BACKGROUND OF THE INVENTION An aneurysm is an abnormal dilation of a blood vessel that poses a risk to health from the potential for rupture, clotting, or dissecting. Rupture of an aneurysm in the brain causes stroke, and rupture of an aneurysm in the abdomen causes shock. The abdominal aortic aneurysm (AAA) is the most common type of aneurysm.

The exact cause of aneurysm development is not known, however several new theories have developed over the last 15 years. It appears that the disease may require a basic genetic susceptibility that is traceable to a single major locus, probably an autosomal dominant gene. The disease unequivocally runs in families, however there are probably other contributing causes, such as smoking and high blood pressure.

Caucasian men over age 55 are at the greatest risk of aneurysm. In fact, aneurysms are among the top ten causes of death among this cohort. By about age 80, over 5% of white males will have developed an aneurysm. AAA's occur less frequently in white women, and they are relatively uncommon in African Americans of both sexes.

AAA's cause many deaths as they are usually silent until a medical emergency occurs. If the patient is thin and has a moderately large AAA, it may be palpable below the rib cage. Many aneurysms are incidentally discovered as a result of medical imaging for other conditions, by ultrasound exams, CAT scans, MRI's, or even by X-ray of the abdomen.

Few patients survive the rupture of an aneurysm. The best predictor of risk of rupture is the size of the aneurysm. The diameter of a normal aorta is about 2 centimeters. Once a AAA has reached 5-6 centimeters in diameter, the risk of rupture is substantial, probably about 50/50 over the next few years. While most vascular surgeons would recommend that a 5-6 centimeter aneurysm should be repaired, the presence of other medical factors in a patient is likely to increase the risk associated with surgical repair. There is less unanimity of opinion about smaller AAA's, since the risk of rupture is much lower.

While the risks associated with surgery may be small, there is a definite risk of death. The risk of death from surgery is related to hospital expertise and experience, the skill of the surgeon, and the basic underlying health of the patient. Mortality rates are frequently reported to be as low as 0 to 2% in academic medical centers with vascular specialists and superior intensive care.

Rates may be higher in small community hospitals without dedicated vascular specialists. Patients without any history or signs of heart disease generally have an uneventful recovery, because heart attack postoperatively is the leading cause of surgical mortality.

After aneurysm repair surgery the average hospital stay is 7-10 days, and most patients take about 6 weeks to recover before returning to work. The vast majority of patients are back on a normal survival curve for life expectancy, consistent with their cohort of persons of similar age and with similar underlying health (e. g. , heart condition, renal function, etc. ). One unfortunate complication of surgery is the possibility of sexual dysfunction in men, usually in the form of "retrograde ejaculation".

Accordingly, while the prior art discloses many forms of surgical repair for aneurysm, there are substantial drawbacks for the patient.

Some control over the progression of an aneurysm may be gained by giving up tobacco, ensuring reasonable blood pressure control, and improving physical fitness. However such measures are only of limited effectiveness.

Another type of aneurysm is the thoracic aortic aneurysm (TAA). A TAA is a localized expansion of the wall of the aorta within the chest cavity, and is often caused by atherosclerosis, hypertension, congenital disorders such as Marfan's syndrome, trauma, or less commonly, syphilis. Atherosclerosis is by far the most common cause.

Most TAA patients have no symptoms until the aneurysm begins to leak or expand. Most non-leaking thoracic aortic aneurysms are detected by chest X- ray or a chest CT scan conducted for other reasons. Chest or back pain may indicate acute expansion or leakage of the aneurysm.

The physical examination is often normal, however a chest X-ray or chest CT scan demonstrate enlargement of the aorta. A chest CT scan identifies the diameter of the aorta and the exact location of the aneurysm. An aortogram (a special set of X-ray images made during injection of dye into the aorta) may also identify the location and extent of the aneurysm and identify any branch arteries of the aorta that are also involved.

The treatment of TAA depends on the location of the aneurysm. For patients with aneurysms of the ascending aorta or aortic arch, surgery to replace the aorta is recommended if the diameter of the aorta measures greater than 5-6 cm. The aorta is replaced with a fabric substitute in an operation that uses a heart-lung machine. If the aortic arch is involved, a specialized technique called "circulatory arrest" (a period without blood circulation while on life support) may be necessary. Of course, this is a procedure with inherent risk.

For patients with aneurysms of the descending thoracic aorta, two options are available. For patients with aneurysms that are larger than 6 cm, an operation for replacement of the aorta with a fabric substitute can be done, or the aorta can be stented.

Stenting involves the use of a tube placed inside the vessel and can be performed without a chest incision, with specialized catheters that are

introduced through arteries at the groin. Not all patients with descending thoracic aneurysms are candidates for stenting, however.

The long-term prognosis for patients with TAA is determined by other medical problems such as heart disease and diabetes, which may have caused or contributed to the condition.

Serious complications after aortic surgery can include heart attack, irregular pulse, bleeding, stroke, paralysis, graft infection, and kidney damage. Death early after the operation may occur in 5-10% of patients.

Aneurysms can also occur in the brain. An intracerebral aneurysm is a small, thin walled dilatation of one of the large blood vessels that supply the brain.

These aneurysms pose a risk to health from the potential for rupture and subsequent bleeding into the brain and/or the fluid-filled spaces that surround the brain (the subarachnoid space). These so-called saccular or"berry" aneurysms occur at the bifurcation of the large blood vessels at the base of the brain.

Intracerebral aneurysms can result from trauma, infection, or neoplastic disease. Most aneurysms, however, result from a developmental abnormality of the inside lining or intima of an artery with abnormal thinning of the vessel at the site of origin. It appears there may be a genetic predisposition to the development of intracerebral aneurysms; the existence in some families runs as high as 10%, approximately 10 times higher than that found in the general population. There are several other causes of intracerebral aneurys, such as infected embolic material from a bacterial infection on one of the heart valves being deposited on one of the arteries in the brain (mycotic aneurym).

Cerebral aneurysmal rupture leads to subarachnoid hemorrhage (SAH) and occurs most often in patients between 40 and 60 years of age with approximately equal sex distribution. Cigarette smoking and excess alcohol use have been shown to increase the risk of rupture. Likewise, the existence of intracerebral aneurysms is associated with other diseases such as polycystic

kidney disease, coarctation of the aorta, and fibromuscular hyperplasia. Other factors such as high blood pressure seem to be less important since aneurysms often occur in persons with normal blood pressure.

Like AAAs and TAAs, cerebral aneurysms are usually asymptomatic prior to rupture. However, an expanding aneurysm can have a"mass"effect causing problems with double vision, loss of vision, numbness in the face, an enlarged pupil size, or a drooping eyelid. Usually patients who have an aneurysm rupture experience sudden onset of a severe headache, frequently accompanied by transient loss of consciousness and sometimes vomiting. A stiff neck often follows. Rupture of an aneurysm usually occur while the person is active rather than during sleep. Occasionally, patients experience a warning or"sentinel" headache which is attributed to a smaller leakage of blood usually preceding a major bleed by several hours to days later. These milder headaches are often associated with nausea and vomiting and are often mistaken for migraine headaches.

Carotid and vertebral angiography is the only definitive means of demonstrating an intracerebral aneurysm, while a CT scan of the head will confirm the presence of blood within the brain or subarachnoid space if an aneurysm has ruptured. Lumbar puncture is sometimes used to evaluate for the presence of blood in the cerebrospinal fluid if the results of the CT scan are equivocal. More recently, non-invasive studies using magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) have shown promise in detection of aneurysms. However, the intracerebral angiogram remains the test of choice.

If rupture of a cerebral aneurysm occurs, only approximately half of patients survive. The best predictor of risk of rupture is the size of the aneurysm. Most aneurysms that rupture have a diameter equal to or greater than 10mm but rupture also occurs with aneurysms of smaller size. A guide to prognosis is provided by the neurologic grade (Hunt and Hess Grades I-V) of the patient determined by his/her level of consciousness and neurologic deficits when first examined upon arrival to the hospital. In a large study of survival of patients from aneurysm rupture, a Grade of I-II (awake with slight to moderately severe

headache and neck stiffness) predicted a low mortality (4%) and an independent life (up to 90%) at follow-up whereas Grades IV-V (stupor with neurological deficits to deep coma) predicted increasingly higher mortality rates (up to 46%) and decreased independent functioning (only about 30%).

The development of cerebral vasospasm, re-bleeding from the aneurysm, swelling of the ventricles in the brain (hydrocephalus), and seizures are complications that may occur after rupture of an intracerebral aneurysm.

Cerebral vasospasm after aneurysmal subarachnoid hemorrhage usually occur within the first 14 days of rupture and is a major cause of morbidity and mortality in survivors of the bleed. Its incidence has varied in different studies between approximately 20 to 80% of all patients with SAH and its occurrence is related to the amount of subarachnoid blood in the brain. Other complications including re-bleeding from an aneurysm and hydrocephalus also contribute to the overall morbidity and mortality. In addition, dangerous cardiac arrhythmias may develop in the acute period following a bleed.

Like AAAs and TAAs, there are risks to surgical repair, however the risks are significantly higher. The timing of surgery is now recognized as an important factor in the prevention of complications associated with aneurysmal rupture.

Successful early surgical clipping of a ruptured aneurysm (within the first 5 days of a bleed) helps to prevent the re-occurrence of bleeding, likely to be an even more catastrophic event when it occurs, and permits the safe treatment of cerebral ischemia due to vasospasm. High morbidity and mortality may occur even in low-risk patients treated with delayed operation because preoperative complications have time to develop. However, such patients operated on within the first 5 days of a bleed usually recover with no or mild neurological deficit and mortality is less than 5%.

As stated above, patients in Grades 1-111 can be operated on safely within 72 hours with good results. In one study of 145 patients with Hunt & Hess Grades l-lil, 81% (117 patients) made a good recovery. The morbidity was 12% (17patients) and the mortality 7% (11 patients). The most common cause of unfavourable outcome was surgical complications. Other factors contributing to

a worse outcome correlated with a higher age, worse Grade, and more severe SAH on CT scan.

The management of asymptomatic aneurysms discovered incidentally remains controversial. A recent study followed 142 patients with unruptured aneurysms for a period of 14 years and found an average annual rupture incidence of 1.4%. The cumulative rate of bleeding from the aneurysms was 10% at 10 years, 26% at 20 years, and 32% at 30 years after the diagnosis. The surgical clipping of an intact and accessible aneurysm is usually a procedure of low risks without the future development of postoperative ischemia or vasospasm, which are often encountered with the clipping of ruptured aneurysms (leading to risk of death or disability). Thus, as for aortic aneurysms there exists a need for a low risk intervention such as drug therapy.

It would clearly be desirable to provide a non-surgical approach to the treatment of aneurysm, or for the prevention of aneurysm. As discussed above, the treatment of aneurysm by surgery is fraught with risk, and may result in significant adverse effects for the patient. To the best of the Applicant's knowledge, the prior art has yet to provide a non-surgical approach proven in a prospective clinical trial to reduce the growth rate of aneurysms in humans.

While propranolol (a beta-blocker) has been shown to reduce the incidence of ruptured aneurysms in turkeys and to delay the growth of aneurysms in mice.

Retrospective studies have suggested that propranolol might be beneficial in human subjects, however proof will await a prospective trial.

Another disorder of the vasculature is that of neointimal hyperplasia. Neointimal hyperplasia is the pathological process that underlies graft atherosclerosis, stenosis, and the majority of vascular graft occlusion. It is a condition that is commonly seen after various forms of vascular injury and a major component of a vein graft's response to harvest and surgical implantation into high-pressure arterial circulation. Smooth muscle cells in the middle layer (i. e. media layer) of the vessel wall become activated, divide, proliferate and migrate into the inner layer (i. e. intima layer). The resulting abnormal neointimal cells express pro- inflammatory molecules, including cytokines, chemokines and adhesion

molecules that further trigger a cascade of events that lead to occlusive neointimal disease and eventually graft failure.

Until very recently, symptomatic restenosis of a stented artery or stenosis of a bypass graft could not be prevented and usually required further vascular reconstruction. A number of drugs have been tried including antithrombotic agents, anticoagulants, ACE inhibitors, and cytotoxic agent, and they have all failed.

Taxol, rapamycin, and radiation have achieved some success in preliminary trials in suppressing intimal hyperplasia, in part because the drugs and radiation are particularly effective when they are delivered or released into the injured artery. This form of delivery targets the vascular bed at risk and prevents systemic toxicity. Unfortunately, none of the aforementioned compounds is specific for the vascular smooth muscle cell, the cell responsible for the intimal lesion, and it is possible that failure of endothelial regeneration may leave the reconstructed vessel vulnerable to late thrombosis as has been shown in irradiated and stented coronary arteries.

The inventors have alleviated a problem of the prior art by providing novel compounds useful in the treatment or prevention of aneurysm and neointimal hyperplasia. Also provided for is the use of certain known compounds that have been found to have efficacy in the treatment or prevention of aneurysm and neointimal hyperplasia.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

IN THE FIGURES Fig. 1 shows the results of an in vitro smooth muscle cell migration assay. Cells were treated with varying concentrations of MCT-1, oxamflatin, or vehicle.

Fig. 2A shows an example of Northern Blot analysis performed on WSR rat VSMC following treatment with Oxamflatin and/or Metacept-1 for 16 hours. mRNA loading was examined under UV illumination.

Fig. 2B is a graphical representation of inhibition of MMP-2 RNA expression by Oxamflatin alone or with Metacept-1 in rat aortic VSMC for 16 hours (n =3).

Fig. 3A shows a membrane probed with a monoclonal antibody to MMP-2 to detect presence of the protein. The lower panel shows the same western blot following staining with coomassie blue dye to demonstrate that loading of conditioned media was balanced.

Fig. 3B is a graphical representation of MMP-2 protein expression inhibition by Oxamflatin and/or Metacept-1 (n = 3).

Fig. 4A shows a membrane probed with a monoclonal antibody to MMP-2. The lower panel shows the same western blot following staining with coomassie blue dye to demonstrate that loading of conditioned media was even.

Fig. 4B is a graphical representation of inhibition of MMP-2 protein expression by Oxamflatin treatment for 16 hours (n = 3).

Fig. 5A shows a membrane probed with a monoclonal antibody to MMP-2. The lower panel shows the same western blot following staining with coomassie blue dye to demonstrate that loading of conditioned media was even.

Fig. 5B is a graphical representation of inhibition of MMP-2 protein expression by MCT-1 treatment for 24 hours in hVSMC (n = 3).

Fig. 6A shows a representative Gelatin zymography-white bands indicate proteolysis of gelatin present in SDS-PAGE gel.

Fig. 6B is a graphical representation of inhibition of MMP-mediated proteolytic activity by Oxamflatin and Metacept-1 (n = 3).

Fig. 7A shows a gelatin Zymogram-white bands indicate sites of proteolytic activity on a gelatin SDS-PAGE gel.

Fig. 7B is a graphical representation of inhibition of MMP2-mediated proteolytic activity by Oxamflatin in hVSMC (n = 3).

Fig. 8A is a gelatin zymogram showing white bands of proteolytic cleavage on a gelatin SDS-PAGE gel.

Fig. 8B is a graphical representation of MCT-1 inhibition of MMP-2 mediated proteolytic activity following 24 hours of treatment (n = 3).

Fig. 9A is a graphical representation of inhibition of recombinant MMP-2 activity when diluted to 1: 1000 with diluted (1: 5) and undiluted conditioned media from human vascular smooth muscle cells (n = 4).

Fig. 10 shows the incidence of aneurysm in a mouse model after treatment with oxamflatin or MCT-1.

Fig 11 shows the effects of oxamflatin and MCT-1 on incidence of AAA in a murine model. Panel A shows percentage incidence taking into account deceased animals, while Panel B excludes deceased animals. Bars correspond to the legend on the right of the histogram (ie, from left to right in Panel A, the first bar is placebo, the second bar is vehicle, the third bar is oxamflatin 1uM etc). Doxycycline is a positive control.

Fig. 12 shows the effect of MCT-1 on neointimal development in a murine model by reference to vessel wall thickness as measured by diameter in um

(Panel A) and % of luminal diameter (Panel B). Placebo animals were not treated with Ang II. Doxycycline is a positive control.

Fig 13 shows neointimal development in a murine model. Transverse sections of the ascending aorta of 12-week old mice treated for 2 weeks were stained with haematoxylin and eosin under 40x magnification. Scale bar represents 20 , um. (1) Vehicle treated wild type (2) Vehicle treated ApoE-/-. The histogram represents intimal and medial growth in a proximal section of ascending aorta in 12-week old wild type mice and 12-week old ApoE~/-mice treated for 2 weeks with vehicle.

Fig 14 shows neointimal development in a murine model. Transverse sections of the ascending aorta of 30-week old mice treated for 2 weeks were stained with haematoxylin and eosin under 40x magnification. Scale bar represents 20 jum. (1) Vehicle treated wild type (2) Vehicle treated ApoE-/-. The histogram represents intimal and medial growth in a proximal section of ascending aorta in 30-week old wild type mice and 30-week old ApoE~/~ mice treated for 2 weeks with vehicle.

Fig 15 shows neointimal development in a murine model. Transverse sections of carotid arteries from 12-week old ApoE~/~ mice treated for 2 weeks were stained with haematoxylin and eosin under 20x magnification. Scale bar represents 20 µm. (1) Ang II (1000ng/kg/min) treated ApoE~/~-contralateral carotid artery. (2) Ang II (1000ng/kg/min) treated ApoE-/--cuffed carotid artery.

The histogram represents wall growth in a section of common carotid artery in 12-week old ApoE-/- mice subjected to cuff placement or not (contralateral) treated for 2 weeks with Ang I I (1 OOOng/kg/min).

SUMMARY OF THE INVENTION Throughout the description and claims of this specification, the word"comprise" and variations of the word, such as"comprising"and"comprises", is not intended to exclude other additives, components, integers or steps.

In one aspect the present invention provides compounds of the formula

wherein Ri is selected from the group consisting of H, C1-C12 alkyl, substituted Cl-Cl2 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C3-C12 cycloalkyl, substituted C3-C12 cycloalkyl, C2-C12 althyryl, substituted C2-C12 althyryl, bicycloalkyl, substituted bicycloalkyl, tricycloalkyl, substituted tricycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl cycloalkyl, substituted alkyl cycloalkyl, cycloalkyl alkyl, substituted cycloalkyl alkyl, alkyl aryl, substituted alkyl aryl, aryl alkyl, substituted aryl alkyl, alkyl heteroaryl, substituted alkyl heteroaryl, heteroaryl alkyl, substituted heteryaryl alkyl, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy ; R2 is selected from the group consisting of H, C1-C12 alkyl, substituted Cl-Cl2 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, Cl-ci cycloalkyl, substituted C3-C12 cycloalkyl, C2-C12 althyryl, substituted C2-C12 althyryl, bicycloalkyl, substituted bicycloalkyl, tricycloalkyl, substituted tricycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl cycloalkyl, substituted alkyl cycloalkyl, cycloalkyl alkyl, substituted cycloalkyl alkyl, alkyl aryl, substituted alkyl aryl, aryl alkyl, substituted aryl alkyl, alkyl heteroaryl, substituted alkyl heteroaryl, heteroaryl alkyl, substituted heteryaryl alkyl, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy ; L is a linking group A is selected from the group consisting of

wherein R3 is selected from the group consisting of H, Cl-Cl2 alkyl, substituted Cl-Cl2 alkyl, C2-C2 alkenyl, substituted C2-C12 alkenyl, C3-C12 cycloalkyl, substituted C3-C12 cycloalkyl, C2-C12 althyryl, substituted C2-C12 althyryl, bicycloalkyl, substituted bicycloalkyl, tricycloalkyl, substituted tricycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl cycloalkyl, substituted alkyl cycloalkyl, cycloalkyl alkyl, substituted cycloalkyl alkyl, alkyl aryl, substituted alkyl aryl, aryl alkyl, substituted aryl alkyl, alkyl heteroaryl, substituted alkyl heteroaryl, heteroaryl alkyl, substituted heteryaryl alkyl, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy ; or a pharmaceutically acceptable salt thereof.

In a preferred embodiment the linking group is such that there are from 4-12 bonds in the shortest direct chain through the linker joining the nitrogen moiety of the sulfonamide with the group A.

In an even more preferred embodiment there are between 6-10 bonds in the chain, more preferably there are 8 bonds. In the examples of linking groups provided immediately below, it may be seen that the shortest chain through the linking group consists of 8 bonds Suitable linking groups are selected from the group consisting of: wherein each R4 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are

attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur. n is an integer from 0 to 3 wherein each R5 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur. m is an integer from 0 to 3 (2) wherein each R6 is independently selected from the growth consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

O is an integer from 0 to 4 R7 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may

together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

X is 0 or S (3) Each R8 is independently selected from the group consisting of Rg is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

Rio is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

Z is O or CH2 Ri, each Ri, is independently selected from the group consisting of q is an integer from 0 to 4 (5)

R12 is independently selected fro the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

R is an integer from 0 to 4 (6)

Each R13 is independently selected from the graph consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

S is an integer from 0 to 4 R14 is selected from the graph consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

Preferably the linker is of the formula selected from the following :

In a preferred form of the invention the compound has the following structural formula

The above compound is referred to herein as Metacept-1 or MCT-1.

In another aspect the present invention provides pharmaceutical compositions including the compounds of the present invention.

In yet a further aspect the present invention provides the use of a compound described herein in the preparation of a medicament for the treatment or prevention of aneurysm or neointimal hyperplasia.

In another aspect the present invention provides a method for treating or preventing aneurysm or neointimal hyperplasia comprising administering to a subject in need thereof a therapeutic amount of a compound or composition as described herein.

DETAILED DESCRIPTION OF THE INVENTION In one aspect the present invention provides compounds of the formula wherein Ri is selected from the group consisting of H, Ci-Cis alkyl, substituted Cl-Cl2 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C3-C12 cycloalkyl, substituted C3-C12 cycloalkyl, C2-C12 althyryl, substituted C2-C12 althyryl, bicycloalkyl, substituted bicycloalkyl, tricycloalkyl, substituted tricycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl cycloalkyl, substituted alkyl cycloalkyl, cycloalkyl alkyl, substituted cycloalkyl alkyl, alkyl aryl, substituted alkyl aryl, aryl alkyl, substituted aryl alkyl, alkyl heteroaryl, substituted alkyl heteroaryl, heteroaryl alkyl, substituted heteryaryl alkyl, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy ; R2 is selected from the group consisting of H, Cl-Cl2 alkyl, substituted Cl-Cl2 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C3-C12 cycloalkyl, substituted C3-C12 cycloalkyl, C2-C12 althyryl, substituted C2-C12 althyryl, bicycloalkyl, substituted bicycloalkyl, tricycloalkyl, substituted tricycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl cycloalkyl, substituted alkyl

cycloalkyl, cycloalkyl alkyl, substituted cycloalkyl alkyl, alkyl aryl, substituted alkyl aryl, aryl alkyl, substituted aryl alkyl, alkyl heteroaryl, substituted alkyl heteroaryl, heteroaryl alkyl, substituted heteryaryl alkyl, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy ; L is a linking group A is selected from the group consisting of wherein R3 is selected from the group consisting of H, Cl-Cl2 alkyl, substituted Cl-Cl2 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C3-C12 cycloalkyl, substituted C3-C12 cycloalkyl, C2-C12 althyryl, substituted C2-C12 althyryl, bicycloalkyl, substituted bicycloalkyl, tricycloalkyl, substituted tricycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl cycloalkyl, substituted alkyl cycloalkyl, cycloalkyl alkyl, substituted cycloalkyl alkyl, alkyl aryl, substituted alkyl aryl, aryl alkyl, substituted aryl alkyl, alkyl heteroaryl, substituted alkyl heteroaryl, heteroaryl alkyl, substituted heteryaryl alkyl, alkoxy, substituted alkoxy, aryloxy, substituted aryloxy ; or a pharmaceutically acceptable salt thereof.

In a preferred embodiment the linking group is such that there are from 4-12 bonds in the shortest direct chain through the linker joining the nitrogen moiety of the sulfonamide with the group A.

In an even more preferred embodiment there are between 6-10 bonds in the chain, more preferably there are 8 bonds. In the examples of linking groups provided immediately below, it may be seen that the shortest chain through the linking group consists of 8 bonds Suitable linking groups are selected from the group consisting of:

wherein each R4 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur. n is an integer from 0 to 3 wherein each R5 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro ; acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur. m is an integer from 0 to 3 (2) wherein each R6 is independently selected from the growth consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy,

aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido ; carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0, 1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

O is an integer from 0 to 4 R7 is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

X is 0 or S (3) Each R8 is independently selected from the group consisting of Rg is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

Rio is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

Z is O or CH2 (4)

Rll each RI, is independently selected from the group consisting of q is an integer from 0 to 4 (5)

R12 is independently selected fro the group consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur. R is an integer from 0 to 4 (6)

Each R13 is independently selected from the graph consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6-membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

S is an integer from 0 to 4 R14 is selected from the graph consisting of alkyl, alkenyl, alkynyl, aryl, fluoro, chloro, bromo, hydroxy, alkyloxy, alkenyloxy, aryloxy, acyloxy, amino, alkylamino, dialkylamino, arylamino, thio, alkylthio, arylthio, cyano, nitro, acyl, amido, alkylamido, dialkylamido, carboxyl, or two optional substituents may together with the carbon atoms to which they are attached form a 5-or 6- membered aromatic or non-aromatic ring containing 0,1 or 2 heteroatoms selected from nitrogen, oxygen or sulfur.

Preferably the linker is of the formula selected from the following :

In highly preferred forms of the invention the compound has the structure selected from the group consisting of

Applicants have shown that compounds as described herein (and especially compound K as shown above (referred to herein as metacept-1 or MCT-1) have efficacy in the treatment and/or prevention of aneurysm or neointimal hyperplasia. Also included in the present invention are derivatives of the compounds detailed above. The skilled person will understand that various modifications may be made to the structures of the compounds defined herein without materially affecting their biological activities.

Methods of synthesising organic compounds are well known in the art. The following text book references will instruct the skilled artisan in appropriate methods for synthesis of the compounds of the present invention, and are incorporated herein by reference: "Advanced Organic Chemistry" (5th ed. ), J. March, Wiley, 1992.

"Comprehensive Organic Transformations"R. J. Larock, VCH, 1989.

"The Logic of Chemical Synthesis", E. J. Corey, X. -M. Cheng, Wiley, 1989.

"Organic Synthesis: the Disconnection Approach"S. G. Warren, Chichester ; New York : Wiley, 1982.

"Some Modern Methods of Organic Synthesis" (3rd ed, ), W. Carruthers, Cambridge University Press, 1978.

"Protective Groups in Organic Synthesis" (2nd ed. ) by T. W. Greene, P. G. M. Wuts, Wiley, 1991.

"Protecting Groups"by P. J. Kocienski, Thieme, 1991.

"Organic Synthesis" (1st ed. ), M. B. Smith. McGraw Hill, 1994.

"Classics in Total Synthesis"K. C. Nicolaou, E. J. Sorensen, VCH, 1996.

As an example, a possible synthetic strategy for compound A is described below :

As another example, a possible synthetic strategy for compound I is described below. As yet a further example, a possible synthetic strategy for compound J is described below.

The compounds of the present invention can be formulated for administration to a subject as a pharmaceutical composition. Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. 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 dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example,

parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes. The

active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutical acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1, 3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.

Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants. The active component is admixed

under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.

The term"pharmaceutically acceptable salts, esters, amides, and prodrugs"as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term"salts"refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention.

These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.

Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, nitrate, acetate, oxalate, valerat, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylat, citrate, maleat, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate and laurylsulphonate salts, and the like. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See, for example, Berge S. M. et al.,"Pharmaceutical Salts,"J. Pharm. Sci. , 1977 ; 66: 1-19 which is incorporated herein by reference.) In another aspect the present invention provides a method for treating or preventing aneurysm or neointimal hyperplasia comprising administering to a subject in need thereof a therapeutic amount of a compound or composition as described above.

The present invention further provides a method for treating or preventing aneurysm or neointimal hyperplasia comprising administering to a subject in need thereof of a therapeutic amount of amiloride or oxamflatin or a derivative thereof. Amiloride hydrochloride is sold under a number of trade names including Kaluril (Alphapharm Pty Ltd; Australia) and Midamore (Merck Sharp and Dohme Pty Ltd).

Compositions containing amiloride have been known in the art for many years, although have only been approved for clinical use in diuresis. The drug has a known potassium conserving role leading to weak natriuretic, diuretic and antihypertensive activities. The main indication for use is to conserve potassium in patients receiving strong diuretics in whom excessive potassium loss is expected. Unexpectedly, amiloride and compounds described herein are proposed to have activity in the treatment and prevention of aneurysm or neointimal hyperplasia.

The present invention also includes the use of other known derivatives of amiloride such as phenamil, benzamil, dichlorobenzamil, ethylisopropyl amiloride, and 5- (N, N-dimethyl) amiloride for the treatment of aneurysm or neointimal hyperplasia.

The Applicants have also proposed that oxamflatin and other derivatives have activity in the treatment and prevention of aneurysm or neointimal hyperplasia.

Oxamflatin ( (2E)-5- [3- (phenylsulfonylamino) phenyl] pent-2-ene-4-ynohydroxamic acid), has the following formula:

This compound has been found in US 5534654 to possess activity against the growth of vascular endothelial cells and the expression of lymphocyte adhesive factors, detransforming activity of cells transformed by ras gene, inhibition of cell growth and have effect on inflammation and on tumors. It has previously been shown to reduce primary tumour growth but has not been indicated to have any benefit in the inhibition of metastatic disease spread. Cell growth inhibition as demonstrated by this compound is quite different to cellular spread that requires an element of mobility not present in cell growth. However, the Applicants have been the first to propose a new use for this compound and its known derivatives for the treatment of aneurysm or neointimal hyperplasia.

US 5534654 also contains a number of other derivatives having activity useful in the context of the present invention. All of these compounds are incorporated herein by reference. One especially active compound has the following structural formula Reference herein to"treatment"and"prevention"is to be considered in its broadest context. The term"treatment"does not necessarily imply that a subject is treated until total recovery. Similarly,"prevention"does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prevention include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term"prevention"may be considered as reducing the severity of onset of a particular condition."Treatment'may also reduce the severity of an existing condition or simply inhibit the further progression of the condition.

As used herein the term"therapeutic amount"means an amount necessary to at least partly attain the desired response.

The term"subject"is intended to include humans, primates, livestock animals (eg horses, cattle. sheep, pigs and donkeys), laboratory test animals (eg mice, rats, rabbits, guinea pigs), companion animals (eg dogs, cats) captive wild animals (eg kangaroos, deer, foxes), poultry birds (eg chickens, ducks, bantams, pheasants) reptiles and fish. Preferably, the subject is a human or a laboratory test animal. In a highly preferred form the subject is a human.

It is contemplated that the present invention is relevant to the treatment of a broad range of aneurysms. Preferably the aneurysm is selected from the group consisting of abdominal aortic aneurysm, thoracic aortic aneurysm and cerebral aneurysm. More preferably, the aneurysm is abdominal aortic aneurysm.

More preferably the compound is administered at a rate of 0.5 to 30 mg per day for a human subject. Extrapolation of the dosages used for the in vivo studies described herein translates to around 0.5mg to 2. 0mg per day for the average human. The compound of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the compound chosen. A broad range of doses may be applicable.

Dosage regimes may be adjusted to provide the optimum therapeutic response.

For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

Preferably the aneurysm is selected from the group including abdominal aortic aneurysm, thoracic aortic aneurysm and cerebral aneurysm. More preferably, the aneurysm is abdominal aortic aneurysm.

The compounds and compositions described herein may be administered by any suitable route including either orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray.

In a preferred form of the invention the compound is administered orally or intravenously.

More preferably the compound is administered at a rate of 5 to 30 mg per day.

Even more preferably the compound is administered at a rate of 20 mg per day.

In yet a further aspect the present invention provides the use of a compound described herein in the preparation of a medicament for the treatment or prevention of aneurysm or neointimal hyperplasia.

Preferably the aneurysm is selected from the group including abdominal aortic aneurysm, thoracic aortic aneurysm and cerebral aneurysm. More preferably, the aneurysm is abdominal aortic aneurysm.

Preferably the medicament is formulated for administration orally or intravenously. More preferably the medicament is formulated to allow administration at a rate of 0.5 to 30 mg per day.

It is further contemplated that a subject may be treated with a combination of any of the compounds described herein, or with any other compound.

The invention further provides the use of a compound described herein in the preparation of a medicament for the treatment or prevention of neointimal hyperplasia.

In another aspect the present invention provides a method of modulating the migration of a vascular smooth muscle cell, said method comprising exposing the cell to an effective amount of a compound or composition as described herein.

Yet a further aspect provides a method of modulating the expression of a matrix metalloproteinase in a vascular smooth muscle cell, said method comprising exposing the cell to an effective amount of a compound or composition as

described herein. Preferably the matrix metalloproteinase is matrix metalloproteinase 2 or matrix metalloproteinase 9.

A further aspect of the present invention provides a method of inhibiting proteolytic activity in a vascular smooth muscle cell, said method comprising exposing the cell to an effective amount of a compound or a composition described herein.

In another aspect, the present invention provides a method of remodelling the wall of a blood vessel, said method comprising exposing the blood vessel wall to an effective amount of a compound or a composition as described herein.

The invention will now be more fully described by reference to the following non-limiting examples.

EXAMPLES EXAMPLE 1: Smooth muscle migration assay.

Rat smooth muscle cells were placed into 3D-culture (collagen, +/-10nM FGF- 9) and place into migration chambers containing a fibronectin coated membrane. The lower chamber contained PDGF-BB as the chemotactic agent.

Migration was then allowed to occur over the next 24 hours. Then SMCs and collagen were removed from the top chamber and SMCs that had migrated completely through the membrane were stained with crystal violet and counted.

Ten microscope fields were counted for each agent/concentration and then averaged (mean +/-SEM). Raw data has been graphed and presented in Fig 1. Overall, it can be seen that MCT-1 inhibits the migration of SMCs from a collagen/growth factor-rich matrix towards a chemotactic factor.

It is known that smooth muscle cells are recruited to the site of an aneurysm. It is therefore proposed that inhibition of smooth muscle cell migration will at least partially inhibit the progression of aneurysm.

EXAMPLE 2: Materials and Methods The materials and methods were utilised in Examples 3 to 9.

Cell culture.

Adherent Cells.

Human vascular smooth muscle cells, Wistar rat aortic vascular smooth muscle cells, and human HT-1080 fibrosarcoma cells were cultured in 90x14mm Nunclon tissue culture dishes containing 5ml of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% (w/v) heat inactivated foetal calf serum, 50units/ml penicillin with 50pg/ml streptomycin and 2mM glutamin.

Cell cultures were grown in a 37°C incubator with 5% (w/v) C02 with 95% (w/v) air mixture.

Prior to stimulation, the medium in each dish was aspirated, the cells washed with PBS, and trypsinised at 37°C, then a haemocytmeter was used to assess cell numbers. 1x 106 cells were seeded onto 60x15mm Nunclon tissue culture dishes containing 3ml DMEM containing serum and incubated at 37°C for approximately 48 hours until confluent.

Freezing cells in liquid nitrogen stocks.

A confluent 90x14mm dish of cells had the media aspirated and washed with 3ml PBS, and incubated with 3ml of warmed trypsin at 37°C for approximately 5 minutes. Cells were collected, placed in 50mi falcon tube with 10ml of fresh DMEM and centrifuged at 2000rpm for five minutes. The media was aspirated from the cell pellet, which was resuspended in 1 ml of freezing solution for each aliquot into cryotubes. Vials were placed on ice for 15 minutes, dry ice for 15 minutes, and finally in liquid nitrogen for long-term storage.

Thawing cells from liquid nitrogen stocks.

Vials transferred from liquid nitrogen stores were warmed in a 37°C water bath for 1 minute, then placed in 10moi of warmed DMEM and centrifuged for 5 minutes at 2000rpm. The supernatant was aspirated and the cell pellet resuspended in 5ml of fresh DMEM, and transferred to a 90x14mm Nunclon tissue culture dish.

Cell treatments.

The media was aspirated from a confluent 60x15mm dish of cells and replaced with 2ml of serum-free, DMEM and 1, u1 of a stock solution of Oxamflatin or Metacept-1 to give final concentrations of 50nM, 500nM and 5pM. The cells were stimulated at 37°C for either 16 hours or 24 hours before harvesting.

Amplification and Maxiprep for DNA extraction.

A 1 00pi aliquot of competent E. coli DH5 cells was thawed from-80°C stock and transformed with the MCHC6-MMP-2 expression vector (see Appendix I).

Immediately thereafter, they were kept on ice for 30 minutes, heat shocked at 42°C for 2 minutes and incubated on ice for 2 minutes. 500µl of LB broth was added to the cells and incubated on a shaker for one hour at 37°C and 225rpm.

The culture was streaked onto a Petri dish containing LB agar supplemented with 50ng/ml Ampicillin and incubated overnight at 37°C.

Single colonies of Ampicillin resistant colonies expressing the transformed MMP-2 plasmid DNA were selected using a toothpick and amplified in 3ml LB broth containing 31ul of Ampicillin overnight on a shaker at 37°C and 225rpm.

The entire 3ml culture was then amplified in a 200moi LB broth containing lOpg/ml Ampicillin overnight at 37°C and 225rpm.

The 200mi culture was centrifuged for 15 minutes at 4°C and 4000rpm. The supernatant was discarded and the bacterial pellet resuspended in 20ml STE buffer, transferred to a 50ml falcon tube and centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was discarded and the pellet resuspended in 9ml of Solution I, then 1ml of lysozyme in 10mM TRIS (pH 8.0). The solution was left at room temperature for 5 minutes then thoroughly mixed with 20ml of freshly prepared Solution II. The solution was incubated at room temperature for 10 minutes, and 15moi of ice-cold Solution III was added, accompanied by vigorous shaking, stored on ice for 10 minutes, then centrifuged for 15 minutes at 4°C and 4000 rpm. The supernatant was filtered through four-layered cheesecloth and cold isopropanol at 0.6 x the volume was added, mixed thoroughly and left at room temperature for one hour. The mixture was centrifuged for fifteen minutes at room temperature and 5000 rpm, the

supernatant discarded, the pellet rinsed with 70% ethanol then dissolved in 3ml of TE buffer (pH 8.0).

Once resuspended, 3ml ice-cold 5M LiC ! was added and the mixture centrifuged for 10 minutes at 10,000 rpm and 4°C to precipitate any RNA. The supernatant was collected, an equal volume of isopropanol was added, and the sample was left at room temperature for 30 minutes, then centrifuged for 10 minutes at 10,000 rpm and 4°C. The supernatant was removed, the pellet rinsed with 70% ethanol, and dissolved in 500pi TE buffer (pH8.0) containing 3ul of 10mg/ml Rnase A, the mixture transferred to an eppendorf tube and left at room temperature for 1 hour. Following this, 500lu1 of 1.6M NaCI with 13% (w/v) polyethylene glycol (PEG 8000) was added and the sample incubated on ice for at least one hour, then centrifuged for 10 minutes at 13,500 rpm at 4°C. The supernatant was removed and again, the pellet was dissolved in 400pI TE (pH 8.0).

The solution was extracted once with equal volume phenol, once with phenol/chloroform and once with chloroform. The upper layer was mixed with 10% of the volume of 3M sodium Acetate (pH5.5) and 2.5x the volume of 100% ethanol.

The DNA was precipitated at-80°C for 1 hour, centrifuged again for 10 minutes at 4°C and 13,500, the supernatant removed and the pellet dissolved in 500 TE buffer (pH 8.0).

The optical density (O. D) of a 1: 100 dilution (in water) of the sample was assessed using a spectrometer at 260nm (1 O. D. unit at 260nm is equal to 50, ug DNA/ml).

Restriction Enzyme Digestion of Plasmid DNA The restriction enzyme digestion reaction consisted of the following : 1. Plasmid DNA of required volume 2. 2pi of 10X BSA 3. 2, of 10X buffer

4. Xhol added to a final concentration of 1 U of restriction enzyme per 1 ug of DNA 5. Remainder of volume consisted of water (usually this reaction volume was 20p1).

The reaction mixture was incubated at 37°C for two hours, 6X DNA loading buffer was added to each sample which were loaded onto a 1% (w/v) agarose gel together with lOpI of lambda standard digested with Hindi ! ! and EcoRI to identify the fragment sizes. The gel was electrophoresed for 2 hours at 100V using 1 X TBE buffer, and the bands visualized under UV. The required DNA fragments were excised out of the gel and purified using QlAquick Gel Extraction Kit according to the manufacturer's instructions.

RNA Extraction.

This was performed following the protocol of Chromczynski and Sacchi (Chromczynski and Sacchi, 1987). Each confluent dish of cells were rinsed with 2ml of PBS, 500pi of Solution D was added, and the mixtures were transferred to separate eppendorf tubes and stored at-20°C until required.

50µl of sodium acetate (pH 4.0) was added to each tube and mixed, then 500pi of water saturated phenol and vortexed, and finally, 200pi of chloroform- isoamylalcohol (25: 1) was added to the solution and vortexed until it was white.

Following this, the samples were centrifuged at 13,200 rpm for 10 minutes and the upper phase transferred to a new eppendorf tube. Equal volume chloroform was added to each sample, vortexed and spun at 13,200 rpm for 5 minutes.

The upper phase was transferred to a new eppendorf tube and equal volume of isopropanol was added in order to precipitate the RNA, mixed and left at-20°C for 30 minutes, then spun for 10 minutes at 13,200 rpm.

The RNA pellet was washed twice in 70% ethanol and resuspended in 12. 5u1 water. 2. 5, u1 of RNA was diluted in 497. 5p1 of water and used to take an O. D measurement via spectrometry at #260 in order to determine the concentration of RNA in each sample (using the formula: OD x dilution (100) x 40 =, ug/ml of RNA.)

Northern Blotting.

1g of agarose was mixed with 70ml milli-Q water and 10ml 10X MOPS and heated until the agarose had dissolved. 1 1 ethidium bromide and 20ml formaldehyde were added in the fume hood and the mixture poured into a prepared gel tray, with a comb 1 cm from the edge of the tray, and left to set.

10p. g of RNA extract was mixed with approximately 20p1 of RNA loading buffer, heated at 65°C for 5 minutes, iced for 5 minutes, and loaded onto the gel, with 1X MOPS as the running buffer. The gel was electrophoresed at 70V and 100mA for 2-3 hours until the dye front had moved approximately 8cm. The gel was de-stained overnight in milli-Q water to remove excess ethidium bromide.

RNA bands were visualised on a UV trans-illuminator to assess loading, and transferred onto a nitrocellulose membrane via capillary action for a minimum of 6 hours.

Following transfer, the membrane was exposed to UV light for 30 seconds to cross-link the RNA, then baked at 80°C for 2 hours to fix RNA to the membrane.

Then the membrane was incubated in 1 owl of 50% formamide pre-hybridization buffer containing 200pg/ml salmon sperm DNA for 2 hours at 42°C.

The MMP-2 probe used in the northern blots was a 2Kb fragment cDNA fragment excised from plasmid DNA, specifically, the pCHC6 vector, using Xho I (Kindly donated by Professor Rik Thompson, St Vincent's Institute of Medical Research, University of Melbourne, Melbourne, Australia) The probe was labelled using the Prime-a-gene@ Labelling system following the manufacturer's instructions. The solution was incubated for one hour at 37°C.

A G-50 sephadex column suspended in STE buffer was pre-washed with STE buffer, then the probe was added. The labelled cDNA probe is larger than the unlabelled probe, so it moves around the G-50 beads more quickly than the unlabelled probe (which must move through the beads) and is eluted sooner from the column. Increments of 3 or 4 drops were collected into approximately

25 eppendorf tubes, and the 3-4 tubes containing the highest counts (as assessed by a Geiger counter) were pooled to form the hybridisation probe.

The probe was strand separated at 100°C for 5 minutes, placed on ice, pulse centrifuged then added directly to the pre-hybridisation buffer of the membrane and incubated overnight at 42°C. The following day, the membrane was rinsed twice with 2 x SSC; twice for 15-20 minutes at 55°C in 2 x SSC/0. 1% (w/v) SDS, and finally washed with 0.1 SSC/0. 1% (w/v) SDS until the background radioactivity was at a satisfactory level (tested crudely via Geiger counter). The membrane was blotted dry, wrapped in plastic covering and exposed to autoradiographic film at-80°C using an intensifying screen.

Western Blot.

25u of media sample was added to 5p1 of loading buffer, heated at 100°C for five minutes and loaded into a dry 10% protein gel. The gel was run at 100V and 100mA for 90 minutes in western running buffer.

Once the gel had run, a piece of immunofilter paper was soaked in methanol for 5 seconds, milli-Q water for 3 minutes, then in western transfer buffer for 5 minutes. The gel contents were transferred to the membrane via electrophoresis at 100V and 100mA for 90 minutes in western transfer buffer.

Following transfer, the membrane was blotted in blot solution for 2 hours at room temperature or overnight at 4°C. The membrane was washed (rinsed twice with 1 x TBS with 0. 1% Tween, followed by a fifteen minute wash (with shaking) and two five minute washes (with shaking) ). The primary antibody was diluted in blot solution (1: 1000 dilution for MMP-2 mAb, 1: 3000 dilution for TIMP-2 mAb), and the membrane was incubated in the diluted antibody overnight at 4°C.

The membrane was washed as previously, then the membrane was incubated (1 ! anti-mouse monoclonal antibody in 5ml blot solution) in diluted secondary antibody for one hour at room temperature. Following a third wash, the membrane was incubated in ECL solution for 60 seconds, blotted dry on paper

towel, wrapped in plastic and placed against exposure film for 30 seconds to 15 minutes.

Western membranes were stained with coomassie blue dye as a loading control. Because all samples were from serum-free media, only one band was clearly present, most likely due to trace amounts of Bovine Serum Albumin.

This gives some indication of protein loading.

Gelatin Zymography.

10p1 of Laemmeli sample buffer was added to 40, of conditioned medium and loaded onto a non-reducing SDS-PAGE gel containing 0.15% (w/v) gelatin. The gel was electrophoresed at 140V in gelatin zymography running buffer for approximately 90 minutes. The stacking gel was removed and the gel washed twice for 30 minutes in 2.5% (v/v) Triton X-100 solution to remove SDS, then placed in Zymography developing buffer and incubated overnight at 37°C.

After incubation, the gel was stained with 0. 1% (w/v) Coomassie Brilliant Blue R-250, then de-stained in 12.5% (v/v) ethanol and 7.5% (v/v) acetic acid until the bands of lysis became clear.

CHEMICOM MMP Gelatinase Assay Kit.

This method was used to directly quantitate total gelatinase activity in conditioned media. Samples were prepared using a 90% confluent dish of hVSMC grown in DMEM containing foetal calf serum, then washed and incubated in serum-free DMEM for 16 hours. A sample of conditioned media (CM) was diluted 1: 5 with serum-free DMEM. Recombinant MMP-2 was diluted to 1: 10,1 : 100,1 : 1000 and 1: 10000 with serum free DMEM, undiluted and diluted CM, and, along with a pure MMP-2 control and a blank control, were tested with the CHEMICOM MMP Gelatinase Assay kit according to the manufacturer's instructions.

Statistical Methods.

Northern Blot, Western Blot and Gelatin Zymography analysis. 5 10 15 20 25 Digitised images of gels and autoradiographic films were taken using the GENE-SNAP computer system software. The corresponding GENE-TOOLS program was used to calculate the area of signal (Northern and Western blots) and proteolysis (gelatin zymography).

Results were analysed using Prism Software by analysis of variance (ANOVA), using the unpaired t-test.

Controls in each experiment were considered 100%, with treated samples calculated as a percentage of the control so as to allow comparison between experiments. Results were considered statistically significant if p<0.05.

MMP Gelatinase Activity Assay Optical density values were obtained using a microplate reader at 450nm. If more than one of each sample had been tested, the average was deemed to be the result. As the readings were inverse to the activity (i. e. the higher the value, the lower the activity), the values were re-calculated as in the following table. 1 2 3 4 Sample Reading 1-Reading (1-Reading)- (1-Blank Reading) Blank 0. 50 0. 50 0 DMEM 0. 25 0. 75 0. 25 CM 0. 40 0. 60 0. 10 Table 1: Representation of calculation of Assay results.

As with the previous studies, the Control sample (i. e. MMP-2 diluted with DMEM) was considered to be 100% and the CM samples were taken as a percentage. These results were analysed by Prism software by analysis of variance (ANOVA), using the unpaired t-test. Statistical significance was given when p<0.05.

EXAMPLE 3: Oxamflatin and Metacept-1 Treatment Modulates MMP-2 mRNA Expression in VSMC

WSR rat vascular smooth muscle cells were treated with 5llM of Oxamflatin, Metacept-1 and Oxamflatin and Metacept-1 combined for 16 hours. Treatment with 5uM of Oxamflatin alone resulted in approximately 45% inhibition of mRNA expression, as determined by Northern blotting (p<0.0005). Blotting also showed that combined treatment of Oxamflatin and Metacept-1 inhibited mRNA expression by approximately 55% (p<0.005). Treatment with Metacept-1 alone did not produce any statistically significant inhibition (Fig. 2).

EXAMPLE 4: Treatment with Oxamflatin and Metacept-1 Modulates MMP-2 Protein Expression in VSMC Effect of Oxamflatin and/or Metacept-1 treatment on MMP-2 protein expression in WSR rat vascular smooth muscle cells.

In WSR rat VSMC, treatments of 5, uM Oxamflatin, 5, uM Metacept-1 and 5uM of both compounds combined each resulted in a decrease in MMP-2 protein expression as shown by western blotting (Fig. 3).

Oxamflatin treatment was the most effective, showing statistically significant inhibition of over 75% of the expression seen in untreated cells (p<0.001). As can also be seen in Fig. 3B, Metacept-1 treatment produced a statistically significant (p<0.05) inhibition that was not as great as that associated with Oxamflatin, while treatment with both drugs resulted in statistically significant inhibition by more than 50% (p>0.001).

Treatment with Metacept-1 (MCT-1) for 24 hours Decreases MMP-2 Protein Expression in Vascular Smooth Muscle Cells (Fig. 5).

The MMP-2 expression of human vascular smooth muscle cells (hVSMCs) is greatly reduced by treatment with MCT-1 for 24 hours, particularly at 5uM concentration, which reduces expression to less than 20% of untreated cells (p<0. 0001).

50nM and 500nM concentrations also reduced MMP-2 expression by approximately 50% and 60% respectively (p<0.001 and p<0.05 respectively).

EXAMPLE 5: Oxamflatin and Metacept-1 Treatment Modulates MMP-2 Mediated Proteolytic activity in VSMC.

Effect of Treatment with Oxamflatin on MMP-2 proteolytic activity alone and in conjunction with Metacept-1 in WSR rat VSMC when treated for 16 hours.

Gelatin zymography results display a significant decrease in MMP-2 mediated proteolytic activity in rat aortic smooth muscle cells following treatment with 5uM Oxamflatin for 16 hours. Treatment reduced activity by approximately 40% and was statistically significant (p<0.05).

Treatment with 5uM Metacept-1 for the same length of time occasionally resulted in inhibition as can been seen in Fig. 6A, however, this result was not statistically significant (see Fig. 6B).

Panel B shows that treatment with 5uM of Oxamflatin and 5uM Metacept-1 together for 16 hours resulted in statistically significant (p<0.05) reduction in MMP-2 proteolytic activity to approximately 65% of the proteolytic activity shown by untreated cells.

The zymogram in Fig. 6A shows a relatively high expression of MMP-2, and little or no MMP-9 expression, a result that was seen in all zymograms performed on rat aortic smooth muscle cells.

Effect of Oxamflatin Treatment for 16 hours on MMP-2 Mediated Proteolytic Activity in Human Vascular Smooth Muscle Cells (hVSMCs).

Zymograph results show a statistically significant decrease in MMP-2 mediated proteolytic activity following treatment with Oxamflatin for 16 hours. 500nM treatment reduced activity by approximately 40%, while 5uM of Oxamflatin decreased activity by 75%. 50nM treatments did not show a significant reduction in activity (see Fig. 7B).

Fig. 7A suggests a small amount of MMP-9 production by hVSMC, due to the band of gelatinolytic activity that corresponds to a protein length of roughly 92kDa, the size of the MMP-9 protein. A band at this position was only occasionally distinguishable on zymograms performed on hVSMC, and the effects of drug treatment could not be determined.

Effect of Treatment with Metacept-1 (MCT-1) for 24 hours on MMP-2 Mediated Proteolytic Activity in Human Vascular Smooth Muscle Cells.

Treatment with Metacept-1 for 24 hours significantly decreased the proteolytic activity of MMP-2 as observed by gelatin zymograms (see Fig. 8). 50nM and 500nM of MCT-1 resulted in inhibition of approximately 30% (p<0.05 and p<0. 001 respectively), while 5uM of MCT-1 induced inhibition of protein activity to around 50% of untreated cells (p<0.001).

EXAMPLE 6: Effect of Conditioned Media (CM) from Human Vascular Smooth Muscle Cells on the Proteolytic Activity of Recombinant MMP-2.

Serum-free DMEM conditioned by human vascular smooth muscle cells for 16 hours was used to dilute recombinant MMP-2 and the activity was assessed using a commercial assay (Chemicon). The human, AMPA-activated MMP-2 was included with the kit.

The conditioned media was tested undiluted and diluted 1: 5 with serum-free DMEM, in addition to serum-free DMEM alone, which acted as a control. Each sample was used to dilute recombinant MMP-2 and the activity tested using the assay kit.

When the recombinant MMP-2 was diluted to 1: 1000, activity was inhibited to approximately 50% of control by both diluted and undiluted conditioned media, although the undiluted media was slightly more effective and had greater statistical significance (p<0.05 for the diluted CM and p<0.001 for the undiluted CM) (see Fig. 9).

EXAMPLE 7: Assessment of efficacy of compounds for the treatment of aneurysm in an animal model.

The following experimental protocol, in an animal model, has been used to evaluate compounds and compositions of the present invention for efficacy in the treatment and prevention of aneurysm.

Experimental animals It has been reported that chronic infusion of angiotensin 11 (Ang II) induces AAA in apoE-deficient mice. The aneurysms are characterized by complex tissue remodeling in the adventitia similar to human disease. In addition, the aneurysmal tissue also showed exacerbated vascular inflammation, such as monocyte/macrophage infiltration in the vascular wall.

Animal preparation and test compounds At approximately 6 months of age, Alzet osmotic mini-pumps (Model 2004) were subcutaneously implanted into the subscapular space of apoE deficient mice. In initial studies, mice received treatment with either: saline (vehicle or placebo group), Ang II alone (at 1. 44mg/kg/day), Ang II co-treated with either 1pM or 511M oxamflatin, or Ang II co-treated with either 1M or 511M MCT-1.

After 28 days, mice were sacrificed and the heart and aorta removed, and connective tissue around the aorta was carefully removed by dissection. The diameter of the abdominal aorta was grossly imaged and calculated using a Moticam-480P Digital Camera (and appropriate Motic Images Plus 2.0 software). The aortic diameter at the location of aneurysm formation was further validated postmortem by direct measurement of cross sections of the suprarenal aorta (See Histopathological Examination). Additional groups of mice will be used to test dose-response relationships of test compounds, as well as the ability of test compounds to regress existing aneurysms.

Noninvasive measurement of systolic blood pressure Systolic blood pressure measurements were taken prior to and at the end of the treatment. On the day of blood pressure determination, mice are placed under a 100 watt lamp for 20 minutes before being placed in a restrainer. Systolic blood pressure is then measured repeatedly using a noninvasive tail cuff device and

recorded on a data acquisition system (PowerLab; ADlnstruments, Australia).

Systolic blood pressure was averaged from five consecutive measurements.

Histopathological Examination Abdominal aortae dissected from all placebo and treatment groups are immersion fixed in Carnoy's fixative (60% alcohol, 30% chloroform, 10% glacial acetic acid). Cross-sections of aorta (2.5 mm in thickness) are made between the superior mesenteric and right renal arteries. A small portion of the right renal artery is left attached to the samples to facilitate orientation of the specimen.

These tissues are dehydrated through a graded ethanol series, cleared with xylene, infiltrated with warm paraffin, embedded in paraffin blocks, cut at 5-um thickness, and stained with hematoxylin and eosin.

Direct measurements of aneurysms from the suprarenal aorta are made after tissue sections have been prepared for histological examination. The lumen and adventitial circumferences at the maximal expended portion of the suprarenal aorta are quantified by image analysis (MCID system) that is then used to calculate the luminal and outer diameters of the vessel. The wall thickness will be calculated from the difference between the luminal and outer diameters.

Quantification of the degree of atherosclerosis exhibited in all treatment groups will be performed on the aortic arch and thoracic aorta. The abdominal aorta will be excluded due to aneurysm formation. The aortic arch and trunk will be cut open for en face staining with Oil Red O. Imaging will be performed using the Moticam-480P Digital Camera and quantitated using appropriate software (Motic Images Plus 2.0). Lesion area will be measured and expressed as a percentage of the total area of the vessel.

The results of the animal trial are present in Fig. 10 and demonstrate efficacy of both Oxamflatin and MCT-1 in decreasing aneurysm incidence.

Panel A of Fig 11 demonstrates the efficacy of oxamflatin and MCT-1 in lowering the incidence of AAA. Control animals demonstrated an incidence of

81 %, with all treated animals showing incidences of 60% or less. It is notable that MCT-1 was able to lower AAA incidence to levels lower than treatment with doxycycline which is the"gold standard"for treatment of AAA in animal models.

Figure 12 shows the efficacy of MCT-1 with reference to wall thickness, and again shows the superiority of this compound to doxycycline and likely decrease in AAA progression.

EXAMPLE 8 : Validation of animal model.

This Example demonstrates that the murine model used to generate experimental data in this application is valid for abdominal aortic aneurysm.

Animals Male B6 Apolipoprotein E-deficient mice with a >99% C57BL/6J background were obtained from Animal Resources Centre (WA) and male C57BL/J6 mice were obtained from Central Animal Services (Monash University). All mice were obtained at 5 weeks of age and weighed between 25-35g at the time of experiments. These animals were housed in standard mouse cages located in the Pharmacology Animal House, Monash University, Clayon, at 215°C with a 12 hour light/dark cycle. Standard food and water was provided ad libitum until 6 weeks of age. After this time they were transferred to a high fat diet containing 0.15% cholesterol and 21% fat (Specialty Feeds, WA) for approximately 6 weeks prior to treatment in young animals and 26 weeks in older animals.

Animals were maintained on the high fat diet throughout the treatment period.

Experimental Groups Treatment groups consisted of: 1. Vehicle control (saline) 2. Angiotensin II (1000ng/kg/min) (note that this group was only carried out in the young ApoE~/~ mice) All drugs were administered via individual osmotic infusion mini-pumps (Alzet, Model 2002) over a 2 week period.

Surgical Procedure Osmotic mini-pumps were prepared prior to the surgery. All drugs were dissolved in saline and using a syringe, injected into the pump. The mice were anaesthetised via isoflurane inhalation. The throat area of the mice was shaved and a small incision (~1cm) was made along this region. The common carotid artery was isolated from surrounding tissue using curved forceps. A 3 mm long piece of polyethylene tubing, with an internal diameter of 0.28mm (Tyco Electronics, USA), was cut longitudinally and placed around the common carotid artery and secured in place by a double knot of 6-0 Dysilk (Dynek Pty Ltd, Australia). The incision was closed with sutures using 4-0 Dysilk. Following this, a small area in the scapular region of the neck was shaved and an incision made between the scapulae, allowing enough room for the osmotic mini-pumps to be inserted. Again, the incision was closed with sutures using 4-0 Dysilk.

Antibiotic powder (Cicatrin, Pfizer) was placed on both incision areas.

Systolic Blood Pressure Measurement Systolic blood pressure of all mice were measured using the non-invasive tail- cuff apparatus (ADlnstruments, Sydney). Prior to taking the systolic blood pressure measurement, the mice were heated under an external light source for 20 minutes. Following this period, the mice were placed in a perspex mouse restrainer, leaving the tail accessible. An inflatable cuff, through which pressure was applied, was placed around the proximal end of the tail with a piezoelectric transducer around the tail distal to the cuff, to detect a pulsing flow signal from the caudal artery. Pressure was measured by a Maclab-8 data acquisition system, via a pressure transducer connected to a Maclab bridge amplifier (ADlnstruments, Sydney). Once a clear pulse was detected, the caudal artery was occluded through inflation of the cuff and thereby abolishing the flow signal.

Pressure was then gradually released until the pulse was redetected. This point was recorded as the systolic blood pressure. This procedure was performed prior to surgery and 2 weeks later at the end of the treatment. Three to five readings were obtained and averaged and expressed as mean standard error of the mean (SEM).

General Dissection Procedure On the day of the study, animals were killed in a glass jar by isoflurane inhalation overdose. The section of the common carotid artery surrounded by the polyethylene cuff, as well as a section of the contralateral common carotid artery, was removed. The heart and whole aorta was then removed and pinned in a glass petri-dish containing a silicone rubber base filled with ice-cold Krebs' solution. This solution had a pH of 7.4 and consisted of (mM): NaCI 118, KCI 4.7, KH2PO4 1.2, MgS04. 7H20 1.2, Caca2 2.5, NaHCO3 25 and glucose 11.7.

Under a binocular dissection light microscope (Olympus SZ40) connective tissue surrounding the aorta was carefully removed.

Vascular Ring Preparation Aortic rings were cut transversely from the abdominal aorta in order to carry out in vitro organ bath studies. Four aortic rings were obtained (-3mm long) from the abdominal aorta. Two stainless steel wires were threaded through the lumen of the aorta with the tissues then being mounted and suspended in vertical 10 mi organ baths containing Krebs'bicarbonate solution at 37°C and bubbled with carbogen (95% 02 and 5% C02). Tension in the aortic rings was set to 0.5g. Isometric tension was measured continuously via a force transducer (Grass FT03) and displayed on a Macintosh using a Maclab data acquisition system (ADlnstruments, Sydney). Tissues were allowed to equilibrate for 90 minutes, with the Krebs'bicarbonate solution being changed every 15 minutes.

Assessment of Neointimal Hyperplasia Haematoxylin and Eosin Staining Carotid artery was dissected and fixed in Carnoy's fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) until used. The tissue was then transferred to 70% ethanol. After 24 hours in 70% ethanol solution, the tissue was put into a biopsy pad encased in a cassette. Tissues in the cassettes were then processed through a series of graded alcohol washes using the automated paraffin processor in the Histology Central Facility (Monash University, Clayon), to dehydrate tissues before being embedded in paraffin blocks. elm cross- sections of the tissues were then obtained using a microtome and placed on a microscope slide. Slides were dried overnight in a 40°C oven. Prior to staining,

slides were heated in a 60°C oven for 20 minutes to melt excess wax. The slides then underwent washing through a series of xylene clearing solutions and dehydrated with alcohol. Tap water was then used to wash the slides for 2 minutes followed by immersion in haematoxylin for 5 minutes. Again, the tissues were washed with tap water for 3 minutes and immersed in Scott's tap water for 1 minute. Slides were then placed in aqueous eosin for 3 minutes and dehydrated with alcohol and placed again in a xylene clearing solution.

Coverslips were then mounted onto the microscope slides with polystyrene (DPX).

Measuring Neointimal Hyperplasia The area of the entire vessel wall and lumen area were measured. This was used to determine a wall : lumen ratio (WLR). WLR was calculated as vessel wall area divided by lumen area.

Analysis of Data Systolic blood pressure (SBP) measurements (mmHg) taken at the end of the 2 week treatment were compared to SBP measured prior to treatment using a one-way analysis of variance (ANOVA) and Bonferroni post hoc test.

Vascular growth was measured using an intima: media ratio (IMR). IMR was obtained by dividing intimal area by medial area. IMRs were analysed using a one-way ANOVA and Bonferroni post hoc test.

Wall : lumen ratios (WLR) were obtained to measure neointimal formation. WLR was obtained by dividing vessel wall area by lumen area. All values are expressed as mean standard error of the mean (SEM). Statistical significance was acknowledged where p<0.05.

FIGS 13 and 14 show neointimal growth in the wall of the aorta in 12 and 30 week APOE knock out mice respectively, whilst FIG 15 shows neointimal formation in the cuffed and uncuffed carotid ateries in 12 week old animals treated with ATI I for 2 weeks. Cuffing the artery induces neointimal hyperplasia.

As can be seen from the data the cuffed artery demonstrates neointimal hyperplasia.

EXAMPLE 9: Assessment of efficacy of compounds for the treatment of aneurysm in humans.

The following experimental protocol is provided to illustrate how the present invention may be tested on humans in a clinical trial. As a prophetic example, the protocol illustrates the evaluation of compounds and compositions of the present invention for efficacy in the treatment and prevention of aneurysm.

Inclusions : Patients aged 18 to 80, having a demonstrable AAA Exclusions: Patients with unstable heart failure.

2. Patients who have had a coronary event of procedure in the previous 4 weeks.

3 Patients having an aneurysm at risk of rupture 3. Patients less than 18 years of age, or more than 80.

4. Patients having IDDM or NIDDM, renal impairment (creatinine >0.22 mmol/L), or hepatic failure.

Where possible, subjects will cease any existing therapy 2 weeks before commencement of the study. After the 2 week"washout"period, all subjects will then be subjected to a chest CT scan to create a baseline measurement.

The test compound will be administered in dosages of 5,15, 20, or 30 mg/d, as follows: Group 1 Group 2 (n=12) (n=12) Vehicle Test Compound Omg/d 5mg/d Omg/d 15mg/d

Omg/d 20mg/d Omg/d 30mg/d Clinic visits will be performed at weeks 2,4, 6 and 12 and an abdominal ultrasound scan performed at each visit. Aortic diameter will be ascertained from the scan. Data collection and statistical analysis will be as described herein.

Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.