WO2017009199A1

WO2017009199A1 - Methods of treating damage after a stroke

Info

Publication number: WO2017009199A1
Application number: PCT/EP2016/066210
Authority: WO
Inventors: Christian Naus; John Bechberger; Rie Schultz Hansen; Ulrik Mouritzen
Original assignee: Zealand Pharma A/S
Priority date: 2015-07-10
Filing date: 2016-07-07
Publication date: 2017-01-19
Also published as: GB201512139D0

Abstract

(2S,4R)-1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-Nh₂; or a pharmaceutically acceptable salt or hydrate thereof, for use in methods of reducing the extent of tissue damage and methods of preventing or reducing cerebral reperfusion injury subsequent to the occurrence of a stroke in a human subject.

Description

METHODS OF TREATING DAMAGE AFTER A STROKE

Field of the invention

The present invention relates, inter alia, to the use of certain peptide compounds disclosed herein in the treatment or prevention of cerebral reperfusion injury following stroke, including stroke of any ischemic origin (i.e. resulting from reduced blood flow in a region of the brain, e.g. subsequent to cerebral infarction) and stroke of hemorrhagic origin [i.e. stroke arising as a result of cerebral hemorrhage (bleeding) and

compression of surrounding tissues as a result thereof).

Background

Stroke ranks second after ischemic heart disease as cause of lost disability-adjusted life-years in high-income countries and as cause of death worldwide (Lopez et al., Lancet 2006;367: 1747-57).

Different clinical pictures are summarized under the term "stroke", which term describes a disturbance in the blood supply to the brain. A stroke is a medical emergency, and treatment must be sought as quickly as possible in order to minimize the physiological consequences of stroke.

It will be appreciated therefore that different clinical pictures are summarized under the term "stroke": either the blood supply becomes blocked or reduced as a result of a cerebral infarction caused by thrombosis or embolism (ischemic stroke), or a blood vessel within the brain ruptures with resulting local compression of surrounding brain tissues (hemorrhagic stroke). Three main types of stroke are recognized; ischemic strokes, hemorrhagic strokes and transient ischemic attacks (TIAs), also referred to as mini-strokes. Strokes are sometimes referred to as cerebrovascular accidents (CVA), cerebrovascular insults (CVIs) or brain attacks.

Ischemia may result in damage to tissues or organs in the affected area as a result of a cascade of events from energy depletion to cell death. Intermediate factors include an excess of extracellular excitatory amino acids, free radical formation and inflammation.

Immediately after arterial occlusion, a central core of very low perfusion is surrounded by an area of dysfunction caused by metabolic and ionic disturbances, but in which structural integrity is preserved. In the first minutes to hours the clinical deficits do not necessary result in irreversible damage, and it is therefore of utmost importance to provide medical intervention as soon as possible.

Summary of the invention

The present invention relates to compounds that are gap junction intercellular communication (GJIC) modulators for use in methods of treatment of stroke patients. Importantly, these compounds can be administered very quickly after the stroke event, improving the chances of tissue salvage. The only proven acute stroke therapy for ischemic stroke is thrombectomy or thrombolytic therapy. This should be initiated within 2-3 hours of the stroke event, and ideally as soon as possible after the stroke event. Evidence suggests that necrosis following stroke occurs over 2 to 3 hours, so intervention in this period may salvage tissue, improving the patient's prognosis. Of course, earlier intervention is preferable.

However, when patients present with clinical signs of stroke it is often not clear whether the stroke is ischemic or hemorrhagic until a computed tomography (CT) scan or similar imaging procedure has been performed. One of the current challenges in the management of stroke is that thrombectomy or thrombolytic therapy cannot be applied until the physician can confidently diagnose the patient as suffering from an ischemic stroke (and not a hemorrhagic stroke). This is because this treatment might increase bleeding and make a hemorrhagic stroke worse.

Bringing the patient to a hospital and performing a CT scan in order to make the diagnosis takes time and is associated with progressive tissue damage as time elapses. When the blood flow is eventually restored, reperfusion injury often occurs as the lack of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress. This reperfusion injury can significantly increase the volume of cerebral infarct, increasing the risk of fatality and poor patient prognosis.

The inventors have surprisingly found evidence that the GJIC modulator compounds disclosed herein are safe to administer immediately following a stroke event, without the need to determine whether the stroke is ischemic or hemorrhagic. The

compounds can be used to prevent or reduce cerebral reperfusion injury. The compounds can be used to reduce the volume of cerebral infarct following a stroke. This indicates that the GJIC modulator compounds described herein can be given to a patient as soon as a stroke is suspected by a medical practitioner, without the need to determine the stroke type and with little or no risk of exacerbating a hemorrhagic stroke. It appears that the GJIC modulator compound can therefore enter and circulate in the blood stream and so is present as the blood flows back to the tissue when circulation is restored. It will also reach tissue surrounding the ischemic zone and within the ischemic zone in the limited cerebral blood flow (via collateral vessels). This not only helps to prevent reperfusion injury when circulation is restored, but is postulated to have a stabilizing effect on the cells during the period of ischemia.

Following first administration of the GJIC modulator compound, the type of stroke may be determined, and if appropriate, a thrombectomy or thrombolysis performed. Importantly, stroke patients are known to sometimes undergo spontaneous partial or complete thrombolysis (the clot may dissolve or dislodge in the case of ischemic stroke, the bleed pressure may relieve in the case of hemorrhagic stroke). For example, circulation may spontaneously be restored in transit to a hospital, or while waiting for a scan, and reperfusion injury may occur during this restoration.

The potential benefits of safe early administration of a compound that prevents or reduces reperfusion injury are evident.

Thus, in a first aspect, the present invention relates to a GJIC modulator compound as described herein for use in a method of reducing the extent of tissue damage subsequent to an occurrence of stroke in a subject, the method comprising

administering to the subject a therapeutically effective amount of said GJIC modulator compound; or a pharmaceutically acceptable salt thereof. Suitably, the GJIC modulator compound is selected from (2S,4R)-1 -(2-aminoacetyl)-4- benzoylamino-pyrrolidine-2-carboxylic acid and Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly- NH2; or a pharmaceutically acceptable salt or hydrate thereof.

Suitably, the subject is human. It will be appreciated that the stroke may be an ischemic stroke or a hemorrhagic stroke. The method may be a method of reducing the volume of cerebral infarct associated with an occurrence of ischemic stroke. The reduction of extent of tissue damage may be a result of the prevention or reduction of cerebral reperfusion injury. Accordingly, the present invention also relates to a GJIC modulator compound as described herein for use in a method of preventing or reducing cerebral reperfusion injury subsequent to the occurrence of stroke in a subject, the method comprising administering to the subject a

therapeutically effective amount of said GJIC modulator compound; or a

pharmaceutically acceptable salt or hydrate thereof.

Accordingly, in a first aspect the present invention may provide a compound selected from:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

or a pharmaceutically acceptable salt or hydrate thereof, for use in a method of reducing the extent of tissue damage subsequent to an occurrence of stroke in a human subject, the method comprising administering to the subject a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt or hydrate thereof.

The tissue damage is typically a result of ischemic brain injury. The extent of injury is typically determined by quantifying the volume of cerebral infarct.

In other words, the present invention may provide a compound selected from:

or a pharmaceutically acceptable salt or hydrate thereof, for use in a method of reducing the volume of cerebral infarct associated with an occurrence of stroke in a human subject, the method comprising administering to the subject a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt thereof. The stroke may be ischemic or hemorrhagic. Importantly, it is not necessary that the type of stroke is determined before administration of the compound. Accordingly, the first administration of the compound may occur prior to a cerebral scan, for example, a CT scan.

Accordingly, suitably the compound is administered at the time of first medical contact with the subject, for example, on arrival of a paramedic who suspects stroke has occurred. Early administration is preferable. In this connection, in the methods of the invention, in order to minimize the degree of lasting or permanent cerebral tissue damage that may result following the stroke occurrence, the administration of the compound (or pharmaceutically acceptable salt or hydrate thereof) to the subject should normally take place at most approximately 3 hours, such as at most approx. 2 hours, after the stroke has occurred. In general it will be highly desirable that administration takes place at most 60 minutes, such as at most 45 minutes, e.g. at most 30 minutes, preferably at most 10 minutes, after the stroke has occurred.

The GJIC compounds described herein may enable cells to protect themselves during ischemic conditions (i.e. before the cause of ischemia, e.g. a clot in the case of ischemic stroke, is removed). Without wishing to be bound to any particular theory, the inventors speculate that the compounds may have stabilizing effects on the cells, reducing the tendency for mitochondria to become leaky and/or reducing the tendency for cells to develop a leaky outer cell membrane. The compounds may result in better coupling between cells, enabling the cells to share available energy (ATP) and also share/dilute toxic substances within the cells.

Importantly, the compounds prevent or reduce cerebral reperfusion injury when circulation is restored following removal of the cause of ischemia.

Accordingly, the present invention further provides a compound selected from:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-Nh ;

or a pharmaceutically acceptable salt or hydrate thereof, for use in a method of preventing or reducing cerebral reperfusion injury subsequent to the occurrence of a stroke in a human subject, the method comprising

administering to the subject a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt or hydrate thereof. In a hemorrhagic stroke, circulation may be restored as a result of reduction of compression when the hematoma is removed or otherwise disperses. In an ischemic stroke, circulation may be restored as a result of spontaneous thrombolysis or medical intervention, for example a thrombectomy or induced thrombolysis (for example, administering a clot-dissolving thrombolytic agent such as tissue plasminogen activator (TPA)). As described above, reperfusion intervention should normally be performed if the stroke is found to be ischemic. The method may therefore include the step of determining the stroke type, for example, by CT scan. Preferably, the compound is administered before the determination step. The method may include a step of performing reperfusion intervention, for example, a

thrombectomy or administration of a thrombolytic compound if the stroke is found to be ischemic.

Accordingly, the method may be a method of preventing or reducing cerebral reperfusion injury during or after thrombolytic therapy performed subsequent to the occurrence of an ischemic stroke in a human subject. In order to ensure a cerebral blood concentration of the compound that is adequate to minimize the risk of cerebral tissue injury as a result of reperfusion of (i.e.

reestablishment of blood flow to) the affected area of the brain it will be generally be desirable that administration of the compound takes place at least 10 minutes, e.g. at least 15 minutes, such as at least 20 minutes, before reperfusion intervention takes place. In the present context, the term "reperfusion intervention" refers to a medical intervention for the purpose of lysis (dissolving, e.g. by thrombolytic treatment) or surgical removal of the infarct (termed thrombectomy) that is the cause of the ischemic event in question. In a further aspect, the present invention provides a pharmaceutical composition comprising a GJIC modulator compound as described herein for use in a method as described herein, the pharmaceutical composition comprising said GJIC modulator compound and a pharmaceutically acceptable excipient. The present invention further relates to use of a GJIC modulator compound as described herein in the manufacture of a medicament for use in a method of reducing the extent of tissue damage subsequent to an occurrence of stroke in a subject, the method comprising administering to the subject a therapeutically effective amount of said GJIC modulator compound; or a pharmaceutically acceptable salt or hydrate thereof. Suitably, the GJIC modulator compound is selected from (2S,4R)-1 -(2-aminoacetyl)-4- benzoylamino-pyrrolidine-2-carboxylic acid and Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly- NH₂; or a pharmaceutically acceptable salt or hydrate thereof.

The present invention further provides use of a compound selected from:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

or a pharmaceutically acceptable salt or hydrate thereof, in the manufacture of a medicament for use in a method of preventing or reducing cerebral reperfusion injury subsequent to the occurrence of a stroke in a human subject, the method comprising administering to the subject a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt or hydrate thereof.

As above, the method may be a method of preventing or reducing cerebral reperfusion injury during or after thrombolytic therapy performed subsequent to the occurrence of an ischemic stroke in a human subject.

The present invention also provides methods of treatment of patients in whom a stroke is suspected. Accordingly, at their broadest, the methods described herein are methods of treating suspected stroke victims.

Thus, in a further aspect, the invention relates to a method of preventing or reducing cerebral reperfusion injury subsequent to an occurrence of stroke in a human subject, the method comprising administering the subject a therapeutically effective amount of a compound selected from the group consisting of:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

or a pharmaceutically acceptable salt or hydrate thereof. Suitably, the administration occurs at the time of first medical contact with the subject. A further aspect of the present invention relates to a method of reducing the extent of tissue damage (for example, the volume of cerebral infarct) associated with an occurrence of stroke in a human subject, the method comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

or a pharmaceutically acceptable salt or hydrate thereof. Suitably, the administration occurs at the time of first medical contact with the subject. Suitably, the stroke is ischemic stroke.

The methods may include a step of diagnosing a suspected stroke in the subject. The methods may include a step of, after diagnosing a suspected stroke or stroke in the subject, a decision to treat the subject according to a method of treatment as described herein. A therapeutically acceptable amount of a compound as described herein is then administered to the subject. The first administration may occur before the type of stroke (i.e. ischemic or hemorrhagic) has been determined. For example, the first administration may occur before an imaging technique (for example, a CT scan) has been used on the subject.

In further embodiments, it may be desirable to administer one or more additional doses of the compound (or pharmaceutically acceptable salt or hydrate thereof) after the initial administration. The dose, or each additional dose, may be provided portion wise, for example, as further injections, or as a continuous infusion. For example, the initial administration may be followed by one or more additional administrations beginning not more than 2 hours after first administration, preferably not more than 1 hour after first administration. In some embodiments, the initial administration is followed by one or more additional administrations beginning not more than 30 minutes after first administration, for example, not more than 10 minutes after first administration. For example, the initial administration may be followed by administration by infusion beginning not more than 2 hours after first administration, preferably not more than 1 hour after first administration. In some embodiments, the initial administration is followed by administration by infusion beginning not more than 30 minutes after first administration, for example, not more than 10 minutes after first administration.

For example, the first administration may be given by emergency services before the patient is transported to hospital, or when the patient first presents at the accident and emergency department of a hospital, or a stroke is otherwise suspected by medical personnel. Subsequent doses may be provided on admittance at hospital, and before, during or after a scan to determine the nature of the stroke event. In some embodiments, the first administration may achieve a concentration of 50 nM to 5 μΜ in the plasma of the subject. Subsequent administration may maintain a

concentration of 50 nM to 5 μΜ in the plasma of the subject for at least 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or even longer.

It will be appreciated that the optional and preferred features described for the first aspect apply similarly to all other aspects, and vice versa. Additional aspects and embodiments of the invention will become apparent from the following disclosure.

Brief description of the figures

Figure 1 summarizes cerebral infarct volume data from a mouse study of

experimentally-induced stroke involving reperfusion 1.0 h after initiation of ischemic stroke. It is clearly apparent that the infarct volume in the treated mice was very significantly reduced (roughly halved) compared to the control (sham-treated) mice. Figure 2 summarizes cerebral infarct volume data from a larger mouse study of experimentally-induced stroke.

Figure 3 shows representative thionine stained brain sections from the smaller mouse study. Figure 3a shows a brain section from a sham group (a. suture was placed under the middle cerebral artery, but no clamp was placed on the common carotid arter), Figure 3b shows a brain section from the group administered saline and Figure 3c shows a brain section from the group administered danegaptide.

Detailed description of the invention

The present invention provides methods of preventing or reducing cerebral reperfusion injury subsequent to an occurrence of stroke in a human subject, the method comprising:

administering the subject a therapeutically effective amount of a compound selected from the group consisting of:

or a pharmaceutically acceptable salt or hydrate thereof.

The stroke in question may be ischemic stroke or hemorrhagic stroke. Suitably, administration occurs at the time of first medical contact with the subject.

The invention further provides methods of reducing the volume of a cerebral infarct associated with an occurrence of stroke in a human subject, the method comprising: administering the subject a therapeutically effective amount of a compound selected from the group consisting of:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

or a pharmaceutically acceptable salt or hydrate thereof. Suitably, administration occurs at the time of first medical contact with the subject. In some embodiments, the stroke is ischemic stroke.

In the context of the invention, the expression "time of first medical contact" refers to the point in time at which the subject that is suspected of having suffered an occurrence of stroke comes under professional medical care, typically by a physician, paramedic or other medically qualified person. Time is of the essence in connection with treatment of subjects who have undergone stroke, and a very important feature of the methods of the present invention is the ability to administer a compound among those disclosed herein to the subject immediately without first having to establish (e.g. by CT or MR imaging in a clinical or hospital environment) whether the subject has undergone stroke resulting from infarction (ischemic stroke) or resulting from hemorrhage. Current treatments of ischemic stroke often involve administration of a thrombolytic agent in order to lyse or dissolve the infarct in the affected cerebral tissue, but such administration to a subject who has undergone hemorrhagic stroke can lead to serious - possibly fatal - exacerbation of bleeding in the affected cerebral tissue. The present applicant has established that the compounds used in the methods of the present invention are highly safe and that following their administration there is little or no risk of exacerbation of hemorrhagic stroke: Thus, using the methodology of the present invention there is no need to delay treatment of the subject until a firm diagnosis of ischemic contra hemorrhagic stroke has been made, thereby saving precious time that may be of crucial importance with regard to the extent to which the subject may recover following the occurrence of stroke. In some embodiments, the administration may take place at most 60 minutes, such as at most 45 minutes, e.g. at most 30 minutes, preferably at most 10 minutes, after the stroke has occurred. In some embodiments, the administration takes place at least 10 minutes before reperfusion intervention.

Various routes of administration may be employed in connection with the methodology of the present invention, including intravenous (i.v.), subcutaneous (s.c),

intramuscular (i.m.), intraperitoneal (i.p.), oral (p.o.), sublingual, nasal, rectal, intracerebroventricular (i.e. v.) and intrathecal (i.t.) administration. Parenteral administration, e.g. intravenous administration, will often be desirable in order to minimize the time necessary to attain a therapeutically effective concentration of the active substance in the cerebral blood supply. In this connection, attainment of a concentration of the administered compound (or pharmaceutically acceptable salt or hydrate thereof) in the plasma of the subject in the range of from 50 nM to 5 μΜ may be desirable.

Definitions

Unless specified otherwise, the following definitions are provided for specific terms.

In the present description and claims the standard three-letter and one-letter codes for natural amino acids are used. The term "peptide" herein designates a chain of two or more amino acid moieties (amino acid residues) that are linked by means of a peptide bond. In general, peptides may contain one or more naturally occurring amino acids and/or one or more non-naturally occurring amino acids.

In the present context, the term "naturally occurring amino acid" refers to one of the following 20 amino acids: Ala (A), Cys (C), Ser (S), Thr (T), Asp (D), Glu (E), Asn (N), Gin (Q), His (H), Arg (R), Lys (K), lie (I), Leu (L), Met (M), Val (V), Phe (F), Tyr (Y), Trp (W), Gly (G), and Pro (P). In naturally occurring peptide molecules these amino acids (with the exception of Gly, which lacks a chiral centre) generally occur in the form of L-amino acid residues, but compounds suitable for use in the the present invention include peptides comprising D-amino acid residues (such as Ac-DTyr-DPro- DHyp-Gly-DAIa-Gly-NH₂).

Amino acid 3 letter codes are as used in the art. Hyp refers to 4-hydroxyproline. The compounds for use in the present invention may contain two or more asymmetric atoms (also referred to as chiral centers), giving rise to the possibility of the

occurrence of diastereomers. Compounds suited for use in the present invention include such diastereomers.

Compounds suited for use in accordance with the invention

An example of a compound well suited for use in accordance with the present invention is 1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid, such as the (2S.4R) diastereomer thereof [i.e. (2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino- pyrrolidine-2-carboxylic acid], or a pharmaceutically acceptable salt or hydrate thereof. An example of an alternative name for this compound is (2S, 4R)-1 -(2-aminoacety!)-4- benzamidopyrrolidine-2-carboxylic acid.

Other diastereomers of the latter compound (i.e. the 2S4S, 2R4R or 2R4S

diastereomers) may also be value for use in the context of the present invention.

Another compound (peptide) suited for use in the present invention is Ac-DTyr-DPro- DHyp-Gly-DAIa-Gly-NH₂ (vide supra). Particular forms of these compounds may also be referred to as danegaptide and rotigaptide, respectively.

In addition to the compounds recited above, further compounds that may be suitable for use in the context of the invention include certain other GJIC modulating

compounds, such as the antiarrhythmic peptides AAP (Aonuma et al., Chem. Pharm. Bull. (Tokyo), 28, 3332-3339 (1980)), AAP10 (Dhein et al., Naunyn Schmiedebergs Arch Pharmacol. , 350, 174-184 ( 994); Muller et al. , Eur. J. Pharmacol. , 327, 65-72 ( 1997)), and HP5 (disclosed in U.S. Patent No. 4,775,743). It will be appreciated that the compounds described herein may be used in

combination. For example, more than one GJIC compound may be administered in the methods described herein, either concurrently or sequentially.

Pharmaceutically acceptable salts

Pharmaceutically acceptable salts of compounds suited for use in accordance with the invention having an acidic moiety can be formed using organic or inorganic bases. Suitable salts formed with bases include metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, or magnesium salts; ammonia salts and organic amine salts, such as those formed with morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine (e.g., ethyl-tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethy!propylamine), or a mono-, di- or trihydroxy lower alkylamine (e.g., mono-, di- or triethanolamine). Internal salts also can be formed. When a compound suited for use in accordance with the invention contains a basic moiety (as in the case of, e.g., 1 -(2-aminoacetyl)-4-benzoylamino- pyrrolidine-2-carboxylic acid and the recited diastereomers thereof), salts may be formed using organic or inorganic acids. For example, salts can be formed from the following acids: acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic or camphorsulfonic. Other known pharmaceutically acceptable acids may also be employed. As already mentioned (vide supra), a preferred salt form of the (2S.4R) diastereomer of 1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid is the hydrochloride monohydrate.

The present teachings may also extend to the use of prodrugs of the compounds disclosed herein as being suited for use in accordance with the present invention. As used herein, "prodrug" refers to a moiety that produces, generates or releases a compound of one of the disclosed types when administered to a mammalian subject, notably a human subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either by routine manipulation or in vivo, from the parent compounds. Examples of prodrugs include compounds as disclosed herein that contain one or more molecular moieties appended (bonded) to a hydroxy, amino, sulfhydryl or carboxy group of the compound, and that when administered to a subject to be treated are cleaved in vivo to form the free hydroxy, amino, sulfhydryl or carboxy group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds disclosed herein for use in accordance with the invention. Examples of preferred prodrugs include oxazolidinone or imidazolidinone prodrugs. Ester prodrugs may be formed with lower alcohols, such as Ci-6 alcohols. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. Pharmaceutical compositions

Compounds, or pharmaceutically acceptable salts or hydrates thereof, employed in accordance with the present invention may be administered in the form of appropriate pharmaceutical compositions, which can be administered via any acceptable method known in the art, either singly or in combination. Pharmaceutical compositions of relevance in the present context may comprise a compound as disclosed herein for use in accordance with the invention in admixture with one or more pharmaceutically acceptable carriers, diluents, vehicles or excipients. Such compositions may suitably be formulated for parenteral administration, including intravenous (i.v. ), subcutaneous (s.c ), intramuscular (i.m.) or intraperitoneal (i.p. ) administration. Other administration routes include intracerebrovascular and intrathecal administration (vide supra).

Useful formulations may include formulations that provide sustained release of the compounds of the present teachings. These may be particularly useful for subsequent administration (after the first administration). The compositions are preferably in the form of liquid formulations, and methods for their preparation are generally described in "Remington's Pharmaceutical Sciences", 17th Ed. , Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S.A., 1985. Such compositions generally contain an effective amount of the one or more active compounds of the present teachings, together with a suitable carrier in order to provide the dosage in a form compatible with the route of administration selected. Preferably, the carrier is in the form of a vehicle, a diluent, a buffering agent, a tonicity adjusting agent, a preservative and/or a stabilizer. The excipients constituting the carrier must be compatible with the active pharmaceutical ingredient(s) and are preferably capable of stabilizing the compounds without being deleterious to the subject being treated.

A form of repository or sustained-release formulation can be used so that

therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following administration of the compound or composition, e.g., by transdermal injection or deposition. Formulations suitable for sustained release may comprise biodegradable polymers, such as L-lactic acid, D-lactic acid, DL-lactic acid, glycolide, glycolic acid, and isomers thereof. Similarly, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. Other sustained release formulations can include, but are not limited to, formulations that include at least one of the compounds disclosed herein combined with liposomes, microspheres, emulsions or micelles and liquid stabilizers. Administration of a compound (or pharmaceutical salt or hydrate thereof) in

accordance with the invention may be conducted in a single unit dosage form (e.g. in the form of a bolus) or as a continuous therapy in the form of multiple doses over time. Alternatively, continuous infusion systems or slow release depot formulations may be employed. Two or more compounds for use in accordance with the invention (or pharmaceutical compositions thereof) may be co-administered simultaneously or sequentially in any order. In addition, the compounds and compositions may be administered in a similar manner for prophylactic purposes, for example if a stroke event is considered likely. Ultimately, the best dosing regimen will be decided by the attending physician for each patient individually.

Therapeutic uses

Conditions which may be treated or prevented in accordance with the present invention using compounds as specified herein include cerebrovascular disease, notably stroke, for example ischemic stroke. The methodology of the present invention may thus be used, inter alia, to prevent or treat ischemic injury in the cerebral tissue of a human subject.

According to the invention, one or more of the compounds, or pharmaceutically acceptable salts or hydrates thereof (e.g. in the form of an appropriate pharmaceutical composition), may be administered to an individual in need thereof in a therapeutically effective amount.

As used herein, "a therapeutically effective amount'' refers to an amount that is capable of reducing the symptoms of a given cerebrovascular condition or pathology, and preferably which is capable of partly or wholly normalizing physiological responses in a subject with the condition or pathology. Reduction of symptoms or normalization of physiological responses can be determined using methods known in the art and can vary with a given condition or pathology. The effective amount will be determined by the skilled person taking into account such factors as potency of the drug, age and constitution of the patient, body weight, pharmacokinetic profile of the drug, and in general the drug will be prescribed for each patient or group of patients. The effective amount of the compound can be at least about 10

body weight/day, such as at least about 100 μg/kg body weight/day, at least about 300 weight/day, and at least about 1000 ng/kg body weight/day. On the other hand, the effective amount of the compound or dimer can be at most about 100 mg/kg body weight/day, such as at most about 50 mg/kg body weight/day and at most about 10 mg/kg body weight/day. It is expected that the effective amount of the compound will be about 100

body weight/day, about 300 body weight/day or about 1000 body weight. Ischemic strokes

Ischemic strokes are the most common form of stroke, constituting around 85% of strokes. They are caused by an artery or arteries supplying blood to the brain becoming blocked or narrowed, resulting in ischemia. Such blockage or narrowing is often caused by the occurrence of a blood clot, which can form either in the artery itself, or be formed at another location before being transported by the bloodstream and then becoming lodged in the artery within the brain. Blockage or narrowing of an artery within the brain can also be caused by fatty deposits within the arteries (often termed plaque). Hemorrhagic strokes

Hemorrhagic strokes are caused by leakage or rupture of a vessel (typically an artery) in the brain, releasing blood directly into the surrounding brain tissues. As the cranial compartment is essentially rigid and non-elastic, the released blood can exert pressure on, inter alia, other blood vessels (notably arteries) and cells within the brain tissue, resulting in possible cell damage as a result of ischemia. Hemorrhagic stroke can occur not only within the brain, but also at or near the surface of the brain. In the latter case, blood may be released into the space between the brain and the skull. Such arterial leakage or rupture may result, for example, from trauma, from an aneurysm (i.e. a weakness in a blood vessel wall), from medical conditions such as hypertension, or from treatment with a blood-thinning medication.

Transient ischemic attack (TIA)

TIAs differ from the aforementioned types of stroke in that the flow of blood to the brain is disrupted only temporarily for a short period of time. They resemble ischemic strokes in that they are often caused by blood clots or arterial blockage or narrowing by other debris, such as plaque (vide supra). A TIA should be regarded as a medical emergency just like stroke per se, even though the cerebrovascular event is of short duration. The occurrence of a TIA should be taken as a warning sign of the possibility of a future stroke. According to the Center for Disease Control and Prevention (CDC), over one third of those subjects who experience a TIA but receive no subsequent treatment go on to have a major stroke within a year, and 10-15% may be expected to have a major stroke within 3 months. Clinical symptoms of stroke

These may include:

Sudden numbness or weakness of the face, or in an arm or a leg (especially on one side of the body)

Sudden confusion, difficulty in speaking or understanding speech

· Sudden problems with vision in one or both eyes

Sudden problems with walking, dizziness, loss of balance or coordination Sudden severe headache with no immediately identifiable cause

Evaluating Strokes

Health care professionals use a variety of imaging techniques to evaluate stroke patients. The most widely used imaging procedure is computed tomography (CT) scanning, also known as computerized axial tomography (CAT). CT scanning creates a series of cross-sectional images of the head and brain. As it is normally readily available round the clock at most major hospitals or clinics and generates images quickly, CT is the most commonly used diagnostic technique for acute stroke. CT also has unique diagnostic benefits. It can quickly rule out the possibility of hemorrhage, and may even reveal evidence of early infarction. Infarctions generally show up on a CT scan about 6 to 8 hours after the start of stroke symptoms. If a stroke is caused by hemorrhage, a CT scan can reveal evidence of bleeding into the brain almost immediately after stroke symptoms appear. The occurrence of hemorrhage is a primary reason for avoiding certain drug treatments for stroke, such as thrombolytic therapy which currently is the only proven acute therapy for ischemic stroke. As explained above, owing to the risk of increased bleeding and exacerbation of existing hemorrhagic stroke, thrombolytic therapy should not be administered to a stroke patient until the physician can confidently rule out the possibility of hemorrhagic stroke as the underlying cause of stroke in the patient. Other imaging techniques include magnetic resonance imaging (MRI).

Mechanisms of tissue damage associated with stroke

The ischemic cascade

The brain is one of the most complex organs in the human body. It contains hundreds of billions of cells that interconnect to form a complex communication network. The brain contains several different types of cells, of which neurons are generally regarded as being particularly important. The organization of neurons in the brain, and the communication that occurs between them, underlay thought processes, memory, cognition and awareness. Other types of brain cells are generally designated collectively as glia (from the Greek word meaning "glue"). These cells provide "scaffolding" and support for the vital neurons, protecting them from infection, toxins, and trauma. Glia make up the blood-brain barrier between blood vessels and the substance of the brain.

Stroke is the sudden onset of paralysis as a result of injury to brain cells caused by disruption in cerebral blood flow. The injury caused by partial or complete blocking of a blood vessel can occur within several minutes and may progress for a period of hours as the result of a chain of chemical reactions that is initiated after the onset of stroke symptoms. Physicians and researchers often call this chain of chemical reactions that lead to the permanent brain injury of stroke the ischemic cascade. Primary cell death

In the first stage of the ischemic cascade, blood flow to a part Of the brain is cut off or reduced (ischemia). This leads to a lack of oxygen (anoxia) and lack of nutrients in the cells of this core area. When the lack of oxygen becomes extreme, the mitochondria (the energy-producing structures within the cell) can no longer produce enough energy to keep the cell functioning. The mitochondria break down, releasing oxygen-based free radical species (known as oxygen free radicals) into the cytoplasm of the cell. These species are highly toxic to the cell, causing destruction of other cell structures, including the cell nucleus. The lack of energy supply to the cell causes the gated channels of the cell membrane that normally maintain homeostasis to open and allow toxic amounts of calcium, sodium and potassium ions to enter the cell . At the same time, the injured ischemic cell releases excitatory amino acids, such as glutamate, into the space between neurons, leading to over excitation and injury to nearby cells. With the loss of homeostasis, water rapidly enters the cell causing swelling of the cell (termed cytotoxic edema) until the cell membrane bursts under the resulting internal pressure.

In this process, the nerve cell is essentially injured which leads to cell death (necrosis and infarction). After a stroke starts, the first cells that are going to die may die within 4 to 5 minutes. The response to the treatment that restores blood flow as late as 2 hours after stroke onset would suggest that, in most cases, the process is not over for at least 2 to 3 hours. After that, with rare exceptions, most of the injury that has occurred is essentially permanent.

Secondary cell death

Due to exposure to excessive amounts of glutamate, nitric oxide, free radicals, and excitatory amino acids released into the intercellular space by necrotic cells, nearby cells have a more difficult time surviving. They receive just enough oxygen from cerebral blood flow (CBF) to stay alive. A compromised cell can survive for several hours in a low-energy state. If blood flow is restored within this narrow window of opportunity, at present thought to be about 2 hours, then some of these cells can be salvaged and become functional again. Researchers have learned that restoring blood flow to these cells can be achieved by administering a clot-dissolving thrombolytic agent (eg. t-PA) within 3 hours of the start of the stroke.

Cell death at time of reperfusion

When absolute or relative ischemia has been present for a period of time, cells will have increasingly switched from aerobic to anaerobic metabolism. The anaerobic metabolism does not meet the demand of aerobic tissues like the brain and, as a consequence, the intracellular ATP levels rapidly fall. In addition, the intracellular acidosis may be enhanced by lactic acid that increases because of the lactate- dependent ATP production. These processes lead to a destabilization of lysosome membranes with the leakage of lysosomal enzymes into the cytoplasm and breakdown of the cell structures. Furthermore, an inhibition of the membrane-bound Na⁺-K⁺-ATPase activity is associated with the relative lack of ATP. The latter process causes a large intracellular increase of Na⁺ ions and water, with consequent edema. Along with Na⁺ ions accumulation into the cell, the intracellular Ca²⁺ levels are also increased associated with the decreased pumping Ca²⁺ out of the cells also associated with the ATP depletion. The calcium overload causes the activation of calcium dependent proteases such as calpains. During ischemia, calpains remain relative inactive because of the acid environment, but may damage the cells after pH normalization at the time of reperfusion. Another effect of Ca²⁺ overload is the generation of reactive oxygen species (ROS) at mitochondrial level during ischemia and particularly at time of reperfusion.

When oxygen is reintroduced at time of reperfusion, a rapid correction of the extracellular environment causes additional and immediate stress to the cells. This, together with the sudden correction of extracellular pH while intracellular Ca²⁺ remains high, causes opening of the mitochondrial transition pore (mPTP), together with massive ROS production and other cytotoxic factors, with apoptosis and cell death as a consequence. The ischemia-reperfusion activates different programs of cell death, which may be categorized in necrosis, apoptosis, and autophagy associated cell- death. Necrosis is characterized by the cell swelling with subsequent rupture of surface membranes. The necrotic cells stimulate the immune system and lead to tissue infiltration of inflammatory-cells with consequent cytokine release. In contrast, the apoptosis, activating a complex caspase signaling cascade, induces a self-limiting program of cell death. Generally the apoptosis process was previously considered as less immunostimulating than the necrosis process, but recent data have documented that the extracellular release of ATP from the apoptotic cells may attract phagocytes.

Inflammation and the immune response

While anoxic and necrotic brain cells are doing damage to still viable brain tissue the immune system of the body is injuring the brain through an inftammatory reaction mediated by the vascular system. Damage to the blood vessel at the site of a blood clot or hemorrhage attracts inflammatory blood elements to that site. Among the first blood elements to arrive are leukocytes, white blood cells that are covered with immune system proteins that attach to the blood vessel wall at the site of the injury. After they attach, the leukocytes penetrate the endothelial wall, move through the blood-brain barrier, and invade the substance of the brain causing further injury and brain cell death. Leukocytes called monocytes and macrophages release

inflammatory substances (cytokines, interleukins, and tissue necrosis factors) at the site of the injury. These substances make it harder for the body to naturally dissolve a clot that has caused a stroke by inactivating anti-clotting factors and inhibiting the release of natural tissue plasminogen activator. Those brain cells that survive the loss of blood flow (ischemia) but are not able to function make up the ischemic penumbra. These areas of still-viable brain cells exist in a patchwork pattern within and around the area of dead brain tissue. It is the clinician's aim to preserve (salvage) as much of this ischemic penumbra as possible.

Cerebral infarct volume

Infarct volume is the standard measure of the extent of ischemic brain injury caused by a stroke event. It is linked to clinical outcome for the patient.

Current limitations in the treatment of stroke:

When patients present with clinical signs of stroke it is often not clear whether the stroke is ischemic or hemorrhagic until a CT scan or similar imaging procedure has been performed. Bringing the patient to a hospital and performing a CT scan in order to make the diagnosis takes time and is associated with progressive tissue damage as time elapses. One of the current challenges in the management of stroke is that thrombectomy or thrombolytic therapy cannot be applied until the physician can confidently diagnose the patient as suffering from an ischemic stroke because thrombolytic therapies, the only proven acute stroke therapy for ischemic stroke, cannot be used until the doctor can confidently diagnose the patient as suffering from an ischemic stroke, as this treatment might increase bleeding and make a

hemorrhagic stroke worse. Transport to the hospital and making this diagnosis takes time, which is critical as thrombectomy or thrombolytic therapies should be initiated within 2-3 hours in order to salvage tissue.

There is increasing recognition that intercellular communication is essential for cellular homeostasis, proliferation and differentiation. Such communication is facilitated by gap junctions. These structures provide a route for coupling cells and permitting cellular "cross-talk." (See generally, Sperelakis N., eds., Cell Interactions and Gap Junctions, CRC Press, Inc. (1989)). Such cross-talk via gap junctions is referred to as "gap junctional intercellular communication" (GJIC).

By modulation of GJIC, peptide compounds as disclosed herein for use in accordance with the invention may protect from reperfusion injury (tissue damage) as a result of cerebral events such as stroke. Without being bound by any theory, it is believed that the GJIC modulating properties of certain peptide compounds and pharmaceutically acceptable salts or hydrates thereof disclosed herein for use in accordance with the invention may play a role in relation to mediating tissue protection by the use of the compounds, salt or hydrates in question in the therapeutic context outlined above.

Phase I studies conducted by the present applicant have demonstrated the safety of the compound (2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid and the compound Ac-DTYR-DPro-DHyp-Gly-DAIa-Gly-NH2, both of which are GJIC modulating compounds. The former compound was demonstrated to increase cellular coupling measured by calcein dye uptake in connexin 43 (Cx43) -expressing cells during simulated ischemia and to increase conduction velocity after gap junction uncoupling as a response to ischemic or metabolic stress. Uncoupling of gap junctions has been described in the experimental setting of ischemic stressed conditions.

Combination therapy

The invention further relates to combination therapy in which a compound as described herein is administered with tissue plasminogen activator (TPA). TPA may also be known as IV rtPA (given intravenously, via the arm). The TPA may be administered contemporaneously with the compound as described herein, or it may be administered separately as part of the method of treatment.

For example, the TPA may be administered after the initial adminisation of a compound as described herein. Where the method includes one or more additional doses of the compound, the TPA may be administered before the first additional dose, with an additional dose, after the last additional dose, or in between additional doses.

The TPA may be admisitered after the stroke type is determined. For example, the method may be a method of reducing the extent of tissue damage subsequent to an occurrence of stroke in a human subject, the method comprising administering to the subject a therapeutically effective amount a compound as described herein, or a pharmaceutically acceptable salt or hydrate thereof; then determining the type of stroke, for example, using a CT scan, then administering a therapeutically effective amount of TPA.

It will be appreciated that the method may include the step of determining the type of stroke and then, if the stroke type is ischemic, administering a therapeutically effective amount of TPA. Preferably, the TPA is administered within 4.5 hours of the stroke occurrence, more preferably within 3 hours.

As the compound described herein and the TPA may be administered concurrently, for example, in a subsequent administration, it may be desirable to formulate the compound described herein and TPA in a single composition, for example, a composition suitable for parenteral administration, preverably intravenous

administration. Accordingly, in a further aspect the invention provides a composition comprising a compound selected from:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

or a pharmaceutically acceptable salt or hydrate thereof, and tissue plasminogen activator (TPA).

EXPERIMENTAL

Preparation of compounds for use in accordance with the invention

Compounds (peptides) for use in accordance with the present invention may suitably be synthesized by means of solid-phase or solution-phase synthesis. In this context, reference may be made, for example, to Fields et al., "Principles and practice of solid- phase peptide synthesis", Synthetic Peptides (2002, 2nd Edition).

With regard to the preparation of 1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2- carboxylic acid, such as the (2S,4R) diastereomer thereof, suitable methods of synthesis and purification thereof are described in WO2007/078990, in which the (2S.4 ) isomer is denoted "Compound 2" (WO2007/078990 is incorporated by reference in its entirety).

A particularly preferred salt form of the (2S.4R) diastereomer is the hydrochloride monohydrate, the preparation of which is described in WO2008/079266, and which is also referred to in the present description as Compound X (WO2008/079266 is incorporated by reference in its entirety). With regard to Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂, the solid-phase synthesis and purification thereof are described in WO01/62775 (in which the compound is denoted "Compound 2"). WO01 /62775 is incorporated by reference in its entirety. Protocol for stroke experiment in male C57BL/6i mice.

Eight male C57BL/6j mice were anaesthetised with 65 mg/kg sodium pentobarbital and divided into two groups of four mice (control/sham-treated group and treatment group, respectively). A hole was drilled through the skull of each mouse, and a 0.150 mm O.D. (outer diameter) suture thread was placed under the right middle cerebral artery (MCA) with the ends of the suture extending over the outer surface of the skull. The right common carotid artery of the mice in the treatment group was clamped with a 70 gram pressure microclamp for 50 minutes. For the control/sham-treated animals, the same procedure was followed but without clamping of the right common carotid artery. Vehicle (saline, pH 7.4) was injected into the tail vein of each of the mice in the control group, while a solution of Compound X in saline (75 pg/kg body weight) was injected into the tail vein of each of the mice in the treatment group. After 60 minutes the clamp on the carotid artery in the treatment group was released, followed by removal of the suture from under the MCA to allow reperfusion. The mice in the treatment group received subsequent IP injections of Compound X in saline (300 pg/kg body weight) at 1 , 2 and 3 hrs, respectively, after the initial injection. Mice in the control group received subsequent I P injections of saline at 1 , 2 and 3 hrs, respectively, after the initial injection. All mice were euthanized at 48 hours post clamping, and the brains were fixed in 10% formalin, sectioned and histologically stained with thionine. The volume of the stroke infarct in each mouse brain was measured using Image J by 2 independent workers who were unaware of whether the mice belonged to the control or treatment group.

The infarct volume data for the treatment and control groups are summarized in Figure 1 , from which it is clearly apparent that infarct volume in the treated mice was very significantly reduced (roughly halved) compared to the control (sham-treated) mice. In Figure 1 , Compound X is labelled danegaptide.

When the number of animals treated was increased, a significant reduction was observed. The procedure was repeated with 10 male mice in the control group, while 1 1 were treated with danegaptide (Compound X). The procedure was as decribed above, except that: • At 40 min after clamping (i.e. before injection at 50 min) 100 μΙ_ of blood was collected for serum testing.

• At 30 min after tail vein injection 100 μΙ_ of blood was collected for serum testing.

• At four hours after initial compound injection 100 μΙ of blood was collected for serum testing (in this case, only for 4 mice from each set).

The results show a significant reduction (p=0.002) in infarct size following

danegaptide treatment.

Representative thionine-stained brain sections from the smaller (n=4 in each group) study are shown in Figure 3.

Claims

1 . A compound selected from:

(2S,4R)-1 -(2-aminoacetyi)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

2. A compound selected from:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;

administering to the subject a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt or hydrate thereof.

3. The compound for use as claimed in claim 1 or claim 2, wherein the stroke is ischemic stroke or hemorrhagic stroke.

4. The compound for use as claimed in any one of claims 1-3, wherein the administration takes place at most 60 minutes, such as at most 45 minutes, e.g. at most 30 minutes, preferably at most 10 minutes, after the stroke has occurred.

5. The compound for use as claimed in any preceding claim, wherein the method further comprises:

- determining that the stroke is ischemic stroke; and then

- reperfusion intervention;

optionally wherein the determining step occurs subsequent to administering the compound.

6. The compound for use as claimed in claim 5, wherein administering the compound takes place at least 10 minutes before reperfusion intervention.

7. The compound for use as claimed in any preceding claim, wherein the administration route is selected among: intravenous (i.v.), subcutaneous (s.c), intramuscular (i.m.), intraperitoneal (i.p.), oral (p.o.), sublingual, nasal, rectal, intracerebroventricular (i.e. v.) and intrathecal (i.t.) administration.

8. The compound for use as claimed in any preceding claim, wherein intravenous administration is employed.

9. The compound for use as claimed in claim 8, wherein a concentration of the administered compound or pharmaceutically acceptable salt or hydrate thereof in the plasma of the subject in the range of from 50 nM to 5 μΜ is attained.

10. The compound for use as claimed in any preceding claim, the method further comprising one or more additional administrations of the compound or

pharmaceutically acceptable salt or hydrate thereof beginning not more than 2 hours after first administration, optionally not more than 1 hour after first administration.

1 1. The compound for use as claimed in claim 10, wherein a concentration of the administered compound or pharmaceutically acceptable salt or hydrate thereof in the plasma of the subject in the range of from 50 nM to 5 μΜ is maintained for at least 30 minutes, optionally at least 60 minutes after first administration.

12. The compound for use as claimed in any preceding claim, the method further comprising administration of tissue plasminogen activator (TPA).

13. A composition comprising a compound selected from:

(2S,4R)-1 -(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; and

Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH₂;