HK1158688A - Periostin-induced pancreatic regeneration - Google Patents

Abstract

A method for regenerating pancreatic tissue using recombinant periostin protein, a nucleic acid encoding said periostin and pharmaceutical compositions comprising said periostin are disclosed. Isolation of a nucleic acid encoding a periostin isoform, panc, is also taught.

Description

Periostin-induced pancreatic regeneration

Technical Field

The present invention relates generally to the use of periostin in the regeneration of pancreatic tissue.

Background

The pancreas produces digestive enzymes, as well as several important hormones, including insulin, glucagon, and somatostatin. Hormone-producing cells are concentrated in the islets of langerhans (islets) that make up approximately 1% -2% of the pancreas. In a healthy pancreas, the β -cells within the islets of langerhans produce insulin in response to elevated levels of blood glucose. There are many diseases that result from or result in the loss of pancreatic tissue. These diseases include diabetes (types 1 and 2) and exocrine pancreatic insufficiency.

Type 1 diabetes (insulin-dependent diabetes mellitus) is an autoimmune disorder in which the body's immune system attacks beta-cells, destroys them, or enough damages them so that little or no insulin is produced. While insulin replacement therapy, strict dietary control, and careful blood glucose monitoring can limit complications associated with diabetes, it is desirable to replace or regenerate the pancreas.

Type 2 diabetes (non-insulin dependent diabetes mellitus) is a metabolic disorder that is initially characterized by insulin resistance and ultimately by the inability of the pancreatic β -cells to adapt insulin production to insulin demand.

Due to the lack of digestive enzymes produced by the pancreas, Exocrine Pancreatic Insufficiency (EPI) does not completely digest food. EPI is found in people with cystic fibrosis and Shu-Diedison Syndrome (Shwachman-Diamond Syndrome). It is caused by the progressive loss of pancreatic cells that produce digestive enzymes. Chronic pancreatitis is the most common cause of EPI in humans. Loss of digestive enzymes results in indigestion and malabsorption of nutrients.

There is a need in the art to develop methods and drugs for regenerating pancreatic tissue.

Surgical transplantation of islets has not proven effective, but pancreatic cells are known to have the ability to regenerate. Pancreatic regeneration-promoting factors such as HIP, INGAP, GLP-1, Exendin-4 (Exendin-4) (e.g., WO 2006/096565, US6,114,307, and USRE39299) have been studied.

Periostin (period)stin) is a secreted protein of about 90kDa, preferentially expressed in the periosteum of bone tissue. (Takeshita et al, (1993) biochem.J., 294: 271-8; Horiuchi et al (1999) J.bone Miner.Res., 14: 1239-49). Periostin comprises NH₂A terminal secretory signal peptide, followed by a cysteine-rich domain, 4 internal homologous repeats, and a COOH-terminal hydrophilic domain. Within each repeat domain, the two regions are highly conserved. Periostin has been recognized in various cancers, and its presence has been considered as a marker and therapeutic target for cancer (Kanno et al (2008) int.j. cancer 122: 1707-18). Periostin has also been shown to be secreted by Pancreatic Stellate Cells (PSC) and to preserve PSC fibrogenic activity while supporting pancreatic tumor cell growth under stress conditions (Erkan, et al (2007) Gastroenterology 132: 1447-64).

Disclosure of Invention

In a first aspect, the invention provides a method of regenerating pancreatic tissue by administering periostin. The regenerated tissue may include beta cells. The methods of the invention can be used to treat diseases caused by or resulting in the loss of pancreatic tissue, such as type 1 diabetes, type 2 diabetes, and EPI.

In another aspect, a nucleotide sequence encoding periostin protein can be administered.

In a further aspect, the present invention provides a nucleotide sequence encoding an periostin protein, the sequence comprising: the sequence panc (FIG. 1); a nucleotide sequence homologous to the sequence panc; or a nucleotide sequence which hybridizes with the complement of the sequence panc.

Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

FIGS. 1A-1C show schematic diagrams of periostin proteins encoded by 5 different nucleotide sequences.

FIG. 1D shows a nucleotide sequence alignment of the 5 periostin nucleotide sequences encoding the variable portions of periostin isoforms shown in FIGS. 1A-1C.

Fig. 2 shows another view of fig. 1D.

FIG. 3 shows the amino acid sequence of recombinant human periostin protein.

Fig. 4A-4B summarize 15 experiments in which pairs of mice were treated with streptozotocin and periostin (at different concentrations), or streptozotocin alone. In fig. 4A, the diamonds represent pairs of mice with normal blood glucose levels in periostin-treated mice, but with STZ-induced diabetes in non-periostin-treated mice; triangles represent pairs of mice with STZ-induced diabetes in the pairs of periostin-treated and non-periostin-treated mice.

FIG. 4B shows that blood glucose levels (mmol/L) from the pair of periostin-treated mice of FIG. 4A are between 40 and 60. mu.g/kg body weight.

FIG. 5A shows a nucleotide sequence encoding an isoform of the periostin protein shown in FIG. 1B.

FIG. 5B shows the amino acid sequence of an isoform of periostin protein shown in FIG. 1B and encoded by the sequence shown in FIG. 5A.

Fig. 6 shows the tissue morphology of the pancreas with transplanted pancreatic astrocytes. Fig. 6A and 6C show infiltration of wild-type donor cells expressing GFP at 3 days and 1 week post-injection. Fig. 6A is an enlarged view of the area indicated by the arrow in fig. 6B. Fig. 6D-6F show that the pancreas injected with mesenchymal cells exhibits: (D) forming a tubular complex expressing epithelial Cadherin (E-Cadherin) and Ngn 3; (E) GFP expressing cells surrounded tubular structures expressing cytokeratin-7; and (F) cells expressing GFP do not express Pdx-1.

Figure 7 shows the effect of direct injection of periostin on pancreatic regeneration and insulin expression. Fig. 7A-7G show: (A) one week after periostin injection, Ngn3+ cells were found near the injection site; (B) insulin expression was observed in the cytokeratin-7 + tubular complex structure; (C) no insulin expression was detected after saline injection; (D and E) after four weeks of injection, insulin expression was observed in and around the tubular structure; (F) the insulin + cluster (cluster) comprises cells expressing glucagon; (G) the insulin + cluster comprises cells expressing Ngn 3.

Detailed Description

In general, the present invention provides a method for regenerating pancreatic tissue using periostin. In one embodiment, the present invention provides a method for regenerating various pancreatic cells within the islets of langerhans using periostin. In a further embodiment, the present invention provides a method for regenerating β -cells in the islets of langerhans using periostin. In another embodiment, the invention encompasses novel isoforms of periostin, including nucleic acids encoding the novel isoforms.

Periostin is present in various isoforms. As used herein, "isoform" is defined as "any one of two or more functionally similar proteins from which different exons have been removed that have similar, but not identical, amino acid sequences and are encoded by different genes or RNA transcripts" (Merriam-Webster's Medical Dictionary one line).

As shown in fig. 1, the nucleic acid sequence encoding periostin consists of a conserved EMI domain (a small cysteine-rich module of about 75 amino acids, first named after its presence in proteins of the EMILIN family was found) and 4 fasciated protein repeats (fasciclin repeats). The carboxy terminus includes multiple exons. How these exons are cleaved off (splice out) changes lead to various isoforms of periostin.

Fig. 1 and 2 show nucleotide sequences encoding 4 known isoforms of murine periostin. Although these sequences are determined from nucleic acids isolated from mice, it is believed that other mammalian species will also contain substantially similar periostin genes. Isoform #1 is the longest known isoform of periostin and includes all 23 exons (encoded by the nucleotide sequence PN1, fig. 2). Isoform #2 (encoded by the nucleotide sequence PN2, fig. 2) is the first isoform of the periostin protein identified and was originally named Osteoblast Specific Factor 2 (OSF-2); it does not include exon 17. Isoform #3 was recently named Periostin-Like Factor (PLF) and includes exon 17 but not exon 21 (encoded by the nucleotide sequence PN3, fig. 2). Isoform #4 is currently the most recently disclosed periostin isoform and it does not include exons 20 and 21 (encoded by the nucleotide sequence PN4, fig. 2).

The present invention encompasses the 5 th novel isoform, referred to herein as PANC (protein identified by all capital letters), which is periostin. PANC is the isoform of periostin most frequently expressed during pancreatic regeneration. PANC is similar in size to isoform #4, but does not include exons 17 and 21, as shown by direct sequencing. FIG. 1 shows an alignment of the variable part of the nucleotide sequence encoding PANC (SEQ ID No: 1, FIG. 2) and the above 4 murine isoforms of periostin. Thus, the present invention encompasses the nucleic acid sequences (DNA identified by all lower case letters) of PANC, and PANC proteins, as well as isolated PANC nucleic acids and PANC proteins.

FIG. 3 shows the amino acid sequence of recombinant human periostin protein. FIG. 5A shows the nucleotide sequence (SEQ ID No: 3) encoding the murine isoform of periostin protein shown in FIG. 1B. FIG. 5B shows the amino acid sequence of the murine isoform of periostin protein shown in FIG. 1B and encoded by the sequence shown in FIG. 5A (SEQ ID No: 2).

In type 1 diabetes, the immune system attacks the pancreas; thus, in one aspect, the periostin molecule can be administered in combination with an immunosuppressive agent.

Defining: the term "treating a disorder or disease" in the context of the present invention refers to preventing, arresting the development of, or delaying the progression of a disorder or disease.

The term "regeneration" encompasses in the context of the present invention an increase in the number of cells (proliferation) as well as differentiation of stem cells into new cells. Regeneration of pancreatic tissue includes proliferation of new pancreatic cells, induction of astrocyte proliferation, and/or tubular complex formation.

Periostin nucleic acid molecule: the periostin nucleic acid molecules of the invention can be cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded (i.e., sense or antisense). Segments of these molecules may also be considered within the scope of the present invention and may be produced by, for example, Polymerase Chain Reaction (PCR) or by treatment with one or more restriction enzymes. Ribonucleic acid (RNA) molecules can be produced by in vitro transcription. Preferably, the nucleic acid molecule encodes a polypeptide that is soluble under normal physiological conditions, regardless of the length of the polypeptide.

The nucleic acid molecules of the invention may comprise naturally occurring sequences, or sequences that differ from those naturally occurring sequences but which, due to the degeneracy of the genetic code, encode the same polypeptide. Furthermore, these nucleic acid molecules are not limited to coding sequences, for example, they may include some or all of the non-coding sequences located upstream or downstream of a coding sequence.

The nucleic acid molecules of the invention can be synthesized by a biological cell (e.g., by phosphoramidite-based synthesis) or obtained from a biological cell, such as a mammalian cell. The nucleic acid can be a human, non-human primate (e.g., monkey), mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat nucleic acid. Combinations or modifications of nucleotides within these types of nucleic acids are also contemplated.

Furthermore, an isolated nucleic acid molecule of the invention encompasses a segment that is not found in its native state as such. Thus, the invention encompasses recombinant nucleic acid molecules (e.g., isolated nucleic acid molecules encoding periostin incorporated into a vector (e.g., a plasmid or viral vector) or incorporated into the genome of a heterologous cell (or the genome of a homologous cell, at a location different from the natural chromosomal location).

Periostin family genes or proteins can be identified based on their similarity to the relevant periostin gene or protein, respectively. For example, the identification can be based on sequence identity. The invention features isolated nucleic acid molecules that are at least 50% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to: (a) the nucleotide sequence of panc (FIG. 2); and (b) a nucleic acid molecule comprising a segment of at least 30 (e.g., at least 50, 100, 150, 150, 200, 250, 300, 350, 400, 500, 700, 900, 1100, 1400, 1700, 2000, 2200, 2250, 2300, or 2310) nucleotides of panc (fig. 2).

Determination of percent identity between two sequences was determined using Karlin and Altschul (1993) proc.natl.acad.sci.usa 90: 58735877, respectively. Such algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al, (1990) j.mol.biol.215, 403410. BLAST nucleotide studies were performed using the BLASTN program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid encoding periostin. BLAST protein studies were performed using the BLASTP program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to periostin polypeptides. To obtain gap alignments for comparison purposes, as described in Altschul et al (1997) Nucleic Acids Res.25: 33893402, using GappedBLAST. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used.

Hybridization can also be used as a measure of homology between two nucleic acid sequences. Nucleic acid sequences encoding periostin, or portions thereof, can be used as hybridization probes according to standard hybridization techniques. Hybridization of the periostin probe to DNA or RNA from a test source (e.g., a mammalian cell) is an indication of the presence of periostin DNA or RNA in the test source. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.16.3.6, 1991. Moderate hybridization conditions are defined as equivalent to hybridization in 2 XSSC/sodium citrate (SSC) at 30 ℃ followed by a wash in 1 XSSC, 0.1% SDS at 50 ℃. Highly stringent conditions are defined as corresponding to hybridization in 6 XSSC/sodium citrate (SSC) at 45 ℃ followed by a wash in 0.2 XSSC, 0.1% SDS at 65 ℃.

The invention also covers: (a) vectors comprising any of the foregoing periostin-related coding sequences and/or their complements (i.e., "antisense" sequences); (b) an expression vector comprising any of the foregoing periostin-related coding sequences operably linked to any transcriptional/translational regulatory elements required for direct expression of the coding sequence; (c) an expression vector encoding a periostin-independent sequence other than a periostin polypeptide, such as a reporter, a marker, or a signal peptide fused to periostin; and (d) a genetically engineered host cell comprising any of the foregoing expression vectors and thereby expressing the nucleic acid molecule of the invention (see below).

The recombinant nucleic acid molecule may comprise a sequence encoding periostin or periostin with a heterologous signal sequence. The full length periostin polypeptide, or fragments thereof, may be fused to such a heterologous signal sequence or to another polypeptide. Similarly, the nucleic acid molecules of the invention can encode the mature form of periostin or a form that includes an exogenous polypeptide that promotes secretion.

Transcriptional/translational regulatory elements mentioned above and described further below include, but are not limited to, inducible and non-inducible promoters, enhancers, operators (operators), and other elements known to those skilled in the art and that drive or otherwise regulate gene expression. Such regulatory elements include, but are not limited to, the cytomegalovirus hCMV immediate early gene, the early or late promoter of SV40 adenovirus, the lac system, the trt system, the TAC system, the TRC system, the major operator and promoter region of phage A, the regulatory region of fd coat protein (control region), the promoter of 3-phosphoglycerate kinase, the promoter of acid phosphatase, and the promoter of yeast α -mating factor (α -mating factor).

Similarly, the nucleic acid may form part of a hybrid gene encoding additional polypeptide sequences, e.g., sequences that function as markers or reporters. Examples of markers and reporter genes include beta-lactamase, Chloramphenicol Acetyltransferase (CAT), Adenosine Deaminase (ADA), glucosaminyl phosphotransferase (neo)^r，G418^r) Dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), Thymidine Kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyl transferase (XGPRT). As with many standard procedures relevant to the practice of the invention, the ordinarily skilled artisan is aware of other useful reagents, e.g., other sequences capable of functioning as markers or reporters. Typically, the hybrid polypeptide comprises a first portion and a second portion; the first portion is a periostin polypeptide and the second portion is a reporter or Ig constant region or a portion of an Ig constant region, e.g., the CH2 and CH3 domains of the IgG2a heavy chain, e.g., as described above. Other hybrids may include an antigen tag or His tag to facilitate purification.

Expression systems that can be used for the purposes of the present invention include, but are not limited to, microorganisms such as bacteria (e.g., E.coli and Bacillus subtilis) transfected with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors comprising a nucleic acid molecule of the present invention; yeast (e.g., saccharomyces and pichia) transfected with a recombinant yeast expression vector comprising a nucleic acid molecule of the invention; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) comprising the nucleic acid molecules of the invention; plant cell systems infected with a recombinant viral expression vector comprising an periostin nucleotide sequence (e.g., cauliflower mosaic virus (CaMV) or Tobacco Mosaic Virus (TMV)) or transfected with a recombinant plasmid expression vector comprising an periostin nucleotide sequence (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells) comprising recombinant expression constructs containing a genome derived from a mammalian cell (e.g., the metallothionein promoter) or a promoter derived from a mammalian virus (e.g., the adenovirus late promoter and the vaccinia virus 7.5K promoter). Primary or secondary cells obtained directly from mammals and transfected with plasmid vectors or infected with viral vectors may also be used as host cells.

Thus, cells transfected or transduced with the expression vectors of the invention can be used, for example, for the large or small scale in vitro production of periostin polypeptides or antigenic fragments thereof by methods known in the art. Essentially, such methods involve culturing the cells under conditions that maximize production of the polypeptide or antigenic fragment and isolating it from the cells or the culture medium.

Periostin protein/polypeptide: periostin proteins/polypeptides of and for use in the present invention include periostin with and without a signal peptide. They also include recombinant forms and isoforms.

The amino acid sequence of the periostin molecule may be identical to the wild type sequence of the periostin molecule. Also encompassed are polypeptides that are substantially identical to the wild-type sequence of periostin. The term "substantial identity" when applied to a protein may mean that two sequences, when optimally aligned, typically share at least about 70% sequence identity, alternatively at least about 80%, 85%, 90%, 95% sequence identity or more, for example by the GAP or BESTFIT programs using default GAP weights. Alternatively, any of the polypeptides may comprise mutations such as deletions, additions, or substitutions. All that is required is that the mutant periostin molecule have at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, or even higher) of the ability of the wild type periostin molecule to bind to an antibody specific for wild type periostin.

For amino acid sequences, amino acid residues that are not identical may differ by conservative amino acid substitutions. The term "conservative substitution" refers to the replacement of an amino acid with another amino acid, where the two amino acids are members of a group (group) of amino acids that have certain common properties. A useful way to determine the common nature between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, g.e. and r.h.schirmer., Principles of Protein Structure, Springer-Verlag). From such analysis, groups of amino acids in which the amino acids within the group are preferentially exchanged with each other can be determined, and thus most similar to each other in their influence on the entire Protein Structure (Schulz, g.e. and r.h.schirmer., Principles of Protein Structure, Springer-Verlag). An example of a set of amino acid groups defined in this way includes: (i) a charged group consisting of Glu and Asp, Lys, Arg and His, (ii) a positively charged group consisting of Lys, Arg and His, (iii) a negatively charged group consisting of Glu and Asp, (iv) an aromatic group consisting of Phe, Tyr and Trp, (v) a nitrogen ring group consisting of His and Trp, (vi) a large aliphatic nonpolar group consisting of Val, Leu and Ile, (vii) a less polar group consisting of Met and Cys, (viii) a small residue group consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, gin and Pro, (ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group consisting of Ser and Thr.

The polypeptide may be purified from a natural source (e.g., blood, serum, plasma, tissue or cells such as pancreatic, lung, placenta, or colon tissue, or any cell that naturally produces an periostin polypeptide in a cancerous cell or a normal cell). Periostin molecules can be those of humans, non-human primates (e.g., monkeys), mice, rats, guinea pigs, cows, sheep, horses, pigs, rabbits, dogs, or cats. Smaller peptides (less than 100 amino acids in length) can also be conveniently synthesized by standard chemical methods. In addition, both polypeptides and peptides can be produced by standard in vitro recombinant DNA techniques and in vivo gene transfer (transgenesis) using nucleotide sequences encoding the appropriate polypeptide or peptide. Methods well known to those skilled in the art can be used to construct expression vectors containing the relevant coding sequences and appropriate transcriptional/translational control signals. See, e.g., in Sambrook et al, Molecular Cloning: a Laboratory Manual (second edition) [ Cold Spring Harbor Laboratory, N.Y., 1989] and Ausubel et al, Current Protocols in Molecular Biology [ Green Publishing Associates and Wiley Interscience, N.Y., 1989 ].

Recombinant periostin can be used in the methods of the invention. An example of recombinant periostin useful in the present invention is provided in FIG. 3.

The proteins and polypeptides of the invention may also be produced from any of the nucleic acid molecules discussed above by techniques known in the art.

The polypeptides of the invention include fragments of full-length periostin, wherein such fragments are also capable of regenerating pancreatic tissue. Such polypeptide fragments may comprise a sequence of at least 15 (or 30, 50, 100 or 150) conserved amino acids of periostin proteins. The polypeptide fragment comprises a portion of periostin that is biologically active in the absence of other portions of the protein. As is known in the art, it is often the case that relatively small amounts of amino acids can be removed from the ends of a protein without destroying activity.

The polypeptide or polypeptide fragment may be part of a larger protein, such as a gene fusion to a second protein or polypeptide. Alternatively, the polypeptide or polypeptide fragment may be bound to a second protein, for example by means of a cross-linking agent.

Periostin or polypeptide portions thereof can be chemically modified by covalent binding to a polymer. This may be done, for example, to increase its circulatory half-life. Polymers and methods for attaching them to peptides are shown in U.S. Pat. nos. 4,766,106, 4,179,337, 4,495,285 and 4,609,546. Examples of polymers are polyoxyethylated polyols and polyethylene glycols (PEG). PEG is soluble in water at room temperature and has the general formula: r (O-CH)₂-CH₂)_nO-R, where R may be hydrogen, or a protecting group such as an alkyl or alkanol group. The protecting group may have 1 to 8 carbons, and may be methyl. The symbol n is a positive integer, typically between 1 and 1,000, and may be between 2 and 500. PEG has a typical average molecular weight between 1000 and 40,000, and may be between 2000 and 20,000, or between 3000 and 12,000. PEG may have at least one hydroxyl group, and may have a terminal hydroxyl group.

Pharmaceutical formulation and route of administration: the pharmaceutical composition comprising periostin protein or fragments thereof may be used for the treatment of pancreatic insufficiency. In another aspect, the pharmaceutical composition may comprise a periostin nucleotide sequence according to the invention.

The compositions of the present invention may be formulated in conventional manner, for example orally, buccally, intranasally, parenterally (e.g. intravenously, intramuscularly or subcutaneously), topically or rectally, or in a form suitable for administration by inhalation, using one or more pharmaceutically acceptable carriers. The composition can be injected directly into the pancreas, circulatory system, or intraperitoneally space. Administration may be surgical insertion of a gel or matrix containing the composition.

When a liquid formulation is used, the polypeptide or nucleic acid may be formulated in different concentrations or using different formulations. For example, these formulations may include oils, polymers; vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Carbohydrates include sugars or sugar alcohols such as mono-, di-, or polysaccharides, or water-soluble glucans. The saccharide or glucan may include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha-and beta-cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose, or mixtures thereof. Sucrose is an example. Sugar alcohols are defined as C having an-OH group₄To C₈Hydrocarbons, and include galactitol, inositol, mannitol, xylitol, sorbitol, glycerolAnd arabitol. Mannitol is an example. These sugars or sugar alcohols mentioned hereinabove may be used alone or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the aqueous preparation. The sugar or sugar alcohol concentration is typically between 1.0 w/v% and 7.0 w/v%, and may be between 2.0 w/v% and 6.0 w/v%. Amino acids include the levorotatory (L) form of carnitine, arginine and betaine; however, other amino acids may be added. The polymer comprises polyvinylpyrrolidone (PVP) having an average molecular weight of between 2,000 and 3,000 or polyethylene glycol (PEG) having an average molecular weight of between 3,000 and 5,000. If they are used, buffers are typically used in the compositions before lyophilization or after reconstitution to minimize changes in the pH of the solution. Most any physiological buffer can be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are typical buffers. The concentration may be 0.01 to 0.3 molar. Surfactants may also be added to the formulation.

Following preparation of the liquid pharmaceutical composition, lyophilization may be performed to prevent degradation and maintain sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. The composition may be reconstituted with a sterile diluent (e.g., ringer's solution, distilled water, or sterile saline) prior to use. After reconstitution, the composition is preferably administered to the subject using methods known to those skilled in the art.

As used herein, "pharmaceutically acceptable excipient" refers to an excipient that can be used to prepare pharmaceutical compositions that are generally safe, non-toxic, and have no biological or other undesirable properties, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. "pharmaceutically acceptable excipient" as used in the specification and claims includes one or more than one such excipient.

Examples

And (4) performing surgical operations. In all surgical procedures, diluted buprenorphine (0.03mg/ml) was administered subcutaneously at a dose of 0.05mg/kg to surgical mice 45 minutes to 1 hour prior to surgery. Mice were induced in an anesthetic box with isoflurane gradually increasing to 5%. The anesthetic agent was delivered by an Ohio Forane vaporizer (induction box) and an isoflurane vaporizer (mask). After completion of anesthesia, mice were transferred to a mask with 1.5% isoflurane. The surgical area was shaved and cleaned with end soap, rinsed with sterile water and surgically prepared with chlorhexidine gluconate solution. BNP eye ointment is spotted in the eyes of the animals to prevent them from dehydrating during anesthesia. 1ml of sterile saline was administered subcutaneously prior to surgery. During surgery, mice were maintained at 1.5% isoflurane levels (increased or decreased if necessary). After completion of surgery, mice were placed in oxygen for about 1 minute and then returned to their cages as soon as they were seen to move.

To remove pancreatic tissue, the abdominal cavity is accessed by performing a median incision. First, a1 to 1.5cm incision was made through the skin at the center of the abdomen using a 10-gauge surgical blade. The skin was carefully separated from the abdominal wall using forceps to expose the midline of the abdomen. The midline was lifted with mouse-tooth forceps and a small incision of less than 1cm was made through the body wall with scissors. After positioning, the splenic pancreas (splenic pancreas lobe) was lifted by incision with forceps. Forceps and cotton rolls (cotton applicator) were used to remove the entire splenic lobe and distal portions of the gastric and duodenal pancreatic lobes by gentle dissection (abrasion) to ensure no damage to the main vein or artery. If excessive bleeding is observed, the site of bleeding is pinched for several minutes to promote clotting. After resection, only a small portion (about 30%) of the pancreas remains around the duodenum. The surgically excised pancreas was approximately 70% of the total pancreas as evidenced by weighing the excised and remaining portions during the pilot study. The body wall was closed with two or three interrupted sutures with surgical suture (Johnson & Johnson). The skin is closed with two or three surgical staples (Fisher). After the surgery was completed, the mice were placed in oxygen for approximately 1 minute and then returned to their cages as soon as they started to move. Blood glucose levels were analyzed every other day and increased glucose blood glucose levels were examined. In addition, one week after surgery, mice were given 0.05mg/kg of buprenorphine subcutaneously per day.

Example 1 pancreatic regeneration Using recombinant periostin

To illustrate the role of periostin in pancreatic regeneration, recombinant periostin protein was injected into the pancreas. The recombinant periostin protein supplied by BioVendor (RD172045100) was resuspended in saline and diluted with saline at a concentration of 10 ng/. mu.l. Recombinant periostin protein (671 amino acid sequence shown in FIG. 3) is a human periostin protein truncated at the C-terminus and is representative of sequences common to all four known isoforms. Mu.l (100ng or 5. mu.g/kg) was injected directly into the pancreas. Direct injection was performed by exposing the pancreas with a median incision and injecting 10 μ l of recombinant periostin solution (50ng/μ l) directly into the pancreas with a Hamilton syringe, as outlined above. Vehicle treated animals received the same amount of buffer diluted in saline. After injection into the pancreas, the body wall was closed with sutures and the skin was closed with wound clips, as was done after pancreatectomy. One week after surgery, mice were monitored daily and given 0.05mg/kg buprenorphine subcutaneously. BrdU (Sigma) was administered at 0.8mg/ml in drinking water after surgery to continuously label dividing cells.

Periostin induced extensive proliferation when compared to saline injection 24 hours after injection. Histological studies showed that this proliferation occurs outside of pancreatic islet, pancreatic duct and glandular cells and is localized to cells expressing vimentin. During regeneration, periostin is located at the regenerative tip of the pancreas surrounding cytokeratin 7+, and Ecad + tubular complexes. These complexes were the source of pancreatic proliferation as shown by Ki67 immunostaining. Periostin mRNA increased approximately 10-fold after 3 days of pancreatectomy relative to other (restating) pancreatic periostin mRNA. This is comparable to only a three-fold increase during fetal development.

The number of cells expressing vimentin increased substantially 3 days after periostin injection. This increase was localized to regions with tubular complexes, but not to other regions of the pancreas expressing normal levels of vimentin. However, after 3 days of periostin injection, no further proliferation occurred in the vimentin-expressing cells, but in the tubular complex expressing cytokeratin 7. The tubular complexes expressed epithelial cadherins as in pancreatic ducts, pancreatic islets and glandular cells, but showed increased proliferation as shown by increased Ki67 immunostaining. The tubular complex also expressed the pancreatic progenitor markers Pdx-1 and Ngn 3. Ngn3+ cells were also found outside the tubular complex, rather than in the region immediately adjacent to the tubular complex. In the distal region of the tubular complex, there was no formation of Ngn3+ cells.

After 1 week of periostin injection, the stroma (stroma) increased as noted by accumulation of epithelial cadherin negative cells relative to the saline-injected pancreas. Although only a few tubular complexes remained in the periostin-injected pancreas, proliferation was extensive compared to the saline-injected controls. However, proliferation is now in cells expressing epithelial cadherin, not just in tubular complexes. Proliferation occurs within the glandular cells expressing amylase, but not in the surrounding matrix. The surrounding matrix exhibited an accumulation of BrdU and a dimensional change, the width of which varied between 10 μm and 300 μm. The maximum accumulation of stroma still contained some epithelial cadherin-positive tubular complexes, however, they were not as much as 3 days after periostin injection. For mice, doses greater than 500 μ g/kg body weight resulted in increased pancreatic cell death, especially in exocrine cells that secrete amylase.

Example 2 pancreatic regeneration using intraperitoneal injection. To determine if periostin could be administered in a less invasive route while still inducing pancreatic regeneration, the recombinant protein was injected intraperitoneally at 50 μ g/kg body weight, as opposed to directly into the pancreas (BioVendor; RD 172045100). One week after periostin injection, an increase in BrdU uptake by islet cells was exhibited relative to saline-injected controls. Furthermore, proliferation in islets was increased as shown by Ki67 staining compared to saline injected controls. FACS analysis using MIP-GFP mice injected with periostin or saline showed an approximately 2-fold increase in the number of insulin-secreting β -cells (n-3). The periostin injected mice showed more than two-fold increase in the number of pancreatic β -cells compared to saline injected cub controls.

Example 3 pancreatic regeneration in STZ-induced diabetes using recombinant periostin. Streptozotocin (STZ) selectively targets and destroys pancreatic β -cells in mammals. It can be used to produce animal models for type 1 diabetes. To induce Diabetes in mice, 100mg/kg of STZ was injected intraperitoneally every other day until the mice became diabetic as described in the C57BL/6J background described in Gross (Gross, J.R. et al (2002) Diabetes, 51: 2227-32) and Craven (Craven, P.A. et al (2001) Diabetes, 50: 2114-25). After the first STZ injection (day 0), blood glucose levels were obtained daily to determine the diabetic status of the mice. In an alternative protocol, diabetes was induced by injecting mice with 50mg/kg of STZ daily for 5 days, wherein blood glucose levels were analyzed every other day. Diabetic mice were treated with insulin needed for improved health and longevity.

To determine whether periostin could prevent STZ-induced diabetes, recombinant periostin (biovector; RD172045100) was injected in mice after STZ treatment. After STZ injection as described above, STZ-treated mice were injected Intraperitoneally (IP) with recombinant periostin at various concentrations ranging from 10mg/kg to 90mg/kg of mouse body weight. Blood glucose levels were analyzed every other day. If periostin-treated mice maintain normal blood glucose levels while their non-periostin-treated sibling control mice develop diabetes, then it is determined that periostin is able to prevent STZ-induced diabetes. When STZ and periostin were injected, 11 of 15 animals were able to maintain normal blood glucose levels (fig. 4). The experiment also suggested that the optimal dose of IP injected periostin in mice appeared to be between 30-70mg/kg body weight. The preferred dose of IP injected periostin in mice appears to be between 40-60mg/kg body weight.

Example 4 isolation of DNA encoding periostin isoform PANC. Pancreatic tissue was flash frozen in liquid nitrogen and flash ground with a freezing mortar and pestle. Prior to thawing, the milled pancreatic tissue was mixed with 1ml of TRIZOL reagent (Invitrogen cat # 15596-018). RNA from pancreatic tissue was then isolated according to the manufacturer's instructions.

RNA samples were reverse transcribed using an RNA PCR Core Kit (Applied biosystems cat # N808-0143) according to the manufacturer's instructions. Oligo d (T) and random hexamer primers provided in the kit were used to prime the Reverse Transcription (RT) reaction to generate cDNA.

The PCR reaction used to amplify the carboxy terminus of periostin from the cDNA generated above included the following reagents: mu.l cDNA, 50nM forward PCR primer AAACTCCTCTATCCAGCAGA (SEQ ID NO: 4), 50nM reverse PCR primer AACGGCCTTCTCTTGATCGTCT (SEQ ID NO: 5), 500nM dNTP, 1mM MgCl₂5. mu.l of 10 × reaction buffer and 0.25. mu.l of TAQ polymerase (Invitrogen cat # 10342-020). The reaction was diluted to 50. mu.l. The conditions for carrying out the PCR reaction were as follows: 25 cycles (60 seconds at 95 ℃,60 seconds at 60 ℃, and 60 seconds at 72 ℃). The reaction was then performed on a 2% agarose gel and the prominent band observed at 462bp was excised from the gel using a clean scalpel. DNA fragments were extracted from the Gel using the QIAquick Gel Extraction kit (Qiagencat #28706) and following the manufacturer's instructions. After gel extraction, the DNA fragments were sequenced in 2 separate reactions using the forward and reverse PCR primers described above. More specifically, 10ng of PCR template was sequenced using an Applied Biosystem3730DNA analyser from Stemcore (OHRI, Ottawa, ON) with 2. mu.M primers.

Example 5 safety of injected periostin. To determine whether periostin can be safely administered, mice were injected with recombinant protein (BioVendor; RD172045100) starting at eight weeks of age, by intraperitoneal injection at 0, 2, 4 or 10 μ g/kg body weight once for each of six weeks. No visible tumors were detected 13 weeks ago. Blood glucose levels were monitored weekly for each mouse. The mean blood glucose levels in mM/L for each group of mice (6 per group) are shown in Table 1 below. The standard deviation ranged from 0.31 to 2.06 mM/L.

TABLE 1

Example 6 astrocytes express periostin and mediate pancreatic regeneration. To determine whether astrocytes are the cell source of periostin during pancreatic regeneration, a highly purified population of periostin-expressing cells was isolated from an adult pancreas. The purified population of periostin-expressing cells is isolated by: (1) in regenerating the pancreas, expression of periostin was observed in cells co-expressing vimentin and stem cell antigen-1 (Sca1/Ly6A), and (2) live cells expressing Sca1 were isolated from the remaining pancreas using standard fluorescent live cell sorting (FACS) protocols (using Sca1 specific antibodies that bind fluorescent dyes; eBioscience 17-5981). Identification of CD31 Using Standard FACS protocols^-Cells (using CD 31-specific antibodies conjugated to fluorescent dye; eBioscience 12-0112) were used to remove endothelial cells that also expressed Sca 1.

FACS purified Sca1 was cultured and expanded in RPMI medium with 10% fetal bovine serum⁺/CD31^-A cell. In culture, Sca1⁺/CD31^-The cells exhibit the morphological characteristics and markers of pancreatic astrocytes (vimentin)⁺Smooth muscle actin⁺Desmin⁺Nestin⁺、GFAP⁺Cytokeratin-7^-Epithelial cadherins^-Amylase^-And insulin^-). Determination of expression of the hybrid periostin-LacZ allele by fluorescein bis (BETA-D-galactopyranoside) (FDG) staining and flow cytometry to restrict it to Sca1⁺A cell. In addition, standard quantitative PCR protocols identified cultured Sca1⁺/CD31^-The cells act as expressed periostin mRNA. These results indicate that pancreatic astrocytes are a cellular source of periostin during pancreatic regeneration.

Example 7 mesenchymal astrocytes induce pancreatic regeneration. Sca1 to be infected with lentivirus-GFP⁺Astrocytes were injected directly into the pancreas of recipient mice (1E4 cells/mouse). Wild-type astrocytes were observed to infiltrate the receptor pancreas and induce tubular complex formation and production of Ngn3 progenitor cells as well as islet cells expressing Pdx 1. Staining with anti-GFP antibody (invitrogen a21311) under standard protocols at 2 weeks post injection showed that the donor cells were interspersed throughout the endocrine tissue. The region containing donor cells expressing GFP also contained a tubular complex expressing Ngn 3. The tubular complex does not contain donor cells that are cytokeratin-7 or Pdx-1 co-localized with GFP.

Fig. 6 shows the histological features of the pancreas with transplanted pancreatic astrocytes. Fig. 6A and 6C show infiltration of GFP-expressing wild-type donor cells 3 days and 1 week after injection, respectively. Fig. 6A is an enlarged view of the area indicated by the arrow in fig. 6B. The scale bar of fig. 6A is 500 μm, while the scale bar of fig. 6B is 1 mm. Fig. 6D-6F show that the pancreas injected with wild-type cells exhibits: (D) forming a tubular complex expressing epithelial cadherin and Ngn 3; (E) a GFP-expressing cell surrounding the tubular structure of cytokeratin-7; and (F) cells expressing GFP but not Pdx-1.

Example 8 periostin-induced pancreatic regeneration and insulin expression. To determine whether periostin induced pancreatic regeneration in STZ-treated diabetic mice, STZ was injected daily for 5 days according to the protocol described above. One week later, recombinant periostin was injected directly into pancreas (5mg/kg body weight) according to the above protocol. One week after periostin injection, tubular complex formation and production of Ngn-3 expressing progenitor cells can be seen, as well as intracellular insulin expression in the pancreatic duct. Insulin expression was found in clusters within and around the pancreatic ducts after 4 weeks of periostin injection using standard histological techniques, with the pancreatic ducts containing insulin-positive and glucagon-positive cells that still expressed Ngn3, indicating that these clusters are immature islets.

Fig. 7A-7G show: (A) one week after periostin injection, Ngn3+ cells were found near the injection route; (B) insulin expression was observed in the cytokeratin-7 + tubular complex structure; (C) no insulin expression was detected after saline injection; (D and E) insulin expression was observed in and around the pancreatic ductal structure 4 weeks after injection; (F) the insulin + cluster comprises cells expressing glucagon; (G) the insulin + cluster comprises cells expressing Ngn 3.

In the previous descriptions, for purposes of explanation, numerous details were set forth in order to provide a thorough understanding of the embodiments of the invention. It will be apparent, however, to one skilled in the art that these specific details may not be required in order to practice the present invention. The above-described embodiments of the present invention are intended to be examples only. Alterations, modifications and variations may be effected to the particular embodiments by those of skill in the art without departing from the scope of the invention, which is defined solely by the claims appended hereto.

Claims (18)

1. A method for regenerating pancreatic tissue by administering periostin.

2. The method according to claim 1, wherein the periostin is a recombinant periostin having the sequence given in figure 3, or having a sequence encoded by nucleotides having the nucleotide sequence panc.

3. The method of claim 1, wherein the regenerated pancreatic tissue comprises beta cells.

4. The method of claim 1, for treating insulin-dependent diabetes.

5. The method of claim 1, wherein the administration is by injection.

6. The method of claim 5, wherein the injection is into the intraperitoneal space, into the circulatory system, or directly into the pancreas.

7. The method of claim 1, wherein said administering is surgical insertion of a gel or matrix comprising said periostin.

8. The method of claim 1, wherein the regenerated tissue releases insulin.

9. A method of regenerating pancreatic tissue by administering a nucleotide sequence encoding an periostin protein.

10. A nucleotide sequence encoding an periostin protein, the sequence comprising:

panc；

a nucleotide sequence homologous to panc; or

Nucleotide sequences which hybridize with the complement of panc.

11. A method of regenerating pancreatic tissue by administering the nucleotide sequence of claim 10.

12. A peptide having a sequence encoded by the nucleotide sequence of claim 10.

13. A method of regenerating pancreatic tissue by administering the peptide of claim 12.

14. A carrier, comprising:

panc；

a nucleotide sequence homologous to panc; or

Nucleotide sequences which hybridize with the complement of panc.

15. A host cell comprising the vector of claim 14.

16. A method of treating, or preventing the onset of, diabetes or exocrine pancreatic insufficiency by administering periostin, the nucleotide sequence of claim 10, or the peptide of claim 12.

17. Use of periostin, the nucleotide sequence of claim 10, or the peptide of claim 12 for treating, or preventing the onset of, diabetes or exocrine pancreatic insufficiency.

18. Use of periostin, the nucleotide sequence of claim 10, or the peptide of claim 12 for the preparation of a medicament for treating, or preventing the onset of, diabetes or exocrine pancreatic insufficiency.

The present invention is as described above.