WO2013075176A1 - Compositions and methods for modulating uterine contractions - Google Patents

Abstract

Provided herein are methods for stimulating or inducing contractility of myometrial tissue, methods for inducing or stimulating uterine contractions, and methods for inducing or assisting labour in pregnant subjects, comprising exposing myometrial tissue to an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

Description

COMPOSITIONS AND METHODS FOR MODULATING UTERINE

CONTRACTIONS

Field of the Disclosure

[001] The present disclosure relates generally to compositions and methods for modulating uterine contractions, in particular by modulating the expression or activity of an ether-a-go-go potassium channel or the gene encoding the channel.

Background

[002] Contractility of the myometrium, the smooth muscle layers of the uterus, is essential for the normal progression of human pregnancy and the initiation of labour. The onset of labour is associated with increasing frequency and intensity of uterine contractions, tightening the uterus and pushing the baby from the uterus into the birth canal for delivery.

[003] A common complication of pregnancy and labour is inadequate uterine contractions. This can lead to a delay or failure in t e initiation of labour or to abnormally prolonged and difficult labour (dystocia). Dystocia can result in an assisted delivery (including forceps delivery or Caesarian section) and may result in fetal health complications such as respiratory or nerve damage, or even fetal death.

[004] Myometrial contractility is also critical after childbirth. Following the separation of the placenta from the uterine wall, blood flow is slowed in the uterus by persistent, regular myometrial contractions. With increased clotting proteins present and blood flow slowed by uterine contractions, myometrial arteries clot. Uterine contractions following birth are generally sufficient to slow the velocity of blood flowing through the uterus to initiate blood clot formation throughout myometrium. However in some women uterine contractions are inadequate. Uncontrolled bleeding following childbirth (post partum haemorrhage) can result in significant blood loss having major health implications for the new mother and even resulting in death. [005] Presently, in cases of inadequate uterine contractions before, during or after labour, contractions may be induced or assisted by the administration of stimulators of contraction such as oxytocin, and prostaglandins. However, such agents typically produce side effects on the vasculature not just in the uterus but throughout the body, and are often contraindicated in patients with hypertension or hypotension. Alternatively, surgical intervention may be required, also carrying risks for both mother and child. In the case of post partum haemorrhage, a hysterectomy may be performed, an operation that leaves the mother infertile.

[006] There is a clear need for the development of alternative, simple to administer treatments to stimulate or induce uterine contractions, either in inducing or assisting labour or in controlling bleeding after labour.

Summary

[007] Aspects disclosed herein are based on the surprising findings, inter alia, that levels of the human ether-a^o-go potassium channel protein (hERG) fall at the onset of labour, and that contractility of human myometrium can be stimulated by the addition of inhibitors of hERG.

[008] Accordingly, a first aspect enabled herein is a method for stimulating or inducing contractility of myometrial tissue, the method comprising exposing the myometrial tissue to an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

[009] In particular embodiments the method comprises administering to a female mammal an effective amount of the agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof. The female mammal may be pregnant, may be in labour or may have given birth. The administration may be provided to stimulate uterine contractions and thereby induce or assist labour. Alternatively the administration may be provided to stimulate uterine contractions post labour to inhibit blood loss. [0010] In particular embodiments the mammal is a human and the ERG potassium channel is hERG.

[0011] The agent may be a potassium channel inhibitor, in particular an inhibitor specific or selective for a voltage-gated potassium channel such as hERG or a homologue or orthologue thereof. In exemplary embodiments the inhibitor may be selected from dofetilide and E-4031 ,

[0012] The agent may be a miRNA, or miRNA antagonist, wherein the miRNA interacts with the ERG gene. The miRNA that interacts with the ERG gene may be hsa_miR_144, hsa_miR_142-3 , hsa_miR_125b, hsa_miR_362-3p, hsa_miR_101, or hsa_miR_133a, comprising the nucleotide sequences set forth in SEQ ID Nos:3-8, respectively. Typically the ERG gene is ERG1. In exemplary embodiments the agent may comprise one or more miRNAs selected from hsa_miR_144, hsa_miR_142-3p, hsa_miR_125b, hsa_miR_362-3p, and hsa_miR_101 . Alternatively or in addition, the agent may comprise an antagonist of hsa_miR_133a.

[0013] The agent capable of inhibiting the expression or activity of an ERG potassium channel may be delivered to the myometrial tissue via a number of means, and may be targeted to the myometrial tissue. In an exemplary embodiment the agent may be delivered to the myometrial tissue in liposomes. The liposomes may be targeted to the myometrial tissue by, for example, conjugating or coating the liposomes with antibodies specific or selective for the oxytocin receptor expressed on the surface of myometrial cells.

[0014] Another aspect enabled herein is use of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, in the manufacture of a medicament for stimulating or inducing contractility of myometrial tissue.

[0015] Another aspect enabled herein is a composition for stimulating or inducing contractility of myometrial tissue, the composition comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0016] Another aspect enabled herein is a method for inducing or assisting labour, comprising administering to a subject in need thereof an effective amount of an agent capable of inliibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

[0017] Also disclosed and enabled herein are compositions for inducing or assisting labour comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, and the use of such an agent in the manufacture of a medicament for inducing or assisting labour.

[0018] Another aspect enabled herein is a method for inducing uterine contractions in a pregnant mammal, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

[0019] Also disclosed and enabled herein are compositions for inducing uterine contractions in a pregnant ' mammal comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, and the use of such an agent in the manufacture of a medicament for inducing uterine contractions in a pregnant mammal.

[0020] Another aspect enabled herein is a method for treating or preventing dystocia, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof. .

[0021] Also disclosed and enabled herein are compositions for treating or preventing dystocia comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, and the use of such an agent in the manufacture of a medicament for treating or preventing dystocia.

[0022] Another aspect enabled herein is a method for treating or preventing post partum haemorrhage, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

^'[0023] Also disclosed and enabled herein are compositions for treating or preventing post partum haemorrhage, the compositions comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, and also the use of such an agent in the manufacture of a medicament for treating or preventing post partum haemorrhage.

Brief Description of the Drawings

[0024] Aspects and embodiments disclosed and exemplified herein are illustrated, by way of non-limiting example only with reference to the following drawings.

[0025] Figure 1. hERG gene ( CNH2) expression levels in non-labouring (NIL), spontaneously labouring (L) and induced-labouring (Liol) human myometrium.

[0026] Figure 2. (A), hERG protein expression in non-labouring and spontaneously, labouring human myometrial biopsies from John Hunter Hospital, Newcastle. (B), Densitometric analysis of hERG protein expression in non-labouring and spontaneously labouring myometrial biopsies.

[0027] Figure 3. hERG protein expression in non-labouring and spontaneously labouring human myometrial biopsies from the KK Women's and Children's Hospital, Singapore.

[0028] Figure 4. Ether-a-go-go protein expression in guinea pig myometrium through gestation. o -SMA, ct-smooth muscle actin. LAB, labour. [00291 Figure 5. Effect of hERG inhibition. (B, dofetilide ; C, E-4031) on contractility of non-labouring human myometrium.

[0030] Figure 6. Effect of hERG activators (B, PD-118057; C, NS1643) on contractility of non-labouring human myometrium.

[0031] Figure 7. Cumulative dose-dependency study of the effect of hERG modulators (A, NS1643 ; B, dofetilide) on contractility of non-labouring human myometrium. ^*

[0032] Figure 8. Time course analysis of the effect of dofetilide on contractility of non-labouring human myometrium.

[0033] Figure 9. Images of hTERT-immortalised myometrial cells treated with 50μ1 of either: A, non-targeted liposomes labelled with Dil; or B, liposomes labelled with Dil, the liposomes conjugated to anti-oxytocin receptor antibodies targeting the liposomes to the myometrial cells. Nuclei of cells were stained with DAPI.

[0034] Figure 10. Time course in spontaneously contracting hTERT-immortalised myometrial cells treated with 10 μΐ of liposomes conjugated to anti-oxytocin receptor antibodies and loaded with 100 nM dofetilide (top panel) or liposomes conjugated with IgG antibodies and loaded with 100 nM dofetilide (bottom panel).

[0035] Nucleotide and amino acid sequences are referred to herein by a sequence identifier number (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:l), <400>2 (SEQ ID NO:2), etc. A sequence listing is provided after the claims. The nucleotide sequence of hERG is set. forth in SEQ ID NO:l, and the encoded amino acid sequence in SEQ ID NO:2. The nucleotide sequences of miRNAs hsa_miR_144, hsa_miR_142-3p, hsa_miR_125b, hsa_miR_362-3p, hsa_miR_101 , or hsa_miR_133a are set forth in SEQ ID Nos:3-8, respectively. Detailed Description

[0036] 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 generalknowledge in the field of endeavour to which this specification relates.

[0037] 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 element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method step or group of elements or integers or method steps.

[0038] As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent, as well as two or more^' agents; reference to "the disclosure" includes a single and multiple aspects described in the disclosure; and so forth. All aspects disclosed, described and/or claimed herein are encompassed by the term "invention". Such aspects are enabled across the width of the present invention.

[0039] It will be understood that as used herein the term "expression" may refer to expression of a polypeptide or protein, or to expression of a polynucleotide or gene, depending on the context. The polynucleotide may be coding or non-coding. Expression of a polynucleotide may be determined, for example, by measuring the production of RNA transcript levels. Expression of a protein or polypeptide may be determined, for example, by immunoassay using an antibody(ies) that bind with the polypeptide.

[0040] In the context of this specification, the term "activity" as it pertains to ERG means any cellulai' function, action, effect or influence exerted by the ERG protein or encoding nucleic acid molecule. The celliilar function, action, effect or influence may be exerted directly or indirectly.

[0041] The terms "agent", "pharmacologically active agent", "medicament" and "drug" may be used interchangeably herein. These terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those agents mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs, mimetics functional equivalents and the like.

[0042] The term "inhibiting" and variations thereof such as "inhibition" and "inhibits" as used herein do not necessarily imply the complete inhibition of hERG expression or activity. Rather, the inhibition of expression or activity may be to an extent, and/or for a time, sufficient to produce the desired effect. Inhibition may be prevention, retardation, reduction or otherwise hindrance of the expression or activity. Such inhibition may be in magnitude and/or be spatial or temporal in nature. In particular contexts, the terms "inhibit" and "prevent", and variations thereof may be used interchangeably.

[0043] In the context of the present disclosure, the term "inhibitor" refers to any agent capable of inhibiting either or both the expression and activity of ERG, either directly or indirectly. Accordingly the inhibitor may operate directly or indirectly on the ERG polypeptide, the corresponding mRNA or gene, or alternatively act via the direct or indirect inhibition of any one or more components of an ERG - associated pathway. Such components may be molecules activated, inhibited or otherwise modulated prior to, in conjunction with, or as a consequence of ERG activity. Thus, the inhibitor may operate to prevent transcription, post-transcriptional, translation, or post-translational processing or otherwise inhibit the activity of ERG or a component of an ERG - associated pathway in any way, via either direct or indirect action. The inhibitor may for example be nucleic acid, peptide, any other suitable chemical compound or molecule or any combination of these. It will be understood that in indirectly impairing the activity of ERG or a component of an ERG - associated pathway, the inhibitor may effect the activity of molecules which regulate, or are themselves subject to regulation or modulation by, ERG of a component of an ERG - associated pathway.

[0044] As used herein the terms "treating", "treatment", "preventing" and "prevention" refer to. any and all uses which remedy a disease, disorder or condition or symptoms, prevent the establishment of a condition or disease, or otherwise prevent, hinder, retard, or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever. 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. Similarly, "prevention" does not necessarily mean that the subject will not eventually contract a particular disease, disorder or condition. Rather, "prevention" encompasses reducing the severity of, or delaying the onset of, a particular disease, disorder or condition. In the context of some conditions, methods of the present invention involve "treating" the disease, disorder or condition in terms of reducing or eliminating the occurrence of a highly undesirable and irreversible outcome of the progression of the condition but may not of itself prevent the initial occurrence of the disease, disorder or condition. Accordingly, treatment and prevention include amelioration of the symptoms of a particular disease, disorder or condition or preventing or otherwise reducing the risk of developing a particular disease, disorder or condition.

[0045] As used herein the tenns "effective amount" and "effective dose" include within their meaning a non-toxic but sufficient amount or dose of an agent or compound to provide the desired effect. The exact amount or dose required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the disease, disorder or condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount" or "effective dose". However, for any given case, an appropriate "effective amount" or "effective dose" may be determined by one of ordinary skill in the art using only routine experimentation. In some embodiments, an effective amount for a human subject lies in the range of about O.lng/kg body weight/dose to lg kg body weight/dose. In some embodiments, the range is about ^g to lg, about lmg to lg, lmg to 500mg, lmg to 250mg, lmg to 50mg, or ^g to lmg/kg body weight/dose. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic dose. For example, several doses may be provided daily, weekly, monthly or other appropriate time intervals.

[0046] The term "subject" as used herein refers to a mammal, typically a human. However, it will be understood by those skilled in the art that aspects disclosed herein have both human and veterinary applications, and hence both human and non-human mammals may benefit from the compositions and methods herein disclosed. Thus in the context of the present disclosure "subject" includes livestock and companion animals such as cattle, horses, sheep, pigs, camelids, goats, donkeys, dogs and cats. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. A "subject" may also be referred to in the present disclosure as an individual, patient, or recipient.

[0047] The ether-a-go-go-related (ERG) gene family, also known as CNH, is comprised of 3 main family members, ERGl, 2 and 3. In addition, splicing variants of ERGl (ERG la and ERG lb) have also been identified. ERG family members encode voltage-dependent potassium channels. The ERG potassium channels exhibit distinctive kinetics that are determined by a C-type inactivation mechanism.

[0048] Of the 3 main family members, ERGl is expressed primarily in cardiac myocytes, whilst ERG2 and 3 are expressed almost exclusively in neuronal cells. Within neuronal cells, ERG2 and ERG3 play a role in regulating the resting membrane conductance of the cells. Within the heart however, ERGl mediates the repolarizing JK_T current in the cardiac action potential, and therefore plays a vital role in regulating the normal electrical activity of the heart. Congenital mutations that impair ERGl function cause a delay in the repolarizing current, which is clinically diagnosed as long QT syndrome, a rare hereditary heart condition that can cause irregular heartbeat and palpitations. Recently ERG1 expression has been described in various smooth muscle tissues from different mammalian species. ERG1 has been detected in myometrial tissue from mice. Prior to the present disclosure human ERG1 (hERG) had not been examined for a role in human myometrium.

[0049] The term "ERG" as used herein refers to the ether-a-go-go voltage-gated potassium ion channel Kvl l .l, also known as ERG1, LQT2 and Eag-related protein 1. Human ERG is referred to herein as hERG, encoded by the KCNH2 gene. In the context of the present disclosure the term "ERG" may be used in reference to both the ERG protein and to the gene encoding the ERG protein. hERG is encoded by the nucleotide sequence set forth in SEQ ID NO: l (GenBank Accession No. NMJ300238). The amino acid sequence of hERG is set forth in SEQ ID NO:2.

[0050] The meaning of the term "homologue" as used herein would be understood by those skilled in the art. Examples of an hERG homologue include gene or protein sequences that share structural and functional similarity to hERG (SEQ ID Nos: I and 2), including gene and protein sequences from non-human mammals. The term "homologue" includes both orthologues, sequences in different species that are structurally similar due to evolution from a common ancestor, and paralogies, similar sequences within the same genome. The terms "ERG" and "hERG" shall be taken to also include ERG and hERG homologues, unless otherwise stated. Homologues also include isoforms, variants and fragments of the ERG protein or nucleic acid sequences encoding such variants and/or fragments.

[0051] Aspects disclosed herein are based on the surprising findings that protein levels of hERG fall at the onset of labour, and that contractility of human myometrium can be stimulated by the addition of inhibitors of hERG.

[0052] Accordingly, disclosed herein is a method for stimulating or inducing contractility of myometrial tissue, comprising exposing the myometrial tissue to an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof. Also disclosed are methods for treating or preventing dystocia or post partum haemorrhage in a subject, comprising administering to the subject an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

[0053] It may, for example, be advantageous to induce uterine contractions in a pregnant mammal in order to induce or assist labour where the pregnancy is difficult, complicated or abnormal in some way and continuing the pregnancy may jeopardise the health of mother and/or child. It may also be advantageous to induce uterine contractions in a pregnant mammal in order to induce labour where the pregnancy has already reached term or is post-term. As used herein, the term dystocia refers to any difficult or obstructed labour, or labour progressing abnormally slowly, including for example shoulder dystocia or a prolonged second stage of labour. Inducing uterine contractions may also be necessary after delivery to minimise blood loss and prevent or reduce post partum haemorrhaging.

[0054] Also enabled herein are compositions for use in accordance with the disclosed methods, the compositions comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

[0055] Aspects and embodiments of the present disclosure provide for the administration of agents capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof. Such inhibitors may directly or indirectly effect ERG expression and may act at the level of the ERG gene or any products thereof including mRNA (precursor or mature message) or polypeptide. The inhibitor may be, for example, a proteinaceous or non-proteinaceous molecule that modulates the transcription and/or translation of the gene or a functional portion thereof (such as a promoter region), or alternatively that modulates the transcription and/or translation of an alternative gene or functional portion thereof, which alternative gene or gene product directly or indirectly modulates the expression of ERG. The inhibitory agent may be an antagonist. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing ERG from ^"carrying out its normal biological functions. For the present purposes, the terms "antagonist" and "inhibitor" may be used interchangeably.

[0056] Those skilled in the art will appreciate that a variety of suitable ERG inhibitors may be employed in accordance with the aspects and embodiments disclosed herein and the scope of the present disclosure is not limited by the selection of any one particular inhibitory molecule or compound. Suitable inhibitors include small molecule and other synthetic or naturally occurring chemical inhibitors, antibodies, such as monoclonal antibodies, antisense nucleic acids which prevent transcription or translation of genes or mRNA and ERG-associated miRNA or antagonists thereof. Methods of identifying suitable ERG inhibitory compounds will also be known to those skilled in the art, including methods disclosed in US patent application no. 20030013136, the disclosure of which is incorporated herein by reference.

[0057} The inhibitor may be a potassium channel inhibitor, in particular an inhibitor specific or selective for a voltage-gated potassium channel such as hERG or a homologue or orthologue thereof. In exemplary embodiments the inhibitor may be selected from dofetilide (N-[4-(2-{ [2-(4-methane sulfonamidophenoxy)ethyl] (methyl)amino}ethyl)phenyt]methanesulfonamide) and E-4031 (N-[4-[l-[2-(6- methylpyridin-2-yl)ethyl]piperidine-4-carbonyl]phenyl]). Other inhibitors of hERG current known to those skilled in the art include, by way of non-limiting example: those disclosed in Yao et al. (2008) (the disclosure of which is incorporated herein by reference), such as verapamil, ziprasidone, amiodarone, cisapride, droperidol, haloperidol, and terfenadine; protease inhibitor antiretroviral drugs such as lopinavir, nelfinavir, ritonavir, and saquinavir; azimilide; propafenone; d-sotalol; flecainide; MK- 499; quinidine; astemizole; pimozide; and risperidone.

[0058] Proteinaceous ERG inhibitors include peptide inhibitors and antibodies. One exemplary peptide inhibitor of ERG is BeKm-1 (Qu et al., 201 1 ; the disclosure of which is incorporated herein by reference). ' Suitable antibodies include, but are not limited to polyclonal, monoclonal, mono-specific, poly-specific (including bi-specific), humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and CDR- grafted antibodies. Various techniques for producing antibodies and preparing recombinant antibody molecules are known in the art. Antibodies may be derived from any species, including, but not limited to, rat, mouse, goat, guinea pig, donkey, rabbit, horse, lama, camel, or any avian species (e.g., chicken, duck). The antibody may be of any suitable isotype, such as IgG, IgM, IgA, IgD, IgE or any subclass thereof. The skilled addressee will appreciate that antibodies produced recombinantly, or by other means, for use in accordance with the methods embodied herein include fragments that are still capable of binding to or otherwise recognizing ERG. Examples include Fab, an F(ab)₂, Fv, scFv fragments. In an embodiment, the antibody is a monoclonal antibody or ERG-binding fragment thereof. The monoclonal antibody can be a humanised or deimmunised form of a non-human antibody, in another embodiment, the monoclonal antibody is a human antibody.

[0059] Suitable antisense constructs for use in accordance with the present invention include antisense oligonucleotides, small interfering RNAs (siRNAs) and catalytic antisense nucleic acid constructs. Suitable antisense oligonucleotides may be prepared by methods well known to those of skill in the art. Typically oligonucleotides will be chemically synthesized on automated synthesizers. Those skilled in the art will readily appreciate that antisense oligonucleotides need not display 100% sequence complementarity to the target sequence. One or more base changes may be made such that less than 100% complementarity exists whilst the oligonucleotide retains specificity for its target and retains antagonistic activity against this target. Suitable antisense oligonucleotides include morpholinos where nucleotides comprise morpholine rings instead of deoxyribose or ribose rings and are linked via phosphorodiamidate groups rather than phosphates.

[0060] An alternative antisense technology, RNA interference (RNAi), may be used, according to known methods in the art (for example Bernstein et al. (2001) Nature 409: 363-366; Elbashir et al (2001) Nature 41 1 : 494-498; WO 99/49029 and WO 01/70949, the disclosures of which are incorporated herein by reference), to inhibit the expression or activity of nucleic acid molecules encoding ERG. RNAi refers to a means of selective post-transcriptional gene silencing by destruction of specific RNA by small interfering RNA molecules (siRNA). The siRNA is generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated. Double- stranded RNA molecules may be synthesised in which one strand is identical to a specific region of the ERG transcript and introduced directly. Alternatively corresponding dsDNA can be employed, which, once presented intracellularly is converted into dsRNA. Methods for the synthesis of suitable molecules for use in RNAi and for achieving post-transcriptional gene silencing are known to those of skill in the art.

[0061] A further means of inhibiting the expression or activity of ERG may be involve introducing catalytic antisense nucleic acid constructs, such as ribozymes, which are capable of cleaving ERG mRNA transcripts. Ribozymes are targeted to and anneal with a particular sequence by virtue of two regions of sequence complementarity to the target flanking the ribozyme catalytic site. After binding the ribozyme cleaves the target in a site-specific manner. The design and testing of ribozymes which specifically recognise and cleave ERG mRNA sequences can be achieved by techniques well known to those in the art (for example Lieber and Strauss, (1995) Mol. Cell. Biol. 15:540-551 , the disclosure of which is incorporated herein by reference).

[0062] Also disclosed herein are miRNA species that interact with hERG, and changes in the expression of which correlate with the changes in expression of hERG at the onset of labour. As exemplified herein, expression of the following miRNAs are significantly upregulated at the onset of labour: hsa_miR_144, hsa_miR_ 142-3p, hsa_miR_125b, hsa_miR_362-3p and hsa_miR_J01. Consequently, disclosed herein is the use of these miRNAs, or precursors thereof, as agents in capable of, directly or indirectly modulating hERG levels in accordance with the disclosed methods and compositions. The mature miRNAs typically comprise the nucleotide sequences set forth in SEQ ID NO:3 (hsa_miR_144), SEQ ID NO:4 (hsa_miR_142-3p), SEQ ID NO:5 (hsa_miR_125b), SEQ ID NO:6 (hsa_miR_362-3p) and SEQ ID NO:7 (hsa_miR_101). Also exemplified herein is the significant reduction in expression of the miRNA hsa_miR_133a at the onset of labour. Consequently, disclosed herein is the use of an antagonist (such as an antagomir) of hsa_miR_133a or a precursor thereof as an agent capable of, directly or indirectly modulating hERG levels in accordance with the disclosed methods and compositions. Methods and techniques for producing antagomirs and other antagonists of miRNAs will be well known to those skilled in the art. The ^'mature hsa_miR_133a miRNA typically comprises the nucleotide sequence set forth in SEQ ID NO:8. Sequences of the miRNAs disclosed herein and of precursors and variants thereof can be found in the miRBase database (www.mirbase.org).

[0063] Disclosed herein are compositions comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0064] By pharmaceutically acceptable carrier, diluent or excipient is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected conjugate without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, colouring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like. Carriers may also include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. -It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

[0065] 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.

[0066] The pharmaceutical compositions disclosed herein may conveniently be presented in unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active agent(s) with the pharmaceutical carrier(s) or excipient(s). The compositions described herein may be formulated into any of many possible dosage forms such as, but not limited to, injectable formulations, and tablets, capsules, gel capsules and liquids. Accordingly, pharmaceutical compositions described herein include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations herein described may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0067] Agents and compositions disclosed herein may be administered via any convenient or

route such as by parenteral (e.g. subcutaneous, intraarterial, intravenous, intramuscular), oral, intranasal or topical routes. In particular embodiments of the present disclosure local or regional administration may be preferred over systemic administration to ensure that the required amount or concentration of the agent is delivered to the myometrial tissue, whilst avoiding exposure of other organs of the body to the compound and thereby potentially reducing side effects. Thus, in particular embodiments intra-uterine delivery of the agent or composition may be advantageous. A variety of suitable intra-uterine drug delivery systems are available and would be known to those skilled in the art, including for example, those described in Wildemeersch et ah (2003) and Wildemeersch (2010), the disclosures of which are incorporated herein by reference.

[0068] A range of delivery vehicles known to those skilled in the art may be employed for the delivery of agents disclosed herein. Suitable delivery vehicles include, but are not limited to, liposomes, micelles such as polymeric micelles, lipoprotein-based drug carriers, nanoparticles, and dendrimers. Further, targeted delivery of agents disclosed herein to the myometrium may be achieved by modifying such delivery vehicles by the addition of ligands to vehicle surfaces, suitable ligands including polypeptides, peptides, antibodies and lectins, that enable increased target specificity. Thus agents disclosed herein may be encapsulated within, or otherwise linked with liposome, micelle, lipoprotein-based drug carrier, nanoparticle, and dendrimer based delivery vehicles which can then in turn be coated or conjugated with a molecule or compound capable of interacting with oxytocin receptors in the myometrium. Suitable molecules and compounds for use in this regard include oxytocin and antibodies that bind oxytocin receptors. The antibody may be specific or selective for an oxytocin receptor. Alternatively, agents disclosed herein may be directly conjugated with a compound capable of interacting with oxytocin receptors in the myometrium such as oxytocin or an oxytocin receptor-binding antibody.

[0069J Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The formulation must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be 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. The proper fluidity can be maintained, for example, 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 superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

[0070] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredients into a sterile vehicle which 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, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

[0071] When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

[0072] Tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as.com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

[0073] The present disclosure further contemplates combination therapies, wherein agents disclosed herein are coadministered with other suitable agents that may facilitate myomterial contractility, uterine contractions or assist in the inducement or progression of labour in some other way. By "coadministered" is meant 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. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. In sequential administration the molecules or compounds may be administered in any order.

[0074] Also provided herein are methods for diagnosing or predicting the onset of labour, comprising obtaining a biological sample from a subject and determining the level of expression and/or activity of ERG in the sample, or the expression of a miRNA selected from hsa_miR_144, hsa_miR_142-3p, hsa_miR_125b, hsa_miR_362-3p, hsa_miR_101 and hsa_miR_133a. Reduced levels of expression of ERG protein or of the miRNA hsa_miR_133a relative to normal endogenous levels in a corresponding non-labouring sample, is diagnostic or predictive of the onset of labour. Increased levels of expression of hsa_miR_144, hsa_miR_142-3p, hsa_miR_125b, hsa_miR_362- 3p or hsa_miR_101 relative to normal endogenous levels in a corresponding non- labouring sample is diagnostic or predictive of the onset of labour.

[0075] Those skilled in the art will appreciate that aspects described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that these aspects include all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0076] Aspects taught herein are now described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples

Example 1 - miRNA and hERG gene expression analysis

[0077] Non-labouring (NIL) and spontaneously labouring (L) myometrial samples were analysed tor differentially expressed miRNA species. Briefly, arrays were performed to compare miRNA expression in 8 ^χ NIL myometrial samples against 8 * L myometrial samples. A series of miRNA species revealed differential expression: approximately 40 species exhibited increased during the onset of labour whilst another 3 species exhibited decreased expression.

[0078] Bioinformatics analysis revealed that at least one of these miRNAs targeted the ERG I gene, and upon further investigation we determined that a total of 6 miRNA species with differential expression targeted the ERG1 gene transcript. Of these miRNAs, 5 were increased with the onset of labour whilst 1 was decreased with the onset of labour (see Table 1).

Table 1: miRNA expression analysis

miRNA species Fold change at labour

hsa_miR_144 5.82

hsa_miR_142-3p 4,73

hsa_miR_125b 4.41

hsa_miR_362-3p 2.86

hsa miRJOl 2.65 hsa_miR_133a 0.44

[0079] Myometrial biopsies were collected during caesarean section from women that •were at term and not in labor, as well as women that were in either spontaneous labor [L(s)] or induced labor [L(iol)]. The myometrial tissue was washed in 4°C saline, blotted to remove excess liquid and then snap frozen using liquid nitrogen. The frozen tissue was then pulverised and 100 mg was homogenised into 1 mL trizol solution for RNA extraction. Total RNA was reverse transcribed into cDNA by Superscript III First-Strand Synthesis System. cDNA from NIL, L(s) and L(iol) samples were amplified using hERG primers. Relative expression of hERG mRNA was then compared, however no difference was observed between NIL, L(s) and L(iol) myometrial samples (Figure 1).

[0080] Given that hERG mRNA expression levels remained unchanged following either the spontaneous or induced onset of labour, the inventor hypothesised that any changes observed in hERG protein expression are mediated by means other than targeted degradation of the hERG mRNA transcripts, i.e. that either altered rates of mRNA translation or altered rates of protein degradation are responsible for any observed changes in hERG protein expression levels.

Example 2 - Myometrial expression of hERG and a Itomologue thereof

[0081] Myometrial biopsies were collected from women at term that were either not in labour (NIL) or in spontaneous labour (L) and undergoing caesarean section at the John

Hunter Hospital in Newcastle, Australia. This data was generated from myometrial tissues collected at John Hunter Hospital as part of the 'To Understand Birth' (TUB) study.

[0082] The myometrial tissue was washed in 4°C saline, blotted to remove excess liquid and then snap frozen using liquid nitrogen. The frozen tissue was then pulverised and 100 mg was homogenised into 1 mL of protein extraction buffer (7 M urea, 2 thiourea, 4% CHAPS, 30 mM Tris) supplemented with PhosSTOP phosphatase inhibitor and Complete Mini Protease inhibitor. The protein extraction homogenate was incubated at 4°C for 30 mins on a rotary mixer before being centrifuged at 1 1 ,000 ^χ g, for 15 min at 4°C. The supernatant was collected and protein content determined by 2D Quant Kit. Protein extracts (20 μg per lane) were loaded onto 4-12% NuPAGE Bis-Tris gels and separated using a Novex Mini-Cell system at constant voltage (200 V for 50 min). Separated proteins were then western transferred onto Hybond-C nitrocellulose membrane using the XCell II Blot Module.

[0083] Membranes were blocked in 5% skim milk in TBS/T (500 mM NaCl, 20 mM Tris, 0.01 % Tween-20) overnight at 4°C. Blocking solution was decanted and primary antibody solution was applied at previously optimised dilutions in 10 mL 1% skim milk in TBS/T for 2 h at RT. For primary antibodies, rabbit anti-K_v 11.1 (hERG)(extracellular)(cat.# APC-109, Alomone Labs, Ltd. Jerusalem, Isreal) was applied at 1 :2000 dilution in the presence or absence of blocking peptide (2 μg per 1 ^ig antibody; Alomone Labs), whilst anti-a-smoofh muscle actin (ct-SMA) (cat.# A5228, Sigma- Aldrich, Castle Hill, NSW) was applied at 1 :20,000. Blots were then washed for 4 x 5 mins with 100 mL of TBS/T. Washed blots were then incubated in horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (cat.# 7074, Cell Signaling) or anti-mouse IgG (cat.# 7076, Cell Signaling) secondary antibody as appropriate. Secondary antibodies were applied at 1 :2500 dilution in 10 mL of 1% skim milk in TBS/T for 1 h at RT. Blots were washed for 4 * 5 min in 100 mL of TBS/T before immunoreactive products were detected with enhanced chemiluminescence (ECL) western blotting reagent. Immunoreactive bands were visualized using the Fujifilm Intelligent Dark Box LAS-3000 Image Reader.

[0084] As shown in Figure 2A, hERG protein expression was readily detectable by immunoblot analysis in the NIL myometrial samples. In contrast, hERG protein detection was significantly reduced, and barely detected, in L myometrial samples. To prove the specificity of the antibody, a blocking peptide control was included to demonstrate that the antibody interacts with a protein band of the appropriate molecular weight for hERG (135kDa) in a specific manner. Addition of the antibody blocking peptide completely abolished detection of hERG in either NIL or L samples, antibody blocking peptide revealed high levels of hERG proptein expression.

[0085] Densitometric analysis of immunoreactive protein bands was performed using Multigauge software (Fuji Photo Film) supplied with the LAS-3000. The optical density of hERG immunoreactive bands was expressed as a percentage of ot-SMA optical density, which was used as a loading control given that a-SMA abundance in myometrium remains unchanged throughout pregnancy. Statistical analysis was conducted using Stata 9.2 software (StataCorp, College Station, TX). The results are shown in Figure 2B.

[0086] Figure 2 (A and B) demonstrates that women at term had high levels of hERG protein expression in their myometrium. Following the onset of labour however, hERG. protein expression was significantly decreased.

[0087] Myometrial biopsies were also collected from women at term that were either not in labour (NIL) or in spontaneous labour (L) and undergoing caesarean section at the K Women's and Children's Hospital in Singapore. The myometrial samples were washed in saline, snap frozen in liquid nitgrogen and transported to the Mothers and Babies Research Centre. Tissue homogenation, protein extraction, protein separation and immunoblotting for hERG protein expression were performed as previously outlined for Figure 2A. In addition to NIL and L myo, hERG protein expression was examined in hTERT-immortalised myometrial cells. The results are shown in Figure 3. Consistent with the TUB study samples from Newcastle, hERG protein expression was readily detectable in NIL myometrium yet was barely detected in L myometrium. hERG protein was also detected in hTERT cells.

[0088] ERG expression in guinea pig myometrium was also examined. Guinea pig myometrial samples were collected during caesarean section at various stages across fetal gestation. The tissue was snap frozen and subjected to protein extraction as previously outlined. Protein was separated by 1D-SDS PAGE and e!ectroblotted onto nitrocellulose membrane. Membranes were probed with anti-ERGl antibody as previously outlined. As shown in Figure 4, across fetal gestation ERGl protein expression peaked at 55 days gestation and subsequently dropped and stabilized approaching labour.

Example 3 - Effect of modulating hERG levels on myometrial contractility

[0089] To examine the role of hERG in human myometrium, the effect of hERG inhibitors and activators on the myometrial contractility was examined in vitro.

[0090] Myometrial samples from non-labouring women were cut into strips (7x2x2 mm) and suspended in organ baths containing 30 ml Krebs-Henseleit buffer with 1.89 mM CaC12. Strips were connected to a Grass FT03C force transducer (Grass Instruments, Quincy, MA) and 1 g passive tension applied. Buffer was replaced five times during the first hour, with strips re-tensioned to 1 g passive tension following each wash. Thereafter strips were maintained at 37°C (pH 7.4) and continuously bubbled with 95% 02/5% C02 until spontaneous rhythmic contractions developed. Data were digitized using a Maclab8E data-acquisition system and contraction status visualised in real time using Chart software (ADI, Melbourne, Australia). Strips were monitored for the development of spontaneous contractions. Contraction baselines were established demonstrating rhythmic contractions with consistent frequency and amplitude. Strips were then treated with the hERG inhibitors dofetilide (Ι μΜ) or E-4031 (ΙμΜ). Given that dofetilide and E-4031 were prepared as lOOOx stocks in DMSO, a separate strip was treated with DMSO only as a control.

[0091] Cumulative treatments with DMSO had no effect on myometrial contractility (Figure 5A) in that contraction frequency and amplitude remained unchanged even at the highest dose (0.2%). Treatment with 1 μΜ dofetilide (Figure 5B) increased the peak contraction time (i.e. the length of time the tissue remain at peak contraction), whilst also producing a decrease in contraction frequency. Similarly, treatment with ΙμΜ E- 4031 (Figure 5C) increased the peak contraction time and decreased contraction frequency. Following E-4031 treatment, the tissue strip received a follow up treatment of the hERG activator NS1643 however the treatment did not negate the effects of the E-4031. These data demonstrate that hERG protein expression in human myometrium plays a role in regulating uterine contractions. Furthermore, hE G function can be manipulated to produce rapid changes in contraction dynamics of human myometrium.

[0092] In addition to hERG inhibitors, the effect of the hERG activators PD-1 18057 and NS 643 was examined. Fluman myometrial strips were prepared for the in vitro contractility bioassay as outlined above. ^'Once consistent baseline contraction traces had been established, strips were treated with the hERG activators PD-1 18057 (10μΜ) or NS1643 (ΙΟμΜ). Treatment with 10μΜ PD-,118057 (Figure 6A) had no effect on human myometrial contractions in that contraction frequency and amplitude remained . unchanged. Similarly, treatment with NS1643 (Figure 6B) had no effect on contraction frequency or amplitude. To confirm the presence of hERG in the tissue, a follow treatment of Ι μΜ Dofetilide was administered to each strip. Dofetilide induced a characteristic increase in peak contraction time and decrease in contraction frequency, thereby demonstrating the strips were indeed expressing hERG.

[0093] Thus, treatment with hERG inhibitors increases peak contraction time and decreases contraction frequency, however treatment with hERG activators had no observed effect. Without wishing to be bound by any one specific theory, these data suggest that hERG potassium channels in the human myometrium are constitutively active, and therefore were unable to further activated by treatment with PD-118057 or NS1643.

[0094] To further investigate the effect of hERG modulators on myometrial contractility, the hERG activator NS1643 and the hERG inhibitor dofetilide were examined across a range of cumulative concentrations. Human myometrial strips were prepared for the in vitro contractility bioassay as outlined above. Once consistent baseline contraction traces had been established, strips were treated with the cumulative doses of the hERG activator NS1643 1 ΟμΜ or dofetilide. At maximum dosage of 30μΜ NS1643 still had no effect on contraction frequency or amplitude (Figure 7 A). A follow up treatment with dofetilide did however produce a characteristic increase in peak contraction time and ' decrease in contraction frequency thereby demonstrating expression of hERG in the tissue. Cumulative doses with dofetilide revealed no effect at 0.1 and 1.1 nM concentrations, however at l l . lnM a slight increase in peak contraction time was evident (Figure 7B). Increased peak contraction time was clearly observed at 1 1 1. ΙμΜ Dofetilide. As dofetilide dose increased contraction amplitude also increased (Figure 7B). These data demonstrate that dofetilide is capable of modulating hERG function at low nanomolar concentrations. Even at such low concentrations dofetilide exerts effects on contraction peak time, frequency and amplitude.

(0095J The effects of dofetilide treatment over an extended timeline were also investigated. Human myometrial strips were prepared for the in vitro contractility bioassay as outlined above. Once consistent baseline contraction traces had been established, strips were treated with a single dose of 1 μΜ Dofetilide and observed for a period of 2.4 h. A separate strip was treated with DMSO (up to 0.2%) as a control and monitored over the same period. DMSO treatment had no effect contraction frequency or amplitude across the 2.4 h time course (Figure 8A). Treatment with Ι μΜ dofetilide once again produced a rapid increase in peak contraction time (Figure 8B). Across the course of the 2.4 h a decrease in contraction frequency was observed in conjunction with an increase in contraction amplitude. 1 μΜ dofetilide therefore produced increased peak contraction time, increased contraction amplitude and decreased contraction frequency.

Example 4 - Targeted delivery of dofetilide to myometrial cells

[0096] The targeted delivery of hERG inhibitors to myometrial cells was investigated using liposomes conjugated with antibodies specific for the oxytocin receptor (OTR- targeted liposomes). The antibody was a rabbit polyclonal antibody (Sapphire Bioscience Pty Ltd) directed against a synthetic peptide corresponding to the N-terminal extracellular domain of the human oxytocin receptor.

[0097] The liposomes were immurioliposomes composed of 1,2-distearoyl-sn-glycero- 2-phosphocholine (DSPC) and cholesterol (molar ratio 2.T ) containing 1,2-distearoyl- . 5/i-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE- PEG(2000) Maleimide) at 1.5 mol percent of DSPC as a coupling lipid and the fluorescent lipophilic dye 1 ,1 '-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) at 0.2 mol% of DSPC, prepared by dried lipid film hydration in PBS buffer (pH 7.4). The resulting multilamellar dispersions were reduced in size and lamellarity by high pressure extrusion. The activated liposome suspension was immediately mixed with thiolated antibody at room temperature. Thiolated antibodies were prepared by conjugating anti-oxytocin receptor monoclonal antibodies or nonspecific rabbit IgG antibodies with a heterobifunctional reagent N-succinimidyl-3-(2- pyridyldithio) propionate (SPDP) (6.25 mg/ml; SPDP/antibody molar ratio = 10: 1). Excess SPDP was removed through a PD-10 column equilibrated with distilled water. Fractions containing pyridyldithiopropionated-antibody (PDP-Ab) conjugates (assessed by absorbance in 280 nm) were pooled and lyophilized to form a solid product, which was then stored at 4°C under nitrogen gas. To produce thiolated antibodies (Ab-SH), PDP-Ab was reduced with 5 mM TCEP for 5 min. Absorbance was checked at 280 nm (protein concentration) and 343 nm (SPDP modification), in order to ^"ensure stability of the compound as described above. The Ab-SH was mixed immediately with liposomes and incubated for 1 h at room temperature with stirring in the dark. Unconjugated antibody was removed using Slide- A-Lyzer dialysis cassettes (10 MWCO). Liposomes were stored at 4°C, under nitrogen gas and in the dark. The size distribution of the liposomal dispersion was determined by dynamic laser light scattering (Zetasizer 3000 ^rM, Malvern, Worcestershire, United Kingdom).

[0098] First, 50μ1 OTR-targeted liposomes labelled with Dil were added to hTERT- immortalised myometrial cells in culture. As shown in Figure 9, treatment of hTERT- immortalised myometrial cells with non-targeted liposomes revealed no internalised Dil labeling (Figure 9A), whereas treatment with OTR-targeted liposomes revealed internalised Dil fluorescence (Figure 9B).

[0099] Having successfully demonstrated that liposomes can be targeted to bind to myometrial cells, the inventors then loaded OTR-liposomes with lOQnM dofetilide and investigated their effect on myometrial cell contractility. Briefly, ΙΟμΙ of dofetilide loaded OTR-liposomes were added to spontaneously contracting human myometrial strips and contractility measured as described in above examples. As shown in Figure 10 (top panel), treatment with OTR-targeted liposomes increased the length of uterine contractions, in the same manner as described above following direct treatment with dofetilide. In contrast, treatment with IgG-targeted liposomes (Figure 10, bottom panel) did not increase the length of uterine contractions.

References

Qu et al. (201 1) BeKrn-1 , a peptide inhibitor of human ether-a-go-go-related gene potassium currents, prolongs QTc intervals in isolated rabbit heart. J Pharmacol Exp Ther 337:2-8.

Wildemeersch et al. (2003) Miniature, low-dose, intrauterine drug-delivery systems. Ann N YAcad Sci 997: 174-184.

Wildemeersch (2010) Intrauterine drug delivery for contraception and gynaecological treatment: novel approaches. Handb Exp Pharmacol 197:267-298.

Yao et al. (2008) Predicting QT prolongation in humans during early drug development using hERG inhibition and an anaesthetized guinea-pig model. Br J Pharmacol

154: 1446-1456.

Claims

1. A method for stimulating or inducing contractility of myometrial tissue, the method comprising exposing the myometrial tissue to an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

2. A method according to claim 1, comprising administering to a female mammal an effective amount of the agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

3. A method, according to claim 1 or 2, wherein the female mammal is pregnant, is in labour or has given birth.

4. A method according to any one of claims 1 to 3, wherein the administration is provided to stimulate uterine contractions and thereby induce or assist labour.

5. A method according to any one of claims 1 to 3, wherein the administration is provided to stimulate uterine contractions post labour to inhibit blood loss.

6. A method according to any one of claims 1 to 5, wherein the mammal is a human and the ERG potassium channel is hERG.

7. A method according to any one of claims 1 to 6, wherein the agent is a potassium channel inhibitor.

8. A method according to any one of claims 1 to 7, wherein the inhibitor is specific or selective for ERG.

9. A method according to any one of claims 1 to 8, wherein the inhibitor is selected from dofetilide and E-4031.

10. A method according to any one of claims 1 to 8, wherein the agent is an miRNA, or miRNA antagonist, wherein the miRNA interacts with the ERG gene.

11. A method according to claim 10, wherein the miRNA that interacts with the ERG gene is selected from hsa_miR_144, hsa_miR_142-3p, hsa_miR_125b, hsa_miR_362-3p, hsa miR lOl , and hsa_miR_133a.

12. A method according to claim 1 1 , wherein the miRNA hsa_miR_144, hsa_miR_142-3p, hsa_mlR_125b, hsa_miR_362-3p, hsa_miR_101, and hsa_ miR_133a comprise the nucleotide sequences set forth in SEQ ID Nos:3-8, respectively.

13. - A method according to any one of claims 10 to 12, wherein the ERG gene is ERG1.

14. A method according to any one of claims 10 to 13, wherein the agent comprises one or more miRNAs selected from hsa_miR_144, hsa_miR_142-3p, hsa_miR_125b, hsa_miR_362-3p, and hsa_miR_101.

15. A method according to any one of claims 10 to 14, wherein the agent comprises an antagonist of hsa_miR_ 133a.

16. A method according to any one of claims 1 to 15, wherein delivery of the agent to myometrial tissue is targeted thereto.

17. A method according to any one of claims 1 to 16, wherein the agent is encapsulated in liposomes for delivery to myometrial tissue.

18. A method according to claim 17, wherein the liposomes are formulated for targeted delivery to the myometrial tissue.

19. A method according to claim 18, wherein the liposomes are conjugated with anti -oxytocin receptor .anti bodies .

20. A method for inducing or assisting labour, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

21. A method for inducing uterine contractions in a pregnant mammal, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

22. A method for treating or preventing dystocia, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

23. A method for treating or preventing dystocia, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

24. A method for treating or preventing post partum haemorrhage, comprising administering to a subject in need thereof an effective amount of an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof.

25. A composition for carrying out a method according to any one of claims 1 to 20, the composition comprising an agent capable of inhibiting the expression or activity of an ERG potassium channel, or a homologue thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.