WO2005118638A1 - Degradation-resistant analogues of pro-insulin c-peptide - Google Patents

Abstract

The present invention provides a C-peptide analogue wherein the C-peptide is modified at one or more leucine and/or one or more valine residues, and wherein the analogue has an increased resistance to proteolytic degradation as compared to un-modified C-peptide. Such analogues may be used in C-peptide therapy, notably in the therapy of diabetes and/or diabetic complications.

Description

Degradation-resistant analogues of pro-insulin C-Peptide

The present invention relates to analogues of proinsulin C-peptide with increased resistance to proteolytic degradation particularly by endopeptidases, and their use in the treatment of diabetes and/or diabetic complications. Insulin-dependent diabetes mellitus (IDDM) , generally synonymous with type 1 diabetes, is the classical, life-threatening form of diabetes, the treatment of which was revolutionized by the discovery of insulin in 1922. The prevalence of IDDM is unfortunately widespread throughout much of the world and hence IDDM represents a serious condition with a significant drain on health resources. The etiology of IDDM is multifactorial and not yet entirely clear. However it is characterised by a partial or complete destruction of the pancreatic beta cells. In the acute phase of IDDM insulin deficiency is thus the dominating pathophysiological feature. After starting insulin treatment many patients enjoy good blood glucose control with only small doses of insulin. There is an early phase, the "honeymoon period" , which may last a few months to a year and which probably reflects a partial recovery of beta cell function. This is, however, a temporary stage and ultimately, the progressive destruction of the beta cells leads to complete cessation of insulin secretion and increasing requirements for exogenous insulin. While the short term effects of hypoinsulinemia in the acute phase of IDDM can be well controlled by insulin administration, the long term natural history of IDDM is darkened by the appearance in many patients of potentially serious complications known as late, or late onset complications . These include the specifically diabetic problems of nephropathy, retinopathy and neuropathy. These conditions are often referred to as icrovascular complications even though microvascular alterations are not the only cause. Atherosclerotic disease of the large arteries, particularly the coronary arteries and the arteries of the lower extremities, may also occur. Nephropathy develops in approximately 35% of IDDM patients, particularly in male patients and in those with onset of the disease before the age of 15 years . Diabetic nephropathy is characterized by persistent albuminuria secondary to glo erular capillary damage, a progressive reduction of the glomerular filtration rate and eventually, end stage renal failure. The prevalence of diabetic retinopathy is highest among young-onset IDDM patients and it increases with the duration of the disease. Proliferative retinopathy is generally present in about 25% of the patients after 15 years duration and in over 50% after 20 years. The earliest lesion of diabetic retinopathy is a thickening of the capillary basement membrane, followed by capillary dilation and leakage and formation of microaneurysms . Subsequently, occlusion of retinal vessels occurs resulting in hypoperfusion of parts of the retina, oedema, bleeding and formation of new vessels as well as progressive loss of vision. Diabetic neuropathy includes a wide variety of disturbances of somatic and autonomic nervous function. Sensory neuropathy may cause progressive loss of sensation or, alternatively, result in unpleasant sensations, often pain, in the legs or feet. Motor neuropathy is usually accompanied by muscle wasting and weakness. Nerve biopsies generally show axonal degeneration, demyelination and abnormalities of the vasa nervorum. Neurophysiological studies indicate reduced motor and sensory nerve conduction velocities . Autonomic neuropathy afflicts some 40% of the patients with IDDM of more than 15 years duration. It may evolve through defects in ther oregulation, impotence and bladder dysfunction followed by cardiovascular reflex abnormalities . Late manifestations may include generalized sweating disorders, postural hypotension, gastrointestinal problems and reduced awareness of hypoglycemia . The latter symptom has grave clinical implications . A number of theories have been advanced with regard to possible mechanism(s) involved in the pathogenesis of the different diabetic complications but this has not yet been fully elucidated. Metabolic factors may be of importance and it has been shown that good metabolic control is accompanied by significantly reduced incidence of complications of all types. Nevertheless, after 7-10 years of good metabolic control, as many as 15-25% of the patients show signs of beginning nephropathy, 10-25% have symptoms of retinopathy and 15- 20% show delayed nerve conduction velocity indicating neuropathy. With longer duration of the disease the incidence of complications increases further. There is thus a significant clinical need for the control and management of these diabetic complications. Proinsulin C-peptide is a part of the proinsulin molecule which, in turn, is a precursor to insulin formed in the beta cells of the pancreas. For a long time it was believed that C-peptide (known variously as C-peptide or proinsulin C-peptide) had no role other than as a structural component of proinsulin, facilitating correct folding of the insulin part. However, it has in more recent years been recognised that C-peptide has a physiological role as a hormone in its own right (Wahren et al . , (2000), Am. J. Physiol. Endocrinol. Metab, 278, E759-E768) . In diabetic patients, it alleviates renal dysfunction, improves blood flow in several tissues, ameliorates nerve functional impairments and is believed to delay or prevent the onset of late complications (Wahren et al., (2000) supra; Wahren and Johansson (1998) , Hor . Metab. Res. 30, A2-A5) . Indeed, C-peptide has been proposed for use in the treatment of diabetes in EP 132769 and in SE460334 for use in combination with insulin in the treatment of diabetes and prevention of diabetic complications . Proinsulin, or large parts of it, is known in 37 different variants, representing 33 different species, ranging from Atlantic hagfish, Myxine glutinosa, to human. Whilst the insulin segments (i.e. the A and B chains of proinsulin) are well conserved between species, C-peptide is much more highly variable, showing not only sequence variation, but also several internal deletions, making the length of C-peptide variable (see Figure 1) . Human C-peptide is a 31 amino acid peptide having the following sequence: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID. NO. 1) . C-peptide can be ascribed a tripartite overall structure, with more conserved N- and C-terminal segments and a more variable mid-sequence, or internal, portion. Thus, in the case of human C-peptide the N- terminal segment can be regarded as residues 1-12, the mid-portion as residues 13-25, and the C-terminal segment as residues 26-31. The C-terminal pentapeptide fragment of C-peptide has been shown to have similar physiological and molecular effects to C-peptide itself, suggesting that this segment is an essential part of C-peptide (Wahren et al . , 2000, supra; Rigler et al . , 1999, PNAS USA 96, 13318-13323; Ohtomo et al., 1998, Diabetologia 41, 287- 291; Pra anik et al., 2001, BBRC 284, 94-98; Shafqat et al , 2002, Cell Mol. Life Sci., 59, 1185-1189). WO 98/13384 proposes the use of this C-terminal pentapeptide, and other C-terminally located peptide fragments of C-peptide in the treatment of diabetes and diabetic complications. The mid-sequence portion also has been shown to have molecular and physiological effects (see e.g. Ido et al . (1997, Science 277, 563- 566) and Ohto o et al . , (1998), supra) and has also been proposed in WO 98/13384 to have clinical utility. Ido et al . , have speculated that the mid-portion may mediate its effects through membrane interactions, although this is still to be confirmed and is not supported by other studies. The N-terminal segment of C-peptide although not active on its own, has also recently been shown to be functionally important and to contribute to C-peptide activity. Accordingly, various N-terminal fragments and derivatives of C-peptide, including in particular variants of C-peptide modified in the N-terminal region, have also been proposed in WO 2004/016647 to have therapeutic utility. C-peptide thus appears to be a somewhat "complex" molecule, which may exert its effects by a variety of different mechanisms, including possibly via interaction with more than one receptor and/or more than one signalling pathway. Direct membranotropic mechanisms may also be involved although, as mentioned above, this is yet to be conclusively established. Thus, not only C-peptide itself (i.e. an intact native or wild-type C- peptide) but also various C-peptide fragments and modified variants or analogues thereof have therapeutic potential in the treatment and/or prevention of diabetes and/or diabetic complications. The use or potential use of all such C-peptides, and fragments and derivatives thereof and is referred to herein as "C-peptide therapy" . C-peptide is known however to have a relatively short half-life in the body (specifically in the circulation or plasma) . The major degradation and removal of C-peptide takes place in the kidneys (Faber et al . , Diabetes, 27, 207-209, 1978; Zavaroni et al . , J. Clin. Endocrinol. Metab., 65, 494-498, 1987), although little is known in the prior art concerning the proteolytic enzymes involved and the mode of degradation. In one study, tests with a neutral metallo-endopeptidase purified from rat kidney revealed that the enzyme can cleave proinsulin at peptide bonds residing in or near the C-peptide moiety of the parent molecule. Incubations of isolated bovine C-peptide with the enzyme followed a similar time-course of digestion to that shown by porcine proinsulin. The pattern of digestion of the isolated C-peptide molecule was not, however, studied. (Varandoni et al . , Biochim. Biophys.

Acta, 661, 182-190, 1981) . In another study, N- terminally ^12SI-tyrosylated human C-peptide was introduced into the maternal circulation of a pregnant Rhesus monkey and it was shown to be degraded in the uterus during transfer to the fetus (Gruppuso et al., J.

Clin. Invest. 80, 1132-1137, 1987). Studies have shown that both C-peptide and its C- terminal pentapeptide are degraded in serum, with a longer half-life for intact C-peptide than for the C- terminal pentapeptide. Preliminary evidence suggests that the two peptides may be degraded in different ways in the serum. (Melles et al., Cell. Mol. Life Sci., 60, 1019-1025, 2003) . The in vivo half life of C-peptide circulating in blood has been reported as approximately 30 minutes (as compared to 4-5 minutes for insulin) , and that of the C-terminal pentapeptide is believed to be even shorter . Studies have shown that a dose of C- peptide injected into a rat would be expected to have disappeared entirely from circulation within 2-3 hours. Thus, in studying C-peptide physiological activity or in C-peptide therapy, it has been customary to administer C-peptide in several daily doses or to use a continuous dose. Similarly insulin, which is derived from the same prohor one (proinsulin) as C-peptide requires administration 3-5 times daily. Hence, a treatment which has longer lasting action and can be administered to a patient less often would be of great clinical importance and utility. The present invention is directed towards this aim, and in particular, towards providing modified variants or analogues of C-peptide which have been modified to increase the in vivo (circulating) half-life of C- peptide so as to provide a longer-lasting and more efficaceous therapeutic agent . More particularly, the present inventors have found that the C-peptide molecule may be degraded by protease, particular endoprotease, action at a number of particular sites. More specifically, it has been found that the enzymes which degrade C-peptide in vivo cleave the molecule at sites positioned N-terminally of a leucine or valine residue. By modifying these sites to protect against protease action the C-peptide molecule may be protected against degradation and its biological half-life may thereby be extended. This finding offers significant practical advantages to doctor and patient alike in the treatment of diabetes and diabetic complications . Thus, in one aspect the present invention provides a C-peptide analogue wherein the C-peptide is modified at one or more leucine and/or one or more valine residues, and wherein the analogue has an increased resistance to proteolytic degradation as compared to unmodified C-peptide. The analogues are preferably more resistant to endopeptidase degradation, particularly an amino acid specific endopeptidase which acts at leucine residues and especially endopeptidase (s) which acts at Leu and/or Val residues. However, the action of non-specific proteases including endopeptidases, is not excluded (for example a non-specific protease or endopeptidase capable of cleaving at Val residues) . The analogue may, thus, have increased resistance to cleavage by one or more proteases, particularly endopeptidases, and particularly to a leucine-endopeptidase and a valine-endopeptidase, or to a leucine-endopeptidase, or to a valine- endopeptidase, or to a leucine-endopeptidase in combination with one or more other protease or endopeptidase enzymes capable of cleaving at a Val residue. More preferably, the analogues are resistant to leucine specific endopeptidases. In particular, the C-peptide analogues of the present invention are modified so as to (or comprise modification (s) which) provide increased resistance to degradation (or protect against cleavage) by an endopeptidase at a site N-terminally of a Leu and/or Val residue. The C-peptide analogues of the invention thus comprise one or more modifications (as compared to the C-peptide prior to the modification, or to a native or wild-type C-peptide sequence) which protect the analogue against endopeptidase cleavage N-terminally of a Leu and/or Val residue. Alternatively viewed, the modification (s) protect (s) at least one Leu and/or Valine residue from endopeptidase cleavage N-terminally thereof . The C-peptide analogues of the present invention may thus be obtained, or prepared, by modifying a C- peptide, or more particularly, by introducing (into a C- peptide) one or more of the "protecting" modifications. The C-peptide which may be "modified" in this way to form the analogues of the invention may be any C- peptide. Thus, the term "C-peptide" as used herein includes all forms of C-peptide (also known as proinsulin C-peptide) , including native or synthetic peptides. Such C-peptides may be the human peptide, or may be from other animal species and genera, preferably mammals. Thus, variants of human C-peptide are included, which may be native variants, or synthetically or artificially derived. C-peptides from a number of different species have been sequenced and are known in the art. It would thus be a routine matter to select a variant being a C-peptide from a species or genus other than human. Several such variants of C-peptide (i.e. representative C-peptides from other species) are shown in Figure 1 (see SEQ ID NOS. 1 and 54-76) . Thus variants and modifications of native human C-peptide are included as long as they retain C-peptide activity. The C-peptides may be in their native form, i.e. as different variants as they appear in nature in different species or due to geographical variation etc., or may be functionally equivalent variants or derivatives thereof, which may differ in their amino acid sequence, for example by truncation (e.g. from the N- or C-terminus or both) or other amino acid deletions, additions, insertions or substitutions . It is known in the art to modify the sequences of proteins or peptides, whilst retaining their useful activity and this may be achieved using techniques which are standard in the art and widely described in the literature e.g. random or site- directed mutagenesis, cleavage and ligation of nucleic acids etc. Thus the C-peptide analogues of the invention may comprise or contain other modifications as compared to a native C-peptide sequence, other than the specific modifications made to protect one or more leucine residues and/or valine residues from degradation. Any such modifications, or combinations thereof, may be made, as long as activity is retained. The C- terminal end of the molecule is believed to be important for activity. Preferably, therefore, the C-terminal end of the C-peptide should be preserved in any such C- peptide variants, more preferably the terminal pentapeptide of C-peptide should be preserved. Modifications to the mid-part of the C-peptide sequence (e.g. to residues 13 to 25 of human C-peptide) allow the production of functional variants of C-peptide and are hence covered. Thus C-peptides which may be modified to form the analogues of the invention (or on which the analogues of the invention are based, or from which they are derived) may have amino acid sequences which are substantially homologous, or substantially similar to the native C- peptide amino acid sequences, for example to the human C-peptide sequence of SEQ ID NO. 1 or any of the other native C-peptide sequences shown in Fig. 1. Such substantially homologous sequences may include those having at least 30% (or more preferably at least 40, 50, 60, 70, 75, 80, 85, 90, 95, 98 or 99%) similarity to any one of SEQ ID Nos. 1 or 54 to 76 as shown in Fig. 1, preferably to the native human sequence of SEQ ID No. 1. Alternatively, the C-peptide may have an amino acid sequence having at least 30% (or more preferably at least 40, 50, 60, 70, 75, 80, 85, 90, 95, 98 or 99%) identity with the amino acid sequence of any one of SEQ ID Nos. 1 or 54 to 76 as shown in Fig. 1, preferably with the native human sequence of SEQ ID No. 1. Although any amino acid of C-peptide may be altered as described above, it is preferred that one or more of the glutamic acid residues at positions 3, 11 and 27 of human C-peptide (SEQ ID NO. 1) or corresponding or equivalent positions in C-peptide of other species, are conserved. Preferably all of the glutamic acid residues at positions 3, 11 and 27 (or corresponding Glu residues) of SEQ ID NO. 1 are conserved. Amino acid sequence identity or similarity may be determined using the BestFit program of the Genetics Computer Group (GCG) Version 10 Software package from the University of Wisconsin. The program uses the local ho ology algorithm of Smith and Waterman with the default values: Gap creation penalty = 8, Gap extension penalty = 2, Average match = 2.912, Average mismatch = 2.003. Thus, functionally equivalent variants of native C-peptide sequences may readily be prepared according to techniques well known in the art, and include peptide sequences having a functional, e.g. a biological, activity of a native C-peptide. Thus a variant of a naturally occurring wild-type or native C-peptide sequence may, for example, differ by 1 to 10, more preferably 1 to 6, or 1 to 4, or 1 to 3 amino acid substitutions, insertions and/or deletions which may be contiguous or non-contiguous as compared to the native or wild-type sequence (e.g. as compared to the sequence of any one of SEQ ID Nos. 1 or 54 to 76, preferably SEQ ID No. 1) . Representative such variants may include those having 1 to 6, or more preferably 1 to 4, 1 to 3 or 1 or 2 amino acid substitutions as compared to SEQ ID No. 1. The substituted amino acid, particularly one of the well known 20 conventional amino acids (Ala (A) ; Cys(C); Asp(D); Glu(E); Phe(F); Gly(G); His (H) ; Ile(I); Lys(K); Leu(L); Met (M) ; Asn(N); Pro(P); Gln(Q); Arg(R); Ser(S); Thr(T); Val (V) ; Trp (W) ; and Tyr (Y) ) . Conservative amino acid substitutions are preferred. Thus, an amino acid may be replaced by another which preserves the physicochemical character of the peptide (e.g. D may be replaced by E or vice versa, N by Q, or L; I by V or vice versa) . Generally, the substituting amino acid has similar properties e.g. hydrophobicity, hydrophilicity, electronegativity, bulky side chains etc. to the amino acid being replaced. Iso ers of the 'native' L-amino acid, e.g. D-amino acids may be incorporated. Additional alterations may include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acids. Longer peptides may comprise multiple copies of one or more of the peptide sequences. C and N-terminal protecting groups may be included. As above, a proviso of such variants is that they retain C-peptide activity. Fragments of native or synthetic C-peptide sequences may also have the desirable functional properties of the peptide from which they derive and are hence also included. Mention may be made in particular of the C- peptide fragments described by Wahren et al., in

W098/13384. Chemical modification of the peptide structure is not precluded, e.g. by glycosylation, as long as the structure of the variant remains essentially peptide in nature. All such variants, derivatives or fragments of C-peptide are included, and are subsumed under the term "C-peptide". As mentioned above, the C-peptide analogues of the present invention have increased resistance to proteolytic degradation as compared to the unmodified C- peptide. Such an "unmodified" C-peptide will generally be the C-peptide prior to the modification (at one or more Leu and/or Val residues) . Thus, the unmodified C- peptide may correspond to the C-peptide analogue absent the modification (s) at the Leu and/or Val residues. In other words, the unmodified C-peptide may be the C- peptide from which the analogue of the invention is derived (or on which it is based) . The unmodified C- peptide may be a wild-type or native peptide, particularly the intact native human C-peptide of SEQ ID. No. 1. Preferably, the C-peptide analogue has an increased biological half-life compared to unmodified C-peptide, . This may include, for example, an increased biological half-life as compared to a native or wild-type C- peptide, particularly human C-peptide. The term "biological half-life" refers to the half-life in vivo, e.g. following administration of the analogue (or C- peptide) to an individual (which may be any human or non-human, preferably mammalian, animal, e.g. a patient) . Thus, the half-life or retention time in the circulation may be increased. Such biological half-life or retention time may be determined using methods known and described in the art . As stated above, the biological half-life or retention time of a C-peptide analogue of the invention can be determined by using unmodi ied C-peptide as a reference point. Hence the half-life or retention time of a C- peptide analogue is determined by comparison to that of unmodified C-peptide. The biological half-life may be at least 45 minutes, for example at least 50 minutes and more preferably at least 55, 60 or 65 minutes. Preferably however, the biological half-life is increased to at least 70 minutes, preferably at least 75 minutes, and more preferably at least 80, 85, 90, 95 or 100 minutes. More preferably the half-life is increased up to 150, 300, 450, 600 or 750 minutes. Alternatively viewed, the biological half-life is increased by at least 15%, preferably by at least 20, 25, 30, 35, 40, 45 or 50%. More preferably, the half- life is increased by up to 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000%. Alternatively viewed, the biological half-life is increased 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 fold. Most preferably the increase in biological half-life is up to 5 fold or up to 10 fold. The improved or increased biological half-life is a manifestation of the increased resistance to proteolytic degradation afforded by the modifications made at the Leu and/or Val residues. Thus, C-peptide analogues of the invention have an improved stability to proteolytic degradation. This increased resistance or stability may also be manifested, and hence tested for, or assayed, in other ways. For example, the analogues may exhibit increased stability, and hence half-life, or resistance to proteolytic degradation, in an in vitro system. Hence, the C-peptide analogues of the invention may have increased resistance to degradation in serum in vitro, and may accordingly have an increased half-life in serum in vitro, particularly human serum, i.e. show a greater stability upon incubation with serum. Thus, for example, the degradation of a C-peptide analogue in serum may be monitored, e.g. by examining the degradation products, or conversely stability may be assessed by monitoring the content of intact analogue, for example at particular time points or over a time course. An in vitro serum degradation assay for C- peptide is described in Melles et al . , 2003, supra. The in vitro half-life of human C-peptide incubated in human serum at 37^DC is 174 hours when diluted serum (at 1:1 with water) is used and when following exposure of the C-peptide to the serum, the proteins are precipitated, for example by adding 70% acetone or by using zinc sulphate or ultracentrifugation. The C-peptide analogues of the invention may exhibit a half-life in human serum at 37°C of at least 175 hours, preferably at least 176, 178, 180, 190, 200, 205, 210, 215, 220, 230, 240, 245 or 250 hours. More preferably, the half-life of the C-peptide analogues in human serum may be increased up to 700, 875, 1050, 1225, 1400, 1575 or 1750 hours (i.e. up to 4, 5, 6, 7, 8, 9 or 10 fold) . Alternatively viewed, the in vitro half-life in human serum at 37°C is preferably increased by at least 5%, preferably by at least 10, 15, 20, 25, 30, 35, 40, 45 or 50%. More preferably, the half-life is incresed up to 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000%. The increased resistance to proteolytic degradation may be assessed by such methods, for example by a serum- incubation assay as described above, (e.g. in Melles 2003, supra) . Alternatively, resistance to proteolytic degradation may be assessed by incubating the analogue with (or exposing the analogue to) a proteolytic preparation or composition, e.g. a proteolytic enzyme preparation which may be a purified or semi-purified enzyme preparation (e.g. a preparation of one or more endopeptidase enzymes) or a tissue preparation (e.g. homogenate) , for example from a tissue known to contain proteolytic enzyme or to degrade C-peptide, e.g. a kidney preparation or placental preparation. Thus, for example, a preparation of rat or mouse kidney homogenate, or of human placental homogenate may be used (e.g. as described in Example 1 below) . The analogue or reference C-peptide (i.e. "unmodified" C-peptide) may be incubated with the enzyme or tissue preparation under appropriate conditions (which may be routinely determined) and the degradation may be monitored or assessed, e.g. by examining or detecting the appearance or content the degradation product or of the intact C- peptide or analogue at one or more appropriate time points, or over a time course etc. This may be done by any convenient or desired method, for example, chromatographically e.g. by HPLC or by mass spectrometry. The degradation of the C-peptide analogue and reference (i.e. unmodified) C-peptide may be compared to determine whether the analogue has increased resistance to degradation. Preferably the analogue has at least 10% greater resistance to degradation than the unmodified C-peptide, more preferably at least 10, 15, 20, 25, 30, 35, 40, 45, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400 or 500% increased resistance to degradation. It is of course important that the C-peptide analogue of the invention retains C-peptide activity (and hence the therapeutic utility of C-peptide) . Preferably the C-peptide analogue retains at least 20% of C-peptide activity. Such C-peptide activity may be assessed relative to the unmodified C-peptide. Preferably, the analogue exhibits at least 20% of C- peptide activity as compared to a native C-peptide, particularly human C-peptide. More preferably, the analogue exhibits at least 25, 30, 35, 40, 50, 55 or 60%, still more preferably at least 70, 75, 80, 85, or 90% of the activity of C- peptide, particularly human C-peptide. Thus the analogues are capable of functioning as a C-peptide, e.g. can substitute for human C-peptide in C-peptide therapy. The term "C-peptide activity" as used herein means any activity exhibited by a native C-peptide, whether a physiological response exhibited in an in vivo or in vitro test system, or any biological activity or reaction mediated by a native C-peptide, for example in an enzyme assay or in binding to test tissues or membranes . Thus, it is known that C-peptide increases the intracellular concentration of calcium. An assay for C- peptide activity can thus be by assaying for changes in intracellular calcium concentrations upon addition or administration of the peptide (e.g. C-peptide analogue) in question. Such an assay is described in for example in Ohtomo et al . , (1996), Diabetologia 39, 199-205, Kunt et al . , Diabetes 47, A30; Shafqat et al (supra). Further, C-peptide has been found to induce phosphorylation of the MAP-kinases ERK 1 and 2 of a mouse embryonic fibroblast cell line (Swiss 3T3) , and measurement of such phosphorylation and MAPK activation may be used to assess, or assay for C-peptide activity, as described for example by Kitamura et al . , (2001),

Biochem. J. 355, 123-129. C-peptide also has a well known effect in stimulating Na⁺K⁺ATPase activity and this also may form the basis of an assay for C-peptide activity, for example as described in WO 98/13384 or in Ohtomo et al . , (1996), supra or Ohtomo et al., (1998), supra. This is the preferred test to establish C-peptide' activity and active analogues will preferably induce NA⁺K⁺ATPase activity in the sciatic nerve by at least 50% over basal levels . An assay for C-peptide activity based on endothelial nitric oxide synthase (eNOS) activity is also described in Kunt et al . , supra, using bovine aortic cells and a reporter cell assay. Binding to particular cells may also be used to assess or assay for C-peptide activity, for example to cell membranes from human renal tubular cells, skin fibroblasts and saphenous vein endothelial cells using fluorescence correlation spectroscopy, as described for example in Rigler et al . , supra, Henriksson et al., (2000), Cell Mol. Life Sci., 57, 337-342 and Pra anik et al., supra. Finally, affinity tests based on measurements of protein binding may be used as activity tests of C-peptide. As mentioned above, a C-peptide analogue of the present invention may be an analogue of a C-peptide from any species (see Figure 1) . Preferably however, the C- peptide analogue is an analogue of a mammalian C- peptide, and particularly a primate C-peptide. More preferably the analogue is an analogue of human C- peptide (SEQ ID NO. 1) . The C-peptides analogues of the present invention are modified at one or more leucine residues and/or valine residues as compared to the unmodified C-peptide. Such modifications include any modification which confers resistance to degradation (cleavage) N- terminally of a leucine and/or valine residue. This may be achieved in various ways and hence the modification may be an amino acid substitution (i.e. the substitution of another amino acid for a leucine and/or valine residue) , substitution of D-leucine or D-valine for a (L-) leucine or (L-) valine (or indeed substitution by any D-amino acid) , a chemical modification of a leucine and/or valine residue (e.g. to introduce a blocking or protecting group, or a modification of the peptide (amide) bond N-terminal of a leucine and/or valine residue (i.e. the bond between a leucine and/or valine residue and the amino acid residue immediately N- terminal thereof, i.e. preceding it). Amino acid substitution is generally preferred and any amino acid may be substituted for one or more of the leucine and/or valine residues in the C-peptide. Such amino acids may include any naturally-occurring amino acid, for example one of the well-known 20 conventional amino acids. However, non-conventional or non-naturally occurring (i.e. unnatural or synthetic) amino acids may also be used (e.g. the unnatural amino acids butylglycine, norleucine, hydroxyproline and others which are commercially available or which have been described in the literature) . Preferably, however the amino acid selected for substitution may preserve the physioche ical character of the peptide. Preferred amino acids for substitution include alanine, methionine, glutamine and isoleucine, particularly isoleucine. Other amino acids may however be used and include glycine. As mentioned above, other substitutions which may be made include substituting D-Leu or D-Val for the natural L-form, or indeed any D-amino acid. Thus, amino acid substitution may serve to increase the resistance of the analogue to degradation by removing (i.e. by modifying) the natural cleavage site, such that the analogue may no longer be recognised by the endopeptidases which degrade the C-peptide in vivo (i.e. is not susceptible or has reduced susceptibility to cleavage in vivo) , ox by incorporating amino acids which are themselves known to confer increased stability to proteolytic degradation e.g. D amino acids or other unnatural amino acids (such as the butylglycine etc. listed above) . As mentioned above, modification by introducing a blocking group to the leucine/valine residues is also contemplated. The blocking group can be any group or entity which prevents/reduces degradation at the residues to which it is bound. Modifications can also be made to the peptide bonds linking the leucine/valine to the adjacent residues in the C-peptide analogue to increase resistance to degradation. For example, the peptide bonds in question can be methylated (particularly N-methylated) to increase their stability. The peptide bond can also be synthesised as a "reduced" peptide bond (-CH₂-NH-bond) . The peptide bonds may also be replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases. Such mimetics, and methods of incorporating them into peptide, are well known in the art. Thus for example, a peptide aldehyde may be used in the formation of a reduced peptide bond (pseudo peptide bond) to form a more stable peptidomimetic. Other types of bond include a urea bond. Any or all of the leucine and/or valine residues in a C-peptide may be protected (e.g. modified) according to the present invention. Preferably, the C-peptide analogue is modified at all leucine and valine residues . Alternatively, the C-peptide analogue may be modified at all leucine residues or all valine residues. Further, the analogue may comprise modifications at any one or any combination of leucine and/or valine residues. Hence, the analogue may be modified at any one or any combination of the leucine residues at positions 5, 12, 21, 24, 26 or 30 in human C-peptide (or the equivalent leucine residues in C-peptide from other species) and/or the valine residues at positions 7 and 10 (or the equivalent valines in another C-peptide, e.g., C-peptide from other species), as discussed above. The equivalent residue may be the residue at the corresponding position in another C-peptide. Encompassed by the present invention are peptides defined by the general formula I:

EAEDX₁QZ₁GQZ₂EX₂GGGPGAGSX₃QPX_iAX_sEGSX₆Q ( SEQ ID NO . 2 ) wherein i- ₆ are a leucine residue or a modified leucine residue which may be the same or different and Z_x and Z₂ are a valine residue or a modified valine residue which may be the same or different, but wherein at least one of X-_.-X_s or Z₁-Z₂ is modified. More particularly, when X₂-X_s = leucine and Zi-Z₂ = valine, then X_x = modified leucine; when X_x + X₃-X_s = leucine residues and Z = valine, then X₂ = modified leucine; when X_x-X₂ and X₄-X₆ = leucine and Z_x-Z₂ = valine, then X₃ = modified leucine; when X_x-X₃ and X₅-X₆ = leucine and Z₁-Z₂ = valine, then X₄ = modified leucine; when X_x-X₄ + X₆ = leucine and Z₁-Z₂ = valine, then X_s = modified leucine; when X-_.-X_s = leucine and Z₁-Z₂ = valine, then X_s = modified leucine; when i-X₆ = leucine and Z - valine then Z₂ = modified valine; and when X-_.-X₆ = leucine and Z₂ = valine, then Z_x = modified valine. Preferably, at least one of

is modified. The modified residue may be substituted by another amino acid (including a D-amino acid) , may be chemically modified (.e.g. carry a blocking or protecting group) or may be modified by virtue of its N-terminal bond being modified. Furthermore, as mentioned above, the modified leucines and/or modified valines may be the same or different . Preferably, when modified, X-_.-X₆ is an amino acid residue other than leucine and Z_x-Z₂ is an amino acid residue other than valine. The amino acid residue may be natural or unnatural . Preferably however it is a natural amino acid and most preferably a natural amino acid selected from one of the 20 conventional amino acids listed above. It may also be a D-amino acid, and in this case preferably Xj_.-X_s is D-Leu and Z_x-Z₂ is D-Val. More preferably,

and Z_x-Z₂ is selected from isoleucine, alanine, glutamine or methionine. Other representative amino acids include glycine. Thus, the same or different modifications may be made to each or any combination of the leucine residues and/or valine residues. Hence, for example, X₃-X_s could all be substituted with isoleucine or X₃ and X₄ could be substituted with isoleucine and X_s with alanine etc. In a preferred embodiment, the analogue is modified at one or more leucines at positions 12, 21, 24 and 26 in C-peptide (or at an equivalent or corresponding position) . Hence preferably leucines 12, 21, 24 or 26 alone are modified, or leucines 12 and 21, 21 and 24, 12 and 24, 12 and 26, 21 and 26 or 24 and 26 are modified. Leucines 12, 21 and 24; 12, 21 and 26; 12, 24 and 26; 21, 24 and 26 can also be modified. More preferably, all leucines at positions 12, 21, 24 and 26 are modified. Having regard to Formula I (SEQ ID NO. 2) , in such a preferred embodiment, one or more of X₂, X₃, X₄ and X_s are modified leucine residues. Preferably one of X₂, X₃, X₄ or X_s is modified, or any two of X₂-X₅, i.e. X₂ and X₃, X₃ and X₄, X₂ and X₄, X₂ and _Sι X₃ and X_s, or X₄ and X₅ are modified. Any three of X₂-X_s, i.e. X₂, X₃ and X₄; X₂, X₃ and X₅, X₂, X₄ and X_s; X₃, X₄ and X₆ can also be modified. More preferably, all of _2ι X₃, X₄ and X_s are modified. Hence the following sequences are particularly preferred embodiments of the present invention:

EAEDLQVGQVEXGGGPGAGSXQPXALEGS Q (SEQ ID NO. 3) EAED QVGQVEXGGGPGAGSXQPLAXEGSLQ (SEQ ID NO. 4) EAED QVGQVEXGGGPGAGSLQPXAXEGS Q (SEQ ID NO. 5) EAEDLQVGQVELGGGPGAGSXQPXAXEGS Q (SEQ ID NO. 6) EAED QVGQVELGGGPGAGSXQPXA EGSLQ (SEQ ID NO. 7) EAED QVGQVEXGGGPGAGSXQPLA EGS Q (SEQ ID NO. 8) EAEDLQVGQVEXGGGPGAGS QPXALEGS Q (SEQ ID NO. 9) EAEDLQVGQVEXGGGPGAGSLQPLAXEGSLQ (SEQ ID NO. 10) EAEDLQVGQVELGGGPGAGSXQP AXEGS Q (SEQ ID NO. 11) EAED QVGQVELGGGPGAGS QPXAXEGSLQ (SEQ ID NO. 12) EAEDLQVGQVEXGGGPGAGSLQPLA EGS Q (SEQ ID NO. 13) EAED QVGQVELGGGPGAGSXQP AIiEGSIiQ (SEQ ID NO. 14) EAEDLQVGQVELGGGPGAGS QPXALEGS Q (SEQ ID NO. 15) EAEDIiQVGQVE GGGPGAGS QPLAXEGSLQ (SEQ ID NO. 16) EAED QVGQVEXGGGPGAGSXQPXAXEGSLQ (SEQ ID NO. 17) EAEDXQVGQVEXGGGPGAGSXQPXAXEGSXQ (SEQ ID NO. 18)

where X is a modified leucine residue, and is preferably a substituted amino acid residue, more preferably an isoleucine, alanine, glutamine or methionine residue. Most preferably X is isoleucine. It may, however, be another amino acid e.g. glycine. Therefore particularly preferred peptides of the invention include:

EAEDLQVGQVEIGGGPGAGSIQPIA EGSLQ (SEQ ID NO. 19) EAEDLQVGQVEIGGGPGAGSIQP AIEGS Q (SEQ ID NO. 20) EAEDLQVGQVEIGGGPGAGS QPIAIEGSLQ (SEQ ID NO. 21) EAEDLQVGQVELGGGPGAGSIQPIAIEGSLQ (SEQ ID NO. 22) EAEDLQVGQVELGGGPGAGSIQPIALEGSLQ (SEQ ID NO. 23) EAED QVGQVEIGGGPGAGSIQPLA EGSLQ (SEQ ID NO. 24) EAEDLQVGQVEIGGGPGAGS QPIALEGS Q (SEQ ID NO. 25) EAED QVGQVEIGGGPGAGSLQPLAIEGSIiQ (SEQ ID NO. 26) EAED QVGQVE GGGPGAGSIQPLAIEGSLQ (SEQ ID NO. 27) EAEDLQVGQVELGGGPGAGSLQPIAIEGSLQ (SEQ ID NO. 28) EAEDLQVGQVEIGGGPGAGSLQPLALEGSLQ (SEQ ID NO. 29) EAEDLQVGQVELGGGPGAGSIQPLALEGS Q (SEQ ID NO. 30) EAEDLQVGQVELGGGPGAGSLQPIALEGSLQ (SEQ ID NO. 31) EAEDLQVGQVE GGGPGAGSLQPLAIEGS Q (SEQ ID NO. 32) EAEDLQVGQVEIGGGPGAGSIQPIAIEGSLQ (SEQ ID NO. 33) EAEDIQVGQVEIGGGPGAGSIQPIAIEGSIQ (SEQ ID NO. 34)

Table 1 sets out which residues in the C-peptide analogue for SEQ ID NOs . 1-34 have been modified. Other representative peptides of the invention include those of SEQ ID NOS . 19 to 34 wherein, rather than by isoleucine, the leucine residues are substituted by glycine. As described previously, the present invention also covers any of SEQ ID NOs. 3 to 34 wherein the valine residue at position 7 and/or the valine residue at position 10 is also modified. Preferably, the valine residue is substituted with an isoleucine, alanine, glutamine or methionine residue. More preferably, the valine residue is substituted with isoleucine. Other representative substitutions include glycine. The modification made to the valine residues can be the same or different to that made to the leucine residues.

Table 1

A further embodiment of the invention concerns a C- peptide analogue wherein the valine residues at positions 7 and/or 10 of C-peptide (or an equivalent or corresponding position) are modified alone. Preferably modification of valine at position 7 and/or 10 is by substitution with isoleucine, alanine, glutamine or methionine. More preferably, valine at position 7 and/or 10 is substituted with isoleucine. Hence, the following peptides are particularly preferred:

EAEDLQVGQZ₂ELGGGPGAGSLQPLALEGSLQ (SEQ ID NO . 35) EAEDLQVGQIELGGGPGAGSLQPLALEGSLQ (SEQ ID NO . 36) EAEDLQZ-GQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO . 37) EAEDLQIGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO. 38) EAEDLQZ₁GQZ₂ELGGGPGAGSLQPLALEGSLQ (SEQ ID NO. 39) EAEDLQIGQIELGGGPGAGSLQPLALEGSLQ (SEQ ID NO. 40) .

Other representative substitutions include glycine. As mentioned above, the C-peptide analogue of the present invention may be derived from a C-peptide which is a fragment of a full-length native or modified C- peptide. Thus, in a further embodiment of the present invention the analogue may be viewed as a fragment of an analogue of an intact C-peptide (e.g. any of the analogues set out in SEQ ID. NOs 2 to 40 above) , wherein the fragment is modified at at least one leucine residue and/or valine residue. Such a fragment should preferably retain the C-peptide activity of the C- peptide (or C-peptide analogue) from which it is derived. Such "fragment" analogues may comprise any or all of residues 15-31 of the C-peptide or C-peptide analogue (or corresponding or equivalent residues) , more especially residues 20-31, wherein one or more of the leucine residues is modified. Representative analogues of the invention may also include peptides which comprise the pentapeptide EGSX_SQ (SEQ ID NO. 41) (corresponding to residues 27-31 of human C-peptide) , and particularly include fragments of a full-length C-peptide analogue which comprise EGSX_SQ (SEQ ID NO. 41) , where X₃ to X_s is a modified leucine residue, preferably an isoleucine, alanine, glutamine or methionine residue, and more preferably an isoleucine residue. X₃ to X_s may, however, also be another amino acid, e.g. glycine. The fragment may vary in size from for example 4 to 30 amino acids. Exemplary such analogues include EGSX_SQ (SEQ ID NO. 41), X₅EGSX_SQ (SEQ ID NO. 42), AX₅EGSX_SQ (SEQ ID NO. 43), X₄AX₅EGSX_SQ (SEQ ID NO. 44), PX₄AX₅EGSX_SQ (SEQ ID NO . 45), QPX₄AX₅EGSX_SQ (SEQ ID NO. 46), and X₃QPX₄AX₅EGSX_SQ (SEQ ID NO. 47) (wherein X₃-X_s are as defined above) . The analogue may also be based on (or derived from) a C-peptide which is an N-terminal fragment of a full- length C-peptide. Thus, the analogue may also comprise an N-terminal fragment of a full length C-peptide analogue, having the sequence EAEDX₁QZ₁GAZ₂EX₂ (SEQ ID NO. 48) or EAEDX_XQVGAVEX₂ (SEQ ID NO. 49) . Alternatively, the analogue may have the sequence EAEDX_XQVGAVEL (SEQ ID NO. 50), EAEDLQVGAVEX₂ (SEQ ID NO. 51), EAEDLQZ_XGAZ₂EX₂ (SEQ ID NO. 52), or EAEDX₁QZ₁GAZ₂EL (SEQ ID NO. 53) . Further, a fragment of SEQ ID NOs. 48-53 comprising one or more modified leucine/valine residues, and 2 acidic amino acid residues capable of adopting a conformation where said 2 acidic amino acid residues are spatially separated by a distance of 9-14A between the -carbons thereof is covered by the present invention. Also included are C-peptide analogues wherein the "C-peptide" sequence or moiety thereof is flanked by one or more extensions or flanking sequences at the N and/or C-terminal thereof. The length of such "extended" analogues may vary but typically they are not more than 50, 30, 25 or 20 amino acids in length. The analogues of the invention may readily and conveniently be synthesised using known and standard techniques such as are widely known and well described in the literature. Suitable methods include e.g. the well known Merrifield solid phase synthesis method and derivatives thereof. Analogues having modified bonds may be synthesised by using appropriately modified or derivatised amino acids (e.g. peptide aldehydes or urea bond peptides) . Also included in the invention are the salts, solvates and esters of the analogues, such as may be prepared and used in accordance with standard pharmaceutical procedures well known in the art. Thus the analogues of the invention may be presented as pharmaceutically or physiologically acceptable salts e.g. acid addition salts. This may include both organic and inorganic salts such as those prepared for example from acids such as hydrochloric, hydrofluoric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic, ascorbic, p- toluenesulfonic, benzene-sulfonic, naphthalenesulfonic, propionic, and the like. Preferably, the acid addition salts are those prepared from hydrochloric acid, acetic acid, or succinic acid. Such salts may be prepared by conventional methods well known to those skilled in the art . Alternatively the analogue may be converted into a carboxylic acid salt, such as an ammonium or alkali metal salt e.g. a sodium, potassium or lithium salt etc. As mentioned above, the analogues of the invention have a utility in C-peptide based therapies, that is in the therapy of (i.e. in combatting) any condition which may be alleviated or improved by, or which responds to, C-peptide administration. "Therapy" and "combatting" in this regard include both treatment and prophylaxis. In particular the peptides of the invention can be used for the therapy of (i.e. for combatting) diabetes and diabetic complications, most notably type 1 diabetes and its complications. As used herein the term "diabetic complications" includes all complications which may be associated with various forms of diabetes, in particular retinopathy, neuropathy and nephropathy. The peptides or fragments may thus be used in treatment of type 1 diabetes patients with one or more of the above- mentioned complications, or for preventing or retarding the development of such complications. Thus, the peptides or fragments may be used in C-peptide replacement therapy of diabetic patients. A further aspect of this invention thus provides an analogue of the invention as hereinbefore defined, for use in therapy, and in particular in C-peptide therapy, (e.g. C-peptide replacement therapy in diabetes) , and also the use of such an analogue in preparing a medicament for use in C-peptide therapy (e.g. for combatting diabetes or diabetic complications) . Alternatively viewed, the present invention provides a method of combatting diabetes or diabetic complications in a patient, said method comprising administering to said patient an analogue of the invention as hereinbefore defined. Further, the invention provides a pharmaceutical composition comprising an analogue of the invention as defined above, together with at least one pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions comprising an analogue of SEQ ID NOs 2 to 18 are particularly preferred. Pharmaceutical compositions comprising more than one analogue of the invention are also contemplated. However, the respective analogues used in such combinations need not both be included in the same composition/medicament and could be administered separately in separate compositions/medicaments simultaneously or sequentially. A further aspect of the invention thus provides a product containing two or more analogues of the present invention as a combined preparation for simultaneous, separate or sequential use in C-peptide based therapy (e.g. in combatting diabetes and/or diabetic complications) . The analogues may also be used in combination or conjunction with other agents active or effective to treat diabetes and/or its complications. Such other active agents include for example insulin. In such "combination" therapies the analogue (s) and second active agent may be administered together in the same composition or separately in separate compositions, simultaneously or sequentially. A further aspect of the invention thus provides a product containing an analogue of the invention as hereinbefore defined, together with a further active agent effective to combat diabetes or diabetic complications, as a combined preparation for simultaneous, separate or sequential use in combatting diabetes and/or diabetic complications. Preferably such a further active agent is insulin. In such combined therapies, where insulin is used, it is to be understood that the term "insulin" encompasses all forms, types and derivatives of insulin which may be used for therapy e.g. synthetic, modified, or truncated variants of the active human insulin sequence . The compositions of the invention may be administered in any convenient way, e.g. orally or parenterally, for example by the subcutaneous, intramuscular or intravenous route. The compositions of this invention may comprise active analogues of the invention, together with a pharmaceutically acceptable carrier therefor and optionally, other therapeutic ingredients, for example human insulin. The total amount of active ingredients in the composition may vary from 99.99 to 0.01 percent of weight. The carrier must be acceptable in the sense that it is compatible with other components of the composition and is not deleterious to the recipient thereof . The compositions may be formulated according to techniques and procedures well known in the art and widely described in the literature, and may comprise any of the known carriers, diluents or excipients. Thus, for example, compositions of this invention suitable for parenteral administration conveniently comprise sterile aqueous solutions and/or suspensions of the pharmaceutically active ingredients (e.g. the peptides of the invention) preferably made isotonic with the blood of the recipient, generally using sodium chloride, glycerin, glucose, mannitol, sorbitol, and the like. In addition, the compositions may contain any of a number of adjuvants, such as buffers, preservatives, dispersing agents, agents that promote rapid onset of action or prolonged duration of action and the like. Compositions of this invention suitable for oral administration may, for example, comprise the analogues in sterile purified stock powder form preferably covered by an envelope or envelopes (enterocapsule) protecting from degradation (dicarboxylation or hydrolysis) of the analogues in the stomach and thereby enabling absorption of the substance from the gingiva or in the small intestine. The envelope (s) may contain any of a number of adjuvants such as buffers, preservative agents, agents that promote prolonged or rapid release giving an optimal bioavailability of the compositions in this invention, and the like. Appropriate representative formulations (i.e. compositions) and dosages etc. are described in WO 98/13384. The invention will now be described in more detail in the following non-limiting Examples and with reference to the drawing in which: Figure 1 is an alignment showing all reported C- peptide amino acid sequences; Figures 2A and B show the degradation products detected in the kidney and placenta incubations at certain times, respectively; reverse phase HPLC of C- peptide (residues 1-31) and its degradation products generated during incubation in (A) mouse kidney homogenate for 60 minutes and (B) human placenta homogenate for 3 hours. Identification was by mass determination using nano-electrospray mass spectrometry. In addition, to ascertain identifications, tandem mass spectrometry or Edman degradations were applied in a few cases; Figure 3 is a schematic showing an overview of the fragments generated during digestion of human C-peptide in kidney and placenta homogenates . Arrows indicate residues found to be susceptible to N-terminal proteolytic cleavage, and lines, the resulting cleavage products now detected using HPLC and mass spectrometry; Figure 4 is a CID spectrum of fragment 645.48 Da generated during incubation of C-peptide in the kidney homogenate (peak 8 , Fig. 1A) corresponding to the hexapeptide LEGSLQ that appeared as a singly charged peptide in the mass spectrum; Figure 5 shows the time-course of degradation of C- peptide (4.3 nmol) in (A) mouse kidney homogenate (26 mg tissue/ml) and (B) human placenta homogenate (280 mg tissue/ml) . Both are linear with an apparent degradation rate for the kidney homogenate that is about twice that obtained in the placenta homogenate; Figure (A) shows C-peptide analogues 80(241), 81(211) and 83 (21G, 24G) and intact C-peptide, as tested for degradation in Example 3. Arrows show sites of degradation. Substituted residues are underlined; Figure 6 (B) shows the time-course of degradation (amount peptide (nmole) vs time (min) ) of C-peptide (♦) and three analogues in kidney extracts: Analogue 80(124) (D) ; analogue 81 (121) (Δ) ; analogue 83(G24; G21) (*) .

Example 1 - Incubation of C-peptide with kidney tissue or placenta

Materials and Methods

Mouse kidneys were dissected and recovered from anesthetized animals (approved by the local ethical committee) and then stored at -80 C. Kidneys were disintegrated in 100 mM HEPES buffer, pH 7.1, using a Potter Elvehjem homogenizer. For one kidney (0.180 g) , 300 μl buffer was used, followed by the addition of 400 μl buffer. Aliquots of 50 μl homogenate were stored at -80 C. Human placenta (immediately frozen upon partus) was obtained from Karolinska Hospital (approved by the local ethical committee) . After thawing, the tissue was cut into pieces and 14 g was disintegrated in 100 mM HEPES buffer, pH 7.1, using a Ultra-turrax T25 mixer resulting in 5 ml homogenate that was stored in 500 μl aliquots at -80 C. For degradation studies, C-peptide (26 nmol,polypeptides) dissolved in 78 μl water was incubated with 40 μl kidney or placenta homogenate and 282 μl HEPES buffer at 37 C which means a 10-fold dilution of the crude ho ogenates. As a blank incubation, 40 μl kidney or placenta homogenate and 360 μl HEPES buffer was used. Aliquots of 66.7 μl corresponding to 4.3 nmol C-peptide were withdrawn at specific time points (0, 15, 20,40, 60 rain and 3 h for the kidney incubations; and 0, 20, 40, 60 min, and 3 and 6 h for the placenta incubations) . The aliquots were acidified (pH 2) by addition of 200 μl 0.13% trifluoroacetic acid (TFA) and then 10 μl 10% TFA, followed by centrifugation at 10 000 rpm for 10 min. After filtration through a 0.2 μm membrane filter (NanoSep, Pall Gelman Laboratory) , the sample was analyzed by reverse phase HPLC (Akta system, Amersham Pharmacia) using a Vydac C₄ column (4.6 x 250 mm) at 1 ml/min. Fractions were collected in Eppendorf tubes, dried under a stream of nitrogen and dissolved in 60% acetonitrile containing 1% acetic acid for direct nano- electrospray mass spectrometry. When necessary, HPLC fractions were desalted using C_1B ZipTips (Millipore) . The fractions were applied to the ZipTips in 0.1% TFA, washed with the same solvent and eluted in 60% acetonitrile containing 1% acetic acid. Mass spectra were recorded using a quadrupole time- of-flight tandem mass spectrometer (Q-TOF, Micromass) equipped with an orthogonal sampling electrospray ionization (ESI) -interface (Z-spray, Micromass) , and metal-coated nano-ESI needles (Protana) which gave a spraying orifice of about 5 μm and a flow of approximately 20-50 nl/min at a capillary voltage of 0.8-1.2 kV. For the acquisition of collision-induced dissociation (CID) spectra, the collision energy was optimized in the range 30-80 eV with argon as the collision gas . Some fractions were also analyzed by Edman degradations using a Procise HT instrument (Applied Biosysterns) . Results and Discussion

The degradation of human C-peptide and the cleavage products generated were studied after incubations in mouse kidney and human placenta homogenates for different times. To decrease the background of proteins and peptides, we found it suitable to dilute the homogenates 1:10 before mixing with C-peptide. The dilution still led to detectable degradation of C- peptide within 15 min in kidney and 20 minutes in placenta. Aliquots were taken at specific time points and were immediately acidified with TFA (pH 2) to stop proteolytic activities. Samples were centrifuged to separate protein precipitates, and the supematants were filtered, followed by injection onto reverse phase HPLC. The C-peptide eluted after 38 minutes while the degradation products eluted earlier according to the hydrophobicity of the respective fragment (Fig. 2) . The fractions containing C-peptide cleavage products (as judged from comparison with a blank run in which an incubation without C-peptide was injected) were collected and analysed by nano-electrospray mass spectrometry to identify the corresponding C-peptide segments . The degradation of human C-peptide in the two model systems, mouse kidney and human placenta homogenates, was established. An early degradation product detected in the kidney incubation (eluting at 32.5 min upon HPLC) revealed a mass of 1868.92 Da, corresponding to the 20- residue segment

resulting from a cleavage at Ser₂₀-Leu₂₁ (Fig. 2A and Fig. 3) . This fragment was also completely confirmed by Edman degradation. In the placenta homogenate, an early degradation product appeared to be a 29-residue fragment with a mass of 2777.40 Da, eluting at 37 min, corresponding to the segment

(Fig. 2B and Fig. 3) . In both these early cleavages, the cleavage site is N-terminal of a Leu residue. Other early fragments detected by HPLC analysis had masses of 2207.16 Da (the 23-residue peptide

in the kidney homogenate (Fig. 2A and Fig. 3), and 2320.16 Da (the 24-residue fragment Glu_x- Leu₂₄) in the placenta homogenate (Fig 2B and Fig. 3) . Here again, the cleavage sites are N-terminal of hydrophobic residues, a Leu-residue in the renal processing, and an Ala-residue in the placental degradation. Other fragments detected in both homogenates consisted of the first 25 residues of C- peptide, with a mass of 2391 Da (Fig. 2) , also with the cleavage site N-terminal of a Leu-residue (Fig. 3) . Subsequent fragments detected in both kidney and placenta homogenates again resulted from cleavages N- terminally of Leu residues, a pattern partly overlapping with that of insulin protease. For kidney, the mass 1215.68 Da was determined, corresponding to an 11- residue fragment starting at the C-peptide N-terminus and ending at Glu^, resulting from a cleavage before Leu₁₂ (Fig. 2A and Fig. 3) , while in the placenta homogenate, a longer segment with mass 2207.06, Glu_x- Pro₂₃, resulting from a cleavage before Leu₂₄ (Fig. 2B and Fig. 3) was detected. This placental degradation product was also detected in the kidney homogenate (above) . Shorter fragments appearing at a later stage consisted of 9-, 8- and 7-residue peptides in the kidney homogenate, and 8-, 7- and 6-residue peptides in the placenta homogenate (Figs. 2 and 3) . The pattern in these late products is different since these cleavage products result from secondary cleavages. Two of these peptides in each set, the 8- and 7-residue peptides, are also N-terminally truncated and identical between homogenates (Fig. 3) . The 8-residue peptide is truncated by one N-terminal residue and starts at Ala₂ while the 7-residue peptide is truncated by two residues and starts at Glu₃ (Fig. 3) . Thus, in addition to an aminopeptidase-like activity, the late occurring fragments result from secondary cleavages, derived from endopeptidase cleavages between Gln₉ and Val₁₀ (kidney) and Gln_s and Val₇ (placenta) . The last f agment detected after incubation with the kidney homogenate consisted of 6 residues, with the mass 645.48 Da and corresponding to the C-terminal segment LEGSLQ (Fig. 2A and Fig. 3) . This peptide, in similarity to the C-terminal pentaneptide EGSLQ, exerts C-peptide-like effects in vitro. In the placenta homogenate, the last fragment detected had the mass 703.35 Da and was identified as the N-terminalhexapeptide segment with the sequence EAEDLQ (Fig. 2B and Fig. 3) . For each fragment, the mass was determined, and where applicable to ascertain the identifications, tandem mass spectrometry or sequencer Edman degradations were employed (Fig. 4) . The degradation rates were estimated by monitoring the disappearance of the HPLC peak corresponding to the intact C-peptide over time. It was found that the degradation was about twice as fast in the kidney homogenate as in the placenta homogenate (Fig. 5) . In conclusion, the degradation patterns detected when C-peptide is incubated in kidney and placenta homogenates are largely similar and overlapping, and a majority of the fragments are generated via cleavages at sites N-terminal of a Leu residue, indicating that the proteolytic enzyme involved in C-peptide processing in both kidney and placenta is amino acid specific.

Example 2 - Substitution of leucine residues at positions 12 , 21, 24 and 26 and valine-10 of C-peptide with, isoleucine

Human C-peptide analogues were manufactured by solid phase peptide synthesis where the following leucine/valine residues were substituted with isoleucine: - 1) Leucine 21 (SEQ ID NO. 30)

2) Leucine 24 (SEQ ID NO. 31)

3) Leucine 21 and leucine 24 (SEQ ID NO. 28)

4) Valine 10 (SEQ ID NO. 36)

5) Leucine 12 (SEQ ID NO. 29)

6) Leucine 26 (SEQ ID NO. 32)

7) Leucine 21, 24 and 26 (SEQ ID NO. 22)

Example 3 - Stability of C-peptide and C-peptide analogues in kidney extracts

Method

For degradation studies, C-peptide (26 nmoles, PolyPeptides) dissolved in 78 μl water was incubated with 40 μl kidney or placenta homogenate and 282 μl HEPES buffer at 37°C which means a 10-fold dilution of the crude homogenates. The peptides tested are shown in Figure 6(A) . It will be noted that analogues 80 and 81 correspond to SEQ ID NOS. 31 and 30 respectively. Analogue 83 corresponds to SEQ ID NO. 23, wherein G is substituted for I at positions 21 and 24. It is termed SEQ ID NO. 77 hereinafter. As a blank incubation, 40 μl kidney and 360 μl HEPES buffer was used. Aliquots of 66.7 μl corresponding to 4.3 nmoles C-peptide were withdrawn at specific time points. The aliquots were acidified (pH 2) by addition of 200 μl 0.13% trifluoroacetic acid (TFA) and then 10 μl 10% TFA, followed by centrifugation at 10 000 rpm for 10 min. After filtration through a 0.2 μm membrane filter (NanoSep, Pall Gelman Laboratory) , the sample was analyzed by reverse phase HPLC (Akta system, Amersham Pharmacia) using a Vydac C₄ column (4.6 x 250 mm) at lml/min. Fractions were collected in Eppendorf tubes, dried under a stream of nitrogen and dissolved in 60% acetonitrile containing 1% acetic acid for direct nano- electrospray mass spectrometry. Result

The results are shown in Table 2 below and in Figure 6(B) .

It will be seen that all three C-peptide analogues show increased half-life over intact native C-peptide in the in vi tro system used. The increase in t% is 20-35% as indicated in Table 2. Isoleucine substituted at position 24 gives the most marked prolongation of t% .

Table 2. Half-life of C-peptide and its analogs in mouse kidney extract

Claims

Claims

1. A C-peptide analogue wherein the C-peptide is modified at one or more leucine and/or one or more valine residues, and wherein the analogue has an increased resistance to proteolytic degradation as compared to un-modified C-peptide.

2. The C-peptide analogue of claim 1 wherein said analogue has an increased biological half-life compared to unmodified C-peptide.

3. The C-peptide analogue of claim 1 or 2 wherein said analogue retains at least 20% of the functional activity of C-peptide.

4. The C-peptide analogue of any one of claims 1 to 3 which is an analogue of a mammalian C-peptide or a functionally equivalent variant thereof.

5. The C-peptide analogue of any one of claims 1 to 4 which is an analogue of human C-peptide (SEQ ID NO. 1) or a functionally equivalent variant thereof .

6. The C-peptide analogue of any one of claims 1 to 5, wherein said analogue is modified at one or more leucine residues .

7. The C-peptide analogue of any one of claims 1 to 6 wherein said analogue is modified at all leucine residues in said C-peptide.

8. The C-peptide analogue of any one of claims 1 to 6 wherein said analogue is modified at one or more leucine residues at or equivalent or corresponding to positions 12, 21, 24 and 26 of human C-peptide.

9. The C-peptide analogue of any one of claims 1 to 6, wherein said analogue is modified at one of the leucine residues at or equivalent or corresponding to positions 12, 21, 24 and 26 of human C-peptide.

10. The C-peptide analogue of any one of claims 1 to 9, wherein said analogue is modified at the valine residue at or equivalent or corresponding to position 10 of human C-peptide.

11. The C-peptide analogue of any one of claims 1 to 10 wherein said analogue has the formula (I)

EAEDX₁QZ₁GQZ₂EX₂GGGPGAGSX₃QPX₄AX_SEGSX₆Q (SEQ ID NO. 2)

wherein Xj- g are a leucine residue or a modified leucine residue which may be the same or different and Z_x and Z₂ are a valine residue or a modified valine residue which may be the same or different, but wherein at least one of X -X₆ or Z₁-Z₂ is modified.

1 . The C-peptide analogue of any one of claims 1 to

11, wherein said modification is an amino acid substitution.

13. The C-peptide analogue of any one of claims 1 to

12, wherein said modification is a substitution with an amino acid selected from alanine, methionine, glutamine or isoleucine.

1 . The C-peptide analogue of any one of claims 1 to 13 for use in therapy.

15. Use of the C-peptide analogue of any one of claims 1 to 7 in the manufacture of a medicament for use in C- peptide therapy.

16. A pharmaceutical composition comprising the C- peptide analogue of any one of claims 1 to 13 together with at least one pharmaceutically acceptable carrier or excipient .

17. A product containing the C-peptide analogue of any one of claims 1 to 13 together with a further active agent effective to combat diabetes or diabetic complications as a combined preparation for simultaneous, separate or sequential use in combatting diabetes and/or diabetic complications.