Patent Application
20090281032
The present invention relates to the regulation of feeding and control of energy metabolism. More particularly the invention relates to the use of peptides to suppress food intake and pharmaceutical preparations for the treatment of obesity and type 2 diabetes.
Obesity and type 2 diabetes are two of the most common metabolic disorders in western societies. The risks to health posed by obesity are considerable, including predisposition to diabetes and its associated long-term complications. Despite this worldwide epidemic, there is currently only a limited number of drugs available to counter these major metabolic diseases. These are largely ineffective in the case of obesity or unable to prevent development of complications in diabetes.
The present invention concerns the discovery of novel modified long-acting analogues of CCK-8 and their potential use for regulation of appetite control and treatment of obesity and related diabetes. The insulin-releasing capability of these analogues is also directly beneficial in terms of improved blood glucose control, thereby making these agents a novel class of antidiabetic agent.
The regulation of food intake is a complex process that is controlled by a system of hunger and satiety signals interacting in complex pathways both peripherally and centrally (Ukkola 2004). Signals from the gastrointestinal tract, pancreas and adipose tissue together with circulating nutrients converge on the hypothalamus to regulate food intake and energy expenditure. The arcuate nucleus (ARC), in particular, is thought to play a pivotal role in the integration of these signals (Wynne et al. 2005). A growing number of peptides have been discovered which elicit the ability to decrease food intake (anorexigenic peptides) or increase food intake (orexigenic peptides) in animals and humans. As a group, they provide a number of leads for potential drug development.
Cholecystokinin (CCK) is a neuropeptide hormone found in the brain and secreted from gut endocrine cells, which was originally identified from its ability to stimulate gall bladder contraction. CCK is now known to play a significant role in many physiological processes including regulation of satiety, bowel motility, gastric emptying, insulin secretion, pancreatic enzyme secretion and neurotransmission. Cholecystokinin is a neuropeptide hormone released postprandially by gut endocrine I cells (Liddle 1994). CCK-8 acts via two major receptor sub-populations CCKA (peripheral) and CCKB (brain) (Innis et al. 1980). CCK exists in multiple molecular forms in the circulation ranging from 58, 39, 33, 22, 8 and 4 amino acids in length (Cantor 1989, Inui 2000). CCK-33 was the original form purified from porcine intestine. The C-terminal octapeptide CCK-8 is well conserved between species and is the smallest form that retains the full range of biological activities (Smith 1984, Crawley & Corwin 1995, Inui 2000). A variety of CCK molecular forms are secreted following ingestion of dietary fat and protein, from endocrine mucosal I cells that are mainly located in the duodenum and proximal jejunum. Once released, CCK-8 exerts its biological action on various target tissues within the body in a neurocrine, paracrine or endocrine manner. These actions are mediated through two major receptor sub-populations CCKA (peripheral subtype) and CCKB (brain subtype). Specific receptor antagonists such as proglumide have aided our understanding of the action of CCK on food intake.
CCK receptors are also present in pancreatic islets. CCK-8 has been shown to reduce feeding dose dependently in a variety of species including man (Gibbs et al. 1973, Morley 1987, Silver et al. 1991). Involvement of CCK in the control of food intake in rodents was recognised in the early 1970's, and since then this peptide hormone has been shown to reduce feeding in man and in several animal species. The induction of satiety is a common feature in different species but the mechanism by which this is achieved is poorly understood. However, many different tissues are known to possess specific receptors for CCK including the vagus nerve, pyloric sphincter and brain, all of which may be implicated in this satiety control mechanism. It has been proposed that CCK stimulates receptors in the intestine that activate the vagus nerve, which relays a message to the satiety centres in the hypothalamus. In support of this concept, it has been found that satiety effects of CCK are eliminated in vagotomized animals. Furthermore, rodent studies have indicated that CCK has a more potent satiating ability when administered by the intraperitoneal route rather than centrally. Intraperitoneal CCK-8 is thought to act locally rather than hormonally. In addition, it is known that CCK-8 does not cross the blood brain barrier.
Nevertheless, other evidence suggests that CCK has a definite neuronal influence on food intake in the central nervous system. Some work in dogs has suggested that circulating levels of CCK were too low to induce satiety effects. However, studies in pigs immunized against CCK revealed that these animals increased their food intake and had accelerated weight gain compared to control animals. In addition CCK receptor antagonists increased food intake in pigs and decreased satiety in humans. Overall the above studies indicate that CCK plays a significant role in regulating food intake in mammals.
CCK-8 has been considered as a short-term meal-related satiety signal whereas the recently discovered OB gene product leptin, is more likely to act as an adiposity signal which may reduce total food intake over the longer term. Indeed some workers have suggested that CCK-8 and leptin act synergistically to control long term feeding in mice.
The present invention aims to provide effective analogues of CCK-8. It is one aim of the invention to provide pharmaceuticals for treatment of obesity and/or type 2 diabetes.
According to the present invention there is provided an effective peptide analogue of the biologically active CCK-8 which has improved characteristics for the treatment of obesity and/or type 2 diabetes wherein the analogue has at least one amino acid substitution or modification and not including Asp1-glucitol CCK-8.
The primary structure of human CCK-8 is shown below:
Asp1Tyr2(SO3H)-Met3Gly4Trp5Met6Asp7Phe8 amide
The analogue may include modification by fatty acid addition (e.g., palmitoyl) at the alpha amino group of Asp1 or an epsilon amino group of a substituted lysine residue. The invention includes Asp1-glucitol CCK-8 having fatty acid addition at an epsilon amino group of at least one substituted lysine residue.
By glucitol is meant
and by Asp1-glucitol is meant the moiety in which a hydroxyl group of glucitol is reacted with the amino group of an amino acid.
Analogues of CCK-8 may have an enhanced capacity to inhibit food intake, stimulate insulin secretion, enhance glucose disposal or may exhibit enhanced stability in plasma compared to native CCK-8. They may also possess enhanced resistance to degradation by naturally occurring exo- and endo-peptidases.
Any of these properties will enhance the potency of the analogue as a therapeutic agent.
Analogues having one or more D-amino acid substitutions within CCK-8 and/or N-glycated, N-alkylated, N-acetylated, N-acylated, N-isopropyl, N-pyroglutamyl, pGluGln amino acids at position 1 are included.
Analogues having one or more D-amino acid substitutions within CCK-8 and/or N-glycated, N-alkylated, N-acetylated, N-acylated, N-isopropyl, N-pyroglutamyl amino acids at position 1 are included.
By pyroglutamic acid is meant:
and by pyroglutamyl is meant the moiety in which the hydroxyl group of the carboxyl group of pyroglutamic acid is reacted with the amino group of another amino acid.
Various amino acid substitutions including for example, replacement of Met3 and/or Met6 by norleucine or 2-aminohexanoic acid. Various other substitutions of one or more amino acids by alternative amino acids include replacing Met3 by Thr, Met6 by Phe, Phe8 by N-methyl Phe.
Other stabilised analogues include those with a peptide isostere bond replacing the normal peptide bond between residues 1 and 2 as well as at any other site within the molecule. Furthermore, more than one isostere bond may be present in the same analogue. These various analogues should be resistant to plasma enzymes responsible for degradation and inactivation of CCK-8 in vivo, including for example aminopeptidase A.
In particular embodiments, the invention provides a peptide which is more potent than CCK-8 in inducing satiety, inhibiting food intake or moderating blood glucose excursions, said peptide consisting of CCK(1-8), or smaller fragment, with one or more modifications selected from the group consisting of:
(i) N-terminal extension of CCK-8 by pGlu-Gln;
(ii) N-terminal extension of CCK-8 by pGlu-Gln with substitution of Met8 by Phe;
(iii) N-terminal extension of CCK-8 by Arg;
(iv) N-terminal extension of CCK-8 by pyroglutamyl (pGlu);
(v) substitution of the penultimate Tyr2 (SO3H) by a phosphorylated Tyr;
(vi) substitution of the penultimate Tyr2 (SO3H) by Phe(pCH2SO3Na);
(vii) substitution of a naturally occurring amino acid by an alternative amino acid including; Met3 and/or Met6 by norleucine or 2-aminohexanoic acid, Met3 by Thr, Met6 by Phe, Phe8 by N-methyl Phe;
(viii) substitution described in (vii) above with or without N-terminal modification of Asp1 (e.g., by acetylation, glycation, acylation, alkylation, isopropylation, pGlu, pGlu-Gln etc);
(ix) modification of Asp1 by acetylation;
(x) modification of Asp1 by acylation (e.g., palmitate);
(xi) modification of a substituted Lys residue by a fatty acid (e.g., palmitate);
(xii) modification of Asp1 by alkylation;
(xiii) modification of Asp1 by glycation in addition to a fatty acid (e.g., palmitate) linked to an epsilon amino group of a substituted Lys residue;
(xiv) modification of Asp1 by isopropyl;
(xv) modification of Asp1 by Fmoc or Boc;
(xvi) conversion of Asp1-Tyr2 bond to a stable non-peptide isostere bond CH2NH;
(xvii) conversion of Tyr2-Met3 bond to a psi [CH2NH] bond;
(xviii) conversion of Met3-Gly4 bond to a psi [CH2NH] bond;
(xix) conversion of Met6-Asp7 bond to a psi [CH2NH] bond;
(xx) conversion of other peptide bonds to a psi [CH2NH] bond;
(xxi) modification of Tyr2 by acetylation (i.e. acetylated CCK-7);
(xxii) modification of Tyr2 by pyroglutamyl (i.e. pyroglutamyl CCK-7);
(xxiii) modification of Tyr2 by glycation (i.e. glycated CCK-7);
(xxiv) modification of Tyr2 by succinic acid (i.e. succinyl CCK-7);
(xxv) modification of Tyr2 by Fmoc (i.e. Fmoc CCK-7);
(xxvi) modification of Tyr2 by Boc (i.e. Boc CCK-7);
(xxvii) D-amino acid substituted CCK-8 at one or more sites;
(xxviii) D-amino acid substituted CCK-8 at one or more sites in addition to an N-terminal modification by, for example, acetylation, acylation, glycation, alkylation, isopropylation, pGlu, pGluGln, etc;
(xxix) reteroinverso CCK-8 (substituted by D-amino acids throughout octapeptide and primary structure synthesised in reverse order); and
(xxx) shortened N- and/or C-terminal truncated forms of CCK-8 and cyclic forms of CCK-8.
The invention also provides a method of N-terminally modifying CCK-8, or analogues thereof, during synthesis. Preferably, the agents would be glucose, acetic anhydride or pyroglutamic acid.
The invention also provides the use of Asp1-glucitol CCK-8, pGlu-Gln CCK-8 and other analogues in the preparation of medicament for treatment of obesity and/or type 2 diabetes.
The invention further provides improved pharmaceutical compositions including analogues of CCK-8 with improved pharmacological properties.
Other possible analogues include truncated forms of CCK-8 represented by removal of single or multiple amino acids from either the C- or N-terminus in combination with one or more of the other modifications specified above.
According to the present invention there is also provided a pharmaceutical composition useful in the treatment of obesity and/or type 2 diabetes which comprises an effective amount of the peptide as described herein, in admixture with a pharmaceutically acceptable excipient for delivery through transdermal, nasal inhalation, oral or injected routes. Said peptide can be administered alone or in combination therapy with native or derived analogues of leptin, exendin, islet amyloid polypeptide (IAPP) or bombesin (gastrin-releasing peptide).
The invention also provides a method of N-terminally modifying CCK-8 and analogues thereof. This 3 step process firstly involves solid phase synthesis of the C-terminus up to Met3. Secondly, adding Tyr(tBu) to a manual bubbler system as an Fmoc-protected PAM resin, deprotecting the Fmoc by piperidine in DMF and reacting with an Fmoc protected Asp(OtBu)-OH, allowing the reaction to proceed to completion, removal of the Fmoc protecting group from the dipeptide, reacting the dipeptide with the modifying agent (e.g., glucose, acetic anhydride, palmitate, etc), removal of side-chain protecting groups (tBu and OtBu) by acid, sulphating the Tyr2 with sulphur trioxide, cleaving the peptide from the resin under alkaline conditions. Thirdly, the N-terminal modified dipeptide can be added to the C-terminal peptide resin in the synthesizer, followed by cleavage from the resin under alkaline conditions with methanolic ammonia, and finally purification of the final product using standard procedures.
According to a first aspect of the present invention there is provided a peptide based on biologically active CCK-8, the peptide having improved characteristics for the treatment of at least one of obesity and type 2 diabetes, wherein the structure of the peptide is:
(Z)-Asp1-Aaa2(X)-Aaa3Gly4Trp5Aaa6Asp7(Y)Aaa8K,
wherein:
Optionally, the structure of the peptide is:
(Z)-Asp1-Aaa2(X)-Aaa3Gly4Trp5Aaa6Asp7(Y)Aaa8K,
Further optionally, said N-terminal modification at position 1 is selected from the group comprising N-alkylation, N-acetylation, N-acylation, N-glycation and N-isopropylation of the amino acid at position 1. Still further optionally, said N-terminal extension is selected from the group comprising pGlu, pGlu-Gln, an acid, a fatty acid, Boc, Fmoc, Arg and attachment of a polymer moiety of the general formula HO—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22.
Optionally, the peptide is further modified by replacement of any amino acid with Lys, with or without fatty acid addition at an epsilon amino group of at least one substituted lysine residue.
Optionally, the peptide is further modified by attachment to Asp7 of a polymer moiety of the general formula HO—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22. Further optionally, the peptide is modified by replacement of any amino acid with an amino acid selected from the group including, but not limited to, lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine and attachment of a polymer moiety of the general formula HO—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22 to at least one substituted amino acid.
Optionally, Z is selected from the group consisting of:
(i) N-terminal extension of the peptide by pGlu-Gln and Aaa8 is Phe;
(ii) N-terminal extension of the peptide by pGlu-Gln and Aaa8 is Met;
(iii) N-terminal extension of the peptide by Arg;
(iv) N-terminal extension of the peptide by pyroglutamyl (pGlu);
(v) modification of Asp1 by acetylation;
(vi) modification of Asp1 by acylation;
(vii) modification of Asp1 by alkylation or glycation;
(viii) modification of Asp1 by isopropylation;
(ix) N-terminal extension of the peptide at Asp1 by Fmoc or Boc;
(x) N-terminal extension of the peptide by attachment of a polymer moiety of the general formula HO—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22; and
(xi) N-terminal extension of the peptide by pGlu-Gln and C-terminal extension of the peptide by attachment of a polymer moiety of the general formula HO—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22.
Alternatively or additionally, the peptide is modified by
(i) D-amino acid substituted CCK-8 at one or more amino acid sites and Z comprises an N-terminal extension or an N-terminal modification;
(ii) reteroinverso CCK-8 (substituted by D-amino acids throughout octapeptide and primary structure synthesised in reverse order); and
(iii) X is PO3H2−.
Optionally, at least one of K, X and Z comprises a polymer moiety covalently bound to Phe8, the polymer moiety being of the general formula HO—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22; further optionally, wherein n is an integer between 1 and about 10; still further optionally, wherein n is an integer between about 2 and about 6.
Optionally, when K comprises a polymer moiety covalently bound to Phe8, the polymer moiety is of the general formula —O—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22, the peptide is further modified by N-terminal extension of the peptide, wherein the peptide is, optionally, modified by N-terminal extension of the peptide by pGlu-Gln.
Optionally, in the peptide of the present invention,
Optionally, in the peptide of the present invention,
Optionally, there is at least one peptide isostere bond is present between amino acid residues at any site within the peptide. For example, an isostere bond may be present between Asp1-Tyr2; between Tyr2-Met3; between Met3-Gly4; or between Met6-Asp7.
Optionally, Z is selected from the group consisting of:
(i) N-terminal extension of the peptide by pGlu-Gln;
(ii) N-terminal extension of the peptide by Arg;
(iii) N-terminal extension of the peptide by pyroglutamyl (pGlu);
(iv) modification of Asp1 by acetylation;
(v) modification of Asp1 by acylation;
(vi) modification of Asp1 by alkylation or glycation;
(vii) modification of Asp1 by isopropylation; and
The invention further provides an effective peptide analogue of the biologically active CCK-8 which has improved characteristics for the treatment of obesity and/or type 2 diabetes, wherein the analogue has at least one amino acid substitution or modification, wherein said at least one amino acid substitution or modification comprises attachment of a polymer moiety to the CCK-8 analogue, or peptide fragment, of the general formula —O—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22. Optionally, the polymer moiety has an average molecular weight of no more than 1000 Da. Preferably, the polymer moiety has an average molecular weight of less than 1000 Da. Preferably, n is an integer between 1 and about 10. More preferably, n is an integer between about 2 and about 6. Optionally, the polymer molecule has a branched structure. The branched structure may comprise the attachment of at least two polymer moieties of linear structure. Alternatively, the branch point may be located within the structure of each polymer moiety. Alternatively, the polymer moiety has a linear structure. Some or all monomers of the polymer moiety can be associated with water molecules. Attachment of the polymer moiety can be achieved via a covalent bond. Optionally, the covalent bond is a stable covalent bond. Alternatively, the covalent bond is reversible. The covalent bond can be hydrolysable. The or each polymer moiety can be attached adjacent the N-terminal amino acid; adjacent the C-terminal amino acid; or to a naturally occurring amino acid selected from the group including, but not limited to, aspartic acid and tyrosine. Alternatively, the peptide analogue further comprises substitution of a naturally occurring amino acid with an amino acid selected from the group including, but not limited to, lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine; the or each polymer moiety being attached to the or each substituted amino acid. Optionally, the or each polymer moiety is attached adjacent the C-terminal amino acid. Further optionally, the or each polymer moiety is attached to the C-terminal amino acid.
Polyethylene glycol (PEG) is a polymer having the general structure: HO—(CH2—CH2—O)n—H, which is produced from the polymerisation of ethylene glycol monomers (C2H4(OH)2), by the interaction of ethylene oxide with water, ethylene glycol or ethylene glycol oligomers, the reaction being catalysed by acidic or basic catalysts. Polymer chain length is determined by the number of ethylene glycol monomers (n), and is dependent on the ratio of reactants.
The covalent attachment of one or more polyethylene glycol molecules (PEGs) to CCK-8 analogues, or peptide fragments, has been investigated with the goal of improving the pharmacokinetic behaviour of therapeutic drugs. The use of peptide-based therapeutic agents, in particular, is hampered by several disadvantages. Primarily, the peptide is often susceptible to proteolytic enzyme degradation, short circulating half-life, low solubility, and rapid clearance by the kidneys. Such peptides also have a propensity to generate neutralising antibodies. The process of PEGylation can circumvent problems associated with the use of peptide-based therapeutics. The resultant pharmacokinetic outcomes of PEGylation can manifest as changes occurring in overall circulation life span, tissue distribution pattern, and elimination pathway of the attached therapeutic molecule, which can ultimately result in improved pharmacodynamic outcomes.
PEGylation can prolong the circulatory half-life of a protein, allowing the protein to be effective over a longer time. The covalent attachment of PEG to a protein can significantly increase the protein's effective size and hydrodynamic volume, and so reduce its clearance rate from the body, especially via the kidneys. Similarly, the attached PEG molecule can act as a physical barrier to proteolytic enzymes, thereby reducing the enzymatic degradation of the PEGylated protein. However, PEGs are typically of a molecular weight of 20-40 kDa whilst, optionally, the polymer moiety used in the present invention has a molecular weight of no more than 1000 Da.
The invention provides a peptide based on biologically active CCK-8, the peptide having improved characteristics for the treatment of at least one of obesity and type 2 diabetes, wherein the structure of the peptide is:
(Z)-Asp1-Aaa2(X)-Aaa3Gly4Trp5Aaa6Asp7(Y)Aaa8K,
wherein:
Further optionally, at least one of X and K is a polymer moiety of the general formula —O—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22 and Z is an N-terminal extension is selected from the group comprising pGlu, pGlu-Gln, an acid, a fatty acid, Boc, Fmoc and Arg. Still further optionally, at least one of X and K is a polymer moiety of the general formula —O—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22 and Z is pGlu-Gln.
The invention further provides a peptide based on biologically active CCK-8, the peptide having improved characteristics for the treatment of at least one of obesity and type 2 diabetes, wherein the structure of the peptide is:
(Z)-Asp1-Aaa2(X)-Aaa3Gly4Trp5Aaa6Asp1(Y)Aaa8K,
wherein:
The invention further provides a peptide based on biologically active CCK-8, the peptide having improved characteristics for the treatment of at least one of obesity and type 2 diabetes, wherein the structure of the peptide is:
(Z)-Asp1-Tyr2(PO3H2−)-Aaa3Gly4Trp5Aaa6Asp7(Y)Aaa8K,
wherein:
The invention further provides a fragment of the peptide of the invention, wherein the structure of the peptide fragment is:
(Z)-Aaa2(X)-Aaa3Gly4Trp5Aaa6Asp7(Y)Aaa8K,
wherein:
Further optionally, the structure of the peptide fragment is:
(Z)-Aaa2(X)-Aaa3Gly4Trp5 Aaa6Asp7(Y)Aaa8K,
Still further optionally, the invention provides a peptide fragment, wherein said N-terminal modification is selected from the group comprising N-alkylation, N-acetylation, N-acylation, N-glycation, or N-isopropylation at Aaa2. Even still further optionally, Aaa2 is Tyr and said N-terminal modification is selected from the group comprising:
(i) acetylation of Tyr2;
(ii) glycation of Tyr2; and
(iii) acylation of Tyr2 by succinic acid.
Still further optionally, the invention provides a peptide fragment, wherein said N-terminal extension is selected from the group comprising pGlu, pGlu-Gln, an acid, a fatty acid, Boc, Fmoc, Arg and a polymer moiety of the general formula —O—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22. Even still further optionally, wherein said N-terminal extension is selected from the group comprising:
(i) modification of Tyr2 by pyroglutamyl;
(ii) modification of Tyr2 by Fmoc; and
(iii) modification of Tyr2 by Boc.
A further aspect of the invention provides use of at least one of the aforementioned peptides and peptide fragments in the preparation of a medicament to at least one of inhibit food intake, induce satiety, stimulate insulin secretion, moderate blood glucose excursions, enhance glucose disposal and exhibit enhanced stability in plasma compared to native CCK-8.
A further aspect of the invention provides use at least one of the aforementioned peptides and peptide fragments or Asp1-glucitol CCK-8 in the preparation of a medicament for the treatment of at least one of obesity and type 2 diabetes.
A further aspect of the invention provides a pharmaceutical composition including at least one of the aforementioned peptides and peptide fragments.
A further aspect of the invention provides a pharmaceutical composition useful in the treatment of at least one of obesity and type 2 diabetes, which comprises an effective amount of at least one of the aforementioned peptides and peptide fragments in admixture with a pharmaceutically acceptable excipient for delivery through transdermal, nasal inhalation, oral or injected routes. Optionally, the pharmaceutical composition further comprises native or derived analogues of leptin, exendin, islet amyloid polypeptide (IAPP) or bombesin.
A further aspect of the invention provides a method for treating at least one of obesity and type 2 diabetes, the method comprising administering to an individual in need of such treatment an effective amount at least one of the aforementioned peptides and peptide fragments.
The invention will now be demonstrated with reference to the following non-limiting examples and the accompanying figures wherein:
The N-terminal modification of CCK-8 is essentially a three-step process. Firstly, CCK-8 is synthesised from its C-terminal (starting from an Fmoc-Phe-OCH2-PAM-Resin, Novabiochem) up to Met3 on an automated peptide synthesizer (Applied Biosystems, CA, USA). The synthesis follows standard Fmoc peptide chemistry protocols utilizing other protected amino acids in a sequential manner used including Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp-OH, Fmoc-Gly-OH, Fmoc-Met-OH. Deprotection of the N-terminal Fmoc-Met will be performed using piperidine in DMF (20% v/v). The OtBu group will be removed by shaking in TFA/Anisole/DCM. Secondly, the penultimate N-terminal amino acid of native CCK-8 (Tyr(tBu) is added to a manual bubbler system as an alkali labile Fmoc-protected Tyr(tBu)-PAM resin. This amino acid is deprotected at its N-terminus (piperidine in DMF (20% v/v)). This is then allowed to react with excess Fmoc-Asp(OtBu)-OH forming a resin bound dipeptide Fmoc-Asp(OtBu)-Tyr(tBu)-PAM resin. This will be deprotected at its N-terminus (piperidine in DMF (20% v/v)) leaving a free α-amino group. This will be allowed to react with excess glucose (glycation, under reducing conditions with sodium cyanoborohydride), acetic anhydride (acetylation), pyroglutamic acid (pyroglutamyl) etc. for up to 24 hours as necessary to allow the reaction to go to completion. The completeness of reaction will be monitored using the ninhydrin test which will determine the presence of available free α-amino groups. Deprotection of the side-chains will be achieved by shaking in TFA/Anisole/DCM. Sulphation of the N-terminally modified dipeptide will be achieved by suspending the peptide in DMF/pyridine and adding sulphur trioxide-pyridine complex with shaking up to 24 hours. Once the reaction is complete, the now structurally modified N-terminal dipeptide, containing the sulphated Tyr, will be cleaved from the PAM resin (under basic conditions with methanolic ammonia) and with appropriate scavengers. Thirdly, a 4-fold excess of the N-terminally modified-Asp-Tyr(SO3H)—OH will be added directly to the automated peptide synthesizer, which will carry on the synthesis, thereby stitching the N-terminally modified-region to the α-amino of CCK(Met3), completing the synthesis of the sulphated CCK analogue. This peptide is cleaved off the PAM resin (as above under alkaline conditions) and then worked up using the standard Buchner filtering, precipitation, rotary evaporation and drying techniques. The filtrate will be lyophilized prior to purification on a Vydac semi-preparative C-18 HPLC column (1.0×25 cm). Confirmation of the structure of CCK-8 related analogues will be performed by mass spectrometry (ESI-MS and/or MALDI-MS).
The following example investigates preparation of Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 together with evaluation of their effectiveness at inducing satiety and decreasing food intake in vivo. The results clearly demonstrate that these novel analogues exhibit substantial resistance to aminopeptidase degradation and increased biological activity compared with native CCK-8.
Cholecystokinin octapeptide (sulphated CCK-8), pGlu-Gln CCK-8 and other analogues will be synthesised using an Applied Biosystems 432 Peptide synthesizer (as described above). HPLC grade acetonitrile was obtained from Rathburn (Walkersburn, Scotland). Sequencing grade trifluoroacetic acid (TFA) was obtained from Aldrich (Poole, U.K.). All water used in these experiments was purified using a Milli-Q, Water Purification System (Millipore Corporation, Millford, Mass., U.S.A.). All other chemicals purchased were from Sigma, Poole, UK.
Preparation of Asp1Glucitol CCK-8 and pGlu-Gln CCK-8.
Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 were prepared by a 3 step process as described in Example 1. The peptides were purified on a Vydac semi-preparative C-18 HPLC column (1.0×25 cm) followed by a C-18 analytical column using gradient elution with acetonitrile/water/TFA solvents. Confirmation of the structure of CCK-8 related analogues was by mass spectrometry (ESI-MS and/or MALDI-MS). Purified control and structurally modified CCK-8 fractions used for animal studies were quantified (using the Supelcosil C-8 column) by comparison of peak areas with a standard curve constructed from known concentrations of CCK-8 (0.78-25 μg/ml).
Molecular Mass Determination of Asp1Glucitol CCK-8 and pGlu-Gln CCK-8 by Electrospray Ionization Mass Spectrometry (ESI-MS).
Samples of CCK-8 and structurally modified CCK-8 analogues were purified on reversed-phase HPLC. Peptides were dissolved (approximately 400 pmol) in 100 μl of water and applied to the LCQ benchtop mass spectrometer (Finnigan MAT, Hemel Hempstead, UK) equipped with a microbore C-18 HPLC column (150×2.0 mm, Phenomenex, UK, Ltd., Macclesfield). Samples (30 μl direct loop injection) were injected at a flow rate of 0.2 ml/min, under isocratic conditions 35% (v/v) acetonitrile/water. Mass spectra were obtained from the quadripole ion trap mass analyzer and recorded. Spectra were collected in the positive and negative mode using full ion scan mode over the mass-to-charge (m/z) range 150-2000. The molecular masses of positive ions from CCK-8 and related analogues were determined from ESI-MS profiles using prominent multiple charged ions and the following equation Mr=iMi−iMh (where Mr=molecular mass; Mi=m/z ratio; i=number of charges; Mh=mass of a proton).
Effects of CCK-8, Asp1Glucitol CCK-8, pGlu-Gln CCK-8 and Other Peptides on Food Intake in Mice.
Studies to evaluate the relative potencies of control CCK-8, Asp1-glucitol CCK-8, pGlu-Gln CCK-8 and other peptides involved in regulation of feeding were performed using male Swiss TO mice (n=16) aged 7-12 weeks from a colony originating from the Behavioral and Biomedical Research Unit, University of Ulster. The animals were housed individually in an air-conditioned room at 22+−2° C. with 12 h light/12 h dark cycle. Drinking water was supplied ad libitum and standard mouse maintenance diet (Trouw Nutrition, Cheshire, UK) was provided for various times as indicated below. The mice were habituated to a daily feeding period of 3 h/day by progressively reducing the feeding period over a 3 week period. On days 1-6, food was supplied from 10.00 to 20.00 h, days 7-14 from 10.00 to 16.00 h and days 15-21 food was restricted to 10.00 to 13.00 h. Body weight, food and water intake were monitored daily.
Mice which had been previously habituated to feeding for 3 h/day were administered a single i.p. injection of saline (0.9% w/v NaCl, 10 ml/kg) in the fasted state (10.00 h) and food was immediately returned following injection. Two days after the saline injection, mice were randomly allocated into groups of 7-8 animals which were administered a single i.p. injection (from 1 to 100 nmol/kg) of either CCK-8, structurally modified CCK-8 analogues and/or other peptide hormones (including, bombesin, leptin and islet amyloid polypeptide (IAPP)). Food intake was carefully monitored at 30 min intervals up to 180 min post injection. In one series of experiments, the ability of CCK-8 and Asp1-glucitol CCK-8 to inhibit feeding activity was studied in overnight fasted adult obese hyperglycaemic (ob/ob) mice. All animal studies were done in accordance with the Animals (Scientific Procedures) Act 1986.
Effects of Mouse Serum on Degradation of CCK-8, Asp1Glucitol CCK-8 and pGlu-Gln CCK-8.
Serum (20 μl) from fasted Swiss TO mice was incubated at 37° C. with 10 μg of either native CCK-8, Asp1-glucitol CCK-8 or pGlu-Gln CCK-8 for periods up to 2 h in a reaction mixture (final vol. 500 μl) containing 50 mmol/l triethanolamine/HCl buffer pH 7.8. The reaction was stopped by addition of 5 μl of TFA and the final volume adjusted to 1.0 ml using 0.1% (v/v) TFA/water. Samples were centrifuged (13,000 g, 5 min) and the supernatant applied to a C-18 Sep-Pak cartridge (Waters/Millipore) which was previously primed and washed with 0.1% (v/v) TFA/water. After washing with 20 ml 0.12% TFA/water, bound material was released by elution with 2 ml of 80% (v/v) acetonitrile/water and concentrated using a Speed-Vac concentrator (AES 1000, Savant). The volume was adjusted to 1.0 ml with 0.12% (v/v) TFA/water and applied to a (250×4.6 mm) Vydac C-18 column pre-equilibrated with 0.12% (v/v) TFA/water at a flow rate of 1.0 ml/min. The concentration of acetonitrile in the eluting solvent was raised from 0 to 31.5% over 15 min, from 31.5 to 38.5% over 30 min, and from 38.5 to 70% over 5 min, using linear gradients monitoring eluting peaks at 206 nm.
Groups of data are presented as means+−SE. Statistical evaluation was performed using analysis of variance, least significant difference multiple comparisons test and Student's unpaired t-test as appropriate. Differences were considered to be significant if P<0.05.
Following incubation, Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 were clearly separated from native CCK-8 on a Vydac C-18 HPLC column. The average molecular masses of CCK-8 (Mr 1064.2), Asp1-glucitol CCK-8 (Mr 1228.4) and pGlu-Gln CCK-8 (Mr 1352.4) were determined by ESI-MS, confirming their structures.
In Vitro Degradation of CCK-8, Asp1Glucitol CCK-8 and pGlu-Gln CCK-8.
The daily food intake of mice during the period before administration of peptides indicated that mean food consumption of the mice allowed 3 h access to food was 3.8+−0.2 g/mouse. Following administration of i.p. saline, there was no significant difference in 3 h voluntary food intake (3.66+−0.1 g) when compared to 3 h food intake alone.
Dose-response effects of CCK-8, Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 on food intake are shown in
The current study examined the effects of CCK-8, Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 on food intake in mice. The present study demonstrated that CCK-8 was effective in reducing food intake up to 90 min after administration compared to saline controls. The effects of Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 on food intake were investigated and revealed that these amino-terminally modified peptides had a remarkably enhanced and prolonged ability to reduce voluntary food intake compared to an equimolar dose of native CCK-8. The alteration in primary structure by N-terminal modification of CCK-8 appears to enhance its biological activity and extend its duration of action in normal animals from 90 min to more than 3 h. Indeed the results also indicate that a potent satiety effect can persist for more than 5 h in obese diabetic (ob/ob) mice. The change in biological activity encountered with Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 extends previous observations that glycation of peptides can alter their biological activities. It is noteworthy that control experiments conducted with glycated tGLP-1 indicate that the presence of a glucitol adduct on the amino-terminus of a peptide, is insufficient on its own to induce satiety in this test system.
The fact that Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 enhance appetite suppression raises the question of a possible mechanism. Since the very short 1-2 min half-life of CCK-8 is generally accepted as the explanation of the transient satiety effect of the peptide, it is possible that modification of the amino terminus of CCK-8 prolongs the half-life by protecting it against aminopeptidase attack thus enhancing its activity. Aminopeptidase A has been shown to directly degrade CCK-8 in vivo by hydrolysing the Asp-Tyr bond. The peptide can also be degraded by neutral endopeptidase 24.11 (NEP), thiol or serine endopeptidases and angiotensin converting enzyme. The present study revealed that Asp1-glucitol CCK-8 and pGlu-Gln CCK-8 were extremely resistant to degradation by peptidases in serum. Thus it seems likely that protection of the amino terminus of CCK-8 with a glucitol or pyroglutamyl-Gln adduct enhances the half-life of glycated CCK-8 in the circulation and thus contributes to enhancement of its biological activity by extending its duration of action in vivo.
Various mechanisms have been proposed to explain the action of CCK in reducing food intake. One hypothesis is that after ingestion of food, gastric distension and nutrient absorption causes release of CCK-8 which ends feeding. It is proposed that CCK-8 both contracts the pyloric sphincter as well as relaxing the proximal stomach which together delays gastric emptying. The gastric branch of the vagus nerve is closely involved in mediating the action of CCK-8. The satiety signal appears to be transmitted from the vagus nerve to the hypothalamus via the nucleus tractus solitarius and the area postrema.
Although much attention has been given to actions and possible therapeutic use of leptin in obesity and NIDDM, Asp1-glucitol CCK-8, pGlu-Gln CCK-8 or other structurally modified analogues of CCK-8 may potentially have a number of significant attributes compared with leptin. Firstly, there is accumulating evidence for defects in the leptin receptor and post-receptor signalling in certain forms of obesity-diabetes. Secondly, CCK-8 has potent peripheral actions, whereas leptin acts centrally and requires passage through the blood-brain barrier. Thirdly, the effects of CCK-8 on food intake are immediate whereas the action of leptin requires high dosage and is protracted. Fourthly, CCK has been shown to act as a satiety hormone in humans at physiological concentrations and a specific inhibitor of CCK degradation demonstrates pro-satiating effects in rats. It is also interesting to note that the effects of CCK-8 administered together with either leptin, IAPP, exendin(1-39) or bombesin on satiety are additive, raising the possibility of complementary mechanisms and combined therapies.
In summary, this study demonstrates that CCK-8 can be readily structurally modified at the amino terminus and that intraperitoneally administered Asp1-glucitol CCK-8 or pGlu-Gln CCK-8, in particular, display markedly enhanced satiating action in vivo, due in part to protection from serum aminopeptidases. These data clearly indicate the potential of N-terminally modified CCK-8 analogues for inhibition of feeding and suggest a possible therapeutic use in humans in the management of obesity and related metabolic disorders.
We have described that N-terminal modification of CCK-8 endows the molecule with resistance to in vivo enzymatic degradation, thereby substantially increasing its potency as a satiety agent and potential therapeutic agent. Claims were made for a range of N-terminal modifications together with beneficial combinations with other obesity or diabetic drugs. Here we present supporting data to exemplify and extend those claims. This work fully supports the utility of analogues of CCK-8 for treatment of obesity and diabetes. These data show stability/effectiveness of another N-terminal modification —N—Ac—CCK-8; illustrate effects going beyond normal mice, i.e., animals with genetic or diet-induced obesity; demonstrate that inhibition of food intake is sustainable and able to induce significant body weight loss; demonstrate absence of toxic or adverse effects on welfare of animals dosed twice per day for up to 34 days; evidence benefit of 2nd generation modification, i.e., using long-acting-PEGylation; show beneficial effects not only on food intake, body weight but on various parameters of blood glucose control; demonstrate that phosphorylated CCK-8 is an unexpected stimulator of insulin secretion—possibly with therapeutic potential; and show that CCK-8 and pGluGln-CCK-8 stimulate insulin secretion.
The invention will now be demonstrated with reference to the following non-limiting examples and the accompanying figures wherein:
Peptide synthesis: CCK-8 peptides (sulphated form, unless indicated otherwise) were sequentially synthesised with an automated peptide synthesiser using standard solid phase Fmoc procedure. Peptides were purified by reversed-phase HPLC using Vydac analytical columns (The Separations Group, Hesperia, USA). The structure of purified peptides was confirmed by mass spectrometry.
Degradation of CCK-8 and related peptides: To assess the susceptibility of CCK-8 peptides to in vivo degradation, serum (20 μl) from fasted Swiss TO mice was incubated at 37° C. with 10 μg of peptide for various times in a reaction mixture (final vol. 500 μl) containing 50 mmol/l triethanolamine/HCl buffer pH 7.8. The reaction was stopped by addition of 5 μl of TFA and the final volume adjusted to 1.0 ml using 0.1% v/v TFA/water. Samples were centrifuged (13,000 g, 5 min) and the supernatant applied to a C-18 Sep-Pak cartridge (Waters/Millipore) which was previously primed and washed with 0.1% v/v TFA/water. After washing with 20 ml 0.12% TFA/water, bound material was released by elution with 2 ml of 80% v/v acetonitrile/water and concentrated using a Speed-Vac concentrator (AES 1000, Savant). The volume was adjusted to 1.0 ml with 0.12% TFA/water and applied to a (250×4.6 mm) Vydac C-18 column pre-equilibrated with 0.12% TFA/water at a flow rate of 1.0 ml/min. The concentration of acetonitrile in the eluting solvent was raised from 0 to 31.5% over 15 min, from 31.5 to 38.5% over 30 min, and from 38.5 to 70% over 5 min, using linear gradients monitoring eluting peaks at 206 nm. The identity of purified peptides was confirmed by mass spectrometry.
Molecular mass determination of CCK-8 peptides: MALDI-TOF mass spectrometry was carried out using out using a Voyager DE-PRO instrument (Applied Biosystems, Foster City, Calif., USA) that was operated in reflectron mode with delayed extraction. The accelerating voltage in the ion source was 20 kV and α-cyano-4-hydroxycinnamic acid was used as matrix. The instrument was calibrated with peptides of known molecular mass in the 2000-4000 Daltons range. The accuracy of mass determinations was ±0.02%.
pGluGlnCCK-8-PEG (PEG is the covalent attachment of a polymer moiety of the general formula HO—(CH2—O—CH2)n—H, in which n is an integer between 1 and about 22)
Structure: pGlu-Gln-Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-PEG
N—Ac—CCK-8 (Ac is acetyl)
CCK-8, where X is PO3H2
Culture of insulin-secreting cells: Clonal rat insulin-secreting BRIN-BD11 cells were cultured in RPMI-1640 tissue culture medium containing 10% (v/v) foetal calf serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose. The production and characterisation of BRIN-BD11 cells are described elsewhere (McClenaghan et al., 1996). Cells were maintained in sterile tissue culture flasks (Corning, Glass Works, UK) at 37° C. in an atmosphere of 5% CO2 and 95% air using LEEC incubator (Laboratory Technical Engineering, Nottingham, UK). Cell monolayers were used to assess insulin release. The cells were harvested with the aid of trypsin/EDTA (Gibco), seeded into 24-multiwell plates (Nunc, Rosklide, Denmark) at a density of 1.5×106 cells per well, and allowed to attach overnight. Prior to acute test, cells were preincubated for 40 min at 37° C. in a 1.0 ml Krebs Ringer bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM NaHCO3, 5 g/l bovine serum albumin, pH 7.4) supplemented with 1.1 mM glucose. Test incubations were performed for 20 min at 37° C. using the same buffer supplemented with 5.6 mM glucose in the absence (control) and presence of various peptide concentrations. The phosphodiesterase inhibitor, IBMX, was added to preserve cyclic AMP and enhance the natural secretory effects of CCK-8. Insulin was measured by radioimmunoassay.
Animal studies: Initial studies to evaluate the effects of CCK-8 peptides on feeding activity were performed using male Swiss TO mice (aged 7-12 weeks). Other studies used adult ob/ob mice (aged 12-16 weeks). The animals were housed individually in an air-conditioned room at 22±2° C. with 12 h light/dark cycle (08.00-20.00 h light). Drinking water was supplied ad libitum and standard mouse maintenance diet (Trouw Nutrition, Cheshire, UK) was provided as indicated. This normal mouse maintenance diet contains 3.5% fat, 14% protein and 63.9% carbohydrate; 4.5% fibre, crude oil 4.00%, ash 4.7%, and various minerals, amino acids and vitamins makes up the remainder and has a total metabolisable energy content is 13.1 kj/g. In other studies, TO mice were fed synthetic energy-rich high fat diet (45% fat, 20% protein and 35% carbohydrate; percent of total energy of 26.15 kj/g; Special Diets Service, Essex, UK) for up to 35 weeks to induce obesity and glucose intolerance. Some feeding experiments were performed using animals maintained on reverse light cycle (08.00-20.00 h dark).
Acute animal studies: Where indicated, TO mice were gradually habituated to a strict daily feeding regime of 3 h/day by progressively reducing the feeding time over a 3-week period. On days 1-6, food was supplied from 10:00 h to 20:00 h; on days 7-14, food was supplied from 10:00 h to 16:00 h; and on days 15-21 food was supplied from 10:00 h to 13:00 h. This was followed by one week of consistent 3 h daily food intake in which mice received a single i.p injection of saline (0.9% w/v NaCl; 10 ml/kg). For food intake studies, mice habituated to the feeding regime of 3 h/day were randomly allocated into groups. All peptides were dissolved in saline and administered intraperitoneally at the doses described in the legends. Food intake was monitored at 30 min intervals following introduction of food.
Long-term animal studies: Mice allowed unrestricted access to food were injected intraperitoneally with either peptide or saline (control) as described in the Figures. Food intake, body weight and indicators of blood glucose control (glucose tolerance, insulin sensitivity etc) were measured as indicated in the Figures and legends. All animal studies were carried out in accordance with the Animals (Scientific Procedures) Act of 1986.
Determination of plasma glucose and insulin: Plasma glucose concentration was measured by means of an automated glucose oxidase method using a Beckman Glucose Analyzer (Beckman Instruments, UK). Insulin was determined by radioimmunoassay.
Statistical analysis: Results are expressed as mean±S.E.M. Data were compared using Student's t-test or ANOVA followed by a Student-Newman-Keuls post hoc test, as appropriate. Groups of data were considered to be significantly different if P<0.05.
Consistent with our previous observations, HPLC combined with MALDI-TOF mass spectrometry revealed the rapid and extensive degradation of naturally occurring sulphated CCK-8 by incubation with mouse plasma for 120 min (
With this further indication to the efficacy of N-terminally modified CCK-8 analogues, a series of experiments was initiated to examine the effectiveness of these analogues in an animal model of genetic obesity-diabetes rather than in normal mice. This showed that daily administration of pGluGln-CCK-8 significantly inhibited food intake for more than 5 hours after injection (
Having shown efficacy of stable CCK-8 analogues in genetic obesity-diabetes, experiments were performed using normal mice previously maintained on a synthetic high fat energy-rich diet to induce obesity, insulin resistance and glucose intolerance. Such a model more closely resembles the obesity syndromes commonly observed in man. As expected, treatment of such animals with twice daily injection of pGluGln-CCK-8 resulted in substantial body weight loss due to decreased food intake over a period of more than 30 days (
The basic observations made using pGluGln-CCK-8 were fully confirmed by a separate series of experiments in high fat fed mice, which were designed to evaluate the effectiveness of a second generation analogue modified further by PEGylation to augment in vivo potency and in particular durability of biological activity. Twice daily administration of pGluGln-CCK-8-PEG reproduced all of the beneficial effects of pGluGln-CCK-8 on feeding activity, body weight, blood glucose control and insulin sensitivity (
All of the peptides tested in these experiments were based on the naturally occurring sulphated form of CCK-8. Thus removal of the sulphate group resulted in substantial loss of biological activity in terms of inhibition of feeding as shown in
These observations not only evidence the ability of these modified CCK-8 peptides to serve as potent stimulators of insulin secretion, but illustrate that phosphorylated CCK-8 and analogues thereof represent a class of potential new CCK drugs with differential effects on feeding and insulin secretory activity. The insulin output induced by these peptides is approximately equivalent to that induced by the therapeutic incretin hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP).
Overall, this research further exemplifies the potential of stable N-terminally modified analogues of CCK-8 for promotion of satiety, body weight loss and improvement of blood glucose control. Molecules such as N—Ac—CCK-8 and pGluGln CCK-8 have been shown to be stable with long-acting biological effectiveness in genetic and diet-induced obesity-diabetes. Further modification by addition of fatty acid side chain or, as demonstrated here, by PEGylation provides the opportunity to further improve attractiveness of the approach by increasing biological durability. This approach may yield a long-acting form for once or twice-weekly injection. These attributes together with the small size of the molecule, which may facilitate trans-cutaneous administration, make peptidergic CCK-8 analogues a particularly attractive means of harnessing the therapeutic power of the CCK receptor for treatment of obesity, metabolic syndrome, glucose intolerance and obesity.
The invention is not limited to the embodiment(s) described herein but can be amended or modified without departing from the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0105069.9 | Mar 2001 | GB | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/GB02/00827 | Feb 2002 | US |
| Child | 10469655 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10469655 | Feb 2004 | US |
| Child | 12177306 | US |
