MXPA97007949A - Peptido-2 similar to glucagon and its use terapeut - Google Patents

Abstract

Glucagon-like peptide-2, a glucagon gene expression product, and glucagon-like peptide-2 analogs, have been identified as gastrointestinal tissue growth factors. Its effects on the growth of the small intestine and pancreatic islets are described. Its formulation is described as a pharmaceutical and its therapeutic use in the treatment of intestinal disorders.

Description

PEPTIDE - 2 SIMILAR TO GLUCAGON AND ITS THERAPEUTIC USE CROSSOVER REFERENCE TO RELATED REQUESTS This request is a continuation of part of the application no. series 08/42 ?, 540, filed on April 14, 1995, the presentation of which is incorporated herein by reference. FIELD OF THE INVENTION This invention relates to glucagon-related peptides that have gastrointestinal tissue growth promoting properties, and to their therapeutic use to treat various medical conditions resulting from impaired growth. or of the loss of gastrointestinal tissue, particularly intestinal and pancreatic tissue. BACKGROUND OF THE INVENTION The expression of the glucagon gene provides a tissue-specific variety of peptide products processed from the 160 residue proglucagon product. The organization of these peptides within the proglucagon precursor was elucidated by the molecular cloning of preproglucan cDNAs from the pejesapo, rat, hamster and bovine pancreas. These analyzes showed that preproglucagon contains not only the glucagon and glicentin sequence, but also two additional glucagon-like peptides (GLP-1 and GLP-2) separated from glucagon and between them by two spacer or intervening peptides (IP- I and IP-II). These peptides are raised by basic, characteristic amino acid pairs. of yes ii-js of disocia »: JÓ? of classical prohormones, suggesting that they may be freed after post-prophylactic processing of proglucagon (Dructer, Pancreas, 1990, 5 (4): 484). An analysis of the peptides released from proglucagon in the pancreatic islets of Langerhans, for example, suggests that the primary pancreatic peptide released is 29-mer glucagon, while glicentin, axintamodulin, IP-II and peptides similar to Glucagon are more prevalent in the small intestine and in the large intestine. This demonstration that glucagon-like peptides are found in the intestine prompted research on the precise structure and on the putative function (s) of these newly discovered intestinal peptides. Most studies have focused on GLP-1, because several lines of evidence suggested that 6LP-1 could be an important novel regulatory peptide. Certainly it has been determined that GLP-1 is the most potent peptidergic stimulant known for the release of insulin, an action mediated in a glucose-dependent manner through interaction with receptors in pancreatic beta cells. GLP-1 and its derivatives are in development for use in the treatment of diabetic patients. The functions of Glicentin and > e the o: < i tomodul i na, known as "enterog lucagopes", are also under investigation, specifically in relation to the regulation of acid secretion and the growth of intestinal cells. The axintamodul ina can inhibit the secretion of gastric acid stimulated by pentagastrin in a dose-dependent manner. The role of glicentin in the mediation of changes in intestinal adaptation and growth of the intestinal mucosa has been investigated and the effect has been investigated. 3 intestinotrófico glicentina and its therapeutic use have been rep »Drta» dos by Matsuno et al. in EP 612,531, published on August 31, 1994. In contrast to GLP-1 and other peptides related to glucagon, the physiological function of glucagon-like peptide (GLP-2) remains poorly investigated despite the isolation and sequencing of several GLP-2 homologs from humans, rats, bovines, swine, rabbits from india, haters, and degu species. Using GLP-2 antisera prepared against synthetic versions of GLP-2, several groups have determined that GLP-2 is present primarily in intestinal extracts in place of pancreatic extracts (see Mojosov et al., J. Biol. Chem., 1986, 261 (25) i 11880; Ors ov et al. in Endocrinology, 1986, 119 (4): 1467 and in Diabetología, 1987, 30: 874 and in FEBS Letters, 1989, 247 (2): 193; Gtíorge et al- FEB5S Letters, 1985, 192Í2): 275). In: a) to its biological function, Hoosein et al report (FEBS Letters, 1984, 178 (1): 83) that GLP-2 na competes with glucagon for its binding on hepatic and rat brain tissues, nor does it stimulate adenylate cyclase production in hepatic plasma membranes, but, in enigmatic form, can stimulate adenylate cyclase in rat hypothatatic and pituitary membranes at concentrations of 30-50 pM. It would obviously be desirable to elucidate the physiological function of GLP-2. COMPENDIUM OF THE INVENTION It has now been determined that GLP-2 acts as a trophic agent to promote the growth of gastrointestinal tissue. The effect of GLP-2 is particularly marked by increased growth of the small intestine, and is therefore referred to herein as a "trophic bowel" effect. Notably, the effects of promoting the growth of GLP-2 also manifest as growth of pancreatic islets, and particularly by an increase and proliferation of the islets. Accordingly, it is a general object of the present invention to express GLP-2 as well as GLP-2 analogs for therapeutic and related purposes. More particularly, and in accordance with one aspect of the present invention, up GLP-2 and a GLP-2 analog are provided in a pharmaceutically acceptable form suitable for the formulation and subsequent administration of ion to patients. In another aspect, the present invention provides a pharmaceutical composition comprising a 6LP-2 or a GLP-2 analog and a pharmaceutically acceptable carrier. In a further aspect, the present invention provides a method for promoting the growth and proliferation of intact gastrointestinal tissue, including small intestine and pancreatic islet tissue, in a patient in need of such promotion, comprising the step of supplying the patient an amount that promotes the growth of the gastrointestinal tissue of a GLP-2 or of an analogue -le 6LP-2. In another aspect of the present invention, there is provided a useful method for identifying novel gut-instrophic GLP-2 analogs, comprising the steps of: 1) obtaining an inotrophic gut-2 GLP-2 analog, co, d >: > g »: - > it has at least one substitution, removal of an amino acid addition or an amino acid with a blocking group; 2) the treatment of a mammal with said analogue by the use of a regimen capable of causing an inotrophic intestinal effect when used for rat GLP-2; and 3) determination of the effect of said analogue on the weight of the small intestine and / or on the height of the crypt plus hairiness and / or the size of the pancreatic illates in relation to a control mammal treated at f »; > Feigned rma, so that said inotrophic peptide is identified with an analogue that causes an increase in said weight and / or said at 1 tura / o d size. In another of its aspects, the present invention provides novel analogs of GLP-2, in the form of intestinotropic analogs of vertebrate GLP-2. These GLP-2 analogues promote the growth and proliferation of gastrointestinal tissue, including small-bowel tissue and pancreatic islet tissue. In another aspect of the present invention, a method is provided in which the treatment of patients to restore gastrointestinal tissue is accomplished by means of the steps of (1) culturing said tissue or cells with an amount of a GLP-2, or a GLP-2 analog to promote tissue growth, and then (2) implant said tissue or cells in the patient to be treated. In a related aspect, the present invention provides a method for the growth of gastrointestinal tissue or gastrointestinal cells, comprising the step of culturing said tissue or said cells in a culture medium supplemented with an amount of GLP-2. or analogous to 6LP-2 to promote growth. BRIEF REFERENCE TO THE DRAWINGS Figure 1 illustrates results of dose response with a GLP-2. Med? C? >-fine effects on the weight of the small intestine (BW-panel A), crypt height plus villus in the proximal jejunum (PJ-panel B), distal jejunum (DT-panel c), and distal ileum »3n (Di-panel D) in? Ni? T? Al »33 injected > Rat GLP-2, each plotted according to the dose of rat GLP-2. Figure 2 illustrates the effect of formulation vehicle on the intestinal-trophic activity of a GLP-2. The weight of the small intestine (BW), and the height of the crypt plus villus in the proximal jejunum (PJJ), jejunum distal! (DJ), and distal ileum (DI) were measured according to the administration of rat GLP-2 in gelatin (6) or saline solution (PBS). A saline solution without rat GLP-2 was used as control (C). Figure 3 illustrates the effect of the administration route on the introphic intestinal activity of up GLP-2. The percentage change in weight of the small intestine was measured after injection of rat GLP-2 either subcutaneously (SC), intramuscularly (IM), or intratrapepically (IP). Bars marked with the letter T indicate rat samples marked with GLP-2; C indicates samples obtained from control rats injected with a saline solution. Figure 4 illustrates the effect of administration sequences on GLP-2 activity. Animals were injected subcutaneously with PBS every 12 hours, with 2.5 μg of rat GLP-2 every 12 hours (ql2h), with 5 μg of rat GLP-2 every day fqd), or with 10 μg of GLP- 2 rat every third day < q »_3»), in accordance with that indicated in the letter of the -. The weight of the small intestine (BW), and the height of the crypt plus villi were measured in the proximal jejunum (PJ), distal jejunum (D-J), and distal ileum (ID) for each administration protocol. Figure 5 illustrates the effect of the duration of 6LP-2 administration as a function of activity. Animals were injected once a day with 5 μg of rat 6LP-2 in 10% gelatin, or with 10X gelatin only for 4 weeks (panel A), 8 weeks (panel B), or 12 weeks (panel C), and then the animals were sacrificed. The effect of GLP-2 treatment compared to control was measured for e) small bowel weight (BW), and crypt height plus villus in the proximal jejunum (PJ), distal jejunum (DJ), as well as as a distal ileum (ID). Figure 6 provides the time course of the intestinotrophic effect of a GLP-2. Female patches injected 2 times a day with 2.5 μg of rat GLP-2 PBS were sacrificed in several days after the start of treatment, and the small intestine weight was evaluated in comparison with a control animal injected with PBS only.

Figure 7 illustrates the age of the container as well as the effects of the genus on the inotrophic intestinal activity of GLP-2. In panels A to H, animals treated with GL.P-2 of corresponding sex (CDI mice) from 4 to 16 weeks of age were compared with their own controls for both the small intestine weight (BW) and the height of the Crypt plus villus in the proximal jejunum (PJ), distal jejunum (DJ), and distal ileum (ID) after treatment with rat GLP-2. Figure 8 illustrates the effect of several different 6LP-2 as well as different GLP-2 analogs in relation to a control. Panels A, C, E, and G present changes in the weight of the small intestine (BW); panels B, D, and F present change in height of the crypt plus villus in the proximal jejunum (PJ). Analogous abbreviations are N-acetyl (peptides of rat GLP-2 blocked at the amino terminus with an acetyl group), Arg + 1 (rat GLP-2 containing an additional residue of Arg at the amino terminus), Arg + 34 (rat GRP-2) containing an additional Arg residue at the carboxyl end »3), C-amido (rat 6LP-2 with an amido group that blocks the carbidoyl end), Arg + 1 + 2 containing two additional residues of Arg at the end Not me). Additionally, the effect of GLP-2 of degu (degu) was tested to determine its inotrophic intestinal effect on mice. Figure 9 illustrates the inotropic intest effect of rat 6LP-2 on the length of the small intestine. The increase in the length of the small intestine was measured in relation to an animal of "control after": ie a treatment C »: J? GLP-2 during l > Jí s. DETAILED DESCRIPTION OF THE INVENTION The invention relates to therapeutic and related uses of GLP-2 and GLP analogues, particularly for promoting the growth and proliferation of gastrointestinal tissue, more particularly tissue of the small intestine or pancreatic icates. Regarding the small intestine tissue, such growth is conveniently measured as a GLP-2 mediated increase in the mass of the small intestine compared to an untreated control. As the results presented here demonstrate, the effect of 6LP-2 on the small intestine also manifests itself as an increase in the height of the most villous crypt. Such activity is known here as an "intestinotrophic" activity. An increase in the proliferation of crypt cells and / or a decrease in apoptosis of the small intestine epithelium was also detected in response to GLP-2. These cellular effects are observed more significantly in relation to the jejunum, including the distal jejunum and particularly the proximal jejunum, and are also observed in the distal ileum. A compound is considered to have an "innate iatrophic effect" if the test animals of at least one vertebrate species that responds to a reference GLP-2 peptide show a significantly increased incineration of the intestine. , an increased height of the crypt axis more v ^ ll »Ds? da, either an increased proliferation of the cells» of the crypt or a diminished apoptosis of the epithelium of the small intestine when they are traced with the compound (or else genetically manipulated to express it themselves).

A suitable model D for determining such gastrointestinal growth is described by Matussumo et al, supra, and is exemplified below in Example 1, morphometric analysis of 1 > : The cellular effects of GLP-2 and GLP-2 analogues on the height of the crypt axis plus villus in the small intestine »D are described below in example 2. As for the pancreatic islets, such growth is indicated for an extension and / or a pr > r > The 6LP-2 mediation of pancreatic eggs, compared to an untreated control. A method for determining the intestinotropic effect of a compound by evaluating the effect of the test compound on the growth of pancreatic islet cells, including both a size and an increased number of pancreatic cells, is exemplified below in example 1. The term "6LP-2 peptide" refers collectively herein to 6LP-2 occurring naturally in vertebrates, and to analogues of forms occurring naturally of GLP-2, said analogue of GLP-7 cause a non-rhophic intestinal effect y = on e =? Structurally altered mind compared to a given vertebrate GLP-2, by at least one addition, removal, substitution of an amino acid or by incorporation of one or more to inocid (s) with a blocking group. The various forms of GLP-2 vertebrates include, for example, rat FLP-2 and its homologs including beef GLP-2, pork GLP-2, degu GLP-2, bovine GLP-2, GLP-2 from guinea pigs from India, hamster GLP-2, human GLP-2, rainbow trout GLP-2, and chicken GLP-2, whose sequences have been reported by numerous authors including Buhl et al in J. Biol. Chem., 1988, 263 (18): 8621, Nishi and Steiner, Mol. Endocrinal., 1990, 4: 1192-8, and Ir? In and Wong, Mol. Endocrinol., 1995, <3): 267-77. The sequences reported by these authors are incorporated herein by reference. Vertebrate GLP-2 analogs can be generated using standard peptide chemistry techniques and can also be evaluated for their inotropic intestinal activity, all in accordance with the guidelines provided herein. Particularly preferred analogs of the invention are those based on a following human GLP-2 sequence: His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Aßn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Aßn- Trp- Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp • where one or several re? Duos di? am? n? »oá > "i eos estío ? ' st i t '? d »33 conservatively or ^ another resi uo > of amino acid, insofar as the analogue continues to maintain its intestinotrophic activity, such as, for example, small intestine growth, growth of the pancreatic islets, and / or increase of the height of the crypts / villi in a vertebrate. Conservative events in any naturally occurring GLP-2, preferably in the human GLP-2 sequence, are defined as exchanges within any of the following 5 groups: I. Ala, Ser, Tur (Pro, Gly) II . Asn, Asp, Glu, Gln III. His, Arg, Lys IV. Met, Leu, lie, Val (Cyß) V. Phe, Tyr, Trp. The invention also encompasses its non-conservative amino acid sequences in any vertebrate GLP-2 sequence, provided that non-conservative substitutions occur at amino acid positions known to vary in GLP-2 isolated from different species. Unconserved residue positions are easily determined by aligning all known vertebrate GLP-2 sequences. For example, Buhl et al., J. Biol. Chem., 1988, 263 > 18): 8621, compared the GLP-2 sequences in human, porcine, rat, hamster, guinea pig and Indian, and bovine. found that the poiitions 13, 16, 19, 27 28 were not conserved (position numbers refer to the analogous position in the human GLP-2 sequence.

Steiner, Mol. Endocrine! , 1990, 4: 1192-8, found that an additional position within the sequence that encodes GLP-2, residue 20 in the sequence of previous human being, also vanaba in degu, a species of indigenous rodent of South America. Therefore, according to this standard, the positions of amino acids that vary in mammals and that can be substituted with non-conservative residues are positions 13, 16, 19, 20, 27 and 28. The additional amino acid residues that vary in the vertebrates and which can also be substituted with non-conserved residues occur at positions 2, 5, 7, 8, 9, 10, 12, 17, 21, 22, 23, 24, 26, 29, 30, 31, 32 and 33. Alternatively, non-conservative substitutions can be made in any position in which the alanine scanning mutagenesis reveals a certain tolerance for the mutation in this amino acid residue substitution with alanine to destroy all the intactinotrophic activity. The technique of alanine scanning mutagenesis is described in Cunningham and Wells, Science, 1989, 24: 1081, and is incorporated herein by reference in its entirety. Since most of the GLF-2 sequences consist of only about 33 to j ">: '' and in Gl F-2 > 1 & human alanine occurs at 4 positions), a specialist in the field could easily test an alanine analogue at each remaining position to determine the intestinotropic effect, in accordance with what is taught in the following examples. By aligning the known vertebrate GLP-2 sequences, a general formula has been constructed that takes into account the significant sequence homology between these GLP-2 species, as well as the known residues by varying between species. This formula can be used to guide the choice of particularly preferred non-conserved residues for substitution, addition, removal or modification by addition of amino acid blocking group. Accordingly, particular analogs of GLP-2 of vertebrates encompassed by the present invention, according to one of its aspects, are the GLP-2 and GLP-2 analogs of vertebrates that comply with the general formula presented below as SEQ ID NO: 1 Rl- [ Y] »- Hie-Ala-Aßp-Gly-Ser-Phe-Ser-A« p-Glu-Met-Asn-Thr-aal-Leu-Asp-aa2-Leu-Ala-aa3-aa4-Asp-Phe- Ile-Asn- Trp-Leu-aa5-aa6-Thr-Lys-Ile-Thr-Aβp- [X) n-R2 where aa. it refers to any amino acid residue, and aal to aa6 are the positions of known residues by varying between GLP-2 sequences obtained from different species and I X and one or two A? T? inoac? d »:? s selected inside > : Group III, such as Arg, Lys or Arg-Arg Y, is one or two amino acids selected within the group III, as by Ar »g, Lys or Arg-Arg m is 0 or 1; n is 0 or 1; R1 is H or an N-terminal blocking group; and R2 is OH or a C-terminal block group. In the embodiments of the invention, aai to a6 are defined as follows: aal is selected "within group IV, aa2 is selected from group I or well II; aa3 is selected from group I, aa4 is selected from group III, aa5 is selected from group IV, aa6 is selected from group II or III, In particularly preferred embodiments of the present invention, aal a a6 they are chosen from the group of residues known to occur in this position in GLP-2 isolated from different species, as follows: ai is lie or Val; aa2 is Asn or Ser; aa3 is Ala or Thr; aa4 is Lys or Arg; a5 is lie or Leu; and human and rat GLP-2 differ from each other only at the amino acid residue at position 19. In the human sequence, this residue is alanine; in rat 6LP-2, position 19 is trespina. Accordingly, GLP-2 or particular 6LP-2 analogs encompassed by the present invention contain a variable residue at position 19. In these embodiments of the invention, the GLP-2 peptide conforms to SEQ ID NO: 2 which It is illustrated below: Rl- [Y] m-Hiß-? La-Asp-Gly-Ser-Phe-Ser Asp-Glu-Met-? ßn- Thr-Ile-L * u-Asp-Asn-l «u-Ala-aa3- ? rg-Asp-Fhe-Ile ~ Aßn- Trp-Lßu-Ile-Gln-Thr-Lyß-Ile-Thr-Aßp- [X) n-R2 where a3, Y, m, X, n, R1 and R2 are as defined above. In specific embodiments of the invention, the GLP-2 peptide is selected from 1) rat GLP-2 having SEQ ID NO: 3 illustrated below. Hie-Ala-Asp-Gly-Ser-Phß-Sßr-Aßp-Glu-Met-Asn-Thr-Ile-l? U-Asp-Asn-Leu-Ala-Thr-Arg-Asp-Phe-Ile-Asn- Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp; Human 6LP-2, which is the equivalent Thrl9 to Alal9 of rat GLP- illustrated below: Hiß-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Le? -Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp -Leu-Ile-Gln-T r-Lys-Ile-T r-Asp; 3) degu GLP-2 which is the equivalent of fllel3 to Val 13, Asnl6 to Hisl6, Lys20 to Arg20) of rat GLP-2; and 4) GLP-2 and GLP-2 analogs, which incorporate an N-terminal blocking group and / or an N-terminal extension such as Arg or Arg-Arg; and / or incorporate a C-end blocking group and / or a C-terminal extension such as Arg or Arg-Arg. The "blocking groups" represented by R1 and P2 are chemical groups routinely employed in the peptide chemistry technique to provide biochemical stability and resistance to digestion by exspeptidase. Suitable N-end protecting groups include, for example, C1-C5 alkanoyl groups, such as acetyl.

Protective groups N also suitable are analogs of amino acids that do not have the amino function. Suitable C-terminus protecting groups include groups which form ketones or amides at the carbon atom of the C-terminal carbokyl, or else groups which form steres at the carboxyl oxygen atom. Ketones and stereos forming groups include alkyl groups, particularly branched or unbranched Cl-C5 alkyl groups, for example, methyl, ethyl and propyl groups, while amide forming groups include functions such as, for example, primary amine, or else function alkylamines such as, for example, monoalkylamino groups C1-C5 and di-1-C1-C5 such as, for example, methylamino, ethyls, d. et i lamino, diethylamino, me i let i the ino and si illa. Amino acid analogs are also suitable for protecting the C-terminus of the present compounds, for example carbaxylated amino acid analogues, such as for example agmatine. The particular form of GLP-2 selected to promote the growth of intestinal gastrointes tissue can be prepared by vain techniques well known for the generation of peptide products? »D» _ >; Vertebral shapes < of 6LP-2 can obviously be obtained by extracting the natural source, using an appropriate combination of protein isolation techniques. As described by Buhl et al., Supra, the isolation and purification of porcine 6LP-2 is achieved from acid-ethanol extracts of ileal mucosa through a combination of size and cloning selection based on HPLC, with the help of antibodies raised against proglucagon 126-159 synthetic to monitor preparation. As an alternative to the extraction of 6LP-2, these forms of GLP-2 that incorporate only L-amino acids, either vertebrate GLP-2 or analogs thereof, can be produced in commercial quantities by the application of recombinant DNA technology. For this purpose, the DNA coding for GLP-2 or an lsg'-i > The desired GLP-2 is incorporated into a deletion vector and transformed into a microbial host, for example, lev3 or other cellular host, which is then cultured under appropriate conditions for the expression of GLP-2. Several gene expression systems have been adapted for this purpose, and typically carry out the expression of the desired gene from expression controls naturally employed by the chosen host. Since 8LP-2 does not require post translational glycosylation for its activity, its production can be more conveniently achieved in bacterial hosts, such as E. cali. For such a position, the DNA coding for the selected GLP-2 peptide can be usefully placed under expression controls of the E. coll, lac, trp or PL genes. As an alternative to the expression of the DNA coding for 6LP-2 per se, the host can adapt to express a GLP-2 peptide as a fusion protein wherein 6LP-2 is releasably linked to a carrier protein. which facilitates the isolation and stability of the expression product. In a universally applicable approach to the production of a GLP-2 or GLP-2 analog selected, and one necessarily employed to produce GLP-2 peptides incorporating non-genetically encoded amino acids and N-terminal and C-derived forms, well-established techniques of automated peptide synthesis are employed, whose general decisions appear, for example, in JM Stewart and T.D. Young, S-olid Phase Peptide Synthesis, 2nd edition, 1984, Pierce Chemical Company, Rockfart, Illinois; and in M. Bodanszky and A. Badanszky, The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York; Applied Biosystems 430A User Manual, 1987, ABI inc., Foster City, California. In these techniques, a GLP-2 peptide is cultured from its C-terminal resin conjugate residue by the sequential addition of appropriately protected amino acids, using either the Fmoc or tBoc protocols, as described for example. by Orskov et al, 1989, supra. For the incorporation of N and / or C blocking groups, conventional protocols for solid phase peptide synthesis methods can also be applied. For the incorporation of C-terminal blocking groups, for example, the synthesis of the desired peptide is typically carried out using, as a solid phase, a support resin that has been chemically modified in such a way that the dissociation of the resin result in a GLP-2 peptide having the desired C-terminus blocking group. To provide peptides wherein the C-terminus carries a primary amino blocking group, for example, the synthesis is carried out using a P-met i lbenxh i dr 11 amine resin.

(MBHA) in such a way that, when the synthesis of the peptide ends, the treatment with hydrofluoric acid liberates the desired knotted peptide C. In the same way, the incorporation of a blocking group of N-met i lamí na at the C-terminus is achieved using a DVB resin derived from N-met i inoeti lo, which when treated with HF releases a peptide carrying a end C N-met i lamidado. The protection of the C-shell by esterification can also be achieved using conventional methods. This requires the use of a resin / blocking group combination that allows the release of side chain protected peptide from the resin, to allow subsequent reaction with the desired alcohol, to form the ester function. FMOC protection groups, in combination with DVB resin derived with alcohol methoxyalkoxybenzyl 1 to an equivalent linker, can be used for this purpose, with dissociation of the support achieved by TFA in dichloromethane. The activation of the carboxylic function suitably activated, for example, with DCC, can then be continued by the addition of the desired alcohol, followed by the deprotection and isolation of the purified GLP-2 peptide. The N-terminal block group incorporation can be achieved while the synthesized GLP-2 peptide is still bound to the resin, for example, by suitable anhydride and nitrile treatment. To incorporate an acetyl blocking group at the N-terminus, for example, the peptide coupled with resin can be treated with acetic anhydride at 20 '/. in acetonitrile. The N-blocked GLP-2 peptide can then be dissociated from the resin, deprotected and subsequently isolated. Once the desired GLP-2 peptide has been synthesized, dissociated from the resin and completely deprotected, the peptide is then purified to ensure recovery of an oligopeptide having the selected amino acid sequence. Purification can be achieved using any of the standard approaches, which include reverse phase high pressure liquid chromatography (RP-HPLC) on alkylated silica columns, eg, C4-silica, C8-silica, or C18-silica . Such column fractionation is generally achieved by linear gradients, for example 10-90 * / *, of increasing percentage of organic solvent, for example, acetoni rile, in aqueous buffer, usually containing a small amount (for example, 0.1 * 4) of an annealing agent such as TFA or TEA. Alternatively, ion exchange HPLC can be used to separate peptide species based on their loading characteristics. Column fractions are collected, and those containing peptide of the desired / required purity are optionally combined. In one embodiment of the invention, the GLP-2 peptide is then treated in the manner established to exchange the dissociated acid (eg, TFA) with a pharmaceutically acceptable acid, such as, for example, acetic, hydrochloric, phosphoric, aleic, tartaric acid. , succinic and the like to generate a pharmaceutically acceptable acid addition salt of the peptide. For administration to patients, the 6LP-2 peptide or its salt is provided, in one aspect of the invention, in pharmaceutically acceptable form, for example, as a sterile filtered preparation, for example, through a 0.22μ filter. , and substantially free of pyrogen. Desirably, the 6LP-2 peptide to be formulated migrates as a unique or indi-tized T? P »3 in HPLC, shows uniform and authentic amino acid composition and sequence when performing its analysis, and otherwise fulfills standards established by the various national bodies that regulate the quality of pharmaceutical products. For therapeutic use, the chosen GLP-2 or analog of 6LP-2 is formulated with a pharmaceutically acceptable carrier and suitable for delivering the peptide by the chosen administration route. Suitable pharmaceutically acceptable carriers are those conventionally employed with peptide-based drugs, such as, for example, excipient diluents and the like. Reference may be made to "Re ington's Pharmaceutical Sciences" 17th edition, Mack Publishing Company, Easton, Penn. , 1985, for guidance on formulations of drugs in general terms. In one embodiment of the present invention, the compounds are formulated for administration by infusion, for example, when used as liquid nutritional supplements for patients on total parenteral nutrition therapy, or by injection, for example subcutaneous, intramuscular or intravenous , and are therefore used as aqueous solutions in sterile, pyrogen-free and opt. iana regulated at a physiologically tolerable pH, for example, a slightly acidic or physiological pH. Accordingly, the compounds can be administered in a vehicle such as for example distilled water or, more preferably in saline, phosphate-buffered saline or 5 '/ solution. of dextrose. The solubility in water of GLP-2 or GLP-2 analog can be increased, if desired, by the incorporation of a solubility enhancer, such as for example acetic acid. The aqueous vehicle can be supplemented for use as injectable solutions with a quantity of gelatin having for deposit of GLP-2 or GLP-2 analog at the injection site or close to said site, for its slow release to the desired site of action. It is considered that gelatin concentrations effective to achieve the effect of deposit »-) are within n» 3? of >; To 20 * / .. Agents to the gel formation agents, as for example hyaluronic acid, can also be useful as depot agents. The GLP-2 and GLP-2 analogs of the present invention can also be formulated as release release implantation devices for extended and sustained administration of GLP-2. Examples of such sustained release formulations include biacompatible polymer compounds with, for example, (polylactic acid), (polylactic-co-1-ylactic acid), methylcellulose, hyaluronic acid, collagen, and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reported in several publications, including, A. Do b et al., Polymers for Advanced Technologies 3: 279-292 U992). An additional guide for selecting and employing polymers in pharmaceutical formulations can be found in the text prepared by M. Chasin and R. langer (eds.), "Biodegradabe Polymers as Drug Delivery Systems," volume 45 of "Drugs and the Pharmaceutical Sciences," M. Defcker, New York, 1990. Liposomes can also be used to provide a prolonged release of a GLP-2 or analogous of 6LP-2. Details as to how to employ and prepare liposomal formulations of drugs of interest may be found, inter alia, in the American patent not. 4,944,948; in the North American patent na. 5,008,050; in the North American patent no. 4,921,706; in the North American patent na. 4,927,637; in the North American patent no. 4,452,747; in U.S. Patent No. 4,016,100; in the North American patent no. 4,311,712; in the North American patent no. , 370,349; in the North American patent no. 4,372,949; in the North American patent no. 4,529,561; in the North American patent no. 5,009,956; in the North American patent no. 4,725,442; in the North American patent no. 4,737,323; in the North American patent no. 4,920,016. Prolonged-release formulations are especially interesting when it is desired to provide a high local concentration of GLP-2 or GLP-2 analog, for example, near the pancreas to promote pancreatic growth, in diabetes, etc. For your. employment in the stimulation of small intestine growth or in the proliferation and increase of pancreatic islet cells in mammals including humans, the present invention provides in one of its aspe < a package in the form of a sterile-filled vial or vial containing an amount to promote tissue growth of GLP-2 or GLP-2 analog, either in unit doses or in multiple dose quantities, where the package incorporates an instruction label to use its content for the pr »: > motion of such growth. In a modality of the present invention, the package contains the GLP-2 or analog of 6LP-2 and the desired vehicle, or a formulation ready for administration. Alternatively, and in accordance with another embodiment of the invention, the package provides the GLP-2 or GLP-2 analogue in a form, such as, for example, a lyophilized form, suitable for its re-use in a suitable vehicle, eat, for example, phosphate-buffered saline. In one embodiment, the pack is a sterile filled vial containing an injectable solution comprising an effective, intestinotrophic amount of GLP-2 or a 6LP-2 analogue dissolved in an aqueous carrier. As an alternative to injectable formulations, 6LP-2 or GLP-2 analog may be formulated for administration by other routes. Oral dosage forms, such as for example tablets, capsules and the like, can be formulated in accordance with standard pharmaceutical practices. In accordance with the present invention, GLP-2 or GLP-2 analog is administered to treat patients who could benefit from gastrointestinal tissue growth. In one aspect, the candidate patients are the patients who could benefit from the growth of small bowel tissue. The effects of the GLP-α peptide - >This work, in accordance with what is evidenced by the results exemplified here, is grammatical and would clearly benefit patients suffering from diseases or conditions marked by abnormalities in the mucosa of the small intestine tract, including: ulcers and inflammatory disorders; congenital or acquired digestion or absorption disorders, including malabsorption syndromes; and diseases and conditions caused by a loss of the function of the intestinal mucosa, particularly in patients who undergo parenteral feeding e, tended to good who, as a result of a surgical intervention, have been submitted to a reception of the intestines and suffer from short bowel syndrome, cul-de-sac syndrome. A therapeutic treatment with GLP-2 peptides is administered to reduce or eliminate the symptoms of the disease in these patients, associated with their mucosal function of the reduced intestinal tract. For example, 6LP-2 or GLP-2 analogue is administered to a patient with a condition of intestinal inflammation in an amount sufficient to improve intestinal discomfort and diarrhea caused by the condition. Additionally, GLP-2 or a GLP-2 analog can be administered to patients with malabsorption disorders to increase the nutptional absorption and to improve the nutptional state of such pa > - n. In general, Dac lenses 'u' 3 could benefit from either an increased small bowel mass and therefore an increased function of the small bowel mucosa are candidates for treatment with GLP-2 or GLP-2 analogue. Particular conditions that can be treated with GLP-2 include the vain forms of sprue including celiac sprue that results from a toxic reaction to wheat 1 to 1-ladine, and is noted by a significant loss of villi of the intestine; tropical sprue that results from an infection and is marked by a partial flattening of the villi; Hypogammaglobul inemic esprue that is frequently observed in patients with common variable immunoglobulinemia or hypoglycemia and is marked by a significant decrease in villus height. The therapeutic efficacy of 6LP-2 treatment can be monitored by enteric biopsy to examine villous morphology, by biochemical evaluation of nutrient uptake, by increasing the patient's weight, or by improving the symptoms associated with these conditions. Other conditions that can be treated with 6LP-2 or 6LP-2 analogue, or for which 6LP-2 or GLP-2 analog may be useful prophylactically, include radium enteritis »pne, infectious enteritis well post-infection =, regional enteritis (Crohn's disease), small intestine damage caused by toxic agents or other chemotherapeutic agents, and patients with short bowel syndrome. In another aspect, candidate patients for treatment with GLP-2 or analogous of 6LP-2 are the patients who could benefit from the growth of the pancreatic icates, and particularly »the lengthening or proliferation or regeneration of pancreatic islets. Such patients include those suffering from diseases or conditions marked by the absence or reduction of pancreatic islets or by the reducing function of the pancreatic icates. Particular candidate patients are those suffering from type one or type 2 diabetes, as well as patients with secondary forms of diabetes due to infiltration, inflammation or destruction of the pancreas. GLP-2 or GLP-2 analogue is administered to these patients in an amount sufficient to at least partially restore pancreatic function, increase the level of endogenous insulin and improve its symptoms. The therapeutic dosage and the most appropriate regimen for the treatment of the patient will obviously depend on the disease or condition to be treated, and the weight of the patient as well as other parameters. The results presented below demonstrate that a dose of GLP-2 »D or 6LP-2 analogue equivalent to approximately 2.5 mg / kg (or less, see below) administered twice a day for 10 days can generate very significant increases of the small intestine mass and the crypt / villus height particularly of the proximal jejunum. It is expected that these small doses, for example, within the range μg / kg and of a shorter or longer duration or frequency of treatment, will also produce therapeutically useful results, ie, a statistically significant increase particularly in that to the mass of the small intestine. The dosage sizes and dosing regimen most appropriate for human use are based on the results presented here and can be confirmed in appropriately designed clinical trials. An effective dosage and treatment protocol can be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects and systematically varying the dosage regimen. Numerous factors must be taken into consideration by a doctor when determining an optimal dosage for a given patient. First, the amount of GLP-2 normally circulating in the plasma, which is of the order of 151 pmol / ml, should be considered. at rest, revealing at 225 pmol / L 'after nutrient intake in the case of healthy adult humans. Orskotn ', C. and Helst, J.J., 1987, Scand, J. Cli. The-'. Invest. 47: 165. Additional factors include the size of the patient, the age of the patient, the general condition of the patient, the particular disease to be treated, the severity of the disease, the presence of other drugs in the patient, the in vivo activity of the GLP-2 peptide and Similar. The assay dosages should be chosen after the configuration of the results of animal studies and the clinical literature.

A person with certain knowledge in the art will note that information such as link constant and Ki derived from in vitro GLP-2 link composition assays can also be used to calculate dasings. A typical human dose of a GLP-2 peptide would be approximately 10% of body weight / day at approximately 10 mg / kg / day, preferably from approximately 50 μg / kg / day to approximately 5 mg / kg / day, and more preferably from 100 μg / kg / day to 1 mg / kg / day. In another of these aspects, the present invention offers the treatment of candidate patients using implanted cells that have been either conditioned in vitro or in vivo by previous incubation or treatment with GLP-2 or analogue e GI.P-2 or pro »Dur?» Jos generically - manipulated to produce it. The conditioning of the ex vivo cells can be accomplished simply by transferring the cells to tissues for transplantation in a medium that has been supplemented with an amount that promotes the growth of GLP-2. or analogous > of GLP-2 and is appropriate for atr-part for the cultivation of these cells. The cells can, after a suitable storage period, be implanted either directly into the patient or encapsulated using established cell encapsulation technology, and then implanted. Another aspect of the present invention encompasses the treatment of live animals with 6LP-2 peptides to promote tissue growth of the small intestine or to increase the size or number of pancreatic islet cells. After subsequent enlargement of the small intestine and / or pancreatic icates, these tissues can be used in an enotransplantation procedure. Such a peptide treatment with GLP-2 may be beneficial prior to xenatransplanting tissue from a non-human animal to a human being because the size of the transplanted organ or tissue often limits the success of this procedure. For example, up animal donor p > -? rc? po can be treated with GLP-2 peptide in order to increase the size of the small intestine before xenotransplantation of porcine non-slim inte.ti tissue in up to human being that requires > - > - =: > t »-1 rij-ipn, Al tet nati vaniept» l, 1 • - =. The cells to be implanted can be grown in vitro from a cell that has been genetically engineered to express either to express excessively either the glucagon gene or, more directly, DNA encoding only GLP-2. The sequence of such DNA can be easily determined from the amino acid sequence of the selected GLP-2, with the limitation that only forms of GLP-2 containing genetically encoded amino acids can be produced in this way. Vary viral vectors, suitable for the introduction of genetic information into human cells, can be used and will incorporate the DNA encoding GLP-2 under functional expression controls in the host cells. Once genetically altered, the engineered cells can be implanted using established procedures in the art. EXAMPLE 1 In a first experiment designed to investigate the effect of glicentin on the growth of the small intestine, two groups of 6 mice were treated (3n) in the following manner (8 weeks, CDL females from Charles River Laboratories) each mouse received injections of 41.5 μg every 12 hours for 10 days. The injections were applied subcutaneously in a final volume of 10 '/. of 'gelatin, with the injection of your cut line »J <; > , ce every 12 hours. Glicentin (rat, 1 s * = easily dissolved in 10 ml of water.) DS control mice received 0.5 c of a 16 * / gelatin solution, but no peptide, every 12 hours. standard food for rats with free access to food and water, up to 12 hours before slaughter, at this time "the food was withdrawn and only water was procured." The weight of the small intestine was determined by removing all The small intestine, and the removal of the stomach (proximal end) and of the appendix / caecum / intestine and non-thick intestine (distal end) .The remaining small intestine was cleansed with a saline solution to remove feces, and weighed. following: Weight of mice (g) Weight of intestine day 0 day 10 thin (gm) day 10 Control 30.0 27.8 1.6 29.8 27.5 1.3 28.7 25.6 1.7 28.8 25.8 1.2 28.0 25.8 0.7 27.9 26.2 1.3 Glicentina 2277..99 26.6 1.6 27. 26.2 1.7 28. 0 2 .6 1.3 8 24.5 1.6 24.7 1.7 26.5 25.8 1.9 With these results indicating that the intestinotrophic effect of glicentin was modest, a second experiment was carried out using the same protocols to investigate the effects of other products derived from the proglucagon gene, including GLP-1 and GLP-2. For this purpose, rat GLP-2 from human SEQ ID NO: 2 and 6LP-1 (7-36 amides) were synthesized by the solid phase approach based on tBoc. Rat 6LP-2 analysis revealed a purity of 95 * 4 by anionic HPLC (20 μl sample of 1.0 mg / ml; 5μ Vydac C18 columns; TFA 0.1 * 4 / 20-60 * 4 CH2CN for 20 minutes at 1.5 ml / min). GLP-2 formulations for injection were prepared in accordance with the following: gelatin was dissolved in hot water to a weight ratio of 16 * /. and 50 ml of solution was placed in an autoclave and cooled to room temperature. A peptide solution was then prepared separately by mixing 5 mg of GLP-2 with water in a volume slightly less than 10 μL and then by adding 1N acetic acid in volume up to 10-20 μl) sufficient to completely dissolve the peptide. The pH was then readjusted to approximately 7.0 by the addition of an equal V »Dlumen, of NaOH IN < 10-20μl), and the volume of the solution was then adjusted to 10 ml by the addition of distilled water. To prepare the formulation for injection, the 10 ml peptide solution and the 50 ml gelatin solution at 16 * /. were combined with mixture and aliquots were withdrawn for injection in a 0.5 ml syringe of insulin. The same procedure was used to formulate GLP-1, with the exception that the acid / base adjustment was necessary given its significantly higher solubility in water. Mice were injected with 0.5 ml of the 16 * 4 gelatin solution, with or without peptide (62.5 μg / dasis). Four groups of four mice (8 weeks old, CDL females from Charles River Laboratories) were injected twice daily for 10 days. The results appear in the following table: Weight of mice (gm) day 0 day 10 control 26.0 25.4 27.0 25.9 26.0 26.7 25.6 24.4 GLP-1 26.6 24.8 23 26 .0 27.0 '• tr. . 1 2 .5 GLP-2 25 * "?. 1 25.7 28 .4 27.1 25, .8 25.4 Weight of the small intestine (gm) day 10 average * 4 control body weight 1.4 1.4 +/- 04 5.47 +/- .14 1.3 1.5 1.4 6LP-1 1.4 1.33 +/-. 04 5.26 +/-. 2 1.3 1.2 1.4 6LP-2 1.8 2.08 +/- 14 8.12 +/-. 40 2.4 1.8 These results demonstrate that, in one dose of approximately 2.5 mg / kg (640 nmol / kg) 6LP-2 shows a statistically significant increase (p less than 0.05) of the small bowel mass after two administrations per day for 10-days, compared to the control group that did not receive peptide and also compared to the group that received another peptide related to glucagon, GLP-1 In comparison with the results presented here for glicentin, it is clear that GLP-2 constitutes an important intestinal tissue growth factor The effects of GLP-2 peptide administration on these mice were further explored, by sectioning the gastrointestinal organs of the 4 mice treated with GLP-2 and the four control mice, using sections placed in paraffin and standard histopatho-gi-cas techniques. The areas of the islands were measured by morphometry analysis. Sections labeled with hematoxylin and eosin were used for quantification. The total pancreatic area and the total islet area were measured in each of the sections. The data indicated that the area of the islets was, on average 0.31 '/. of the total pancreatic area in the control group. On the other hand, the islets of the group treated with GLP-2 represented 0.76 * /. of the total pancreatic area, which represents an increase in the area of the islets of more than double in the group treated with 6LP-2. In addition to the size of the islets, an increase in the number of islets was observed. EXAMPLE 2 The effects of GLP-2 peptide on small intestine growth were further investigated, pallidly to assess tissue response as a function of dose, time, route of administration and frequency, type of formulation, as well as as depending on the gender and age of the recipient. These effects were measured in the context not only of an increased small bowel mass, but also in the context of increased crypt height and villi. For these purposes, rat GLP-2 was prepared in accordance with that described in example 3. Rat 6LP-2 was formulated either in a phosphate buffered saline or in the form of a reservoir formulation containing gelatin . To prepare the phosphate regulated saline, the GLP-2 peptide was prepared in the following manner. A solution of 10X mother PBS was prepared first, using 80 g of NaCl (BDH ACS 783), 2 g of KcL (BDH ACS 645), 11.5 g of Na2HP04 (Anachemia AC-8460), and 2 g of H2P05 (Malinckrodt AR7100) , which was taken to a total volume of one liter with sterile distilled water, the final work solution was obtained by 10: 1 dilution of the stock solution with sterile distilled water and adjusted to a pH of 7.3-7.4, if necessary , using several microliters of NaOH ION (sodium hydroxide). The working solution was then autoclaved for 30 minutes. In the final work PBS solution, the concentrations were 137 M NaCl, 2.7 M KCl, 4.3 M Na2HPo4. 7H20, and 1.4 M H2P04. P: -tra 'g nerate the peptide GLP-2 formula'dc) with PBS, the p >; ? d »: > in p '^ lvo it was given to the solution > of PBS working in accordance with that required for formulations having the desired concentrations of peptide. For example, to generate a peptide solution of PBS at 130 mg / L, 4.2 mg of GLP-2 was dissolved in 40 ml of PBS to provide a concentration of 6LP-2 of 130 ug / l. To give a dose of 2.5 mg / kg to a mouse, 0.5 ml of this solution should be injected twice a day. To generate the gelatin-based formulation, a gelatin solution was first prepared by dissolving 12 grams of gelatin (Sigma, G-8150 Lot No. 54H07241 type A porcine skin (9000-70-8) 300 Bloom) in 100 g. mL of distilled water. The gelatin solution was then autoclaved, heated to 37 ° C and the GLP-2 peptide previously dissolved in a phosphate buffered saline according to the above described was added to achieve specific concentrations of the desired peptide. The gelatin-based GLP-2 formulations were then prepared at the desired GLP-2 concentration by mixing the GLP-2 formulated with PBS with the gelatin solution prepared in accordance with the above-described. For example, to generate a solution of PBS based on GLP-2 gelatin at a concentration of 130 mg / L, 10 ml of a PBS solution prepared with 4.2 milligrams of GLP-2 was diluted with 30 ml of the working gelatine solution at 20 * 4 prepared in accordance with the above described. The solution was mixed by soft pipetting to provide a final solution of 130 mg / L of GLP-2 in PBS with 15 * 4 of gelatin. As in Example 1, the vessels were CDI mice obtained from Charles River Laboratory (Ontario, Canada). CDl mice were female at the age of injection at the time of injection (n = 3-4 per group) of 6 weeks of age, unless otherwise specified. The animals were left for a minimum of 24 hours to acclimate to the laboratory facilities before the beginning of each experiment. The animals were identified by marking on the ear. The mice were not restricted in terms of diet or activity during the experiments. The light / dark cycle was 12 hours between 6 pm and 6 am. Most of the injections used 12 * / of gelatin or FBS as vehicle. The controls were animals of sex and corresponding age < N = 3-4) that were injected with PBS or gelatin formulation. Each peptide was prepared in a specific concentration dissolved in 0.5 ce of vehicle. The peptides were injected subcutaneously and the mice were monitored daily in the laboratory facilities. The animals were sacrificed 14 days after injection, and fasted 20-24 hours before slaughter. The mice were anesthetized with C02 and then their blood was removed by cardiac perforation. The blood was collected in 75 μl of TED (Trasysol, EDTA (5000 KlU / ml: 1.2 mg / ml, Dipratin-A), and the blood was centrifuged at 14 kxg for 5 minutes and the plasma was stored at a temperature of -70 ° before the analysis The small intestine was removed from the peritoneal cavity, from the pylorus to the caecum, washed, dried and measured.For purposes of comparison, sections of each animal were obtained from the position identical anatomy.Fragments measuring each 1.5-2.0 cm in length were obtained 8 +/- 2 cm, 18 +/- 2 cm, 32 +/- 2 cm pylorus for histomorphometry representing the proximal jejunum, distal jejunum, and ileum Each fragment of the small intestine was opened longitudinally at its antisense border in a block of tissue and then placed in 10 * 4 formalin (vol. / vol.) overnight and then transferred to ETOH at 70 * 4. For micrometry and orphometric analysis, and particularly to evaluate the effects of GLP-2 s At the height of the crypt / hairiness sections of 5 μ thickness were cut, which were marked with hematoxylin and eosinin. An intestinal micrometry was carried out using a microscope with a video camera (Leitz, Wetzar, Germany), connected to a computer monitor. The microscope was calibrated with magnifications of 4, iOx, 25? and the same my roscope was used for all evaluations. The height of the crypt plus villus was measured by examining at least 20 longitudinally oriented villi from the base of the crypt to the tip of the villi from each stage to the jejunum pro.; - imma and distal as well as distal ileum and is expressed in μm + S.E.M. The results of vain analyzes appear in the attached Figures 1-7 and 9, and are summarized below with reference to these figures: DOSE RESPONSE: Figure 1 illustrates the response to rat GLP-2 med? »D > 3 as weight of small intestine (BW-panel A) and as to crypt ura more villus in proximal jejunum (PJ - panel B), distal jejunum (DJ - panel O and distal ileum (DI - penis D) depending on the dose of rat GLP-2 in a 12 * 4 gelatin formulation (ie, GLP-2 in PBS) administered sc The rat GLP-2 peptide was administered subcutaneously.The results are expressed as a percentage change compared to the Controls that re? r? b? erort only gelatin at 12 * 4 as a vehicle The asterisks indicate statistically significant differences H? comparison with controls <* ~ p 0.005, • ** - p .oi, • * - * 4- ~ p 0.O01). S > - will observe from the results presented in Figure 1 that the injection of GLP-2 peptide results in statistically significant increases in weight of the small intestine in a dose of 1.0 to 5.0 μg. The desired effect on the height of the crypt / hairiness is done in doses of 0.25 μ. EFFECT OF THE FORMULATION: Figure 2 illustrates the results obtained using the formulation based on gelatin (G) or the formulation of FBS (PBS) when administered subcutaneously in a dose of GLP-2 of 2-5 μg 2 times up to date. Panel A illustrates differences in the effect observed in the weight of the small intestine in grams, and panel B illustrates differences in observed effect in crypt height / velocity in μm observed for proximal jejunum (PJ) and distal (DJ). as a distal ileum (ID). It will be observed that both types of formulation caused a statistically significant increase. Probably because of its ability to release GLP-2 more sustainably from the injection site, the gelatin-based formulation elicited a slightly greater response than the PBS formulation. EFFECT OF THE ADMI VIATION ISTRATION: Figure 3 illustrates the percentage increase in intestinal weight (BW) when injecting GLP -2 d »? rat, in a dose of 2.5ug twice a day, either »subcutaneously (SO, intramuscularly (IM) or well abducted 1 < 1F) in the phosphate-buffered saline vehicle. It will be noted that a significant response to the GLP-2 peptide was caused independently of the selected route of administration compared to a control group which received subcutaneously only the PBS vehicle. The subcutaneous injection provided the greatest response. EFFECT OF ADMINISTRATION FREQUENCY: Figure 4 illustrates the results of a small bowel weight assessment and the height of the crypt / villus as a function of the frequency of GLP-2 administration. GL.P-2 s.c. in the indicated dose, either every 12 hours (ql2h) once a day (qd) or once every third day (qod). It will be observed from the illustrated results that all administration frequencies caused a significant increase in the percentage change in weight of the small intestine, compared to a control group that received PBS alone. The greatest increase was caused, for the weight of the small intestine and for the height of the crypt / villus by the most frequent dosing scheme, that is, every 12 hours. EFFECT OF LONG-TERM ADMINISTRATION: Rat GLP-2 in a 10 'gelatin formulation. was administered »subcutaneously in a single dose of 5μg once a day continuously for 4 weeks (panel A), for 8 weeks (panel B), or for 12 weeks (panel C). Figure 5 illustrates the effects mediated by GLP-2 on the weight of the small intestine (BW) and on the heights of crypt plus hairiness fPJ5 DJ and DI) in treated groups' T), compared to a control (P) that received Gelatin at 10 * only. The results for intestinal weight indicate that the small intestine weight increase mediated by GLP-2 is induced and sustained during the period of administration examined, as well as the height of the crypt / ve junitary jejunum. A similar response was also observed for the distal jejunum. All animals received a complete autopsy at the time of sacrifice and no histological abnormality was observed in any of the animals. EVALUATION WITH THE PASSAGE OF TIME: Figure 6 presents the percentage change in the weight of the intestine measured as measured in female CD-1 mice treated subcutaneously with PBS alone (control) or with 2.5 μg of rat GLP-2 administered in PBS twice a day during the indicated number of consecutive days. It is clear from the results that a significant result is obtained after the fourth day of administration, and that this effect is sustained with continuous administration. In other studies, it is also clear that the increased weight of the small intestine achieved with the administration of GLP-2 had exited around days after treatment. However, the effects of GLP-2 on the villus hyperplasty did not return completely, paricularly in the older mice (24 months old) suggesting a slower rate of regeneration of intestinal tissue in these slower vessels . Therefore, it is appropriate to maintain the containers in a maintenance dose regimen during GLP-2 therapy. EVALUATION OF THE GENRE AND AGE OF THE CONTAINERS: Figure 7 illustrates the results obtained with CD-1 mice with corresponding sex treated with 2.5 μg of GLP-2 twice a day from 4 to 16 weeks of age, in comparison with their own controls for the weight of the small intestine as well as histology. It will be observed from the results presented that the effects of GLP-2 on the small intestine are achieved regardless of the gender of the recipient. In a related experiment, female C57BL mice (Charles River, USA) from an age of 6 months to 2 years treated with GLP-2 were evaluated and it was found that GLP-2 was effective in promoting the growth of the small intestine in 6-year-old mice. months to 2 years old. EVALUATION OF THE EFFECTS OF GLP-2 ON THE LENGTH OF THE SMALL INTESTINE: Figure 9 illustrates the effect of the administration of GLP-2 on the length of the small intestine. Patones CD-1 were treated with PBS (ontrol) or rat GLP-2 (2.5μ / j 2 times a day in PBS for 1"days, after which the mice were srified and measured in centimeters the length of the small intestine from the stomach to the ileacecal valve The lengthening of the villi observed in response to GLP-2 may arise from either a GLP-2 effect on cell proliferation or on the inhibition of senescence. To examine these two possibilities, sections of small intestine paraffin from stimulated tissues and control tissue were examined to detect proliferative cell nuclear antigen (PCNA), as a measure of proliferation, and to detect apoptotic cells using the TUNNEL method for apoptosis analysis Proliferation velocities in the proximal jejunum of GLP-2 treated mice were increased (124%) compared to control mice '46 .0 +/- 1.0 * 4 in control, 57 +/- 5.5 * 4 in treated animals). In control mice, proliferation was limited to the small intestine crypt compartment; the villi did not contain p »Ds? t? vas cells for PCNA. In the group treated with GLP-2, prolific cells were detected in the villi, and in the union of the crypt-villi. The apoptotic villi in the proximal jejunum of mice treated with GLP-2 decreased compared to control mice. Cells ap'-iptót ícas in control mice were found ptrirt ipa 3 mind in 3 tip or b »Drde of the villi; none were found in the compartment of the intestine crypt. In mice treated with GLP-2, the distribution of apoptotic cells was similar, but their number was lower. EXAMPLE 3 The results observed with rat GLP-2 in recipient mice, it was thought that several homologs and vertebrate analogues of rat GLP-2 could also mediate an intestinotritic effect. For this purpose, several GLP-2 and GLP-2 analogue were synthesized and evaluated, in accordance with what is described above. A synthesis of solid-phase peptides (SPP) in 300-milliliter (L) container on a scale of 3 millimoles (mmoles) was carried out manually using 6 grams (g) of chloromethylamide resin (Merrifield) (for peptides of C-free free acid, with a substitution of 0.5 ml 1 iqqui valent (meq) per gram, amino acids were protected with the t-butyloxycarboni lo (tBoc) group The side chains of the amino acids were protected with the protection groups of side chains of benzyla (Bz for serine and threonine) benci loximeti lo (BOM, piara histidine), 2-bro obencloxicarboni lo (2-BrZ, for tyrosine), 2- benzyl lo ic rboni lo (2-C1Z, for lysine ), cyclohexyl (cHex, for aspic and glutaric acids) and tosi lo, (for rina) and resin of lorontt't i lo < Merpfe] d). The first amino acid was coupled to the ethyl acetate resin through the amino acid state protected in the presence of potassium fluoride (KF). C-terminal amide peptides were synthesized in a 4-meth i lbenxidpamine resin (MBHA) on a scale of 3 mmoles using 6 g of resin with a substitution of 0.5 ml 1 lequi alentes / g. The first amino acid was coupled with the MBHA ream according to the procedure described for peptide elongation. The deprotection of the amino groups was carried out using 50 * / t lumoacetic tpf acid (TFA). in CH2C12, followed by neutralization using two washes of tpeti lamina at 10 * / (Et3N) in CH2C12. The elongation of peptides carried out using N, N-dichlorohexycarbodiomide / 1-hydraxybenzotriazole (DCC / HOBt) in CH2C12 / d? Met? lfor amide (DMF). The growing peptide chain after each elongation step with acetic anhydride at 20 * / (Ac20) in dichloromethane (CH2C12). The peptide-resipa was washed after each elongation step, capped and deprotection with isopropanol dPrOH) and with methanol (MeOH). The washes were repeated once. N-terminal acetyl peptides were prepared by acetylation of the aminoter inal group with Ac 0 in CH 2 C 12. Resin-bonded products were routinely dissociated by a procedure ba o-alfca usocl > _ > hydrogen fluoride «? F) containing dimethyl sulfide (DMS) and p-cresol co or scrubbers. Crude peptides were purified by preparative high pressure liquid chromatography (HPLC) using a Vydac C18, pore width 15-20μm, 5.08cm x 30.48cm, reverse phase silica column using gradient elution with 0.1 TFA * 4 in water modified with acetoni tp lo. The elution was monitored at 220 nanometers (nm). Each fraction collected was analyzed for purity by analytical HPLC using a Vydac C18, 5μm, 4. 6 x 254 millimeters (m), reversed phase silica column P »DG gradient elution with 0.1 * TFA in water modified with acetanitplo, and onitared at 215 nm. The fractions that showed a purity higher than 95 * / were combined and 1 iodized 1. Acetate salts of the peptides were prepared from TFA salts by dissolving the lyophilized powder in water, with addition of acetonitoplo to aid in the dissolution, if necessary. The solution was passed through a protonated Bio-Rex cation exchange resin. The resin was washed with 5 volumes of water, and the peptide bound to the resin was replaced with 50% acetic acid in water. The eluent was diluted with water and lyophilized.

The p »: > The final lyophilized pH was analyzed to determine its purity: by the analytical reverse phase HPLC methods usau'Jü '.tn Vydac C18, column of 5 μm, column of silica ú * -? Reverse phase of 4.6: • 254 m. The solvent systems employed were a gradient of water adjusted to a pH of 2.25 with triethylamine phosphate, modified with acetonitrile, and a gradient of 0.1 * TFA in water modified with acetonitrile. The column eluent was monitored at 215 nm. The identity of each product was confirmed by means of amino acid analysis and electro-spraying mass spectrometry. By means of this method, the following GLP-2 or GLP-2 analogs Dgos were produced in the form of acetates: a) rat GLP-2 of SEQ ID NO: 3; b) 6LP-2 rat N-acetyl, where the. amino end of rat 6LP-2 was blocked with an acetyl group; c) rat GLP-2 of (Arg + 1), which is rat GLP-2 modified by an additional Arg residue at the amino terminus; d) Rat GLP-2 C-amido which is rat GLP-2 with an amido group added at the carboxyl end (a solution of 1 mg in VA acetic acid (110 μl) was obtained, neutralized with 450 μl of 5N NaOH); e) rat GLP-2 (Arg + 1, + 2) which is rat GLP-2 modified by two additional Arg residues present at the amino terminus f) human GLP-2 (Arg + 34) which is human GLP-2 with a residue Ar »:» added after residue 33; and g) Gl P-7 degu. E or peptides were completely soluble in water at room temperature unless indicated 1:: > contrary.

The inotropic intestinal effect of these GLP-2 and GLP-2 analogs was evaluated in the manner described in example 2. Particularly, peptides were formulated in PBS in a dose of 2.5 μg per injection of 0.5 ml, and administered subcutaneously to female CD-1 mice every 12 hours for 10 or 14 days. The weight of the intestine and the height of the crypt was compared with dysfunctionally treated mice (PBS alone). The results of these experiments are presented in Figure 8. Peptides that were modified from GLP-2 by addition of chemical groups to the amino terminus, specifically (N-acetyl) -GLP-2, or that contained amino acid <; s) adíonal (es) at the N end, (Arg + 1) -GLP-2, or at e! Carboxylic end, as for example (Arg + 34) ~ GLP-2 resulted in GLP-2 derivatives that continued to show properties of small intestine growth factor n vivo, in accordance with what is exemplified by its effectiveness in promoting the growth of the intestine thin and increased crypt height plus hairiness (compared to controls treated with saline) in a 1-day experiment in mice (Figure 8, AF panels). In addition, molecules related to GLP-2 are effective if properties of the small intestine growth can be revealed by the use of information derived from the sequences of related molecules similar to GLP-2 from several species. For example, the degu GLP-2 sequence also demonstrates activity similar to the gut growth factor of »gad» D in a 10-day experiment in mice, with an increase in the weight of the small intestine almost comparable to that observed with GLP -2 rat (both peptides) supplied subcutaneously and 2.5 μg twice a day). These data demonstrate that these modifications of the GLP-2 peptide structure, in accordance with what is illustrated herein, result in molecules that exhibit properties similar to native GLP-2 in vivo. In contrast, when the exocarb oxido region of the molecule was modified by the addition of a blocking group to ino, (C-amido) -GLP-2, the resulting peptide did not exhibit significant biological activity of 6LP-2. alive (figure 8C-D). EQUIVALENTS The above specification is sufficient to enable an expert in the field to practice the invention. Obviously, various modifications of the means described above to carry out the invention that are obvious to a person skilled in the art of molecular biology, medicine or related fields are within the scope of the following claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Drucker, Daniel J. ii) TITLE OF THE INVENTION: PEPTI O - 2 SIMILAR TO GLUCAGON AND ITS THERAPEUTIC USE (iii) SEQUENCE NUMBER: 3 (iv) ADDRESS FOR THE CORRESPONDENCE: (A) RECIPIENT: Pennie% > . Edmonds (B) STREET: 1155 Avenue of the A ericas (C) CITY: New York (D) STATE: N.Y. (E) COUNTRY: USA (F) POSTAL CODE: 10036-2711 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Soft disk (B) COMPUTER: compatible with IBM personal computer (OR OPERATIVE SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Reléase No. 1.0, Version No. 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) SUBMISSION DATE: - (C) CLASSIFICATION: - (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: (202) 638-5000 ( B) TELEFAX: (202) 638-4810 (2) INFORMATION FOR SEQ ID NO: l: < i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (C) HEBREW CONFORMATION: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS: (A) NAME / KEY: modified site (B) LOCATION: 13 (D) OTHER INFORMATION: / note = "ONE AMINO ACID NEUTRAL / POLAR / LARGE / NON AROMATIC "(ix) FEATURES: (A) NAME / KEY: modified site (B) LOCATION: 16 (D) OTHER INFORMATION: / note =" AN AMINOACIDO NEUTRAL / POLAR / " (ix) CHARACTERISTICS: (A) NAME / KEY s modified site (B) LOCATION: 19 (D) OTHER INFORMATION: / note = "A NEUTRAL AMINOACIDO" (ix) FEATURES: (A) NAME / KEY: modified site (B) ) LOCATION: 20 (D) OTHER INFORMATION: / note = "ONE NEUTRAL AMINO" (ix) CHARACTERISTICS: (A) NAME / KEY: modified site (B) LOCATION: 27 ID) OTHER INFORMATION: / note = "ONE AMINO ACID NEUTRAL / PQLAR / GRANDE / NO AROM TICO "(ix) CHARACTERISTICS: (A) NAME / KEYPLACE modified site (B) LOCATION: 28 (D) OTHER INFORMATION: / note =" A NEUTRAL OR BASIC AMINO ACID "(i?) CHARACTERISTICS : (A) NAME / KEY: modified site (B) LOCATION: 34 (D) OTHER INFORMATION: / note = "AN AMINO ACID SELECTED BETWEEN ARG, LYS OR AN ARG-LYS CHAIN" (xi> SEQUENCE DESCRIPTION: SEQ ID N0: 1: Ble Ala Asp cly Ser Phß ß »r? Ap clu Mßt Aßn Thr Xaa Lau Aap Xa * i 1 5 10« Lau? Xa Xaa? ßp Phß llß? ßn Trp Leu Xaa x «a Thr Lyß Ile Thr 20 26 30 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 ami nocides (B) TYPE: am i noce i do < C) CONFORMATION OF HEBRAí only (D) TOPOLOGY: linear Ui) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS: (A) NAME / KEY: yes io modified (B) LOCATION: 19 (D) OTHER INFORMATION: / note = " A NEUTRAL AMINO (ix) CHARACTERISTICS: (A) NAME / KEY: modified site (B) LOCATION: 34 (D) OTHER INFORMATION: / note = "AN AMINO ACID SELECTED BETWEEN ARG, LYS OR THE ARG-LYS CHAIN" (xi) DESCRIPTION OF SEC * UENCE: SEQ ID N0: 2: Hiß? the Aßp ßly and Ser Phe Ser? ßp Glu.Met Aßn Thr Xlß Leu? ap Aßn 1 5 10 15 Leu? La xaa? Rg? Ap Phe Xlß Aßn Trp Leu lie Gln Thr Lyß Xle Thr 20 25 30 (2) INFORMATION FOR SEQ ID N0: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) HEBREW CONFORMATION: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (? I) SEQUENCE DESCRIPTION: SEQ ID N0: 3: Hi *? La? Ap ßly BFX Ph? Ser? ßp Glu M? T? ßn Thr lie Leu? ßp? ßn 1 5 10 is Leu? The Thr? Rg ? ßp Phß Xlß? ßn Trp Leu Xle Cln Thr Lyß Xlβ Thr 20 25 30? ßp

Claims (8)

1. CLAIMS 1. A pharmaceutical composition, comprising a Gl P-2 peptide by selecting between a vertebrate GLP-2 or an ipotrophic intestinal analog of a vertebrate GLP-2, analogue differs from a vertebrate 6LP-2 insofar as it incorporates at least one addition, removal, amino acid substitution or an amino acid with an N-terminal or C-amino acid blocking group, and a pharmaceutically acceptable carrier. 2. A pharmaceutical composition according to claim 1, wherein the GLP-2 peptide is of the formula:
2. Rl- [Ylm-Hiß-Ala-Asp-Cly-Sßr-.p eS * r-Aßp-ßlu-M # t-? Í5n- Tbr-aal-Lßu-Aßp-ßa2- eu-Ala-aa3-aa4- ? «P-Phe-Ile-Aen- Trp-Leu-aa5-ßa6-Thr-LyB-ll € -T r-Aßp- (X] n-R2 where aal, aa2, aa3, aa4, aa5, and aa6 refer to any amino acid residue, and: X is one or two amino acids selected within the group
3. III; And it's one or two amino acids selected within the group
4. III? m is either 1 j n is either lj Rl is H or an N-end blocking group; and R2 is OH or a C-terminal blocking group. 3. The pharmaceutical composition > from rei ication 2, where: aal is selected from group IV; aa2 is selected from group I or II; aa3 is selected within group I; aa4 is selected from group III; aa5 is selected within group IV; aa6 is selected within group II or III. 4. A pharmaceutical composition according to claim 3, wherein the GLP-2 peptide has the amino acid sequence: Rl- [Yjn-Hiβ-Ala-Aβp-Cly-Ser-Phe-Sßr-Aip-Glu-Mβt.- Asn- Thr-Ilß ^ eu-Aßp-Aßn-Leu-Ala-áa3-Arg-Asp-P ß-Ile-Asn-rrp-Leu-llß-5ln-T r-yß-lle-Thr-Aßp- [X ] n-R2.
5. 5. A pharmaceutical composition according to claim 1, wherein the peptide 8LP-2 is a vertebrate GLP-2.
6. 6. A pharmaceutical composition according to claim 5, wherein the GLP-2 peptide is a rat GLP-2.
7. 7. A pharmaceutical composition according to claim 5, wherein the GLP-2 is a human GLP-2.
8. 8. A pharmaceutical composition according to claim 1, wherein: GLP-2 is present in an inotrophic amount. c ?. A method to remove the intestinal recynthesis in the patient who is relieved of such promotion > from. growth, comprising step 3 of administering to the patient a pharmaceutical composition according to claim 8. 10. A pharmaceutical composition according to claim 1, wherein the GLP-peptide is present in a effective amount to promote the growth of pancreatic islets. 11. A method for promoting the growth of pipcrectic islets in a patient that requires said growth promotion, comprising the step of supplying the patient with a pharmaceutical composition in accordance with that claimed in claim 10. 12. A salt of pharmaceutically acceptable addition of a GLP-2 peptide. 13. A pharmaceutically acceptable acid addition salt of a GLP-2 peptide according to claim 12 wherein said GLP-2 peptide is a vertebrate GLP-2 peptide. 14. A useful method for identifying novel intestinotropic peptides, comprising the steps of: a) obtaining an analogue of a vertebrate GLP-2 peptide in vitro, the analogue having a) less substitution, r »amore »_ ?? n, -idition, of snunoscido, or an amino acid with a rub db] ouo; b) traf-a mammal with lii ho nsl 'jü using a réirenep capar to provoke up n testinotrófico effect when used for rat GLP-2; and c) determining the effect of said analogue on the weight of the small intestine and / or on the height of the crypt plus villi and / or the size of the pancreatic islets compared to a control mammal treated in a feigned manner, d > Wherein said intestinal and non-trophic peptide is identified as an analogue that causes an increase of d? ch »3 weight / or said height and / or said size. 15. A method for treating a patient to restore gastrointestinal tissue, the gastrointestinal tissue selected from pancreatic islets and intestinal tissue, comprising the step of implanting in said patient cells that have been conditioned by culture in a medium containing GLP-2 peptide. 16. A method for the treatment of a gastrointestinal disease, where the gastrointestinal disease is selected within the group consisting of ulcers, digestion disorders, malabsorption syndrome, short bowel syndrome, cul de sac syndrome, disease inf 1 amatoria of the intestines, esprue cel laca, tropical sprue, eaprue h ipogammag lobul i né ic, enteritis, regional enteri is Crohri disease), small bowel damage prav >;: - > > : for other therapeutic agents, and other therapeutic agents, and short bowel syndrome, and where the method comprises administering to a patient having gastrointestinal disease, a therapeutically effective amount of a GLP-2 or either a GLP-2 analogue together with a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the gastrointesal disease. 17. A method for the treatment of diabetes, comprising administering to a patient having diabetes a therapeutically effective amount of a GLP-2 or a GLP-2 analog together with a pharmaceutically acceptable carrier to increase the levels of insulin in the patient.