Title: Insulin C-peptides
United States Patent: 6,610,649
Issued: August 26, 2003
Inventors: Wahren; John (Djursholm, SE); Johansson; Bo-Lennart (Uttran, SE); Jornvall; Hans (Stockholm, SE)
Assignee: Creative Peptides Sweden AB (SE)
Appl. No.: 269439
Filed: July 8, 1999
PCT Filed: September 26, 1997
PCT NO: PCT/GB97/02627
PCT PUB.NO.: WO98/13384
PCT PUB. Date: April 2, 1998
The invention features peptides that are fragments of the human insulin C-peptide, which peptides include the sequence ELGGGPGAG or a fragment thereof, or the sequence EGSLQ or a fragment thereof. The peptides have the ability to stimulate Na+ K+ ATPase activity. Also provided are biomimetic organic compounds exhibiting activation of Na+ K+ ATPase activity and/or cellular binding to renal tubule cells and fibroblasts. Such peptides and compounds are useful in combating diabetes and diabetic complications, or for stimulating Na+ K+ ATPase activity.
BRIEF SUMMARY OF THE INVENTION
The present invention is based on the discovery of a group of peptides from the middle portion and the C-terminal part of the C-peptide molecule which are characterized by a remarkable ability to stimulate Na+ K+ ATPase activity. These peptides are all small fragments of the C-peptide molecule. C-peptide itself is able to stimulate Na+ K+ ATPase via activation of a G-protein, increase in the intracellular Ca2+ concentration and activation of protein phosphatase 2B (7). However, the smaller peptides' stimulatory effect on Na+ K+ ATPase activity is similar to or greater than that of C-peptide itself. There is both in vitro and in vivo evidence to indicate that upon administration of one of these peptides together with regular insulin treatment, renal function improves, early signs of retinopathy regress and the function of somatic and autonomic nerves improves. Treatment with these specific peptides, optionally in combination with conventional insulin therapy is thus useful in preventing or substantially retarding the development of late diabetic complications. A potential advantage that the small peptides possess over C-peptide is that they may be administered orally instead of by injection as in the case of C-peptide and insulin.
In one aspect, the present invention thus provides a peptide being a fragment of the human insulin C-peptide, said peptide comprising the sequence ELGGGPGAG (SEQ ID NO. 2) (hereinafter "peptide A") or a fragment thereof, or the sequence EGSLQ (SED ID NO. 3) (hereinafter "peptide E"), or a fragment thereof, and having the ability to stimulate Na+ K+ ATPase activity.
In a more particular embodiment, the present invention provides a peptide having the sequence ELGGGPGAG (SEQ ID NO. 2) or EGSLQ (SEQ ID NO. 3), or a fragment thereof.
Especially, the invention provides such peptides for use in therapy and more particularly for use in combatting diabetes and diabetic complications.
In another aspect the present invention provides a pharmaceutical composition comprising a peptide of the invention or a fragment thereof as hereinbefore defined together with at least one pharmaceutically acceptable carrier or excipient.
A yet further aspect of the present invention provides the use of a peptide of the invention, or a fragment thereof, as hereinbefore defined, in the manufacture of a medicament for combatting diabetes or diabetic complications.
As used herein the term "combatting" includes both treatment and prophylaxis.
The present invention thus relates to the use of the following peptides which all are fragments of C-peptide: Peptide A (amino acid sequence ELGGGPGAG) (SEQ ID NO. 2) or components thereof, for example Peptide B (ELGG)(SEQ ID NO. 4), Peptide C (ELGGGP) (SEQ ID NO. 5) or Peptide D (GGPGA) (SEQ ID NO. 6). In addition, the invention includes Peptide E (EGSLQ)(SEQ ID NO. 3) and parts thereof, for example Peptide F (GSLQ) (SEQ ID NO. 7). All are intended for the manufacture of a medicament for treating type 1 diabetes.
Fragments of the invention have been proven to stimulate Na+ K+ ATPase activity to varying extent. Thus, studies involving renal tubule cells under in vitro conditions indicate that Peptides A-D stimulate Na+ K+ ATPase activity to an extent comparable to that for the whole C-peptide molecule. As much as 90% of the effect is achieved within 3 minutes. Moreover, Peptides E and F possess a stimulatory effect on Na+ K+ ATPase of renal cells which is comparable to or greater than that for the whole molecule. Combinations of Peptides A-D with Peptides E or F result in a stimulation of the enzyme activity that is greater than that for either peptide alone. For detailed examples of the stimulatory effects of the above peptides, see Example 1, below.
C-peptide exhibits specific binding to the surface of several cell types, notably renal tubule cells and fibroblasts. When fluorescently labelled C-peptide is incubated with cells it binds to the cell surface. The specificity of the binding is illustrated by the fact that preincubation with unmarked C-peptide prevents binding of the fluorescently labelled C-peptide. When preincubation with the fragments of the invention, particularly with either of fragments E or F was made, the fragments were found to prevent binding of the fluorescently marked C-peptide, demonstrating that the fragments bind specifically to the same binding site on the cell surface as C-peptide itself. For a detailed example of the binding of Fragment E see Example 28, below.
As mentioned above, included within the scope of the invention are peptides comprising the sequences of not only peptides A and E, but also their fragments. In the case of the nonapeptide A, such fragments may be 8 to 2 amino acids in length. In the case of the pentapeptide peptide E, such fragments may be 4 to 2 amino acids in length. Exemplary fragments B, C and D (for peptide A) and F (for peptide E) are listed above, but other fragments are also included.
In the case of peptide A certain studies on Na+ K+ ATPase activity, studying the ability of the peptide fragments to stimulate the activity of Na+ K+ ATPase of rat renal tubule segments, have shown that one or more of the central tri-glycine residues may be important, and preferred peptide fragments, where peptide A is concerned, thus include at least one, and more preferably, at least two, of the central tri-glycine residues. Thus, in addition to peptides B, C and D mentioned above, representative exemplary peptide fragments include GGGPGAG (SEQ ID NO. 8), GGGPG (SEQ ID No. 9), GGGP (SEQ ID NO. 10), GGP and GGPG (SEQ ID NO. 11).
Furthermore, it has been found that peptides containing non-natural D-amino acid isomers may also be active, including for example the dipeptide D-LG or D,L-LG. Thus, included within the scope of the invention are "non-native" isomers of the "native" L-amino acid C-peptide sequences. Insofar as peptide A is concerned, it is believed that the presence of at least one (if D-peptide) or two (if L-peptide) of the central tri-glycine residues may be important in a 9 amino acid or less peptide segment.
In the case of peptide E, exemplary representative fragments include not only the tetrapeptide, peptide F, but also SLQ and LQ. The C-terminal Q residue is believed to be of importance. Likewise, non-native isomers or derivatives of the peptides e.g. peptides including D-amino acids are included within the scope of the invention.
The invention encompasses peptides comprising the sequences of peptides A and E. Thus, also included within the scope of the invention are peptides having N- and/or C-terminal extensions, or flanking sequences, to the sequences of peptides A and C. Such peptides may include additional amino acids which may either be those provided in the corresponding position in the native human insulion C-peptide or other amino acids (excluding of course the possibility of reconstituting the entire insulin C-peptide). The length of such "extended" peptides may vary, but preferably the peptides of the invention are no more than 25 or 20, especially preferably not more than 15 or 10 amino acids in length. Exemplary peptides include octa-, hepta and hexa-peptides including the sequence of peptide E, e.g. LALEGSLQ (SEQ ID NO. 12), ALEGSLQ (SEQ ID NO. 13) and LEGSLQ (SEQ ID NO. 14).
The peptides of the invention can be used for the treatment of diabetes and diabetic complications, most notably type 1 diabetes and its complications. As used herein the term "diabetic complications" thus includes all complications known in the art to be associated with various forms of diabetes. Whilst not wishing to be bound by theory, the utility of the peptides is believed, as explained above, to be linked to their ability to stimulate Na+ K+ ATPase activity. A further aspect of the invention thus includes the peptides for use in, and their use in preparing medicaments for use in stimulating Na+ K+ ATPase activity in a subject.
Na+ K+ ATPase activity may readily be assayed using techniques known in the art and described in the literature and thus the effect of the peptides in stimulating Na+ K+ ATPase activity may readily be determined (for example, see reference 7).
Thus, the peptides can be used for the manufacture of a medicament for stimulation of Na+ K+ ATPase activity, for treating type 1 diabetes patients with retinopathy, for treating type 1 diabetes patients with nephropathy, for treating type 1 diabetes patients with neuropathy and for retarding the development of late diabetic complications. The medicament may comprise insulin. The invention also relates to the method for treatment or prevention of the above given indications.
The peptides of the invention may be used singly or in combination and thus a pharmaceutical composition or medicament may be prepared comprising one or more of the peptides. As mentioned above, a synergy has been observed between peptide A or peptides based on or derived from peptide A (the "peptide A group") and peptide E or peptides based on or derived from peptide E (the "peptide E group"). Thus, synergistic combinations of a peptide from the peptide A group, with a peptide from the peptide E group represent a preferred embodiment of the invention.
The peptides 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 exammple insulin. In such "combination" therapies the peptide(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 a peptide of the invention, or a fragment thereof, 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 orally or parenterally by the subcutaneous, intramuscular or intravenous route. The compositions of this invention comprise active fragments/peptides of the C-peptide molecule (e.g. Peptides A-F), together with a L pharmaceutically acceptable carrier therefor and optionally, other therapeutic ingredients, for example human insulin. The total amount of active ingredients in the composition varies 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. Peptides A-F) 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 active fragments/peptides of the C-peptide molecule (e.g. Peptides A-F) in sterile purified stock powder form preferably covered by an envelope or envelopes (enterocapsule) protecting from degradation (decarboxylation or hydrolysis) of the active peptides in the stomach and thereby enabling absorption of these substances 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.
In addition, the present invention relates to non-peptide compounds showing the same stimulatory effects as displayed by their C-peptide-derived counterparts. Such peptidomimetics or "small-molecules" capable of mimicking the activity of the naturally occurring proteins or peptides are likely to be better suited for e.g. oral delivery due to their increased chemical stability (8,9).
It is now commonplace in the art to replace peptide or protein-based active agents e.g. therapeutic peptides with such peptidomimetics having functionally-equivalent activity. Various molecular libraries and combinatorial chemistry techiques exist and are available to facilitate the identification, selection and/or synthesis of such compounds using standard techniques (10). Such standard techniques may be used to obtain the peptidomimetic compounds according to the present invention, namely peptidomimetic organic compounds which show substantially similar or the same activation of Na+ K+ ATPase and/or cellular binding characteristics as the peptides of the invention, e.g. as described herein in the Examples.
A further aspect of the invention thus provides a biomimetic organic compound based on the peptides of the invention, characterised in that said compound exhibits activation of Na+ K+ ATPase and/or cellular binding characteristics to renal tubule cells and fibroblasts at at least the level exhibited by the peptides and peptide fragments of the invention as hereinbefore defined.
Claim 1 of 27 Claims
What is claimed is:
1. A pharmaceutical composition comprising a peptide that (a) comprises the sequence EGSLQ (SEQ ID NO:3), (b) is up to 15 amino acids in length, and (c) has the ability to stimulate Na+ K+ ATPase activity.