CN100408096C - Gastrin compositions and methods of use and preparation - Google Patents

Abstract

An embodiment of the invention provided herein is a pharmaceutical composition comprising a gastrin compound having an extended activity upon administration to a subject in comparison with native gastrin. Methods are provided of conjugating portions of the amino acid sequence of gastrin having functional ability to bind to the gastrin/CCK, receptor, to various carrier moieties, including the use of amino acid spacer regions, and use of bifunctional cross-linking reagents. Methods of treating a diabetes patient with the compositions are provided.

Description

Gastrin compositions and preparation and use thereof and preparation method

Invention field

The present invention provides gastrin compositions, preparation method with activity in vivo function longer than gastrin peptide and the method for using gastrin combination treatment diabetes with multiple embodiments.

Background of invention

Therapeutic agent, for example peptide of using in the disease treatment and low molecular weight protein are subjected to a lot of restrictions.These therapeutic agents obtain getting rid of by kidney usually at short notice, or obtain degraded by protease, have therefore limited their bioavailability, cause short plasma half-life and than the required lower drug level of curative effect.Keep long high serum levels with regard to the acquisition greatest treatment efficacy with regard to needs, the high clearance rate of medicine is not optimal.Increase dosage or raising administration frequency bring higher curative effect usually, but also bring higher side effect risk, have therefore limited input dosage or have thrown in frequency.

The half-life of some peptide hormones in blood flow is extremely short, causes just having lost biological activity soon after the administration.Confirmed that gastrin is the peptide hormone that other somatomedin of associating are treated diabetes effectively.Yet when throwing in gastrin separately, curative effect is restricted.In addition, found that gastrin has the relatively shorter half-life.For example gastrin-17 has about 5-9 minute circulating half-life, and gastrin-34 has about 35 minutes circulating half-life.

The compositions that therefore need contain gastrin compounds with prolongation or long lasting effect.

Summary

Feature embodiment of the present invention provides the pharmaceutical composition that comprises gastrin compounds, and this gastrin compounds is compared with natural gastrin, has the activity of prolongation after the patient is given in administration.Gastrin composition in this embodiment contains and is selected from following aminoacid at least: the 29-34 position among the 29-34 position among the SEQ IDNO:1, the SEQ ID NO:2, the 12-17 position among the SEQ ID NO:3 and the 12-17 position among the SEQ ID NO:4, and gastrin is further associated with protein, polymer, lipid or carbohydrate.

Optionally the feature embodiment provides and has comprised Z-Y _m-X _n-AA ₁-AA ₂-AA ₃-AA ₄-AA ₅-AA ₆Gastrin compounds, AA wherein ₁Be Tyr or Phe, AA ₂Be Gly, Ala or Ser, AA ₃Be Trp, Val or Ile, AA ₄Be Met or Leu, AA ₅Be Asp or Glu, and AA ₆Be Phe or Tyr, AA ₆By amidatioon; Wherein Z is a polymer, and when this polymer was protein, Z was this proteinic aminoacid sequence; Y _mIt is the spacer of choosing wantonly, the amino acid residue that comprises m micromolecule neutral amino acid, and X is selected from following continuous part arbitrarily: the 1-28 position residue of SEQ ID NO:1, the 1-28 position residue of SEQ ID NO:2, the 1-11 position residue of SEQ ID NO:3, with the 1-11 position residue of SEQ ID NO:4, condition is that this gastrin compounds is in conjunction with gastrin/cck receptor.AA for example ₁-AA ₂-AA ₃-AA ₄-AA ₅-AA ₆Be Tyr-Gly-Trp-Met-Asp-Phe.Perhaps AA ₁-AA ₂-AA ₃-AA ₄-AA ₅-AA ₆Be Tyr-Gly-Trp-Leu-Asp-Phe.In this general formula, Z is a protein, and for example, Z is the human serum albumin.

Y is the aminoacid sequence that comprises m base, and it has alternative glycine and alanine, for example [Gly-Ala] ₅, perhaps have the random sequence of glycine and alanine.When m greater than 1 the time, in the Y amino-terminal end, or when m was 0, in the X amino-terminal end, this gastrin compounds also had cysteine residues.Gastrin compounds also comprises the bi-functional cross-linking agent that is used for bonding Z.Usually, m is 0 to about 20 residues.In certain embodiment, if m is 0, Xn-AA wherein ₁-AA ₂-AA ₃-AA ₄-AA ₅-AA ₆Also comprise the bi-functional cross-linking agent that is used for bonding Z.

In certain embodiment, X is selected from following sequence: 1 to 11 of SEQ ID NO:3; 1 to 11 of SEQ ID NO:4; 2 to 11 of SEQ ID NO:3; And 2 to 11 of SEQ ID NO:4.Wherein Z is that proteinic gastrin compounds can produce with recombination form.

Another embodiment that the application provides is to encode wherein that Z is the nucleotide sequence of proteinic gastrin compounds.The cell that carries this nucleotide sequence also is provided.This cell is antibacterial or yeast cells.When this cell was bacterial cell, it can be, for example, and the cell of the kind of Escherichia, bacillus or streptomyces.When this cell was yeast cells, it can be the cell of Saccharomyces, Kluyveromyces, Schizosaccharomyces or Pichia sp. kind.

Gastrin compounds contains minimum gastrin composition usually, and this composition is the 29-34 amino acids of SEQ IDNO:2 or the 12-17 amino acids of SEQ ID NO:4 at least.They are arranged in the carboxyl terminal of the gastrin that circulation occurs, and in multiple embodiments, for example can have the additional aminoacid from gastrin.

Component of polymer is not necessarily limited to protein, can also be the synthetic polymer of Polyethylene Glycol (PEG) or glucosan for example.When polymer was protein, in multiple embodiments, it was a serum albumin, for example serum albumin, for example human serum albumin.

Another embodiment of the gastrin compounds that the application provides has C-Y _m-X structure, wherein C is Cys or Lys, Ym is the spacer of the amino acid residue that comprises m neutral p1 amino acid chosen wantonly, X is at least six amino acid residues, and it comprises the sequence in the site of 29-34 at least of the sequence of the site 12 at least-17 that is selected from gastrin-17 (SEQ ID NO:3 and 4) and gastrin-34 (SEQID NO:1 and 2).Gastrin compounds also carries out coupling with polymer, for example, carries out coupling with Polyethylene Glycol (PEG) or glucosan.Gastrin compounds also optionally with the protein coupling.In certain embodiment, gastrin compounds also comprises bi-functional cross-linking agent, wherein terminal the and C covalent bond of first reaction of cross-linking agent.Second reaction terminal and the polymer or the protein covalent bond of cross-linking agent.C-Y _m-X can be with recombination form production, or synthesized by method of peptide synthesis.

In certain embodiment, any gastrin compounds that the application provides is provided with effective dose.The gastrin compounds that the application provides also comprises immunosuppressant.The gastrin compounds that the application provides also comprises Hypoylycemic agents.The gastrin compounds that the application provides also comprises pharmaceutically acceptable carrier.The gastrin compounds that the application provides also comprises somatomedin.For example, in certain embodiment, somatomedin is the glucagon-like peptide 1 receptors ligand.Perhaps, in certain embodiment, somatomedin is the EGF receptors ligand.

The application also provides embodiment of the present invention, and the preparation method that it comprises the medicine that is used for the treatment of the patient who suffers from diabetes comprises arbitrary gastrin compounds that preparation is described according to description, and gastrin compounds is delivered medicine to the patient.In certain embodiment of this method, the administration frequency of gastrin compounds is less than the administration frequency of natural gastrin.This method also comprises measures the regenerated physiological indicator of islets of langerhans, for example, measures fasting glucose (FBG).This method comprises the dependency that reduces insulin.

Another embodiment of the invention also provides the preparation method of gastrin compounds, and this method is that the aminoacid sequence of gastrin and a kind of carrier components compositions are associated.Correspondingly, before gastrin and carrier association, modify gastrin and make it comprise cysteine replacement or additional cysteine residues.It is that pyroglutamic acid is replaced that cysteine is replaced.The gastrin aminoacid sequence comprises at least and being selected from: 29-34 position residue among the aminoacid sequence SEQ ID NO:1; 29-34 position residue among the aminoacid sequence SEQ ID NO:2; 12-17 residue among the aminoacid sequence SEQ ID NO:3; And the 12-17 residue of aminoacid sequence SEQ ID NO:4.In certain embodiment, cysteine is the amino-terminal end in gastrin; Perhaps, before gastrin and carrier association, this method comprises that modifying gastrin makes it further comprise bi-functional cross-linking agent.

Another embodiment of the invention also provides the preparation method of the medicine that is used for the treatment of diabetic, this method comprise preparation can with the gastrin compounds of the modification of serum albumin covalent reaction, and the gastrin of modifying for patient's administration.The gastrin of modifying comprises can be in conjunction with gastrin/cck receptor and cysteine or the terminal natural gastrin sequence of lysine amino acid.Correspondingly, natural gastrin sequence is selected from: the 29-34 position residue among the aminoacid sequence SEQ ID NO:1; 29-34 position residue among the aminoacid sequence SEQ ID NO:2; 12-17 position residue among the aminoacid sequence SEQ ID NO:3; And the 12-17 position residue among the aminoacid sequence SEQ ID NO:4.

The application also provides with the serum levels of the peptide of the aminoacid sequence with gastrin and has compared, be used for keeping for a long time the preparation method of medicine of the gastrin serum levels of growth, this method comprises the aforesaid gastrin compounds of preparation definition, and this gastrin compounds of administration.

The application also provides the test kit that comprises as the application's gastrin compounds that describe, at least a effective dose.

Brief description

After Fig. 1 was presented at processing in 14 days, the gastrin of unmodified was to the influence of the fasting blood glucose level of the NOD mice of nearest diabetes outbreak.

After Fig. 2 was presented at processing in 14 days, the gastrin of unmodified was to the influence of the pancreas insulin level of the NOD mice of nearest diabetes outbreak.

The invention enumeration

Use the gastrin compounds of long-acting, can cause reducing the clearance rate of gastrin or the degraded of minimizing gastrin by enzyme, thereby therefore the half-life of the Plasma Gastrin of long term maintenance high concentration and/or raising gastrin causes curative effect to be improved. By using than low dosage and/or reducing the frequency of diabetic administration gastrin is improved dosage. In addition, when gastrin and carrier yoke close, might some carriers also can shield gastrin and not by immune system recognition, therefore reduce or suppress the gastrin challenge, and improve the half-life of serum gastrin and/or keep the serum gastrin of high concentration.

Conventional embodiment of the present invention provides the composition with gastrin sample activity, refers to gastrin compounds herein. Term " gastrin compounds " as used herein, mean in conjunction with, stimulate or with the interactional preparation of gastrin/cck receptor. Gastrin compounds comprise can with the interactional gastrin derivative of gastrin/cck receptor and conjugates and peptide homologue. Term used herein " derivative " and " conjugates " are equivalent, and it is used to indicate the composition of chemical property association, and it is prepared by method synthetic, biological, restructuring or chemistry.

In a plurality of embodiments, preparation " modification " gastrin also is used for the treatment of the patient who suffers from diabetes. Term " diabetes " as used herein, refer to generation or the excessive any physiology sign of blood sugar of insulin deficit, AIA, perhaps comprise any mammal of experimental animal model and comprise for example any obvious diabetic symptom of people's pattern of I type and type ii diabetes that the symptom in early stage of early diabetes and diabetes reduces insulin or the gentle blood sugar level that improves as feature take gentleness. As used herein, term " mammal " has mammiferous any a member, and comprises people's its ordinary meaning.

The gastrin of modifying can be gastrin derivative or analog, the minmal sequence that comprises 6 amino acid (terminal from C-), and the reactive group that also can add the cysteine residues (relating to SEQ ID 1-4) that for example can stand addition reaction. In a plurality of embodiments, gastrin may extend to 34 amino acid (" greatly " gastrin or gastrin-34), wherein at least 1 reactive amino acid, and for example cysteine residues or lysine residue are added or replace at the N-end. For example the interpolation of the reactive amino acid of cysteine can be distinguished endways, and in relevant embodiment, spacer region can randomly be positioned at before the reactive amino acid of interpolation. For example, the part that spacer region can be used as the gastrin amino acid sequence by biosynthesis or can chemical bond on the amino acid sequence of gastrin, form and have the structure of gastrin sequence-spacer region-cysteine. For example, the amino acid sequence that formed by several for example alanine or glycine of spacer region. Amino acid sequence can be amino acid (such as glycine/alanine) or non-replacing alternately, namely can be random sequence or specific sequence. Sequence is comprised of an amino acid at least.

In embodiment optionally, bi-functional cross-linking agent as reactive ingredients is added to the gastrin of modification, particularly add to amino-terminal end have interpolation reactive group (as, cysteine), or add in the modification gastrin with spacer region, the gastrin that generates the modification with mercaptan for example or amino reactive group at end by the same difunctional or isodigeranyl funtion part of crosslinking agent (for example, listed such as following carboxyl terminal to form, have the gastrin-spacer region of the reactive group of exposure-cys-crosslinking agent-carrier, gastrin-cys-crosslinking agent-carrier, gastrin-spacer region-cys/-crosslinking agent, and have the gastrin of the reactive group of exposure-cys-crosslinking agent).

Then in this case the gastrin of modifying is expelled among the patient, perhaps before injection, external it is coupled to one or more plasma fractions of the serum of the whole plasm that for example obtains or fractionation from patient, or in conjunction with the haemocyanin of one or more purifying of albumin, transferrins or immunoglobulin (Ig) (matter) for example, or in conjunction with lipid/lipophilicity part/hydrophobic parts or the combination polymer support of glucan or PEG for example. As used herein with claim in the term polymer, comprise polymer, synthetic polymer (for example PEG) of amino acid, sugar, nucleosides and composition thereof. For example before combination, can or pass through other chemical method activated polymers by bi-functional cross-linking agent. Compare with the natural gastrin of administration, gastrin compounds or the gastrin conjugates of administration activation cause improving the half-life of serum and/or the high blood concentration of long term maintenance gastrin.

General embodiment of the present invention provides the gastrin composition with part, described part is gastrin compounds, and for example polymer or fusion associate or to associate with the form of the fusion of other peptide compounds with amino acid sequence in non-covalent mode or with covalency conjugates form with large molecule. Compare with natural gastrin form, the gastrin compounds of mentioning herein has longer circulating half-life in infected animal or patient, and/or keeps higher gastrin compounds bulk concentration. In addition, the present invention provides the method for composition and preparation and experiment gastrin compounds in other embodiments, both can offer separately patient or unites at least a growth factor, associating Hypoylycemic agents or combined immunization inhibitor and treat diabetes. The example of growth factor includes but not limited to for example EGF receptors ligand, the GLP-1 receptors ligand of for example GLP-1, for example the growth hormone receptor part of the prolactin receptors ligand of prolactin and for example growth hormone of EGF. The example of immunodepressant includes but not limited to cyclosporin, FK506, rapamycin and daclizumab. The unrestricted type example of Hypoylycemic agents comprises sulfonylurea, meglitinide, biguanides, thiazolidinedione and Alpha-glucosidase inhibitor.

In one embodiment, gastrin compounds can be in blood in conjunction with larger structure or a plurality of structure, and still keep ability in conjunction with target protein, for example gastrin/cck receptor. Usually, gastrin, it is fast degradation otherwise in vivo, is incorporated into carrier protein; Use said composition, can obtain more long-term drug effect. Perhaps gastrin compounds can be coupled to polymer support for example on polyethylene glycol (PEG) or the glucan, can reach similar purpose.

In certain embodiment, the chemical modification of gastrin is used for providing the compound that can covalently or non-covalently react with carrier protein or polymer support in external or body. In relevant embodiment, noncovalent interaction is static or hydrophobic. In other embodiment, modify gastrin in described mode so that when injection it the time have in blood flow affinity to the enhancing of carrier. In another embodiment, need not carrier protein, in vivo or externally prepare long-acting gastrin compounds by chemical modification.

In certain embodiment, carrier protein is plasma protein. In relevant embodiment, plasma protein is the composition of albumin or immunoglobulin (Ig) or immunoglobulin (Ig). Before combination, can modify or the composition of excalation immunoglobulin (Ig) or immunoglobulin (Ig). In certain embodiment, polymer support is polyethylene glycol or glucan. For example, by the amino in the gastrin compounds, the PEG that activates is incorporated into gastrin compounds, and (Vernonese, FM. Biomaterials 22 (2001)-405-417).

In other embodiments, before injection, the genetic recombination technology of Application standard will be carried out the genetic engineering fusion as gastrin compounds and the carrier protein of amino acid sequence, and wherein said albumen is also as amino acid sequence. With gastrin and the carrier protein that has or do not have crosslinking agent/spacer region, for example comprise the neutral uncharged amino acid restructuring of little molecule fusion. The nucleic acid of coding gastrin can be recombinated with all or part of carrier protein and be merged or directly synthetic, and this nucleic acid construct or fusion, codified or insert many plus Amino Acids as the spacer region between two albumen. The fusion of restructuring can be expressed in the bacterial system of yeast (saccharomyces, Pichia pastoris) or standard, perhaps can use mammality or insect cell system. After the standardization program of expression and/or purifying, fusion can be used in the treatment. In the structure of fusion, if necessary, can introduce the modification to the peptide sequence of gastrin compounds.

In one embodiment, modify gastrin compounds, with for example introduce those be present in amino acid for example on lysine or the cysteine reactive group, so that also with the contacted reactive group of other compounds of the polymer of for example carrier protein or carrier nonprotein, can form covalent interaction with carrier protein or polymer. For example, use succinimide base propionic acid-3-2-pyridine disulfide group ester (SPDP), add the reacting thiourea alcohol radical to the gastrin molecule by the amino at lysine, reduce to discharge active sulfur alcohol radical ((" Protein thiolation and reversible protein-protein conjugation.N-Succinimidyl 3-(2-pyridyldithio) propionate; a new heterobifunctional reagent. " Carlsson J with DTT subsequently, Drevin H, Axen R.Biochem J 173,723-737 (1978)). In addition, after having added cysteine or lysine, also can add bifunctional group so that reactive terminal of crosslinking agent and cysteine/lysine reacts, simultaneously another reactive terminal of another end keep exposing or with the carrier coupling.

By EDAC mediation and reaction cystamine, use subsequently DTT reduction disulphide, also can insert mercaptan (" Introduction of sulffiydryl groups into proteins at carboxyl sites. " Lin CM at carboxyl, Mihal KA, Krueger RJ.Biochim Biophys Acta 1038,382-385 (1990). In nonrestrictive embodiment, trans by making-4-(maleimide methyl) cyclohexylamine-1-carboxylic acid succinimido ester (" Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide. " Yoshitake S, Yamada Y, Ishikawa E, Masseyeff R.Eur J Biochem 101,395-399 (1979)), with the amino reaction on the lysine residue in the gastrin, introduce the thiol-reactive group at the amino acid sites of gastrin, described gastrin is subsequently with carrier protein or without the cysteine residues reaction of the activated polymer of mercapto.

Gastrin compounds-carrier complexes comprises additional pattern composition, and it contains easy preparation or separates spacerarm or element or other compositions gastrin compounds-carrier complexes or that strengthen or keep the functional activity of gastrin compounds. Spacerarm can be one or more amino acid, peptide, polypeptide analogies or organic molecule, and can comprise with difunctional or isodigeranyl functional cross-link agent or chitin live alone as a widow thing or polyethylene glycol or relevant polymer.

In another embodiment, carrier and gastrin compounds can with or do not carry out covalent cross-linking with spacerarm.The example of non-spacerarm (cross-linking agent of distance of zero mark degree) comprises EDC.For example, the same bi-functional cross-linking agent that generates spacerarm is suberic acid two succinimido esters, and the isodigeranyl functional cross-link agent that generates spacerarm is 2-imino group tiacyclopentane, 6-succinimido [3-(2-pyridine disulfide group) propionamido] caproic acid (LC-SPDP) and 4-(N-methyl maleimide ylmethyl) thiacyclohexane-l-carboxylate (SMCC).

In a plurality of embodiments, gastrin compounds can with bigger carrier part such as polymer, for example protein association.Because associating can be covalently or non-covalently, protein can be considered to carrier protein.The kind of carrier protein can have non-antigenic character, that is, be natural people's albumen, and can keep maintenance in circulation.Ideal carrier protein is a kind of ordinary matter of finding in people's blood circulation usually.

As used herein, term " gastrin/cck receptor part " comprises combination, stimulation or any chemical compound that reacts to each other with the gastrin cck receptor.United States Patent (USP) 6,288,301 (calendar year 2001s JIUYUE 11 days open) provided the example of described gastrin/cck receptor part, this example comprises the gastrin of variform, for example gastrin 34 (big gastrin), gastrin 17 (little gastrin or short gastrin), gastrin 14, gastrin 13, gastrin 10 and gastrin 8, pentagastrin, tetra gastrin; The cholecystokinin of variform is as CCK58, CCK33, CCK22, CCK12 and CCK8; And other gastrins/cck receptor part.Usually, the total carboxyl terminal aminoacid sequence Trp-Met-Asp-Phe-amide of gastrin/CCK part.Aforesaid methionine (Met) can be replaced by leucine.Expection can also be activatory analog, fragment and other above trims, comprises the local agonist of peptide and non-peptide agonists or gastrin/cck receptor, for example A71378 (Lin etc., Am.J.Physiol.258 (4Pt1): G648,1990).

Synthetic by peptide, can prepare for example gastrin 17 of little gastrin form economically, and synthetic peptide is commercial available.Synthetic people's gastrin 17 for example 15 has methionine or leucic people's gastrin 17 in the site, also can be from Bachem AG, and Bubendorf, Switzerland and from Researchplus, obtaining.The gastrin peptide that occurring in nature is found is the amidated peptide of carboxyl terminal, and the amino acid whose amidatioon of carboxyl terminal is in this article in the scope of gastrin compounds.

Gastrin/cck receptor part also includes the trim of active analog, fragment and other above parts, the aminoacid sequence of its total endogenous mammal gastrin for example, has 60% sequence homogeneity, or 70% homogeneity, or 80% homogeneity.Described part also comprises can promote the excretory chemical compound of endogenous gastrin, from the cholecystokinin in tissue storage site or similarly activate peptide.These examples are gastrin releasing peptides, suppress the gastrin excretory omeprazole of acid and promote the soybean trypsin that CCK stimulates.

The sequence that has shown big gastrin 34 and little gastrin 17 herein.Big gastrin-34 is the extension form that has the little gastrin-17 of additional aminoacid sequence at N-terminal basically.Big gastrin is eliminated in vivo to discharge gastrin-17.Symbol " Glp " at N-terminal is the pyroglutamic acid residue, and it is the natural cyclisation form of glutamic acid.In a plurality of embodiments, replace pyroglutamic acid by using glutamic acid or glutamine, or the disappearance pyroglutamic acid, modify gastrin, to contain N-terminal cysteine or lysine residue with N-terminal pyroglutamic acid residue.In addition, each gastrin 34 and gastrin-17 with the site shown in SEQ ID No:1-2 herein 32 respectively or respectively the site 15 shown in SEQ ID No:3-4 have methionine or leucic modification form is used.

N-terminal Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (SEQ ID NO:1).

N-terminal Glp-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-

Gln-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe-NH ₂

(SEQ ID NO：2)。

N-terminal Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-' Trp-Met-Asp-Phe-NH ₂(SEQ ID NO:3).

N-terminal Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe

-NH ₂(SEQ ID NO：4)。

In certain embodiment, as the gastrin aminoacid sequence with at the fusion rotein of the aminoterminal optional amino acid spacer region of gastrin sequence and have the gastrin compounds of protein carrier, can be applied to the patient by transgenic.Embodiment at the nucleic acid construct that is used for the described fusion rotein of transgene expression, by being similar to U.S. Patent number 5,885,956 disclosed technology, the peptide precursor gene that people's gastrin is former are blended in the proteic gene of code carrier that has or do not have spacer.

Treatment needs the method for the diabetic individual of treatment to comprise administration group of individuals compound, and said composition provides gastrin compounds and for example FACGINT of EGF, GLP-1, prolactin antagonist and growth hormone.The derivant, analog and the conjugates that also comprise these FACGINT.

As used herein, term " FACGINT " is meant and replenishes the factor that gastrin is used for insulin regeneration therapy.Word " FACGINT " also refers to " one or more FACGINT " or " at least one FACGINT " as used herein.

Term " FACGINT " comprises the part and the effector of the variant of a large amount of somatomedin and growth hormone, the preparation of regulating one or more hormone factors, one or more receptors, described receptor relates to and the combining of these growth hormone and somatomedin, usually be appreciated that, illustrate these terms, but it is not limited to: the EGF receptors ligand; PTHrP (PTHrP for example; Garcia-Ocana, A. etc., 2001, PTH associated protein (PTHrP) receptors ligand J.clin.Endocrin.Metab.86:984-988); HGF (HGF for example; Nielsen, J. etc., 1999, J Mol Med 77:62-66) hepatocyte growth factor (HGF) receptors ligand; For example the fibroblast somatomedin of FGF, for example keratinocyte growth factor of KGF (KGF) receptors ligand; The nerve growth factor of NGF (NGF) receptors ligand for example; The Gastric inhibitory polypeptide of GIP (GIP) receptor for example; Transforming growth factor (TGFss) receptors ligand of TGFss (U. S. application patent on June 13rd, 2002/0072115,2002 open) for example; The laminine receptors ligand of laminine for example; The islets of langerhans of INGAP regeneration associated protein (INGAP) receptors ligand for example; Bone morphogenetic factor (BMP) receptors ligand of BMP-2 for example; The vasoactive intestinal peptide of VIP (VIP) receptors ligand for example; The glucagon-like peptide-1 receptor part of GLP-1 and Exendin-4 for example; The glucagon-like peptide 2 of GLP-2 (GLP-2) receptors ligand for example; The dipeptide amido peptidase TV mortifier, its by suppress the enzyme relevant with its integrity come remote-effects GLP-1 level (Hughes, T. etc., 2002, Am Diabetes Assoc Abstract 272-or); The proteic REG receptors ligand of REG for example; The for example growth hormone of GH (GH) receptors ligand, for example prolactin antagonist (PRL) receptors ligand of PRL and Placenta Hominis lactogen (PL); The insulin like growth factor of IGF1 and IGF2 (1 type and 2 types) receptors ligand for example; For example EPO ( Http:// www.drinet.ort/html/august_2002_.htm) erythropoietin (EPO) receptors ligand; Betacellunin (thinking that also it is the member of EGF family); The activin A receptors ligand of activin A for example; The VEGF of VEGF (VEGF) receptors ligand for example; Bone morphogenetic factor (BMP) receptors ligand of BMP-2 for example; The vasoactive intestinal peptide of VIP (VIP) receptors ligand for example; The VEGF of VEGF (VEGF) receptors ligand for example; For example the pituitary adenylate cyclase of PACAP activates peptide (PACAP) receptors ligand; The granulocyte colony-stimulating factor of G-CSF (G-CSF) receptors ligand for example; For example the granulocyte of GM-CSH-huge is had a liking for colony-stimulating factor (GM-CSF) receptors ligand; The platelet derived growth factor of PDGF (PDGF) receptors ligand for example; The secretin receptors ligand of secretin for example.

For any somatomedin, enzyme, enzyme inhibitor, peptide, protein and hormonal compounds that shows typical FACGINT herein, and all known analogs and derivant, no matter be natural existence or by the mutation preparation or design syntheticly, all should be considered as the equivalent of FACGINT.It is conjugates that these equivalents also can be considered, that is, and and by adding the deutero-compositions of one or more chemical groups, and composition thereof.Can change encoding gene, for example by instructing the oligonucleotide that sudden change takes place to generate the FACGINT analog, as people's analog of recombinating.

In addition, take place, can change the identity or the position of more than one amino acid residue by target mutation.Proteinic one-level aminoacid sequence can be increased by coupling, as by glycosylation, acyl groupization or add any other complementary molecule, for example one or more lipids, phosphoric acid, sulphuric acid and/or acetyl group.In addition, the single amino acids residue in the chain can be modified by Oxidation, reduction or other derivatizations.Can shear FACGINT to obtain to keep active any fragment.Prodrug or the metabolite of FACGINT are equivalent to FACGINT.Complete polypeptide or protein or any fragment can with any other peptides or protein, for example immunoglobulin and other cytokines merge.Conjugates comprises, for example, contains the compositions of the FACGINT of the polymer that the coupling non-natural exists, and described polymer contains polyalkylene glycol moiety.Term also comprises the derivant that the one or more amino acid residues by chemical modification parent peptide prepare, and described chemical modification for example is alkylation, acyl groupization, ester formation effect or amide formation effect.Perhaps, can consider to induce the preparation of effect of the formation of FACGINT or simulation FACGINT as equivalent chemical compound." FACGINT " of single form means the chemical compound of any one or a plurality of typical as shown here FACGINT.Term " derivant " and " conjugates " are equivalent as used herein, and be used to represent chemically relevant, and the compositions by synthetic, biology or reorganization or chemical method preparation.

Term " receptors ligand " as used herein, it is receptor related with concrete part, means and receptors bind, stimulation or interactional arbitrary composition.

Recipient embryo comprises the range of definition of receptor stimulating agent, and for the receptor of concrete arbitrarily FACGINT, no matter whether agonist is relevant with the structure of FACGINT.

Term " prolactin antagonist " means any peptides that has similar in appearance to the basic sequence of endogenous mammals prolactin antagonist as used herein, and this term is the active protein factor of prolactin antagonist that has well known in the art, for example, and people's prolactin antagonist.Endogenous people prolactin antagonist is 199 amino acid whose polypeptide that produced by hypophysis.This term comprises that the endogenous prolactin antagonist lacks, inserts or replace sudden change and keeps active prolactin antagonist analog, and comprises the prolactin antagonist from other species and naturally occurring variant.Functional prolactin antagonist comprises, as U.S. Patent number 6,333, the disclosed compositions that the prolactin antagonist receptor is had agonist activity of 031 (activated amino acid sequence) and 6,413,952 (metal composite receptors ligand agonist), G120RhGH (Mode etc. as the human growth hormone's of prolactin antagonist agonist analog, 1966, Endocrinol.137 (2): 447-454) with as United States Patent (USP) 5,506,107 and 5,837, the part of 460 disclosed prolactin antagonists.

PRL, GH and PL are the members of polypeptide hormone family, its total identical structure, immunology and biological function (referring to: " Pancreatic Growth and Regeneration ", Ed.N.Sarvetnick, Ch.1.Brejie, T. etc., 1997), therefore refer to PRL/GH/PL family here.PRL and GH are secreted by vertebrate antepituitary.PRL is relevant with the biological function of wide model, comprises Osmoregulation, reproduction, lactogenic and immunomodulating.GH with relate to the growth relevant with morphogenetic physiological process.Relevant receptors ligand refers to " PRL/GH/PL " receptors ligand.Based on peptide and proteinic structural similarity, about the functional similarity of gastrin complementary action, about functional similarity in conjunction with one or more receptors, FACGINTs can be divided into many groups, and these groups are respectively in the scope of multiple embodiments of the present invention.

Pancreas hyperglycemia sample peptide-1 is synthetic in enteral secretory cell with GLP-1 molecular forms (residue with the conventional site 7-36 of appointment), and this GLP-1 is the amide similar in appearance to GLP-1 (7-37).The original research of GLP-1 biologic activity, the purposes (1-37 and 1-36, the latter are amide) of the extend type of the total length N-terminal of discovery GLP-1.Big GLP-1 molecule lacks biological activity usually.Found afterwards before removing, behind the six amino acid, will to bring GLP-1 molecule than short-form with abundant enhanced biological activity.

The majority that has among the active GLP-1 of circulating biological is found that with GLP-1 (7-36) amide form more a spot of biological activity GLP-1 (7-37) form is also arranged simultaneously.Referring to Orskov, C. etc., Diabetes 1994, and two kinds of peptides of 43:335-339. have all showed same biological function.GLP-1 is secreted in endocrine cells of digestive tract, and described cellular response is in nutrition intake, and brings into play multiple action in the metabolism dynamic equilibrium after alimentation.By the shearing in the site 2 of alanine residue of dipeptidyl peptidase (DPP-IV) mediation, the N-terminal of the peptide of degrading is realized the adjusting of GLP-1.Total summary can be referring to DPP-IV.The biologic activity of GLP-1 comprises the biosynthesis that stimulates glucose dependent form insulin secretion and insulin, glucagon suppression secretion and gastric emptying, and suppress food intake.GLP-1 presents many additional effects at gastrointestinal tract and central nervous system, referring to Drucker, and D., Endocrin 142:521-527,2001.Typical GLP-1 compositions comprises: BIM 51077 (the GLP-1 analog of anti-DPP-IV digestion, available from Beaufour Ipsen), AC2592 (GLP-1, available from Amylin, San Diego CA), ThGLP-1 (GLP-1, modified amino acid and fatty acid appurtenance, available from Theratechnologies, Saint-Laurent, Quebec, Canada), DAC:GLP-1 (Conjuchem, Montreal, Quebec, Canada), CJC-1131 or DAC ^TM: GLP-l (is used for the albuminised GLP-1 genetic engineering of covalent coupling analog, Conjuchem), LY315902 and lasting LY315902 (the GLP-1 analog of DDP-IV resistance that discharges, available from Eli Lilly, Indianapolis, IN), low-molecular-weight GLP-1 analogies, Albugon (albumin: GLP-1 fusogenic peptide, available from Human Genome Sciences, Rockville, MD), Liraglutide or NN2211 (the long-acting GLP-1 derivant of the acylation preparation by the GLP-1 molecule, enter blood flow after, the broad incorporation albumin, described albumin can protect it not degraded by DPPIV, and reduces its renal clearance; Elbrond etc., Diabetes Care 2002 Aug 25 (8): 1398-404).

Exendin-the 4th from the new peptide of Heloderma susspectum (Gila monster) venom, has 53% homology with GLP-1 (7-36) amide.Because the degraded of its anti-DDP-IV, so it plays the long-acting proteic effect of glucagon-like peptide 1 (GLP-1) receptor.Exendin-4 has the character that is similar to GLP-1, and regulates gastric emptying, insulin secretion, food intake and slucagon secretion.The example of Exendin-4 comprises the exenatide (synthesized form that is called as AC2993, Amylin), exenatide LAR (long-acting form), the ZP10 (Exendin of modification-4, it has additional six lysine residues, Aventis/ZealandPharma) and AP10 (long-acting preparaton, Alkermes, Cambridge MA).Physiologic Studies shows that the continuous expression of Exendin-4 in transgene mammal can not disturb dynamic equilibrium, cell mass or the food intake (Biaggio of glucose, L. etc., J Biol Chem 275:34472-34477,2000), so that do not understand the physiological role of Exendin-4 fully.

DPP IV (DPP-IV) mortifier is meant the active chemical compound that suppresses DPP-IV and 766 amino acid whose peptidases relevant with film, and described aminoacid comprises its substrate GLP-1, GLP-2 and GIP.The inactivation of DPP-IV mediation GLP-1 is the bioactive determiner of GLP-1 in the body.The example of DPP-IV mortifier comprises isoleucine thiazole amide, valine purrolidide, NVP-DPP738 (Novartis, Cambridge, MA), LAF237 (Novartis), P32/98 (Probiodrug AG, Halle, Germany) and P93/01 (Probiodrug).

As used herein, term " EGF receptors ligand " comprises the chemical compound that stimulates the EGF receptor, and therefore when gastrin/cck receptor is upset in same or adjacent tissue or same individual, the islet cells that produces insulin will be induced.The example of described EGF receptors ligand comprises it being the length EGF of EGF1-53D, and also comprises EGF1-48, EGF1-49, EGF1-52, and fragment and active analogue thereof.Other examples of EGF receptors ligand are TGF forms, comprise 1-48,1-47,1-51, amphiregulin and poxvirus somatomedin, and the EGF receptors ligand of any performance and the synergistic activity that gastrin/the cck receptor part is identical.These comprise above activation analog, fragment and variant.Also can referring to: Carpenter and Wahl, Chapter 4, in Peptide Growth Factors (Eds.Sporn andRoberts), Springer Verlag, 1990.

This group chemical compound comprises the EGF receptors ligand, further comprises " EGF of modification ", and it comprises variant common or wild type EGF.Variant demonstrates can influence one or more biologic activity, for example the clearance rate of EGF.This term comprises the peptide that has substantially similar in appearance to the aminoacid sequence of people EGF, for example, has replaced one or several amino acid whose peptide in a plurality of residues site.

Made up reorganization EGF form, changed structure and activity, for example, described the EGF that has replaced the methionine (U.S. Patent number 4,760,023) at 21 places, site with leucine residue by genetic engineering.Recombined human EGF (hEGF) with 51 residues, promptly, lack at two C-terminal residues at hEGF site 52 and 53 places and have the replacement of 51 neutral amino acids, in the process of production microorganism and behind the administration patient, keep the active and protease inhibitor degraded of EGF in the site.Many nucleic acid molecules are described to the protein family that coding is very similar to EGF and TGF α (WO 00/29438).Also described in the site 16 the EGF mutain (EGF of sudden change) (WO 93/03757) that neutrality or acidic amino acid replace histidine takes place, described form keeps active when low pH.The chemical analog of EGF and TGF α and fragment have kept the ability (U.S. Patent number 4,686,283) in conjunction with the multiple member of EGF receptor family.The multiple variant of EGF or TGF α can be given influences favourable character, external and intravital stability and the intravital activity that one or more recombiant proteins are produced.As used among the embodiment, the EGF receptors ligand of typical recombinant modified is that length is the C end disappearance form of 51 amino acid whose people EGF and the agedoite (being called EGF51N herein) with site 51, and it has kept total length I.N.T. substantially ^TMActivity, and have in the body and/or vitro stability, promptly stability is at least greater than common or wild type hEGF (on May 15th, 2003 is open, all is incorporated herein by reference for S.Magil etc., PCT/US02/33907) herein.

Term " somatomedin " comprises total identical with endogenous mammals growth hormone primary amino acid sequence and any peptides with biologic activity of mammals growth hormone as used herein.The human growth hormone is contained 191 amino acid whose polypeptide in the strand, molecular weight is about 22 kilodaltons (Goeddel etc., 1979, Nature 281:544-548; Gray etc., 1985, Gene 39:247-254).This term comprises the analog that has disappearance, insert or replace and from the growth hormone of other species and naturally occurring variant.Referring to Cunningham etc., 1989, Science 243:1330-1336 and 1989, Science 244:1081-1085; With WO 90/05185 and U.S. Patent number 5,506,107.

Term " treatment " or " improvement " as used herein means one or more symptoms that alleviate or eliminate diabetes.Term " diabetes " as used herein, the generation or the excessive any physiology sign of blood glucose that refer to insulin deficit, anti-insulin antibody, perhaps comprise any mammal of experimental animal model and comprise for example any tangible diabetic symptom of people's pattern of I type and type ii diabetes that the symptom in early stage of early diabetes and diabetes reduces insulin with gentleness or gentle to improve blood sugar level be feature." symptoms in early stage of diabetes " are described to suspect to suffer from diabetes or related indication mammal, for example, be not diagnosed as diabetes, but show the corresponding symptom of insulin or glucose level, and because family history or genetic predisposition suffer from diabetes or relevant disease easily or about the obesity of type ii diabetes or suffered from diabetes in the past or associated conditions and the risk of recurrence is arranged now.

As used herein, term " immunosuppressant " or " being used for the immunosuppressant preparation " mean any preparation that suppresses immunne response.Table 1 shows typical immunosuppressant, and thinks that any derivant of these preparations or function equivalent is applicable to the embodiment in the present invention described herein and claims.Immunosuppressant or other equivalent preparations in the table 1 that the manufacturer is provided carry out administration according to the known weight in patients standard of the technical staff of pharmaceutical field.For example, Tacrolimus is usually by injection or orally carry out administration, and Sirolimus oral administration normally.

Table 1. is used for the preparation and the commercial source of immunosuppressant exemplary

Hypoylycemic agents or medicine are the enhancers of insulin replies, are generally used for the glucemia control of diabetic.These preparations include but not limited to that sulfonylurea is (as acetohexamide; chlorpropamide; first sulphur nitrogen grass urea; tolbutamide; glyburide; glipizide; glutathione); the meglitinide class is (as repaglinide; Nateglinide); biguanide (as metformin); thiazolidinedione is (as pioglitazone; rosiglitazone); alpha-Glucosidase mortifier (as miglitol); glucagon antagonist; the potassium-channel opener; euglycemic agent; the liver enzyme inhibitor; glucose uptake adjustment thing is regulated the chemical compound of lipid metabolism and the chemical compound of reduction food intake.These preparations of mentioning herein are to use with the mode of gastrin compounds combination.

As used herein, term " mammal " should comprise the mammals that is not subjected to any restriction, for example important animal or barren sow, goat, sheep, horse on people, ape, rodent such as mice or rat, Canis familiaris L., cat, the agricultural, or ape such as gorilla or chimpanzee.As describe in detail herein, indivedual mammals are ND, pre-diabetes patient or diabetics.

The whole body mode of administration includes, but are not limited in the transdermal, sheath, intramuscular, peritoneum, vein, subcutaneous, intranasal and oral route.With any approach easily chemical compound is carried out administration, for example, by inculcating or bolus injection, absorption by epithelium or mucocutaneous lining (as oral mucosa, mucous membrane of rectum, vaginal mucosa, nasal mucosa, intestinal mucosa, etc.), and can unite the other biological active ingredient and carry out administration.Typical route of administration is a whole body, for example, passes through subcutaneous injection.If associating FACGINT or immunosuppressant or Hypoylycemic agents administration, then gastrin compounds can carry out administration with single unitized dose, and perhaps these compositions can carry out individually dosed with any order.

The preparation of compositions method

Synthesizing of gastrin peptide

Come production gastrin peptide by any suitable method, for example in the host cell of reorganization, express or by chemosynthesis production.For the latter, for example the gastrin peptide is synthesized into synthetic by use solid phase Fomc (fluorenylmethyloxycarbonyl) peptide on poly dimethyl acrylamide gel resin.Then by the standard method purified polypeptide.

Coupling counter pair/carrier

Coupling counter pair/carrier comprises: plasma fraction for example be attained at patient's serum, the serum albumin of purification (matter) as albumin, transferrins or immunoglobulin, erythrocyte albumen for example alpha-Glycophorins and AE-1, carbohydrate-binding protein for example enzyme, phosphate and the sulfate of hemagglutinin, passivation is conjugated protein, cholic acid, lipid binding protein, lipid/lipotropy part; Polymer support is glucosan or Polyethylene Glycol for example.When gastrin coupling during in lipid/lipotropy part, in case injected, might these conjugates and the interaction of serum albumin generation noncovalent interaction or covalency, described serum albumin for example is the albumin known with conjugated fatty acid, or the lipid binding protein in the serum for example.For the purpose of compositions herein and method, can habitually serum and blood plasma be exchanged use.For with the gastrin coupling in carrier, at first need carrier to be activated by introducing reactive group.In some cases, carrier has comprised at least one reactive group, for example albuminised cysteine 34.Behind activated carrier (if desired), carrier and gastrin compounds are carried out coupling.Usually, by the reactive group in the protein chain, mercapto, α-and epsilon-amino, carboxyl or aromatic rings for example, carrier can with the albumen covalent bond, wherein all reactive groups all exist, or use known molecular biology method to be added by proteic chemical modification in advance, promptly in chemosynthesis, added, or added by existing gastrin peptide of chemical modification or the aminoacid sequence by modified protein.For example, but the epsilon-amino of the mercapto of the lysine residue that is positioned at N-terminal of carrier conjugated protein or cysteine residues.Perhaps, make carrier and protein bound by the isodigeranyl function or with bi-functional cross-linking agent.

Plasma protein

Plasma protein is in conjunction with being the effective ways that improve the pharmacokinetic property of other short life molecules such as gastrin.The one side of plasma protein is the natural molecule amount of filtering boundary (45kDa) greater than kidney, and therefore the holdup time of prolongation is arranged in blood plasma.Plasma protein includes but not limited to: albumin, α-1 acidoglycoprotein, alpha-1-antichymotrypsin analogues, α-1-antitrypsin, α-2-antiplasmin, α-2-HS-glycoprotein, α-2-macroglobulin, proangiotensin, Antithrombin III, apolipoproteins AI (HDL), apolipoproteins All (HDL), apolipoproteins B (LDL), apolipoproteins CI (VLDL), apolipoproteins CII (VLDL), apolipoproteins CIII (VLDL), apolipoproteins E (VLDL), apotransferrin, Cl esterase mortifier, the plasma copper ferritin, ferritin, Fibrinogen, the GC globulin, hoptoglobin (mixed type), hematochrome, immunoglobulin A, immunoglobulin A 1, immunoglobulin A 1, immunoglobulin A 2, immunoglobulin D, IgE, immunoglobulin G, the Fab fragment, immunoglobulin G, the Fc fragment, immunoglobulin G, immunoglobulin G 1, immunoglobulin G 2, immunoglobulin G 3, immunoglobulin G 4, IgM, the μ chain, IgM, FC _{5 μ}IgM, heavy chain immunoglobulin (H), light chain immunoglobulin (L)-K-light chain, γ-light chain, IgF ab fragment, the immunoglobulin Fc fragment, lactotransferrin, lipoprotein a, [Lp (a)], lipoprotein (high density), lipoprotein (low-density), lipoprotein (extra-low density), prelbumin, haemoglutinin, prothymosin-α, rheumatoid factor, steroid binding protein, transcortin, thyroxine-binding globulin, siderophillin and α-fetoprotein.The tabulation of plasma protein can be referring to Anderson and Anderson, Molecular and CellularProteomics 2002,1.11:845.

Human serum albumin's molecular weight is 67kDa, and the half-life in circulation is 19 days, is albumen the abundantest in the human plasma.Albumin is made up of 585 aminoacid that form single polypeptide chain.Albumin and a large amount of chemical compound interact, and the physiology's part, certain therapeutic agent that comprises long-chain fatty acid for example be warfarin and valproate and inorganic part calcium for example for example.

Antibody/immunoglobulin comprises complete immunoglobulin or its fragment, and wherein immunoglobulin comprises multiple type and isotype, for example IgA, IgD, IgE, IgGI, IgG2a, IgG2b and IgG3, IgM etc.Its fragment comprises Fab, Fv and F (ab ') ₂, Fab ' and analog.Can use fragment, agglutinator, polymer or immunoglobulin conjugates when in addition, suitable.Antibody is monoclonal or polyclonal antibody, technology of using by routine such as host immune method and serum collecting method (polyclone) or by the successive hybrid cell line of preparation with collect secretory protein (monoclonal), maybe can prepare antibody by the saltant sequence that clone and express nucleic acid sequence or its are encoded at least specifically in conjunction with the required aminoacid sequence of natural antibody.

Polymer support

Coupling pairing body can comprise polymer, and it is naturally occurring or synthetic in a plurality of embodiments.The example of polymer comprises for example glucosan of protein, glycopeptide, polysaccharide, for example glycosaminoglycan or carboxyl methyl glucosan, for example dextran sulfate, hetastarch, cellulose fibre element are former with the biopolymer derivant of glucosan, comprise methylcellulose and carboxymethyl, starch and starch derivatives inulin, heparin, heparin fragment; Synthetic polymer is alkyl diol (PAG) for example, for example PEG and derivant thereof, polyoxyethylene polyols (POP) is polyoxyethylene glycerol (POG) for example, polytrimethylene glycol (PTG), polypropylene glycol (PPG), poly hydroxy ethyl acrylate, polyvinyl alcohol (PVA), polyacrylic acid, the Ju ethyl oxazoline, polyacrylamide, polyvinylpyrrolidone (PVP), polyamino acid, polyurethane and polyphosphazene, poly-(lactic acid-altogether-ethylene glycol) PLA-PEG, poly-(D, L-lactic acid-altogether-glycolic) PLGA, poly-(ortho esters), poly-(lactic acid-altogether-Acetic acid, hydroxy-, bimol. cyclic ester)-block-poly-(ethylene glycol), polyoxyethylene polyols, polyvinylpyrrolidone, poly hydroxy ethyl acrylate, polyvinyl alcohol, with polyurethane alcohol)-block-poly-(lactide-co-glycolide) is (PLGA-PEG-PLGA), poly-(N-N-isopropylacrylamide, polyoxyethylene polyols, poly hydroxy ethyl acrylate, N-(2-hydroxypropyl) Methacrylamide (HPMA); The synthetic copolymer of hydrazone, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-Methacrylamide, polypropylene glycol, poly-alkane glycol and derivant thereof; The copolymer of poly-alkane glycol and derivant thereof, polyvinyl ethylether and α, [(2-ethoxy)-DL-N, poly-(N-acryloyl morpholine) are (PacM) for β-poly-.Usually, synthetic polymer can be homopolymer or copolymers linear or ramose, that replace or unsubstituted, two or more different synthons.

Peptide and proteinic Pegylation can cause when keeping biological function, and curative effect is strengthened.By increasing bulk of molecule and the degraded by proteolytic enzyme, with PEG coupling (or with other polymer, as glucosan), can reduce that kidney filters or the process of immunne response, all these can strengthen stability in vivo.PEG (Polyethylene Glycol) is the repeated monomer of normal linearity, can carry out chemical activation to it on free C-terminal.Described activation can bring the reaction with a large amount of functional groups, comprises sulfydryl and amino.In order to suppress cross-linking agent, the available methoxyl group (mPEG) in two terminal hydroxyls adds medicated cap.In order to allow to have the site of a plurality of binding peptides, also can synthesize to have a plurality of forks or ramose PEG at single PEG molecule.

By using amine-modified at least one mPEG of maleimide, the peptide (as cysteine, using the abbreviation Cys of 3 alphabetical aminoacid symbols) that contains free sulfhydryl groups will be easy to take place alkylated reaction, and form stable thioester bond.With regard to gastrin, can use to have that half Guang glycosides peptide is introduced is not the synthetic gastrin compounds of the essential peptide zone of its biologic activity (for example, at its N end).

The peptide (as lysine or N-terminal amino) that contains unhindered amina can react with the PEG that is modified by the succinimide ester, to produce stable amido link.Owing to lack the specificity of chemical reaction, as gastrin-34 with regard to containing 3 lysine residues, any or more lysine residue can react with the PEG that modifies, and cause the heterogeneous mixture of the coupling peptide of chemical property or functional character, so its purification are not inessential.In peptide is synthetic, be linked in lysine residue (describing) on the epsilon-amino acid side chain by introducing PEG wherein as Felix (1997), realize the site-specific Pegylation of special lysine residue easily.Felix，A.M.(1997)Site-Specific Poly(ethylene glycol)ylation of Peptides.In″Poly(ethylene glycol).Chemistry and Biological Ap plications.″Harris，J.M.&Zalipsky S.Eds.

" activatory PEG " (or " Polyethylene Glycol chemical preparation ") is any PEG derivant, and it can be used as the albumen instrumentality, because it contains the functional group that can react with some functional groups in the proteins/peptides, to produce PEG-proteins/peptides conjugates.Activatory PEG comprises the PEG of alkylating PEG, acidylate and has the PEG of amino acid arm.(as mPEG-maleimide-20000, come from ShearwaterCorporation, Huntsville AL) can carry out Pegylation on cysteine residues by using the PEG maleimide.Amino PEG can be used for carboxy polyethylene glycolization.The example of activatory PEG comprise the inferior amide succinate of methoxy poly (ethylene glycol), diaminourea methoxyl group-Polyethylene Glycol, methoxy poly (ethylene glycol)-p-nitro-benzene base carbonate, methoxy poly (ethylene glycol) succinum, methoxy poly (ethylene glycol) tresylate, methoxyl group-polyoxyethylene amine (amino PEG), methoxy polyoxyethylene-carboxylic acid, mono methoxy-PEG right-Nitrobenzol carbonic ester, N-N '-N,N'-carbonyldiimidazole-activatory PEG and methoxy polyoxyethylene imidazoles-carbonyl.The PEG of different molecular weight can be from commercial acquisition.The scope of the mean molecule quantity of reactant PEG arrives between about 100,000 dalton between about 5,000.Method of attachment is not conclusive, but does not preferably change, or only minimally changes the activity of biologically active molecules.Preferred method of attachment is to be linked in polypeptide by N-terminal.Referring to Veronese, Biomaterials 22 (5):: 405 (2001), it has summarized the different activation strategy to PEG.Perhaps, by before coupling, earlier sugared Pegylation being taken place in gastrin and derivation with glycan molecule, can be with the PEG coupling in peptide/protein (that is, glycosyl Pegylation method be as, use Neose ' s Glyco Pegylation technology).

Glucosan is naturally occurring polymer, and it mainly is made up of the linear polysaccharide with the recurring unit of the D-glucose of glycosidic bond link each other.Have branch point in dextran polymer, branch pattern and branch degree are according to species and different.Glucosan can be used for the coupling gastrin compounds.The average molecular weight range of soluble glucan is between about 10,000 to 500,000 dalton.Dextran polymer contains adjacent hydroxyl on each glucose monomer, described monomer by sodium metaperiodate oxidation discharge can with functional group's aldehyde radical of amino reaction.By Schiff's base formation reduction amination taking place subsequently does in order to make up stable combination, can be with the coupling of metacetaldehyde glucosan on amino, for example on the ε amino on the lysine residue.By with chloroacetic acid reaction to produce the carboxymethoxyl glucosan, make glucosan be able to carboxymethoxylization, its with the hydrazides condensation in can form glucosan-hydrazides, this hydrazides can with carbonyl or aldehyde radical reaction.The reactive glucan derivative of sulfydryl can prepare by using the isodigeranyl functional cross-link agent, and described cross-linking agent for example contains pyridyl disulfide, maleimide or iodoacetyl so that coupling reaction is oriented on the sulfydryl at an end.

Coupling with the lipotropy part

Lipophilic substituent can be incorporated into amino acid whose reactive group at the N-terminal of gastrin compounds, or randomly is incorporated into by isodigeranyl function or the reactive group that generates with the bi-functional cross-linking agent group.For example, the carboxyl of lipophilic substituent can react with the amino on the lysine.For example, lipophilic substituent specifically is the long chain alkyl group that contains just like 8-40 carbon atom.For example, lipophilic substituent can be the acyl group of straight or branched alkyl, straight or branched fatty acid, the acyl group of straight or branched alkane alpha, omega-dicarboxylic acid.For example, the lipophilic derivatives of gastrin can be by being synthesized with amino chemical bond, for example uses multiple fatty acid to be connected described fatty acid with the amino of N-terminal and comprise lauric acid (just-dodecylic acid), myristic acid (just-tetradecanoic acid), Palmic acid (just-hexadecanoic acid), palmitoleic acid (just-gaidic acid), stearic acid (just-octadecanoid acid), oleic acid (just-octadecenic acid), acetic acid, linoleic acid and arachidonic acid.

Perhaps, the hydrophobic parts of rigid conformation (promptly have two keys, triple bond is saturated or unsaturated ring) can be incorporated into the gastrin peptide.For example, the long-chain fatty acid that comprises carboxyl can be incorporated into the amino that for example is present on the lysine residue.

Bi-functional cross-linking agent

The example of cross-linking agent includes but not limited to following: amino directed congenerous cross-linking agent comprises: imidodicarbonic diamide ester (diimine acid esters), for example, acetimide acid methyl ester-HCl, suberoyl imidic acid dimethyl ester-2HCl; Two-N-butanimide radical derivative, for example, two (thiosuccimide base-suberates) (BSSS), succinic acid two-(N-hydroxyl-butanimide) ester; Difunctional aryl halide, for example, 1,5 ,-two chloro-2,4-dinitro benzene; Difunctional acylating agent, for example, vulcabond and diisothio-cyanate, as, 1,6 ,-hexylidene diisocyanate; Difunctional sulfonic acid halide, for example, phenol-2,4 ,-disulfonic acid chloride; The dinitro benzene phenolic ester, for example, carboxylic acid two-(right-Nitrobenzol) ester; With difunctional acyl azide, for example, tartaroyl two nitrine; Dialdehyde, for example, glutaraldehyde; Diketone, for example, 2, the 5-acetyl butyryl; For example benzoquinone, 2-imino group tiacyclopentane, tetramethylolmethane two carbonic esters, mucobromic acid, mucochloric acid, chloro ethyl formate, carbonochloridic acid p-nitrophenyl ester, 6-diazanyl nicotinic acid succinimido ester, acetone hydrazone and 4-formoxyl benzoic acid succinimide ester (are for example at first modified amine with the hydrazine joint with other; next, is cross-linked to each other together to the acetaldehyde joint then with amine-modified).

Another group bi-functional cross-linking agent is the same bi-functional cross-linking agent of sulfydryl orientation, and it comprises: mercury reagent, for example, 1,4 ,-two (bromo mercuri) butane; Disulphide forms agent, for example, and polymethylene two (methyl mercapto sulfonate); BMI, for example, N, N '-di-2-ethylhexylphosphine oxide maleimide, two (N-maleimide methyl) ester, two-maleimide ethane, 1,4-pair-dimaleoyl imino butane, 1,4-two-dimaleoyl imino-2,3 ,-dihydroxy butane, two-dimaleoyl imino hexane, 1,8-two-maleimide triethylene glycol.Also having another group is alkylating agent, two-acetyl halide for example, and as 1, the 3-dibromoacetone; Two (alkyl halides), for example, two (2-chloroethyl) sulfide; Sym-triazine, for example, 2,4 ,-two chloro-6-methoxyl group-s-triazine; Aziridine, for example, 2,3,4 ,-three (ethylidene imino group)-s-triazine; With two-epoxide, for example, 1,2,3,4-diepoxy butane; And other, as vinyl sulfone(Remzaol.

The same bi-functional cross-linking agent of other group orientations is that the chemical cross-linking agent field is known, the directed reagent of carboxyl for example, as, the bisazo hexane; Phenates and imidazole radicals directed reagent, for example bigeminy aniline; Arginine reagent, for example, the two Biformyls of right-phenylene; With other reagent, (be used for alpha2-macroglobulin as cis-dichloro two ammino platinum (II) with crosslinked at one or more methionine residues in recognition site or proximity identification site, the complementary strand of the adipic dihydrazide of crosslinkable DNA, and crosslinked glycoprotein, acid phosphatase and saccharase), N, N '-two (b-glycine)-tartramide.

The isodigeranyl functional cross-link agent of group selectivity also is well known in the art, it comprises: difunctional dose of amino and sulfydryl orientation, for example, 3-(2-pyridine disulfide group propanoic acid)-N-succinimido ester (SPDP) and analog thereof (LC-SPDP, sulfo--LC-SPDP), succinimido oxygen base carbonyl-Alpha-Methyl-α-(2-pyridine disulfide group) toluene (SMPT) and analog thiosuccimide-6-{ Alpha-Methyl-α thereof-(2-pyridine disulfide group) toluyl amido } and alkyl caproate (sulfo--LC-SMPT), succinimido-4-(N-maleimide ylmethyl) cyclohexane extraction-1-carboxylate (SMCC) and analog thereof, sulfo-succinyl di-imidogen-4-(N-maleimide ylmethyl) cyclohexane extraction-1-carboxylate (sulfo-MCC), between-dimaleoyl imino benzoyl-N-hydroxyl-succinimide ester (MBS) and analog thereof between-dimaleoyl imino benzoyl-N-hydroxyl-thiosuccimide ester (sulfo--MBS), N-succinimido (4-iodoacetyl)-Aminobenzoate (SIAB) and analog thiosuccimide base thereof (4-iodo acetyl group-Aminobenzoate (sulfo--SIAB), succinimido-4-(right-the dimaleoyl imino phenyl) butyrate (SMBP) and analog thereof, thiosuccimide base-4-(right-the dimaleoyl imino phenyl) butyrate (sulfo-SMBP), N-γ-dimaleoyl imino butyryl-oxygen base succinimide ester (GMBS) and analog thereof (sulfo--GMBS), succinimido-6-[(iodo acetyl group)-and amino] alkyl caproate (SIAX) and analog succinimido-6-[6-((iodo acetyl group) amino)-caproyl thereof] amino] caproate (SIAXX), succinimido-4-(((iodo acetyl group) amino) methyl)-cyclohexane extraction-1-carboxyl ester (SIAC) and analog succinimido-6-thereof ((((4-(iodo acetyl group) amino) methyl)-cyclohexane extraction-1-carbonyl) amino) alkyl caproate (SIACX), N-succinimido-4-dimaleoyl imino butyrate; Carboxyl and sulfydryl or amino directed bifunctional reagent, for example, diazoacetic acid is right-the nitrobenzophenone ester; The bifunctional reagent of carbonyl and sulfydryl orientation, for example, 1-(amino oxygen base)-4-[(3-nitro-2-pyridine radicals) disulfide group)] butane; 4-(4-N-dimaleoyl imino phenyl) butanoic acid hydrazonium salt hydrochlorate (MPBH), 4-(N-maleimide ylmethyl) cyclohexane extraction-l-carboxyl-hydrazine (M ₂C ₂H), 3-(2-pyridine disulfide group) propiono hydrazine (PDPH) and other 2-methyl-N '-benzenesulfonyl-N for example ⁴-bromo acetyl group quinone two inferior amide, N-hydroxy-succinamide base-to formoxyl-benzoate, methyl-4-(6-formoxyl-3-azido-phenoxy group) butyryl imidoether HCl, acrylic aldehyde.

Comprise the group that in embodiments suitable crosslinking agent also has the cross-linking agent of distance of zero mark degree in addition, it comprises: carboxyl activator, carbodiimides for example, as 1-ethyl-3-(3-dimethylaminopropanecompounds)-carbodiimide hydrochloride (EDC) (activation can with NH ₂The carboxyl that group is crosslinked); The oxazole derivant; The carbonochloridic acid ester; Carbonyl dimidazoles; With N-carbalkoxy dihydroquinoline; Disulphide forms agent, as cupric two (1,10 phenanthrolene); Enzyme, for example T-5398, peroxidase (between lysine residue), xanthine oxidase (formation disulfide bond); And other, for example pyrro-quinoline quinone (PQQ) (with lysine be converted to can with half acetaldehyde of other lysine residues reaction).

Pharmaceutical composition

The invention provides pharmaceutical composition in multiple embodiments, it comprises the independent gastrin compounds for the treatment of effective dose or the combination of FACGINT, or contains the Hypoylycemic agents of gastrin compounds.All pharmaceutical compositions of Miao Shuing can use or not use and be used for the immunosuppressant preparation herein, use or do not use the composition or the device that are used for lasting release, part or whole body administration to prepare.Can add pharmaceutically acceptable carrier or excipient.Described carrier includes but not limited to saline solution, buffer salt solution, glucosan, water, glycerol, ethanol, and combination.Dosage form should be suitable for administering mode.Term used herein in this case " effective dose " is the combination that is enough to reach the therapeutic agent or the preparation of the discernible medical purpose that can treat diabetic symptom.As described herein, the technical staff has the existing method of related parameter according to measurement, can determine effective dose by rule of thumb.

Compositions herein also comprises wetting agent or emulsifying agent, or the pH buffer agent.Compositions can be liquid solution, suspension, emulsion, tablet, pill, capsule, slow releasing preparation, or powder.Binding agent by routine and carrier be triglyceride for example, compositions can be mixed with suppository.Oral formulations can comprise standard vector, for example mannitol of pharmaceutical grade, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc.Multiple drug-supplying system is known, and can be used for administration compositions of the present invention, and for example, it is medium to be wrapped in liposome, microgranule, microcapsule.

In exemplary embodiment, according to the conventional method that is suitable for pharmaceutical composition, for example be used for people's subcutaneous administration method, can prepare compositions herein.Typically, the compositions that is used for subcutaneous administration is the water buffer solution of sterile isotonic.If desired, compositions also comprises solubilizing agent and is used to alleviate the local anesthetic of injection site pain.Usually, these compositions or provide individually or to be mixed in unit dosage forms together, for example, the dry powder in sealed container, freeze-dried powder or do not have aqueous concentrate, described sealed container such as ampoule or as indicate the flat capsule of activator quantity.If by the transfusion administration composition, then can be by containing aseptic medicinal level water, buffer or saline solution compositions formulated.If by drug administration by injection compositions, the sterilized water that can be provided for injecting or the ampere of saline solution, so that mixed these compositions before the administration.Compositions herein can be formulated into suppository with multiple composition, and it contains calculates by weight the active component that is about 0.5% to 10% scope; Oral preparaton preferably contains calculates by weight the active component that is about 10% to 95% scope.Daily dose carries out administration with single dose, or is divided into a plurality of less divided doses and carries out the every day of administration repeatedly.

As used herein, the dosage timetable is meant the medication that is used for any compositions, wherein the gastrin compounds of the modification that for example provides herein is provided compositions, or the modification gastrin of associating FACGINT or associating Hypoylycemic agents and/or immunosuppressant, each compositions is with effective dose administration simultaneously or administration in independent separately interval, described interval is for example in separately one day, or as combination preparation administration or individually dosed, this method also is included in the amount of the compositions of administration in the time per unit of every day for example, and each compositions of administration time or cycle time of continuing.

In one aspect, invention is provided for suppressing or treating the method for diabetes, this method comprises the mammal to the compositions that needs gastrin compounds, administration gastrin compounds or associating FACGINT or associating Hypoylycemic agents and/or the administration of combined immunization inhibitor individually, each dosage is enough to improve the quantity of the cell of excreting insulin β in the mammal; And the regenerated sum of definite islets of langerhans, thereby treatment or inhibition diabetes.Determine that the regenerated sum of islets of langerhans is to measure to be selected from following parameter: blood glucose, serum glucose, blood glycated hemoglobin, insulin β cell lump, serum insulin and content of insulin.With blood sugar detection before the administration composition relatively, administration compositions described herein can reduce blood glucose, for example compares with the blood sugar detection before the administration composition, it is about 50% that these compositionss of administration can reduce blood glucose, or 70%.With the concentration ratio of the glycated hemoglobin of before administration composition, measuring in the mammal, the concentration of glycated hemoglobin is minimized.Compare with the serum insulin concentration of measuring before administration composition in the mammal, serum insulin concentration is improved.Compare with the insulin concentration of measuring before administration composition in the mammal, insulin concentration is improved.

Compositions of the present invention can be mixed with neutrality or salt form.Pharmaceutically acceptable salt comprises the salt that forms with free amine group, for example be derived from the salt of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., and with the salt that free carboxy forms, for example be derived from the salt of sodium, potassium, ammonium, calcium, hydrated ferric oxide., 2-aminopropane., triethylamine, 2-ethamine ethanol, histidine, procaine etc.

Can effectively treat the amount of the therapeutic agent of the present invention of the special disease or the state of an illness, depend on the person's character of the disease or the state of an illness, and can be determined by the clinical technology of standard.Used accurate dosage also depends on the order of severity of route of administration, disease or disease in the preparation, and is determined according to doctor's judgement and each patient's details.Those of ordinary skills can determine the blood levels of insulin or C peptide, and the conventional determining of the empty stomach level of glucose or glucose stimulation.By being derived from dose response curve external or the animal model test macro, the those of ordinary skill of pharmaceutical field can be inferred effective dose.Use normal data, described data comprise the toxicologic standard of receptors ligand of the embodiment of absorption, distribution, half-life kinetics and this paper in circulation, metabolism, the drainage, to delivering medicine to patient's composition dosage, regulate at species to the known variant of species.Suitable dosage scope, be about 0.01 microgram to 10 of every kg body weight every day normally to each reactive compound, 000 microgram, for example, every day, about 0.01 microgram to 1 microgram/kg, about 0.1 microgram to 10 microgram/kg, about 1 microgram to 500 microgram/kg, about 10 micrograms were to 10mg/kg.Therefore suitable normally about 0.01 microgram of dosage scope/kg body weight/day is to the 10mg/kg body weight/day.

The invention provides medicine bag or test kit in other embodiments, they comprise one or more containers that are filled with one or more compositions of pharmaceutical composition of the present invention.In described parcel or test kit, can find the container of the unit dose that contains the gastrin compounds that enlarges purposes.What be associated with this container can be multiple writing material, as description, perhaps manage the bulletin of government organs' defined form of medicine and biological product manufacturing, use or sale, this bulletin has reflected manufacturing, use or has sold the allowance of the government organs of human drugs.

Unless otherwise defined, all herein technology and scientific terminology can normally be understood as by those skilled in the art in the invention and have identical implication.Be similar to or be equivalent to those methods and the material described herein, can be used for realizing the present invention.The present invention is able to abundant description by multiple embodiments, and additional embodiment can be used as subsequently embodiment and the example of claim describe, wherein they are not meaned as further restriction and make an explanation.The list of references content of all references is all quoted as a reference at this.

Embodiment

Embodiment 1. by intravenous injection to male Rhesus Macacus administration after, the pharmacokinetics embodiment of the gastrin of unmodified

By independent intravenous injection to male Rhesus Macacus administration after, carry out this embodiment with the assessment unmodified gastrin (refer to the compd B in the literary composition; Referring to table 3) pharmacokinetics (PK) curve chart.Used 17 amino acid whose gastrin analog have in the site 15 monamino acid sudden change, and wherein methionine is replaced by leucine.Though therefore compd B is different from naturally occurring gastrin, all vaild evidences show that it is the function equivalent of gastrin.

Following defining with PK analyzed the relevant term of term:

C _MaxThe maximum of-the plasma concentration that observes.

t _Max.-Cmax time corresponding.

The area of AUC-curve below is as to being exposed to total the measuring of medicine in a period of time.

Blood plasma t _1/2-measure medicine rest in the blood reduce to until blood drug level 50% how long required.

The administration of gastrin

Use buck in the present embodiment.Every animal is accepted gastrin by intravenously administrable (3 μ g/kg, 10 μ g/kg and 30 μ g/kg).Be used for intravenous dosage solution with single group dosage, carry out administration by the dosage of 1ml/kg by saphena.Behind dosed administration, use the salt water lotion of 0.2ml in order to ensure the administration of all dosage volume.The actual volume that delivers medicine to every animal depends in the nearest body weight of animal.

Blood collecting

By arm vein or femoral vein, collect successive blood sample (about every time point 0.5ml): 0 (before the administration), 1,3,5,10,15,30 minute, 1,2 and 4 hour (after the administration) by following time point.

Every duplicate samples is collected in the test tube that contains EDTA, placed on ice until centrifugal.With 1,500g (RCF) is the minimum frozen centrifugation of sample 10 minutes, changes over to gained blood plasma in another test tube and places dry ice.To be used for all freezings of all samples that PK as described below analyzes.

Analyze the gastrin level

By formulating competition radioimmunoassay perfect, that be used for the quantitative assay gastrin, detect the gastrin (Russell et al., Postgraduate MedicalJournal 52:645,1976) in the plasma sample.Antibody used in the detection produces in the anti-synthetic people's gastrin I of rabbit, and described gastrin I passes through the carbodiimides coupling in bovine serum albumin.Use the 125I labelled antigen, and in the presence of this is antigenic, use gamma radiation counter to survey antibody response.

Pharmacokinetic analysis

In order to calculate pharmacokinetic parameter, with the gastrin blood levels data input Graphpad Prism 3.0 that is equipped with time point of every animal (GraphPad Software, San Diego, California, USA, www.graphpad.com).

After table 2 intravenous injection, the summary sheet of the mean P K parameter of the gastrin of primates

Administration compd B (μ g/kg)	t 1/2 (min)	C max (pg/ml)	T max (min)	AUC (ng.ml/min)
					3	5	27000	2	251
10	4	127487	1	939
					30	5	267625	1	2526

The data of table 2 show the shorter blood plasma t of compd B ^1/2, it on average is about 4-5 minute for three administration groups.Behind single group dosed administration, compd B shows from the primates blood flow and is eliminated soon.Raising along with the intravenously administrable dosage of compd B can observe C _MaxWith the raising of AUC value, yet improve dosage to t ^1/2Value is influence not.The dosage that these data show strengthen compd Bs can not strengthen the short life cycle of this peptide in circulation.In a word, these data show that gastrin was eliminated from blood flow in several minutes.Therefore, because the quick removing of gastrin in the serum has limited the existence of bioactive compound in the serum.

The gastrin of embodiment 2. unmodifieds is to the fasting blood glucose level of the NOD mice of recent outbreak diabetes and the influence of insulin content.

Determine the morbidity of diabetes according to fasting glucose (FBG) level＞6.6mmol/l, thereby monitor the female Mus of the diabetes (NOD) of not showing effect.After the diabetes outbreak, with (i) excipient (n=4); Or (ii) gastrin (compd B listed as table 3) is pressed the dosage of 3ug/kg/day, and be administered once every day (n=5) totally 14 days handles mice.Mice is not accepted the insulin supply and handles.The fasting blood glucose level and the insulin content of (after stopping to handle the 21st day) two processed group of assessment the 0th day and the 35th day.

Fig. 1 shows the fasting blood glucose level (FBG) of the control animal that excipient is handled after 35 days.On the contrary, the blood sugar level that the processing of gastrin can suppress in some diabetes NOD mices rises, yet FBG residue level is higher than viewed level in common mice (about 3-7mM) significantly.These data show that for level is reduced to normal level, people need improve the effect of gastrin.

Because beta cell is destroyed, the insulin level of vehicle-treated group descended at the 35th day, yet compared with pretreatment values, the rising that the animal of handling with gastrin shows insulin level significantly.Referring to Fig. 2.Yet after handling with the compd B of 0.6 μ g/ml, the rising of insulin level still is starkly lower than the level (12 μ g/ pancreas) of common mice.These data are together with the pharmacokinetic analysis among the embodiment 1, show that the gastrin of using unmodified handles the effect of diabetes NOD mice, are subjected to the restriction (5 minutes) of the shorter life cycle of blood plasma gastrin.Therefore, utilize long-acting gastrin can more effectively stimulate the new life of islet cells, improve the progress of diabetes in insulin and the inhibition NOD mice.

Embodiment 3: the peptide of gastrin peptide is synthetic

Use the standard technique of solid-phase peptide, Steward for example, J.M.and Young, J.D. (1984) in " Solid Phase Peptide Synthesis ", 2 ^NdEd., Pierce ChemicalCompany describes, and any those of ordinary skill can easily synthesize the gastrin peptide in this area.Use standard technique, for example use reverse hplc and by the H of 0.1%TFA ₂The volatility double element system that the acetonitrile of O and 0.1%TFA is formed can carry out the purification of gastrin peptide.Monitor eluting by uv absorption,, then with its lyophilization, as required with its dissolving, be used for administration or test subsequently, or be used for further coupling reaction to collect the peptide of purification.

The synthetic gastrin is synthesized peptide, and it contains any continuous part of the 1-28 residue except that the 29-34 residue of SEQ ID NO:1 or 2, or contains any continuous part of the 1-11 residue except that the 12-17 residue of SEQ ID NO:3 or 4.In addition, synthetic has the gastrin peptide of the spacer of N-terminal, and described spacer comprises for example neutral p1 amino acid residue of Gly and Ala.Also can synthesize the synthetic peptide of the gastrin with N-terminal Cys residue, this peptide has or does not have spacer.

Table 3 has been listed some and has been synthesized the summary sheet of the gastrin peptide that is used for the gastrin compositions, and comprises " greatly " gastrin-34 (A), " little " or " weak point " gastrin-17 (B), and gastrin-13 (C).

The summary sheet of table 3. gastrin compositions and composition thereof

Chemical compound	Polymer	N-terminal Cys residue	Cross-linking agent	The gastrin peptide	Residue is from SEQ ID NO:
						A	Do not have	Do not have	Do not have	1-34	2
B	Do not have	Do not have	Do not have	1-17	4
						C	Do not have	Have	Do not have	5-17	4
D	Do not have	Have	Do not have	2-34	2
						E	Do not have	Have	Do not have	2-17	4
F	Do not have	Have	Do not have	5-17	4
						G	Do not have	Have	(GA) 5	2-17	4
H	Do not have	Have	(GA) 5	5-17	4
						I	PEG	Have	Do not have	2-34	2
J	PEG	Have	Do not have	2-17	4
						K	PEG	Have	Do not have	5-17	4
L	PEG	Have	(GA) 5	2-17	4
						M	PEG	Have	(GA) 5	5-17	4
N	PEG	Have	Do not have	2-34	2
						O	HAS	Have	Do not have	2-17	4
P	HSA	Have	Do not have	5-17	4
						Q	HAS	Have	(GA) 5	2-17	4
R	HSA	Have	(GA) 5	5-17	4

PEG is poly-(ethylene glycol)-20000; HAS is the human serum albumin; (GA) ₅It is the Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala spacer.

Embodiment 4: the coupling of gastrin and PEG-20000

The gastrin peptide (Compound D in the table 3, E, F, G and H) that N end is modified by Cys, near under the neutral condition with three [2-carboxyethyl] phosphonium salt hydrochlorate (TCEP of molar excess; Reducing agent with non-activity of maleimide amine moiety) incubation 30 minutes (buffer pH 6.5-7.5) is together gone back ortho states to guarantee that the Cys residue is in, and can be used for reaction.

MPEG-maleimide-20000 (the mean molecule quantity 20000 that progressively adds molar excess; From Shearwater Corporation, Huntsville AL, U SA obtains), mixing promotes dissolving simultaneously, after abundant dissolving, with the extra mixing of solution 1 to 4 hour, to finish coupling reaction.Use anionite, the Q-agarose of neutral pH for example, carry out the purification of conjugates and combine closely up to conjugates and any unreacted gastrin because the theoretic isoelectric point, IP (pI) of gastrin is 3.4, and neutral unreacted mPEG-maleimide can not in conjunction with.For further purification, based on the greatest differences of their molecular weight, use the size exclusion chromatography method, Sephadex G-5 and be suitable for the buffer of therapeutic use for example, for example PBS separates conjugates at an easy rate from unreacted gastrin.

These reactions cause the generation of Compound I in the table 3, J, K, L and M.Use is at the biuret reaction of peptide content with at the colorimetry (Habeeb, A.E S.A. (1966) Anal.Biochem.14:328-336) of PEG content, and the Electrospray Mass Spectrometry of definite conjugates total molecular weight, checks the success of coupling reaction.

Embodiment 5: with human serum albumin's coupling

Serum albumin contains single come-at-able cysteine reduction residue (Cys-34), and described residue is topmost reactive mercapto among the human albumin (Pedersen etc. (1980) Eur.J.Biochem.106:291-295).This character can allow to comprise that the part coupling specifically of peptide is in serum albumin.

With the human serum albumin in down and three [2-carboxyethyl] phosphonium salt hydrochlorate (TCEP of molar excess near neutrallty condition; Reducing agent with non-activity of maleimide amine moiety) incubation 30 minutes (buffer pH 6.5-7.5) is together gone back ortho states to guarantee that the Cys residue is in, and can be used for reaction.Two-dimaleoyl imino ethane of molar excess (have short spacer and to the reactive same bi-functional cross-linking agent of sulfydryl) is added with activation Cys-34.In independent reaction, add the gastrin peptide (Compound D in the table 3, E, F, G and H) that the N end is modified by Cys, with the extra mixing of gained solution 1 to 4 hour, to finish coupling reaction.Based on the greatest differences of molecular weight, use the buffer of size exclusion chromatography method and use therapeutic use, for example PBS separates conjugates at an easy rate from unreacted gastrin (with any gastrin dimer that forms).From the HAS of gastrin peptide coupling, can't further separate unreacted HAS.

These reactions cause the generation of compound N in the table 3, O, P, Q and R.Use the Electrospray Mass Spectrometry of measuring the conjugates total molecular weight, check the success of coupling reaction.

Embodiment 6. by intravenous injection to the administration of Wistar rat after, pharmacokinetics between the gastrin compounds of unmodified and modification is relatively

Introduce this enforcement side to assess to behind the Wistar rat intravenous injection pharmacokinetics curve chart of the gastrin derivant/conjugates of unmodified and modification.

The administration of gastrin

Rat (three groups) is accepted as synthetic multiple test compounds among the embodiment 3, and the gastrin equivalent is carried out intravenously administrable by the dosage level of 10 μ g/kg.Below used test compounds all be the chemical compound that above table 3 is listed.Details see also table 3.

Blood collecting

Collect successive blood sample (about every time point 0.4ml) by following time point: in injection 5,60,180,480 minutes behind all animals.At each time point, gather blood from 3 animals of every group.

Every duplicate samples is collected in the test tube that contains EDTA, placed on ice until centrifugal.With 1,500g (RCF) is the minimum frozen centrifugation of sample 10 minutes, changes over to gained blood plasma in another test tube and places dry ice.The freezing all samples is analyzed in order to PK.

Analyze the gastrin level

Use is from R﹠amp; D systems, people's gastrin 1 (G-17) immunoassay kit of catalog number DE3400 by the ELISA method, is measured compd B level in the blood plasma.This is measured based on the competition combination technology, wherein is present in the site on the gastrin I competition rabbit polyclonal antibody of gastrin I and the alkali phosphatase enzyme mark of quota in the sample.In incubation, the antibodies goat anti-rabbit antibody, and be embedded on the microtest plate.Washing is removed after excessive conjugates and the unconjugated sample, adds substrate solution to determine the activity of desmoenzyme in culture hole.The concentration of gastrin I is inversely proportional in the brightness of color and the sample.

Pharmacokinetic analysis

Use PK Functions for Microsoft

Excel (Usansky JI, Desai A, Tang-Liu D, Department of Pharmacokinetics and Drug Metabolism, Irvine CA), analyzes the blood drug level of gastrin (compd B) and different derivant/conjugates thereof.In order to assess the PK curve chart, calculate multiple test compounds and following PK value: C _Max, t _Max, AUC and t _1/2

Because gastrin (as compd B) but be naturally occurring in blood with detection level, deduct the base value that obtains before the administration the blood plasma level value that therefore behind the administration compd B, obtains.Data are represented with meansigma methods ± SD.

Data show is compared with natural gastrin, has the gastrin of the modification of cysteine (as the functional group of N-terminal), or in the modification gastrin compounds of N-terminal coupling in PEG or HAS, the half-life with remarkable length.Compare with natural gastrin 17 or gastrin 34, the gastrin compounds/conjugates of modification with the high concentration long-term existence in serum.These data show that also AUC also can promote the chemical modification of gastrin molecule.Can envision, but maximum duration keep the gastrin derivant/conjugates of the modification of Cmax, the desired effect of adjusting for the pancreatic cell regeneration and the glucose level of diabetic animal has maximum potentiality.

The gastrin compounds that embodiment 7. relatively modifies and the gastrin of unmodified suppress the diabetes progress of the NOD mice of outbreak diabetes in the recent period

In this embodiment, in the NOD mice of diabetes that shows effect in the recent period, the therapeutic effect of the gastrin of inspection unmodified and the gastrin compounds/conjugates of modification, to determine the comparing insulin content whether the gastrin derivant/conjugates of multiple modification more is effective in the serious hyperglycemia of inhibition and improves the NOD mice of the diabetes of showing effect in the recent period with the gastrin of unmodified.Gastrin compounds/the conjugates of modification of using shown below (details are referring to table 3): compd B, it is to be prepared to have 17 amino acid residues and have the gastrin of synthetic people's gastrin I of Leu and compd E, chemical compound G, chemical compound J, compound L, chemical compound O, compound Q at amino acid sites 15 simultaneously.

Female Mus to the non-obese diabetic (NOD) in age in 12-14 week, monitor the progress (fasting glucose＞8.0 are to 15mmol/l) of its diabetes outbreak, and in 48 hours of symptom occurring, on the same group mice is not carried out the processing of 3 or 10 μ g/kg/ days gastrin equivalent separately, by every day lumbar injection carry out administration and handle.

Treatment is to carry out administration 14 days.Monitor fasting glucose (FBG) level of animal weekly.Stopping to take food afterwards about 12 hours and injecting back 24 hours, measure the FBG level at nearest peptide or excipient.In case stop treatment, just in next 4 weeks (2-6 week) in, detect the FBG level of all mices, after definite stopped treatment processing, the prevention to hyperglycemia is continued.Stopped to handle at the 14th day.

Method be included in the 6th week to these mice repeated samplings, gather the blood be used to analyze FBG and plasma C peptide, and put to death mice with measure insulin and to the islets of langerhans inflammation (insulitis) keep the score.When initial the processing, mice had not both been accepted insulin replacement therapy and had not accepted immunosuppressant yet.Assessment following parameters: the existence of survival rate, insulin level, insulitis and fasting blood glucose level.

Data show the long half-lift of having more and the gastrin derivant/conjugates of the modification of AUC, more are effective in the hyperglycemia that suppresses diabetes NOD mice.In some cases, the gastrin compounds/conjugates of modification reverses glucose level fully to normal level, has shown that at this model moderate stimulation the islets of langerhans of significant level is regenerated.

The gastrin compounds that embodiment 8. relatively modifies and the gastrin of unmodified are together with GLP-1 suppress to show effect the in the recent period diabetes progress of NOD mice of diabetes

In this embodiment, NOD mice to recent outbreak diabetes, the therapeutic effect of the combination of the gastrin of detection GLP-1 and unmodified and the combination of GLP-1 and modification gastrin compounds/conjugates, whether suppress serious hyperglycemia with the administration of determining GLP-1 and gastrin, and whether improve the insulin content in the mice of recent outbreak diabetes.Used GLP-1 is that (compare with coming free its this segmental precursor of processing, it has the residue of site 7-36 as the GLP-1 of the bioactive fragment of people/mice GLP-1; Be attained at BachemH6795).Below be the gastrin compounds/conjugates of used modification: compd B-compd B-gastrin, as have 17 amino acid residues and have the SHG I of Leu and compd E, compound Q simultaneously at amino acid sites 15 places.

Female Mus to the non-obese diabetic (NOD) in age in 12-14 week, monitor the progress (fasting glucose＞8.0 are to 15mmol/l) of its diabetes outbreak, and in 48 hours of symptom occurring, four groups of mices are carried out following processing separately: only handle for one group with excipient; Other group associating GLP-1 (100 μ g/kg/ days) and gastrin compounds (3 μ g/kg/ days gastrin equivalents) are handled, by every day lumbar injection each group is carried out the administration processing.

Treatment is to carry out administration 14 days.Monitor fasting glucose (FBG) level of animal weekly.Stopping to take food afterwards about 12 hours and injecting back 24 hours, measure the FBG level at nearest peptide or excipient.In case stop treatment, just in next 4 weeks (2-6 week) in, detect the FBG level of all mices, after definite stopped treatment processing, the prevention of hyperglycemia is continued.Stopped to handle at the 14th day.

Method be included in the 6th week to these mice repeated samplings, gather the blood be used to analyze FBG and plasma C peptide, and put to death mice with measure insulin and to the islets of langerhans inflammation (insulitis) keep the score.During initial the processing, mice had not both been accepted insulin replacement therapy and had not accepted immunosuppressant yet.Assessment following parameters: the existence of survival rate, insulin level, insulitis and fasting blood glucose level.

Data show with the GLP-1 that combines natural gastrin (compd B) to be compared, and the GLP-1 of gastrin compounds/conjugates (compd E or Q) combination of the modification with have more the long half-lift can more effectively reduce the blood sugar level of diabetic animal.These data acknowledgements have the gastrin compounds of more long-life modification/with the purposes of the conjugates of GLP-1 or other somatomedin.

Sequence table

<110>WARATAH PHARMACEUTICALS，INC.

CRUZ，Antonio

＜120〉gastrin compositions and preparation and use thereof and preparation method

<130>82629-3

＜140〉also unspecified

<141>2003-11-21

<150>US 60/428,100

<151>2002-11-21

<150>US 60/428,562

<151>2002-11-22

<150>US 60/530,590

<151>2002-12-03

＜150〉U.S. is also unspecified

<151>2003-11-14

<160>8

<170>PatentIn version 3.2

<210>1

<211>34

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic peptide

<220>

＜221〉mixed features

<222>(1)..(1)

＜223〉Xaa=pyroglutamic acid

<220>

<221>MOD_RES

<222>(34)..(34)

＜223〉amidation

<400>1

Xaa Leu Gly Pro Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys

1 5 10 15

Lys Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met

20 25 30

Asp Phe

<210>2

<211>34

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic peptide

<220>

＜221〉mixed features

<222>(1)..(1)

＜223〉Xaa=pyroglutamic acid

<220>

<221>MOD_RES

<222>(34)..(34)

＜223〉amidation

<400>2

Xaa Leu Gly Pro Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys

1 5 10 15

Lys Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Leu

20 25 30

Asp Phe

<210>3

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic peptide

<220>

＜221〉mixed features

<222>(1)..(1)

＜223〉Xaa=pyroglutamic acid

<220>

<221>MOD_RES

<222>(1)..(1)

＜223〉amidation

<400>3

Xaa Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp

1 5 10 15

Phe

<210>4

<211>17

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic peptide

<220>

＜221〉mixed features

<222>(1)..(1)

＜223〉Xaa=pyroglutamic acid

<220>

<221>MOD_RES

<222>(17)..(17)

＜223〉amidation

<400>4

Xaa Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Leu Asp

1 5 10 15

Phe

<210>5

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic peptide

<400>5

Tyr Gly Trp Met Asp Phe

1 5

<210>6

<211>6

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic peptide

<400>6

Tyr Gly Trp Leu Asp Phe

1 5

<210>7

<211>4

<212>PRT

＜213〉artificial

<220>

＜223〉aminoacid sequence of the carboxyl terminal of common gastrin/cck receptor part

<220>

<221>MOD_RES

<222>(4)..(4)

＜223〉amidation

<400>7

Trp Met Asp Phe

1

<210>8

<211>10

<212>PRT

＜213〉artificial

<220>

＜223〉spacerarm

<400>8

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10

Claims (6)

1. gastrin compounds, it comprises aminoacid sequence Z-Y by amino terminal _m-X-Tyr-Gly-Trp-Leu-Asp-Phe-NH ₂, wherein

Z is the human serum albumin;

Y is (Gly-Ala);

M is 0 or 5;

X is Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala; And

When m was 5, the amino terminal of Y had cysteine residues, and perhaps when m was 0, the amino terminal of X had cysteine residues;

Prerequisite is gastrin compounds and gastrin/CCK _BReceptors bind.

2. the gastrin compounds of claim 1, wherein m is 0.

3. the gastrin compounds of claim 1, wherein m is 5.

4. pharmaceutical composition that is used for the treatment of diabetes, it comprises among the claim 1-3 each gastrin compounds.

5. each gastrin compounds is used for the treatment of purposes in the medicine of diabetes in preparation among the claim 1-3.

6. the test kit of pharmaceutical composition that contains the claim 4 of at least a effective dose.

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