GB2455186A - C-peptide for use in treating macrovascular complications of diabetes - Google Patents

Abstract

The use of C-peptide for the prevention and/or treatment of macrovascular complications in diabetic patients are disclosed. The composition of C-peptide may be administered sequentially or simultaneously with insulin. Associated methods of treatment using C-peptide and medicaments containing C-peptide are also claimed. The macrovascular complications can include primary atherosclerotic disease, coronary heart disease, and intimal hyperplasia in vein grafts. C-peptide may exert its effects by preventing the insulin induced proliferation of vascular smooth muscle cells. Use of C-peptide for enhancing endothelial cell proliferation and/or endothelial cell regeneration, and for the treatment of endothelial injury are also claimed.

Description

I

COMPOSITIONS AND METHODS FOR REDUCING MACROVASCULAR

COMPLICATIONS IN DIABETIC PATIENTS

The present Invention relates to compositions and methods for reducing or ameliorating macrovascular complications in diabetic patients, especially macrovascular complications such as atherosclerotic coronary heart disease. The compositions and methods of the present invention are particularly, but not exclusively, for use in controlling or reducing intimal hyperplasia and for reducing the risk of graft failure in diabetic patients undergoing coronary artery bypass surgery.

BACKGROUND

Diabetes mellitus is a major risk factor for cardiovascular disease, with coronary heart disease (CHD) being the leading cause of death among adults with diabetes. Diabetic subjects, as compared to non-diabetic subjects, have a higher frequency of myocardial infarction, a greater occurrence of multi-vessel CHD, increased frequency and severity of complications arising during treatment and a poorer overall prognosis. Although revascularisation, for example by balloon angioplasty, stenting or coronary artery bypass grafting (CABG) may remedy such conditions, revascularisation of the diabetic patient is problematic and the long- term outcomes are disappointing. Diabetic patients are at increased risk of restenosis Irrespective of the mode of revascularisation.

Although diabetis mellitus is a risk factor in association with surgical revascularisation, CABG is considered as the treatment of choice in this group of patients. CABG using the autologous saphenous vein (SV) is routinely used to revascularise atherosclerotic coronary arteries. However, occlusions in SV grafts are common, with only -50% of grafts remaining competent after 10 years. The rate of SV graft failure is higher in diabetic patients, most likely duo to exaggerated intimal hyperplasia (IH) which causes late lumen loss and vessel occlusion. lH is a complex process initiated In the vessel wall as a consequence, in part, of endothelial damage during SV harvesting and grafting.

Endothelial injury exposes the normally quiescent underlying smooth muscle cells (SMC) to a variety of growth factors and cytokines produced by circulating blood cells and the damaged endothelium. These factors stimulate secretion of matrix-degrading proteases (MMPs, particularly MMP-2 and MMP-9) from the SMC, thus permitting intima-directed invasion and proliferation of SMC.

Blood glucose in patients with Type I diabetes or advanced Type 2 diabetes is routinely controlled by administration of insulin. However, insulin itself may play a causative role in the formation of atherosclerotic lesions in diabetic patients, through stimulation of SMC proliferation and migration. In addition, insulin can promote neointima formation following arterial injury. Thus, the pro-proliferative and pro-migratory effects of insulin on SV-SMC may contribute to the pathogenesis of 1K in diabetic patients. These adverse side effects pose a quandary in the treatment of diabetic patients since insulin is essential to control the diabetes condition consequently diabetics must tolerate the undesired side effects of insulin.

Human proinsulin C-peptide is a 31-amino acid peptide that links the A and B chains of proinsulin, ensuring its correct folding. C-peptlde is cleaved from proinsulin during insulin biosynthesis and subsequently released into the circulation with insulin in equimolar concentrations. Indeed, measurement of the plasma concentration of C-peptide is routinely used as a marker of insulin synthesis and secretion. The normal mean plasma concentration of C-peptide is 0.5-1.5 nM. Patients with Type I diabetes have low or absent C-peptido levels due to impaired proinsulin synthesis and secretion. However, in contrast, C-peptide levels are initially elevated in Type 2 diabetic patients, but these patients also eventually become insulin-dependent.

Early studies to address any physiological effects of proinsulin C-peptide concluded that it performed no biological functions. This was supported by the lack of conservation of the C-peptide sequence between species, both in terms of chain length and amino acid composition. However, it is now becoming increasingly apparent that human C-peptlde Is a biologically active peptide hormone that can stimulate specific intracellular processes and modulate cellular function. Current opinion is that C-peptide administration would be beneficial only to Type I diabetic, and not type 2 patients (Waharen et al 2007, Diabetologia, 50, 503-509). For example it has been reported in the prior art that chronic administration of "replacement doses" of C-peptide can ameliorate the microvascular complications of Type 1 diabetes, for example diabetic neuropathy, nephropathy and retinopathy (or animal models of these). Most recent opinions are that C-peptide has beneficial effects on the complications of Type I diabetes on the kidneys, nerves and eyes as evidenced by: Improvement of renal structure and function (clinical studies) * Retardation or reversal of renal structural abnormalities, reduction of hyperfiltration and reduction of urinary albumin excretion (animal models) * Stimulation of nerve NaIK-ATPase activity, increase in endoneurial blood flow, stimulation of neurotrophic factors hence preventing or reversing nerve structural changes.

* C-peptide administration to type I diabetic patients and animal models of type I diabetes results in increases in blood flow in several tissues.

However, whilst the above evidence supports a positive role for C-peptide treatment in microvascular complications of Type I diabetes the effect of C-peptide on macrovascular complications is negative. Li et al 2003, Diabetes Metab. Res. Rev. 19 375-385, reported that C-peptide acted synergistically with insulin, thereby increasing cell proliferation in an SH-SY5Y neural cell line and that C-peptide, in the presence of insulin, had a synergistic effect on insulin cell signalling intermediaries. . Using concentrations of C-peptide comparable with those described hereinafter, they showed that C-peptide exhibited growth promoting effects and enhanced the effect of insulin on cell prolIferation.

Further studies have claimed that C-peptide has a pro-atherogenic effect and that it plays an active role in the development of atherosclerotic lesions by its deposition in the arteries of type 2 diabetic patients and that it also acts as a chemoattractant for 1-lymphocytes to invade the blood vessel wall (early stages of atherosclerosis) (Waicher et al 2004, Diabetes 53, 1664-1670). This group also showed that C-peptide induced migration in vitro (using the same Boyden chamber technique as hereinafter described) and have gone on to show that C-peptide promotes proliferation of arterial smooth muscle cells in culture (Walcher et al 2006, Circ Res, 99, 1181-1187). They further demonstrated that in arterial SMC (from human and rat aorta) C-peptide at concentrations of 0.1, 1 and 10 nM increased 8MG proliferation and that C-pepide activates a number of intracellular signalling pathways linked to proliferation (Src, P1-3 kinase, ERKI/2 and AKT)..

In summary, it is known from the prior art that C-peptide has a beneficial effect on microvascuiar complications of Type I diabetes but is not of benefit to Type 2 sufferers and that C-peptide has a detrimental effect on macrovasculature by increasing cell proliferation and promoting arterial disease by playing an active role in the development of atherosclerotic lesions.

The present invention however is based on the surprising and contrary observations that in human SV-SMC, C-peptide-induced proliferation was not observed. Rather what was observed, was an abolition of insulin-induced proliferation. Moreover, this inhibitory response of C-peptide was found to be specific to the growth-promoting effect of insulin and not other growth factor-induced proliferation. Contrary to the prior art showing a detrimental effect of C-peptide by the potential to promote arterial disease, the present invention proposes a beneficial effect of C-peptide in preventing occlusion in human saphenous vein grafts. In the results presented hereinafter using the Boyden chamber assay comparable to Walcher et al 2004, Diabetes 53, 1664-1670, data are provided which shows that insulin promoted SMC migration and that C-peptide did not and more importantly C-peptide significantly Inhibited the insulin-induced migration. This is in direct

contrast to the prior art observations.

Therefore, a composition and method which could reduce or ameliorate macrovascular complications in diabetic patients such as atherosclerotic coronary heart disease complications would offer immediate advantage to the medical profession and sufferers alike. It is envisaged that the compositions and methods of the present invention are of particularl utility in the control and reduction of insulin induced intimal hyperplasia and may be of particular utility in reducing the risk of graft failure in patients and especially diabetic patients undergoing coronary artery bypass surgery or lower limb vein graft procedures.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the invention there is provided C-peptide for the prevention and/or treatment of macrovascular complications in diabetic patients.

According to a second aspect of the invention there is provided use of proinsulin C-peptide for the manufacture of a medicament for the prevention and for treatment of macrovascular complications in diabetic patients.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Throughout the description the terms "proinsulin C-peptide' and "C-peptide" are interchangeable.

Reference herein to "proinsulin 0-peptide" or "C-peptide' is intended to preferably include a synthetic or naturally derived human peptide having the peptide sequence EADLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 1) or pharmacologically acceptable salts of the peptide or a derivative or biologically active analogue or fragment thereof that retains the biological function of the human proinsulin C-peptide.

A biologically active fragment of human proinsulin C-peptide denotes a fragment according to patent application WO 98/13384. A biologically active analogue of C-peptide denotes a peptide produced by conservative amino acid substitution from human C-peptide, while retaining the biological activity of human C-peptlde. A biologically active derivative of C-peptide denotes a peptide which is obtained from human proinsulin C-peptlde by the modification of a side chain while retaining the biological activity.

Reference to a "macrovascular complication" is intended to include a disease of any large (macro) blood vessel in the body, such as those found in the heart. For example, macrovascular complication include, without limitation, atherosclerosis, coronary heart disease, stroke, and peripheral vascular disease.

Preferably, the macrovascular complication is selected from the group comprising primary atherosclerotic disease, coronary heart disease and intimal hyperplasia in vein grafts.

Preferably, the diabetic patient is insulin dependent and more preferably is a Type 2 sufferer.

Preferably, the Cpeptide further includes, when in formulation, a suitable diluent, excipient or carrier.

In one embodiment of the invention, the C-peptide is administered as a separate agent simultaneously, concurrently or sequentially with insulin, that is to say prior to1 after or in conjunction with insulin.

As used herein, the administraon of C-peptide "in conjunction with" insulin means that the two compounds are administered closely enough in time that the presence of one alters the biological effects of the other. The two compounds may be administered simultaneously (concurrently) or sequentially. Simultaneous administration may be carried out by mixing the compounds prior to administration, or by administering the compounds at the same point in time but at different anatomic sites or using different routes of administration.

The phrases "concurrent administration", "simultaneous administration' or "administered simultaneously1' as used herein, means that the compounds are administered at the same point in time or immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time.

Methods of administering C-peptide are as would be known to one skilled in the art. C-peptide may be administered, for example, parenteraily by injection (intravenously intramuscularly, intraperitoneally, subcutaneously), in nebulised form, dermally or orally.

In an alternative embodiment of the invention, C-peptide may be administered as a combined medicament with insulin. In this aspect of the invention the C-peptide may be formulated along with insulin so as to form a single medicamentation or it may be administered as a single peptide which can be metabolised in vivo to the component parts of C-peptide plus insufln.

Accordingly the invention provides the use of C-peptide either as a separate agent or a combined preparation with insulin for the prevention and/or treatment of rnacrovascular complications in diabetic patients. The basis for the invention resides in the fact that in the course of experimentation, no growth-promoting effects of C-peptide were ever observed either alone or in combination with insulin under any condition, which Is in complete contrast to the prior art observations. Moreover, studies have shown that C-peptide prevents insulin-induced proliferation.

According to a third aspect of the invention there is provided C-peptide for reducing the risk of graft vessel failure and/or graft vessel narrowing and/or vessel restenosis in a diabetic patient undergoing coronary artery bypass grafting surgery or a lower limb vein grafting procedure.

According to a fourth aspect of the invention there is provided use of C-peptide for the manufacture of a medicament for reducing the risk of graft vessel failure and/or graft vessel narrowing and/or vessel stenosis in a diabetic patient undergoing coronary artery bypass grafting surgery or a lower limb vein grafting procedure.

Reference herein to graft narrowing is intended to include any flow impairment in the grafted vessel whether it be a total or partial occlusion.

Preferably, the grafted vessel is a saphenous vein or a similar vein.

Preferably, the third and fourth aspects of the invention further include any one or more of the features of the first and second aspects of the invention.

Coronary heart disease is the leading cause of death in diabetic patients. Although the saphenous vein (SV) is frequently used to bypass diseased coronary arteries, graft failure rates are high, particularly in diabetic individuals. The primary cause of graft failure is intimal hyperplasia (lH)1 a process characterised by smooth muscle cell (SMC) migration and proliferation. In diabetic patients with impaired insulin synthesis and secretion, hyperglycaemia is controlled by insulin administration. However, insulin itself can directly modulate SMC function, thereby augmenting IH. Data shows that proinsulin C-peptide (that is cleaved from proinsulin during insulin's natural biosynthesis) appears to reverse the adverse effects of insulin on human SV-SMC function. This is important because diabetic patients receiving insulin therapy do not receive C-peptide.

According to a fifth aspect of the invention there is provided a method of preventing and/or treating macrovascular complications in a diabetic patient comprising administering a therapeutically effective amount of C-peptide to the diabetic patient.

According to a sixth aspect of the invention there is provided a method of reducing the risk of graft failure and/or graft narrowing andior restenosis in a diabetic patient undergoing coronary artery bypass grafting or a lower limb vein grafting procedure comprising administering a therapeutically effective amount of C-peptide to the diabetic patient.

Preferably, the fifth and sixth aspects of the invention further include any one or more of the features of the first and second aspects of the invention.

According to a seventh aspect of the invention there is provided use of C-peptide for enhancing endothelial cell proliferation and/or endothelial cell regeneration.

According to an eighth aspect of the invention there is provided use of C-peptide for the treatment of endothelial injury.

Preferably, the endothehal injury may be caused by mechanical damage, exposure to radiation, Inflammation, heart disease or cancer.

Preferably, the seventh and eighth aspects of the Invention further include any one or more of the features of the first and second aspects of the invention.

It will be appreciated that feature ascribed to any one particular aspect of the invention apply mutatis mutandis to any other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows histologically the effects of insulin and proinsulin C-peptide on neointima formation of human saphenous vein tissue and neointimal thickness in human organ-cultured SV. Representative histological sections of pre-cultured or 14-day cultured SV segments in 30% FCS alone (control) or supplemented with 100 nM insulin alone or together with human C-peptide (10 nM). Sections are stained with H&E. Scale bar = 30 NI = neointima, I = intima, M = media.

Figure 2 shows the results of image analysis which was performed along the full length of SV sections and wherein the mean neointlma thickness was calculated. Data shown are mean � SEM from 9 different patients. Paired t-test: control vs insulin, **o.oo47; insulin vs insulin + C-peptide, P=0.0032. SV segments were cultured for 14 days in medium containing 30% FCS alone (control) or supplemented with 100 nM insulin alone or together with human C-peptide (10 nM).

Figure 3 shows the effect of insulin and proinsulin C-peptide on human saphenous vein smooth muscle cells (SV-SMC) and internal mammary artery smooth muscle cells (IMA-SMC); Figure 3A shows Increasing cell numbers of SV-SMC against increasing insulin concentration with and without C-peptide; Figure 3B shows cell numbers (note no increase in cell numbers) of IMA-SMC from the same patients as Figure 3A against increasing insulin concentration with and without 0-peptide and; Figure 3C shows cell numbers of SV-SMC against increasing C-peptide concentration with and without insulin.

Figure 4A shows the effect of insulin and proinsulin C-peptide on SV-SMC migration, the migration assay was performed using Boyden chamber technique. Migration of SV-SMC from 6 different patients towards a 100 nM insulin stimulus, in the presence of 0, 1 or 10 nM C-peptide. Data are normailsed to control (no insulin, no C-peptide) for each patient.

Statistical analysis performed using paired ratio f-test (n=6).

Figure 4B shows individual data n = 11, illustrating increased migration of SV-SMC with insulin, attenuated by co-incubation with 0-peptide. Results demonstrate migration of SV-SMC from 11 separate patients towards a 100 nM insulin stimulus, in the presence or absence of 10 nM C-peptide. Paired t-test: control vs insulin, *P=0.0004; insulin vs insulin + C-peptide, ***p_ 0008 Figure 5A shows Western blots of SV-SMC that have been exposed to insulin and/or C-peptide against AKT or phosphorylated AKT.

Figure 58 shows the ratio of phosphorylated-AKT to AKT of SV-SMC that have been exposed to insulin and/or C-peptide.

Figure 6A shows the effect of endothelial cell proliferation in response to the presence of insulin.

Figure 6B shows the effect of endothelial cell proliferation in response to the presence of C-peptide.

DETAILED DESCRIPTION

Tissue Fresh, undistended saphenous vein or internal mammary artery tissue was obtained from non-diabetic patients (those receiving neither insulin nor oral therapy) undergoing coronary artery bypass grafting (CABG).

Savhenous Vein Orqan Culture Organ-cultured human SV is a well-validated in vitro model of SV graft intimal hyperplasia and an established technique. In this model, the neointima that develops in vitro closely resembles the stenotic lesion that develops in vein grafts in viva. Rings of freshly harvested veins are cultured for 14 days in Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 30% foetal calf serum (FCS, optimal growth medium), with medium changed every 2-3 days. Each evaluation is performed on sequential segments from a single vein.

ProliferatIon and A�oi.tosis Studies In order to assess proliferation, vein cultures are supplemented with 5-bromodeoxyundine (BrdU) to label replicating DNA, as described In Porter at al 1996, Eur. J. Endovasc. Surgery 11; 48-58. Specimens are processed and sectioned to 4 j.&ni before immunostalning and measurement of neointimal thickness and determination of proliferation index using computerised image analysis. Further slide-mounted sections immediately adjacent to those used for BrdU are used to assess apoptosis in the same vessels. This is performed using an end-labelling technique (TUNEL) with the ApopTag apoptosis detection kit (Chemicon).

Gelatinase Activity To assess functional activity of gelatinases in organ-cultured veins (ie. the net effect of metalioproteinase (MMP) and tissue inhibitor of MMP (TIMP) activity) frozen tissue is required. In-situ zymography is performed essentially as described previously Goodall et al 2001, Circulation, 104, 304-3-9. Rings are embedded and frozen without fixation, sectioned to 10 tm and applied to silane-coated slides. Slides are then incubated in fluoresceinated gelatin in a humidified atmosphere at 37°C for 18 h, using 4-aminophenylmercuric acetate (APMA)-treated and 1,1 0-phenanthroline-treated sections as positive and negative controls, respectively. Gelatinolysis is visualised under mercury vapour lamp illumination as black zones against fluorescent substrate. The precise location of this activity is determined by comparing serial sections under light illumination.

Insulin Studies Initial data indicates that 100 nM insulin augments neointirna formation in vein segments cultured in 30% FCS and 25 mM (high) glucose. In order to investigate the concentration dependency of this effect, and whether It is modulated by C-peptide, the following protocol is adpoted: (a) 0, 1, 10 and 100 nM insulin (4 segments).

(b) 0, 1 and 10 nM C-peptide (3 segments).

(c) From (a) and (b), a single insulin concentration is selected and the effects of C-peptide investigated (1 and 10 nM) on insulin- induced neointimal thickness (4 segments).

For all the above core experiments neointimal thickness, proliferation and apoptosis is measured. In experiments where greater lengths of vein are available, the effects of low (5.5 mM) glucose (made osmotically equivalent to 25 mM glucose using mannitol) and scrambled C-peptide (Sigma Genosys) on these parameters is also assessed.

Effects of C-Pept/de and Insulin on SV-SMC Function, Proliferation and A�ootosis Smooth muscle cells (8MG) are cultured from explants of SV and IMA specimens from non-diabetic patients, characterised as described previously and maintained in full growth medium (DMEM containing 25 mM glucose and 10% FCS).

Assays were performed to determine the effects of insulin and C-peptide on SV-SMC and IMA-SMC cell number over a 7-day period. SV-SMC were plated into 24-well culture plates at a density of 1x104 cells/well in full growth medium. After 48-hr serum-starvation, cells were incubated with fresh medium containing appropriate supplements and cell number determined in triplicate over a 7-day period using Trypan Blue and a haemocytometer.

SV-SMC proliferation assays are performed in 96-well culture plates by incubating quiescent SV-SMC with 10-100 nM insulin in the absence or presence of 10 nM C-peptide in minimal growth medium (both high and low glucose) for 8-24 h. BrdU labelling reagent are then added to the cells for a further 18-24 hr and incorporated BrdU quantified by ELISA (Amersham Biosciences).

SV-SMC are exposed to insulin with or without C-peptide (or scrambled C-peptide) as above for time periods up to 7 days before analysing apoptosis by two separate methods; measurement of caspase-3 activation and analysis of DNA fragmentation.

Caspase-3 cleavage (activation) is determined by immunoblotting whole cell homogenates with antibodies specific to total and Asp175-cleaved caspase-3 (Cell Signaling Technology, CST). DNA fragmentation analysis is by agarose gel electrophoresis using a commercially available kit (AxxoraTM).

Molecular Mechanisms of Proliferation /Aoootosis To asses whether C-peptide reduces the insulin-induced increase in SV-SMC cell number by reducing cell proliferation or increasing apoptosis the underlying molecular mechanisms of either or both of these effects was investigated.

Cell cycle progression requires a coordinated increase in expression of specific cyclin proteins and reduced expression of Cdk inhibitors. Apoptosis is modulated by the Bcl-2 family of proteins that act by controlling the opening of the mitochondrial permeability transition pore (PTP). Thus, cellular fate is a balance between expression of anti-apoptotlc (Bcl-2, Bcl-xL) and pro-apoptotic (Bad, Bax) members of the Bcl-2 family. Bad Is also modulated by AKT-and ERK-mediated phosphorylation (at Ser-1 36 and Ser- 112), with phosphorylation promoting cell survival.

The effects of insulin and C-peptide on cell cycle and/or apoptosis proteins is studied by using time courses (8-36 h) to examine the profile of expression and phosphorylation of specIfic proteins in response to 10-100 nM insulin in the absence or presence of 10 nM C-peptide. Expression of cell cycle proteins (such as Cyclin A, Cyclin D1..3, Cyclin E, p21 and p27) and apoptosis proteins (such as Bcl-2, Bct-xL, Bad and Bax), as well as phosphorylation of Bad (Ser-122 and Ser-136) will be determined by immunoblotting SV-SMC homogenates with commercially available antibodies (CST).

Effects of Insulin and C-pentide on SV-SMC Migration Initial studies showed that insulin (100 nM) was chemotactic for SV-SMC and increased their rate of migration in a modified Boyden chamber assay. Moreover, 1-10 nM C-peptide prevented the pro-migratory properties of insulin. Thus, in addition to attenuating insulin-Induced SMC proliferation, C-peptide also reduces insulin-induced SMC migration, both of which may contribute to a reduction in lH.

SV-SMC Migration Assays Using the Boyden chamber migration assay (Porter et a) 2002, J Vasc Surg, 36, 150- 157) the dose-dependency of the effect of insulin (10-100 nM) on SV-SMC migration, alone or in combination with other relevant chemotactic stimuli (eg PDGF) is investigated. In addition, the ability of 0-peptide to modulate these effects is also assessed.

Effects of C-Peytjde on Insulin-Induced Signalling in SV-SMC Insulin acting at the insulin receptor (IR) stimulates two major signalling pathways; the Manti-apoptotlc PI3K/AKT pathway and the "pro-proliferativ& ERK pathway. Although the cellular receptor for C-peptide has not been identified, there is increasing evidence that C-peptide can activate an array of intracellular second messengers and signalling pathways. However, it is not known how human C-peptide signals in human SV-SMC.

Initial data showing that C-peptide can prevent the proliferative and migratory effects of insulin suggests that C-peptide is modulating insulin signalling pathways. Using sri IR-specific neutralising antibody it is confirmed that 10 nM insulin stimulated AKT phosphorylation specifically via IR activation. In contrast, the response to 100 riM insulin was not markedly reduced by the anti-IR antibody, suggesting other receptors (e.g. IGF receptor) are Involved. Importantly, our initial data showed that the inhibitory effects of C-peptide on insulin-induced SV-SMC proliferation occurred at both 10 nM and 100 nM insulin concentrations, indicating that C-peptide can modulate lR-specific responses.

Extending these findings, the effects of C-peptide on "classicar IR signalling pathways by Western blotting whole cell homogenates with commercially available expression and phospho-specific antibodies (CST) Will be assessed by phosphorylatiori of AKT and ERK in response to 10 nM insulin over a 5-120 mm time course.

Power Calculations and Statistics Power calculations for organ culture and cell-based assays are based on SD values from previous experiments from our laboratory, and were calculated using GraphPad StatMate (www.graphpad.com). Assuming a correlation coefficient of 0.75 for paired data, a sample size of 7 in each group gives a 90% power of detecting a 20-30% difference in means with a significance level of 0.05 (two-tailed). Hence, all organ culture and cell-based assays will be performed on tissue and cells from 7 different patients.

Data will be analysed using paired ratio t-tests, except organ culture data that will be analysed using a Wilcoxon paired rank test for non-parametric data.

EXAMPLE I

Insulin has a stimulatory effect on neointima formation in human saphenous vein tissue as verified histologically and by quantification see Figure 1 and Figure 2. Insulin increased rieointimal thickness over that of control veins, a response that was attenuated by C-peptide.

Data has also demonstrated that the increase in SV-SMC cell number observed in full growth medium (10% FCS) could be further increased by co-treatment with 100 nM insulin. Interestingly, although human C-peptide (1-10 nM) had no modulatory effect on its own, it prevented the mitogenic effect of insulin in a dose-dependent manner. As FCS is a cocktail of growth factors and cytokines, this phenomenon was further investigated in a more defined culture medium that elicited a minimal growth response. Cells cultured in "minimal growth medium" (0.2% FCS + 10 ng/ml PDGF) underwent a modest level of proliferation (48% increase) over the 7-day period. Again, cell number was significantly increased by insulin in a concentration-dependent manner with a maximum increase of 228% observed with 100 nM insulin. Importantly, 10 nM C-peptide inhibited both the 100 nM and 10 nM insulin-induced increase in proliferation, without affecting proliferation In the absence of insulin.

To examine the effects of insulin and C-peptide on human SV-SMC and IMA-SMC, cells were seeded in parallel (10,000 celIsIweil), quiesced and then exposed to medium containing 0.2% FCS + 10 ngfnil PDGF supplemented with insulin and/or C-peptide. Cell counts were performed after 7 days. Figure 3A shows the results of SV-SMC exposed to 10-100 nM insulin alone (empty bars) or together with 10 nM C-peptide (filled bars).

ANOVA analysis: P<0.0001 for effect of insulin (***P<0.001 post-hoc Newman-Keuls test); P=0.3084 for effect of insulin + C-peptide (n4). Figure 3B shows the results of IMA-SMC from same patients as Figure 3A, exposed to 10-100 nM insulin alone (empty bars) or together with 10 nM C-peptide (filled bars). ANOVA analysis: P0.3743 for effect of insulin; P0.7783 for effect of insulin + C-peptide (n=4). Figure 3C shows the results of SV-SMC exposed to 0.1-10 nM C-peptide alone (empty bars) or together with nM insulin (filled bars). Paired t-test: ep<o.o5; NS, not significant for effect of insulin (n4).

These results show that insulin dose-dependently increases SV-SMC proliferation, and that a single concentration of C-peptide (10 nM) completely negates the effect of insulin.

Conversely, IMA-SMC (from the same patients from which the SV tissue was obtained) shows that insulin does not cause a proliferative response at all in these cells, moreover addItion of C-peptide (10 nM) has no effect. Together this data confirms that the effect of C-peptide is specific to the Insulin response.

From the results (Figure 3C) of SV-SMC and a single concentration of insulin (50 nM) with a concentration range of C-peptide from 0.1 to 10 nM it can also be concluded that insulin induces the usual proliferation In the absence of C-peptide, and C-peptide even at the lowest concentration is able to abrogate the proliferative effect of insulin.

EXAMPLE 2

It has been demonstrated with reference to Figures 4A and 4B that C-peptide (at a concentration of I and 10 nM) and on its own does not have any effect on smooth muscle migration. Insulin alone (100 nM) induces migration that is inhibited completely when C-peptide is included in the medium data is provided from six separate experiments (see Figure 4A) and from 11 different separate patients (Figure 4B).

Turning to Western blot analysis, SV-SMC were exposed to 50 nM insulin andIor 10 nM C-peptide for 15 mm before preparing whole cell homogenates and immunoblotting for phospho-AKT and AKT. Bar chart depicts the ratio of phospho-AKT to AKT (densitometry) from 5 separate experiments. Paired t-test: mPc0.001 **1<0.01 for effect of insulin. NS, not significant for effect of C-peptide. These Western blot analysis of AKT signalling pathway (classical pathway stimulated by insulin) shows no effect of C-peptide either as a stimulus or an inhibitor of insulin-stimulated AKT activation (Figures 5 A and 5B). Therefore the effect of C-peptide on the cell functions under investigation are not at this level and must be further "downstream" of receptor stimulation.

EXAMPLE 3

The effect of insulin and proinsulin C-peptide on human saphenous vein endothelial cell proliferation was investigated. Figure 6A shows SV-EC proliferation where cells were seeded in parallel (10,000 ceilsIwell), quiesced and then exposed to medium containing 10% FCS supplemented with 0-100 nM insulin. Cell counts were performed after 5 days in the presence of insulin and/or C-peptide. Cell counts were performed after 5 days.

ANOVA analysis: P= 0.077 for effect of insulin (n=10). Figure 6B shows the results of SV-EC proliferation where cells were exposed to 10 nM C-peptide alone or in combination with 1-100 nM insulin for 5 days before counting cells. Paired t-test: *O.o5 for the effect of C-peptide; ANOVA analysis P0.287 for the effect of C-peptide in the presence of insulin (n=10).

These data provided shows endothellal cell proliferation In response to Insulin and/or C-peptide and surprisingly the results indicate that insulin (1-100 nM) did not influence EC proliferation. On the other hand, C-peptide (10 nM) increased EC proliferation and this has been shown not to be modulated by the presence of insulin. This is belived to represent a new clinical use for C-peptide as it assists the endothelium to regenerate and insulin does not affect this property. Accordingly, C-pepiide may be used therapeutically for assisting endothelial regeneration and/or proliferation and also for the treatment of endothelial injury and diseases associated with endothelial injury.

Claims (20)

1. Use of C-peptide for the prevention and/or treatment of macrovascular complications in diabetic patients.

2. C-peptide according to claim 1 wherein the C-peptide is a synthetic or naturally derived human peptide having the peptide sequence EADLQVGQVELGGGP GAGSLQPLALEGSLQ (SEQ ID NO:1) or a pharmacologically acceptable salts of the peptide or a derivative or biologically active analogue or fragment thereof that retains the biological function of human proinsulin C-peptide.

3. C-peptide according to either claim I or 2 wherein the macrovascular complication is selected from the group comprising primary atherosclerotic disease, coronary heart disease and intimal hyperplasia in vein grafts.

4. C-peptide according to any preceding claim wherein the diabetic patient is Insulin dependent.

5. C-peptide according to claim 4 wherein the diabetic patient is a Type 2 sufferer.

6. C-peptide according to any preceding claim further including, when in formulation, a suitable diluerit, excipient or carrier.

7. C-peptide according to any preceding claim that is administered as a separate agent sequentially or simultaneously with insulin.

8. C-peptide according to claim 7 wherein the C-peptide is administered prior to, after, in conjunction or mixed with insulin.

9. C-peptide according to any one of claims I to 8 wherein C-peptide is provided as a combined preparation with insulin.

10. C-peptide according to claim 9 wherein the combined preparation comprises a peptide.

11. Use of proinsulin C-peptide for the manufacture of a medicament for the prevention and/or treatment of macrovascular comphcations in diabetic patients.

12. Use according to claim 11 further including any one or more of the features of claims 2 to 10.

13. C-peptide for reducing the risk of graft vessel failure and/or graft vessel narrowing and/or vessel restenosis in a diabetic patient undergoing coronary artery bypass grafting surgery or a lower limb vein grafting procedure.

14. C-peptide according to claim 13 wherein the grafted vessel is a saphenous vein.

15. C-peptide according to either claim 13 or 14 further including any one or more of the features of claims 2 to 10.

16. Use of C-peptide for the manufacture of a medicament for reducing the risk of graft vessel failure and/or graft vessel narrowing and/or vessel restenosis in a diabetic patient undergoing coronary artery bypass grafting surgery or a lower limb vein grafting procedure.

17. Use according to claim 16 wherein the grafted vessel is a saphenous vein.

18. Use according to either claim 16 or 17 further including any one or more of the features of claims 2 to 10.

19. Use of C-peptide for enhancing endotheiial cell proliferation and/or endothelial cell regeneration.

20. Use according to any one of claims 19 to 21 further including the features of either claim 2 or 6.

20. Use of C-peptide for the treatment of endothelial injury.

21. Use according to claim 20 wherein the endothelial injury is as a result of mechanical damage, exposure to radiation, inflammation, heart disease or cancer.