioid receptor.

PBS Phosphate-buffered saline. pcDNA3 Eukaryotic expression vector.

PVN Paraventricular nucleus.

RC Reporter component.

RET Resonance energy transfer.

Rluc Renilla luciferase.

Rlucδ An improved Renilla luciferase.

TYFP Topaz Yellow Fluorescent Protein.

Venus An improved Yellow Fluorescent Protein

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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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).

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

It will be apparent to a person skilled in the art that association of the alpha- adrenergic receptor with 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 an alpha-adrenergic receptor-related compound.

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

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

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

In one embodiment, the alpha-adrenergic receptor-related compound modulates the production of a catacholamine. In one embodiment, the alpha-adrenergic receptor-related compound is a catecholamine binding agent, or a catecholamine binding fragment thereof.

In one embodiment, the catacholamine 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.

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

In the context of alpha-adrenergic receptor-related compounds that modulate the production of a compound that interacts with the alpha-adrenergic receptor, the phrase "selective for the alpha-adrenergic 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 alpha-adrenergic receptor to a greater extent than it modulates the production of angiotensin.

In the context of alpha-adrenergic receptor-related compounds that are catecholamine binding agents, or catecholamine binding fragments thereof, the phrase "selective for the alpha-adrenergic receptor relative to the angiotensin receptor" is to be understood to mean that the catecholamine binding agent, or the catecholamine binding fragment thereof, binds the catecholamine selectively relative to angiotensin. In one embodiment, the aipha-adrenergic receptor-related compound is coadministered 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 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 alpha-ad renergic-related ailment by administering a therapeutically effective amount of an angiotensin receptor-related compound.

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 alpha-adrenergic 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 alpha- adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 a catacholamine.

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 alpha-adrenergic receptor is to be understood to mean that the angiotensin binding agent, or the angiotensin binding fragment thereof, binds angiotensin selectively relative to catecholamine.

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

In one embodiment, the alpha-adrenergic receptor-related compound modulates the production of a catecholamine.

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

In one embodiment, the catecholamine 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 an alpha-adrenergic receptor-related compound.

In one embodiment, the medicament contains an angiotensin 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 alpha-adrenergic- related ailment by administering a therapeutically effective amount of an angiotensin 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.

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 alpha-adrenergic-selective binding agent, or fragment thereof.

The alpha-adrenergic 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 alpha-adrenergic-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-idiotypic antibody.

The present invention further encompasses a method for the treatment of a patient suffering from an alpha-adrenergic-related ailment or an angiotensin- related ailment by administering a therapeutically effective amount of an alpha- adrenergic 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 an alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer selective agonist, selective inverse 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 alpha-adrenergic-related ailment or an angiotensin-related ailment.

In one form of the invention, the angiotensin receptor / alpha-adrenergic 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 angiotensin receptor / alpha- adrenergic receptor hetero-dimer/-oligomer by a factor of at least 10. In one form of the invention, the angiotensin receptor / alpha-adrenergic receptor hetero- dimer/-o!igomer 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 angiotensin receptor / alpha-adrenergic receptor hetero-dimer/-oligomer by a factor of at least 100. In one form of the invention, the angiotensin receptor / alpha-adrenergic 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 angiotensin receptor / alpha-adrenergic 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 alpha-adrenergic-related ailment by administering a therapeutically effective amount of a selective angiotensin receptor / alpha- adrenergic 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 selective angiotensin receptor / alpha-adrenergic 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 alpha-adrenergic receptor-related compound.

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

In one embodiment, the selective angiotensin receptor / alpha-adrenergic receptor hetero-dimer / -oligomer agonist, selective inverse agonist, selective partial agonist, selective antagonist or other molecule that selectively interacts with the hetero-dimerAoligomer, 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 / alpha- adrenergic receptor hetero-dimer / -oligomer agonist, 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 selective angiotensin receptor / alpha-adrenergic 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 alpha-adrenergic receptor-related compound.

In one embodiment, the selective angiotensin receptor / alpha-adrenergic 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 alpha-ad renergic- related ailment comprising use of a therapeutically effective amount of a selective angiotensin receptor / alpha-adrenergic 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.

In one embodiment, the medicament contains an alpha-adrenergic receptor- related compound. In one embodiment, the alpha-adrenergic receptor-related compound is an alpha-adrenergic 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 / alpha-adrenergic 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 / alpha-adrenergic 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.

Alpha-adrenergic-related ailments include conditions that are related to increased or decreased production of catecholamines, and/or increased or decreased responsiveness of cells to catecholamines. An alpha-adrenergic-related ailment should also be understood to mean a condition in which alpha-adrenergic receptors display aberrant characteristics or are the target of a particular intervention. The following list provides some examples of alpha-adrenergic- related ailments: - Alzheimer's;

- Asthma;

- Atherosclerosis/ ischemia;

- Chronic heart failure;

- Chronic obstructive pulmonary disease (COPD);

- Depression;

- Hypertension/cardiac hypertrophy;

- Parkinson's;

- Rheumatoid arthritis;

- Sickle cell disease;

- Stroke;

- Thyrotoxicosis

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

Known alpha-adrenergic receptor-related compounds include; adrenaline (also known as adrenalin or epinephrine), noradrenaline (also known as noradrenalin or norepinephrine), butaxamine, cirazoline, clonidine, guanfacine, guanabenz, guanoxabenz, guanethidine, isoprenaline, lofexidine, methoxamine, methyl- noradrenaline, oxymetazoline, phenylephrine, ritodrine, tizanidine and xylazine.

Known antagonists of alpha-adrenergic receptors include; alfuzosin, butoxamine, carvedilol, celiprolol, dihydroergotamine, doxazosin, ergotamine, idazoxan, indoramin, labetolol, phentolamine, phenoxybenzamine, prazosin, terazosin, tamsulosin and yohimbine.

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 (AT-iR; market name Teveten®, Abbott Laboratories USA), Losartan (AT-|R; market name Cozaar®, Merk & Co), Valsartan (AT-iR; market name Diovan®, Novartis), Telmisartan (AT-iR, market name Micardis®, Boehringer Ingelheim), lrbesartan (AT-iR, market name Avapro®, SanofiAventis), Olemsartan (AT-iR, market name Benicar®, Daiichi Sankyo Inc), PD123319 (AT₂R, Tocris), ZD-7115 (AT₁R), Saralasin ((Sar¹- Ala⁸)Angll), Sarthran ((Sar¹-Thr⁸)Angll) and DuP753 (AT₁R).

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

Early research examining the ability of the angiotensin and adrenergic systems to regulate sympathetic tone determined the effect of CA treatment upon AT₁R regulation in neuronal cell cultures (Sumners, C. et al. (1986) Alpha 1 -adrenergic receptor-mediated downregulation of angiotensin Il receptors in neuronal cultures. J Neurochem 47:1117-1126). Both dopamine and NA treatment caused a significant decrease in Angll binding, which occurred due to a reduction in the number of ATiRs present. This effect could be abrogated by co-administration of an alpha-1a adrenergic receptor (αi_aAR) antagonist, suggesting this receptor subtype mediates the impact of CAs upon Angll signalling. In an effort to further understand these results, the same research group conducted a follow-up study (Yang, H. et al. (1996) Lack of cross talk between alphal -adrenergic and angiotensin type 1 receptors in neurons of spontaneously hypertensive rat brain. Hypertension 27:1277-1283). The authors were able to demonstrate that the down-regulation of AT₁R expression occurred as a result of inhibition at the transcriptional level, which interestingly, was not observed in spontaneously hypertensive animals.

Similarly, the effect of Angll upon αAR signalling was investigated in cultured rat cardiac myocytes (Li, HT. et al. (1997) Cross talk between angiotensin AT1 and alpha 1 -adrenergic receptors: angiotensin Il downregulates alpha 1a-adrenergic receptor subtype mRNA and density in neonatal rat cardiac myocytes. Circulation Research 81:396-403). While Angll treatment had no effect upon the expression of either α-i_bAR or α-i_dAR subtypes, α_1aAR mRNA expression was significantly decreased, an effect that was mediated by AT₁R and associated with decreased α_1aAR mRNA stability. The potential for a functional and physiological relationship between AT₁R and βAR was also examined in cardiac myocytes (Barki- Harrington, L. et al. (2003) Dual inhibition of beta-adrenergic and angiotensin Il receptors by a single antagonist: a functional role for receptor-receptor interaction in vivo. Circulation 108:1611-1618). Results showed that administration of an AT₁R antagonist resulted in signalling inhibition of βARs and vice versa. This frans-inhibitory effect that was found to be due to a direct physical interaction between angiotensin and β-adrenergic receptors, providing further evidence that the angiotensin and adrenergic receptor systems can interact at the molecular level.

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 an alpha-adrenergic receptor-related compound selected from the group: adrenaline (also known as adrenalin or epinephrine), noradrenaline (also known as noradrenalin or norepinephrine), butaxamine, cirazoline, clonidine, guanfacine, guanabenz, guanoxabenz, guanethidine, isoprenaline, lofexidine, methoxamine, methyl-noradrenaline, oxymetazoline, phenylephrine, ritodrine, tizanidine, xylazine, alfuzosin, butoxamine, carvedilol, celiprolol, dihydroergotamine, doxazosin, ergotamine, idazoxan, indoramin, labetolol, phentolamine, phenoxybenzamine, prazosin, terazosin, tamsulosin and yohimbine.

In one form of the invention, the alpha-adrenergic receptor-related compound is an alpha-adrenergic 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 alpha-adrenergic-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 (AT₁R; market name Teveten®, Abbott Laboratories USA), Losartan (AT-iR; market name Cozaar®, Merk & Co), Valsartan (AT₁R; market name Diovan®, Novartis), Telmisartan (ATiR, market name Micardis®, Boehringer Ingelheim), lrbesartan (ATiR, market name Avapro®, SanofiAventis), Olemsartan (AT₁R, market name Benicar®, Daiichi Sankyo Inc), Candesartan (market name Atacand®, AstraZeneca) PD123319 (AT₂R, Tocris), ZD-7115 (AT₁R), Saralasin ((Sar¹-Ala⁸)Angll), Sarthran ((Sar¹- Thr⁸)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.

In one embodiment, the present invention provides a method for the treatment of a patient suffering from Parkinsonian syndromes in combination with hypertension or raised blood pressure, including diagnosis of patients suffering from vascular parkinsonism, arteriosclerotic Parkinsonism. An overview of vascular Parkinsonism is provided in ThanviB, Lo N., et al. (Vascular Parkinsonism - an important cause of Parkinsonism in older people. Age Ageing 2005; 34:114-9). Vascular Parkinsonism is assumed to be caused by a vascular disease of the brain and control of hypertension is considered appropriate for managing symptoms. Frontline treatments for hypertension include angiotensin Il Type 1 receptor blockers as monotherapies and in combination with some other classes of drugs such as ACE inhibitors and diuretics. Control of hypertension through a combination of an angiotensin Il Type 1 receptor blocker and an alphal b adrenergic receptor antagonist is not a front line treatment. In fact, the combination of an angiotensin Il Type 1 receptor blocker and an alphal b adrenergic blocker has been shown to be no more effective than an angiotensin Il Type 1 receptor blocker monotherapy when evaluated based on control of blood pressure while attenuating the effect on the development of hypertensive cardiac hypertrophy (Asai T, Kushiro T, Fujita H and Kanmatsuse K, Different effects on inhibition of cardiac hypertrophy in spontaneously hypertensive rates by monotherapy and combination therapy of adrenergic receptor antagonists and/or angiotensin Il Type 1 Receptor Blocker under Comparable Blood Pressure Reduction, Hypertens Res Vol. 28 No 1 (2005) 79-87). There is no systematic study to ascertain effective control mechanisms for hypertension in patients with vascular Parkinsonism.

Patients with vascular Parkinsonism will typically have lesions involving the substantia nigra. Alpha-1b adrenergic receptor knock-out mice have been shown to be protected against induced nigro-striatal degeneration (Battaglia, G, Fornai F, Buscetti CL, Lembo G, Nicoletti F and De Blasi A, Alpha-I B adrenergic receptor knockout mice are protected against methamphetamine toxicity, Journal of neurochemistry, 2003, 86:413-421 ). Based on the association of the angiotensin receptor and the alpha adrenergic receptors, and more specifically the angiotensin Il Type 1 receptor and the alpha 1 b adrenergic receptor or the angiotensin Il Type Il receptor and the alpha 1b adrenergic receptor, this embodiment is a method for treating a patient with Parkinsonism symptoms and a raised blood pressure response. In this embodiment, treatment of the patient is by administering a therapeutically effective amount of (i) an angiotensin receptor antagonist; and (ii) an alpha adrenergic receptor antagonist simultaneously or within 14 days of each other to produce a therapeutic benefit to the patient. In one embodiment, the two compounds are any angiotensin Il Type 1 receptor antagonist and an alphalb specific receptor antagonist.

Pharmacological blockade of adrenergic receptors with an alpha 1 adrenergic antagonist prazosin has been shown to protect against methamphetamine induced nigro-striatal degeneration (Battaglia, G, Fornai F, Buscetti CL, Lembo G, Nicoletti F and De Blasi A, Alpha-IB adrenergic receptor knockout mice are protected against methamphetamine toxicity, Journal of Neurochemistry, 2003, 86 413-421). Based on the association of the angiotensin receptor and the alpha adrenergic receptors, and more specifically the angiotensin Il Type 1 receptor and the alpha 1b adrenergic receptor or the angiotensin Il Type Il receptor and the alpha 1b adrenergic receptor, this embodiment is a method for prophylactic treatment of vascular Parkinsonism by controlling blood pressure while protecting against Parkinsonism syndromes. The invention includes the prophylactic treatment of vascular Parkinsonism through control of blood pressure through a combination of a therapeutically effect amount of (i) an angiotensin receptor antagonist; and (ii) an alpha adrenergic receptor antagonist simultaneously or within 14 days of each other provided on a basis over time required to produce a therapeutic benefit to the patient. In one embodiment for prophylactic treatment, the two compounds are any angiotensin Il Type 1 receptor antagonist and an alphalb specific receptor antagonist.

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 an alpha-adrenergic receptor; b) assessing whether and/or the extent to which the activity of the alpha- adrenergic receptor is modulated compared to activity of the alpha- adrenergic 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 an alpha-adrenergic receptor; b) assessing the extent to which the activity of the alpha-adrenergic 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 alpha-adrenergic receptor with said test compound; detecting a change in alpha-adrenergic receptor activity.

Methods for assessing the extent to which the activity of an alpha-adrenergic 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 alpha-adrenergic-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 alpha-adrenergic-related ailment.

The present invention also includes a method for screening a test compound for potential therapeutic activity against an alpha-adrenergic-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 alpha-adrenergic-related ailment.

In one embodiment, the method for screening a test compound for potential therapeutic activity against an alpha-adrenergic-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 alpha-adrenergic-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 alpha-adrenergic 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 alpha-adrenergic receptor; and b) determining whether, and/or the extent to which the test compound interacts with the angiotensin receptor in the absence of the alpha- adrenergic 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 alpha-adrenergic receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

The present invention comprises a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor; and b) if the test compound interacts with the angiotensin receptor while the angiotensin receptor is associated with the alpha-adrenergic receptor, determining whether, or the extent to which the test compound interacts with the angiotensin receptor in the absence of the alpha-adrenergic 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 alpha-adrenergic receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for alpha- adrenergic 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 alpha- adrenergic 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 alpha-adrenergic receptor with said test compound; detecting a change in angiotensin receptor or alpha-adrenergic receptor activity.

The present invention comprises a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor while the alpha-adrenergic receptor is associated with the angiotensin receptor; and b) if the test compound interacts with the alpha-adrenergic receptor while the alpha-adrenergic receptor is associated with the angiotensin receptor, deteriming whether, or the extent to which the test compound interacts with the alpha-adrenergic 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 alpha-adrenergic receptor while the alpha- adrenergic receptor is associated with the angiotensin receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

The present invention comprises a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor while the alpha-adrenergic receptor is associated with the angiotensin receptor compared to whether, and/or the extent to which the test compound interacts with the alpha- adrenergic 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 alpha-adrenergic receptor while the alpha- adrenergic receptor is associated with the angiotensin receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for alpha- adrenergic 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 alpha- adrenergic 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 alpha-adrenergic receptor with said test compound; detecting a change in angiotensin receptor or alpha-adrenergic receptor activity.

In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor; iv). an agonist of the angiotensin receptor, the alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero- dimer/-oligomer; c) if the test compound is an antagonist or partial agonist of the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an antagonist or partial agonist of the alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic or partial agonistic properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha- adrenergic 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 alpha-adrenergic receptor; iv). an agonist of the angiotensin receptor, the alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/oligomer.

In one embodiment, the method for screening a test compound for alpha- adrenergic 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 alpha- adrenergic 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 alpha-adrenergic receptor with said test compound; detecting a change in angiotensin receptor or alpha-adrenergic receptor activity. In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero- dimer/-oligomer; c) if the test compound is an antagonist or partial agonist of the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an antagonist or partial agonist of the alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic or partial agonistic properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha- adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for alpha- adrenergic 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 alpha-adrenergic receptor with said test compound; detecting a change in angiotensin receptor or alpha-adrenergic receptor activity.

In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha- adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer; c) if the test compound is an inverse agonist of the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an inverse agonist of the alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha- adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic / angiotensin receptor hetero-dimer/-oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha- adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic / angiotensin receptor hetero-dimer/- oligomer.

In one embodiment, the method for screening a test compound for alpha- adrenergic 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 alpha- adrenergic 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 alpha-adrenergic receptor with said test compound; detecting a change in angiotensin receptor or alpha-adrenergic receptor activity.

In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer; c) if the test compound is an inverse agonist of the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, determining whether, or the extent to which the test compound is an inverse agonist of the alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha- adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer.

In one aspect of the invention, there is provided a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic receptor coupled to a first reporter component; N). 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

The present invention further provides a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor; iv). an agonist of the angiotensin receptor, the alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic / angiotensin receptor hetero-dimer/-oligomer.

The present invention further provides a method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

In one embodiment, the method for screening a test compound for alpha- adrenergic 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 alpha- adrenergic 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 alpha-adrenergic receptor with said test compound; detecting a change in angiotensin receptor or alpha-adrenergic receptor activity.

In a preferred embodiment of the invention, the step of determining whether, and/or the extent to which, the test compound interacts with the alpha-adrenergic receptor while the alpha-adrenergic 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 alpha-adrenergic 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 alpha- adrenergic receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the alpha-adrenergic receptor while the alpha-adrenergic 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 alpha-adrenergic receptor; and/or the step of determining whether, and/or the extent to which, the test compound interacts with the alpha- adrenergic receptor while the alpha-adrenergic 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)₁ 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 alpha-adrenergic 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 alpha-adrenergic receptor/angiotensin receptor hetero-dimer/-oligomer. Thθ present invention comprises a cell, or fraction of a cell, in which both an alpha-adrenergic receptor and an angiotensin receptor are over-expressed.

The present invention comprises a cell, or fraction of a cell, in which an alpha- adrenergic 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 alpha- adrenergic receptor.

Throughout this specification, unless the context requires otherwise, the phrase "fraction of a cell" 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 an alpha-adrenergic receptor and an angiotensin receptor are over-expressed.

The present invention comprises a cell in which an alpha-adrenergic 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 alpha-adrenergic 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 adrenergic-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 alpha-adrenergic receptor agonist, inverse agonist, antagonist or allosteric modulator with the alpha-adrenergic receptor or of angiotensin receptor/ alpha-adrenergic receptor hetero-dimer/oligomer-specific agonist, inverse agonist, antagonist or allosteric modulator with an angiotensin receptor/ alpha-adrenergic receptor hetero-dimer/oligomer.

In the context of aspects of the invention where both an alpha-adrenergic receptor-related compound, such as and without limitation an alpha-adrenergic 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 an alpha-adrenergic receptor-related compound or a therapeutically effective amount of an angiotensin receptor-related compound in combination may be lower than therapeutically effective amounts of an alpha- adrenergic receptor-related compound or angiotensin receptor-related compound when administered alone. That is, the administration of an alpha-adrenergic receptor-related compound and an angiotensin receptor-related compound in combination may generate a therapeutic effect at what would otherwise be subtherapeutic 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, intraventricular^, intracranially, intracapsularly, intraspinally, intracistemally, 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, is a 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 alpha-adrenergic 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 L₁ 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 1 mm. 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 (alphaAR 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 alphaAR-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 ⁰C 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 normalised 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 ALPHA-ADRENERGIC RECEPTORS WITH ANGIOTENSIN RECEPTORS

Referring now to Figures 4 and 5, eBRET signals were measured from cells transiently expressing AT1 R/Venus (Figure 4) or AT2RΛ/enus (Figure 5) and barr2/Rluc8 with either pcDNA3 or wild-type ARs following treatment with N- phenylpiperazine (NPP). In parallel, eBRET signals were measured from cells transiently expressing barr2/Rluc8 with pcDNA3 following treatment with N- phenylpiperazine (NPP) as a negative control.

Prior to ligand treatment (added at 0 minutes), a baseline eBRET signal was recorded for each of the combinations. Negligible BRET signals were observed with the negative controls of NPP-treated barr2/Rluc8 and pcDNA3, or NPP- treated ATR/Venus, barr2/Rluc8 and pcDNA3. NPP treatment of cells co- expressing AT1 R/Venus and barr2/Rluc8 with alphalaAR, alpha2aAR or alpha2bAR resulted in an increase in eBRET signal above that observed with the negative controls. Furthermore, a substantially larger eBRET signal was observed following NPP treatment of cells co-expressing AT1 RΛ/enus and barr2/Rluc8 with alphalbAR. NPP treatment of cells co-expressing AT2RA/enus and barr2/Rluc8 with alpha2aAR resulted in an increase in eBRET signal above that observed with the negative controls. Furthermore, a substantially larger eBRET signal was observed following NPP treatment of cells co-expressing AT2RA/enus and barr2/Rluc8 with alphal bAR. This example demonstrates that a signal resulting from the proximity of RC1 and RC2 is detected specifically for the combination where the angiotensin receptor 1 (AT1 R) or angiotensin receptor 2 (AT2R) is IG1 , Venus is RC1 , beta-arrestin 2 (barr2) is IG2, Rlucδ is RC2 and an alpha-adrenergic receptor (alphaAR) is IG3, and when the modulator, in this case NPP, 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 alpha-adrenergic receptor with the angiotensin receptor.

Without wishing to be bound by theory, BRET signal resulting from NPP treatment of cells co-expressing ATR/Venus and barr2/Rluc8 in the absence of exogenously-expressed adrenergic receptor may result from the activation of endogenously-expressed adrenergic receptor. This appears to be particularly relevant for AT2R where it is notable that these signals are lower than when adrenergic receptor is exogenously expressed, but higher than in the absence of AT2R/Venus (Figure 5).

EXAMPLE 2 MEASUREMENT OF DETECTABLE SIGNALS INDICATIVE OF THE MOLECULAR ASSOCIATION OF THE ALPHA-ADRENERGIC RECEPTOR WITH THE ANGIOTENSIN RECEPTOR IN A DOSE-DEPENDENT MANNER

Referring now to Figure 6, eBRET signals were measured from cells transiently expressing AT1 R/Venus or AT2RΛ/enus with barr2/RIuc8 and wild-type alphalbAR following treatment with various doses of noradrenaline (NA). Data is expressed as percentage of maximum response.

NA treatment of cells co-expressing AT1 R/Venus or AT2R/Venus with barr2/Rlucδ and wild-type alphalb adrenergic receptor (alphal bAR) results in a dose- dependent increase in BRET signal with similar EC_5O values for both combinations. This example demonstrates that a signal resulting from the proximity of RC1 and RC2 is detected specifically for the combination where the angiotensin receptor 1 (AT1 R) or angiotensin receptor 2 (AT2R) is IG1 , Venus is RC1 , beta-arrestin 2 (barr2) is IG2, Rlucδ is RC2 and wild type alpha-1b adrenergic receptor (alphalbAR) is IG3, and when the modulator, in this case NA, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.

This example provides further demonstration that the inventors have identified the molecular association of the alpha-adrenergic receptor with the angiotensin receptor.

This example demonstrates that the ligand-dependent increase in BRET signal reporting the molecular association of the alpha-adrenergic receptor and the angiotensin receptor occurs with NA, in addition to NPP.

EXAMPLE 3 MEASUREMENT OF SUMMATION WITH DUAL TREATMENT INDICATIVE OF THE MOLECULAR ASSOCIATION OF ALPHA-ADRENERGIC RECEPTORS WITH ANGIOTENSIN RECEPTORS

Referring now to Figures 7 and 8, eBRET signals were measured from cells transiently expressing AT1 R/Venus (Figure 7) or AT2R/Venus (Figure 8) and barr2/Rluc8 with pcDNA3 or wild-type alphalbAR following treatment with noradrenaline (NA) or angiotensin Il (ATM) alone or in combination (NA + ATII).

Prior to ligand treatment (added at 0 minutes), a baseline eBRET signal was recorded for each of the combinations. Negligible BRET signals were observed with the negative control of NA-treated ATR/Venus, barr2/Rluc8 and pcDNA3. NA treatment of cells co-expressing AT1R/Venus and barr2/Rluc8 with alphalbAR resulted in an increase in eBRET signal above that observed with the negative control. A substantially larger eBRET signal was observed following ATlI treatment, however, the signal was greater still following treatment with NA and ATII in combination. ATII treatment of cells co-expressing AT2RΛ/enus and barr2/Rluc8 in the presence or absence of wild type alphal bAR did not result in an increase in BRET signal. This reflects the inability of AT2R to interact with barr2. Consistent with this observation, treatment of cells co-expressing AT2R/Venus, barr2/Rluc8 and pcDNA3 with NA or both NA and ATII resulted in a similar BRET signal. Furthermore, treatment of cells co-expressing AT2R/Venus, barr2/Rluc8 and alphalbAR with NA or both NA and ATII resulted in similar, substantially greater BRET signals.

This example demonstrates that a signal resulting from the proximity of RC1 and RC2 is detected specifically for the combination where the angiotensin receptor 1 (AT1 R) or angiotensin receptor 2 (AT2R) is IG1 , Venus is RC1 , beta-arrestin 2 (barr2) is IG2, Rlucδ is RC2 and an alpha-adrenergic receptor (alphaAR) is IG3, and when the modulator, in this case NA, modulates the association of IG2 and IG3 as a result of interacting specifically with IG3.

This example again demonstrates that the inventors have identified the molecular association of the alpha-adrenergic receptor with the angiotensin receptor.

This example also demonstrates the additive effect of combined treatment with IG1 ligand (ATII) and IG3 ligand (NA; modulator). This provides further and distinct evidence for the molecular association of the alpha-adrenergic receptor with the angiotensin 1 receptor, as this 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 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 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.

Claims

The Claims Defining the Invention are as Follows:

1. A hetero-dimeric or hetero-oligomeric receptor, comprising at least one alpha- adrenergic 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 an alpha- adrenergic receptor-related compound.

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

4. A method for the treatment of a patient suffering from an alpha-adrenergic- 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 alpha-adrenergic 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 an alpha-adrenergic receptor; b) assessing whether and/or the extent to which the activity of the alpha- adrenergic receptor is modulated compared to activity of the alpha- adrenergic 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 alpha-adrenergic-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 alpha-adrenergic-related ailment.

8. A method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor compared to whether, and/or the extent to which the test compound interacts with the angiotensin receptor in the absence of the alpha-adrenergic 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 alpha-adrenergic receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

9. A method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor while the alpha-adrenergic receptor is associated with the angiotensin receptor compared to whether, and/or the extent to which the test compound interacts with the alpha-adrenergic 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 alpha-adrenergic receptor while the alpha- adrenergic receptor is associated with the angiotensin receptor is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

10. A method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor; iv). an agonist of the angiotensin receptor, the alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

11. A method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha- adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater antagonistic, partial agonistic or negative allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/- oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

12. A method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha- adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic / angiotensin receptor hetero-dimer/- oligomer.

13.A method for screening a test compound for alpha-adrenergic receptor / angiotensin receptor het8ro-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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater inverse agonistic properties when interacting with the alpha- adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer.

14. A method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor; iv). an agonist of the angiotensin receptor, the alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic / angiotensin receptor hetero-dimer/-oligomer.

15. A method for screening a test compound for alpha-adrenergic 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 alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer, by contacting said test compound with a system comprising: i). a first agent, comprising the alpha-adrenergic 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 alpha-adrenergic receptor and/or the alpha-adrenergic 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 alpha-adrenergic 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 alpha-adrenergic receptor in the absence of the angiotensin receptor and the angiotensin receptor in the absence of the alpha-adrenergic receptor; such that a test compound that exhibits greater positive allosteric modulator properties when interacting with the alpha-adrenergic receptor / angiotensin receptor hetero-dimer/-oligomer is selective for the alpha-adrenergic 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 alpha-adrenergic receptor/angiotensin receptor hetero-dimer/-oligomer.

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

18. A cell, or fraction of a cell, in which an alpha-adrenergic 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 alpha-adrenergic receptor.