CN100508957C - Improved oral delivery of peptides - Google Patents

Abstract

Bioavailability of peptide active agents to be administered orally is enhanced by a pharmaceutical composition providing an active peptide that is amidated at a site that is not naturally amidated.

Description

The improvement of oral delivery of peptides

Related application

The application is based on the 60/441st, No. 856 U.S. Provisional Application of submitting on January 21st, 2003 and require the priority of above-mentioned application.

Background of invention

Field of the present invention

The present invention relates to peroral administration peptide pharmaceutical products, its reactive compound comprises several amino acids and at least one peptide bond in their molecular structures, and relates to the method that improves the peroral administration bioavailability of this peptide reactive compound.

Description of related art

Many people's hormones, neurotransmitter, cytokine, somatomedin and other important biologic artifact are all with the substantial portion of peptide as their molecular structures.It is the level that improves these peptide compounds in patient's body that numerous disease is replied actual.These biological related peptides of treatment effective dose can be applied to the patient by many modes.Yet as discussed below, preferred oral administration is very difficult for such reactive compound.

For example, salmon calcitonin see calcimar is a kind of peptide hormone, and it can reduce the release of calcium from skeleton.When being used to handle bone photo related disorders or calcium disease (such as osteoporosis, Paget (Paget), malignant hypercalcemia etc.), its helpful effect of keeping bone density.Many kinds of calcitonins (human calcitonin, salmon calcitonin see calcimar, Anguillar japonica calcitonin, elcatonin (elkatonin), pig calcitonin and chicken calcitonin) have been separated to.Between these different calcitonin types, has significant structure non-homology.Such as, between the aminoacid that constitutes human calcitonin and salmon calcitonin see calcimar, have only 50% concordance.Although variant on molecular structure, salmon calcitonin see calcimar can be used to the above-mentioned calcium of human treatment and reply disease.

The example of another peptide hormone is parathyroid hormone (PTH).PTH is produced by parathyroid gland, is the main regulator of calcium level.PTH is a peptide species, and synthetic polypeptide can prepare (The Proteins, volumes such as Neurath with the method that Erickson and Merrifield disclose, Academic Press, New York, 1976, the 257th page), and with the method for Hodges etc. (1988, Peptide Research 1,19) or Atherton, E. and Sheppard, the method for R.C. (Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989) modify.

When blood calcium was brought down below normal level, parathyroid gland discharged PTH, and by the absorbing again of bone calcium, heavily absorb calcium the nascent urine in renal tubules and come the increasing blood calcium level by increasing from intestinal absorption calcium and by increasing.Can remove calcium from skeleton although inject low-level PTH constantly, inject same low dosage in fact off and on and can promote bone growth.

Tregear is at United States Patent (USP) the 4th, 086, described people PTH analog in No. 196, and claimed that aminoacid preceding 27 to 34 in the cell in vitro test is the most effective on the stimulation adenyl cyclase.Rosenblatt is at United States Patent (USP) the 4th, 771, disclosed the character of hPTH analog in No. 124, wherein Trp ₂₃Replaced by phenylalanine, leucine, nor-leucine, valine, tyrosine, β-naphthylalanine or α-naphthylalanine, can be used as the PTH antagonist.The hPTH analog of these modifieds has 2 to 6 end amino acids to be removed, and loses most important antagonistic activity when causing being used to treat osteoporosis.These analog are designed to the inhibitor of the relevant peptide class of PTH or PTH.These analog are advocated to be used for the treatment of the hypercalcemia relevant with some tumors.

Pang etc. (WO93/06845, on April 15th, 1993 is open) have described the hPTH analog, and it relates to numerous aminoacid replacement Arg ²⁵, Lys ²⁶, Lys ²⁷, comprise alanine, agedoite, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.It is said that they can effectively treat osteoporosis, and to the minimum that influences of blood pressure and smooth muscle.

PTH works by activating two kinds of second messenger systems: G _sAdenyl cyclase of protein activation (AC) and G _qThe phospholipase C of protein activation _βThe latter causes the activity of activated membrane bindin kinase C (PKC).The activity of known PKC needs PTH residue 29-32 (Jouishomme etc., (1994) J.Bone Mineral Res.9, (1179-1189)).Confirmed the increase of bone growth, i.e. useful effect in osteoporotic treatment, to improve the active ability of AC related with peptide sequence.Natural PTH sequence is proved has all these activity.People hPTH-(1-34) sequence ordinary representation is as follows:

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His

Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu

Gln Asp Val His Asn Phe-OH(SEQ ID NO：1)

United States Patent (USP) 5,955,425 and 6,110,892 have disclosed different PTH analog.Following linear analog (tack hPTH), hPTH-(1-31)-NH ₂Have only the AC stimulating activity, and to be proved in the skeleton of ovariectomized mouse model is rebuild be active fully (Rixon, R.H. etc., (1994) J.Bone Miner.Res.9,1179-1189; Whitfield etc., (1996), Calcified Tissue Int.58,81-87; With Willick etc., United States Patent (USP) 5,556,940).

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His

Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu

Gln Asp Val-NH ₂(SEQ ID NO：2)。

The peptide medicament that uses in the former technology is usually by injection or nose administration.Insulin is a kind of example that passes through the peptide medicament of administrated by injection usually.As if a kind of preferred and peroral administration more easily mode is existing problems, because peptide class reactive compound is very sensitive to the gastrointestinal degraded.For example, reported in the former technology when salmon calcitonin see calcimar and parathyroid hormone oral administration to obtain the repeatably ability of blood level that such level is very low.This is considered to because these peptide hormonees lack enough stability in gastrointestinal tract, and the situation that is transported in the blood through intestinal wall is very poor.Yet injection and nose administration are obviously inconvenient more compared with oral administration, and the patient is caused bigger discomfort.Usually, this inconvenience and discomfort cause the patient that therapeutic scheme is not obedient to.Therefore, be necessary to picture insulin, salmon calcitonin see calcimar, parathyroid hormone and other peptide medicament is more effective and repeatably the oral administration mode here discuss in more detail.

The Proteolytic enzyme endonuclease capable of harmonization of the stomach intestinal degraded peptide, make they before absorbing intravasation by inactivation.Any amount of peptide of surviving under pepsin (having acid optimum pH scope typically) hydrolytic degradation will meet with the enzyme (having neutral optimum pH scope to alkalescence typically) of Small Intestine Protease and pancreatic secretion afterwards.Special difficulty result from peptide as the salmon calcitonin see calcimar have big relatively molecular weight with and the distribution of carrying electric charge.This may make salmon calcitonin see calcimar be difficult to more permeate the mucus that distributes along intestinal wall, perhaps passes the IBB film and enters blood.

A kind of mode that improves the effectiveness of peroral administration peptide is to protect them to exempt from the gastrointestinal protein hydrolytic enzyme, improves their absorptions from enteral again simultaneously, thereby improves their bioavailability.Improving oral effectiveness is very important on several reasons.At first, peptide and protein are very expensive, no matter be with chemosynthesis or recombinant DNA technology production.Therefore, can increase the method for bioavailability, the dosage that needs in the medicine Orally administered composition is few more.

Secondly, the bioavailability of oral peptide is high more, and the variation of the daily dose of individual bulk absorption is just more little.

The 3rd, the bioavailability of oral peptide is high more, and is just few more for the worry of peptide product degraded, because such catabolite can be as peptide in conjunction with the agonist or the antagonist that excite bioactive receptor.

Summary of the invention

Therefore, an object of the present invention is to provide the effective combination of oral medication of a kind of treatment so that reliably defeated drug delivery peptide, physiologically active peptide reagent for example is such as insulin, salmon calcitonin see calcimar, parathyroid hormone, vassopressin or its analog and other material discussed here.

Another object of the present invention provides the Therapeutic Method of the bioavailability that improves these peptides.

A further object of the present invention provides by Orally administered salmon calcitonin see calcimar or PTH 1-31NH ₂The method of treatment bone photo related disorders and calcium disease.

On the one hand, the invention provides a kind of pharmaceutical composition, be used for that oral delivery is a kind of the bioactive peptide reagent of physiologically active, form by described bioactive peptide at the treatment effective dose, wherein said bioactive peptide in the amidated site of a kind of non-natural by amidatioon.

Preferred peptide activating agent includes but are not limited to insulin, vassopressin, salmon calcitonin see calcimar, glucagon-like peptide 1 or 2, parathyroid hormone, luteinizing hormone releasing hormone, erythropoietin and analog thereof.Particularly preferably be parathyroid hormone and analog thereof.

On the other hand, this aspect provides a kind of method of bioavailability of the therapeutic peptide activating agent that improves a kind of oral delivery, and described method comprises the described peptide reagent of amidatioon.

Be sure of that the present invention can reduce the peptide reactive compound by the probability of proteolytic degradation; this is by protecting peptide by (1) pepsin simultaneously; it is most active under acid pH usually, and (2) intestinal or pancreas protease (to neutral pH vigor being arranged in alkalescence most usually) Proteolytic enzyme is attacked.

Equally, be sure of the present invention, continue the protection peptide simultaneously and avoid proteolytic degradation because amidated existence can promote the process that peptide enters blood by the IBB film.

The acid-resisting protective finish protection peptide activating agent of capsule or tablet exempts from the stomach by the proteasome degradation of acid activation.Thereafter, after medicament enters the so not tart intestinal of pH, the enteric coating dissolving, the inclusions of medicament discharges.A large amount of acid (peptide activating agent and its mix) reduces neutral protease to alkaline activation, and (for example, the protease of luminol or digestible protein enzyme and brush border membrane) vigor discharges the pH in site to the optimum activity scope that is lower than them by reducing medicament.

Its its feature of the present invention and advantage will be more obvious in the going through below.

Detailed Description Of The Invention

According to the present invention, need to adopt the patient of peptide active component treatment, be given a kind of oral pharmaceutical composition (with proper dosage), and nonessential but preferably with the tablet or the capsular form of the common size of medical industry.The dosage of administration and frequency will go through below.The patient who is benefited is that any increase has the patient who suffers disease of sound response to the peptide containing compound level.For example, according to the oral salmon of the present invention the calcium element can be used to treat the patient who meets with calcium disease or skeletal diseases.For example, by oral calcitonin, oral parathyroid hormone, more preferably oral hPTH 1-31NH ₂With hPTH 1-34NH ₂The present invention can be used to treat osteoporosis, Paget, malignant hypercalcemia and similar disease.

According to the present invention, salmon calcitonin see calcimar is owing to numerous reasons are preferred preferred active component that use.For example it in addition have than human calcitonin and more to many advantage, even as the pharmaceutical agent of treatment human diseases.Utilizing salmon calcitonin see calcimar to replace the half life that the osteoporotic numerous advantages of human calcitonin treatment people comprise enhanced potentiality, analgesia and raising.Salmon calcitonin see calcimar is more effective than human calcitonin in treatment, because it is littler than the dosage of human calcitonin needs.Have tangible non-homology between salmon and human calcitonin, the aminoacid sequence of two kinds of calcitonins has only 50% concordance.

Be not limited to any theory, pharmaceutical composition of the present invention be sure of to overcome a series of difficulties and the incoherent natural cover for defense of bioavailability.The heterogeneity effect of pharmaceutical composition overcomes barrier by the mechanism that is suitable for separately, causes the cooperative effect of peptide active component on bioavailability.

This peptide reactive compound can oral administration.According to the present invention, the existence of at least one amide groups can protect peptide or protein to avoid proteoclastic degraded, has therefore improved bioavailability.Amide groups also can improve the membrane permeability that protein passes the enteral chamber.Also has other because the existence of amide groups improves the mechanism of bioavailability.

The technology that has many recombinant production peptide products promptly comprises the amino acid whose any chemical compound that connects by peptide bond in a large number in the molecular structure.

The summary of preferred expression carrier

At United States Patent (USP) 6,210, preferred expression vector has been described in 925, the document is merged in this paper as a reference.A kind of example of carrier of preferred expression salmon calcitonin see calcimar is presented at United States Patent (USP) 6,210, among Fig. 9 of 925.For the expression of other peptide product, the nucleic acid of a coding purpose peptide prod is used to replace the nucleic acid of salmon calcitonin see calcimar of encoding.

Preferred expression vector is made of a coding region and a control zone.The control zone operably is connected to the coding region, is made of a plurality of promoteres and at least one ribosome land, and wherein at least one promoter is selected from tac and lac.

Preferably, carrier comprises the box of transcribing of a plurality of palindrome placements, and each box has a control zone of the present invention and coding region.Be sure of that so dual-gene carrier or multigene carrier can provide better expression than bicistronic mRNA or polycistron expression vector.This is to bicistronic mRNA or the polycistronic surprised improvement of people in addition, be sure of not hinted in this former technology.

Carrier is also chosen wantonly and is comprised the nucleic acid that coding is prevented peptide, and it can suppress and the promoter of one or more control zones, a transcription pausing subregion, a selected marker zone and/or at least one relevant operon in secretion enhancing peptide zone of coding.

Many commercially available carriers can be as the initial promoter of the preferred promoter of the present invention.The favored area of some promoteres of the present invention may be included in initial promoter, obtains promoter of the present invention like this and need carry out improved number of times appropriateness relatively.

The control zone

The control zone operably is connected to the coding region, is made of a plurality of promoteres and at least one ribosome land, and wherein at least one promoter is from being selected by the group that comprises tac and lac.Before a single control zone, significantly increase the output (as going through) of product that the coding region produces interior in conjunction with a promoter.Other promoter is known in technology, and can be used in combination with a tac or lac promoter.Such promoter includes but not limited to lpp, ara B, trp E, gal K.

Preferably, the control zone is made of two promoteres closely.When one of them is tac, so preferred tac promoter is in 5 ' of another promoter of control zone.When one of them promoter is lac, then the lac promoter is preferably in 3 ' of another promoter of control zone.Equally preferably, the control zone is made of a tac promoter and a lac promoter, and preferred lac promoter is in 3 ' of tac promoter.

The coding region

The coding region comprises the nucleic acid that the interested peptide prod of coding in the frame is read in the nucleic acid downstream that is connected the coded signal peptide, so the peptide of coding region coding comprises signal and peptide prod respectively from the N-terminal to the C-terminal.Be not limited only to theory, be sure of that this signal can provide some protections for peptide prod, make it exempt from proteolytic degradation, in addition also participate in its secretion to pericentral siphon.

Many peptide signal sequences are known and can be used for the present invention.Comprising the host cell outer membrane protein signal sequence of portrayal in detail with anyly can insert to the sequence of pericentral siphon and the sequence of being translated the back cutting as the result who shifts by host cell to peptide prod.Useful signal peptide includes but are not limited to Omp A, pel B, Omp C, Omp F, Omp T, β-la, Pho A, Pho S and Staph A.

Peptide prod preferably should be enough little, and Technology needs a kind of fusion partner because it utilizes in the past.Usually, the molecular weight of such peptide prod should be less than 10KDa.More preferably, the C-of peptide prod end is a glycine, and as precursor the glycine of C-end is converted into other amide groups in enzymic catalytic reaction, causes a kind of amidated peptide like this.Such conversion will more go through once.Many biologically important peptide hormonees and neurotransmitter are such amidated peptides.Such as, the peptide prod of coding region coding may be salmon calcitonin see calcimar precursor or the relevant peptide precursor of calcitonin gene, the two all has the terminal glycine of C-, and the two can the enzyme catalysis amidatioon become sophisticated salmon calcitonin see calcimar or sophisticated calcitonin-gene-related peptide.

Analogs of parathyroid hormone also can be according to production of the present invention.For example, a kind of have preceding 34 the amino acid whose peptides of parathyroid hormone and can give parathyroid hormone itself similar function, the same with 34 amino acid analogue amidatioons.According to one or more expression systems described herein, the latter can produce 34 aminoacid that parathyroid hormone begins by expressing, and next is the 35th glycine.Enzyme catalysis amidatioon discussed here can be converted into amide groups to glycine.

Other of preferred vector of the present invention or other carrier of will be in same host expressing as carrier repressor of the present invention optional aspect

Randomly, preferred carrier may comprise the nucleic acid that a kind of coding is prevented peptide, can suppress to express by at least one promoter control.Perhaps, however coding prevents the nucleic acid of peptide to be positioned to carry on the plasmid that separates in the host cell of the present invention.For many operons suitable repressor is arranged in present technique.More preferably, nucleic acid coding a kind of lac repressor of on preferred entity of the present invention, encode of coding repressor, because its inhibition comprises the lac operon of tac and lac promoter, one of them operon is to be positioned at forever on the preferred vector of the present invention.

Selected marker

Any being positioned on the carrier of (such as the gene of coding kalamycin resistance) is preferred among many selected markers.This carries out suitable clear and definite screening with allowing, and obtains those and is effectively transformed by novel plasmid of the present invention or the host cell of transfection

Secretion strengthens peptide

The nucleic acid that at least a secretion of encoding strengthens peptide should be positioned on the plasmid of the present invention.Perhaps, the coding secretion nucleic acid that the strengthens peptide plasmid independently on the plasmid that can be arranged in same host and expression of peptides product.Preferably, secretion enhancing peptide is selected from SecY (prlA) or prlA-4.It may be noted that SecY and prlA are consistent, these two speech are used as synonym in present technique.PrlA-4 is the well-known improvement to prlA, and has similar function.It is SecE that another one is preferably secreted the enhancing peptide, also is called " prlG ", and this speech is used as the synonym of " SecE ".Most preferably, many secretions strengthen peptide and can be encoded, and one of them is SecE, and another one is selected from SecY (prlA) and prlA-4.It is believed that these two help peptide prod migration from the Cytoplasm to the pericentral siphon that interacts.There is no need to be limited to theory, these secretions strengthen peptide and can help to protect peptide prod to avoid the effect of cytoplasm protein enzyme, improve their secretory function in addition.

Peptide and proteinic amidatioon preferably at C-terminal, have given significantly improving of oral administration biaavailability in the demonstration hereinafter.Technology is in the past pointed out stability (Eipper etc., Annu.Rev.Neurosci., 15:57-85,1992 that the natural amidatioon of biologic activity peptide can help to improve receptors bind and improve these peptides; Merkler, Enzyme Micob.Technol., 16:450-456, especially the 51st page, 1994).The bioavailability that amidatioon by these peptides gives significantly by being unexpected because present knowledge point out peptide and protein bioavailability rise main conclusive be site, secondary and tertiary structure and the electric charge of molecule.

Usually, eukaryotic plasma membrane is impermeable for big peptide or albumen.Yet, some hydrophobic primitive, as aminoacid, fatty acid and bile acid, be called ferry-boat peptide or film transposition sequence or primitive with different titles, when merging with protein that function is arranged or peptide, particularly with N or C-terminal, can be as protein called membrane transporters, and mediate the transportation that these albumen enter living cells.These protein called membrane transporters (MTs) can be cut by blood or lymphoid protease to small part in the present invention.

According to another aspect of the present invention, the existence of at least a protein called membrane transporters, preferred two kinds of MT, more preferably two kinds of peptide MT can cross the membrane permeability in enteral chamber and give improved bioavailability with the peptide that MT merges.Since the bioactive peptide that is connected with MT can be cut by blood or lymphoid enzyme action, the bioactive peptide that stays freely reaches target spot.

Equally, according to the present invention, peptide and protein called membrane transporters are reduced by gastric enzyme (wherein great majority have activity in the acid pH scope) or pancreas protease (wherein great majority have activity in neutrality to alkaline pH scope) hydrolytic degradation.

Once more, be not limited only to theory, seem be, according to the present invention, peptide transportation under the vectorial protection of antacid protectiveness that is fit to prevents fully that by stomach salmon calcitonin see calcimar other bioactive peptide alive from contacting with any pepsin that may degrade it.In case having passed through stomach, pharmaceutical composition of the present invention enters the intestinal zone, wherein neutrality to alkaline pH is preponderated, it is the most suitable to neutral pH that the protease there trends towards alkalescence, enteric coating other vehicle release peptide alive and acid or protease inhibitor (very approaching mutually).

Acid is believed to reduce intestinal local pH (place that activating agent discharges) to being lower than many protease and the only scope of other intestinal enzyme.The reduction of pH has reduced the proteolytic activity of intestinal enzyme, thereby for peptide and protein called membrane transporters provide protection, exempts from potential degraded.The vigor of these protease is reduced by temporary transient sour environment provided by the invention.It is preferred that enough acidity is provided, and makes the partial pH of intestinal temporarily reduce to 5.5 or lower, preferably reduces to 4.7 or lower, preferably reduces to 3.5 or lower.The sodium bicarbonate test that describes below (is in the part of " pH depressant " at title) is the indication to the amount of required acid.Preferably, the situation that reduces intestinal pH will continue one section time enough, and protection peptide reagent and protein called membrane transporters are avoided proteolytic degradation, have an opportunity to pass through intestinal wall until one of them a little peptide reagent and enter blood flow.For salmon calcitonin see calcimar, evidence is worked as the active component direct injection and is advanced duodenum, ileum and the colon of Mus, and the Tmax that salmon calcitonin see calcimar is up to the standard in blood is 5-15 minute.

Perhaps, protease inhibitor is believed to reduce the proteolytic activity of erepsin, thereby provides protection for peptide and protein called membrane transporters exempt from immature potential reduction.

Compositions of the present invention can be chosen wantonly and comprise absorption enhancer.Absorption enhancer of the present invention under the situation that reduces the Proteolytic enzyme vigor, can be worked in coordination with the absorption of promotion peptide and enter blood.

Mechanism of the present invention is considered to finish the target that improves bioavailability, and being has active component to realize by discharging as far as possible simultaneously with pharmaceutical composition.To the end, preferably keep the volume of enteric coating low as far as possible, to exempt from pepsin consistent with protection is provided for this.May disturb peptide to discharge thereby enteric coating is less, perhaps disturb in time the release of other component that discharges near peptide.Usually the amount of enteric coating should be less than 30% of remaining pharmaceutical composition (that is to say the other medicines compositions except that enteric coating).Suitable, it should be less than 20%, and more suitably, enteric coating adds between the composition weight 10% to 20% that account for coating not.

Absorption enhancer should a kind of solubility enhancer and/or transportation reinforcing agent (as hereinafter describing in detail), help peptide reagent to enter blood from the intestinal transportation, and can promote this process, so it is preferably in the time appearance that reduces intestinal pH and reduce Erepsin hydrolysis vigor.Many surfactants can be promptly as dissolution enhancers, again as transportation (picked-up) reinforcing agent.If be not limited only to theory once more, be sure of to strengthen the aqueous part that dissolubility can provide (1) active component of the present invention more side by side to discharge into intestinal, (2) make peptide dissolve better and transport at the intestinal mucosa layer.In case the peptide active component reaches intestinal wall, a kind of absorption enhancer can provide the brush border membrane of passing through intestinal better to enter blood, via iuntercellular or the other transportation of cell.

The preferred composition of each of medical compounds of the present invention will separate discussion hereinafter.The combination of a plurality of pH depressants or a plurality of reinforcing agents can be used, also single pH depressant and/or reinforcing agent can be only used.Some preferably make up also and will discuss hereinafter.

The peptide active component

Divide according to those peptides that will be benefited in oral delivery of the present invention active title, comprise any therapeutic agent and a large amount of aminoacid that physiologically active is arranged, and at least one peptide is combined on the molecular structure, a site can be by amidatioon.The amidatioon of peptide can reach by chemistry or enzyme method, perhaps by the two combination.Amidated method for optimizing is the peptide-glycine amidatioon behavior by monooxygenase.

More preferably, peptide extends at the C-terminal of peptide when producing by recombinant technique and adds a glycine, C-terminal by enzyme reaction by amidatioon.Perhaps, also can carry out amidatioon with suitable amino acid side chain by chemical reaction.

Equally preferably, these peptide active component are connected to a MT sequence, promote their absorptions from enteral.These MT must the protected cutting that exempts from the harmonization of the stomach erepsin before being absorbed.Yet in case be absorbed, MT should be able to be removed by protease so that allow bioactive peptide gain freedom to small part.

MT can be made up of an aminoacid sequence, preferably by a signal peptide or signal sequence." signal peptide " here be one usually but nonessential be about 10 to 50 or more amino acid residue, wherein many (usually approximately 55-60%) residue is hydrophobic, they have hydrophobic, a fat-soluble part like this.This fat-soluble part is common, the major part of signal peptide, often is the core of signal peptide when secreting from cell.Signal peptide is one section aminoacid sequence, can promote the output of cytoplasm protein.Signal peptide of the present invention as what find here, " is fit to enter " equally, promptly can the outside permeates cell membranes enter cell interior.Amino acid residue can be made a variation and/or be modified (just can form mimetic), as long as modify the transposition-medium capability that does not influence peptide.Therefore " peptide " this speech comprises mimetic, and " aminoacid " this speech comprises the aminoacid of modification, as used herein, and the aminoacid of abnormal aminoacid and D-form.Among the present invention the signal peptide of all competent imports of indication have adjusting from the extracellular to cell in the function of transmembrane transport.They have also kept them to allow protein to enter the ability of external environment condition from cell output.A kind of generally acknowledged signal peptide can be tested the import vigor easily according to the technology that bottom provides here, comprises the specificity of testing any cell type.

The aminoacid sequence that following table 1 is enumerated, wherein each can both be used as MT.

The aminoacid sequence and the source thereof of some MT peptides of table 1

Sequence	Sequence is derived	The source
			ALA-ALA-VAL-ALA-LEU- LEU-PRO-ALA-VAL-LEU- LEU-ALA-LEU-LEU-ALA- PRO-VAL-ASN-ARG-LYS- ARG-ASN-LYS-LEU-MET -PRO(SEQ ID No：3)	Signal peptide from the Kaposi fibroblast growth factor	United States Patent (USP) 5,807,746
TYR-GLY-ARG-LYS-LYS- ARG-ARG-GLN-ARG-ARG- ARG(SEQ ID No：4)	The albumen transformational structure territory of HIV TAT	Schwarz etc., (1999) Science 285:1569
			VAL-THR-VAL-LEU-ALA- LEU-GLY-ALA-LEU-ALA- GLY-VAL-GLY-VAL-GLY (SEQ ID No：5)	People Integrin β 3 signal sequences	Zhang etc., (1988) PNAS 95:9184
The protein of 38kDa	HSV-VP22 albumen	Phelan etc., Protein (1998), Nature Bio-technology 16:440
			ALA-ALA-VAL-LEU-LEU- PRO-VAL-LEU-LEU-ALA- ALA-PRO(SEQ ID No：6)	Modify from 16 residues of the signal peptide hydrophobic region of Kaposi fibroblast growth factor and to obtain	Rojas etc., Nature Biotechnology 16:370

MT can be made up of fatty acid and/or bile acid equally.Such molecule, in the time of use, with bioactive peptide by being connect by the aminoacid bridging of protease cutting in the blood plasma.Perhaps, MT can be connected with bioactive peptide by non-peptide connected mode, under these circumstances in vivo and attachable enzyme can be the enzyme that is different from protease.The aminoacid bridge must be the cutting target spot of at least one plasma proteinase.Plasma proteinase and their target sequence are known in technology.Table 2 illustrates some and their the concrete target spot in these enzymes.

Table 2 plasma proteinase and their special target spot

Protease	Special target spot	Estimate
			Guang winter enzyme-1	Tyr-Val-Ala-Asp-Xaa *(SEQ ID No：7)
Guang winter enzyme-3	Asp-Xaa-Xaa-Asp-Xaa(SEQ ID No：8)
			Preceding albumen (Proprotein) invertase 1	Arg-(Xaa) n-Arg-Xaa(SEQ ID No：9)	N=2,4 or 6
	Lys-(Xaa) n-Arg-Xaa(SEQ ID No：10)	N=2,4 or 6
				Arg-Arg-Xaa
	Lys-Arg-Xaa
			Preceding convertase 2	The same with preceding convertase 1
Preceding convertase 4	Gly-Arg-Thr-Lys-Arg-Xaa(SEQ ID No：11)
			Preceding convertase 4PACE4	Arg-Val-Arg-Arg-Arg-Xaa(SEQ ID No：12)
	Capryl-Arg-Val-Arg-Arg-Xaa (SEQ ID No:13)
			Prolyl oligopeptidase	Pro-Xaa
Endothelium peptide after the dipeptide amido peptidase TV is sheared enzyme	Trp-Val-Pro-Xaa(SEQ ID No：14) Trp-Val-Ala-Xaa IV(SEQ ID No：15)
			Signal peptidase		Near depending on aminoacid
Neutral lyase after the dipeptide amido peptidase TV	Xaa-Phe-Xaa-Xaa(SEQ ID No：16)	Wide specificity, greatest length=40 aminoacid
				Xaa-Tyr-Xaa-Xaa(SEQ ID No：17)
	Xaa-Trp-Xaa-Xaa(SEQ ID No：18)
			Feritin after the dipeptide amido peptidase TV	Asp-Arg-Tyr-Ile-Pro-Phe-His-Leu- Leu-Val-Tyr-Ser(SEQ ID No：19)	Replace Val and Ser with Pro or Ala

* the amino acid whose N-terminal of runic is the special cleavage site of protease.

The present invention suppress active component by proteasome degradation, and this protease is with one or more peptide bonds of possibility cleavage activity peptide by several mechanism.

According to the present invention, synthetic and natural peptide can both oral delivery.The peptide reactive compound that comprises among the present invention comprises but is not to only limit to insulin, vassopressin, calcitonin (not only comprise preferred salmon calcitonin see calcimar, also comprise other calcitonin) and parathyroid hormone and its analog.Other example comprises calcitonin gene relevant peptide, luteinizing hormone-releasing factor (LRH), erythropoietin, organizes the plasminogen catalyst, human growth hormone, short adrenal gland's skin element (adrenocorticototropin), various interleukin, enkephalin glucagon-like peptide 1 and all analog.

When using salmon calcitonin see calcimar, it is more suitable for being made of the relative weight of the 0.02-0.2% of whole pharmaceutical composition (except that enteric coating) gross weight.Salmon calcitonin see calcimar can obtain (for example from BACHEM, Torrence, Calif.) from the commercial channel.Perhaps, it also can be synthetic with known method, and the some of them method will concise and to the point below argumentation.Other bioactive peptide should give higher or lower concentration, rely on the reactive compound target in blood middle concentration and it bioavailability in oral delivery of the present invention system.

Salmon calcitonin see calcimar can be with the method that the is re-combined into manufacturing in chemical method or the technology.The active propeptide of other amidatioon can be made with similar mode.Reconstitution cell production is considered to have tangible cost performance.Be converted into activated salmon calcitonin see calcimar by known method in the technology by the amidation process precursor equally.For example, the amidatioon of enzyme is at United States Patent (USP) 4,708,934 and european patent application 0308067 and 0382403 in description is arranged.Reconstitution cell production all is preferred to the enzyme that precursor and catalyged precursor become salmon calcitonin see calcimar.Such reconstitution cell production is at Biotechnology, and volume 11 (1993) pp.64-70 discuss, and to have further described a precursor conversion be a kind of amidated product.Here Bao Dao reconstitution cell product and natural salmon calcitonin see calcimar and synthetic with solution and solid state chemistry peptide be consistent.

When MT was connected to bioactive peptide of the present invention, it can be finished by known reconstitution cell in chemosynthesis or the technology is synthetic.Here " bonding " meaning that adopts is that biologically active peptide is connected by this way with MT, and when MT passed cell membrane, bioactive peptide also can pass through cell membrane.The example of such connected mode comprises that (A) is bonded together MT and bioactive peptide by peptide bond, and just, two peptides (peptide moiety of MT closes bioactive peptide) can be synthesized together; (B) by non-peptide covalent bond MT is bonded to bioactive peptide (for example using cross-linking reagent signal peptide and protein bound together); (C) use the chemical bond method between the such MT carboxyl terminal of bioactive peptide and image signal peptide, to produce covalent bond.

Below the example of method (A) demonstration is arranged, one of them peptide is known standard method synthetic (Merrifield, J.Am.Chem.Soc.85:2149-2154,1963 in the employing technology; With Lin etc., Biochemistry27:5640-5645,1988), and aminoacid sequence and a bioactive amino acid sequences of beginning to comprise a kind of signal peptide (MT), can be cut by plasma proteinase from amino terminal with linear mode.Such peptide can be produced by the reconstitution cell technology equally, express generation peptide (Sambrook etc. from the described amino acid whose recombination to construct of encoding, Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

To method (B), perhaps utilize as described peptide bond, perhaps use non-peptide covalent bond, MT is connected with biologically active peptide, polypeptide or protein.This non-peptide covalent bond can form by the standard method in the technology, for example MT and peptide, polypeptide or protein is passed through a kind of cross-linking reagent, as glutaraldehyde, couples together.Such method is standard (Walter etc., Proc.Natl.Acad.Sci.USA 77:5197 in technology; 1980).

For method (C), the chemical bond method of standard, for example chemical cross-linking agent can be used to interact with the carboxyl terminal of signal peptide.Such method is standard (Goodfriend etc., Science143:1344 in technology; 1964, with water miscible imidodicarbonic diamide as binding reagents), can easily be connected the carboxyl terminal of signal peptide with any bioactive molecule of selecting.

For example, the salmon calcitonin see calcimar precursor that can be in escherichia coli extends as the glycine of soluble fusion protein with glutathione-S-transferase production of preferred reconstitution cell salmon calcitonin see calcimar (rsCT) with make.The precursor molecule structure that glycine extends, consistent with active salmon calcitonin see calcimar except that the C-end (salmon calcitonin see calcimar is with-pro-NH ₂Finish, and precursor finishes with-pro-gly.The α that describes in the described publication-amidating enzyme catalyged precursor changes into salmon calcitonin see calcimar.This enzyme is more suitable for recombination method production, for example in Chinese hamster ovary (CHO) cell), as what mention in the Biotechnology article of quoting in the above.Other gas also can be produced with similar approach to other amidated peptide.

Not natural amidated peptide also can enough similar modes be produced, according to the present invention's amidatioon in a similar fashion.

PH depressant and protease inhibitor

The total amount of the pH reduction chemical compound that gives when at every turn using salmon calcitonin see calcimar preferably when it is released into intestinal, should be enough to intestinal pH significantly is brought down below the optimum pH of protease there.The amount that needs should necessaryly change according to Several Factors, comprises the type (being discussed below) of pH depressant of use and the proton of the equivalence that specific pH depressant provides.On the reagent, the required amount that the good biological availability can be provided should be that in adding the 10ml0.1M sodium bicarbonate solution, the pH of sodium bicarbonate solution reduces to and is not higher than 5.5, preferably reduces to not to be higher than 4.7, more preferably reduces to not to be higher than 3.5.In aforementioned test, in some embodiment, perhaps to pH be reduced to and not be higher than 2.8.In the present invention, the pH depressant that uses in the medical compounds is 300mg at least preferably, more preferably 400mg at least.Aforesaid parameter is selected relevant with all pH depressant gross weights, and such reagent may be two to unite use to more kinds of.Oral compositions should not comprise the alkali of any amount, will stop the pH of described sodium bicarbonate test to reduce to 5.5 or lower when chemical compound discharges when it reduces with pH.

PH depressant of the present invention should be acceptable chemical compound on any pharmacopedics, and is nontoxic in the gastrointestinal tract kind, and can fail the high hydrogen example content of passing hydrion (a kind of traditional acid) or reducing local environment.Also can be these combination of compounds.The pKa that plants preferred at least a pH depressant in the present invention is not higher than 4.2, preferably is not higher than 3.0.At room temperature to be at least every 30g/100ml water be suitable to the dissolubility of pH depressant in water.

Cause the example of the chemical compound of higher hydrogen ion concentration to comprise aluminum chloride and zinc chloride.The acceptable classical acid comprises but is not limited only to amino acid whose acid salt (for example, amino acid salts hydrochlorate) or related derivatives on the pharmacopedics.These examples have following acid salt: acetylglutamate; alanine; arginine; agedoite; aspartic acid; betanin; carnitine; carnosine; citrulline; sarcosine; glutamic acid; glycine; histidine; hydroxylysine; hydroxyproline; hypotaurine; isoleucine; leucine; lysine; methylhistidin; nor-leucine; ornithine; phenylalanine; proline; sarcosine; serine; taurine (taurine); threonine; tryptophan; tyrosine and valine.

Other comprises that the useful pH of carboxylic acid reduces chemical compound as aspirin, acetic acid, ascorbic acid, citric acid, Fumaric acid, glucuronic acid, 1,3-propanedicarboxylic acid, glyceric acid, glycolic (glycocolic acid), glyoxalic acid, 1-Hydroxy-1,2,3-propanetricarboxylic acid., isovaleric acid, lactic acid, maleic acid, oxaloacetic acid, oxalosuccinic acid, propanoic acid, acetone acid, succinic acid, tartaric acid, valeric acid etc.

Other useful pH depressant has, and may not be called as " acid " in technology usually, and however still they are useful according to the present invention, phosphate ester (for example, fructose 1,6 diphosphonic acid, glucose 1,6 diphosphonic acid, phosphoglyceric acid and diphosphoglyceric acid), (trade mark of BF Goodrich) and the polymer as the Polycarbophil also can be used in and reduce pH.

The combination of any pH depressant can not be higher than 5.5, can use requiring pH to reduce in described sodium bicarbonate test.A kind of embodiment preferred is utilized the pH depressant at least a pharmaceutical composition, a kind of acid that is selected from citric acid, tartaric acid and amino acid salts.

When salmon calcitonin see calcimar as the peptide activating agent, determine that the pH depressant of ratio and salmon calcitonin see calcimar are proved effective especially.Preferred pH depressant surpasses 200:1 with the weight ratio of salmon calcitonin see calcimar, is preferably 800:1, more preferably 2000L:1.

As the alternative method of using the pH depressant or the additional inhibitor that is to use protease inhibitor, particularly erepsin.Under tabulate and 3 enumerate some known erepsin inhibitor.

Table 3 erepsin and their specific site

Protease	Specific site	Optimum pH	Comment
				Insulin	Lys-Xaa	8
	Arg-Xaa
				Chymase	Try-Xaa	7.0-9.0
	Phe-Xaa
					Trp-Xaa
Elastoser	Ala-Xaa	8.8
					Val-Xaa
	Leu-Xaa
					Ile-Xaa
	Gly-Xaa
					Ser-Xaa
Kallikrein	Arg-Xaa	7.0-8.0
					Phe-Arg-Xaa		Preferably
	Leu-Arg-Xaa		Preferably
				Carboxypeptidase	Xaa-Xaa	7.0-9.0	From C-terminal

Optional member-absorption enhancer

When using, absorption enhancer preferably accounts for the 0.1-20% of weight on amount, with respect to the total weight (except that enteric coating) of pharmaceutical composition.Surfactant during preferred absorption enhancer, can either have as solubility enhancer can be as absorption enhancer.In general, " solubility enhancer " not only improved component among the present invention in their initial water environments that discharges but also improve dissolving in the outer field lipophillic environment of mucosa of intestinal wall, and perhaps the two all improves." transportation (picked-up) reinforcing agent " (following usually as the same surfactant of solubility enhancer) is that those can help peptide reagent to pass through intestinal wall.

One or more absorption enhancer can only be carried out a function (for example dissolubility), and perhaps one or more absorption enhancer can only be carried out a function (for example picked-up), does not exceed scope of the present invention.Same possible is the mixture of some chemical compounds, and some of them provide improved dissolubility, and some provide improved picked-up and/or some to finish this two tasks.If be not limited only to theory, believe that the effect of picked-up reinforcing agent is the hydrophobic region disorder that (1) improves the intestinal wall adventitia, allow the iuntercellular transportation that increases; Or the iuntercellular that (2) elimination memebrane protein causes increasing transports; Or the radius in (3) expansion iuntercellular hole increases the other transportation of cell.

Surfactant is considered to not only can be used as solubility enhancer but also can be used as absorption enhancer.For example, detergent is used for (1) is dissolved in their initial aqueous environments that discharges all active components fast, (2) lipotropy of raising component of the present invention, peptide activating agent particularly, help it to enter and pass through intestinal mucosa, (3) improve the ability that normal polarity peptide activating agent passes through brush border membrane epithelium barrier, (4) improve iuntercellular or the other transportation of cell, and are as indicated above.

When surfactant was used as absorption enhancer, preferably they were free-pouring powder, promoted mixed and capsular filling in manufacture process.Because the inherent character of salmon calcitonin see calcimar and other peptide (for instance, their isoelectric point, IP, molecular weight, aminoacid are formed, or the like), preferably some surfactant and some peptide interact.Really, some can be undesirably interacts with the electrically charged part of salmon calcitonin see calcimar, stops it to absorb, the therefore beyond thought reduction that causes bioavailability.Be preferably in when attempting improving the bioavailability of salmon calcitonin see calcimar and other peptide, any surfactant as absorption enhancer is selected from the group of these formations: (1) is anion surfactant, and described anion surfactant is cholesterol derivative (as a bile acid); (2) cationic surfactant (as fatty acyl carnitine, phospholipid and analog); (3) nonionic surfactant; (4) chemical compound of anion surfactant (particularly those have the straight-chain hydrocarbons zone) and negative charge nertralizer.The negative charge nertralizer includes but not limited to fatty acyl carnitine, cetyl pyridinium hydrochloric acid and analog.Equally preferably absorption enhancer can dissolve under acid pH, particularly in the 3.0-5.0 scope.

Good particularly suitable combination of effect is that salmon calcitonin see calcimar and cationic surfactant and anion surfactant (described anion surfactant is a cholesterol derivative) is mixed, and the two all is a solubility at acid pH.

A particularly suitable combination is sour dissolved bile acid and a kind of cationic surfactant.Fatty acyl carnitine and sucrose ester are a kind of good combinations.When using a kind of independent absorption enhancer, its preferably a kind of cationic surfactant.Fatty acyl carnitine (as the dodecyl carnitine), phospholipid and bile acid all are good especially absorption enhancer, particularly fatty acyl carnitine.In some embodiment, also use the such anion surfactant of cholesterol derivative.The purpose that these parameters are selected is the interaction of avoiding with peptide reagent, the absorption of disturbing peptide reagent to enter blood.

For reducing the probability of side effect; preferred detergent, when as absorption enhancer of the present invention, should or biodegradable or (for example can absorb again; but the biologically chemical compound of recirculation such as bile acid, phospholipid and/or fatty acyl carnitine), more preferably biodegradable.It is particularly useful in improving the other transportation of cell that fatty acyl carnitine is considered to.When bile acid (anionic detergent of perhaps other shortage straight-chain hydrocarbons) is used in combination with a kind of cationic detergent, salmon calcitonin see calcimar can betransported transportation better and enter and pass intestinal wall.

Preferred absorption enhancer comprises: (a) salicylic acid; as sodium salicylate; 3-methoxyl group salicylic acid; 5-methoxyl group salicylic acid and homovanillic acid salt (homovanilate); (b) bile acids; as cattle sulphur bile acid; cattle sulphur deoxidation bile acid; the deoxidation bile acid; bile acid; glycolic (glycholic); lithobilic acid; goose bile acid; the acid of ursodeoxycholic juice; Fel Ursi acid; the dehydrogenation bile acid; fusidinic acid or the like; (c) non-ionic surface active agent; as polyoxyethylene ether (as Brij 36T; Brij 52; Brij 56; Brij 76; Brij 96; Texaphor A6; Texaphor A14; Texaphor A60 or the like); p-t-octyl phenol polyoxy ethylene (Triton X-45; Triton X-100; TritonX-114; Triton X-305 or the like); Nonylphenoxy polyoxyethylene (as Igepal CO series); polyoxyethylene sorbitan ester is (as Tween-20; Tween-80 etc.); (d) anion surfactant; as dioctyl sodium sulphosuccinate; (e) lysophosphatide; as LYSOLECITHIN SUNLECITHIN A and lysophosphatidyl ethanolamine; (f) fatty acyl carnitine; acyl group choline and acylamino acid; as the lauroyl carnitine; the myristoyl carnitine; the palmityl carnitine; the lauroyl choline; the myristoyl choline; the palmityl choline; cetyl lysine; N-acyl group phenylalanine; N-acylglycine or the like; (g) aqueous phospholipid; (h) medium chain triglycerides; be single; two; the mixture of triglyceride; the fatty acid that contains the medium chain degree is (sad; capric acid and lauric acid); (i) ethylenediaminetetraacetic acid; (j) cationic surfactant; as the cetyl pyridinium choline; (k) derivative of fatty acid of Polyethylene Glycol; as Labrasol; Labrafac or the like; (1) alkyl sugar is as the lauryl alcohol maltoside; lauroyl sucrose; myristoyl sucrose; palmityl sucrose or the like.

In some preferred embodiments, there is no need to be limited to theory, cationic ion exchange reagent (as detergent) is included in, and provides dissolubility to increase by other possible mechanism.Especially, they may stop salmon calcitonin see calcimar or other peptide activating agent to be attached to mucus.Preferred cation ion exchange reagent comprises protamine chloride or any other polycation.

Other optional ingredients

Preferably, a kind of water solublity barrier separates protease inhibitor and/or pH depressant and acid-resisting protection carrier.A kind of medicament capsule of routine can be used in this purpose, and this barrier is provided.Known but water solublity barrier that be not limited only to these comprises HYDROXY PROPYL METHYLCELLULOSE and conventional medicinal gel in many literary compositions.

In some preferred embodiments, other peptide (for example albumin, casein, soybean protein, other plant or animal protein and analog) is included in to reduce non-specific absorption (for instance, peptide is attached on the casing slime barrier), reduced the necessary concentration of expensive peptide activating agent in this respect.When adding, peptide preferably accounts between the 1.0-10.0% of relative weight of pharmaceutical composition gross weight (not comprising the protectiveness carrier).Second peptide is more preferably does not have physiologically active, preferably a kind of food peptide, as soybean peptide or similarly.Be not limited only to theory, second peptide also may strengthen bioavailability, by as the protease street cleaner, with the interaction of protease in compete with the peptide activating agent.Second peptide also can help reactive compound to pass through liver.

All pharmaceutical compositions of the present invention can be chosen wantonly and comprise common drug diluent, fluidizer, lubricant, gel capsule, antiseptic, coloring agent and analog, with them common size and amount.

The protectiveness carrier

Any vehicle or carrier, it can help peptide activating agent and pepsin to isolate, and dissolving so other composition of the present invention can be discharged in the intestinal, all is suitable then.Known many enteric coatings in technology, and be useful according to the present invention.These examples comprise the copolymer of cellulose acetate phthalate ester, hydroxypropyl methyl ethyl cellulose succinate, hydroxypropyl methyl cellulose phthalate, carboxymethylethylcellulose and methyl-prop diluted acid-methyl methacrylate.In some embodiments, active phthalein, absorption enhancer are included in a kind of enough heavy-gravity guarantor's property understood syrup as dissolving and/or picked-up reinforcing agent and pH reduction chemical compound, allow component protectiveness of the present invention to pass through stomach.

Suitable enteric coating is used to protect peptide reagent contact pepsin, such as, should be applied to capsule later on by remaining in the present invention component filling capsule.In other embodiments, enteric coating is coated in the outside of tablet, perhaps is coated in the particulate outer surface of active component, be pressed into the tablet shape then or be loaded into capsule, and it preferably also is coated with the last layer enteric coating.

The effect of wanting very much is that all components of the present invention discharges and dissolves the intestinal environment from vehicle or carrier as far as possible simultaneously.Preferably carrier or carrier discharge active component at small intestinal, improve the uptake enhancer of the other transportation of iuntercellular or cell here, discharge at colonic than same uptake enhancer to cause that the probability of unnecessary side reaction is little.In any case, be stressed that the present invention is proved at colonic and effectively same in small intestinal.Many carriers or carrier are except that described, known in this article.Keeping enteric coating low is (particularly when optimization component of the present invention discharges simultaneously) of desirable.Preferably, the enteric coating of interpolation is no more than 30% (" residue " refers to get rid of the pharmaceutical composition of enteric coating self) of residual drug composition weight.More preferably, add to be no more than 20%, particularly account for coating not compositions 12% to 20%.Enteric coating should be enough, to prevent that pharmaceutical composition of the present invention is in 0.1N HCl disintegrate after two kinds hours, be elevated to 6.3 in the water-bath when pH is dissolving then, rotate with the speed of 100rpm, can allow all inclusions of pharmaceutical composition to discharge at 30 minutes in compositions described in the water-bath.

Other parameter

Preferably pH depressant and/or protease inhibitor and absorption enhancer (as words) weight ratio arrive 20:1 at 3:1, be more preferably 4:1 to 12:1, be preferably in 5:1 to 10:1.The gross weight of the gross weight of all pH depressants and/or protease inhibitor and all absorption enhancers is included in the aforesaid suitable proportion in specific pharmaceutical composition.For example, if a kind of pharmaceutical composition comprises two kinds of pH depressants and three absorption enhancers, described ratio will be by two kinds of bonded weight of pH depressant and three bonded total weight of absorption enhancer.

Preferably pH reduction and/or protease inhibitor, peptide activating agent and absorption enhancer (when the words that exist) (no matter being the unification compound or the chemical compound lot of each kind) are evenly distributed in the pharmaceutical composition.In one embodiment, pharmaceutical composition is by the granulometric composition that comprises a kind of medicine adhesive, and peptide activating agent, pH depressant and absorption enhancer uniform mixing are in described binding agent.Preferred granule should be made up of a kind of acid core equally, around the layer of even organic acid, one deck reinforcing agent and one deck peptide by the organic acid of outside around.Granule can be from the aqueous mixture manufacturing, and mixture is made of with pH reduction of the present invention, absorption enhancer and peptide activating agent medicine adhesive such as polyvinylpyrrolidone or hydroxypropyl emthylcellulose.

Manufacture process

Preferred pharmaceutical composition of the present invention comprises a kind of capsule of 00 size, uses the 0.25mg salmon calcitonin see calcimar, the 400mg graininess Fructus Citri Limoniae that are connected with MT to calculate (for example can obtain from Archer Daniels Midland Corp), 50mg cattle deoxidation bile acid (for example can obtain from SIGMA), 50mg fatty acyl carnitine (SIGMA) filling.

All compositionss preferably encapsulation are at last advanced gelatine capsule, and preferably Powdered so that add blender to any state.Thereafter, blender was opened three minutes, thoroughly mixed until powder.Blended then powder packing advances a big end of gelatine capsule.Add the other end then, then quick closedown capsule, 500 or more capsule can join a kind of plater, and (for example, Vector LDCS 20/30 Laboratory DevelopmentCoating System (can be available from Vector Corp., Marion, Iowa))

Enteric coating solution is by following method preparation: weighing 500g EUDRAGIT L30D-55 (copolymer of a kind of methacrylic acid and methyl methacrylate, a kind of enteric coating can be from RHM Tech Inc., Maidan, Mass acquisition).Add 411g distilled water, 15g triethyl group citric acid and 38g Pulvis Talci.The capsule coating of enough 500 00 sizes of Liang material like this.

Capsule is weighed and is placed the tympanum of coating machine then.Open machine and rotate tympanum (comprising capsule now inside) with 24-28rpm.The temperature of inlet nozzle is preferably in about 45 ℃.Delivery temperature is at about 30 ℃.The capsule temperature of coating is not preferably in about 25 ℃.Air mass flow is 38 cubic feet of per minutes.

Stretch out the coating solution that a pipe inserts described method preparation from machine then.Opening pump is plater supply solution.Automatically carry out coating then.Machine can at any time stop, and whether the amount of weighing capsule decision material is enough.Usually coating was allowed to carry out 60 minutes.Closed pump then about 5 minutes, and machine is still at the capsule of running with the help dry coating.Closing machine then, capsular coating is finished, though recommend capsule air drying two days.

Because enhanced bioavailability provided by the invention, the concentration of expensive salmon calcitonin see calcimar can keep relatively low in this aspect pharmaceutical composition.The example of particular formulations will illustrate hereinafter.

Patient's treatment

When selecting salmon calcitonin see calcimar, recommend the administration of employing cycle as the osteoporotic active component of treatment.Salmon calcitonin see calcimar is by tachymetabolism after subcutaneous administration in the man, and half life is approximately 20-40 minute.Yet its useful effect longer duration many on osteoclast can continue to surpass 24h, although level descends rapidly in the blood.Usually surpassing two hours with routine dose injection salmon calcitonin see calcimar will detect less than blood level.Therefore preferred dosage is weekly 5 days administration cycle.Subcutaneous administration salmon calcitonin see calcimar (100IU) usually causes that peak concentration is 250pg/ml in blood plasma.Nose administration calcitonin (200IU) is proved to be effective to osteoporosis when peak value is low to moderate 10pg/ml.Some patient is reported on the peak value level (for example, or surpass 200pg/ml) some gastrointestinal upsets.Therefore, in the blood plasma peak value of salmon calcitonin see calcimar preferably between 10-150pg/ml, more preferably between 10-50pg/ml.Blood plasma level can be with known radioimmunoassay commercial measurement.The doctor in charge should detect replacement labelling (as the pyridinoline or the Deoxypyridinoline of urine), particularly Zhi Liao starting stage (1-6 month) of patient's reaction, salmon calcitonin see calcimar level or skeletal diseases.He should change dosage a little according to the metabolism and the reaction of individual patient.

Preferably use a kind of single capsule, because single capsule discharges when can give polypeptide, pH depressant and absorption enhancer best at every turn.This is very worth because when acid in time with the release of polypeptide very near the time, can reduce best the unnecessary proteins hydrolysis of polypeptide is attacked.Near release simultaneously, can be by all components of the present invention administration in single pill or capsule be realized best.Yet the present invention comprises that also for example, the aequum with acid and reinforcing agent when using splits in two or more capsules, and these capsules can be used together, and they just can provide the requirement of all the components like this." pharmaceutical composition " comprises a kind of complete dosage that especially is fit to give human patients here, and it can arbitrarily segment, as long as in fact can use simultaneously.

The amidatioon of embodiment 1-carboxyl terminal is to the influence of oral salmon calcitonin (sCT) bioavailability

On a kind of dog model, carried out a research with the more oral defeated sCT (sCTgly) and amidated sCT (sCT-NH that glycine extends that pass ₂) pharmacokinetic parameter.

8 the bull beagles of body weight between the 12-16 kilogram are used to this research.Canis familiaris L. is in the peptide fasting evening before yesterday of oral administration test, but permission is free near water.The gastric lavage time of every Canis familiaris L. before testing was at least for 1 week.Every Canis familiaris L. per os has the gelatine capsule of enteric coating, and the 1st week wherein comprised 1.11mg sCTgly, and second week was 1.11mg sCT-NH ₂Each capsular total composition is presented in the table 4.Before giving capsule, collect blood sample with a kind of intravenous (IV) conduit insertion arm vein of 20 specifications.From the arm vein collect every Canis familiaris L. take medicine for twice before the blood sample of 3ml.

Table 4 sCT-gly and sCTNH ₂Capsular composition

Capsule	Graininess citric acid (mg)	LLC(mg)	sCT-gly(mg)	sCTNH 2(mg)	Talc(mg)
						The sCT-gly capsule	596	62	1.11		30
sCTNH 2Capsule	576	58		1.11	29

After giving capsule, collect the 3ml blood sample from the arm vein, after administration 240 minutes with 15 minutes intervals.Blood sample is collected the Monovette sampling syringe of into new heparinization.Sample places on ice, then centrifugal 10 minutes of 2-8 ℃ of about 2750rpm.The blood plasma supernatant goes in the little centrifuge tube of color-code, sticks the label of time point, is analyzing definite sCTgly or sCT-NH ₂Concentration before stored frozen at-20 ℃.

The concentration of sCTgly uses the breadboard RIA test kit of Peninsula to determine with the radioimmunoassay method in the blood plasma.SCT-NH in the blood plasma ₂Concentration use the test kit of Diagnostic Systems Laboratories company to measure with the sandwich ELISA method of immunity.SCTgly or sCT-NH from blood plasma ₂The pharmacokinetics curve, Cmax (peak plasma concentration pg/ml) and AUC (area under curve) are determined.The numerical value of all mensuration is standardized on 1mg dosage for any in two kinds of peptides.The meansigma methods of each is presented at table 5 in these parameters.

Table 5

Peptide	Mean Cmax * ± SEM (pg/ml)	Average A UC* ± SERM (pg/ml)
			sCTgly	485±106	25,125±5525
SCT-NH 2	3,199±602	140,000±18,200

C-terminal is that the mean Cmax of the sCT of amide groups is 6.6 times of sCTgly.Average A UC is the indirect measurement of bioavailability, for sCT-NH ₂Be 5.9 times of sCTgly.So to these two kinds of peptides, they are consistent except that have a kind of glycine or a kind of amide groups at C-terminal, the measurer of the peptide that is measured in the blood plasma after the oral delivery has very big difference, and this can directly advise because of in the existence of C-terminal amide groups.

The comparison of embodiment 2-parathyroid hormone amidatioon and non-amidated analog bioavailability

On a kind of dog model, carry out two researchs that separate, determine the pharmacokinetic parameter of oral delivery PTH analog.In first test, adopt PTH1-34-OH.In second test, adopt a kind of less analog PTH1-31NH ₂Remove little difference in size, these two kinds of main differences of molecule are that 1-34 peptide has free acid at C-terminal, and the 1-31 peptide at C-terminal by amidatioon.

8 the bull beagles of weight between the 12-16 kilogram are used to the PTH1-34-OH test.At PTH1-31NH ₂In the test, use 6 same Canis familiaris L.s.The peptide fasting evening before yesterday that Canis familiaris L. is tested at oral administration, but be allowed to free near water.The gastric lavage time of every Canis familiaris L. before testing was at least for 1 week.Every Canis familiaris L. per os has the gelatine capsule of enteric coating, wherein comprises 2.64mg PTH1-34-OH in first test, comprises 2.38mg PTH1-31NH in second test ₂Each capsular all the components is presented at table 6.Before giving capsule, a kind of intravenous support of 20 specifications inserts the arm vein to collect blood sample.Each 3ml sample from the arm vein before twice administration of collection.

Table 6 PTH1-34-OH and PTH1-31NH ₂Capsular composition

Capsule	Graininess citric acid (mg)	LLC(mg)	sCT-gly(mg)	sCTNH 2(mg)	Talc(mg)
						The PTH1-34-OH capsule	472	47	2.64		24
PTH1-31NH 2Capsule	576	58		2.38	29

After giving capsule, collect the 3ml blood sample from the arm vein, after administration 240 minutes with 15 minutes intervals.Blood sample is collected the Monovette sampling syringe of into new heparinization.Sample places on ice, then centrifugal 10 minutes of 2-8 ℃ of about 2750rpm.The blood plasma supernatant goes in the little centrifuge tube of color-code, sticks the label of time point, is analyzing definite PTH1-34-OH or PTH1-31NH ₂Concentration before stored frozen at-20 ℃.

The concentration of PTH1-34-OH uses the breadboard RIA test kit of Peninsula to determine with the radioimmunoassay method in the blood plasma.PTH1-31NH in the blood plasma ₂The competitive ELISA method of immunity of use Unigene Laboratories exploitation quantitative.PTH1-34-OH or PTH1-31NH from blood plasma ₂The pharmacokinetics curve, Cmax (peak plasma concentration pg/ml) and AUC (area under curve) are calculated.The meansigma methods of each is presented at table 7 in these parameters.

Table 7

Peptide	Mean Cmax * ± SEM (pg/ml)	Average A UC* ± SERM (pg/ml)
			PTH1-34-OH	314±117	11,893±3,366
PTH1-31-NH 2	2,155±602456	116,400±32,100

* be adjusted to 1mg dosage

PTH1-31NH ₂The Cmax meansigma methods be 6.25 times of PTH1-34-OH.Average A UC is the indirect measurement of bioavailability, for PTH1-31NH ₂Big 9.8 times.Although PTH1-31NH ₂Little 3 aminoacid of molecular proportion PTH1-34-OH, these two kinds of peptides can not be explained their difference on bioavailability in the little difference on the molecular weight (being respectively 3718 Daltons and 4118 Daltons).Therefore, the important difference between these two kinds of peptides be the C-terminal amide groups existence whether.

Embodiment 3-relatively has or does not have the bioavailability of aminoterminal parathyroid hormone PTH1-34 analog with the Mus of intraduodenal administration

Female Sprague-Dawley Mus (250-275g) is (for PTH1-34-OH n=6, for PTH1-34NH ₂N=7) he orders and xylazine anesthesia with gram before carotid artery inserts sleeve pipe.Sleeve pipe is installed into a kind of valve of threeway, can gather blood and use physiologic saline for substitute by it.Do midline incision at abdominal part, the duodenum that the 0.5ml medicament is advanced to expose by direct injection.Contain citric acid (0.5M), lauroyl carnitine (10mg/ml), salmon calcitonin see calcimar (as inner marker) and PTH1-34-OH or PTH1-34NH in each peptide formulations ₂(0.5mg/ml).5,15,30,60 and 120 minutes collection blood (0.5ml) before administration and after the administration.Centrifugal 10 minutes of blood sample 2600g, the blood plasma supernatant that obtains are stored in-20 ℃.The concentration of peptide is measured with competitiveness enzyme-linked immunoassay (ELISA) in the blood plasma.The absolute bioavailability intravenous dosages of each peptide (promptly with respect to) is from PTH1-34-OH and PTH1-34NH ₂The area under curve of the curve that the plasma concentration function time obtains calculates.

PTH1-34-OH and PTH1-34NH ₂Administration after 5 minutes from Mus duodenum fast Absorption.The Cmax of PTH1-34-OH is 3.05ng/ml, PTH1-34NH ₂Be 26.7ng/ml, be PTH (1-34) free acid form near 9 times.After 60 minutes, PTH1-34NH ₂Concentration remain PTH1-34-OH near 9 times (table 8).PTH1-34NH ₂Absolute bioavailability be 3.68%, PTH1-34-OH's is 0.45%.After these results showed that the OH group of C-terminal is replaced by amide groups, maximum peptide concentration had improved 8.75 times in the blood plasma, and the absolute bioavailability of PTH1-34 improves 8.2 times.

The amidatioon of table 8 C-terminal is to the influence of PTH1-34 pharmacokinetic curve

Time	PTH1-34-OH	PTH1-34NH 2
			Minute	Ng/ml ± standard error	Ng/ml ± standard error
0	0.00	0.00
			5	2.69±1.35	26.70±7.84
15	3.05±1.31	21.03±4.07
			30	1.90±0.81	13.13±3.36
60	0.62±0.38	5.39±3.08
			120	0.81±0.35	1.18±1.08
			Absolute bioavailability (%)	0.45±0.18	3.68±0.76

The influence that embodiment 4-C terminal amino group absorbs at the Mus duodenum LHRH

For C-terminal LHRH-NH2 (LHRH-NH ₂) tested by the duodenal absorption of anesthesia Mus.In this test, compare LHRH-NH ₂(the amidated decapeptide of a kind of C-terminal of natural generation) and LHRH-COOH (a kind of and LHRH-NH ₂Aminoacid sequence identical decapeptide except that C-terminal, but the C-terminal of LHRH-COOH is gly-COOH rather than gly-NH ₂) the absorption feature.12 Mus are anaesthetized and are implanted a sleeve pipe at carotid artery, are used at the different time blood sample collection.6 Mus are with No. 27 LHRH-NHs of pin in duodenum injection 0.5M citric acid and fatty acyl carnitine (10mg/mL) ₂(5mg/mL), 6 Mus are injected 0.5mLLHRH-COOH with same prescription) (5mg/mL).Blood sample is giving LHRH-NH ₂Or collected in 5,15,30,60 and 120 minutes before the LHRH-COOH and after the administration.The plasma sample that obtains is analyzed LHRH-NH ₂Or LHRH-COOH, with peptide concentration in the HPLC detection blood plasma of equipment fluorescence detector.Administration detects amidatioon and the Cmax of amidatioon LHRH (Cmax) not in the blood plasma after 5 minutes.Although the LHRH of two kinds of forms, detected the amidatioon LHRH of many 5 times of specific ionization acid form LHRH-COOH, LHRH-NH with identical amount administration mice in blood plasma after 5 minutes ₂(table 9).Area under curve (AUC), to the measurement of peptide degree of absorption and bioavailability, big 6 times (table 9) of LHRH of amidated LHRH specific ionization acid form.These results show amidated preparation, comprise a kind of acid and a kind of reinforcing agent, and are bigger than the bioavailability of non-amidated peptide.

The amidatioon of table 9 C-terminal absorbs the influence of LHRH to the Mus duodenum

Minute	LHRH-NH 2	LHRH-COOH
			0	ng/ml±sem	ng/ml±sem
5	0	0
			15	3276±893	654±103
30	2897±612	391±81
			60	1282±282	163±68
120	382±103	56±19
Cmax	3276±893	654±103
			AUC	109350±23652	17731±4002

Although described the present invention according to specific embodiment, many other change and modify and other purposes is clearly for those experienced operators.Therefore the present invention is not limited in clear and definite announcement here, and is only limited to claim.

Claims (62)

1. the pharmaceutical composition of an oral delivery physiologically active peptide reagent, it comprises the described bioactive peptide for the treatment of effective dose, wherein, described bioactive peptide in an amidated site of non-natural by amidatioon.

2. pharmaceutical composition as claimed in claim 1 further comprises at least a pharmaceutically acceptable pH depressant and/or protease inhibitor.

3. pharmaceutical composition as claimed in claim 2 further comprises a kind of acid-resisting protectiveness carrier, and this carrier can effectively transport the stomach of described pharmaceutical composition by the patient, prevents that simultaneously described bioactive peptide reagent from contacting with pepsin.

4. pharmaceutical composition as claimed in claim 1, wherein, described bioactive peptide reagent at C-terminal by amidatioon.

5. pharmaceutical composition as claimed in claim 4, wherein, described peptide is prepared to the precursor of glycine extension and is converted to the C-terminal amide group subsequently.

6. pharmaceutical composition as claimed in claim 1, wherein, described bioactive peptide comprises a kind of aminoacid that contains the amidatioon side chain.

7. pharmaceutical composition as claimed in claim 2, wherein, described pH depressant exists with a certain amount of in described pharmaceutical composition, if add the sodium bicarbonate solution of 10 milliliters of 0.1M in described compositions, described amount is enough to make the pH of described solution to reduce to and is not higher than 5.5.

8. pharmaceutical composition as claimed in claim 2, wherein, described pH depressant exists with a certain amount of in described pharmaceutical composition, if add the sodium bicarbonate solution of 10 milliliters of 0.1M in described compositions, described amount is enough to make the pH of described solution to reduce to and is not higher than 3.5.

9. pharmaceutical composition as claimed in claim 2, wherein, described protease inhibitor is a kind of stomach and/or erepsin inhibitor.

10. pharmaceutical composition as claimed in claim 2, wherein, described protease inhibitor suppresses a kind of enzyme that is selected from following enzyme: pepsin, trypsin, chymase, elastoser, kallikrein and carboxypeptidase.

11. pharmaceutical composition as claimed in claim 1, wherein, described bioactive peptide reagent is connected to the protein called membrane transporters that can partly be cut by enzyme in vivo at least.

12. pharmaceutical composition as claimed in claim 3, wherein, the amount of described protectiveness carrier is no more than 30% of described residual drug composition weight.

13. pharmaceutical composition as claimed in claim 3, wherein, the amount of described protectiveness carrier is no more than 20% of described residual drug composition weight.

14. pharmaceutical composition as claimed in claim 3, wherein, the amount of described protectiveness carrier described residual drug composition weight 10% to 20% between.

15. pharmaceutical composition as claimed in claim 3; wherein; described protectiveness carrier is enough to prevent the disintegrate in 0.1N HCl at least 2 hours of described pharmaceutical composition, rises to all release in 45 minutes of 6.3 backs but allow all inclusions of described pharmaceutical composition to change in the decomposition water-bath of rotating with per minute 100 in described compositions at pH.

16. pharmaceutical composition as claimed in claim 3 further comprises the absorption enhancer of the described activating agent bioavailability of at least a effective promotion.

17. pharmaceutical composition as claimed in claim 16, wherein, described absorption enhancer is a surfactant.

18. pharmaceutical composition as claimed in claim 17, wherein, described surfactant is can absorb or biodegradable.

19. pharmaceutical composition as claimed in claim 17, wherein, described surfactant is selected from fatty acyl carnitine, phospholipid and bile acid.

20. pharmaceutical composition as claimed in claim 19, wherein, described reinforcing agent is a fatty acyl carnitine.

21. pharmaceutical composition as claimed in claim 20 further comprises a kind of sucrose ester.

22. pharmaceutical composition as claimed in claim 16, wherein, described absorption enhancer is selected from: (i) a kind of reagents for anion, described reagents for anion are cholesterol derivatives, the mixture of (ii) a kind of anion nertralizer and anion surfactant, (iii) non-ionic surface active agent and (iv) cationic surfactant.

23. pharmaceutical composition as claimed in claim 16, wherein, described absorption enhancer is selected from cationic surfactant and anion surfactant, and described anion surfactant is a cholesterol derivative.

24. pharmaceutical composition as claimed in claim 16, wherein, described pharmaceutical composition comprises at least two kinds of absorption enhancers, and wherein a kind of is cationic surfactant, another is an anion surfactant, and described anion surfactant is a cholesterol derivative.

25. pharmaceutical composition as claimed in claim 24, wherein, described anion surfactant is acid soluble bile acid.

26. pharmaceutical composition as claimed in claim 1 further comprises a certain amount of second kind of peptide, this peptide is not the physiologically active peptide that effectively improves described peptide activating agent bioavailability.

27. pharmaceutical composition as claimed in claim 3 further comprises a kind of water solublity barrier that can isolate described pH depressant and described protectiveness carrier.

28. pharmaceutical composition as claimed in claim 2, wherein, described compositions comprises at least a pKa and is not higher than 4.2 pH depressant.

29. pharmaceutical composition as claimed in claim 2, wherein, the dissolubility of at least a pH depressant in water is that every 100ml water dissolves 30g at least under the room temperature.

30. pharmaceutical composition as claimed in claim 3, wherein, all the components uniform distribution except that the protectiveness carrier.

31. pharmaceutical composition as claimed in claim 30, wherein, described pharmaceutical composition comprises the granule that contains medicine adhesive and is evenly distributed on the described pH depressant in the described binding agent, described absorption enhancer and described peptide activating agent.

32. pharmaceutical composition as claimed in claim 16, wherein, described compositions is solid dosage forms, and wherein, the weight ratio of described pH depressant and described absorption enhancer is between 3:1 to 20:1.

33. pharmaceutical composition as claimed in claim 16, wherein, described compositions is solid dosage forms, and wherein, the weight ratio of described pH depressant and described absorption enhancer is between 5:1 to 10:1.

34. pharmaceutical composition as claimed in claim 2, wherein, described pH depressant is selected from citric acid, tartaric acid and amino acid whose acid salt.

35. pharmaceutical composition as claimed in claim 2, wherein, described pH depressant is no less than 300mg on amount.

36. pharmaceutical composition as claimed in claim 35, wherein, described pH depressant is no less than 400mg on amount.

37. pharmaceutical composition as claimed in claim 1, wherein, described peptide reagent is human glucagon-like-peptide 1, human glucagon-like-peptide 2 or its analog.

38. pharmaceutical composition as claimed in claim 1, wherein, described peptide reagent is a salmon calcitonin see calcimar.

39. pharmaceutical composition as claimed in claim 1, wherein, described peptide reagent is an insulin.

40. pharmaceutical composition as claimed in claim 1, wherein, described peptide reagent is human parathyroid hormone or its analog.

41. pharmaceutical composition as claimed in claim 1, wherein, described peptide reagent is human parathyroid hormone analog PTH1-31NH ₂

42. as claim 1 or the described pharmaceutical composition of claim 3, wherein, described peptide reagent is human parathyroid hormone analog PTH1-34NH ₂

43. pharmaceutical composition as claimed in claim 3, wherein, described protectiveness carrier is heavy-gravity protectiveness syrup.

44. pharmaceutical composition as claimed in claim 3, wherein, a kind of water solublity barrier separates described pH depressant and described protectiveness carrier.

45. the method for the bioavailability of a physiologically active peptide reagent that improves oral delivery, described method comprises:

(A) at the described peptide reagent of the amidated site amidatioon of the non-natural of peptide; With

(B) the described peptide reagent of dosage forms for oral administration.

46. method as claimed in claim 45; wherein; by behind described patient's the oral cavity stomach function regulating, described peptide activating agent is discharged into patient's enteral with at least a pH depressant and/or protease inhibitor selectivity under the protection of the acid-resisting protection carrier that described peptide activating agent, pH depressant and/or protease inhibitor can fully stop pepsin to contact with described peptide reagent.

47. method as claimed in claim 45, wherein, described bioactive peptide reagent at C-terminal by amidatioon.

48. method as claimed in claim 47, wherein, glycine is prepared to glycine and extends precursor and be converted to the C-terminal amide groups subsequently.

49. method as claimed in claim 45, wherein, described bioactive peptide is in the amino acid side chain amidatioon.

50. method as claimed in claim 46, wherein, described pH depressant exists with a certain amount of in described pharmaceutical composition, if add the sodium bicarbonate aqueous solution of 10 milliliters of 0.1M in described compositions, described amount is enough to make the pH of described solution to reduce to and is not higher than 5.5.

51. method as claimed in claim 46, wherein, described pH depressant exists with a certain amount of in described pharmaceutical composition, if add the sodium bicarbonate aqueous solution of 10 milliliters of 0.1M in described compositions, described amount is enough to make the pH of described solution to reduce to and is not higher than 3.5.

52. method as claimed in claim 46, wherein, described protease inhibitor is a kind of stomach and/or erepsin inhibitor.

53. method as claimed in claim 46, wherein, described protease inhibitor suppresses a kind of enzyme that is selected from following enzyme: pepsin, trypsin, chymase, elastoser, kallikrein and carboxypeptidase.

54. method as claimed in claim 46, wherein, it is to carry out when the absorption enhancer of the bioavailability of the described peptide activating agent of at least a effective promotion exists that described peptide activating agent is discharged into patient's enteral.

55. method as claimed in claim 45, wherein, described bioactive peptide is human glucagon-like-peptide 1, human glucagon-like-peptide 2 or its analog.

56. method as claimed in claim 45, wherein, described peptide reagent is a salmon calcitonin see calcimar.

57. method as claimed in claim 45, wherein, described peptide reagent is an insulin.

58. method as claimed in claim 45, wherein, described peptide reagent is human parathyroid hormone or its analog.

59. method as claimed in claim 58, wherein, described peptide reagent is human parathyroid hormone analog PTH1-31-NH ₂

60. method as claimed in claim 58, wherein, described bioactive peptide is human parathyroid hormone analog PTH1-34-NH ₂

61. method as claimed in claim 45, wherein, described bioactive peptide is a luteinizing hormone releasing hormone.

62. method as claimed in claim 45, wherein, the raising of described bioavailability is that enteral absorbs enhanced results.