Long lasting glucagon-like peptide 2 (glp-2) for the treatment of gastrointestinal diseases and disorders   

FIELD OF THE INVENTION

This invention relates to glucagon-like peptide 2 (GLP-2) derivatives. In particular, this invention relates to GLP-2 peptide derivatives having an extended in vivo half-life, for the treatment or prevention of gastrointestinal disorders or diseases such as inflammatory bowel disease and other gastrointestinal functions, from any segment of the gastrointestinal tract, from the oesophagus to the anus.

BACKGROUND OF THE INVENTION

GLP-2 is a 33 amino acid peptide expressed in a tissue-specific manner from the pleiotropic proglucagon gene, and thus part of the glucagon super-family of peptide hormones. Alternative post-translational processing of proglucagon occurs in pancreas, intestine and brain. Enzymatic cleavages in proglucagon produce numerous multifunctional peptide hormones involved in nutrient metabolism. The major bioactive hormones derived from proglucagon are in the pancreatic α-cells, and GLP-1 and GLP-2 in the intestinal L-cells and brain. It was first discovered to possess potent intestinotropic properties by Drucker et al. (see Proc. Natl. Acad. Sci., 1996, 93, (115), 7911-7916). GLP-2, as a natural intestinal-derived peptide, has been demonstrated to have a significant reparative activity for the mucosal epithelium of the small and large intestine. It has also been demonstrated to increase the ability of the intestine to digest and absorb nutrients, suggesting a potential therapeutic role in the treatment of intestinal insufficiency. Indeed, several studies have now confirmed that GLP-2 administration reduces or prevents intestinal damage in rodent models of colitis, enteritis, total parenteral nutrition and massive resection. Very recently, phase 2 clinical trials of GLP-2 have also been reported, in which patients with short bowel syndrome were demonstrated to exhibit an enhanced ability to absorb enteral nutrients after 30 days of GLP-2 administration, with apparently no undesirable side effects.

The principal metabolic pathway for GLP-2 clearance is through enzymatic degradation. GLP-2 has been shown to be rapidly degraded through the removal of its two N-terminal amino acids by dipeptidylpeptidase-IV (DPP-IV), which represents a major limitation because it leads to the complete inactivation of the peptide. As a result, the half-life of GLP-2 is thus quite short, and current GLP-2 treatment necessitates infusion or frequent injections. Renal clearance has also been shown to be involved in the clearance of GLP-2. The major action of GLP-2 involves stimulation of cell growth, and the mechanism coupling GLP-2 receptor activation, directly or indirectly, to cell is proliferation has not been examined.

It has been shown that peptide analogs of native GLP-2 possess enhanced trophic activity at the small intestine as GLP-2 receptor agonists (see for example U.S. Pat. No. 5,990,077).

Although very useful, a critical disadvantage of GLP-2 peptides and analogs, as stated above, is their very short half-lives in vivo, which is typically not more than 2 minutes.

Inflammatory Bowel Disease (IBD) is a group of chronic disorders that cause inflammation or ulceration in the small and large intestines. It may even be life threatening, and there is currently no known cure. IBD commonly refers to ulcerative colitis (UC), limited to the colon and Crohn\'s disease (CD) which can involve the entire gastrointestinal tract, resulting in a chronic cycle of remissions and flares. Many pharmaceutical products are known to treat IBD, for example to suppress inflammation, to prevent flare-ups, to control symptoms such as pain or diarrhea, or to replace or supplement essential nutrients that are poorly absorbed because of extensive disease or surgery. Current treatment options include a wide variety of pharmaceutical products like aminosalicylates, corticosteroids, immune modulators, and anti-TNF-α agent, and are designed to reduce inflammation and relieve symptoms in addition to replacing lost fluids and nutrients. Most of these products however have a limited use in IBD because of their undesirable side effects on the body in general.

Approximately one million patients are treated for IBD every year in the United States and Europe, most of them generally suffering from either Crohn\'s disease or ulcerative colitis. In fact, it is not uncommon for subjects suffering from IBD to undergo radical surgery involving the removal of major parts of the intestine. Direct annual healthcare costs of IBD are approximately $US 700 million, and the total economic impact of both direct and indirect costs approximate between $2 and $3 billion a year worldwide. Existing treatments, while often providing relief, have some shortcomings.

U.S. Pat. No. 5,789,379 teaches GLP-2 analogs among which one has been developed as a long-acting compound (ALX-600™) and is currently in clinical trials. However, even if DPP-IV degradation of GLP-2 appears to be somehow prevented, its half-life remains limited by renal clearance, and does not exceed 2 minutes, as stated above.

A chimeric antibody (Remicade™) has been developed to bind specifically to human tumor necrosis factor alpha (TNF-α) for the short-term treatment of Crohn\'s. This antibody is indicated for the reduction of the symptoms of moderate to severe Crohn\'s disease in patients who have had an inadequate response to conventional therapy with corticosteroids, other immunosuppressants and/or antibiotics. Nevertheless, serious side effects are observed with such treatment. For example, it has been associated with hypersensitivity reaction, serious infections including sepsis, as well as fatal infections. Its administration could further predispose patients to infections through TNF-blocking.

With the prevalence of IBD increasing in recent years, it would therefore be highly desirable to develop GLP-2 peptide derivatives or analogs capable of substantially maintaining the same level of activity, low toxicity and therapeutic advantages as GLP-2, but with a much longer in vivo half-life, thus avoiding the necessity for continuous administration thereof in the treatment of various diseases such as inflammatory bowel disease, Crohn\'s disease and ulcerative colitis representing the two major inflammatory bowel diseases.

SUMMARY

In accordance with the present invention, there is now provided a GLP-2 gastrointestinal tissue growth promoter derivative having an extended in vivo half-life when compared with the corresponding unmodified GLP-2 gastrointestinal growth promoter. More specifically, the GLP-2 derivative comprises a reactive entity coupled thereto and capable of reacting with available functionalities on a blood component, either in vivo or ex vivo, to form a stable covalent bond. The covalent bonding formed between the GLP-2 derivative and the blood component prevents undesirable cleavage of the GLP-2 by enzymes such as dipeptidylpeptidase IV, thereby extending its in vivo half-life and activity. The reactive entity may be on the N-terminal of the GLP-2 peptide, the C-terminal of the GLP-2 peptide, or on any other available site along the peptidic chain.

Preferred blood components comprise proteins such as immunoglobulins, including IgG and IgM, serum albumin, ferritin, steroid binding proteins, transferrin, thyroxin binding protein, α-2-macroglobulin, haptoglobin etc., serum albumin and IgG being more preferred, and serum albumin being the most preferred.

Preferred reactive entity are capable of forming a covalent bond with the blood component by reacting with amino groups, hydroxy groups or thiol groups present thereon, either in vivo or in vitro (or ex vivo). In a most preferred embodiment, the functionality on the protein will be a thiol group and the reactive entity will be a Michael acceptor, such as acrolein derivatives, haloacetates, haloacetamides, α,β-unsaturated ketones, α,β-unsaturated esters, α,β-unsaturated amides, α,β-unsaturated thioesters, and the like, maleimide or maleimido-containing group such as γ-maleimide-butyrylamide (GMBA) or maleimidopropionic acid (MPA), MPA being the most preferred.

In another aspect of the invention, there is provided a pharmaceutical composition comprising the present GLP-2 gastrointestinal tissue growth promoter derivative in combination with a pharmaceutically acceptable carrier. Such composition is useful for the treatment or prevention of bowel disorders or diseases such as inflammatory bowel disease and other gastrointestinal functions. The composition may also be used for gene therapy to induce cells to endogenously produce the gastrointestinal tissue growth promoter peptide derivative that may then be implanted in a subject to produce the desired biological effect. Finally, the composition may also be used for manufacturing pharmaceutical or veterinary compositions for the enhancement of large intestine tissue growth.

In a further embodiment of the present invention, there is provided a method for the treatment of prevention of bowel disorders or diseases such as inflammatory bowel lo disease, and gastrointestinal functions. The method comprises administering to a subject, preferably a mammal, animal or human, an effective amount of the present GLP-2 gastrointestinal tissue growth promoter derivative or a conjugate thereof, alone or in combination with a pharmaceutically acceptable carrier.

In a further aspect of the present invention, there is provided a conjugate comprising the present GLP-2 gastrointestinal tissue growth promoter derivative covalently bonded to a blood component.

In a further aspect of the present invention, there is provided a method for extending the in vivo half-life of a GLP-2 gastrointestinal tissue growth promoter in a subject, the method comprising covalently bonding the GLP-2 gastrointestinal tissue growth promoter derivative to a blood component. The covalent bonding may take place in vivo or in vitro.

Preferred gastrointestinal tissue growth promoter compounds are peptides such as GLP-2 and GLP-2 analogs, GLP-2 fragments, provided that such analog or fragment possesses gastrointestinal tissue growth promoting activity. Details of the sequences of these peptides, analogs and fragments are illustrated below.

A further use of the present compound derivative may be the determination of the intestinotrophic activity of a hormone when used in combination with the present compound derivative, and particularly when the compound is GLP-2, a GLP-2 analog or a GLP-2 fragment. Such method comprises the steps of: (a) coadministering the hormone with an intestinotrophic amount of the GLP-2 derivative to a test subject; (2) assessing the subsequent growth of small and large intestine tissue in the test subject; and (3) determining whether the growth of small and/or large intestine tissue in the test subject is enhanced relative to control subjects treated with unmodified GLP-2, GLP-2 analog or GLP-2 derivative.

If a linking group is present, it is preferably defined as, without limitation, a straight or branched C1-10 alkyl; a straight or branched C1-10 alkyl partly or perfluorinated; a C1-10 alkyl or fluoroalkyl wherein one or more carbon atom is replaced with O, N or S to form an ether or a thioether, o-, m- or p-disubstituted phenyl wherein the substituents are the same or different and are CH2, O, S, NH, NR wherein R is H, C1-10 alkyl or C1-10 acyl; or disubstituted heterocycles such as furan, thiophene, pyran, oxazole, or thiazole.

FIG. 1 illustrates the changes in small intestine wet weight in mice treated with saline or 5 μg of the compounds of Examples 1-8 twice daily, for 10 days (n=10/group). Results are expressed as mean delta weight vs. control±SEM.

FIG. 2 illustrates changes in small intestine and large intestine wet weight in mice treated with saline or 5, 25 or 50 μg of the compounds of Examples 5, 7 and 8, twice daily, for 10 days (n=10/group). Results are expressed as mean weight±SEM.

FIG. 3 illustrates the mean plasma concentrations for the compounds of Examples 5 and 8 in Sprague-Dawley rats following a single 500 nmol/kg intravenous or subcutaneous dose (n=4/group).

FIG. 4 illustrates detection of the compound of Example 8 conjugated to rat plasma proteins using a polyclonal antibody anti-GLP-2 antibody and comparison to the pattern obtained with an anti-rat albumin.

DETAILED DESCRIPTION

In vivo bioconjugation is the process of covalently bonding a molecule, such as the present gastrointestinal tissue growth promoter compound derivative, within the body, to target blood components, preferably proteins, in a manner that permits the substantial retention, or increase in some instances, of the biological activity of the original unmodified GLP-2 gastrointestinal tissue growth promoter peptide therein, while providing an extended duration of the biological activity though giving the GLP-2 derivative the biophysical parameters of the target blood component.

For the purposes of the present invention, the terms “analog” or “fragment” are meant to include amino acid sequences comprising peptides with different amino acid sequences from the native sequence, such as the GLP-2 sequence, but with similar or comparable activity. Such analogs preferably have an amino acid sequence at least 60%, and more preferably at least 80%, and most preferably at least 95% the same as that of either GLP-2 or a fragment of GLP-2 having the same number of amino acid residues.

In a more preferred embodiment, the present gastrointestinal tissue growth promoter peptide derivative comprise a GLP-2 gastrointestinal tissue growth promoter peptide that has been modified by coupling thereto a reactive entity, either directly or via a linking group, the reactive entity being capable of forming a covalent bond with a blood component, preferably blood proteins. The reactive entity must be stable in an aqueous environment, and preferred embodiments thereof comprise carboxy group, a phosphoryl group, an imidate group, or an acyl group either as an ester or a mixed anhydride. The covalent bond is generally formed between the reactive entity and an amino group, a hydroxy group, or a thiol group on the blood component. The amino group preferably forms a covalent bond with reactive entities like carboxy, phosphoryl or acyl; the hydroxy group preferably forms a covalent bond with reactive entities like activated esters; and the thiol group preferably forms a covalent bond with reactive entities like esters or mixed anhydrides. The preferred blood component comprises mobile blood components like serum albumin, immunoglobulins, or combinations thereof, and the preferred reactive entity comprises anhydrides like maleimide groups. In a most preferred embodiment, the blood component is serum albumin.

The blood components are preferably mobile, which means that they do not have a fixed situs for any extended period of time, generally not exceeding 5 minutes, and more usually one minute. These blood components are not membrane-associated and are present in the blood for extended periods of time in a minimum concentration of at least 0.1 μg/ml. Preferred mobile blood components include serum albumin, transferrin, ferritin and immunoglobulins such as IgM and IgG. The half-life of mobile blood components is at least about 12 hours.

The present gastrointestinal tissue growth promoter derivative is a GLP-2 peptide, and therefore protective groups may be required during the synthesis process of the GLP-2 derivative. These protective groups are conventional in the field of peptide synthesis, and can be generically described as chemical moieties capable of protecting the peptide derivative from reacting with other functional groups. Various protective groups are available commercially, and examples thereof can be found in U.S. Pat. No. 5,493,007 which is hereby incorporated by reference. Typical examples of suitable protective groups include acetyl, fluorenylmethyloxycarbonyl (FMOC), t-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), etc. Table 1 provides both the three letter and one letter abbreviations for amino acids.

TABLE 1 NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS 3-letter 1-letter Name abbreviation abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The present GLP-2 derivative forms a peptidase-stabilized peptide after conjugation to a blood component. It is also contemplated that one or more additional amino acids may be added or substituted to the peptide prior to addition of the reactive entity, to facilitate the coupling thereof to the peptide. Such addition or substitution may be made at the N-terminal, the C-terminal, or therebetween. The thus obtained peptide derivative may be administered to a subject, animal or human, such that conjugation with blood components occurs in vivo, or they may be first conjugated to blood components of the subject, animal or human, in vitro, and the resulting conjugate, or peptidase-stabilized peptide as defined below, administered to the subject.

Any amino acid, present, substituted with or added to, the GLP-2 sequence, may be D-amino acids or L-amino acids or combinations thereof. L-amino acids are generally preferred. A glycine substitution at position 2 of the native GLP-2 sequence represents a preferred embodiment, because it confers to the analog a greater resistance to DPP-IV enzyme. Glycine may also be replaced with D-alanine or proline for that same purpose. In addition, a N-α-methyl aspartic acid substitution at position 3 of the native GLP-2 sequence can achieve the same result, as well as other peptide mimetics such as methyl amino, hydroxyl ethyl, hydrazino, ethylene or sulfonamide as isosteric replacement of the amide bond.

The invention also includes GLP-2 fragments which, although containing a sequence that is substantially homologous to that of a naturally occurring GLP-2 peptide, may lack one or more additional amino acids at their amino and/or their carboxy termini that are naturally found on a GLP-2 native peptide. Thus, the invention pertains to polypeptide fragments of GLP-2 that may lack one or more amino acids that are normally present in a naturally occurring GLP-2 sequence provided that such polypeptides have gastrointestinal tissue growth promoting activity which preferably at least substantially equals that of GLP-2.

The invention also encompasses the obvious or trivial variants of the above-described analogs or fragments which have inconsequential amino acid substitutions (and thus have amino acid sequences which differ from that of the natural sequence) provided that such variants have gastrointestinal tissue growth promoting activity which is substantially similar to that of GLP-2. Examples of obvious or trivial substitutions include the substitution of one basic residue for another (i.e. Arg for Lys), the substitution of one hydrophobic residue for another (i.e. Leu for Ile), or the substitution of one aromatic residue for another (i.e. Phe for Tyr), etc. Further, other trivial variants include analogs wherein conservative substitutions resulting in a substantial structural analogy of the original sequence are obtained. Examples of such conservative substitutions, without limitation, include glutamic acid for aspartic acid and vice-versa; glutamine for asparagine and vice-versa; serine for threonine and vice-versa; lysine for arginine and vice-versa; or any of isoleucine, valine or leucine for each other.

A peptidase-stabilized GLP-2 derivative is more stable in the presence of peptidases in vivo than the corresponding non-stabilized GLP-2 analog. The peptidase stability is determined by comparing the half-life of the native GLP-2 analog in serum or blood to the half-life of the corresponding derivative containing the reactive entity in serum or blood. Half-life is determined by sampling the serum or blood after administration of the derivative and the non-modified peptide, and determining the activity of each compound.

In greater details, the present invention is directed to the modification of GLP-2 and analogs and fragments thereof to improve its bioavailability, extend in vivo half-life and distribution through selective conjugation onto a blood component while substanitally maintaining or improving their remarkable therapeutic properties.

Human GLP-2 is known to have the following sequence:

His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr- Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn- Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp.

The invention relates to therapeutic and related uses of GLP-2 derivatives having an extended half-life in vivo, particularly to promote the growth of small and/or large intestine tissue;

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