AU753771B2 - Glucagon-like peptide-2 analogs - Google Patents

Description

GLUCAGON-LIKE PEPTIDE-2 ANALOGS The present application is a divisional application from Australian patent application number 733857 (25002/97) the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION This invention relates to glucagon-related peptides having intestinal tissue growth promoting properties, and to their use therapeutically to treat various medical conditions resulting from the impaired growth or loss of such tissue.

BACKGROUND TO THE INVENTION Expression of the glucagon gene yields a tissue-determined variety of peptide products that are processed from the 160 residue proglucagon product.

The organization of these peptides within the proglucagon precursor was elucidated by the molecular cloning of preproglucagon cDNAs from the rat, hamster and bovine pancreas. These analyses revealed that preproglucagon contains not only the sequence of glucagon and glicentin, but also two additional glucagon-like peptides (GLP-1 and GLP-2) separated from glucagon and each other by two spacer or intervening peptides (IP-I and IP-II). These peptides are flanked by pairs of basic amino acids, characteristic of classic prohormone cleavage sites, "suggesting they might be liberated after posrtranslanonal processing of proglucagon (Drucker, Pancreas, V1990, 5(4):484).

Analysis of the peptides liberated from proglucagon in the pancreatic islets S 25 of Langerhans, for instance, suggests the primary pancreatic peptide liberated is the 29-mer glucagon. whereas glicenin, oxyntomodulin, IP-II and the glucagon-like peptides are more prevalent in the small and large intestines. This demonstration that the glucagon-like peptides are found in the bowel has prompted research into the precise structure and putative function(s) of these newly discovered gut 30 peptides. Most studies have focussed on GLP-1, because several lines of evidence suggested that GLP-1 may be an important new regulatory peptide. Indeed, it has been determined that GLP-1 is one of the most potent known peptidergic stimulus for insulin release, an action mediated in a glucose-dependent manner through interaction with receptors on pancreatic B cells. GLP-1 and its derivauves are in development for use in the treatment of diabetics.

The physiological roles of glicentin and oxyntomodulin, the so-called "enteroglucagons". are also under investigation, particularly with respect to regulation of acid secretion and the growth of inesdinal cells. Oxyntomoduln is capable of inhibiting penragastrin-stimulated gastric acid secretion in a dosedependentm inannr. The role of glicemnin in mediating the changes of intestinal adaptation and growth of the intesrinal mucosa has been investigated, and the inestinorrophic effect of glicendin and its therapeutic use have recently been reported by Matsuno et al in EP 612,531 published August 31, 1994.

In contrast to GLP-1 and other glucagon-related peptides, the physiological role of glucagon-like peptide GLP-2 remains poorly understood despite the isolation and sequencing of various GLP-2 homologues including human, rat.

bovine, porcine, guinea pig and hamster. Using GLP-2 antisera raised against synthetic versions of GLP-2, various groups have determined that GLP-2 is present primarily in intestinal rather than pancreatic extracts (see Mojsov et al, J. Biol.

Chem., 1986, 261(25):11880; Orskov et al in Endocrinology, 1986, 119(4)-1467 and in Diabetologia, 1987, 30:874 and in FEBS Letters, 1989, 247(2)-193.

George et al, FEES Leaers. 1985, 192(2):275). With respect to its biological role, Hoosein et al report (FEBS Letters, 1984, 178(1):83) that GLP-2 neither competes with glucagon for binding to rat liver and brain tissues, nor stimulates adenylate cyclase production in liver plasma membranes, but, enigmatically, can stimulate adenylate cyclase in both rat hyopthalamic and pituitary tissue, at 30-50pM concentrations. An elucidation of the physiological role of GLP-2 would clearly be 25 desirable.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.

SUMMARY OF THE INVENTION There have now been discovered analogs of GLP-2 which promote growth of small bowel tissue. It is accordingly a general object of the present invention to 30 provide such GLP-2 analogs and to provide for their use therapeutically and for related purposes.

In one aspect of the invention, the GLP-2-analogs exhibit intestinotrophic acuvity and conform to the structural Formula 1 (SEQ ID NO: 1): 3 Rl-(YI)m-X1-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(Pl)-Leul4-Aspl5-Asnl6- Leul7.Alal8-X19-X)20-Asp21-Phe22-(P2)-Trp25-Leu26-Ile27-CGn28-Thr29- Lys30-(P3)-(Y2)n-R2, wherein X1 is His or Tyr; X2. .s Ala or an Ala-replacement amino acid conferring on said analog resistance to DPP-IV enzyme; X3 is Pro, HPro, Asp or Glu; X4 is Gly or Ala; P1 is Glu-XlO-Asn-Thr-Ule or Tyr-Ser-Lys-Tyr; is Met or an oxidatively stable Met-replacement amino acid; X19 is Ala or Thr; is Arg, Lys, His or Ala; P2 is Ile-Asn, Ile-Ala or Val-Ghn P3 is a covalent bond, or is le, Ile-Thr or Ile-Thr-Asp; RI is H or an N-terminal blocking group; R2 is OH or a C-terminal blocking group; Y1 is one or two basic amino acids selected from the group Arg. Lys, and His; Y2 is one or two basic amino acids selected from the group Arg, Lys, and His; and m and n, independently, are 0 or 1: and wherein at least one of Xl, X2, X3, X4, P1, X10, X19, X20, P2 and P3 is other than a wild type, mammalian CLP-2 residue.

25 Particularly preferred analogs according to Formula 1 (SEQ ID NO: 1) are those which are rendered resistant to cleavage by human DPP-IV enzyme by replacing the Ala at position X2 with an alternative amino acid. Other analogs of the invention are those which replace the oxidatively sensitive Met at position .with an amino acid residue which is oxidatively stable. In this manner, the analog 30 peptides have increased stability compared to GLP-2 peptides with the wile-type Met residue at this position. Yet another preferred embodiment of the invention is 'the incorporation at position X20 of a basic amino acid selected from His or Lys.

This substitution is advantageous when the CLP-2 analogs are chemically *synthesized. The Arg residue which 11o11a11Y occ3 at this Position tends to strongly bind solvents used in peptide synthesis procedures. Substitution of the Arg allows easier formulation of the synffheticallY produced GI.P-2 analogs into pharmaceutirallY acceptable coinpOStitOlis.

More particularly, and according to one aspect of the invenion, there are provided analogs of a GLP-2 peptide selected fronm a mammalian GLP-2 species and N- and/or C-teriinally modified forms thereof, the analogs having intestinorophic activity and incorporating, relative to said mamralian GLP-2 peptide, as least one amin acid substitution at a position which is conserved in mammalian GLP-2's. In a preferred aspect. the GLP-2 analogs incorporate a substitution selected from: 1) incorporation at position 2 or at position 3 a replacement amino acid conferring on said analog resistanice to Dipeptidyl Peptidase-IV (hereinafter referred to as l)PP-IV; and 2) incorporation at position 10 of an oxidatively stable Met-replacement amino acid; and 3) incorporation at X2.0 of a replacen~t amino acid other thani Ag.

In still another of its aspects, te invention provides intestinotrophic analogs of a mammalian GLP-2, preferably human CII--2, in which ether the N- and/or .*20 C-termnus is modified .by a blocking group, or additional amino acids, or the substitution of a modifid or alternative amino acid. In yet another aspect, tere arm provided intestinotrophic analogs of mnammalian GLP-2, preferably human GLP-2, in which positions Xl, X5, X7, X16-X18, X25, X30, or X33 incorporate a replacenmn amino acid other than that found in naturlly occurrig mammalian GLP-2's. optionally, in a fther aspect, the invention provides intstinorrophic, analogs in which from 3 to 8 residues are deleted from t C-=erinus.

In another f its aspects, the invention provides a pharmaceutical composition comprising a GLP-2 analog of the present invention in a thierapeutically effective amount and preferably in an inetnotrophic am~ount, and a pharmaceutically acceptable carrier.

In a fther aspect, the invention provides a metho for promoting growth of small bowel tissue in a patient in need thiereof, comprising the step of delivering 1 to the patient an intestinoTrophic amount of a GLP-2 analog of the present invention.

Besides promoting bowel growth, in another of it aspects the invention provides a method for treating a gastrointestinal disease by administering to a patient suffering from gastrointestinal disease a therapeutically effective amount of a GLP-2 analog of the invention, together with a pharmaceutically acceptable carrier, in order to reduce a pathological effect or symptom of the gastrointestinal disease.

In still another aspect of the invention, there is provided a method useful to identify intestinotrophic analogs of GLP-2, comprising the steps of.

1) obtaining a GLP-2 analog conforming to Formula 1 (SEQ ID NO: 1) represented above; 2) treating a mammal with said analog using a regimen capable of eliciting an intestinotrophic effect when utilized for rat GLP-2; and 3) determining the effect of said analog on small bowel weight relative to a mock treated control nummal, whereby said intestinotophic analog of GLP-2 is identified as an analog which elicits an increase in said weight.

DETAILED DESCRIPTION CF THE INVENTION The invention relates to therapeutic and related uses of CLP-2 analogs, "particularly for promoting growth of tissue of the small bowel. The effect on growth elicited by the present GLP-2 analogs manifests as an increase in small bowel weight, relative to a mock-treated control. In particular, GLP-2 analogs are 25 considered to have 'intestinotrophi" activity if, when assessed in the muine "model exemplified herein, the analog mediates an increase in small bowel weight of at least 10% relative to a control animal receiving vehicle alone. Particularly suitable for therapeutic use are those analogs which mediate an increase of at least 20% in small bowel weight; preferred for therapeutic use are those which mediate an increase in small bowel weight of 50% or more. Intestinotrophic -activity is noted most significantly in relation to the jejun=m, including the distal "jejunum and particularly the proximal jejunum, and is also noted in the ileum.

In addition to exhibiting intestinotrophic activity as just defined, the GLP- 2 analogs of the present invention incorporate an arnino acid substitution at one or more sites within a GLP-2 peptide "background", which is either a mammalian GLP-2 species per se, or is a variant of a mammalian GLP-2 species in which the C-terminus and/or the N-terminus has been altered by addition of one or two basic residues, or has been modified to incorporate a blocking group of the type used conventionally in the art of peptide chemistry to protect peptide termini from undesired biochemical attack and degradation inaivo. Thus, the present peptides incorporate an amino acid substitution in the context of any mammalian GLP-2 species,.incuding but not limited to human GLP-2, bovine GLP-2, rat GLP- 2, degu CLP-2,.ox GLP-2, porcine GLP-2, guinea pig GLP-2 and hamster GLP-2, the sequences of which have been reported by many authors, including Buhl er al, J. Biol. Chem., 1988,263(18):8621.

In one aspect of the invention, the intestinotrophic analogs of GLP-2 conform to the sequence of Formula 1 (SEQ ID NO: 1) as follows: R1-(Yl)m-Xl-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(Pl)-Leul4-Aspl5-Asnl6- Leul7-Alal8-X19-X20-Asp21-Phe22-(P2)-Trp25-Leu26-Ile27-Cln28-Thr29- Lys30-(P3)-(Y2)n-R2, wherein 20 X1 is His or Tyr; X2 is Ala or an Ala-replacement amino acid conferring on said analog resistance to DPP-IV enzyme; X3 is Pro, HPro, Asp or Glu; X4 is Gly or Ala; P1 is Clu-X1O-Asn-Th-Ulle or Tyr-Ser-Lys-Tyr, X10 is Met or an oxidatively stable Met-replacement amino acid; X19 is Ala or Thr; X20 is Arg, Lys, His orAla; P2 is Ile-Asn, Ile-Ala or Val-Gin; P3 is a covalent bond, or is le, Ile-Thr or Ile-ThrAsp; Ri is H or an N-terminal blocking group; R2 is OH or a C-terminal blocking group; YI is one or two basic amino acids selected from the group Arg, Lys, and His; Y2 is one or two basic amino acids selected from the group Arg, Lys, and His; and m and n, independently, are 0 or 1; and wherein at least one of XI, X2, X3, X4, P1, X10, X19, X20, P2 and P3 is other than a wild type, mammalian GLP-2 residue.

Wild type mammalian GLP-2 residues which occur at a specific position are determined by aligning the sequences of GLP-2's isolated from different mammalian species and comparing the sequence to the human sequence (SEQ ID NO: reproduced below, for convenience: His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn- 1 5 Thr-11e-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phle- 15 lle-Asn-Trp-Leu-le-Gln-Thr-Lys-Ile-Thr-Asp The amino acid residues which, for purposes of this application, are known to occur at specific positions in wild type mammalian CLP-2's are the following: 2 0 position X13 may be lie or Val; position X16 may be Asn or Ser; position X19 may be Alanine or Threonine; position X20 may be Arg or Lys; position X27 may be lie or Leu; and position X28 may be Gin or His.

The present GLP-2 analogs may incorporate desired amino acid substitutions into a "background" which is an N-terminally or C-terminally modified form of a mammalian GLP-2 peptide. Such analogs are represented in Formula I (SEQ ID NO: 1) as those in which RI constitutes an N-terminal blocking group, and/or when m is I then Y1 is one or two basic amino acids such as Arg or Lys; and/or R2 is a C-terminal blocking group; and/or when n is 1I then Y2 is independently, one or two basic amino acids such as Arg or Lys.

In preferred embodiments of the invention, the GLP-2 analog is an analog of full length GLP-2, ie., GLP-2(1-33), and P3 is accordingly the sequence Ile-Thr- Asn. Alternatively, the GLP-2 analogs may be C-terminally truncated, to yield o GLP-2(1-32) forms in which P3 is lle-Thr, or G1.P-2(1-31) forms in which P3 is le, or GLP-241-30) forms in which P3 is a covalent bond.

The 'blocking groups" represented by RI and R2 are chemical groups that are routinely used in the art of peptide chemistry to confer biochemical stability and resistance to digestion by exopeptidase. Suitable N-terminal protecting groups include, for example, CI- 5 aUkanoyl groups such as acetyl. Also suitable as N-termintal protecting groups are amino acid analogues lacling the amino function, for example, desamino-Tyri. Suitable C-ternminal protecting groups include groups which form ketones or amnides at the carbon atom of the C-terminal carboxyl, or groups which form esters at the oxygen atom of the carboxyl. Ketone and ester-forming groups include alkyil groups, particularly branched or unbranched

C

1 5 alkyl groups, methyl, ethyl and propyl groups, while amideforming groups include amino functions such as primary amine, or alkylamino7 functions, mnono-C 1 5 alkylaino and di-C 1 5 alkylairiino groups such as methylamnino, ethylamino, dintethylarrino, diethylainino, rnethylethylamino and the likce. Amino acid analogues are also suitable for protecting the C-terminal end of the present compounds, for example, decarboxylated amino acid analogues such as agmnatine.

Embodiments of the invention specifically include such analogs in which m.

isO0 and RI is a blockcing group such as acetyl; and analogs in which mis 0Oand R2 is a C-terminal blockIng group such as an an-dde or an amidne, -Nli2.

In a preferred aspect of the invention, the GL.P-2 analogs are analogs of either human GLP-2 or of rat GLP-2. Hun-an GLP-2 is herein referred to interchangeably as hGLP-2(1-33). Rat GLP-2 has the amidno acid sequence of human GLP-2, but incorporates at position 19 a Thr residue instead of an Ala residue. Rat GLP-2 is accordingly referenced herein either as rGLP-2(1-33) or as the Thin" analog of human Gt.P-2, as [r'jhGLP-2Cl-33).

in particularly preferred embodiments of the invention, with respect to both the Formula I (SEQ ID NO: 1) analogs and the more specific human or rat ~3 LP-2 analogs, the GL?-2 analogs incorporate an ammno acid substitution selected .***from; 1) incorporation at X2 and/or at X3 of a replacement am-ino acid which renders said analog resistant to cleavage by DPP-IV enzyme; 2) incorporation at XIO of an oxidatively stable Met-replacement amino acid; and 3) incorporation at X20 of a replacement amnio acid othier than Arg.

In still another of its aspects, the invention provides unestinotrophic analogs of a mammalian GLP-2, preferably human GLP-2. in which either the N- andlor c-termius; is modified by a blocing group, or additional amino acids, or the substitution of a modified or alternative amino acid. In yet another aspect, there are provided intestinotrophic analogs of manmmalian GL.P-2, preferably human GLP-2, in which positions; X1, X5, X7, X16-X1S, X25, X30, or X3 3 incorporate a replacement amino acid other than that found in naturally occurring mammalian GLP-2's. Optionally, in a further aspect, t invension provides intestinoirophic analogs in which from 3 to 8 residues are deleted from the C-termns The D)PP-IV-resistant class of GLP-2 analogs possess part *icularly advantageous properties. As is demonstrated herein, mammn~alian GLP-2 species have been found to be sensitive to cleavage by DPP-IV enzyme. It has also been found that this sensitivity to DPP-IV is t result of the recogniton sequence: Alae As%? found in all mammalian forms of GLP-2. There are accordingly provided by the present invention a class of GLP-2 analogs which incorporate at X2 and/or X3 a replacemezit amino acid. which confers on the GLP-2 analog relative resistance to DPP-IV mediated cleavage, as deterined by any convenient in vkr or in vivo assessment technique that is able to detect the presence of GLP-2 digestion products. A DPP-IV resistant GLP-2 analog is revealed as that GLP-2 analog which is processed or degraded at a rate that is measurably slower than the rate at which human GLP-2 is processed or degraded, under the same conditions.

assay suitable for assessing DPP-IV sensitivity and resistance is described below in Example 3, in the contex of results actually obtained.

*.25 The X2 class of GLP-2 analogs is preferred herein. These Ala 2 -substinited GLP-2 analogs can incorporate at X2 a sictrally wide variety of Alareplacement amino acids to achieve relative resistanc to DPP-IV digestion. A simailarly wide variety of Ala-replacement amino acid allow also for the retention by the analog of intstinotrophic activity. For purposes of identifying thos DPP- **30 IY-resistant X2 analogs that also retain intestinotrophic activity, the X2 analogs showing DPP-WV resistanc are screened in the assay of inemntohc activity described below in Example 4.

in embodimntsr of the present invention, the Ala replacements include stereoisorners of amino acids that would otherwise be substrates for PP-IV, for example D-Ala, D-IiPr, PAla, t-butyl-Gly, L-penicillaniin, a-aminobutyric acid, airtinoisobuyric acid, norvaline, L-phernyl-Gly, and D-Pro, and naturally occurring amino acids, for example, Glu, Lys, Ie, Ser, Asp, Phe, Lys, Met, Msn, Pro, Gin, ThrT qy and Val. fn specific embodiments of the invention, there are provided the following Ala 2 substituted GlP-2 analogs: [D-Ala 2 rGL.P-2(1-33), [G19]jrGLP-2(l-33), [Val]rGLP-2(1-33), [GIJ1hGLP-201-33), (rBuGly'jhGLP-2, [Asp']hGLP-2C1-33), [Glu 2 ]hGLP-2(l-33), [Phe ]hGLp-21-33), [His 2 ]hGLp-2(j-33), [11e 2 ]hGLP-2C1-33), [Lys7]hGLP-2C1-33), [Meet 2 hGLP-2CI-33), (Asn ]hGLP-2(l-33), [Pro~jhGL-P-2(1-33), [GInjhCP-2(1-33), [Ser~lhGLP-2(1-33), [Thr 2 )hCILP-2(1-33), [Val 2 ]hGLP-2(1-33), [Tyr 2 JhGLP-2(1-33), ID-Ala 2 ]hG;LP-2t1-33), [Pen 1hGL-P-2(1- 33), [bAla 2 ]hGLP-2(1-33), IaAbie3hGILP-2(1-33), [Nval 2 ]hGLP-2(1-33), (PhClyjhGLP-2C1-33), and [AibilhGt.P-2(1-33).

The X2 GL.P-2 analogs may incorporate amino acid replacemenrs at other positions. In ewboaixnent of the invention, such analogs include those carrying amino acid substitutions also at one or more of positions Xl, X3, X4, X10, X19, X1 and X2A, and therefore include those which, according to Formula I (SEQ ID NO: include at least one of the following substitutions: X1 is Tyr, X3 is Glu; X4 is **20 Ala; PI is Glu.XIO-Asn-Thr-Ile where X10 is other than Met or PI is Tyr-Ser-Lys- Tyr X10 is an oxidatively stable Met-replacement amnino acid; X19 is Thr, X20 is ~.Lys or Ala; P2 is Val-Gin and P3 is a covalent bond, le, or lle-Thr or lle-Thr-Asn.

In embodimients of the present invention, the X2 analogs of GLP-2 include those which also incorporate one of the following substitutions: X1 is Ala; X3 is Ala; X4 is Ala; X1.0 is an oxidatively stable Met-replacement amino acid such as Val, Ile, Msn, GIx, Tyr, Phe, and preferably Leu, Mle, Ala, ser, and Gly; and P2 is Ile-Ala. In specific embodiments of the invention, there are provided the following GLP-2 analogs: (D-AIa 2 Thr"]hGLP-2C1-33); [Giy2Wjl 9 hGLP-2Cl-33); p/al2Thr'jhGLP-2(1-33); !G1?Ala24hGLP-2(1-33); [Gly2Ala'jhGLP-2-(1-33); [Ala'GIy 2 jhGLF-2(l-33); IGl91AIa 3 JhGLP-2(l-33) and JGl~i'2A 4 ]hGLP-2(1-33).

a related embodiment, the X2 analogs include those which include the following substitutions: GLP-2[Gly 2 AWa]hGLP-2; [GI?Ala 1 'IhGLP-2; jGI9'A~ajhGLP-2; [Cly 2 AIa 9 hGL-P-Z EC~lAla"jhGLP-Z; [Gly9Ala"]hGLP-2; EGlAlau]hGLP-2; (Gly 2 .Ala 5 jhLP-2; [Gl9AAla? 1 )hGLP-2 [GVyAla 27 ]hGLP-2; [Gly 2 AlaulhGL-P-2, [GI?1Ala 3 ]hGLP-2; [Gly 2 Ala']hGLP-2; [Gl? Ala']hGLP-2; [Se? 2 Gln~hGLP-2(1-33); [Gly2, Ala21jhGLP-2(1-33); (GI9', Alan 6 hGLP-2(1-3S); [Gi?2, Ala"IjhC;LP-ZUZ-33); [Gly9 1 AlanjhGLP-2(1-33); [G19 Ala~nhGLP-2(1-33); ITyrr', G19]hGLP-2(1-33); [Gly 2 ArguhGLP-2(1-34); and [Gl9, Tyr 3 hGLP-2(1-34).

Alternatively, in the case where X2 is Ala, "ir or Pro, DPP-IV resistance can be conferred by replacing Asp 3 with an Asp-replacement amino acid, X3 which is Pro or hPr.

The present invention also provides, as another class of GLP-2 analogs, those analogs in which X10 represents an oxidatively stable, Me t-replacernent amino acid.- It has been found that the intestinotrophic activity of mamrmalian CLP-2 is reduced when the Met in position 10 is oxidized, and also that 7 intestinotrophic activity is retained when replacement amino acids insensitive rb oxidation are substituted for Met" 0 replacement. Such Met'R-substituted GLP-2 analogs are accordingly more stable during synthesis, work-up and storage. In embodiments'of 'the present invention, such X10 analogs of GLP-2 are analogs of human GLP-2- In a specific eznbodimi-nis of the invention, such analogs include: [Ser t0 ]hGLP-2(1-33); (Nle' 0 3hLP-2(1-33); (Ala' 0 ]hCLP-2(1-33); [Leu 10 )rGt.P-2(1- 33); (Met(qoV 0 lratGLP-2C1-33); and (Nle' 0 jratGLP-2(1-33).

**20 the X10 analogs may also incorporate amino acid substitutions at one or :17 more positions other than X10. As noted above, such analogs include, for instance, those incorporating substitutions at X2, and those in which PI is Tyr- Ser-Lys-Tyr, as well as those in which any one of the X substituents or the P substituents represented in Formula I (SEQ ID NO: 1) are other than wild type residues or sequences. In specific embodiments, the GLP-2 analogs include (GlyLAla' hCLP-2(1-33) and [TySer'0Lys"Tyr'desIe 3 ]hGLp_ 2 (1-33).

in other embodiments of the invention, the GLP-2 analogs incorporate single amino acid substitutions in the context of the mammalian GLP-2 peptide background in which they are introduced. Such analogs include, for example, those mammalian GLP-2 analogs and particularly the human GLP-2 analogs in which X1 is Tyr X3 is Glu; X4 is Ala; P1 is Tyr-Ser-Lys-Tyr-(des lle); P2 is le-Ala or ValGCln; and P3 is a covalent bond, or is Ile or 1ke-Thn..

Still other *GLP-2 analogs of the invention incorporate alterations of the Nor C-terminus by the addition of amino acids, by blocking groups, or by substituted amino acids. Ther are also provided tfltestinotrophic analogs of manmmalian GLP-2, preferably human, GLP-2, in which positions X1, X5, X7, X 16-X1 8, X25, X30, or X33 incorporate a replacement amino acid other than that found in natural ly occuring mammnalian GLP-2's. Optionally, the invention provides inernrohcanalogs in which from 3 to 8 residues are deleted from the C-terminus. Specific embodiments of such inzestinorrophic; analogs include: Ac-raiGLP-2(1-33); raiGL.P-2( 1-30); ratOLP-2U1-2S); ratGLP-2l1-33)amide; (Arg- ~,rg-'rarGP-2(-33);, [Pro'JhGLP-2(l-33); [Glnn]hGtP-2(l-3) Ap3GP 2(1-33); (TyrT'hGL21 jdesNH2Tyr']hGLP-2(l-33); [TbrjhGLP-2( 1-33); [SerI6,ArgI7,.Argl]hGLP-2(l-33); [Age1 3 hGLP-2(1-34); [Arg~jhGLP-2(1-33); (Alas, Ala~jhGLP-2(1-33); [Glu9IiGLP-2(1-33); [Phe3SGLP-2(l-33); and (Tyr~JhGIP-2(l-33).

The present GLP-2 analogs can be synthesized using standard techniques of peptide chemistry and can be assessed for intestinotrophic activity, all according to te guidane provided herein. With respect to syntheis, the selected GLP-2 analog can be prepared by a variety of techniques well known for generating peptide products. 'Those GLP-2 analogs that incorporate only L-amino acids can be produced in cormmeal quantities by application of recombinant DNA technology. o hsproe DNA coding for the desired GLP-2 analog is incorporated into an expression vector and transformed into a microbial, e.g., yeaSt, or other cellular host, which is tn cultured under conditions appropriate for GLP-2 expression. A variety of gene expression systems have been adapted for This purpose, and typically drive expression of the desired gene from expression regulatory elements used naturally by The chosen host. Because GLP-2 does not require post translational glycosylation for its activity, its production may most conveniently be achieved in bacterial hosts such as E. coli. For such production, DNA coding for t selected GLP-2 peptide may usefully be placed under expression controls of'the lac, urp or PL genes of E. coi. As an alternative to expression of DNA coding for the GLP-2 per se, the host can be adapted to express GLP-2 peptide as a fusion protein int which the GLP-2 is linked releasably to a car-ier protein that facilitates isolation and stability of the expression produc.

In an approach universally applicable to the production of a selected GLP-2 analog, and one used necessarily To produce GLP-2 analogs that incorporate nongenetically encoded amino acids and N- and C-terminally derivatized forms, the well established techniques of automated peptide synthesis are employed, general descriptions of which appear, for example, in J.M. Stewart and U)D. Young, Solid phase Peptide Synthesis, 2nd Edition, 1984. Pierce Chemical Company, Rockford, Illinois; and-in M. Bodanszky and A. Bodanszky, The Practice of Pcptide Synthesis. 1984, Springer-Verlag, New York; Applied Biosysterns 430A Users Manul, 1987, ABI Inc., Foster City, California. In these techniques, GLP-2 analog is grown from its C-terminal. resin-conjugated residue by the sequential addition of appropriately protected amino acids, using eithe rhPFioc or tBoc protocols. as decribed for insanc by Orskov et al, 1989, sWpra For fth incorporation of N- and/or C- blocking groups, protocols conventional to solid phase peptide synthesis mnethods can also be applied. For incorporation of. C-terminal blocking groups, for ezample, synthesis of the desired pepride is typically performed using, as solid phase, a supporting resin that has been chemically mo dified so that cleavage from the resin results in a GLP-2 pcptide having the desired C-terminal blocking group. To provide peprides in which t C-terminus bears a primary amino blocking group, for instance, synthesis is performed using a p-metbylberazhydrylamirte (MBHA) resin so that, when peptide synthesis is comnpleted, treatment with hydrofluoric acid releases the desired C-terminally arnidated peptide. -Similarly, incorporation of an Nrnethylarnine blocking group at the C-teminus is achieved using Nxnethylaminoetbyl-derivatizcd DVB rsin, which upon HF treaumet releases peptide bearing an N-rnethylamiclated C-teminus. Protection of t C-terminus by esterification can also be achieved using conventional procedures. This exitails use of resin/blocing group combination that permits release. of side-chain protected peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function. FMQC protetig groups, in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this purpose, with cleavage from the support being effected by TFA in ~dichloromethan. Fsrificauion of the suitably activated carboxyl function. e.g., with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterifled GLP-2 peptide.

Incorporation of N-terminal blocking groups can be achieved while the synthesized GLP-2 peptide is still artached to Th resin for instance by treatment with suitable anhydride and nirrile. To incorporate an acetyl blacing group at the N-termainus, for instance, the resin-coupled peptide can be treated with 20% acetic anh~ydride in acetonirrile. The N-blocked GLP-2 analog can then be cleaved from the resin, deprotected and subsequently isolated.

Once the desired GLP-2 analog has been syndhesize4, cleaved from the resinl and fully deprorected, the peptide is then purified to ensure t recovery of a single oligopeptide having the selected amino acid sequence. Purification cart be achieved using any of the standard approaches, which include reversd-phase highpressure liquid chromatography (RP-l{PLC) on alkcylated silica-columns,

C

1 or C, 5 silica. Such colun fractionation is generaly accomplished by running linear gradients, 10-90%, of increasing organic solvent, e.g., acetonitrile, in aqueous buffer, usually containing a small amount 0. of pairing agent such as TFA or TEA. Alternatively, ion-exchange HPLC can be employed to separate peptide species on the basis of their charge characteristics.

Column fractions are collected, and those contining peptide of the desired/required purity are optionally pooled. In one embo diment of the invention, the GLP-2 analog is then treated in the established manne to exchange the cleavage acid TFA) with a pharmaceutically acceptable acid, such as acetic, :hydrochloric, phosphoric, maleic, tartaric, succinic and fth like, to generate a pharniacetically acceptable acid addition salt of the peptide.

For administration to patients, the GLP-2 analog or its salt is desirably 0 Ott:provided in pharmaceutically acceptable form, as a preparation that is sterilefiltered, through aO0.22pu filter, and substantially pyrogen-fee. D~esirably, the GLP-2 analog to be formulated migrates as a single or individualized peak on IIPLC, exhbiW uniform and authentic amino acid composition and sequence upon analysis thereof, and otherwise meets standards set by the various national authoritics which regulate quality of pharmaceutical products.

For therapeutic use, the chosen GLP-2 analog is formulated with a carrier that is Pharmaceutically acceptable and is appropriate for delivering the peptide by the chosen route of administaton. Suitable pharmnaceutically acceptable carriers are those used convenionally with peptide-based* drugs, such as diluents, excipients an tihe Reference may be made to "Retnington t s Pha naceuzical Sciences'.

171h Ed., Mack Publishing Company, Easton, Penn-, 1985, for guidac on drug formulations generally. In one embodiment of the invention, the compoun&s are formulated for administration by infuion, when used as liquid u=ritonal supplements for patients on total parezueral nutition terapy, Or by injection, e.g., sub-cutanously, inurmuscUlarly or intravenously, and are accordingly utiized as aqueous Solutions in sterile and pyrogen-fee form and Opdonally buffered ro physiologically tolerable PH, a slighdy acidic or physiological pH. Thus, the compounds may Ibe administered in a vehicle sutch as distilled water or, more desirably, in saline, hosphate buffered saline or 5 dextrose solution.- Water solubility of the GLP-2 a nalog may be enhanced, if desired, by incorporating a solubility enhancer, such as acrtic acid.

The aqueous carrier or vehicle can be supplemented for use as injecrables with an amount of gelatin that serves to depot the GLP-2 analog at or near the site of injection, for its slow release to the desired site of action. C'oeurTions of gelatin effective to achieve the depot effect are expected to lie in the ran ge from 10-20%.' Alternative gelling agents, such as hyaluronic acid. may also be useful as depoting agents.

9*0Th7e GLP-2. andlos of the invention may also be formulated as a slow release implantation device for extended and sustained administaion of GLP-2 analog. Examples of such sustained release fonnuatons include composites of biocompatibic polymers, such as poly(lactic acid), poly(lactic-co-glycolic acid).

metbyleellulose, byahtronic acid, collagen, and the like. The sucwe, selection and use of degradable polymers in drug delivery vehicles have been reviewed in *.***several publications, including, A. lDomb er al., Polymmr for Advanced Is4Ioggje 1:279-292 (1992). Additional guidance in selecting and using polymers in pharmaceutical formulations can-be found ia the text by M. Chasin too. 30 and R. 1Langer "Biodegradable Polymers as Drug Delivery Systems, 'Vol.

of "Drugs and the Pharmaceutical Sciences,* M. Dekker, New York, 1990.

*was Liposomes may also be used to provide for the sustained telease of a GT.P-2 analog. Details concerning how to use and make liposotnal formulations of drugs of interest can be found in, among otr places, U.S. Pat. No 4,944,948; U.S.

Pat. No. 5,008,050; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pa.

No. 4,45:2,747; U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,311,712; U.S. Pat. No.

4,370.349; U.S. Pay. No. 4.372,949; U.S. Pat. No. 4,529,561; U.S. Pal. No.

5,009,956; U.S. Pat. No. 4,725,442; U.S. Pat. No. 4.737,323; U.S. Pat. No.

4,920,016. Sustained release formulations are of paricular~ interest when it is desirable to Provide alhih local coucentation of a GLP-2 analog.

Ile GLP-2 analog can be utilized in the form of a sterile-filled vial or ampoule, That contains an intestinotropitic amount of the peptide, in eithier unit dose or multi-dose amounts. IThe vial or ampoule may contain the QLP-2 analog and the desired carrier, as an administration-ready formulation. Alternatively, the vial or amopoule may contain the GLP-2 pepfide in a form, such as a Iyophilized form, suitable for reconstitution in a suitable carrier, such as phosphate-buffered saline.

As an alternative to injectable formulations, the GLP-2 analog may be formulated for administration by other routes. Oral dosage forms, such as tablets.

capsules and the like,- can be formulated in acrdance with standard pharmaceutical practice. According to the present invention, the GLP-2 analog is administered to treat patients that would benefit from growth of small bowel fissue.

The effects of GLP-2 analog on tis tissue, as evidenced by the results exemplified herein, is dramatic and would clearly benefit those patients suffering from diseases or conditions marked by abnormialities in the small intestinal tract mucosa, which include ulcers and inflammatory disorders; congenital or acquir ed digestion and .absorption disorders including maasrto syndromes; and diseases and conditions caused. by loss of small bowel inucosal function particularly in patients undergoing extende pauenrfal feeding or who, as a result of surgery, have undergone resection of the small bowel and suffer from short-gut syndrome and cul-de-sac syndrome. Therapeutic treatnent with GLP-2 analog is administered so as to reiduce or eliminate the disease symptoms and/or improve the nutrional status in these patients associated with their reduced intestinal tract nucosal S~os *30 function. For example, GLP-2 analog is administrated to a patient with an Goes inflammatory bowel condition in an amount sufficient to ameliorate the intestinal discomfort and diarrhea caused by the condition. Additionally, GUM- analog may be administered to Patients with malabsorprion disorders so as to enhance the nutritional absorption and thereby improve the nutritional status of such patient.

in general, patients who would beneft from increase small intestinal mass and consequent increased small bowel mucosal function are candidates for treatment with GLP-2 analog. Particular conditions [hat may be treated with GLp- 2 analog include t various forms of sprue including celiac spitte which restilts from a toxic reaction to a-liadin from wheat, and is marked by a tremendous loss of villae of t small bowel; tropical sprite which results from infection and is marked by partial flattening of the villae; hypogamglobulinemic sprite which is osrdco onyipatienrs with common variable tarnunodeficiency or hypogamxnaglobulilenia and is marked by significant decrease in villus height.

The therapeutic efficacy of the GLP-2 analog treatment may be monitored by enteric biopsy to examine the villus morphology, by biochemical assessment of nutrient absorption, by patient weight gain, or by amelioration of [he symptoms associated with thee conditions. Other conditions that may be treated with GLP-2 analog, or for which GLP-2 analog may be useful prophylactrically, include radiation enteritis, infectious or post-infectious eneritis, regional eazeritis (Crohn's disease), small intestinal damage due to toxic or othe chemotherapeutic agents, and patients with short bowel syndrome.

The therapeutic dosing and regimen most appropriate for patient treatmenT will of course vary with the disease or condition to be treated, and according to the patient's weight and other parameters. The results presented hereinbelow demonstrate that a dose of GLP-2 poptide equivalent to about lODIsg/kg (or less) administered twice daily over 10 days can generate very significant increase in 25 small bowel mass. It is expected that much smaller doses, in the guS&lk range. and shorter or longer duration or frequency of treatmen, will also produce therapeutically useful results, a statistically significant increse particularly in small bowel mass. Also, it is anticipated that the therapeutic regimen will include the administration of maintenanc doses appropriate for reversing tissue regression 30 that occurs following csaonof initial =rame=. The dosage sizes and dosing regimen most appropriate for human use are guided by the results herein presented, and can be confirmed in properly designed clinical trials.

An effective dosage and treatmenft protocol may be determined by conventional means, starting with a low dose in laboratory animals and tenm increasing the dosage while monitOring t effects, and systemnatically varying the dosage regime as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Primary among these is The amount of GLP-2 normally circulating in the plasma, which is on. the order of 151 pmolimJ in the resting state, rising to 225 pmollrnl after nutrient ingestion for healthy adult humans. Orsow, C. and Heist, J4., 1987, Scand. J.

Cin. Lav. Invest. 47:165. Additional factors include the size of the patient, the age of the paient, the general condition of the patient, the particular dises being treated, t severity of the diseas, the presence of other drugs in the patient, the in yjvo activity, of lbe.GLP-2 analog and the like. The trial dosages would be chosen after consideration of the results of animal studies and the clinical literature. it will be appreciated by t person of ordinary skill in the art that informtnaionl such as binding constants and Ki derived from in~ Alm GLP-2 binding competition assays may also be used in calculating dosages, as well as the calculated half-life of the GLP-2 analog in vivo.

A typical human dose of a GLP-2 peptide would be from about 10 pg/lcg body weightday to about 10 mg/kg/day, preferably from about 50 ,sg/kg/day to about 5 mg/kg/day, and most preferably about 100 pgkg/day to I mg/kg/day. As the GLP-2 analogs o f the invention can be up to 10 to even 100 times more potent than GLP-2. a typical edose of such a GLP-2 analog may be lower, for example, from about lOOPg/kg body weight/day to lmgilcg/day, preferably 1p~&g/gday to 500jzg/kS/day, and even more preferably 1lsg/kg/day to 1lOjpg/icg/day.

Similarly, the intestinotrophic GLP-2 analogs of the invention are also useful for both augmentng bowel growth and treating inflammatory disorders in livestock and pets.

FxapNUVI GLL-2 analg -synthesis Solid phas peptide syntheis (SPPS) is carried out manually in a 300 milliliter (ml) vesiel on a 3 raillmnole (inmole) scale using 6 grams of 0 chloromethYl (Merrifield) resin (for C-terminal free acid peptides) wit a substitution of 0.5 milliequivalent (meq) per gram. Amino acids are protected at 19 itie amino-terminus with the z-buryloxycarbonyl (tBoc) group. The side-chains of triunctional amino acids are protected with the benzl (Bz, for serine and threonine), benzyloxymetyl (BOM, for histidine), 2-bromobenzloxycarbonyl (2- BrZ, for tyrosine), 2-chlorobenzyloxycatbonyl (2-CiA for lysine), cyclohexyl (cHex, for aspartic and giwamnic acids), and tosyl (Tos, for arginine) grups.Th first amino acid is coupled to the chloromethyl resin through esterification of the proteted amino acid in the presence of potassium fluoride (IME. C-terminal amide peptides are synthesized on a 4-methyl enzhydiylamine (MMHA) resin on a 3 mniol scale using 6 g of resin with a substitution of 0.5 rneq/g. The firstain acid is coupled to t MBA resin according to the procedure described for peprde elongation.

Amino-group deprotection is carried out using 50% tifluoroaceric acid (TEA) in dichloromerliane (CHC12). followed by neutralization using two washes of 10 t-ieThylaniine (EtN) in Cfl 2

C

2 Peptide elongation is carried out using N,N-dicyclohexylcarbodiiznide/l-hydroxybenzotriaole (DCC/HOBt) activation in

CH{

2 Cl/diznetbylforxnamide (DMF). ThU growing pepde chain is capped after each elongation step with 20% Ac 2 Q in CH 2

CI

2 The peptide-resin is washed after each elongation, capping and deprotection step with isopropanol (iPrOH) and mcthanol (MeGH). The washes are repeated once. N-terminal aceryl peptides are prepared by acetylation of the terminal amino-group with 20% AcQ in CI{ 2 C1 2 after deprorecton and neutralization as described. Resin-bound products are routinely cleaved by a low-high procedure using hydrogen fluoride (HF) containing dimetbylsulfide (PMS) and p-cresol as scavengers.

*Crude peptides arc purified by preparative high pressure liquid chrouiatography (HPLC) using a Vydac CIS, 15-20 pan wide-pore, 2 inch x 12 inch. reverse-phase silica column using gradien elution with 0. 1% TEA in water modified with aceronitrile. Elution is monitored at 220 naometers Each fraction collected is analyzed for purity by analytical IiPLC using a Vydac CIS, pm, 4.6 x 254 millimieter reverse-phase silica column by gradient elution 30 using 0. 1% TEA in water modified with aceronitrile, and monitored at 215 rm.

Fractions eostaiggreater than 95 purity are combined and lyophilized.

Acetate salts of t peprides are prepared from the TEA salts by dissolution of the lyophilized powder in water, with addition of acetonitrile to aid in dissolution where necessary. The solution is passed through a protonated Bio-Rex-70 cation exchange resin. T'he resin is washed with 5 bed-volumes of water. and the resinbound petide is eluted with 50% actic acid in water. The elueni is diluted with water and lyopilized.

The final lyophilized powder is analyzed for purity by two analytical reverse-phase EPLC methods using a Vydac CIE, 5 pm, 4.6 x 2S4 :mm~ reverephase silica column. Thr two solvent system used are a gradient of water adjusted to pH 2.25 with triethylanmine phosphate, modified with acetoitrile, and a gradient of 0.1% TFA in water, modified with acetonitrile. The column eluent is io monitored at 215 in. heidntt of each product is confirmed by amino acid analysis and by electrocopy-rnass spectroscopy.

7Ue GLP-2 analogs are next formulated as described below in Example 2.

Each of the GLP-2 analogs is fully soluble in water at room temperature unless otherwise noted.

The GLP-2 analogs were formulated for injection eiiber.in phosphate buffered salmine r as a gelatin-containing depot formulation. For the PBSformulated GLY-2 analog preparations, a lOX stock PBS solution, was first prepared, using* 80gNadi (BDH ACS 783), 2g KCl (EPH ACS 645), 1 I.Sg Na.HPO, (Anacheini a AC-8460), and 2g KHzPO4 (Malinckrodt AP.7100), which was brought to a total volume of one itre with sterile distilled water. The final working solution was obtained by 10: 1 dilution of the stock solution with sterile distilled water and adjusted to pH 7.3-7.4 if necessary, using sufficient volumes of ION Na OH. The working solution was then autoclaved for 30 minutes. la the final working PBS solution, concentrations were 137 mM NaCI. 2.7 mM KCI, 4.3 mM Na 2

HPO

4 .7H2O, and 1.4mM KH 2

PO.

The GLP-7 analog, as a powdered peptide, is added to te working PBS solution as required to geneate formulations having t d-sired pepude concentrations. For exarmle, to generate a PBS solutiou of GLP-2 analog at 130 mg/l, 5.2mg of GLP-2 analog is dissolved in 4Oral of PBS to yield a GLP-2 concentration of 130,sg/mlJ. 0.5 ml is injected twice daily.

21 To generate the gelatin-based GLP-2 analog formulations, a gelatin solution was first prepared by dissolving 12 gramn of gelatin (Sigma, G-8 150 Lot #54H07241 Type A from Porcine skin [9000-70-83 300 Bloom) in 100 Wl distilled water. The gelatin solution was then autoclaved, warmed at 37*C, and the GL.P-2 pepiide previ ously dissolved in phosphate buffered saline as described above was then added to achieve specific, desired peptide coiicentrations. For instance, to generate a gelatin-based PBS solution of the GLP-2 at a concentration of 130mg/1, 10 ml of a PBS solution prepared with 5.2 mng of GLP-2 was diluted with 30 ml of the 20% wodrig gelatin solution as first described above. The solution was mixed by gentle pipeting, to yield a final solution 1.of 130O/Il GLP-2 in PBS/15% gelatin.

Example 3 Assay-for Resistance to Dipepidyi Pepridse IV The following peptides were tested for resistance to dipeptidyl peptidase IV (DPP-1V): a control peptide, rGL.P-2; the [D-Ala 2 3rGLP-2 anialog; and t [Gl9J)GLP-2. analog. "To perform the assay, 2.5 microliters of a solution of human'placental DPP-1V (Calbiochein, La Jolla, CA, cat. 9 317624) containiing 0.125 inilliunirs (mU) of enzyme in 50% glycerol, 10 mM Tris, pH 7.8, EDTA and 0.02% NaN 3 was added to 50 pl of a solution of the ums peptide prepared at a concentration of 0.2 mg/mI in PBS at pH 7.4. The mixture was incubated at 370C in a circulating water bath for 24 hours. The incubation was quenched by the addition of 50 1&1 of a solution of diprorin A prepared at a concentration of 4 :mg/mi in PBS. Each peptide was tested in duplicate.

Each sample was analyzed by reverse-phase (RP) HPLC as folows: 90 jd of t quenched incubation mixtue was injected onto a Rainin Dynamax 300 A CIS, 5 mnicron, 4.6 x 250 millimeter column. The samples were eluted with 0.1% trifluoroacetic: acid (WFA) in water modified with 0. 1% mceonitrile using a lina gradient and a flow rate of I ml per minute. Sample components were detected at 214 nanometers (nmn). The ent of cleavage was measured by relative integration of fth peak corresponding to the cleavage product compared To that of the remaining undigested parent peptide. The cleavage product of the conitrol peptide, zGLP-2(1-33), which should be rGLP-2(3-33). was confirmed to have result from cleavage between residues pAjW and Asp 3 by comparison of the retention tume of this component to that of a synthetic peptide standard, rGt.P-2(3-33), and by collection of the product from the IIPLC and analysis by mass spectrometry.

After the 24 hour incubation, 22% of the control peptide, rGt.P-2, was cleaved by DPPIV. No cleavage products were detected for the poptidec (1) Ala 2 ]rGJLP-2 and [Gy~rGLP-2 after 24 hours.

E,~aplC4 -GLP. anlofassessrIeCtT Recipients were CDI mice obtained from Charles River lAboratory (Onitario, Canada). The CDI mice were aged-matchied females at time of injection (n= 3 -4 per group), 6 weeks of age, unless otherwise specified. The animals we= allowed a minimum of 24 hours to acclimatize to the laboratory facility before t initiation of each experiment. Anim als; were idenified by ear punch. The mice were not restricted by diet or activity during the experiments. The light/dark cycle was 12 hours, between 6pin to 6 am. Contols were age- amnd sex-matched (n=3- 4) *=au 4 L. MMit wcat ;aeed subcutaneosly, twice a day %vitL peptide in a total volume or u.3 cc of PBS and were monitorco anty in mie laboratory facility. Airumcil were sacrificed 10 or 14I daye after injeotion, and were fasted at least 20 hours before sacrifice.

The micc were aiiaesthetise with CO 2 anid exsanguinated by cardiac' puncture. Blood was collected in 75 dI of TMl (Trasysol; EPTA (5000 KIU/ml: 1.2 mg/rn); Diprotin-A), and the blood was centrifuged at 14 k x g for 5 minutes and the plasma was stored at -70 prior to analysis. The small bowel was removed frm h peritoxal cavity, from pylorus tocecum. cleaned weighed n measured.

Frcomparative purpose, sections frmeach animal were obtained frmthe identical anatomical position. Fragmets each measuring 1.5-2.0 cmn in lenjgth were obtained 8±2 cm, 18*2 cm, 32±2 cm from pylorus; for bistomnorpliomctry representing proximal jejunumn, distal jejurtun and distal ilewnii. Each small bowel fragment was opened longitudinally on its mntimesenteric border in a tissu block and then placed on 10% forinalin (vol.Ivol)) ovcrwight, then transferred to

ETOH.

Percentage change in smiall bowel weight was calculated by dividing the mean change in bowel weight of anaog treated mice, relative to mmice treated with vehicle only. by the mean bowel weight of mice trated witb vehicle only, and multiplying Nhs figure by 100.

Results of inestinotophic activity assessment are shown in Table 1: TABLE GLP-2 analog Iucrase in (1-3 unlss nted)small bowel weight 1 rGLP-2 2 ITWvr'lrLP-; 37 3 IP-AlaITGLP-2 86 4 [Gl9]TGLP-2 [Val21rGLP-2 6 [Gly2)bGLP-2 59 -7 [Gl9Ala~jhGLP-2 46 8 [GI9'AI' 0 GLP-2 33 9 [Ala Glyi)hGLP-2 23 (GI9-Ala 3 lhGLP-2 18 11 [GI92AIhGLP-2 36 12 [Gluj3rGLP-2 33 13 (Ala'JrGi-P-2 14 [Tyr 9 SeroLys"lTyla(desi)eu)hGLP-2 41 [LeuwqrGLP-2 26 16 ffNleulrGLP-2 52 17 [Met S'qrGLP-2 8 18 [Lys"IrG1LP-2 62 19 [Va1=GWn~jhGLP-2 36 20 Amidated C-term 23 21 [Gly 2 Ala 2 IhGLP-2 67 22 [Gly 2 la')hGLP-2 33 23 (Gly 2 Ala"ThGLP-2 42 24 [Gl9;AWa 21 hG1LP-2 p 25 [Gly 2 Ala'jhGLP-2 31 a 26 (Gly2Ala'"] hGI.P-2 3 T'7 rGh, 2 A1a'?ThGLP-2 36I-

S

1u 28 [GI92A~auIhGLP-2 31 29 [GI9A~a3JhGLP-2 38 30 [Gl2Al 3 1 hGILP-2 28 31 [Gly 2 Aa~hGLP-2 22 32 [Gl?2Ala' 2 hGL-P-2 18 33 [Gly 2 Ala"1hflLP-2 34 (GI9'Ala 7 ]GLP-2 22 It can be reaily seen from the above table that analogs of mamnmalian GLP-2 molecules can have enhanced itstinotrophic activity. Furthermore, it is clear that dcpei*dW~ on t S9iution made, various levelrs of ifltcftiflotrophic activity are manifest. for example, [Gl9JrG1LP-2 has a grcally enhanced intsiirphc activity compared to the naturally occuring molecule; (GlyijhGLP-2 has a substantially increased intestinotrophic activity compared with rGLP-2 and (L-u 2 jGLP-2 has less than a 50% increase in intestinotrophic activity.

The follawig expepiments were condu=ctc to confirm that the increase in bowel weight seen in experimental animals trated with DPP-IV resistant analogs of GLP-2, compared with animals tuawd with wild-type GLP-2, was uLtribuibla in pant to the DPP-IV resistant nature of the molecules. This experiment took advantage of the availability of a DPP-1V deficient rat strain, Fisher 334 DPP-IVrats. Thbe effect of rGLP-2 injections on small bowel weight in the Fisher DPP-1V deficient rats was compared to That in normal Sprague-Dawley rats.

In the following experiments the Sprague-Dawley rats were injected s.c.

twice daily with rGLP-2 in the Gelatin formulation. The Fisher rats were injected s.c. twice daily with GLP-2 peptides (both rGLP-2, and rGLP-2 analogs) in PBS.

Normal Spraguc-Dawicy rats treated with rCILP-2, 2.51s& b.i14. or 2Spug showed no change in small bowel weight compared to control animals given vehicle alone. However, Fisher 334 DPP-1V-rats (DPP-IV deficient animals) demonstrated approximately a 40-50% increase in change in small bowel weight when treated with 20jug b.i.d. rGL.P-2 compared to sknimals treated with vehicle only. Moreover, DPP-IV deficient animals treated with 20 jsg [GlyrGLP-2 showed a 50-60% increase in change in small bowel weight compared with animals given vector alone. Further, Fisher 344 wild-type rats showed 75-85 increas in small bowel weight, over control animals given vehicle alon, when treated with 20j&g b.i.d. of [Gly1irGLP-2.

The above results strongly indicate that GLP-2 is inactivated in vivo in normal rans by cleavage of the two N-terminal residues by a DPP4-I-ie enzyme.

Furthermore, thes results demonstrate that a GLP-2 analog modifed so as to be resistan to cleavage by DPP-IV caused a substantial increase in bowel weight in normal rats, presumably as a result of increased GPL-2 half-life il in As the t)PP-IV cleavage site is conserved in all known naturally occurring formas of GLP-2 it seem likely that M' mamnmals DPP-IV cleavage is an important, and probably the primnary mechanism whereby GLP-2 is inactivated in vivo.

Importantly, GLP-2 analogs resistant to DPP-IV cleavage have enhanced intestinotrophic activity compared to native farm.

Example Experiments assessing the small bowel inducing activity of various analogswas repeated as described above for Example 4. In thee experimnents. the small bowel inducing activity of each analog in mice was calculated relative to that of nAtive rat CLP-2(1-33) (cxprc3scd aq 100% Pf Pefivity). Ttesim=ne of =nlogo to DPP-IV cleavage was performed as described above in Example 3. Results are ~.tabulated below in Table 2.

*.25 TABLE 2.

Ideltificazlon tDcsenption of Sequence Acriy Cleaved Rel. to rGLP-2 by DPP-IV AL.E.0109 mGLP-2(1-33) Native rat sequaec 100%/ Savo raiGLP-2(4-33) N-3; 3 N-zerznba re. removed 6% Ac-tGt-P-201-33) N-Acetyl 44% 0% manefih GLP-) Native angIafish 7% Arg-l)raTOLP-2(-1-33) Arg added to N-urinus 11% 0%/ rag0L.P-201-30) C-3; 3 C-twnal res. removed 23% Ta:GI.P-2(l.25) C48; 8 C-tcrumizal res. removed 8% raUzLP-2(l-33)auiide C-terinal amide 560% [Ar&g.2,AZS-llratGLP-2(2.33) Arg.Arg added wo N-teininus 43% 0% EDAII2]rtMGLP 2 -33) [Gly2]raGL- 2 (-33) [Tyri JraxLP-2( 3-33) 1 A3$a4)aGLP-2( -33) [Glu3])rGL- 2 (l-33) IL~tuI 0]raLGLP-2(1 -33) [Niel 0)rmGLP-2(l- 3 3 [(Lys2OjrRIGLP-2(1-3 3) [Scr2,Gbn33hG1-2- 2 33 Iva123.01fl4]hGLP- 2 1-33) pfaI2)GL-P-2(1-33) Degu GL.P-2 rTyr9.Scrl,LyslI ,yrl2, dcsl1s3bGLP2633) [Gly2,A138Th 0

-P

2 1 33 jlY2.,AI 1ThGLP-20 -33) (Gy,la2)hGLP-2(l133) IGly2,A~i24]GLP2(lI33) 1Gly2.AIWSjhGLP 2

(I-

3 3 [GIY2AI&26]hGP-P 2

(I-

3 3 IGly2,Ala27]hG1LP-2(1-3 3 iGly2,AIS9ThGLP-20 -33) (Gly2,Alal ]hGP-2(133) [Gly2Aa2IhGLFP 2 (l 33 (Giy2,Ala13IhGLP-201-33) fGly2,A1U14)bGOLP-2( -33) [CGy2,Ala1 6JhGLP-2(l-33) jGly2,Ali20hGLrP20 33) jIy2.AI&23bGLP20 -33) [Gly2,Ala28]hG1LP-2(l-33) IGly2,AII3OjhGLPZ(1-33) (Gly2,Al*31hG1LP-2(1 -33) (Gly2]hGL-20 -33) (AIal,G1y2jh01-P-2(l- 3 3 (Gly2413*3]hGLP2(1-33) jGly2&1a&4]hGLP-2(1-33) [G1y2,AlaSjhGLP-2(1-3'3) fGly2,Al&6hGLP2(1433) iGiy2ZAi&7)hG-2(l-3 3 jProllhGLP-2(133) [Me%(O)1 O~raGLP-2(1 -33) rGlaD2ObGLP-2(1-33) [Asp1ihOLP-20-33) It~uGly2PbGU'- 2 [Tyr34]hGI.P-2(1-34) IAsp2hG1P-2(1-33) [Ghu2]hG3-P-2(l- 3 3) 50 (Plhe2)hGLP-2(1-33) [His2ThGLP-2(l 3 3 jIlc23bGLP2(l- 3 3) [Lys2bG1P.2(133) Ice!2hGLP-20)-3 3 IAsu2]hGLP-2(1 3 3 fpro2IhGL-P-20l-33) tGln~]hGLP2(l-33) Ala2->D-A182 Ala2->01Y 2 HiSl->TyrI Gly4->Alu4 ASp3->011a 3 MetlO-'Ltu10 Arg20->Lys20 AIB2->SUt2. Asp3-)Glu 3 l1e23.>Va323, Asfl24-"01n24 A1a2->V12, TPA Natve dcgu scqizcc GLu9->Tyr9, Mal0->Scr1 0, Asni 1->Lysl1, TcrI2->Tyri2, and deletion of Dc43 Ak2Z-"01y2. Asp3-'A118 Aha2-'>3y2, ASUI I ->AIAI I A142-4G1Y2 Asp2l-->Ala2l Ala2->GIY2, A=n24->AI&24 Ala2->Gly2, Trp25->AI&2S AI&2->GIy2Z Ltu26->Ala26 AIa2-)01y2, I1e27->Ala27 Ala2->GThr2, GIU9->Alag AI&2->Cily2, Meti O->Alal 0 Ala2-'GIY 2 Thz12->Ata12 Ala2->G1y 2 lid 3-'>Ala13 Ala2-'Gly2, LeiuI4->AI&14 A112-)Giy2, Asni 6->AI16 Ala2-"01Y2, Leul7-->Aial17 Ala2-7Gly2, AWD)-'AIa2O AIa2-'Gly2, 11e23-'Alm23 Ah2-'Gly2. GkQiS->Ata28 Aha2->Gly2, Lys3O->Aia3O AI&2->GIY 2 lc3-'AI&3 1 AI&2.'Gly2 His I .)Alal. Ala2-'Oly2 Aka2-X31y2, A~p3->Alfl3 A1&2-)Gly2. 01y4-'Ala4 Ala2->'01y2, Ser5-'Ala5 Ala2-'Giy2, Pbc6->Aka6 AI&2.)Gly2. Ser7->AI&7 His 1->Pro I -Met 10 sulfaxide Ar&2O->GiL2o Hisl-)Asp Ai&2->-buyI.GIY2 Addition or C-ucnzinal Tyr Ala2-)Asp2 Ala2->Glu2 AIa2->Phe2 Ala2->ijis2 Ala2->11e2 AI&2-'>Ly,2 Ala2->Met2 Ala2->Asri2 Ala2-)Pr2 Ala2->GWn 210% 0%/ 196% 0%/ 81% 100%/ 590/. 72% 65%! 51% 100% 120%/ 11% 0% 82% 145% 0%/ 94%, 120%/ 150%/ 125% 139% 0% 18% 28% 112% 82%/ 67% 67%/ 309/ 130% 130%/ 105% 24%/ 130% 24%/ 1 00%/ 300%/ 0% 53% 0% 44% 0% I100%/ 0% 136%/ 0% 67% 82% 129/ 0%/ 18% 809/ 600% W/% 123% 0%/ 135% 93% 0%/ 93% 00/6 50%/ 0% 00/. 0%/ 90%M 0%/ W/o 0% 170% 00/ 679/ 0%/ 16% 1000/ 0%/ 4 [Scr2)hGLP-2( 1-33) [Tbr2ThGLP-2(1-3 3 jVaI12]hGL-P-2C) -33) (Tyr23hGLP-2(1-33) [P-Aia2jhGLP-2()-3 3 hGLP-2(l -33) [Tbhr5]hGLP-2( 1-33) jSel6ArS17,A&I

B)

hGLP-2C1- 33 [Asn33]hGLP-2C1 33 [Pcn2)hGL?-2(1-33) [bAja2)bGLP- 2 1-33) [aAbu2IhGLP-2( 1-33) [Nval2]hGLP-2(1-33) EphGWy2ThOLP-2(l-33) [Agm34)hGILP- 2 (l -34) A.r&3O]hWI.- 2 (1- 33 [Pro3)bGLP-2(1-33) [Ala, MAs7ThGLP-2(1-33) ETyrI, Gly2]hGLP-2(1-33) (Gju33hGLP-2(1 -33) (Phc2SjhGt.P-2(1 -33) [Tyr25JhGL-P-2(1-33) [Gly2, Tyr34]hGLP-2(1-3A4) [Aib2]bG1LP-2C1-33) [G1y2, Arg34ThGLP-2(1-34) AIa2->ScT2 Ala2->Tht2 Ala2->Va12 A132->TYr2 Aia2->D-A2 Naxive human stqUcncC H is l->dcsamin0-TYT I SerS-'ThrS ASnl6->Scrl 6 LcuI7-'Argl7, Alal S.>ArgI 8 Asp33->Asn3 3 Ala2->L-eniIIaIntl~l 2 Aia2->bcaA-Ala2 AW-2>pha-unfinobunrrc arid2 AI8->NV842 (norvaline) Ala2->L-PhcnY1GIY2 Additian Of C-.Qvmizia aVnzin (Agrn is des-COOH Mrg) Ly s-30->Ar93 0 Asp3->Pro3 SerS->AWL. Ser7->Ala7 Hijsj-'TyTI, Ala2-'Gly2 Asp33->Gju33 Trp2S-.>Phe2S TrP25 ->TYr2S Ala2->Gjy 2 -rC-wicnnnl TyT Ala2-)Imnoisobut)11c =Qd A132->'Gly 2 C-meminal Arg 167%/ 117% 180% 57% 130% ]DO% 6 125% 1 73% 59% 91% 58% 2 119%/ 84% 4 111% 37% 9716 103% 140% 108% 241% 121% 114% 64% 297% 202% -250% 3%/ 0% 0% 0%.

00/a 77% 0%a 549/9 00/0 0/ 0 0 0 ~~QIALf Tie foregoing wz-irtn specification is sufficieU to enable one skilled in the art to pr actice the invention. Intdeed. various modifications of the above-described 35 rneawn for carrying out the invention which are obvious to those siild in the field of molecular biology, protein cbemnisry, medicine or related fields are intended to be within the scope of the Molowing claims.

Throughout the description and claims of the specification the word "scomprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

0* *0* 0*

Claims (31)

1. A GLP-2 analog which is characterised by intestinotrophic activity and which conforms to Formula 1: R1-(Y1)m-X1-X2-X3-X4-Ser5-Phe6-Ser7-Asp8-(P1)-Leu14-Aspl5- Asn 6-Leu17-Alal8-X19-X20-Asp21-Phe22-(P2)-Trp25-Leu26-lle27-GIn-28- Thr29-Lys30-(P3)-(Y2)n-R2, wherein X1 is His orTyr X2 is Ala or an Ala-replacement amino acid conferring on said analog resistance to DPP-IV enzyme; X3 is Asp or Glu; X4 is Gly or Ala; P1 is Glu-X10-Asn-Thr-Ile or Tyr-Ser-Lys-Tyr; is Met or an oxidatively stable Met-replacement amino acid; X19 is Ala or Thr; is Arg, Lys, His or Ala; P2 is Ile-Asn, Ile-Ala or Val-GIn; P3 is a covalent bond, or is lie, Ile-Thr or Ile-Thr-Asp; R1 is H or an N-terminal blocking group; R2 is OH or a C-terminal blocking group; Y1 is one or two basic amino acids selected from the group Arg, Lys, and His; 25 Y2 is one or two basic amino acids selected from the group Arg, Lys, and His; and m and n, independently, are 0 or 1; and wherein at least one of X1, X2, X3, X4, P1, X10, X20, P2 and P3 is other than a wild type, mammalian GLP-2 residue. 30 2. A GLP-2 analog according to claim 1, wherein X2 is selected from the group consisting of D-hPr, D-Pro, D-Ala, Val, Lys, and lie. X:\ElisabethlPJC\NODELETE\733857.doc 29

3. A GLP-2 analog according to claim 1, wherein X2 is an Ala-replacement amino acid, and at least one of X1, X3, X4, X10, X20, P1, P2, P3, Y1, Y2, R1 and R2 is other than a wild type, mammalian GLP-2 residue.

4. A GLP-2 analog according to claim 3, wherein X2 is selected from D-hPr, D-Pro, D-Ala, Val, Lys, and lie. A GLP-2 analog according to claim 3, wherein X2 is Gly.

6. A GLP-2 analog according to claim 1, wherein X2 is Ala.

7. A GLP-2 analog according to any one of claims 1-6, wherein X10 is an oxidatively stable, Met-replacement amino acid.

8. A GLP-2 analog according to claim 7, wherein X10 is selected from Val, lie, Asn, Glu, GIn, Tyr, Phe, Leu, NIe, Ala, Ser and Gly.

9. A GLP-2 analog according to claim 8, wherein X10 is selected from NIe, Leu, Ala and Gly.

10. A GLP-2 analog according to any preceding claim, wherein X20 is His or .Lys.

11. A GLP-2 analog according to any preceding claim, wherein X1 is His.

12. A GLP-2 analog according to any preceding claim, wherein X3 is Asp.

13. A GLP-2 analog according to any preceding claim, wherein X4 is Gly. 30 14. A GLP-2 analog according to any preceding claim, wherein P1 is Glu- A GLP-2 analog according to any preceding claim, wherein P2 is lie Asn. X:\Elisabeth\PJCNODELETE\733857.doc

16. A GLP-2 analog according to any preceding claim, wherein P3 is lle-Thr- Asp.

17. A GLP-2 analog according to any preceding claim, wherein m is 0.

18. A GLP-2 analog according to any preceding claim, wherein n isO0.

19. A GLP-2 analog according to any preceding claim, wherein R1 is H.

20. A GLP-2 analog according to any preceding claim, wherein R2 is OH.

21. A GLP-2 analog according to any preceding claim, which is an analog of human GLP-2.

22. A GLP-2 analog according to claim 1 selected from the group consisting of: [Tyrl]rGLP-2; [Ala4]rGLP-2; [VaI23Gln24I]hGLP-2 and [Asn33]hGLP-2(1- 33).

23. A GLP-2 analog according to claim 1, wherein the analog is selected from the group consisting of: [Gly2]hGLP-2; [D-Ala2Thrl9]hGLP-2; [Gly2Thrl 9]hGLP-2; [Ala2Gly2]hGLP-2; [Gly2Ala3]hGLP-2; [Gly2Ala4]hGLP-2; [Gly2Ala5I]hGLP-2; [Gly2Ala6]hGLP-2; IGIy2Aa7]hGLP-2; [Gly2Ala8]hGLP-2; [Gly2Ala9]hGLP-2; [GyAap.L l11h L ll ]G P2 25 [Gly2Alal 0]hGLP-2; [Gly2Alal6I]hGLP-2; [Gly2Alal 2]hGLP-2; [Val2Thrl 9]hGLP-2; [Gly2Ala2O]hGLP-2; [Gly2Ala2l1]hGLP-2; [Gly2Ala24]hGLP-2; [Gly2Ala27I]hGLP-2; [Gly2Ala28]hGLP-2; and [Gly2Ala3l ]hGLP-2.

24. A GLP-2 analog according to claim 1, wherein the analog is selected **:from the group consisting of [SerlO]hGLP-2(1-33); [NlelO]hGLP-2(l-33); [AlalI ]hGLP-2(1 -33); [Leu 1 0]rGLP-2(1 [Nlel 0]rat GLP-2(1 [Gly2Alal O~hGLP-2(1 -33); X:\E is ateth\PJC\NO D ELETE\7 33857.doc I 31 [Met(O)1 0]ratGLP-2(1 -33) and [Tyr9Serl OLysi 1 Tyri 2(desllel 3)]hGLP-2(1 -33). A GLP-2 analog which is characterised by intestinotrophic activity and is selected from the group consisting of [Pro3]hGLP-2; [HPr3]hGLP-2; [Glu3Thrl9]hGLP-2; and [Thr19Lys2O]hGLP-2.

26. A GLP-2 analog which is characterised by intestinotrophic activity and is selected from the group consisting of: [Gly2, Ala25]hGLP-2(1-33); [Gly2, Ala26]hGLP-2(1-33); [Ser2,Gln3]hGLP-2(1-33); [Gly2, Alal4]hGLP-2(1-33); [Gly2, Ala23IhGLP-2(1-33); [Gly 2 Ala30]hGLP-2(1-33); [Tyri, Gly2]hGLP-2(1-33); [Gly2, Arg34]hGLP-2(1-34); [Gly2, Tyr34]hGLP-2(1 [tBuGly2]hGLP-2; [Asp2]hGLP-2(1 -33); [Glu2]hGLP-2(1 [Phe2]hGLP-2(1 [His2]hGLP-2(1 -33); [1e2]hGLP-2(1 [Lys2]hGLP-2(1 [Met2]hGLP-2(1 -33); [Asn2]hGLP-2(1 [Pro2]hGLP-2(1 [Gln2]hGLP-2(1 -33); [Ser2]hGLP-2(1 [Thr2]hGLP-2(1 [Val2]hGLP-2(1 -33); [Tyr2]hGLP-2(1 [D-Ala2]hGLP-2(1 [Pen2]hGLP-2(1 -33); [bAla2]hGLP-2(1-33); [aAbu2IhGLP-2(1-33); [Nval2]hGLP-2(1-33); [PhGly2]hGLP-2(1-33); and [Aib2]hGLP-2(1-33). An intestinotrophic GLP-2 analog selected from the group consisting of: ratGLP-2(4-33); Ac-ratGLP-2(1 ratGLP-2(1 ratGLP-2(1 ratGLP-2(1 -33)amide; [Arg 2,Arg-']ratGLP-2(2-33); [Prol ]hGLP-2(1 -33); [Gln2OI]hGLP-2(1 [Aspi ]hGLP-2(1 [Tyr34IhGLP-2(1 -34); [desNH2Tyrl]hGLP-2(1 [Thr5]hGLP-2(1 [Sen 6,Argl 7,Argl 8IhGLP- 2(1-33); [Agm34]hGLP-2(1 [Arg3O]hGLP-2(1 [Ala5, Ala7]hGLP-2(1 -33); [Glu33]hGLP-2(1 [Phe25]hGLP-2(1 and [Tyr25]hGLP-2(1 -33). *28. The peptidle [Gly2IhGLP-2.

29. The peptide according to claim 28, in the form of a lyophilised powder. X AEfis abeth\PJC\NODE LETEW 3 385 7 .doc t 32 The peptide according to claim 28, in a form that is pyrogen-free.

31. A pharmaceutically acceptable salt of the peptide according to claim 28.

32. A pharmaceutical composition comprising a therapeutically effective amount of GLP-2 analog according to any preceding claim, and a pharmaceutically acceptable carrier.

33. A pharmaceutical composition according to claim 32, wherein said therapeutically effective amount is an amount effective in the treatment of a gastrointestinal condition, disorder or disease.

34. A pharmaceutical composition according to claim 33, wherein said therapeutically effective amount is an amount effective to promote the growth of small bowel tissue. A pharmaceutical composition according to claim 33, wherein said therapeutically effective amount is an amount effective to inhibit apoptosis of small bowel tissue.

36. A pharmaceutical composition according to any one of claims 32-35, in the form of a liquid suitable for administration by injection or infusion.

37. A pharmaceutical composition according to any one of claims 32-35, 25 formulated to cause the slow release of said GLP-2 analog following administration thereof.

38. Use of a GLP-2 analog according to any preceding claim in the manufacture of a pharmaceutical for promoting the growth and/or function of 30 gastrointestinal tissue.

39. Use of a GLP-2 analog according to claim 38, for promoting the growth and/or function of small bowel tissue. X \Ehsabeth\PJCNODELETE\733 8

57.doc 33 Use of a GLP-2 analog according to claim 38, for the prophylaxis or treatment of a gastrointestinal condition, disease or disorder. 41. Use of a GLP-2 analog according to claim 40, wherein the gastrointestinal disease is selected from the group consisting of ulcers, digestion disorders, malabsorption syndromes short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac spree, tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), small intestinal damage due to toxic or other chemotherapeutic agents, and short bowel syndrome. 42. Use of a GLP-2 analog according to claim 41, for the prophylaxis of small intestine damage due to a chemotherapeutic agent. 43. Use of [Gly2]hGLP-2 in the preparation of a pharmaceutical for the treatment of short bowel syndrome. 44. A method of identifying intestinotrophic analogs of GLP-2, comprising the steps of: a) obtaining a GLP-2 analog according to claim 1; s. b) treating a mammal with said analog using a regimen capable to eliciting an intestinotrophic effect when utilised for rat GLP-2; and c) determining the effect of said analog on small bowel weight and/or on 5 crypt plus villus height relative to a mock treated control mammal, whereby said to* 25 intestinotrophic analog of GLP-2 is identified as an analog which elicits an increase in said weight and/or said height. *0000* DATED: 29 August, 2001 PHILLIPS ORMONDE FITZPATRICK 30 Attorneys for: ALLELIX BIOPHARMACEUTICALS, INC. **00**0 X %ElisabethkPJCV40DELETEk733857.doC