WO2022246503A1

WO2022246503A1 - Methods for mediating vasoconstriction

Info

Publication number: WO2022246503A1
Application number: PCT/AU2022/050495
Authority: WO
Inventors: Scott AYTON; Ashenafi BETRIE; Ashley Bush; Christine Wright; James Angus
Original assignee: The Florey Institute Of Neuroscience And Mental Health; The University Of Melbourne
Priority date: 2021-05-24
Filing date: 2022-05-24
Publication date: 2022-12-01

Abstract

Provided herein are methods for inducing vasoconstriction in a blood vessel, typically an artery, comprising administering to a subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion. Also provided are methods for the treatment and prevention of vascular diseases and disorders, and for the modulation of vascular tone, comprising administering to a subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

Description

Methods for mediating vasoconstriction

Field

[0001] The present invention relates generally to the use of a zinc chelator for mediating vasoconstriction. More particularly, the present invention relates to the administration of a zinc chelator to mediate vasoconstriction and thereby treat or prevent a vascular disease or disorder amenable to treatment by mediation of vasoconstriction.

Background

[0002] Vascular diseases and disorders may arise form abnormal vascular tone regulation. Vascular tone refers to the contractile activity of vascular smooth muscle cells in the walls of blood vessels. Since the vasculature extends throughout the human body, diseases and disorders associated with abnormal vascular tone regulation can have a local effect (i.e. at the immediate site) and a broader effect on a range of organs and tissues.

[0003] Given the extent and connectivity of the vasculature, the desire to modulate vascular tone in one region often leads to the same effect in other areas. This presents difficulties since global vasoconstriction or global vasodilation is often unfavourable, when a localised effect is desired.

[0004] Zinc is an abundant transition metal and is essential for many proteins that serve structural, functional and signalling functions in cardiovascular biology. Proteins such as nitric oxide synthase, phosphodiesterase, angiotensin converting enzyme, superoxide dismutase, neprilysin and angiotensin II either directly bind to zinc or depend on the presence of zinc for activity. It has been shown that zinc supplements decrease systolic blood pressure, while a deficiency of zinc is associated with high blood pressure. Cellular concentrations of zinc are controlled by 24 zinc transporter channels that have varied expression in different organs and cells. While some effects of zinc on vascular tone are known, the exact mechanism by which zinc mediates these effects is unknown. Furthermore, the receptors that are targeted by zinc to modulate vascular tone and the expression profile of these receptors have not been confirmed.

1

SUBSTITUTE SHEETS (RULE 26) [0005] There remains a need for new agents that are able to control vascular tone by inducing vasoconstriction in a site-specific manner without unwanted side effects.

Summary of the disclosure

[0006] The present invention relates to the finding that vascular tone can be modulated by the administration of a zinc chelator, reducing or ameliorating the effect that zinc has on vascular tone.

[0007] According to a first aspect, the present invention provides a method for inducing vasoconstriction in at least one blood vessel, the method comprising administering to a subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

[0008] In some embodiments, the at least one blood vessel to be targeted is an artery. In some embodiments, the blood vessel is an artery that undergoes vasoconstriction upon administration of the chelator. In some embodiments, the artery is a cerebral artery. In other embodiments, the artery is a coronary artery. In other embodiments, the artery is a basilar artery. In other embodiments, the artery is a saphenous artery.

[0009] In other embodiments, the blood vessel to be targeted is a vein. In some embodiments, the blood vessel is a vein that undergoes vasoconstriction upon administration of the chelator. In other embodiments, the vein is a saphenous vein.

[0010] The finding presented herein indicate that the targeted or selective treatment of various vascular diseases and disorders can be achieved by taking into account the level of endogenous zinc in the vascular tissue that is to be targeted and where appropriate administering to a subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

[0011] Accordingly, a second aspect of the present invention provides a method for the treatment or prevention of a vascular disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion. [0012] In an embodiment, the vascular disease or disorder comprises or is associated with haemorrhage. In an embodiment, the vascular disease or disorder is a headache, optionally migraine.

[0013] According to a third aspect, the present invention provides a method for the modulation of vascular tone in a subject, the method comprising administering to the subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

[0014] Typically, the modulation of vascular tone results in vasoconstriction.

[0015] In some embodiments of the above aspects, the chelator is selective for zinc ions.

[0016] Typically in accordance with the above aspects the metal chelator coordinates intracellular zinc. Accordingly, in accordance with the above aspects the metal chelator is typically cell membrane permeable.

[0017] In some embodiments of the above aspects, the chelator is an organic ligand. In some embodiments, the chelator may be an organic ligand containing one or more heteroatoms, such as an organic ligand containing one or more nitrogen heteroatoms. In some embodiments, the chelator is an organic ligand containing one or more pyridine groups.

[0018] In exemplary embodiments of the above aspects, the chelator is tris(2- pyridylmethyl) amine (TPA). In other embodiments, the chelator is A,/V,A’,/V’-tetrakis(2- pyridinylmethyl)- 1 ,2-ethanediamine (TPEN) .

[0019] Also provided herein is the use of a metal chelator or a salt thereof in the manufacture of a medicament for inducing vasoconstriction, wherein the metal chelator coordinates a zinc ion.

[0020] Also provided herein is the use of a metal chelator or a salt thereof in the manufacture of a medicament for the treatment or prevention of a vascular disease or disorder, wherein the metal chelator coordinates a zinc ion. [0021] Also provided herein is the use of a metal chelator or a salt thereof in the manufacture of a medicament for the modulation of vascular tone, wherein the metal chelator coordinates a zinc ion.

Brief description of the figures

[0022] Embodiments of the present invention are described herein, by way of non-limiting example only, with reference to the following drawings.

[0023] Figure 1. Contractile effects of TPA on resting tone in isolated vessels from rats. Response is % of the KPSS (124 mM K⁺)-evoked contractions . Error bars are SEM (those not shown are contained within the symbol) n = number of arteries isolated from individual rats. TPA induced contraction of middle cerebral, basilar, coronary and saphenous arteries.

[0024] Figure 2. Contractile effects of TPEN on resting tone in isolated vessels from rats. Contractile effects of TPA on resting tone in vessels from rats. Response is % of the KPSS (124 mM K⁺)-evoked contractions. Error bars are SEM (those not shown are contained within the symbol) n = number of arteries isolated from individual rats. TPEN induced contraction of middle cerebral, basilar, coronary and saphenous arteries.

[0025] Figure 3. Contractile effects of TPEN on resting tone in vessels from humans. Response is % of the KPSS (124 mM K⁺)-evoked contractions. Error bars are SEM (those not shown are contained within the symbol) n = number of arteries isolated from individual humans. TPEN caused contraction of human saphenous veins, but not human internal mammary arteries.

[0026] Figure 4. Effect of zinc addition after contraction of middle cerebral arteries of rats with TPEN. Response is % of the KPSS (124 mM K⁺)-evoked contractions. Error bars are SEM (those not shown are contained within the symbol) n = number of arteries isolated from individual rats. **p < 0.01 mixed-effects 1-way ANOVA with Dunnett’s post-test compared to TPEN tone. Contraction by TPEN in middle cerebral arteries was abolished by zinc coadministration. [0027] Figure 5. Concentration of zinc in rat arteries. Whiskers are min to max values and boxes are 25th to 75th percentile, where the line inside the box is the median and the + sign is the mean n = number of arteries isolated from individual rats. Rat coronary arteries contained lower zinc levels than mesenteric and pulmonary arteries 'p < 0.05, ^{: :} 'p < 0.001, Kruskal- Wallis test with Dunn’s post-test.

Detailed description

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0029] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0030] In the context of this specification, the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.

[0031] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0032] The term "optionally" is used herein to mean that the subsequently described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiments in which the event or circumstance occurs as well as embodiments in which it does not.

[0033] As used herein the terms "treating", "treatment", “preventing”, “prevention" and grammatical equivalents refer to any and all uses which remedy the stated neurodegenerative disease, prevent, retard or delay the establishment of the disease, or otherwise prevent, hinder, retard, or reverse the progression of the disease. Thus the terms "treating" and “preventing” and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a patient is treated until total recovery. Where the disease displays or a characterized by multiple symptoms, the treatment or prevention need not necessarily remedy, prevent, hinder, retard, or reverse all of said symptoms, but may prevent, hinder, retard, or reverse one or more of said symptoms.

[0034] The term "subject" as used herein refers to mammals and includes humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), performance and show animals (e.g. horses, livestock, dogs, cats), companion animals (e.g. dogs, cats) and captive wild animals. Preferably, the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human.

[0035] The present invention is predicated on the inventors’ surprising finding that vascular tone can be modulated by the administration of a zinc chelator, reducing the intracellular availability of free zinc, and thereby reducing or ameliorating the effect that zinc has on vascular tone. The present inventors have found that the administration of a zinc chelator leads to vasoconstriction in selected major arteries. Without wishing to be bound by theory, the inventors suggest that the physical properties of the zinc chelator allow for movement of the chelator through a cell membrane and into the cytoplasm of a cell (for example, smooth muscle cells), where the chelator is subsequently able to coordinate free zinc ions. Accordingly, the chelator is typically cell membrane-permeable. Since the presence of zinc in smooth muscle tissue leads to vasorelaxation, the administration of a chelator able to coordinate zinc leads to a reduction in the concentration of zinc and thereby results in vasoconstriction. [0036] Further, without wishing to be bound by theory, the present inventors suggest that the blood vessels that display vasoconstriction upon administration of a chelator are those that have a lower level of endogenous zinc in associated smooth muscle cells. Where the cells have a lower concentration of endogenous zinc, the administration of a chelator selective for zinc ions may therefore lead to the coordination of a greater proportion of zinc and lessen the extent of the basal vasorelaxation mediated by the free zinc ions present. Where the cell tissue of a particular artery has a lower concentration of zinc, the administration of a chelator results in vasoconstriction, whereas a cell tissue with a higher concentration of zinc may not display the same vasoconstriction upon administration of the metal chelator. The present inventors believe that the amount of endogenous zinc present in tissue at a specific site affects the extent of vasorelaxation. For example, where the concentration of zinc is lower, the administration of a metal chelator can coordinate a greater proportion of the zinc present and reduces the basal vasorelaxation mediated by the presence of free zinc ions. Where the concentration of zinc is higher, the administration of the same amount of a metal chelator coordinates a smaller proportion of the zinc ions, which may not reduce the basal vasorelaxation mediated by the presence of zinc ions. This therefore limits the subsequent vasoconstriction observed.

[0037] Accordingly, provided herein are methods for inducing vasoconstriction and for modulating vascular tone in a subject, comprising administering to the subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

[0038] Also provided herein is a method for the treatment or prevention of a vascular disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

[0039] As used herein, the term “vascular disease or disorder” refers to disease or disorder that affects the vasculature or blood vessels, i.e. the arteries, veins and/or capillaries, of a physiological system and the flow of blood through these vessels. In particular, vascular diseases and disorders that may be treated or prevented in accordance with the present invention are those wherein a subject to be treated will benefit from vasocontraction. [0040] For example, haemorrhage (i.e. bleeding) at a particular site may be treated by inducing local vasoconstriction to reduce blood flow and thereby limit bleeding. Other diseases and disorders include headaches such as migraine, which is typically associated with vasodilation in cerebral arteries. Administration of a metal chelator as disclosed herein may lead to local vasocontraction and subsequent relief of migraine.

[0041] As used herein, the term “therapeutically effective amount” refers to an amount sufficient to effect a beneficial or desired result. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease or disorder to be treated. An effective amount may be administered in one or more administrations. The exact amount required will vary from subject to subject and the nature of the disease or disorder. A “therapeutically effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

[0042] In some embodiment, methods described herein for inducing vasoconstriction and for modulating vascular tone and for treating or preventing vascular diseases or disorders may further include the step of determining the level of endogenous zinc in smooth muscle cells associated with the affected blood vessels or the blood vessels in which vasoc

[0043] As used herein, the term “metal chelator” refers to a compound that acts as a ligand to a metal, specifically to a chelator that coordinates a zinc ion. The ligand binds or chelates zinc by forming one or more bonds between atoms of the ligand and the zinc. The moiety formed after binding occurs between the ligand and zinc may be referred to as a “coordination complex”, since the metal is considered to be “coordinated” to the ligand. The bonds formed between the ligand and the zinc may be between a heteroatom on the ligand and the zinc centre. Where the ligand has multiple heteroatoms, one ligand may form multiple bonds with a single zinc centre, such that the ligand is a polydentate ligand. In some embodiments, the ligand may be a polydentate ligand. In an embodiment, the ligand is a bidentate ligand. In another embodiment, the ligand is a tetradentate ligand. In another embodiment, the ligand is a hexadentate ligand. In yet another embodiment, the ligand is an octadentate ligand. In some embodiments, the ligand contains one or more nitrogen heteroatoms. In other embodiments, the ligand contains one or more oxygen heteroatoms. In further embodiments, the ligand contains one or more sulfur heteroatoms. In some embodiments, the ligand contains both nitrogen and oxygen heteroatoms. In other embodiments, the ligand contains both nitrogen and sulfur heteroatoms. The zinc that is chelated by the ligand may be a single atom or an ion of the zinc.

[0044] Chelators suitable for the chelation of zinc are well known to those skilled in the art. The chelator may be specific for zinc. The chelator may be selective for zinc. Typically the chelator is a cell membrane-permeable chelator. Examples of zinc chelators, (not all of which are membrane-permeable) include penicillamine, trientine, N,N'-diethyldithiocarbamate (DDC), 2,3,2'-tetraamine (2,3,2'-tet), neocuproine, N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine (TPEN), 1,10-phenanthroline (PHE), tetraethylenepentamine (TEPA), triethylene tetraamine and tris(2-carboxyethyl) phosphine (TCEP), bathophenanthroline disulfonic acid (BPADA), ethylene glycol (bis) aminoethyl ether tetra acetic acid (EGTA), nitrilotri acetic acid, TIRON™, N,N-bis(2-hydroxyethyl)glycine (bicine); 0,0'-bis(2-aminophenyl ethylene glycol) ethylenediamine-N,N,N',N'-tetraacetic acid (BAPTA), trans- 1,2-diamino cyclohexane-ethylenediamine-N,N,N',N'-tetraacetic acid (CyDTA), l,3-diamino-2-hydroxy-propane-ethylenediamine-N,N,N', N'-tetraacetic acid (DPTA-OH), ethylene-diamine-N,N'-dipropionic acid dihydrochloride (EDDP), ethylenediamine-N,N'-bis(methylenephosphonic acid) hemihydrate (EDDPO), ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid) (EDTPO), N,N'-bis(2- hydroxybenzyl)ethylene diamine -N,N'-diacetic acid (HBED), 1,6-hexamethylenediamine- N,N,N', N'-tetraacetic acid (HDTA, or HEDTA), N-(2-hydroxyethyl)iminodiacetic acid (HID A), iminodiacetic acid (IDA), l,2-diaminopropane-N,N,N', N'-tetraacetic acid (methyl - EDTA), nitriltriacetic acid (NTA), nitrilotripropionic acid (NTP), nitrilotris (methylenephosphonic acid) trisodium salt (NTPO), triethylenetetramine-N,N,N',N'',N''- hexaacetic acid (TTHA), bathocuproine, bathophenanthroline, TETA, citric acid, salicylic acid, and malic acid, and analogues and derivatives, including hydrophobic derivatives and pharmaceutically acceptable salts thereof.

[0045] Non-limiting examples of suitable cell membrane-permeable chelators include N,N,N’J\f ’-tetrakis(2-pyridinylmethyl)- 1 ,2-ethanediamine (TPEN), tris(2- pyridylmethyl) amine (TPA), lO-O-phenanthroline and diethyldithiocarbamate (DEDC). Exemplary chelators include: N,N,N Ά ’-tetrakis(2-pyridinylmethyl)- 1,2- tris(2-pyridylmethyl)amine (TPA) ethanediamine (TREN)

[0046] Embodiments of the present invention provide methods for the in vivo and ex vivo reduction in free zinc levels in cells, and in particular in the cytosol. For example, in vivo methods contemplate the administration of a chelator as described herein to a subject. Alternatively, a blood sample may be obtained from a subject and the blood sample exposed to a chelator as described herein in order to chelate zinc and deplete the blood of free zinc. The zinc-depleted blood sample may then be re-introduced to the subject, or another individual. Such ex vivo embodiments find application, for example during surgery and in blood transfusions more generally.

[0047] Embodiments of the present disclosure contemplate the formulation of a chelator as described herein in the form of pharmaceutical compositions, which compositions may comprise one or more pharmaceutically acceptable carriers, excipients or diluents. For administration to a subject, such compositions may be administered via any convenient or suitable route such as by parenteral (e.g. intraperitoneal, subcutaneous, intraarterial, intravenous, intramuscular, intrathecal, intracerebral, intraocular), oral (including sublingual), nasal, transmucosal or topical routes. In circumstances where it is required that appropriate concentrations of the molecules are delivered directly to the site in the body to be treated, administration may be regional rather than systemic. Regional administration provides the capability of delivering very high local concentrations of the molecules to the required site and thus is suitable for achieving the desired therapeutic or preventative effect whilst avoiding exposure of other organs of the body to the vectors and molecules and thereby potentially reducing side effects.

[0048] As will be appreciated by those skilled in the art, the choice of pharmaceutically acceptable carrier or diluent will be dependent on the route of administration and on the nature of the condition and subject to be treated. The particular carrier or diluent and route of administration may be readily determined by a person skilled in the art. The carrier or diluent and route of administration should be carefully selected so as to ensure activity of the metal chelator upon arrival at the site of action.

[0049] Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example, ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

[0050] A person skilled in the art will readily be able to determine appropriate formulations to be administered using conventional approaches. Techniques for formulation and administration may be found in, for example, Remington (1980) Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition; and Niazi (2009) Handbook of Pharmaceutical Manufacturing Formulations, Informa Healthcare, New York, second edition, the entire contents of which are incorporated by reference.

[0051] Identification of preferred pH ranges (where appropriate) and suitable excipients is routine in the art, for example, as described in Katdare and Chaubel (2006) Excipient Development for Pharmaceutical, Biotechnology and Drug Delivery Systems (CRC Press).

[0052] In some embodiments, the chelator may be formulated for oral administration in a dosage form such as a tablet, pill, capsule, liquid, gel, syrup, slurry, suspension, lozenge and the like for oral ingestion by a subject. In particular embodiments, the compound or agent is formulated for oral administration in a solid dosage form, such as a tablet, pill, lozenge or capsule. In such embodiments, the pharmaceutically acceptable carrier may comprise a number of excipients including, but not limited to, a diluent, disintegrant, binder, lubricant, and the like.

[0053] Suitable diluents (also referred to as “fillers”) include, but are not limited to, lactose (including lactose monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, compressible sugar, isomalt, microcrystalline cellulose, powdered cellulose, starch, pregelatinised starch, dextrates, dextran, dextrin, dextrose, maltodextrin, calcium carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate, magnesium carbonate, magnesium oxide, poloxamers, polyethylene oxide, hydroxypropyl methyl cellulose, silicates (e.g. silicon dioxide), polyvinyl alcohol, talc, and combinations thereof.

[0054] Suitable disintegrants include, but are not limited to, sodium carboxymethyl cellulose, pregelatinised starch, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, sodium starch glycolate, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, sodium alginate and combinations thereof. Suitable binders include, but are not limited to, microcrystalline cellulose, gelatine, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose and combinations thereof. Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, polyethylene glycol and combinations thereof.

[0055] Pharmaceutical formulations for parenteral administration include aqueous solutions of a suitable compound or agent in water-soluble form. Additionally, suspensions of the compound or agent may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilisers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0056] Sterile solutions may be prepared by combining the metal chelator as described herein in the required amount in the appropriate solvent with other excipients as described above as required, followed by sterilization, such as filtration. Generally, dispersions are prepared by incorporating the various sterilised active compounds into a sterile vehicle which contains the basic dispersion medium and the required excipients as described above. Sterile dry powders may be prepared by vacuum- or freeze-drying a sterile solution comprising the active compounds and other required excipients as described above.

[0057] The pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for the preparation of sterile injectable solutions. Such forms should be stable under the conditions of manufacture and storage and may be preserved against reduction, oxidation and microbial contamination. For injection, compositions of the invention may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks’ solution, Ringer’s solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0058] It will be understood that the specific dose level of the chelator for any particular subject will depend upon a variety of factors including, for example, the activity of the chelator, the half-life of the chelator, the age, body weight, general health and diet of the individual to be treated, the time of administration, rate of excretion, and combination with any other treatment or therapy. Single or multiple administrations can be carried out with dose levels and pattern being selected by the treating physician. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of agent may be administered per kilogram of body weight per day. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

[0059] In exemplary embodiments of the present disclosure it is contemplated that the chelator may be administered to a subject daily or less than daily, for example every second day or every third day for the duration of treatment required to achieve the desired outcome. Administration may be continuous, for example on a daily basis or every second day, or may be intermittent with spacing between administrations determined by the treating medical professional depending on response of the subject to treatment and progress of the subject during the course of treatment. [0060] The present invention also contemplates combination therapies, wherein the chelator as described herein is coadministered with other suitable agents that may facilitate the desired therapeutic or prophylactic outcome. The term “coadministered” mean simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. The term “simultaneously” means that the active agents are administered at substantially the same time. The term “sequential” administration means a time difference of from seconds, minutes, hours or days between administration of the agents. Administration may be in any order.

[0061] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

[0062] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Examples

[0063] The following examples are illustrative of the disclosure and should not be construed as limiting in any way the general nature of the disclosure of the description throughout this specification.

General methods

[0064] Data analyses. All data are expressed as mean ± SEM, with n being the number of rats/mice/humans or arteries isolated from separate animals unless stated otherwise. Each sigmoidal concentration-response curve was fitted using Prism 8 (GraphPad Software, USA). In some cases, the last data point (i.e. highest concentration) was imputed (replication of the next highest concentration) for better sigmoidal fitting. The pEC50 values (the negative loglOM of drug concentration that decreases the response by 50%) and Emax (maximum response) were determined for each tissue and averaged. [0065] Two-tailed Student’s paired and unpaired t test were used to analyze the differences between two variables and one-way analysis of variance (1-way ANOVA) with Dunnett’ s post test was used to compare means between three or more variables. Repeated measures 1-way ANOVA (mixed-effects if there were missing values) with Dunnett’ s post-test was used to test concentration-dependent changes from baseline values within the same group. The p values from the post-tests are reported. Values of p<0.05 were considered statistically significant.

[0066] Metal measurements using Inductively Coupled Plasma Mass Spectrometry ( ICP-MS ). Metal levels from isolated tissues were measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS; Agilent 7700 series, Agilent Technologies, Santa Clara, CA, USA) under routine multi-element operating conditions using a helium reaction gas cell. Tissue samples were weighed, freeze-dried, and then resuspended in 69% nitric acid (ultraclean grade, Aristar) overnight. The samples were then heated for 20 min at 90°C, and equivalent volume of hydrogen peroxide (30%, Merck) was added for further 15 min incubation at 70°C. The samples were diluted in double-distilled water and assayed by ICP-MS. Each tissue sample was measured in triplicate and the concentrations determined from the standard curve were normalized to wet tissue weight.

Example 1 — Vasoconstriction in rat and human blood vessels

[0067] Functional in vitro protocols in isolated arteries. Human internal mammary artery and saphenous vein or a range of vessels from rats and mice (left main or anterior descending coronary arteries [250-400 pm internal diameter, i.d.], second order pulmonary interlobar arteries [300-600 pm i.d.], second or third order mesenteric arteries [200-350 pm i.d.], interlobar renal arteries [250-350 pm i.d.], saphenous arteries or veins [300-600 pm i.d.]), or the thoracic aorta were dissected and placed in ice-cold PSS-A with the following composition (mmol/L, mM): NaCl 119; KC1 4.69; MgS04.7H20 1.17; KH2P04 1.18; glucose 5.5; NaHC03 25; CaC12.6H20 2.5 saturated with carbogen (02 95%; C02 5%) at pH 7.4. In addition, rat middle cerebral (250-400 pm i.d.) and basilar (250-450 pm i.d.) arteries were used and for these vessels the PSS-A contained 1.5 mM CaCh to minimize the occurrence of spontaneous contractions.

[0068] After isolation of vessels, ~2 mm length segments of arteries were mounted in myograph chambers (Model 610M and 620M; Danish Myo Technology, Denmark) containing PSS-A at 37°C for isometric force measurement as described previously. Contractile and relaxation responses using rat and mice isolated vessels were recorded with LabChart 7 and a PowerLab 4/30 A/D converter (AD Instruments Pty Ltd, Australia) while those including human isolated vessels were recorded with LabChart 8 (AD Instruments) and Myodaq and Myodata 2.01 (Maastricht University, Maastricht, Netherlands). To normalize the basal conditions, the vessels were passively stretched according to a normalization protocol and adjusted to a diameter setting of 90% of that determined for an equivalent transmural pressure of 100 mmHg (30 mmHg for veins). After allowing the tissues to equilibrate for 30 min, the arteries were exposed to a potassium depolarizing solution (124 mM K⁺ replacing Na⁺ in PSS; termed KPSS) and noradrenaline (10 mM) for 2 min (chemicals, their source and preparation are provided in the online supplement). A second exposure to KPSS solution (only) was used to provide a reference contraction.

[0069] Figures 1 and 2 show the contraction of isolated rat arteries upon exposure to varying concentrations of TPA and TPEN, respectively. The administration of zinc chelators resulted in contraction of selected arteries, i.e. middle cerebral, basilar and coronary arteries and saphenous veins. Without wishing to be bound by theory, the inventors suggest that the cerebral and coronary arteries have a low level of endogenous zinc, such that the addition of a chelator reduces the amount of zinc present sufficiently to ameliorate vasorelaxation.

[0070] Figure 3 shows the contraction of isolated human vessels (i.e. saphenous vein) after exposure to TPEN. Significant contraction of human saphenous vein was observed, however internal mammary arteries did not show the same effect.

Example 2 - Vasoconstriction in isolated arteries is dependent on the presence of zinc

[0071] To further probe the role of zinc in vasocontraction, varying amounts of zinc were added to isolated rat middle cerebral arteries after inducing vasocontraction by administration of TPEN. Vasocontraction attributed to the effect of TPEN was abolished upon administration of zinc, as shown in Figure 4. Higher concentrations of zinc resulted in greater reduction in vasocontraction. Example 3 - Susceptibility of isolated arteries to vasocontraction is dependent on the amount of zinc present

[0072] The regional differences to zinc chelators described in Examples 1 and 2 were speculated to be related to the zinc content in the vascular tissue. The concentration of zinc in each tissue was determined by ICP-MS.

[0073] As shown in Figure 5, the arteries that displayed vasocontraction with administration of a zinc chelator (coronary and saphenous arteries) had lower levels of zinc than tissues that did not display a similar vasocontraction (mesenteric and pulmonary arteries). This suggests that arteries that are susceptible to vasocontraction with exposure to a zinc chelator will have a lower concentration of zinc in the tissue.

Claims

1. A method for inducing vasoconstriction in at least one blood vessel, the method comprising administering to a subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

2. A method according to claim 1, wherein the at least one blood vessel is an artery.

3. A method according to claim 2, wherein the artery is a cerebral artery, a coronary artery, a basilar artery or a saphenous artery.

4. A method according to claim 1, wherein the at least one blood vessel is a vein.

5. A method according to claim 4, wherein the vein is a saphenous vein.

6. A method for the treatment or prevention of a vascular disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

7. A method according to claim 6, wherein the vascular disease or disorder comprises or is associated with haemorrhage, or headache, optionally migraine.

8. A method for the modulation of vascular tone in a subject, the method comprising administering to the subject a therapeutically effective amount of a metal chelator or a salt thereof, wherein the metal chelator coordinates a zinc ion.

9. A method according to claim 8, wherein the modulation of vascular tone comprises vasoconstriction in at least one blood vessel of the subject.

10. A method according to any one of claims 1 to 9, wherein the chelator is selective for zinc ions.

11. A method according to any one of claims 1 to 10, wherein the chelator is cell membrane permeable.

12. A method according to any one of claims 1 to 11, wherein the chelator coordinates intracellular zinc.

13. A method according to any one of claims 1 to 12, wherein chelator is an organic ligand containing one or more heteroatoms.

14. A method according to claim 13, wherein the organic ligand contains one or more nitrogen heteroatoms or one or more pyridine groups.

15. A method according to any one of claims 1 to 14, wherein the chelator is tris(2- pyridylmethyl) amine (TPA) or /V,/V,/VyV’-tctrakis(2-pyridinylmcthyl)-l ,2-cthancdiaminc (TPEN).

16. Use of a metal chelator or a salt thereof in the manufacture of a medicament for inducing vasoconstriction in at least one blood vessel, wherein the metal chelator coordinates a zinc ion.

17. Use of a metal chelator or a salt thereof in the manufacture of a medicament for the treatment or prevention of a vascular disease or disorder, wherein the metal chelator coordinates a zinc ion.

18. Use of a metal chelator or a salt thereof in the manufacture of a medicament for the modulation of vascular tone, wherein the metal chelator coordinates a zinc ion.