CN101233151A - Tight junction modulating peptide components for enhancing mucosal delivery of therapeutic agents - Google Patents

Abstract

Compounds and components including sequences for mucosal epithelial transport of an active agent are given. Tight junction modulating peptide components are described for use in transport and delivery. Permeability can be enhanced with reversibility. Compounds and components for enhanced delivery may be peptide or protein variants, conjugates, or other analog types and structures.

Description

Strengthen the tight junction modulating peptide components of the mucosal delivery of therapeutical agent

Background of invention

Basic care is to guarantee the therapeutical agent safety of medicine and be delivered to the experimenter effectively in the treatment of any disease or the patient's condition.The conventional route of therapeutic agent delivery comprises intravenous injection and Orally administered.Yet these delivering methods have shortcoming, and therefore need alternative delivery system.

A main drawback by the injection drug administration is need come drug administration by the personnel that trained usually.In addition, when by the injection drug administration, the personnel that trained are in the danger.For oneself-medicine of using, many patients reluctantly or can not be to themselves injection on regular basis.Injection is also relevant with the risk of infection that increases.Other shortcoming of medicine injection comprises the mutability of sending the result between the individuality, and pharmaceutically-active unpredictable intensity and time length.

Although have these shortcomings, for many important treatment compounds, the mode of sending that injection is is still checked and approved for unique process.These comprise conventional medicine, and fast-developing peptide and the tabulation of protein biology therapeutical agent.These compounds are for example oral by alternative route of administration, nose and sending of other mucous membrane approach are ideal, but lower bioavailability may be provided.For the macromole kind, for example, peptide and protein for treatment compound, alternative route of administration may be subjected to the restriction of the low absorption of easy inactivation and process mucous membrane barrier.

Because first pass metabolism (hepatic first-pass metabolism) and/or the degraded in gi tract of liver, the Orally administered of some therapeutical agents showing low-down bioavailability and great time lag on.

The mucous membrane of treatment compound is used with injecting to compare with other method of application some advantage is provided, and for example, about the convenience and the speed of sending, and reduces or eliminates compliance issues and side effect.Yet the mucosal delivery of bioactive agent is subjected to the restriction of mucous membrane barrier action and other factors.

Epithelial cell is formed the mucous membrane barrier, and the important interface between outside atmosphere and mucous membrane and mucous membrane undertissue and the extracellular compartment is provided.A kind of most important function of mucosal epithelium cell is to determine and adjusting mucous membrane perviousness.In this case, epithelial cell produces the selective permeation barrier between different physiological compartments.Selective permeability is molecule is transported the perviousness (paracellular pathway (paracellular pathway)) of (transcellular pathway) and the adjusting of iuntercellular spatial by cytoplasmic regulation and control result.

Iuntercellular between the known epithelial cell connects keeping of participation epithelial cell barrier action and regulates and control, and cell-cell adhesion.The tight connection (TJ) of epithelium and endotheliocyte connects particularly important for the infiltrative cell of regulating and control paracellular pathway-cell, and cell surface is divided into top and substrate outside compartment.Closely be connected between the epithelial cell to form between the successive peripheral cell and contact, and produce the regulation and control barrier of the parietal cell motion of water, solute and immunocyte.By the exchange of the membrane lipid between restriction top and the basolateral membrane structural domain, they also provide second type the barrier that constitutes cell polarity.

Think that tight connection participates in epithelial barrier and defense function directly, seal the one-level barrier that spreads by paracellular pathway at solute to generate by producing iuntercellular respectively, and participate in to produce and to keep cell polarity by the border that act as between the plasma structure territory, top and the substrate outside.Closely connection also participates in white corpuscle and arrives moving of inflammatory site.Reply chemical inhibitor, white corpuscle moves from blood by passing endotheliocyte, and in the situation of mucosa infection, passes the epithelial cell of inflammation.Move mainly and take place along paracellular pathway, and as if by closely being connected to regulate and control with closing so that hight coordinate and reversible mode are open.

Identified that many albumen are relevant with TJs, comprised inside and outside plasmalemma protein.Understanding to these proteic complex structures and interactional function still is limited at present.With many albumen of epithelial cell join dependency in, identified the transepithelial membranin of some kinds, it may work in the physiological regulating control that epithelial cell connects.These comprise many " connection adhesion molecules " (JAMs) be called closed albumen, close albumen and connect the relevant molecule of proteic other TJ-.

JAMs, closed albumen and close albumen extend to the parietal cell space, and predict that especially these albumen are the material standed fors that form the epithelium barrier between the adjacent epithelial cell and pass through the passage of epithelium layer.In a kind of model, propose that closed albumen, close albumen and JAM interact as having a liking for same sex binding partners, with the barrier of the regulation and control of the parietal cell motion that between epithelial cell, produces water, solute and immunocyte.

In the situation that medicine is sent, medicine do not sent-help of toughener and as if see through the ability of epithelium layer of mucous membrane surface relevant with many factors, comprise molecular size, fat-soluble and ionization.Usually, be lower than about 300-1,000 daltonian small molecules often can see through the mucous membrane barrier, yet along with molecular size increases, perviousness reduces fast.Owing to these reasons, the mucous membrane medicament administration is typically used than injection needs more substantial medicine.Other treats compound, comprises macromolecular drug, is difficult to pass through mucosal delivery usually.Except low intrinsic permeability, big macromolecular drug stands limited diffusion usually, and inner chamber and cell enzymatic degradation and in the quick removing in mucous membrane site.Therefore, in order to send these bigger molecules with the treatment significant quantity, need the cell permeability toughener to help them and pass these mucous membrane surfaces, and enter systemic circulation, they can promptly act on destination organization there.

Mucous membrane tissue freely spreads the barrier that provides basic for macromolecular, and the enzymatic activity that in mucous membrane secretory product, exists limit treatment agent seriously, particularly peptide and proteic bioavailability.In some mucous membrane site, such as nasal mucosa, because mucociliary clearance fast, the typical residence time of the albumen of sending and other macromole kind is limited, for example, and about 15-30 minute or still less.

Exist permanent and outstanding demand to the method for pharmaceutical preparation and administering therapeutic compound in this area, wherein said treatment compound provides the enhanced mucosal delivery, comprises by the tissue of target and physiological compartment as in central nervous system.

More specifically, in the art, be used for having demand at the safe and reliable method and the composition of the treatment compound of mammalian subject treatment disease and other unfavorable patient's condition for mucosal delivery.There is relevant demand in the method and composition of effectively sending for the macromolecular drug that therapeutic dose is provided by one or more mucous membrane approach, described method and composition effect is quick, easy to implement, and have limited adverse side effect such as mucosa irritation or tissue injury.

In the art, there is demand in the method and composition of mucosal delivery that also overcomes the biotherapy compound of mucosal epithelium barrier cell mechanism for enhancing.Before this, the selective permeability of mucosal epithelium cell is described to the therapeutic macromole, comprises the major obstacle of biologically active peptides and proteic mucosal delivery.Therefore, in the art, there is outstanding demand for the novel method and the instrument that promote biotherapy compound mucosal delivery.Especially, in the art, there is demand in the novel method and the preparation of the mucosal delivery of the biotherapy compound that promotes proof before this to be difficult to send by the mucous membrane barrier.

The accompanying drawing summary

Fig. 1 illustrates PN159 to PTH _1-34The influence of infiltration, its use has other toughener (Me-β-CD, DDPC, PN159 EDTA).

Fig. 2 illustrates PN159 to PTH _1-34The influence of infiltration, it uses the PN159 that does not have other toughener.

Fig. 3 illustrates the influence of PN159 to permeating in the peptide YY body.

Fig. 4 illustrates the influence of PN159 to the infiltration of MC-4 receptor stimulant.

Fig. 5 shows that 25-100 μ M PN159 is to the external influence that sees through the epithelial cell individual layer of 40mg/ml lactic acid lycoremine.

Fig. 6 shows the TJM peptide (A) 5 ℃, (B) 25 ℃ and (C) 40 ℃ chemical stability.About pH 4.0, the data of pH 7.3 and pH 9.0 are expressed as solid diamond, open squares and black triangle respectively.

Fig. 7 is shown in every kind of tight junction modulating peptide (TJMP) and has the penetration kinetics of FITC-dextran MW4000 down.The PYY preparation act as positive control, and the negative contrast of phosphate-buffered salt (PBS).Handling cell with TJMP and FITC-dextran MW4000 after 15 minutes and handle cell after 60 minutes, detect Premeabilisation of cells.Pictorialization infiltration depends on the length of the time that TJMP contacts with epithelial cell, and shows that all TJMPs that detected strengthen the infiltration of FITC-dextran MW4000.

Fig. 8 is shown in PN159 and PEG-PN159 and handles that transepithelial electrical resistance (TER) reduces after 1 hour.

Fig. 9 is shown in the perviousness increase of handling back FITC dextran 3000 with PN159 and PEG-PN159.

Figure 10 illustrates the infiltration ratio of PN159 and PEG-PN159.

The pegization (pegylation) that Figure 11 illustrates PN159 reduces toxicity (LDH detection).

Figure 12 is shown in nose and uses the average blood plasma PYY3-36 concentration that peptide PN529 (PEG-PN159) back of PEGization increases.

Figure 13 is shown in nose and uses the average blood plasma PYY3-36 concentration (logarithm-linear graph) that peptide PN529 (PEG-PN159) back of PEGization increases.

Detailed Description Of The Invention

The present invention satisfies aforementioned need by the novel pharmaceutical combination thing is provided, and realize other purpose and advantage, described novel pharmaceutical combination thing comprises that the tight connection-opening peptide of latest find strengthens the novelty application of the mucosal delivery of bioactive agent in mammalian subject.

One aspect of the present invention is a kind of pharmaceutical preparation, the tight junction modulating peptide (TJMP) that it comprises biologically active agent and strengthens the significant quantity of mucosal delivery, described tight junction modulating peptide reversibly strengthen the mucosal epithelium transhipment of biologically active agent in mammalian subject.

Preferably, the tight junction modulating composition comprises by 2-500 amino-acid residue or 2-100 amino-acid residue or 2-50 peptide or the protein part that amino-acid residue is formed.Tight junction modulating peptide or albumen can be monomer or oligomer, for example, and dimer.

Tight junction modulating peptide can be produced by reorganization consistent with the technology known to the skilled in suitable field or chemosynthesis mode.

The previous peptide that to regulate and control the tight linkage function of epithelial cell (Johnson, P.H. and S.C.Quay, Expert.Opin.Drug Deliv. (expert's viewpoint of sending about medicine) 2:281-98,2000) of having described.Especially, show a kind of tight junction modulating (TJM) peptide of novelty, PN159 reduces and passes the transepithelial electrical resistance (TER) of organizing barrier, and increase has 3 of low cytotoxicity and the high retentivity of cell viability, the parietal cell transhipment of 000Da MW dextran.

In embodiment preferred of the present invention, described TJMP is selected from the group of being made up of following:

The NH2-KLALKLALKALKAALKLA-acid amides

The NH2-GWTLNSAGYLLGKINLKALAALAKKIL-acid amides

The NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-acid amides

The NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-acid amides

The NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-acid amides

The NH2-RQIKIWFQNRRMKWKK-acid amides

The NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-acid amides

The NH2-KLWSAWPSLWSSLWKP-acid amides

The NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-acid amides

Maleimide-GLGSLLKKAGKKLKQPKSKRKV-acid amides

The NH2-KETWWETWWTEWSQPGRKKRRQRRRRPPQ-acid amides.

In other preferred embodiment of the present invention, described TJMP is selected from the group of being made up of following:

CNGRCGGKKKLKLLLKLL

LRKLRKRLLRLRKLRKRLLR。

In one aspect, the present invention describes therapeutic small molecules, peptide and the proteic preparation that is suitable for striding mucosal delivery, and wherein the existence by tight junction modulating peptide promotes to stride mucosal delivery, and wherein said peptide and water-soluble polymers are puted together.Preferably, described water-soluble polymers is the polyalkylene oxide that is selected from by the following group of forming: the polyalkylene oxide derivative of alpha-substitution, alkyl-end capped polyethylene oxide, two-polyethylene oxide, poly-(ortho ester) is as poly-(breast-be total to-glycollide) and derivative thereof, polyoxyethylene glycol (PEG) homopolymer and derivative thereof, polypropylene glycol homopolymer and derivative thereof, the multipolymer of poly-(oxirane), and segmented copolymer or its activatory derivative of poly-(oxirane).Preferably, described polyalkylene oxide has about 200 to about 50,000 molecular weight.More preferably, described polyalkylene oxide has about 200 to about 20,000 molecular weight.Particularly preferred polyalkylene oxide is polyoxyethylene glycol and polyethylene oxide.

TJMP can put together with the water-soluble chain more than.In a preferred embodiment, described poly-(oxirane) chain is polyoxyethylene glycol (PEG) chain, and it can have about 0.2 and the molecular size of about 200 kilodaltons (kDa).

Water-soluble polymers can be puted together by spacerarm and tight junction modulating peptide.This connection can be a reversible.Described water-soluble polymers can be linear maybe side chain can be arranged.

In one embodiment, peptide and single poly-(oxirane) chain are covalently bound.In a relevant embodiment, poly-(oxirane) has and is lower than 2.00 or be lower than 1.20 polydispersity value (Mw/Mn).Described poly-(oxirane) chain can be side chain arranged or do not have a side chain.

With water-soluble polymers such as poly-(ethylene glycol) (PEG) and the derivative of PEG put together as improving the particularly strategy (Caliceti of the transformation period of injection type of albumen, P and F.M.Veronese, Adv.Drug Deliv.Rev. (senior medicine is sent summary) 55:1261-77,2003).Comprise chemistry (Diwan with polymkeric substance such as PEG modified peptides and proteic other potential benefit, M. and T.G.Park, Int.J.Pharm. (Inpharm magazine) 252:111-22,2003) and biochemical stability (Na, D.H. etc., J.Pharm.Sci. (pharmaceutical science magazine) 93:256-61,2004) and immunogenic minimizing (Yang, Z. etc., Cancer Res. (cancer research) 64:6673-78,2004).

Be conjugated to the most common example that the PEG on the albumen uses and be described PEG chain have sufficiently long molecular weight with the situation of giving above-mentioned effect in.Especially, the PEG of 20kDaMW has at least been described.For example, (Holtsberg such as Holtsberg, F.W. etc., J.Control Rel. (controlled release magazine) 80:259-71,2002) show, for the protein-arginine deiminase of puting together with PEG (deiminase), when PEG is 20kDa or when bigger, the pharmacokinetics of preparation and drug effect characteristic increase when using in the mouse medium sized vein.When PEG MW is lower than 20kDa, almost there is not effect.In another example, in rat, to bioavailability in the mono-pegylated nose that causes improving of peptide salmon calcitonin see calcimar, described raising and PEG molecular weight (the MW) (Lee that is inversely proportional to, K.C. etc., Calcif.Tssue Int. (international calcified tissue) 73:545-9,2003, and Shin, B.S. etc., Chem.Pharm.Bull. (Tokyo) (chemistry and pharmacy bulletin (Tokyo)) 52:957-60,2004), it is incorporated into this by reference fully.

Some preferably gather (oxirane) and select by the following group of forming: poly-(oxirane) derivative of alpha-substitution, poly-(ethylene glycol) is homopolymer and derivative thereof (PEG), poly-(propylene glycol) be homopolymer and derivative thereof (PPG), poly-(oxyethane) is polymkeric substance and derivative thereof (PEO), two-poly-(oxyethane) and derivative thereof, the multipolymer of poly-(oxirane), segmented copolymer with poly-(oxirane), poly-(lactide-co-glycolide) and derivative thereof, or their activated derivatives.Described water-soluble polymers can have about 200 to about 40000Da, and preferably about 200 to about 20000Da, or about 200 to 10000Da, or about 200 to 5000Da molecular weight.

Usually, the conjugate between tight junction modulating peptide and PEG or other water-soluble polymers can be resisted physiological action, comprises proteolysis, enzyme effect or hydrolysis.Alternatively, described conjugate can pass through biological degradation, for example precursor-medicine approach and cracking.Preferably, described molecule and single poly-(oxirane) chain are covalently bound, and described poly-(oxirane) chain can be unbranched or side chain arranged.The method of puting together is normally known to the those of ordinary skill, for example, and U.S. Patent number 5,595,732; U.S. Patent number 5,766,897; U.S. Patent number 5,985,265; U.S. Patent number 6,528,485; U.S. Patent number 6,586,398; U.S. Patent number 6,869,932; With U.S. Patent number 6,706,289.

In another aspect of the present invention, TJMP reduces to pass the resistance of mucous membrane tissue barrier.In a preferred embodiment, being reduced to of resistance used at least 80% of the preceding resistance of penetration enhancers.In a relevant embodiment, TJMP increases the perviousness that molecule passes the mucous membrane tissue barrier, preferably twice at least.In another embodiment, the perviousness of described increase is a parietal cell.In another embodiment, the perviousness that is increased is caused by close-connected modification.In an alternate embodiment, the perviousness that is increased is transcellular, or strides the combination of cell and parietal cell.

In another aspect of the present invention, the mucous membrane tissue layer comprises epithelium layer.In a preferred embodiment, epithelial cell is selected from by tracheal cell, segmental bronchus cell, alveolar cell, nose cell, pneumonocyte, gastrointestinal cell, the group that epidermic cell or Stomatocyte are formed, preferred nose cell.

In another aspect of the present invention, active agent is peptide or albumen.Described peptide or albumen can have 2-1000 amino acid.In a preferred embodiment, described peptide or albumen comprise 2-50 amino acid.In another embodiment, described peptide or albumen are annular.In another embodiment, described peptide or albumen form dimer or high-grade oligomer more by physics or chemical bonding.

In a preferred embodiment, described peptide or albumen are selected from by GLP-1, PYY _3-36, PTH _1-34The group of forming with the inhibitor-4 of glucagon-like peptide.In another embodiment, biologically active agent is an albumen, preferably is selected from the group of being made up of any analogue of beta-interferon, alpha-interferon, Regular Insulin, erythropoietin, G-CSF and GM-CSF, tethelin and they.

Perviousness peptide of the present invention comprises PN529, and it comprises sequence WEAALAEALAEALAEHLASQPKSKRKV (SEQ ID NO 57).

Another aspect of the present invention is the method that molecule is administered to animal, and it comprises any above-mentioned preparation of preparation, and makes described preparation contact with the mucous membrane surface of described animal.In a preferred embodiment, described mucous membrane surface is in the nose.

Another aspect of the present invention is the formulation that comprises any above-mentioned preparation, and wherein said formulation is a liquid, preferably droplet form.Alternatively, described formulation can also be a solid, and reconstruct in liquid is used as powder before using, perhaps with capsule, tablet or gel form.

Another aspect of the present invention is the molecule that reversibly strengthens the mucosal epithelium transhipment of biological reagent in mammalian subject, and it has tight junction modulating composition peptide (TJMP), TJMP analogue, the conjugate of TJMP or TJMP analogue, or its mixture.

Perviousness peptide of the present invention comprises PN159, and it has sequence NH2-KLALKLALKALKAALKLA-acid amides.What comprise among the present invention is the analogue of PN159 disclosed herein, any derivative, variant, fragment, analogue body or the fusion molecule of the combination of these analogues and PN159.

The transhipment of the reversible increase mucosal epithelium of described perviousness reagent parietal cell, it typically increases by mucosal epithelium surface modulation epithelial cell tight connecting device and/or physiological in the experimenter.This effect typically comprises by perviousness reagent and suppresses homotype or special-shaped combination between the adjacent epithelial epithelial membrane adhesion protein.The target point protein that is used for this homotype or special-shaped combination retardance can be selected from various relevant connection adhesion molecule (JAMs), closed albumen or close albumen.

Epithelial cell biology

The clones coding mouse connects the cDNA of adhesion molecule-1 (JAM-1), and the I type transmembrane protein (comprising single membrane spaning domain) of its corresponding a kind of prediction, molecular weight (Williams with about 32-kD, Deng, Molecular Immunology (molecule rabbit epidemiology) 36:1175-1188,1999; Gupta, etc., IUBMB Life (IUBMB life) 50:51-56,2000; Ozaki, etc., J.Immunol. (Journal of Immunology) 163:553-557,1999; Martin-Padura, etc., J.Cell.Biol. (cytobiology magazine) 142:117-127,1998).The extracellular fragment of described molecule comprises two Ig-spline structure territories, and it is described to aminoterminal " VH-type " and carboxyl terminal " C2-type " carboxyl terminal β-sandwich folding (Bazzoni etc., Microcirculation (microcirculation) 8:143-152,2001).Mouse JAM-1 also comprises two and is used for the glycosylated site of N-and a tenuigenin structural domain.JAM-1 albumen is a member of immunoglobulin (Ig) (Ig) superfamily, and is positioned at the tight junction of epithelial cell and endotheliocyte.Hyperstructure studies show that JAM-1 is limited in comprising the diaphragm area of closed albumen and close proteic protofibril (fibrils).

Another JAM family member is called " vascular endothelial cell connection-associated molecule " (VE-JAM), comprises two extracellular immunoglobulin like domain, a membrane spaning domain, relative short tenuigenin afterbody with.VE-JAM mainly is positioned at the cell inner boundary (Palmeri, etc., J.Biol.Chem. (journal of biological chemistry) 275:19139-19145,2000) of endotheliocyte.VE-JAM is expressed by venular endotheliocyte height, and is expressed by the endotheliocyte of other blood vessel.The JAM family member of another report, JMA-3 has the aminoacid sequence of prediction, and it shows 36% and 32% homogeny respectively with JAM-2 and JAM-1.JAM-3 shows tissue expression widely, and obviously higher level is arranged in kidney, brain and placenta.On cell levels, the JAM-3 transcript is expressed in endotheliocyte.Reported that JAM-3 and JAM-2 are binding partners.Especially, report JAM-3 ectodomain is in conjunction with JAM2-Fc.JAM-3 albumen raises on peripheral blood lymphocyte after activating.(Pia Arrate, etc., J.Biol.Chem. (journal of biological chemistry) 276:45826-45832,2001).

The film adhesion protein of striding of the another kind of participation epithelial cell tight junction modulating of proposing is closed albumen.Closed albumen is the II type transmembrane protein of a kind of about 65-kD, and it holds the cytoplasmic structure territory to form that (Furuse is etc., J.Cell.Biol. (cytobiology magazine) 123:1777-1788 (1993) by 4 membrane spaning domains, 2 born of the same parents' outer shrouds and big C-; Furuse, etc., J.Cell.Biol. (cytobiology magazine) 127:1617-1626,1994).When observing by immunity-freeze-fracturing Electronic Speculum (immuno-freezefracture electron microscopy), closed albumen directly concentrates on (Fujimoto in the tight connection protofibril, J.Cell Sci. (cell science magazine) 108:3443-3449,1995).

Other two kinds closely connect the conformity membrane albumen, and close albumen-1 and close albumen-2 are by identifying (Furuse, etc., J.Cell.Biol. (cytobiology magazine) 141:1539-1550,1998) from the rich film that connects of the direct biochemical fractional separation of chicken gizzard.Find close albumen-1 and close albumen-2 and closed albumen co-purify, as the wide gel band of an about 22-kD on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.From two of mouse cDNA library clone very relevant proteic sequence predictions of inferring 4 transbilayer helixs are arranged, the extracellular loop of 2 weak points and short tenuigenin N-and C-end.Although similar to closed albumen topology, they do not enjoy sequence homology.Afterwards, 6 other close protein gene products (close protein-3 is to close albumen-8) have been cloned, and show and be positioned at tight connection protofibril, (Morita as determined by immune golden freeze-fracturing mark, Deng, Proc.Natl.Acad.Sci.USA (Proc. Natl. Acad. Sci.USA) 96:511-516,1999).Suppose to keep barrier when not having closed albumen, so close albumen-1 is regarded as the material standed for of the main sealing-formation element of extracellular space to close albumen-8.

Other cytoplasm protein that is positioned at the epithelial cell junction comprises even albumen, couple albumen (symplekin), band albumen and 7H6.Connecting albumen is reported as and closed proteic tenuigenin afterbody bonded cytoplasm protein.What represent this protein family is " ZO-1, ZO-2, and ZO-3 ".The company's of inferring albumen is people's albumen analogue of the tight connection toxin (ZOT) in Asiatic cholera vibrios (Vibrio cholerae) source.

Connecting albumen may be at growth, physiology and pathological process--comprise that tissue morphology takes place, the motion between an enteric cavity and a matter of fluid, macromole and white corpuscle, and work in the tight junction modulating in the disorderly process of inflammatory/autoimmunity (referring to, for example, Wang, Deng, J.Cell.Sci. (cell science magazine) 113:4435-40,2000; Fasano, etc., Lancet (lancet) 355:1518-9,2000; Fasano, Ann.N.Y.Acad.Sci. (NYAS's annual) 915:214-222,2000).In the acute phase of celiaca, connecting protein expression increases in intestinal tissue, and described celiaca is a kind of clinical patient's condition that closely connection is opened and perviousness increases.In external non-human primates intestinal epithelial cells, connecting protein induced tight connection disintegration and intestines perviousness subsequently increases.

Connect amino acid in the albumen active Asiatic cholera vibrios (V.cholerae) ZOT fragment and people and relatively identified the receptors bind structural domain of inferring in these two kinds of albumen n end zones.ZOT biologically active structure territory increase intestines perviousness, this activates the intracellular signal conduction subsequently by interacting with mammalian cellular receptor, causes the interior closely connection of born of the same parents to disintegrate and carry out.Locate to described proteic carboxyl terminal in described ZOT biologically active structure territory, and consistent with the split product of the prediction that is produced by the Asiatic cholera vibrios.This structural domain and company's albumen, the similar thing of ZOT Mammals, sharing has the acceptor-binding motif of inferring.The ZOT active fragments and connect between the albumen amino acid relatively, combine with the gene site-directed mutagenesis experiment, show facing to the aminoterminal of the ZOT of processing and connect proteic N-terminal octapeptide acceptor-binding domains.(Di Pierro, etc., J.Biol.Chem. (journal of biological chemistry) 276:19160-19165,2001).Report ZO-1 is in conjunction with Actin muscle, AF-6, ZO-associated kinase (ZAK), fodrin, and α-Lian albumen.

Be used for perviousness peptide of the present invention and comprise peptide natural or synthetic, treatment or prophylactic activity (comprising two or more covalently bound amino acid), albumen, peptide or protein fragments, peptide or albumen analogue, peptide or albumen stand-in and bioactive peptide or proteic chemically modified derivative or salt.Therefore, when being used for this paper, term " perviousness peptide " is intended to comprise all these reactive species usually, that is, and and peptide and albumen, peptide and protein fragments, peptide and albumen analogue, peptide and albumen stand-in and bioactive peptide or proteic chemically modified derivative and salt.Usually, perviousness peptide or albumen are to be easy to replace, add or delete the amino acid in naturally occurring or natural (for example, wild-type, naturally occurring mutant or allele variant) peptide or the protein sequence and the mutain that obtains by part.In addition, comprise native peptides or proteic bioactive fragment.The derivative of described sudden change and fragment keep the biological activity of native peptides or proteic needs basically.In peptide with carbohydrate chain or proteic situation, the biological activity variant of mark is also included within the present invention by the change in these carbohydrate species.

The perviousness peptide, albumen, analogue and the stand-in that are used for method and composition of the present invention are formulated in pharmaceutical composition usually, described pharmaceutical composition comprises perviousness peptide, albumen, analogue or the stand-in of mucosal delivery-enhancing or infiltration significant quantity, and it reversibly strengthens the transhipment of mucosal epithelium parietal cell by regulation and control epithelial cell syndeton and/or physiology in mammalian subject.

Biologically active agent

Method and composition of the present invention is at the mucous membrane such as the intranasal delivery that increase the broad-spectrum biological activity agent, to obtain treatment, prevention or other ideal physiology result in mammalian subject.When being used for the application, term " biologically active agent " comprises any material that produces physiological response when the method and composition mucous membrane according to this paper is administered to mammalian subject.In this case, useful biologically active agent is included in treatment or the prevention reagent of using in clinical medical all main fields, and nutrition, cofactor, enzyme (endogenous or external source), antioxidant or the like.Therefore, described biologically active agent can be water miscible or water-insoluble, and can comprise more high-molecular weight albumen, peptide, carbohydrate, glycoprotein, lipid and/or glycolipid, nucleosides, polynucleotide and other promoting agent.

Useful medicament comprises medicine and macromole treatment or the preventive that contains the wide spectrum compound in method and composition of the present invention, and it comprises small-molecule drug, peptide, albumen and vaccine reagent.It is bioactive being used for representative medicaments of the present invention, the disease or the patient's condition that are used for the treatment of or prevent to select among the experimenter.In this case, biological activity can be defined as about physiological parameter, mark or the clinical symptom relevant with the experimenter's disease or the patient's condition any significant (promptly, detectable, remarkable satisfactorily) effect, it comprises that by detection system in the suitable external or body actual patient, cell cultures, sample detection or acceptable animal model assess.

Method and composition of the present invention provides unexpected benefit for disease and other patient's condition in the treatment mammalian subject, for example, described benefit is by providing treatment and preventing speed, time length, fidelity or the control of the raising of compound mucosal delivery to regulate and control, (for example to arrive the physiological compartment selected among the experimenter, enter or pass nasal mucosa, enter systemic circulation or central nervous system (CNS), perhaps arrive target spot organ, tissue, fluid or cell or the extracellular compartment of any selection in the experimenter).

In various representative embodiment, method and composition of the present invention can be in conjunction with being selected from one or more following biologically active agents:

Opioid (opiods) or opioid antagonists, such as morphine, hydromorphone, oxymorphone, Lip river cut down coffee promise (lovorphanol), levallorphan, morphine monomethyl ether, Nalmefene, nalorphine, naloxone (nalozone), TREXUPONT; buprenorphine, butorphanol, and nalbuphine (nalbufine);

Kendall compound, such as cortisone, hydrocortisone, fluohydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone (dexamethoasone), Betamethasone Valerate (betamethoasone), paramethasone (paramethosone), and fluocinolone acetonide;

Other antiphlogiston, such as colchicine, Ibuprofen BP/EP, indomethacin, and piroxicam; Antiviral agent is such as acyclovir, ribavirin (ribavarin), trifluorothymidine (trifluorothyridine), Ara-A (ARA-A), acyl group guanosine, Nordeoxyguanosine (nordeoxyguanosine), Zidovodine, DIDEOXYADENOSINE, and zalcitabine; Antiandrogen such as spironolactone;

Male sex hormone is as testosterone;

Oestrogenic hormon is as estradiol;

Progesterone;

Muscle relaxant is as Papaverine;

Vasodilator, as pannonit, the gene peptide that vasoactive intestinal peptide is relevant with thyrocalcitonin;

Antihistaminic agent is as Cyproheptadine;

Reagent with Histamine Receptors site blocking activity, as doxepin, imipramine, and Cimitidine Type A/AB;

Antitussive is as Dextromethorphane Hbr; Neuroleptic such as Clozaril; Anti-arrhythmic;

Antiepileptic drug;

Enzyme is such as superoxide dismutase and neural enkephalinase (neuroenkephalinase);

Anti-mycotic agent, such as amphotericin B, grisovin, miconazole, KETOKONAZOL, tioconazole, itraconazole, and fluconazole;

Antibacterial agent, such as penicillin, cynnematin, tsiklomitsin, aminoglycoside, erythromycin (erythromicin), gentamicin, PXB;

Carcinostatic agent, such as 5 FU 5 fluorouracil, bleomycin, Rheumatrex, and hydroxyurea, didanosine, floxuridine, Ismipur, Dx, daunorubicin, idarubicin (I-darubicin), safe element and taxol;

Antioxidant, such as vitamin-E, retinoids, carotenoid, ubiquinone, metal-chelate mixture, and phytic acid;

Anti-arrhythmic is such as Quinidine; With

Antihypertensive drug such as Prazosin, verapamil, nifedipine, and diltiazem Anodyne such as paracetamol and acetylsalicylic acid;

Mono-clonal and polyclonal antibody comprise humanized antibody and antibody fragment;

Antisense oligonucleotide; With

RNA, DNA and the virus vector that comprises coding treatment peptide and proteic gene.

Except these representational promoting agent kind and classification, method and composition of the present invention comprises above or any physiological agents of other local described or this area of this paper known in addition, and the combination of any multiple actives, its in method and composition of the present invention, be effective to alone or in combination selected disease or the patient's condition in treatment or the prevention mammalian subject (referring to, Physicians ' DeskReference (reference on doctor's table), by Li Tong Industrial Co., Ltd (Litton Industries, medical economics company of branch office Inc) (Medical Economics Company) publishes).

No matter the kind of used compound, be used for biologically active agent of the present invention and should be present in the compositions and methods of the invention with sufficient amount, described amount is enough to provide for the experimenter physiological action of needs, and does not have significant, unacceptable toxicity or other disadvantageous side effect.Those skilled in the art should determine the suitable dosage level of all biologically active agents easily, and need not undo experimentation.Because method and composition of the present invention provides the sending of biologically active agent of raising, so can successfully use the dosage level that significantly is lower than the routine dose level.Usually, described active substance will be with about 0.01 weight % of preparation in total nose to about 50 weight %, the amount of the about 20 weight % of about 0.1 weight %-and general about 1.0 weight %-5 weight % or 10 weight % be present in the composition usually, and this depends on used concrete material.

When being used for this paper, term biological activity " peptide " and " albumen " comprise the polypeptide of all size, and do not limit the invention to the aminoacid polymers of any concrete size.The present invention includes from little be the peptide of several amino acid to length, albumen to any size, and peptide-peptide, protein-protein syzygy and protein-peptide syzygy, as long as described albumen or peptide in the situation that excites special physiology, immunity, treatment or prophylactic action or reaction be have bioactive.

The invention provides the preparation and the collaborative application process of the novelty that is used to improve biologically active peptides and proteic mucosal delivery.Being used for treatment peptide of the present invention and proteic representative example includes, but are not limited to: organize profibr(in)olysin activation factor (TPA), Urogastron (EGF), fibroblast growth factor (the acid or alkalescence of FGF-), Thr6 PDGF BB (PDGF), transforming growth factor (TGF-α or β), vasoactive intestinal peptide, tumour necrosis factor (TNF), hypothalamic releasing factor, prolactin antagonist, thyrotropic hormone (TSH), thyroliberin (ACTH), Rat parathyroid hormone 1-34 (PTH), follicle stimulating hormone (FSF), gonadotropin releasing hormone (LHRH), endorphin, hyperglycemic-glycogenolytic factor, thyrocalcitonin, pitocin, carbetocin, aldoetecone, enkephalin (enkaphalins), somatostin, tethelin, somatomedin, gonad-stimulating hormone, oestrogenic hormon, progesterone, testosterone, α-melanotropin, the opioid that non-natural exists, lignocaine, Ketoprofen, sufentanil, terbutaline, droperidol, Scopolamine, gonadorelin, ciclopirox, buspirone, thyrocalcitonin, sodium cromoglycate or midazolam, ciclosporin, lisinopril, captopril, delapril, Cimitidine Type A/AB, Ranitidine HCL, famotidine, superoxide dismutase, asparaginase, arginase, the arginine desaminase, adenosine deaminase rnase, trypsinase, and papoid chemistry trypsin chemotrypsin).The example of the peptide that other is useful includes, but not limited to bombesin, P material, beta-hypophamine, alpha-globulin, transferrin, factor I, beta lipoprotein, betaglobulin, prothrombin, ceruloplasmin, α ₂-glycoprotein, α ₂-sphaeroprotein, Pp63 glycophosphoproteins, α ₁-lipoprotein, α ₁-sphaeroprotein, albumin, prealbumin, and other biological activated protein and recombinant protein product.

In aspect the present invention is more detailed, be provided for improving the method and composition of the mucosal delivery of special, biologically active peptides or protein for treatment agent, to treat the existing disease or the patient's condition (promptly, eliminate or reduce the seriousness of its generation or symptom), the perhaps outbreak of the preventing disease or the patient's condition among the experimenter in being accredited as the danger that is in the described disease or the patient's condition.The biologically active peptides and the albumen that are used for these aspects of the present invention include, but not limited to hematopoietic; Anti-infective; Dementia resisting medicine (antidementia); Antiviral agent; Antitumour drug; Febrifugee; Anodyne; Antiphlogistic drug; Anti-ulcerative drug; Anti-allergic agent; Thymoleptic; Antipsychotic medicine; Cardiotonic drug; Anti-arrhythmic; Vasodilator; Antihypertensive drug such as hypotensor diuretic(s); Antidiabetic drug; Anti-coagulant; Pravastatin; The therapeutical agent that is used for osteoporosis; Hormone; Microbiotic; Vaccine; Deng.

The biologically active peptides and the albumen that are used for these aspects of the present invention include, but not limited to cytokine; Peptide hormone; Somatomedin; Act on the factor on the cardiovascular systems; The cell adhesion factor; Act on the factor on maincenter and the peripheral nervous system; Act on the factor on body fluid ionogen and the blood organic substance; Act on bone and bone growth or the physiological factor; Act on the factor on the gastro-intestinal system; Act on the factor on kidney and the urinary organ; Act on the factor on reticular tissue and the skin; Act on the factor on the sensory organ; Act on the factor on the immunity system; Act on the factor on the respiratory system; Act on the factor on the reproductive organ; With various enzymes.

For example, the hormone that can use in method and composition of the present invention comprises male sex hormone, oestrogenic hormon, prostaglandin(PG), tethelin, gonad-stimulating hormone, interleukin, steroid and cytokine.

The vaccine that can use in method and composition of the present invention comprises bacterium and virus vaccines, such as the vaccine that is used for hepatitis, influenza, respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, canary pox (canary pox), varicella (chicken pox), measles, mumps, rubella, pneumonia and Human Immunodeficiency Virus (HIV).

The bacterial toxoid that can use in method and composition of the present invention comprises diphtheria, tetanus, pseudomonas (pseudonomas) and mycobacterium tuberculosis (mycobactrium tuberculosis).

Be used for special example cardiovascular or thrombus dissolving reagent of the present invention and comprise r-hirudin (hirugen), hirulos and leech element (hirudine).

The antibody reagent of effectively using with the present invention comprises monoclonal antibody, polyclonal antibody, humanized antibody, antibody fragment, syzygy and polymer and immunoglobulin (Ig).

When being used for this paper, term " conserved amino acid replacement " is meant the common interchangeability of the amino-acid residue with similar side chain.For example, interchangeable amino acid group with aliphatic lateral chain is L-Ala, Xie Ansuan, leucine and Isoleucine usually; Amino acid group with aliphatic series-hydroxyl side chain is Serine and Threonine; Amino acid group with the side chain that contains acid amides is l-asparagine and glutamine; Amino acid group with aromatic side chains is phenylalanine, tyrosine and tryptophane; Amino acid group with basic side chain is Methionin, arginine and Histidine; With the amino acid group with the side chain that contains sulphur be halfcystine and methionine(Met).The conservative example that replaces comprises nonpolar (hydrophobicity) residue such as the replacement each other of Isoleucine, Xie Ansuan, leucine or methionine(Met).Similarly, the present invention considers the replacement of polarity (wetting ability) residue, such as the replacement between arginine and the Methionin, and the replacement between glutamine and the l-asparagine, and the replacement between Threonine and the Serine.In addition, also consider replacement or acidic residues such as aspartic acid or the L-glutamic acid replacement each other each other of alkaline residue such as Methionin, arginine or Histidine.Representational conserved amino acid replacement group is: Val-Leu-Isoleucine, phenylalanine-tyrosine, Methionin-arginine, L-Ala-Xie Ansuan, and l-asparagine-glutamine.

Term biologically active peptides or albumen analogue also comprise the form of native peptides or proteic modification, comprise 20 kinds of amino acid whose steric isomers of routine (for example, D-amino acid), or non-natural amino acid, as α, α-disubstituted amino acid, N-alkyl amino acid, lactic acid.These and other unconventional amino acid can also replace or be inserted in the present invention used native peptides and albumen.Unconventional amino acid whose example comprises: 4-oxyproline, Gla, ε-N; N, N-trimethyl lysine, ε-N-ethanoyl Methionin; the O-phosphoserine; N-ethanoyl Serine, N-formylmethionine, 3-Methyl histidine; the 5-oxylysine; ω-N-methylarginine, and other similar amino acid and imino-acid (for example, 4-oxyproline).In addition, biologically active peptides or albumen analogue comprise single or polysubstituted, the deletion of carbohydrate, lipid and/or protein part and/or add, described carbohydrate, lipid and/or protein part are as described peptide or natural existence of proteic constituent or artificial the existence, perhaps with described peptide or protein binding or association in addition.

In one aspect, be used for peptide of the present invention (comprising polypeptide) by modifying with the one or more naturally occurring side chain of the amino acid (or D amino acid) of 20 kinds of genetic codings of other side substitution, for example, use such as alkyl, low alkyl group, ring 4-, 5-, 6-, to 7-unit alkyl, acid amides, acid amides low alkyl group, acid amides two (low alkyl group), lower alkoxy, hydroxyl, carboxyl and lower member ester derivative thereof with have 4-, 5-, 6-, to 7-unit heterocyclic group, to produce peptide mimics.For example, can prepare proline analogs, the wherein ring size of proline residue variation from 5 yuan to 4,6 or 7 yuan.Cyclic group can be saturated or unsaturated, and if undersaturated, can be aromatic base or non-aromatic base.Heterocyclic radical can comprise one or more nitrogen, oxygen and/or sulfur heteroatom.Such examples of groups comprises furazan base (furazanyl), furyl, imidazolidyl, imidazolyl, imidazolinyl, isothiazolyl ， isoxazolyl, morpholinyl is (for example, morpholino) ， oxazolyl, piperazinyl (for example, 1-piperazinyl), piperidyl (for example, piperidino, piperidino-(1-position only)), pyranyl, pyrazinyl, pyrazolidyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolidyl are (for example, the 1-pyrrolidyl), pyrrolinyl, pyrryl, thiadiazolyl group (thiadiazolyl), thiazolyl, thienyl, thio-morpholinyl (for example, thiomorpholine generation), and triazolyl.These heterocyclic radicals can be substituted or not be substituted.In the substituted situation of group, substituting group can be alkyl, alkoxyl group, halogen, oxygen or replacement or unsubstituted phenyl.

Peptide and albumen, and peptide and albumen analogue and stand-in can also with one or more covalent attachment in many non-protein polymers such as polyoxyethylene glycol, polypropylene glycol or the polyoxyalkylene (polyoxyalkenes), in conjunction with in U.S. Patent number 4,640,835; U.S. Patent number 4,496,689; U.S. Patent number 4,301,144; U.S. Patent number 4,670,417; U.S. Patent number 4,791,192; Or the mode that proposes in the U.S. Patent number 4,179,337 is carried out.

Other peptide in the present invention and albumen analogue and stand-in comprise the glycosylation variant and with the covalency of other chemical part or assemble conjugate.Covalence derivative can be by means commonly known in the art with functionality be arranged in amino acid side chain or be positioned at N-or the group of C-end is connected and prepares.These derivatives can comprise; but be not limited to; carboxyl terminal or contain the aliphatic ester or the acid amides of the residue of carboxylic side-chain, contain hydroxyl residue O-acyl derivative and aminoterminal amino acid or contain amino residue such as Methionin or arginic N-acyl derivative.Acyl group is selected from the group of the alkyl-part that comprises the normal alkyl of C3-C18, forms alkyloyl aroyl kind thus.Can also use and the carrier proteins covalent attachment of immunogenicity part for example.

Except these are modified, can carry out biologically active peptides and proteic glycosylation and change, for example, undertaken by the glycosylation pattern of modified peptides in and the course of processing synthetic or in other procedure of processing at it.The particularly preferred mode of finishing this modification is, by described peptide being exposed to the glycosylase of the cell that derives from the processing that normally provides such, for example, Mammals glycosylase and carrying out.Also successfully use deglycosylating enzyme, to produce the peptide and the albumen of modification useful in the present invention.The form that also comprises natural one-level aminoacid sequence, it has other less modification, comprises the amino-acid residue of phosphorylation, and for example, Tyrosine O-phosphate, phosphoserine or phosphothreonine, or other parts comprise ribosyl or cross-linking reagent.

Peptide mimics can also have by phosphorylation, sulfonation, biotinylation or adding or remove other parts, particularly has those parts of the shape of molecule similar to phosphoric acid fat (or salt) base and the amino-acid residue of chemically modified.

Can cyclisation be used for bioactive peptide of the present invention, perhaps at the end of described peptide in conjunction with deaminizating or decarboxylation residue so that do not have terminal amino group or carboxyl, to reduce susceptibility, perhaps to limit the conformation of described peptide to proteolytic enzyme.C in peptide analogs of the present invention and stand-in end functional group comprise acid amides, acid amides low alkyl group, acid amides two (low alkyl group), alkoxyl group, hydroxyl and carboxyl such as low and its lower member ester derivative, with and pharmaceutical salts.

The invention provides various additives, thinner, matrix (base) and send vehicle, it effectively controls water-content to improve protein stability.Effectively these reagent and the carrier substance as anti--aggregating agent prepared therefrom comprises on this meaning, for example, the polymkeric substance of various functionality, such as polyoxyethylene glycol, dextran, diethylamino ethyl dextran and carboxymethyl cellulose, it increases significantly and mixes with it or connected peptide and proteic stability and reduce solid phase and assemble.In some instances, proteic activity or physical stability can also strengthen by the various additives at the aqueous solution of peptide or protein drug.For example, can use additive, such as polyvalent alcohol (comprising sugar), amino acid, albumen, such as collagen protein and gelatin, and various salt.

Some additive, particularly sugar and other polyvalent alcohol are returned exsiccant such as freeze dried albumen is given significant physical stability.These additives can also be used for the present invention so that not only at freeze-drying process, also in the process with the drying regime storage, prevent protein aggregation.For example, in the solid phase incubation process under various conditions, the provide protection that sucrose and Fei Ke 70 (polymkeric substance with sucrose unit) show significant anti-peptide or protein aggregation.These additives can also improve the stability that is embedded in the solid protein in the polymeric matrix.

Yet other additive, for example sucrose is assembled at the anti-solid of temperature stabilize proteins in moist atmosphere that raises-state, as what can take place in the preparation of some slow constant release of the present invention.Albumen such as gelatin and collagen protein also act as stablizer or weighting agent to reduce unsettled in this case proteic sex change and gathering.These additives can be incorporated in the polymer melted course of processing and composition of the present invention in.For example, can prepare polypeptide particle by the simple freeze-drying of solution or the spraying drying that will contain above-mentioned various stable additive.Can obtain not accumulative peptide and albumen thus in the slow constant release of the time phase that prolongs.

The invention provides various other prepared compositions and method, and specific additive preparation, its generation is used for mucosal delivery and is easy to accumulative peptide and proteic preparation, wherein said peptide or protein stabilized be pure basically, accumulative form not.Consideration is used for these method and formulations with the composition and the additive of certain limit.These anti-examples of assembling reagent are cyclodextrin (CDs) dimers that connect, and it is optionally in conjunction with the hydrophobic side chain of polypeptide.Have been found that these CD dimers are with the protein-bonded hydrophobic fragment of remarkable inhibition accumulative mode.About the described CD dimer that relates to and albumen the two, this inhibition is optionally.The selectivity of described protein aggregation is suppressed in the intranasal delivery method and composition of the present invention extra benefit is provided.Other reagent that is used for this situation comprises having variable geometric CD tripolymer and the tetramer (Breslow etc. that are subjected to blocking peptide and proteic accumulative joint control specifically, J.Am.Chem.Soc. (Journal of the American Chemical Society) 118:11678-11681,1996; Breslow etc., PNAS USA (NAS's journal) 94:11156-11158,1997).

Electric charge is modified and pH value control agent and method

(for example pass biologically active agent that hydrophobic mucous membrane barrier sends in order to improve to be used to improve, macromolecular drug, peptide or albumen) transport features, the present invention also is provided for technology and the reagent that the electric charge of selected biologically active agent or as herein described sending-toughener is modified.In this, macromolecular relevant perviousness is relevant with their partition ratio usually.The degree of ionization of molecule, it depends on the pK of described molecule _aWith the pH value on mucous membrane surface, also influence the perviousness of described molecule.Be used for the infiltration of the biologically active agent of mucosal delivery and permeate agent and distribute and to promote by the electric charge change of described promoting agent or permeate agent or charge distribution, for example, described electric charge changes or charge distribution passes through to change charged functional groups, by modifying the pH value of sending vehicle or solution of active agent delivery, perhaps by the coordination electric charge-or pH value-change agent and using of described promoting agent realize.

Sanitas

Between sanitas such as butylene-chlorohydrin, methyl p-hydroxybenzoate, propylparaben, Sodium Benzoate (0.5%), phenol, cresols, right-chloro--cresols, phenylethyl alcohol, phenylcarbinol, Phenylmercuric Acetate, phenylmercuric borate, Phenylmercurinitrate, Thiomersalate, Sorbic Acid, Benzethonium Chloride or benzylkonium chloride, can join in the preparation of the present invention, to suppress microorganism growth.

PH and buffering system

The pH value uses damping fluid to regulate as the system that comprises citric acid and Citrate trianion such as Trisodium Citrate usually.Other suitable buffering system comprises acetic acid and acetate system, succsinic acid and succinate system, oxysuccinic acid and malate system and glyconic acid and gluconate system.Alternatively, can use the buffering system that comprises mixing acid/salt system, such as acetic acid and sodium citrate systems, citric acid, sodium-acetate system and citric acid, Trisodium Citrate, Sodium Benzoate system.For any buffering system, can add other sour example hydrochloric acid, and other alkali such as sodium hydroxide, be used for the final pH value and regulate.

Be used to regulate epithelial cell syndeton and/or physiological other reagent

The tight connection of epithelial cell can not see through the molecule of the radius with about 15 dusts usually, unless handle with connect open connection physiology control agent substantially as stimulation provided by the present invention.Act as in method and composition of the present invention in " secondary " tight junction modulating composition of useful target of secondary physiology regulation and control, ZO1-ZO2 heterodimer mixture itself shows that be easy to be subjected to can be easily and change the physiology regulation and control of the infiltrative exogenous agent of parietal cell of mucosal epithelium cell effectively.A kind of such reagent of fully having been studied is called " closely connecting toxin " (ZOT) for the bacteriotoxin from the Asiatic cholera vibrios.Also referring to, WO 96/37196; U.S. Patent number 5,945,510; 5,948,629; 5,912,323; 5,864,014; 5,827,534; 5,665,389; With 5,908,825.Therefore, the reagent of ZOT and other regulation and control ZO1-ZO2 mixture can be used with one or more biologically active agent formulated in combination or coordination.

Prepare and use

Mucosal delivery preparation of the present invention comprise typically with one or more pharmaceutical carriers and randomly other therapeutic component combine biologically active agent to be administered.Compatible with other composition of described preparation and do not excite in the experimenter on the meaning of unacceptable deleterious effect, described carrier must be " medicinal ".Described carrier is that this paper is described hereinbefore, perhaps is that the technician of pharmaceutical field is known in addition.Ideally, described preparation should not comprise known described biologically active agent and uses inconsistent material such as enzyme or oxygenant therewith.Described preparation can be prepared by the known any method of pharmaceutical field.

The compositions and methods of the invention can be administered to the experimenter by many mucous membrane methods of application, and bag is by in the oral cavity, rectum, vagina, nose, in the lung or transdermal delivery, perhaps by local delivery to eye, ear, skin or other mucous membrane surface., and can disperse with Sprayable usually using according to composition of the present invention by the whole bag of tricks known to those skilled in the art as the aqueous solution of nose or lung's sprays.Be used to disperse vote as the liquid of nasal spray at U.S. Patent number 4,511, open in 069.Such preparation can be expediently by will according to composition dissolves of the present invention in water producing the aqueous solution, and make that described solution is aseptic and prepare.Described preparation may reside in the multi-dose container, for example, is present at U.S. Patent number 4,511, in 069 in the disclosed sealing distribution system.Other suitable nasal spray delivery system is in Transdermal SystemicMedication (transdermal system pharmacological agent), and Y.W.Chien compiles, Elsevier press, New York, 1985; With at U.S. Patent number 4,778, obtain in 810 describing.Other aerosol delivery form can comprise, for example, pressurized air-, jet-propelled-, ultrasonic-and piezoelectric type atomizer, it sends the biologically active agent that is dissolved or suspended in drug solvent such as water, ethanol or their mixture.

Nose of the present invention and lung's spray solution typically comprise medicine or medicine to be sent, randomly use the preparation of tensio-active agent such as nonionogenic tenside (for example, Tween-80) and one or more buffer reagents, stablizer or tonicity agents (tonicifiers).In some embodiments of the present invention, described nose spray solution also comprises propelling agent.The pH value of described nose spray solution randomly between about pH3.0 and 7.2, but when needs, adjust the pH value and make charged macromole kind (for example, treating albumen or peptide) send optimization with the state of unionization basically.Applied pharmaceutical solvents can also be weakly acidic aqueous buffer solution (pH value 3-6).Be used for the suitable buffer reagent of these compositions as indicated above or as this area in addition known to.Can add other composition, to improve or to keep chemical stability, described other composition comprises sanitas, tensio-active agent, dispersion agent or gas.Suitable preservatives include, but not limited to phenol, methyl p-hydroxybenzoate, p-Hydroxybenzoate ,-cresols, Thiomersalate, benzalkonium chloride (benzylalkonimum chloride) etc.Suitable tensio-active agent comprises, but is not limited to oleic acid, anhydrosorbitol trioleate, polysorbate, phosphatidylcholine, phosphatidylcholine (phosphotidyl cholines) and various long-chain triglyceride and phosphatide.Suitable dispersion agent includes, but not limited to ethylenediamine tetraacetic acid (EDTA) etc.Suitable gas includes, but not limited to nitrogen, helium, chlorofluorocarbon (CFCs), hydrogen fluorohydrocarbon (HFCs), carbonic acid gas, air etc.Suitable stablizer and tonicity agents (tonicifying agent) comprise sugar and other polyvalent alcohol, amino acid and organic and inorganic salt.

The liquid preparation capable of permeating skin can be used as that drops for example installs (installation) or as droplet (spraying) use.Spraying can produce by pump, atomizer or by described other method in this area.For pulmonary delivery, the liquid droplets that is used for going deep into pulmonary deposition shows the granular size that is suitable in the sedimentary minimum of pulmonary passages, usually approximately less than 10 μ m mass median equivalent air kinetic diameter (mass median equivalent aerodynamic diameter, MMEAD), usually approximately less than 5 μ m MMEAD, usually approximately less than about 2 μ m MMEAD.Send for nose, described liquid droplets granular size is usually approximately less than 1000 μ m MMEAD, usually less than 100 μ m MMEAD.

In alternative embodiment, the mucous membrane preparation is used as dry powder formulations, and it comprises with suitable granular size or the exsiccant within the suitable granular size scope biologically active agent of lyophilized form normally, is used for intranasal delivery.For pulmonary delivery, the powder particle that is used for going deep into pulmonary deposition shows and is suitable in the sedimentary smallest particles size of pulmonary passages, usually approximately less than 10 μ m mass median equivalent air kinetic diameters (MMEAD), usually approximately less than 5 μ m MMEAD, usually approximately less than about 2 μ m MMEAD.Send for nose, described powder particle size is usually approximately less than 1000 μ mMMEAD, usually less than 100 μ m MMEAD.Can suck powder in the nose in these magnitude range can be by productions such as various routine techniquess such as jet grinding, spraying drying, solvent deposition, supercutical fluid condensations.The dry powder of these suitable MMEAD can be administered to the patient by conventional Diskus (DPI), the breathing of described conventional Diskus dependent patient, when lung or snuffing fashionable, so that described powder is dispersed into aerosolized amount.Alternatively, described dry powder can be used by the air supplementary unit, and described air supplementary unit uses extra power, so that described powder is dispersed into aerosolized amount, for example uses piston pump to carry out.The drug powder particle can be mixed with the particle that is gathered into macrobead (＞100um MMEAD) in drying regime, and it comprises appropriate carriers, and such as lactose, wherein when distributing described powder, the aggregate of drug particles and carrier granule is destroyed.

The dry powder device typically needs the powder quality of about 1mg-20mg scope, to produce single aerosolized dosage (" aerosol spraying ").If the needs of biologically active agent or ideal dosage be lower than this amount, the powdered promoting agent will be typically filled (bulking) powder combinations with pharmaceutical drying so, so that the total powder quality that needs to be provided.Preferred dry powder filler comprises sucrose, lactose, dextrose, N.F,USP MANNITOL, glycine, trehalose, human serum albumin (HSA) and starch.Other suitable dry-packing powder comprise cellobiose, dextran, trisaccharide maltose, pectin, Trisodium Citrate, sodium ascorbate, or the like.

In order to prepare the composition that is used for mucosal delivery of the present invention, described biologically active agent can be with various medicinal additives and the matrix or the carrier combinations that are used to disperse described promoting agent.The ideal additive comprises, but is not limited to, and pH value control agent is such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid etc.In addition, (for example can comprise local anesthetic, phenylcarbinol), isotonic agent (for example, sodium-chlor, N.F,USP MANNITOL, Sorbitol Powder), absorption inhibitor (for example, tween 80), solubility enhancer (for example, cyclodextrin and derivative thereof), stablizer (for example, serum albumin) and reductive agent (for example, gsh).When the composition that is used for mucosal delivery is liquid, when detecting as tension force with reference to 0.9% (w/v) normal saline solution that is considered as unification, the tension force of preparation typically is adjusted to such value, promptly, at described tension value, in using the nasal mucosa in site, will can not induce basic, irreversible tissue injury.Usually, with the tension adjustment of solution to about 1/3 to 3, or 1/2 to 2, or 3/4 to 1.7 value.

Described biologically active agent can be dispersed in matrix or the vehicle, and it can comprise the hydrophilic compounds of the ability with the described promoting agent of dispersion and the additive of any needs.Described matrix can be selected from the appropriate carriers of wide region, include but not limited to, the multipolymer of poly carboxylic acid or its salt, carboxylic acid anhydride (for example, cis-1), with other monomer (for example, methyl (first) acrylate, vinylformic acid etc.) multipolymer, hydrophilic vinyl polymer such as polyvinylacetate, polyvinyl alcohol, polyvinylpyrrolidone, derivatived cellulose such as Walocel MT 20.000PV, hydroxypropylcellulose etc. and natural polymer such as chitosan, collagen protein, sodiun alginate, gelatin, hyaluronic acid and their non-toxic metal salt.Usually, select biodegradable polymkeric substance as matrix or carrier, for example, poly(lactic acid), poly-(lactic acid-ethanol) multipolymer, multi-hydroxybutyrate, poly-(hydroxybutyric acid-oxyacetic acid) multipolymer and their mixture.Alternatively or additionally, synthetic fatty acid ester such as polyglycerol fatty acid ester, sucrose fatty acid ester etc. can be used as carrier.Hydrophilic polymer and other carrier may be used singly or in combin, and can give the structural integrity that described carrier improves by partial crystallizationization, ionic bonding, crosslinked etc.Carrier can provide with various forms, comprises, and fluid or stickiness solution, gel, paste, powder, microsphere and film, they are directly used in nasal mucosa.In this case, use the carrier of selecting to cause promoting the absorption of biologically active agent.

Described biologically active agent can make up according to the whole bag of tricks with matrix or carrier, and the release of described promoting agent can be undertaken by diffusion, the disintegration of carrier or the association preparation of water channel.In some cases, described promoting agent be dispersed in by in suitable polymkeric substance the microcapsule (microsphere) or Nano capsule (nanometer spheroid) that for example the 2-isobutylcyanoacrylate is made (referring to, for example, Michael, Deng, J.Pharmacy Pharmacol. (pharmacy pharmacology magazine) 43:1-5,1991), and be dispersed in the biocompatibility dispersion medium that is used for nasal mucosa, it produces along with the slow constant of time that prolongs is sent and biological activity.

In order further to strengthen the mucosal delivery of medicament of the present invention, the preparation that comprises described promoting agent can also contain the wetting ability low-molecular weight compound as matrix or vehicle.Described wetting ability low-molecular weight compound provides channel media, by its water-soluble active agent as physiologically active peptide or albumen can by as described in matrix be diffused into absorb as described in the body surface of promoting agent.Described wetting ability low-molecular weight compound randomly absorbs from mucous membrane or uses the moisture of atmosphere, and dissolving water-soluble active peptide.The molecular weight of described wetting ability low-molecular weight compound is no more than 10000 usually, and preferably is no more than 3000.Representational wetting ability low-molecular weight compound comprises polyol compound, such as few-, two-and monose such as sucrose, N.F,USP MANNITOL, lactose, L-arabinose, D-erythrose, D-ribose, D-wood sugar, D-seminose, D-semi-lactosi, milk ketose, cellobiose, gentiobiose (gentibiose), glycerine and polyoxyethylene glycol.The example that is used as other wetting ability low-molecular weight compound of carrier in the present invention comprises N-Methyl pyrrolidone and alcohol (for example, few vinyl alcohol, ethanol, ethylene glycol, propylene glycol etc.).These wetting ability low-molecular weight compounds separately use or combination with one another or with described nose in other activity or the non-activity composition of preparation be used in combination.

Composition of the present invention can alternatively comprise the medicinal carrier substance of suitable physiological condition needs, such as pH value conditioning agent and buffer reagent, tension regulator, wetting agent etc., for example, sodium acetate, Sodium.alpha.-hydroxypropionate, sodium-chlor, Repone K, calcium chloride, Span 20, Emulphor FM etc.About solids composition, can use conventional nontoxicity pharmaceutical carrier, for example, it comprises pharmacy level N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, talcum, Mierocrystalline cellulose, glucose, sucrose, magnesiumcarbonate etc.

In certain embodiments of the invention, described biologically active agent is used in time release formulation, for example, uses in comprising the composition of slow release polymers.Described promoting agent can be with the preparing carriers that prevents snap-out release, and for example, the vehicle of sustained release is as the delivery system or the biological gel that binds of polymkeric substance, little encapsulation.The prolongation of described biologically active agent in various compositions of the present invention send can be by in described composition, comprising delayed absorption reagent for example monostearate aluminium hydrogel and gelatin cause.

When being used for this paper, term " experimenter " means any mammalian subject that can use composition of the present invention.

Test kit

The present invention also comprises test kit, packing and the many container units (unit) that contains aforementioned pharmaceutical compositions, activeconstituents, and/or being used to use its mode, these test kits, packing and many container units and/or method of application are used in mammalian subject prevention and treatment disease and other patient's condition.In brief, these test kits comprise container or the preparation that contains one or more biologically active agents that are formulated in the pharmaceutical preparation that is used for mucosal delivery.Described biologically active agent randomly is included in fills in distribution container (bulk dispensing container) or unit or the multiple-unit formulation.For example, lung or nose internal spraying medicator can provide optional distribution means.Wrapping material comprise that randomly the medicament that indication wherein packs can use by mucous membrane, and for example, the nose planted agent is in order to treatment or prevent the mark or the explanation of the concrete disease or the patient's condition.

Improve the polypeptide that polynucleotide are sent

In other embodiments of the present invention, select or appropriate design improves polypeptide that polynucleotide send to comprise amphipathic aminoacid sequence.For example, the polypeptide that can select useful enhancing polynucleotide to send, it comprises a plurality of nonpolar or hydrophobic amino acid residues that forms hydrophobic sequence structural domain or motif, is connected with a plurality of charged amino-acid residue that forms electrically charged sequence domains or motif, produces amphipathic peptide.

In other embodiments, select to comprise the polypeptide that the raising polynucleotide of nexin transduction domain or motif and fusogenic peptide structural domain or motif are sent.Nexin transduction domain is can insert and the peptide sequence by cell membrane transporter preferably.Fusogenic peptide is to make for example plasma membrane or around the unsettled peptide of film of endosome, it can be enhanced in low pH value of lipid film.Representational fusion structure territory or motif for example find in the fibroblast growth factor 4 (FGF4) at extensive multifarious virus amalgamation protein and other albumen.

For the polypeptide that appropriate design raising polynucleotide of the present invention are sent, nexin transduction domain is entered the motif of cell by plasma membrane as promotion nucleic acid.In certain embodiments, the nucleic acid of transhipment will be encapsulated in the endosome.The inside of endosome has low pH value, causes the fusogenic peptide motif to make the film instability of endosome.The instability of endosome film and destruction allow siNA to be discharged in the tenuigenin, and here siNA can associate and its target spot mRNA that can lead with the RISC mixture.

The example that is used for randomly being attached to the nexin transduction domain of the polypeptide that raising polynucleotide of the present invention send comprises:

1.TAT nexin transduction domain (PTD) (SEQ ID NO:1) KRRQRRR;

2.Penetratin PTD(SEQ ID NO：2)RQIKIWFQNRRMKWKK；

3.VP22 PTD(SEQ ID NO：3)DAATATRGRSAASRPTERPRAPARSASRPRRPVD；

4. Ka Boxi (Kaposi) FGF signal sequence (SEQ ID NO:4) AAVALLPAVLLALLAP and SEQ ID NO:5) AAVLLPVLLPVLLAAP;

5. people β 3 integrin signal sequences (SEQ ID NO:6) VTVLALGALAGVGVG;

6.gp41 fusion sequence (SEQ ID NO:7) GALFLGWLGAAGSTMGA;

7. caiman cayman (Caiman crocodylus) Ig (v) light chain (SEQ ID NO:8) MGLGLHLLVLAAALQGA;

8.hCT-peptide (the SEQ ID NO:9) LGTYTQDFNKFHTFPQTAIGVGAP in source;

9.Transportan(SEQ ID NO：10)GWTLNSAGYLLKINLKALAALAKKIL；

10.Loligomer(SEQ ID NO：11)TPPKKKRKVEDPKKKK；

11. arginine peptide (SEQ ID NO:12) RRRRRRR; With

12. amphipathic pattern peptide (SEQ ID NO:13) KLALKLALKALKAALKLA.

The example that is used for randomly being attached to the viral fusogenic peptide fusion structure territory of the polypeptide that raising polynucleotide of the present invention send comprises:

1. influenza HA 2 (SEQ ID NO:14) GLFGAIAGFIENGWEG;

2. Sendai virus (Sendai) F1 (SEQ ID NO:15) FFGAVIGTIALGVATA;

3. respiratory syncystial virus F 1 (SEQ ID NO:16) FLGFLLGVGSAIASGV;

4.HIV gp41 (SEQ ID NO:17) GVFVLGFLGFLATAGS; With

5. ebola virus (Ebola) GP2 (SEQ ID NO:18) GAAIGLAWIPYFGPAA.

Yet in other embodiments of the present invention, provide and improve the polypeptide that polynucleotide are sent, it is in conjunction with promoting the polypeptide-siNA mixture in the method and composition of the present invention to form and/or strengthen DNA-binding domains or the motif sent of siNAs.Representational in this case DNA binding domains comprises many " zinc refers to " structural domain, as about DNA-in conjunction with modulin with as described in other albumen of hereinafter identifying (referring to, for example, Simpson, Deng, J.Biol.Chem. (journal of biological chemistry) 278:28011-28018,2003).

Table 1:

The protein-bonded representative zinc-finger motif of different DNA-

^*This table shows the conservative zinc-finger motif of double-stranded DNA bonded, it is characterized in that C-x (2,4)-C-x (12)-H-x (3)-H motif pattern, itself can be used for selecting and designing the polypeptide of sending according to raising polynucleotide of the present invention.

^*In the table 1 about Sp1, Sp2, Sp3, Sp4, DrosBtd, DrosSp, the sequence shown in CeT22C8.5 and the Y4pB1A.4 is numbered SEQ ID NOs 19,20,21,22,23,24,25 and 26 respectively at this paper.

Be used to make up the alternative DNA binding domains that the present invention improves the polypeptide that polynucleotide send and comprise, for example, the part of HIV Tat protein sequence (referring to, embodiment, as follows).

In the following representative embodiment of the present invention of this paper, by any aforementioned structure element, structural domain or motif are combined in the single polypeptide, can reasonably design and make up and improve the polypeptide that polynucleotide are sent, send to the enhanced of target cell to regulate and control siNAs effectively.For example, the nexin transduction domain with the TAT polypeptide is fused in 20 amino acid of the proteic N-end of the influenza virus hemagglutinin that is called HA2, to produce the polypeptide that a kind of representational raising polynucleotide of the present invention are sent.The polypeptide construct that provides various other raising polynucleotide to send in the disclosure of invention, this shows that notion of the present invention is widely applicable for the various set that produce and be used to improve the polypeptide that effective raising polynucleotide that siNA sends send.

The polypeptide that other representational raising polynucleotide are sent in the present invention can be selected from following peptide: WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO:27); GKINLKALAALAKKIL (SEQ ID NO:28), RVIRVWFQNKRCKDKK (SEQID NO:29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO:30), GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO:31), poly-Lys-Trp, 4: 1, MW 20,000-50,000; With poly-Orn-Trp, 4: 1, MW 20,000-50,000.The polypeptide that other raising polynucleotide that are used for the compositions and methods of the invention are sent comprises all or part of of mentioned (mellitin) protein sequence.

Embodiment

The present invention is illustrated by following embodiment, and described embodiment is not limited in the scope of describing in the claim of the present invention.

Embodiment 1

Mucosal delivery-penetration kinetics and cytotoxicity

The organotypic model

Following method is generally used for assessing about mucosal delivery parameter, kinetics and the side effect of biologically active treatment agent with the perviousness peptide of the significant quantity that improves mucosal delivery, and the perviousness peptide of the significant quantity of described raising mucosal delivery reversibly improves the transhipment of mucosal epithelium parietal cell by epithelial cell syndeton and/or physiology in the regulation and control mammalian subject.

EpiAirway ^TM(ashland, MA) exploitation is as the interior model that is lining in the pseudostratified epithelium of respiratory tract by Ma Te Tyke (MatTek) company in system.Epithelial cell growth is that this causes cytodifferentiation to become the form of high degree of polarization on the cell cultures inset of bottom with the porous-film the liquid-gas interface place.End face has cilium, has the microvillus superstructure, and epithelium produce mucus (by immunoblotting verified mucinous existence).The diameter of inset is 0.875cm, and 0.6cm is provided ²Surface-area.Cell is layered on the inset in 3 weeks of precontract in transportation in factory.One " test kit " is made up of 24 unit.

A. when arriving, described unit is placed on the aseptic upholder in the 6 hole microtest plates.Proprietary (proprietary) substratum of 5mL is accepted in each hole.This substratum based on DMEM does not contain serum, but has replenished Urogastron and other factor.Detect always and think any cytokine that is used for intranasal delivery or the endogenous levels of somatomedin in the described substratum, but the factor that except that Regular Insulin, does not contain all cells factor and studied up to now.The volume of 5mL is enough to contacting bottom unit self is provided just, but allows the end face of epithelium to keep directly contacting with air.In this step and comprising in all subsequent steps of the unit being transferred in the hole of containing liquid and all use aseptic nipper, to guarantee between described unitary bottom and substratum, not having entrapped air.

B. will in incubator, in air, contain 5%CO in the unit in their flat board ₂Atmosphere in kept 24 hours at 37 ℃.In this time end, replace described substratum with fresh culture, and incubator is put back in described unit continue to keep 24 hours.

Experimental program--penetration kinetics

A.24EpiAirway ^TMUnitary " test kit " can conventional be used to assess 5 kinds of different preparations, and every kind of preparation all is applied in the quadruplicate hole.Each hole is used for determining penetration kinetics (4 time points), transepithelial electrical resistance (TER).Another group hole is with comparing, and they carry out the vacation processing in definite penetration kinetics process, but other the unit that contains specimen with being used for definite transepithelial cell impedance and survival ability is handled equally.

B. in all experiments, with mucosal delivery preparation to be studied with the volume applications of 100 μ L to each unitary end face, described volume is enough to cover whole top.Reserve the test formulation (the needed 100 μ L that are no more than usually) of the concentration that is applied to end face of proper volume, to determine the concentration of active substance subsequently by ELISA or other specified detection.

C. described unit is placed in the 6 hole flat boards of no pallet and is used for this experiment: the 0.9mL substratum is contained in every hole, and it is enough to contact with described unitary porous-film bottom, still described unit is not produced any fluid pressure that makes progress significantly.

D. minimize and avoid the formation of any concentration gradient for the potential source with error, each time point is under study for action transferred to another hole with described unit from a hole of containing 0.9mL.Be applied to the constantly zero of end face based on the test substances with 100 μ L volumes, these transfers are carried out at following time point: 15 minutes, and 30 minutes, 60 minutes and 120 minutes.

E. between time point, will remain in the unit in their flat board in 37 ℃ of incubators.The flat board that also every hole is contained the 0.9mL substratum remains in the incubator, so that in the minimum change of working as occurrence temperature in the short period of time of flat board being shifted out and with aseptic nipper the unit is transferred to from a hole another hole.

When F. each time point is finished, substratum is shifted out from shifting each unitary hole, and whole assigning in two pipes (managed for one and received 700 μ L, receive 200 μ L with another pipe), to be used for the concentration of definite test substances of permeating, and in described test substances is in the Cytotoxic situation, to be used for discharging tenuigenin enzyme, serum lactic dehydrogenase from epithelial cell.To in 24 hours, carry out if detect, so these samples be left in the refrigerator,, and, once detect up to thawing-80 ℃ of freezing preservations perhaps with the inferior whole branch of described sample.Avoid the circulation of multiple freeze-thaw.

G. for error minimize, before beginning to test with all pipes, flat board and hole mark in advance all.

H. when time point finished in 120 minutes, every hole is transferred to from the hole of containing 0.9mL at last in the unit contain the 24 hole microtest plates of 0.3mL substratum.This volume also is enough to the osculating element bottom, but can not apply fluid pressure upwards to described unit.Before detecting the impedance of transepithelial cell, described unit is put back in the incubator.

Experimental program--transepithelial electrical resistance

A. airway epithelial cell closely is connected with external formation in vivo, and limits solute thus and pass flowing of tissue.These are connected in the respiratory tissues of excision and give hundreds of ohm * cm ²Transepithelial electrical resistance.At Ma Te Tyke (MatTek) EpiAirway ^TMIn the unit, the manufacturer claims that transepithelial electrical resistance (TER) is usually at about 1000 ohm * cm ²This paper established data shows, the false contrast EpiAirway that exposes in the sequence of steps in penetration study ^TMLow slightly (700-800 ohm * the cm of unitary TER ²), still, because micromolecular infiltration and TER's is reciprocal proportional, so this value is still enough high, being enough to provides basic barrier for infiltration.On the contrary, not celliferous is that the unit at the end only provides minimum membrane resistance (the about 5-20 ohm * cm that strides with the porous-film ²).

The accurate mensuration of B.TER the electrode of ohmmeter need be placed on the film and film under significant surface-area on, and the distance from described film to electrode should be controlled reproducedly.Determine the method use from WorldPrecision Instruments by the TER that MatTek recommends and all experiments of the present invention are all used, Inc. (world's precision instrument company), Sarasota, " EVOM " of FL ^TMEpithelial cell voltohmmeter and " ENDOHM " ^TMTissue resistance measuring chamber (" ENDOHM " ^TMTissue resistance measurement chamber).

C. before measuring TER, described chamber is begun to be full of Dulbecco (Dulbecco ' s) phosphate-buffered saline (PBS) at least 20 minutes, with counter electrode.

D. with the 1.5mL PBS in the described chamber and measured be that 350 μ LPBS in the unit at the end measure TER with the film.Top electrodes is adjusted to just in time in the position on a unitary film top that does not contain cell (but containing 350 μ L PBS), fixing then, reproducibly place guaranteeing.Not celliferous unitary resistance typically is 5-20 ohm * cm ²(" background resistance ").

E. in a single day described chamber is ready to and has write down background resistance, and the 24 hole flat boards that will be used for penetration test so just now shift out from incubator, and is placed on separately and carries out TER in the described chamber and measure.

F. at first each unit is transferred in the culture dish that contains PBS, moistening to guarantee the film bottom.The aliquots containig of 350 μ L PBS is added in the described unit, suck carefully then in the pipe of mark with the rinse end face.Then described unit application 350 μ L PBS are washed once more, and PBS is drawn in the same collection tube.

G. placing described chamber (containing fresh 1.5mL PBS aliquots containig) before, with described unit outside surface gently trace remove too much PBS.Before top electrodes being positioned on the described chamber, 350 μ L PBS aliquots containigs are added in the described unit, and on the EVOM meter, read TER.

H. in the ENDOHM chamber, read after the described unitary TER, shift out described unit, sucking-off PBS and preservation, and described unit is put back into the 24 hole flat boards that the 0.3mL substratum is contained in every hole, have air interface at described unitary end face.

I. described unit reads in the following sequence: all false contrasts of handling are the sample of all preparation-processing then, then are the TER readings second time of each false contrast of handling.All TER value reportings are the function of organization table area.

TER is calculated as:

TER＝(R ₁-R _b)×A

R wherein _IBe resistance with inset of film, R _bBe the resistance of blank inset, and A is the area (0.6cm of film ²).Comprise strengthen intranasal delivery strengthen reagent for example the effect of the pharmaceutical preparation of perviousness peptide by passing EpiAirway ^TMThe TER of cytolemma (mucosal epithelium cellular layer) detects.The perviousness peptide is applied to EpiAirway with the concentration of 1.0mM ^TMCytolemma.The TER value is with respect to control value (contrast=about 1000 ohm-cm ²Be normalized into 100) the increase with the mucosal epithelium cell permeability of reducing that reduces to show cytolemma resistance.

Experimental program--LDH detects

Measure the necrocytosis amount by using CytoTox 96 cytotoxicity detection kit (Pu Luomaige (Promega) company, Madison, the state of Wisconsin) to detect from the loss of the serum lactic dehydrogenase (LDH) of cell.Sample on the 50 microlitre samples is detected on the flat board to 96 holes.Fresh not celliferous substratum is as blank.In each hole, add 50 μ l substrate solutions, and with flat board in the dark room temperature incubation 30 minutes.Behind the incubation, in each hole, add 50 μ l stop baths, and with described flat board on the optical density (OD) plate reader at the 490nm reading.

Experimental program--EIA method

The EIA test kit (p/n S-1178 (EIAH6101) available from laboratory, peninsula company (PeninsulaLaboratories Inc.BACHEM branch, San Carlos, California, 800-922-1516).17 * 120mm polypropylene conical tube (p/n 352097, Fa Ouken (Falcon), lake, Franklin (Franklin Lakes), New Jersey) is used for all samples preparation.It is quantitative that 8 kinds of standard substances are used for PTH.It is identical with the test kit inset that remaining detects step.

Embodiment 2

Strengthen the epithelium osmosis by PN159

The following embodiment of the present invention shows that osmosis of the present invention strengthens peptide, is example with PN159, strengthens the mucous membrane infiltration that the peptide medicine comprises PTH and peptide YY.This infiltration enhanced activity of the peptide of the present invention that is shown by PN159 may be equivalent to, perhaps greater than strengthening by the epithelium infiltration of using one or more small molecules penetration enhancers to obtain.

Peptide YY _3-36(PYY _3-36) be a kind of 34 amino acid whose peptides that become the theme of many clinical trials.The mucosal delivery of this biologically active peptides can be enhanced in the preparation that comprises the small molecules penetration enhancers.Therefore, this research assessment is whether the infiltration of the present invention of example strengthens peptide and can replace the small molecules penetration enhancers to promote the effect of the mucosal delivery of peptide YY with PN159.These researchs comprise that external assessment PN159 reduces the effect of the infiltration of transepithelial electrical resistance (TEER) and increase mark substance, and the proof relevant body interior research consistent with described in vitro results.

The combination of PN159 and PTH is described in the present embodiment.PTH can be that full-length peptide (1-84) or fragment are as (1-34).Described preparation can also be the combination of PTH, perviousness peptide and one or more other penetration enhancers.Described preparation can also comprise buffer reagent, tonicity agents, pH value conditioning agent and peptide/protein stabiliser such as amino acid, sugar or polyvalent alcohol, polymkeric substance and salt.

Design this research and make up the effect that PTH is permeated with assessment PN159 self or with other penetration enhancers.The PN159 concentration of assessment is 25,50 and 100 μ M.Other penetration enhancers is 45mg/ml M-β-CD, 1mg/ml DDPC and 1mg/ml EDTA.Sorbyl alcohol is adjusted to 220mOsm/kg as tonicity agents (146-190mM) with the osmolarity (osmolarity) with preparation.Preparation pH value is fixed on 4.5.Select PTH as model peptide in the present embodiment.2mg/ml PTH and PN159 and or not with the combination of other penetration enhancers.Use external epithelium model to detect described combination, to detect and the cytotoxicity of monitoring PT H infiltration, transepithelial electrical resistance (TER) and described preparation by LDH.

Transepithelial electrical resistance

The result who detects from the TER of this research shows to reduce greater than 80% TER and is caused by PN159.Observing higher TER with the PN159 concentration that increases reduces.The substratum that is applied to end face does not reduce TER, and the group that triton X handles shows significant TER minimizing as estimating.

Cytotoxicity

This research shows about the data of LDH, when handling cell with 25-100 μ M PN159, does not observe significant cytotoxicity.The substratum that is applied to end face does not show cytotoxicity, and the group that Triton X handles shows significant cytotoxicity as estimating.

Infiltration

About there being and not having the PTH of the PN159 of other toughener _1-34Permeation data shows in Fig. 1 and 2 respectively.When having PN159, observe the remarkable increase of PTH infiltration.25,50, and do not observe the significant difference that % permeates between the 100 μ M PN159.PN159 is suitable to the effect of the effect of PTH infiltration and 45/1/1mg/ml M-β-CD/DDPC/EDTA.Use 45/1/1mg/mlM-b-CD/DDPC/EDTA and PN159 combination to observe the extra increase of PTH infiltration.

Embodiment 3

PN159 equals or exceeds the small molecules infiltration to infiltration enhancement in the body of peptide hormone therapeutical agent to be strengthened The enhancement of agent

The male New Zealand rabbit of 20 3-6 monthly ages and heavy 2.1-3.0kg is divided into 5 treatment group at random, every group of 4 animals.With test animal with 15 μ l/kg and by the suction pipe intranasal administration.The composition of 5 kinds of different administration groups of following table 5 expressions.

For administration group 1 (seeing Table 2), use the PYY clinical preparation that comprises the small molecules penetration enhancers.Small molecules toughener in these researchs comprises methyl-beta-cyclodextrin, didecyl acyl phospholipids phatidylcholine (DDPC) and/or EDTA.Administration group 2 accepts to be dissolved in the PYY in the salt solution (PBS) of phosphoric acid buffer.For administration group 3-5, the PN159 of different concns is added in the administration group 2, so that each group among the administration group 3-5 to be by PYY, PN159 and PBS form.

Table 2

Group	Animal	Penetration enhancers	Administration concentration (mg/ml)	Administration volume (ml/kg)	PYY dosage (μ g/kg)
						1	4M	The small molecules penetration enhancers	13.67	0.015	205
2	4M	Do not have	13.67	0.015	205
						3	4M	25μM PN159	13.67	0.015	205
4	4M	50μM PN159	13.67	0.015	205
						5	4M	100μM PN159	13.67	0.015	205

Collect successive blood sample (every part of about 2ml) by directly carrying out direct venipuncture, collect and contain in the blood collection tube of EDTA as anti-coagulant at auricular vein.0,2.5,5,10,15,30,45,60 and 120 minutes collection blood samples after administration.Collect after the blood, pipe shakes several times gently, with anticoagulation, adds 50 μ l bovine pancreatic trypsin inhibitor solution then.About 4 ℃ with about 1,600xg centrifugal blood 15 minutes, and plasma sample is assigned in the duplicate aliquots containig, and in approximately-70 ℃ freezing preservation.

Average all 4 animals in a treatment group, detect the plasma concentration (table 3) of following PYY:

Table 3

	Group 1	Group 2	Group 3	Group 4	Group 5
						Time, minute	The small molecules penetration enhancers	No penetration enhancers	25μM PN159	50μM PN159	100μM PN159
0	183.825	257.3	228.675	424.4	294.225
						2.5	1280.7	242.8	526.375	749.975	1748.225
5	1449.425	273.675	1430.15	1293.4	3088.2

10	8251.8	372.05	6521.7	12517.2	14486.6
						15	13731.2	398.225	12563.075	34455.3	20882.725
30	19537.55	476.475	15222.6	35294.375	25470.475
						45	13036.075	340.7	9081.125	21582.225	16499.55
60	7080.875	283.825	4843.15	9461.925	10676.625
						120	1671.9	192.575	1224.2	2337.775	1891.275

Be presented at the following table 4 from the pharmacokinetic data of above-mentioned data computation:

Table 4

Compare with group 2 (no toughener) preparation, determine following relative enhancing ratio (table 5):

Table 5

Group	Preparation	Relative Cmax	Relative AUClast
1	The small molecules penetration enhancers	38x	27x
				3	PN159，25μm	30x	21x
4	PN159，50μm	79x	46x
				5	PN159，100μm	54x	38x

Aforementioned data is pictorialization in Fig. 3, and shows, compares with the small molecules penetration enhancers, is that the perviousness peptide of the present invention of example can will infiltration be strengthened to equal or bigger degree in the nose of people's hormone peptide therapeutics in vivo with PN159.Observe the maximum effect of described peptide in 50 μ M concentration.100 μ M concentration cause low slightly infiltration, although the two all causes the perviousness higher than small molecules penetration enhancers.

Embodiment 4

PN159 is to the infiltration enhancement of oligopeptides therapeutical agent

Present embodiment shows a kind of representative peptide of the present invention, and PN159 strengthens a kind of ring pentapeptide, melanocortin 4 receptor stimulants (MC-4RA), the effect that a kind of epithelium that is used for the pattern oligopeptides agonist of mammalian cellular receptor permeates.In this embodiment, the combination of one or more perviousness peptides and MC-4RA is described.Useful in this case preparation can comprise the combination of oligopeptides therapeutical agent, perviousness peptide and one or more other penetration enhancers.Described preparation can also contain buffer reagent, tonicity agents, pH value conditioning agent and peptide/protein stabiliser such as amino acid, sugar or polyvalent alcohol, polymkeric substance and salt.

Assessment PN159 is to the effect of MC-4RA infiltration in this research.MC-4RA be a kind of have about 1, the methane sulfonates of 100Da molecular weight, the activity of its regulation and control MC-4 acceptor.The PN159 concentration of assessment is 5,25,50 and 100 μ M.45mg/ml M-β-CD is as the stablizer of all preparations, to obtain the peptide concentration of 10mg/ml.Assessment PN159 self or with the effect of EDTA (1,2.5,5, or 10mg/ml) combination.The pH value of preparation is fixed on 4, and osmolarity is 220mOsm/kg.

The HPLC method

Analyze the concentration of MC-4RA in the basolateral part substratum by RP-HPLC, described RP-HPLC uses the C18RP chromatography, 25 ℃ of flow velocity 1mL/ minute and column temperatures.

Solvent orange 2 A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN

Volume injected: 50 μ L

Detect: 220nm

Working time: 15 minutes

MC-4RA and 5,25,50 and 100 μ M PN159 combination, and at pH value 4 and osmolarity～220mOsm/kg.Use external epithelium model to detect described combination, to detect and the cytotoxicity of monitoring PT H infiltration, transepithelial electrical resistance (TER) and described preparation by MTT and LDH.

The result of study of MC-4RA infiltration is presented among Fig. 4.These studies show that except the mucous membrane infiltration that strengthens the peptide hormone therapeutical agent, PN159 also significantly strengthens the epithelium infiltration of oligopeptides therapeutical agent.

Embodiment 5

PN159 is to the infiltration enhancement of small-molecule drug

Present embodiment shows a kind of representative peptide of the present invention, and the PN159 enhancing is the effect that the epithelium of the small-molecule drug of example permeates with acetylcholinesterase (ACE) inhibitor lycoremine.The combination of one or more perviousness peptides and small-molecule drug is described in the present embodiment.Useful in this case preparation can comprise the combination of small-molecule drug, perviousness peptide and one or more other penetration enhancers.Described preparation can also comprise buffer reagent, tonicity agents, pH value conditioning agent, stablizer and/or sanitas.

The present invention is with lycoremine and PN159 combination, to strengthen the infiltration that lycoremine passes nasal mucosa.Because lycoremine is a kind of small molecules that can see through nasal epithelial membranes independently, so the increase of this drug osmotic is unexpected.Therefore, the lycoremine that mediates by the vehicle that adds the infiltration of enhancing peptide is surprising by the remarkable enhancement that epithelium permeates, and this is based on, and not expecting described vehicle significantly increases the infiltration that lycoremine passes the epithelium layer usually.Therefore, the present invention will promote the nose of lycoremine and other small-molecule drug to send by the bioavailability that increases them.

In this research, the lycoremine of 40mg/ml lactic acid salt form and 25,50 and 100 μ MPN159 in solution, pH 5.0 and osmolarity～270mOsm combination.Use external epithelium model to detect described combination, to detect the cytotoxicity of monitoring lycoremine infiltration, transepithelial electrical resistance (TER) and described preparation by LDH mentioned above and MTT.The infiltration of lycoremine detects by standard HPLC analysis to be undertaken, as follows.

HPLC analyzes

Use no gradient LC (Waters Alliance (this associating of water)) method and UV and detect, determine in the preparation and the lycoremine concentration in substrate outside substratum (infiltration sample).

Post: this symmetrical posts of water (Waters Symmetry Shield), C18,5 μ m, 25 * 0.46cm

Moving phase: the 5%ACN in the 50mM ammonium formiate, pH 3.0

Flow velocity: 1ml/ minute

Column temperature: 30 ℃

Working curve: 0-400 μ g/ml bromination lycoremine

Detect: in the ultraviolet ray (UV) of 285nm

Based on aforementioned research, PN159 improves the micromolecular mucosal delivery of striding.Lycoremine is selected as the model low-molecular-weight drug, and thinks that the result of this a part is the active expection of perviousness peptide that is used for other small-molecule drug.In order to assess the perviousness activity in this situation, with the lycoremine of 40mg/ml lactic acid salt form and 25,50 and 100 μ M PN159 in solution, pH 5.0 and osmolarity～270mOsm combination.Use external epithelium model to detect described combination, to detect the cytotoxicity of monitoring lycoremine infiltration, transepithelial electrical resistance (TER) and described preparation by LDH and MTT.

In described vitro tissue model, add PN159 and cause medicine to pass the remarkable increase of the infiltration of barrier cell.Particularly, at the P of 40mg/ml lycoremine _AppIn have 2.5-3.5 increase doubly.(Fig. 5)

In the presence of lycoremine, PN159 reduces TER, described in example II.

In the presence of the lactic acid lycoremine and PN159 of all detectable levels, the cell survival ability keeps high (＞80%).On the contrary, as detecting by LDH, when having PN159 and lactic acid lycoremine, cytotoxicity is low.These detections show that all PN159 does not have toxicity to epithelial membrane.

Sum up The above results, this paper has shown that PN159 increases the epithelium infiltration as the lycoremine of model low-molecular-weight drug surprisingly.PN159 is joined the infiltration that remarkable enhancing lycoremine passes the epithelial cell individual layer in the lycoremine solution.Evidence shows that temporary transient minimizing of PN159 passes the TER of epithelial membrane, and does not damage the cell in the film, as measuring by high cell survival ability and low cytotoxicity.Therefore, PN159 is a kind of representative peptide that strengthens the bioavailability of lycoremine and other small-molecule drug in vivo, and it strengthens by the identical mechanism of using the external model proof in this article.Further predict PN159 and also strengthen the infiltration of lycoremine in higher concentration.

Chemical stability

Under the relevant condition of storage of treatment, determine the chemical stability of PN159.Use the HPLC method of expression stability.Solution (50mM) is kept under different pH value (4.0,7.3 and 9.0) and temperature (5 ℃, 25 ℃, 35 ℃, 40 ℃ and the 50 ℃) condition.Sample in pH value 4 contains the 10mM citrate buffer solution.Sample in pH value 7.3 and 9.0 contains the 10mM phosphoric acid buffer.Typical stability in storage data (comprising Arrhenius graphing method (Arrhenius plot)) are presented among Fig. 6.As can be seen, chemically be the most stable at low temperature and pH value PN159.For example, at 5 ℃ and pH 4.0 or pH7.3, for 6 months storage, existing basically, 100% PN159 recovered.When storage temperature is elevated to 25 ℃, after 6 months, there is 7% and 26% natural PN159 loss respectively for sample at pH 4 or pH 7.In the temperature of pH 9 and/or rising, for example, 40-50 ℃, ensue the quick consumption of PN159.4.0-7.3 pH value scope and freezing temperature range to envrionment temperature be maximally related for preparation in the nose.Therefore, these data support that PN159 can keep chemical integrity under the condition of storage relevant with the IN preparation.In the speed of medicine relative time infiltration, there be significant increasing.These data are used for calculating perviousness constant (P _App), be presented in the table 6.

Table 6

The P that uses the vitro tissue model to detect _App

^aPH is 5.0.

When not having PN159, the P of lycoremine _ApP is about 2.1 * 10 ^-6Cm/s.When existence 25,50 and 100mM PN159, P _AppDividing is 5.1 * 10 in addition ^-6, 6.2 * 10 ^-6And 7.2 * 10 ^-6Cm/s.Therefore, at the P of this pattern low-molecular-weight drug _AppIn, PN159 provides 2.4-to 3.4-increase doubly.

Having determined that PN159 is used for the purposes that low-molecular weight compound is striden the mucous membrane preparation, importantly is to distinguish whether these observations can be extrapolated to bigger molecule, for example therapeutic peptide and albumen.For this purpose,, the salmon calcitonin see calcimar as the mode treatment peptide is carried out vitro tissue research not having and exist 25,50 and during 100mM PN159.When not having PN159, for the P of thyrocalcitonin _AppBe about 1 * 10 ^-7Cm/s is lower than the P of lycoremine _AppAbout order of magnitude infers that this is because the difference of molecular weight causes.When having PN159, the remarkable increase of the bright thyrocalcitonin infiltration of data sheet is compared with the situation of having only thyrocalcitonin, at P _AppIn reach 23-and doubly increase (table 6) to 47-.

In order to study the ubiquity of these discoveries, when not existing and having PN159, two kinds of other peptides of check, i.e. human parathyroid hormone 1-34 (PTH in described external model _1-34) and people's peptide YY3-36 (PYY _3-36) (P _AppData presentation is in table 6).When not having PN159, the P of these two kinds of peptides _AppP with thyrocalcitonin _AppConsistent.At PTH _1-34Situation in, PN159 exists in P _AppIn about 3-5 is provided increase doubly.When in the presence of PN159, preparing PYY _3-36The time, Papp increases about 12-to 17-doubly.The ubiquity of our discovery of these data acknowledgements--PN159 has enhancing and strides the purposes that the mucous membrane medicine is sent--.

Embodiment 6

The D-amino acid form of PN159

The PN159 peptide of the D-aminoacid replacement that synthetic and purifying is listed in table 7, and use described in the above-described embodiments method to detect their to strengthen TER and infiltrative ability.

Table 7

The D-aminoacid replacement

Significant perviousness improves but PN407 shows less statistics.The PN159 of anti-(retro inverso) form of all D and regurgitation shows the TER that reduces and recovers, and this shows that the longer TER that can be used for sending in the body reduces effect.D replaces (PN434) and can reduce to strengthen with perviousness at TER and cause invalid activity on the two at random.

Embodiment 7

The PN159 length variations

The PN159 peptide synthetic and purifying is listed in table 8 with length variations, and use described in the above-described embodiments method to detect their enhancing TER and infiltrative abilities.

Table 8

Different sizes

Peptide	Sequence	Describe	TER(x)/ TER(159) +/-SEM ＊	Perm(x)/ Perm(159) +/-SEM *
					PN159	The NH2-KLALKLALKALKAALKLA-acid amides	The pattern peptide	1.00+/- 0.14	1.00+/- 0.13
PN417	NH2-KLALKLALKALKAA-acid amides (SEQ ID NO:39)	Foreshorten to 14 amino acid	0.19+/0.01	0.04+/- 0.01
					PN418	NH2-KLALKLALKALKAALK-acid amides (SEQ ID NO:40)	Foreshorten to 16 amino acid	1.05+/- 0.05	0.64+/- 0.08
PN419	NH2-KLALKLALKALKAALKLALK-acid amides (SEQ ID NO:41)	Extend to 20 amino acid	1.23+/- 0.01	0.74+/- 0.13
					PN420	NH2-KLALKLALKALKAALKLALKLA-acid amides (SEQ ID NO:42)	Extend to 22 amino acid	0.77+/- 0.05	0.24+/- 0.05
PN421	NH2-KLALKLALKALKAALKLALKLALK-acid amides (SEQ ID NO:43)	Extend to 24 amino acid	0.74+/- 0.11	0.17+/- 0.06
					PN422	NH2-KLALKLALKALKAALKLALKLALKAL-acid amides (SEQ ID NO:44)	Extend to 26 amino acid	0.47+/- 0.07	0.07+/- 0.01

^*From repeated mean value

The result shows that the length of PN159 is very important for its TER minimizing and enhanced permeability activity.PN159 is extended to 20 amino acid increase TER minimizing effects still reduce the perviousness effect.It is slower that TER recovers.PN159 is shortened to 16 amino acid TER is reduced not effect, but reduce the perviousness effect.PN159 is shortened to 14 amino acid significantly reduce perviousness, this length that shows PN159 is extremely important to perviousness.Opposite with the perviousness effect, PN159 length more relaxes the effect that TER reduces.

Embodiment 8

Tryptophane among the PN159 and arginine replace

The PN159 peptide synthetic and purifying is listed in table 9 with aminoacid replacement, and use described in the above-described embodiments method to detect their enhancing TER and infiltrative abilities.

Table 9

Aminoacid replacement

The result shows that arginine guanidine head group (headgroup) is more effective than Methionin and Histidine.Tryptophane is the preferred amino acid on water-membrane interface.But PN407 shows less statistics to perviousness to be improved significantly.Arginine replaces Methionin and significantly reduces perviousness, but minimizing has seldom influence to TER, and this shows that the importance of Methionin is perviousness.Eliminated perviousness at the single substituted lactamine of amino acid/11 0 usefulness l-asparagine, this shows that the α spiral is to the active importance of PN159.

Embodiment 9

Hydrophobicity among the PN159 changes

The PN159 peptide synthetic and purifying is listed in table 10 with aminoacid replacement, and use described in the above-described embodiments method to detect their enhancing TER and infiltrative abilities.

Table 10

Hydrophobic surface

Peptide	Sequence	Describe	TER(x)/TER(1 59)+/-SEM *	Perm(x)/Perm(1 59)+/-SEM *
					PN159	NH2-KLALKLALKALKAA LKLA-acid amides	The pattern peptide	1.00+/-0.14	1.00+/-0.13
PN424	NH2-KALKLKAALALLAK LKLA-acid amides (SEQ ID NO:53)	Non-amphipathic	0.59+/-0.07	0.20+/-0.04
					PN441	NH2-KLAAALLKKAKKLA AALL-acid amides (SEQ ID NO:54)	200 ° of hydrophobic surfaces	0.54+/-0.04	0.35+/-0.04
PN442	NH2-KALAALLKKAAKLL AALK-acid amides (SEQ ID NO:55)	180 ° of faces	0.93+/-0.03	0.81+/-0.03
					PN444	NH2-KALAALLKKLAKLL AALK-acid amides (SEQ ID NO:56)	180 ° of faces	0.82+/-0.05	0.41+/-0.08

^*From repeated mean value

PN159 has 280 ° hydrophobic surface.The result shows, the reducing of hydrophobic surface can cause that PN159 is active and reduce.The amphipathic activity for it of PN159 also is important.

In vitro method and scheme

Measure transepithelial electrical resistance (TER), TER recovery, cytotoxicity (LDH) and the sample infiltration (EIA) of each TJMP.Be used for the cell culture condition of every kind of detection and scheme explained in detail hereinafter.

Embodiment 10

In vitro method and scheme

Tight junction modulating peptide or TJMPs are such peptides, that is, described peptide can be with effect infringement (compromising) close-connected integrity that produces perforate between epithelial cell and the barrier function that therefore reduces epithelium.Can see through the resistance and the degree level of system of people's nasal epithelium organize models by test sample, and the state of vitro detection tight conjunction integrity.The minimizing of resistance and enhanced infiltration show that closely connection is compromised, and produces perforate between epithelial cell.Effectively, will induce the minimizing of passing tissue film's resistance of minimizing of detectable being called (TER), and promote small molecules to be categorized as TJMPs by the peptide of the enhanced infiltration of tissue film.In addition, also assess cytotoxicity level, whether can for example send that (IN) medicine act as tight junction modulating peptide in sending in the nose what medicine passed mucous membrane surface to determine these peptides about TJMPs.

The detection that is used to screen the representational peptide of the present invention (table 23 of reference example 25) is described in the present embodiment.These detections comprise transepithelial electrical resistance (TER), cytotoxicity (LDH) and sample infiltration.Present embodiment is also described used reagent and cell culture condition.

Table 11 illustrates the sample reagent that is used for subsequent embodiment.

Table 11

Sample reagent

Reagent	Grade	The manufacturer	The city, the state	Lot#	MW
						1X DPBS++	TC	Base wheat (Gibco)/hero (Invitrogen) TM	The Carlsbad, the California	1213061
The water of aseptic, nuclease free		Em Bean (Ambion) TM	Austin, the Texas	065P053618A
						The fluorescence dextran		Molecular probe/hero (Invitrogen) TM	The Carlsbad, the California	111105	3000
Air-100 substratum TM	TC	Ma Te Tyke (MatTek) TM	Ashland, MA	11110565
						Air-196 inset TM		Ma Te Tyke (MatTek) TM	Ashland, MA	7118
Cytotoxicity 96 detects TM		Pu Luomaige (Promega) TM	Madison, the state of Wisconsin	210634

The TC=tissue culture

Cell cultures

EpiAirway ^TM(ashland, MA) exploitation is as the interior model that is lining in the pseudostratified epithelium of respiratory tract by Ma Te Tyke (MatTek) company in system.Epithelial cell growth is on the cell cultures inset at the end with the porous-film the liquid-gas interface place, and this causes cytodifferentiation to become the form of high degree of polarization.End face has cilium, has the microvillus superstructure, epithelium produce mucus (by immunoblotting verified mucinous existence).Cell is layered on the inset in 3 weeks of precontract in transportation in factory.

Receive EpiAirway that day before experiment beginning ^TMCulture membrane.They transport not containing phenol red and do not contain in the Eagle substratum (DMEM) of Dulbecco (Dulbecco ' s) improvement of hydrogenation cortisone.Cell has cilium and is pseudostratified, grows on the detection system of the many sieves of the Mi Sibo that comprises the polycarbonate filtering system (Millipore) (Multiscreen) Caco-296-hole and converges.When receiving, the inset system does not break a seal 4 ℃ of preservations, and/or cultivates 24 hours at 37 ℃/5%CO2 in every hole 250 μ l minimum mediums (do not contain phenol red and do not contain the Eagle substratum (DMEM) of the Dulbecco improvement of hydrogenation cortisone) before use.

This model system is used for assessing TJMPs regulation and control TEER, influences cytotoxicity and improves the effect of the infiltration of epithelial cell individual layer.

(ashland, clone MA) are tracheae/bronchial epithelial cell (EpiAirway normal, that derive from the people in Ma Te Tyke (MatTek) company ^TMOrganize models) source.It is inset that cell is provided, and it grows on Mi Sibo (Millipore) cell (the Milicell)-CM filter that comprises transparent wetting ability teflon (PTFE) and converges.When receiving, before using with described film in 1ml minimum medium (do not contain phenol red and do not contain the Eagle substratum (DMEM) of the Dulbecco improvement of hydrogenation cortisone) 37 ℃/5%CO2 cultivation 24-48 hour.Nutrition is provided for inset in the every day of recovering.

With the cell inoculation of Madin-Darbey Madin-Darby canine kidney(cell line) (MDCK), human intestinal epithelial's cell (Caco-2) and human bronchial epithelial cell (16HBE14o-) on many sieves (Multi-Screen) Caco-296-hole inset from Mi Sibo.These cells are grown as individual layer and under the condition similar to the EpiAirway epithelial cell.

Peptide is synthetic

Using Nova biological chemistry (NovaBiochem) TGR resin to carry out peptide with 50 micromolar scales on Rainin Symphony synthesizer synthesizes.Go provide protection by handling twice, 10 minute with 20% the piperidines in DMF.After going protection, resin is washed once (30s) with the DMF that 10mL contains 5%HOBt, and wash 4 times (30s) with 10mL DMF.Send isopyknic activator soln in the reaction vessel then by 5 times of excessive Fmoc amino acid in DMF are delivered to, described activator soln contains 6.25 times of excessive N-methylmorpholines and 5 times of excessive HCTU, and carries out coupling.The coupling time of in building-up process, using 40 minutes.After first time linked reaction, in beginning for the second time before the coupling step, with resin with 10mL DMF washed twice (30s).For the peptide of PEGization, when finishing peptide when synthetic, remove N end Fmoc group, and artificial 2 normal O-(N-Fmoc-2-amino-ethyl)-O '-(2-carboxy ethyl)-11 ethylidene glycols in DMF that in reaction vessel, add.Although with manual type, 2 normal activator solns are delivered in the reaction vessel, and allow coupling to spend the night to carry out.Usually, obtain coupling efficiency, and any unreacted peptide is by the diacetyl oxide end-blocking greater than 97%.

By send TFA that 10mL contains 2.5%TIS, 2.5% water then gently nitrogen stirred 3 hours, in single reaction vessel, carry out cracking.Cracked solution is collected in the conical tube automatically, combined, and reduce volume by vapourisation under reduced pressure.The solution that obtains is ground with excessive ice-cold ether, filter and thoroughly wash with ice-cold ether.After the drying, thick peptide is absorbed in Mi Sibo (Millipore) water, and freeze-drying is to dry.

FITC (fluorescein-5-lsothiocyanates)-dextran infiltration detects

Use the dextran (FD3) of the FITC mark of molecular weight 3000 to assess the effect of individual TJMP to the epithelial cell mono-layer osmotic.Contain on the 96-hole reception flat board of 200 μ l DPBS++ as minimum medium organizing the inset flat board to transfer to.The end face of each tissue culture inset with the single detection formulation samples of 20 μ l (about the table 24 of the details reference example 25 that detects preparation) 37 ℃ in the dark vibrator (～100rpm) went up incubation 1 hour.At 1 hour incubation after the time, the minimum medium sample below each tissue culture inset is captured in, and temporarily at room temperature be kept at the dark place, up to quantizing the FD3 level by fluorescence spectroscopy.Measure about FD3, with 150 μ l minimum medium sample transfer in 96-hole flat board black, clearly bottom.Use is measured 485/20 from the FLx800 fluorescence plate reader of rich Tyke difficult to understand instrument (Biotek Instrument) and is excited the back in 528/20 fluorescent emission.

Infiltration calculation is:

The formula term about infiltration of definition:

Cb: basolateral part concentration

Ca: top concentration

Vb: basolateral part volume

Va: top volume

SA: filter surface-area

Dt: elapsed time

Each organizes inset to place to contain the single hole of 1ml Ma Te Tyke (MatTek) minimum medium.At the end face of inset, will use 25 μ l according to research and design and detect preparation, and with sample be placed on vibrator (～100rpm) go up 37 ℃ 1.5 hours.The dextran solution top of FITC-mark is joined in the inset, and carry out fluorescence measurement from substrate outside substratum after the time at incubation.The concentration of FITC-dextran is expressed as the percentage ratio that is applied to the initial substance on the cell.Dextran with FITC mark of molecular weight 4000 (MW4000) is used for assessing load (cargo) the size restriction about individual TJMP infiltration.Note, can obtain the dextran of the FITC mark of all size, to carry out big or small restricted research.

Transepithelial electrical resistance (TER) and TER recover

(FL) the Endohm-12 tissue resistance measuring chamber of Lian Jieing is finished TER and is detected for world's precision instrument (World Precision Instruments), Sarasota with the EVOM epithelial cell voltohmyst with electrode cable in use.Before check calibration, powered-down is with electrode and the blank inset of tissue culture balance at least 20 minutes in the substratum of Ma Te Tyke (MatTek).Measure background resistance with 300 μ l substratum in 1.5ml substratum in the Endohm tissue compartments and the blank inset.Adjust top electrodes like this, so that it is just in time approaching, but does not contact inset film end face.The background resistance of blank inset should be about 5-20 ohm.Measure for each TER, 300 μ l Ma Te Tyke (MatTek) substratum are joined in the inset, be placed at then in the Endohm chamber.All TER values are reported as the function of organization table area.

TER is calculated as:

TER＝(R _I-R _b)×A

R wherein _IBe resistance with inset of film, R _bBe the resistance of blank inset, and A is the area (0.6cm of film ²).The TER value is with respect to control value (contrast=about 1000 ohm-cm ²Be normalized into 100) the increase with the mucosal epithelium cell permeability of reducing that reduces to show cytolemma resistance.

Recover the 1st, 3,5 and 21 hours measurement TER ' s after processing for TER.TER percentage ratio is calculated as:

%TER=(TER T _{After the processing}/ TER T ₀)/(TER T _{After the processing}/ TER T ₀For control medium).

In some embodiments, (world's precision instrument (World Precision Instruments), Sarasota FL) carry out TER and measure to use the REMS self-actuated sampler with electrode cable.Before check calibration, powered-down is with electrode and the blank inset of tissue culture balance at least 20 minutes in the Air-100TM substratum of Ma Te Tyke (MatTek).By repeatedly measuring blank inset flat board, determined the background resistance of inset system, and same value has been used for each detection on this platform.Measuring zero moment TER (TER0) with inset and before detecting the preparation incubation.Adjust top electrodes like this, so that it is just in time approaching, but does not contact the end face of inset film.The background resistance of blank inset should be about 5-20 ohm.Measure for each TER, 100 μ l Ma Te Tyke (MatTek) Air-100TM substratum are joined in the inset, and 250 μ l are joined in the base bore, place the Endohm chamber then.All TER values are reported as the function of organization table area.Resistance meter is shown the percentage ratio of ohm * cm2 and initial TER value.

The TER value is calculated as:

Standard resistance, Ohm*cm ²=(TERt-blank) * 0.12

The formula term about TER calculating of definition:

TER0: the TER in zero moment measures

TERt: the TER that carries out at moment t after detecting the preparation incubation measures

Blank: the background measurement of resistance

The TER value is with respect to the increase with the mucosal epithelium cell permeability of reducing that reduces to show cytolemma resistance of control value.

Cytotoxicity (LDH mensuration)

Measure the necrocytosis amount by using CytoTox 96 cytotoxic assay test kits (Pu Luomaige (Promega) company, Madison, the state of Wisconsin) to measure from the loss of the serum lactic dehydrogenase (LDH) of cell.With sample on the 50 microlitre samples to 96 hole assay plates.Fresh not celliferous substratum is as blank.In each hole, add 50 μ l substrate solutions, and with flat board in the dark room temperature incubation 30 minutes.Behind the incubation, in each hole, add 50 μ l stop baths, and with described flat board on the optical density (OD) plate reader at the 490nm reading.The LDH that is discharged in the substratum of the substrate outside measures the relative cytotoxicity of representing sample.To have 0.3% octylphenol polyethylene (glycol ether) x (TritonX-100) contrast inset as 100% cracking, allowing the LDH value representation is total cracked percentage ratio.

Alternatively, can use WST-1 to measure and measure cytotoxicity.WST-1 measures based on the plastosome metabolic activity and measures the cell survival ability.Handle at peptide, washing and after back 10 minutes TER of processing measure, with the end face of cell monolayer with WST-1 reagent (Luo Shi (Roche)) 37 ℃ of incubations 4 hours.Use micro-plate reader to measure top cell conditioned medium liquid at OD 450nm.% value=sample _{OD 450}The contrast of/substratum _{OD 450}

In some embodiments, by using CytoTox 96 cytotoxicity detection kit (Pu Luomaige (Promega) company, Madison, the state of Wisconsin) detect serum lactic dehydrogenase (LDH) from cell to the substratum of top release and measure the necrocytosis amount.Be diluted in 1% octylphenol polyethylene (glycol ether) x (the Triton X-100 in the salt solution (PBS) of phosphoric acid buffer ^TM) in cultured cells, cause 100% cracking, and at the positive control of this paper as the LDH detection.Total liquid volume of each inset is being reached the final volume of 200 μ l with detecting preparation (about the table 24 of the details reference example 25 that detects preparation) incubation with substratum after 1 hour time.Then, by with the hyperchannel transfer pipet pressure-vaccum 4 times that is set at 100 μ l volumes, and the top substratum is mixed.After the mixing, will be in new 96-hole flat board from 100 μ l sample transfer of each inset end face.The top media samples is sealed with dull and stereotyped sealer, and be kept at room temperature and be used for analyzing on the same day, perhaps spending the night 4 ℃ of preservations is used for analyzing in second day.In order to detect the LDH level, 5 μ l in the 100 μ l top media samples are diluted among the 45 μ l DPBS in new 96-hole flat board.Fresh not celliferous substratum is used as blank.In each hole, add 50 μ l substrate solutions, and with its in room temperature away from direct light incubation 30 minutes.Behind 30 minutes incubations, in each hole, add 50 μ l stop baths.Use absorbs the optical density(OD) (OD) of plate reader detection at 490nm from the uQuant of rich Tyke difficult to understand instrument (Biotek Instruments).The LDH that is discharged in the substratum of top detects the relative cytotoxicity of representing sample.Deduct the absorbancy (the substrate level that LDH discharges) of the PBS contrast of mensuration by the absorbancy that detects preparation from the individuality of measuring, then with described value divided by for 1%Triton X-100 ^TMThe absorbancy that positive control is measured multiply by 100, and calculates every kind of cytotoxicity percentage ratio that detects preparation.

The formula that is used to calculate cytotoxicity percentage ratio is as follows:

Osmolarity

By model 20200 test sample from senior instrument company (Advanced Instruments Inc.) (Nuo Wude, Massachusetts).

Embodiment 11

The peptide that closely connects and strengthen the epithelium layer infiltration at external regulation and control epithelium

Table 12 is presented at external regulation and control tight junction protein and strengthens the aminoacid sequence of 11 kinds of peptides of epithelium layer infiltration, and it is measured by TER detection and penetration kinetics.For the purpose of these embodiment, because their similar activity, so select PN27 to represent PN27 and PN28.

Table 12

Peptide	Aminoacid sequence
		PN159	The NH2-KLALKLALKALKAALKLA-acid amides
PN161	NH2-GWTLNSAGYLLGKINLKALAALAKKIL-acid amides (SEQ ID NO:63)
		PN202	NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-acid amides (SEQ ID NO:64)
PN27	NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-acid amides (SEQ ID NO:65)

PN28	NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-acid amides (SEQ ID NO:66)
		PN58	NH2-RQIKIWFQMRRMKWKK-acid amides (SEQ ID NO:67)
PN73	NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-acid amides (SEQ ID NO:68)
		PN228	NH2-KLWSAWPSLWSSLWKP-acid amides (SEQ ID NO:69)
PN250	NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-acid amides (SEQ ID NO:70)
		PN283	Maleimide-GLGSLLKKAGKKLKQPKSKRKV-acid amides (SEQ ID NO:71)
PN183	NH2-KETWWETWWTEWSQPGRKKRRQRRRRPPQ-acid amides (SEQ ID NO:72)

Embodiment 12

Tight junction modulating peptide reduces TER

Present embodiment is assessed the effect of the tight junction protein of various peptides in external regulation and control epithelial cell individual layer, and it detects by the TER minimizing.The summary of the TER data that obtain from the experiment carried out the EpiAirway epithelial cell for every kind of TJMP is presented in the table 13.Grid representative outstanding in described table reduces for TJMP the highest observed TER in the detectable level scope.

Table 13

Peptide	1000 μM	500 μM	250 μM	125 μM	100 μM	50 μM	25 μM	10 μM	2.5 μM	1 μM
											PN159							94％	89％	79％	54 ％
PN161					84％		73％	43％	11％
											PN202		95％			95％	57％		3％
PN27		94％	91％	81％
											PN283	92％	86％			39％
PN250		84％	79％		58％		17％	3％
											PN228		82％			9％
PN73		83％			38％	8％
											PN58	88％	64％	-6％
PN183	55％	41％			25％

PN159, PN202, PN27 and PN283 reduce TER and surpass 90%, and PN161, PN250, PN228, PN73 and PN58 reduce TER and reach 82%-88%.PN28 does not show, but its function equates with Pn27.Last PN183 has 55% TER minimizing.These data show that the TJMPs of all detections can closely connect at external infringement epithelial cell.

In addition, carrying out TER recovers to analyze to determine handling the speed that back EpitAirway epithelium layer recovers with TJMPs.Surprisingly, the result shows, in the TJMPs of all detections, and PN250, PN202 and PN161 have the fastest time of recovery.These data show that TJMPs is of short duration to the effect of epithelium layer in essence.

Embodiment 13

The body outer osmotic kinetics of tight junction modulating peptide

In the present embodiment, detect the effect of TJMPs regulation and control EpiAirway epithelial cell infiltration.Following table 14 shows the penetration kinetics summary of the every kind of TJMP that represents with infiltration percentage ratio.Grid representative outstanding in described table is for this TJMP observed maximum infiltration in the detectable level scope.

Table 14

Peptide	1000 μM	500 μM	250 μM	125 μM	100 μM	50 μM	25 μM	10 μM	2.5 μM	1 μM
											PN159							12.5 ％	6.5％	1.9％	0.6％
PN161					7.1％		2.9％	1.6％	0.3％
											PN202		5.9％			2.8％	1.5 ％		0.2％
PN27		8.4％	7.3％	7.7％
											PN283	5.2％	3.9％			0.7％
PN250		4.2％	3.3％		1.7％		0.5％	0.3％
											PN228		1.7％			0.2％
PN73		0.8％			0.2％	0.1 ％
											PN58	6.3％	4.5％	0.9％		0.3％	0.3 ％
PN183	0.6％	0.5％			0.2％

These data show that the TJMPs of all detections can be in the infiltration of external enhancing epithelial cell individual layer.Usually, the perviousness degree is relevant with the ability that peptide reduces TER.

Embodiment 14

Tight junction modulating peptide can not cause significant cytotoxicity

Present embodiment is evaluated at and is exposed to TJMPs afterwards to epithelial cytotoxic effect.Carrying out LDH after handling 15 minutes and 60 minutes with every kind of peptide detects.In all situations, after handling in 15 minutes, almost do not observe LDH and discharge.After handling in 60 minutes, cytotoxicity level difference in the peptide that is detected, but within acceptable level, this peptide that shows all detections can not cause significant cell injury.

Embodiment 15

In the epithelial cell type of all detections, it is consistent that tight junction modulating peptide reduces TER

In order to determine whether observed TER result represents other epithelial cell types in EpitAirway epithelial cell culture systems, with MDCK, Caco-2 and 16HBE14o-cell are handled with TJMps, and detect TER.In all scenario, use the observed TER result of these cell types and use the observed TER result of EpiAirway epithelial cell consistent, this shows that these TJMPs have the ability that reduces the TER in all epithelial cell types.

Embodiment 16

Arrange tight junction modulating peptide based on performance

Arrange 9 kinds of TJMPs, and according to their perviousness level, TER value, TER recovery rate and the cytotoxicity that is displayed in Table 15, and be divided into 4 kinds of different performance grades.Do not comprise PN183 and PN28 in the table 15.Following table is summarised in described peptide processing EpiAirway epithelial cell and handles the EpitAirway epithelial cell after 60 minutes after 15 minutes and with described peptide, the optimum concn of every kind of TJMPs (that is, the maximum TER relevant with hypertonicity level reduces and do not show remarkable cytotoxicity) and corresponding infiltration percentage ratio.In addition, every kind of peptide is shown LDH value (cytotoxicity) about 15 minutes and processing in 60 minutes.Show that also TER recovers.TER recovery rate directly related with slope value (that is, bigger slope value recovers relevant with TER faster).

Table 15

Embodiment 17

Tight junction modulating peptide strengthens the infiltration that FITC-dextran MW4000 passes the epithelial cell individual layer

In the present embodiment, study the penetration kinetics of FITC-dextran MW4000 when determining to have every kind of TJMP.Whether this experimental evaluation infiltration depends on the time of peptide with epithelial cell individual layer incubation, and whether infiltration is that payload is dependent.Handling cell with TJMP and FITC-dextran MW4000 after 15 minutes and handle cell and detect Premeabilisation of cells (Fig. 7) after 60 minutes.The PYY preparation is as positive control, and phosphate-buffered saline (PBS) is as negative control.Reduce degree and do not show significant Cytotoxic concentration and detect described peptide showing the maximum TER relevant with hypertonicity level.

For with a kind of TJMP, 60 minutes processing list reveals the remarkable higher penetration degree of processing than 15 minutes.Surprisingly, PN161, PN127 and PN228 show the level of interpenetration (about 7.5%) that equates with PN159.With cell incubation after 60 minutes, TJMPs PN250, PN283, PN202, PN58 obtain about 5% infiltration, described infiltration does not reach just by PN161, PN127, the infiltration that PN228 and PN159 obtain.These data show that the TJMPs of all detections can both strengthen the infiltration of FITC-dextran MW4000, and this enhancement depends on the length of peptide and epithelium layer duration of contact.

Above-mentionedly experiment showed, that the TJMPs that is detected can be in the infiltration of external enhancing epithelial cell individual layer.

Embodiment 18

Tight junction modulating peptide at external enhancing perviousness and observed enhanced permeated height in vivo mutually Close

Carry out linear regression analysis, with determine in external EpiAirway epithelial cell model system observed TJMP penetration kinetics whether with for relevant with the observed drug disposition dynamic metabolism of a kind of TJMP data.In order to determine the body outer osmotic data whether as the good indication of effect in the body, area under curve-final (last) value (AUC-last) that will obtain from the drug disposition dynamic metabolism research of using PYY and TJMPs to carry out is at the epithelial cells in vitro mono-layer osmotic research drawing of using PYY and TJMPs to carry out.Body outer osmotic is expressed as percentage ratio, and AUC-last (AUC-is final) is expressed as a minute * pg/ml.Study in the external and body about 10 kinds of different TJMPs and draw, and carry out linear regression.Obtain 0.82 R ²Value (82% dependency), this shows the dependency that has height for the perviousness percentage ratio that derives from intravital AUC value and observation in vitro.Surprisingly, when between get rid of detecting during (inter-assay) mutability, obtain 0.996 R ²Value (basically 100%), this shows have direct dependency between the effect in body outer osmotic and body.Therefore, body outer osmotic can be used for effect in the predictor.

Embodiment 19

TJMP equals or exceeds the small molecules infiltration to infiltration enhancement in the body of peptide hormone therapeutical agent to be strengthened The enhancement of agent

The male New Zealand rabbit of 20 3-6 monthly ages and heavy 2.1-3.0kg is divided into 5 treatment group at random, every group of 4 animals.With test animal with 15 μ l/kg and by the suction pipe intranasal administration.The composition of 5 kinds of different administration groups of following table 19 expressions.

For administration group 1 (seeing Table 16), use the PYY clinical preparation that comprises the small molecules penetration enhancers.Small molecules toughener in these researchs comprises methyl-beta-cyclodextrin, didecyl acyl phospholipids phatidylcholine (DDPC) and/or EDTA.Administration group 2 accepts to be dissolved in the PYY in the phosphate-buffered saline (PBS).For administration group 3-5, the PN159 of different concns is added in the administration group 2, so that each group among the administration group 3-5 to be by PYY, PN159 and PBS form

Table 16

Group	Animal	Penetration enhancers	Administration concentration (mg/ml)	Administration volume (ml/kg)	PYY dosage (μ g/kg)
						1	4M	The small molecules penetration enhancers	13.67	0.015	205
2	4M	Do not have	13.67	0.015	205

3	4M	25μMPN159	13.67	0.015	205
						4	4M	50μM PN159	13.67	0.015	205
5	4M	100μM PN159	13.67	0.015	205

Collect successive blood sample (every part of about 2ml) by directly carrying out direct venipuncture, collect and contain in the blood collection tube of EDTA as anti-coagulant at auricular vein.0,2.5,5,10,15,30,45,60 and 120 minutes collection blood samples after administration.Collect after the blood, pipe shakes several times gently, with anticoagulation, adds 50 μ l bovine pancreatic trypsin inhibitor solution then.About 4 ℃ with about 1,600xg centrifugal blood 15 minutes, and plasma sample is assigned in the duplicate aliquots containig, and in approximately-70 ℃ freezing preservation.

Average all 4 animals in a treatment group, detect the plasma concentration (table 17) of following PYY:

Table 17

	Group 1	Group 2	Group 3	Group 4	Group 5
						Time, minute	The small molecules penetration enhancers	No penetration enhancers	25μM PN159	50μM PN159	100μM PN159
0	183.825	257.3	228.675	424.4	294.225
						2.5	1280.7	242.8	526.375	749.975	1748.225
5	1449.425	273.675	1430.15	1293.4	3088.2
						10	8251.8	372.05	6521.7	12517.2	14486.6
15	13731.2	398.225	12563.075	34455.3	20882.725
						30	19537.55	476.475	15222.6	35294.375	25470.475
45	13036.075	340.7	9081.125	21582.225	16499.55
						60	7080.875	283.825	4843.15	9461.925	10676.625
120	1671.9	192.575	1224.2	2337.775	1891.275

Be presented at the following table 18 from the pharmacokinetic data of above-mentioned data computation:

Table 18

Compare with group 2 (no toughener) preparation, determine following relative enhancing ratio (table 19):

Table 19

Group	Preparation	Relative Cmax	Relative AUC last
				1	The small molecules penetration enhancers	38x	27x
3	PN159，25μm	30x	21x
				4	PN159，50μm	79x	46x
5	PN159，100μm	54x	38x

Aforementioned data shows, compares with the small molecules penetration enhancers, and TJMP will infiltration be strengthened to equal or bigger degree in the nose of people's hormone peptide therapeutics in vivo.Observe the maximum effect of described peptide in 50 μ M concentration.100 μ M concentration cause low slightly infiltration, although the two all causes the perviousness higher than small molecules penetration enhancers.

Embodiment 20

TJMP is to the infiltration enhancement of oligopeptides therapeutical agent

Present embodiment shows a kind of representative peptide of the present invention, and PN159 strengthens a kind of ring pentapeptide, melanocortin-4 receptor agonist (MC-4RA), a kind of pattern oligopeptides agonist that is used for mammalian cellular receptor, the effect of epithelial cell infiltration.In this embodiment, the combination of one or more perviousness peptides and MC-4RA is described.Useful in this case preparation comprises the combination of oligopeptides therapeutical agent, perviousness peptide and one or more other penetration enhancers.Described preparation can also contain buffer reagent, tonicity agents, pH value conditioning agent and peptide/protein stabiliser such as amino acid, sugar or polyvalent alcohol, polymkeric substance and salt.

Assessment PN159 is to the effect of MC-4RA infiltration in this research.MC-4RA be a kind of have about 1, the methane sulfonates of 100Da molecular weight, the activity of its regulation and control MC-4 acceptor.The PN159 concentration of assessment is 5,25,50 and 100 μ M.45mg/ml M-β-CD is as the solubilizing agent of all preparations, to obtain the peptide concentration of 10mg/ml.Assessment PN159 self or with the effect of EDTA (1,2.5,5, or 10mg/ml) combination.The pH value of preparation is fixed on 4, and osmolarity is 220mOsm/kg.

The HPLC method

Analyze the concentration of MC-4RA in the basolateral part substratum by RP-HPLC, described RP-HPLC uses the C18RP chromatography, 25 ℃ of flow velocity 1mL/ minute and column temperatures.

Solvent orange 2 A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN

Volume injected: 50 μ L

Detect: 220nm

Working time: 15 minutes

MC-4RA and 5,25,50 and 100 μ M PN159 combination, and at pH value 4 and osmolarity～220mOsm/kg.Use external epithelium model to detect described combination, to detect and the cytotoxicity of monitoring PT H infiltration, transepithelial electrical resistance (TER) and described preparation by MTT and LDH.

The result of study of MC-4RA infiltration shows, this TJMP except the mucous membrane infiltration that strengthens the peptide hormone therapeutical agent, also significantly strengthens the epithelium infiltration of oligopeptides therapeutical agent.

Embodiment 21

TJMP is to the infiltration enhancement of small-molecule drug

Present embodiment shows a kind of representative peptide of the present invention, and the PN159 enhancing is the effect that the epithelium of the small-molecule drug of example permeates with acetylcholinesterase (ACE) inhibitor lycoremine.The combination of one or more perviousness peptides and small-molecule drug is described in the present embodiment.Useful in this case preparation can comprise the combination of small-molecule drug, perviousness peptide and one or more other penetration enhancers.Described preparation can also comprise buffer reagent, tonicity agents, pH value conditioning agent, stablizer and/or sanitas.

The present invention is with lycoremine and PN159 combination, to strengthen the infiltration that lycoremine passes nasal mucosa.Because lycoremine is a kind of small molecules that can see through nasal epithelial membranes independently, so the increase of this drug osmotic is unexpected.Therefore, the lycoremine that mediates by the infiltrative vehicle of adding enhancing peptide is surprising by the remarkable enhancement that epithelium permeates, and this is based on, and not expecting described vehicle significantly increases the infiltration that lycoremine passes the epithelium layer usually.Therefore, the present invention will promote the nose of lycoremine and other small-molecule drug to send by the bioavailability that increases them.

In this research, the lycoremine of 40mg/ml lactic acid salt form and 25,50 and 100 μ MPN159 make up among pH 5.0 and the osmolarity～270mOsm at solution.Use external epithelium model to detect described combination, to detect the cytotoxicity of monitoring lycoremine infiltration, transepithelial electrical resistance (TER) and described preparation by LDH mentioned above and MTT.The infiltration of lycoremine detects by standard HPLC analysis to be undertaken, as follows.

HPLC analyzes

Use no gradient LC (Waters Alliance (this associating of water)) method and UV and detect, determine in the preparation and the lycoremine concentration in substrate outside substratum (infiltration sample).

Post: this symmetrical posts of water (Waters Symmetry Shield), C 18,5um, 25 * 0.46cm

Moving phase: the 5%ACN in the 50mM ammonium formiate, pH 3.0

Flow velocity: 1ml/ minute

Column temperature: 30 ℃

Working curve: 0-400 μ g/ml bromination lycoremine

Detect: in the ultraviolet ray (UV) of 285nm

Based on aforementioned research, PN159 improves the micromolecular mucosal delivery of striding.Select lycoremine as the model low-molecular-weight drug, and think that the result of this a part is the active prediction of perviousness peptide that is used for other small-molecule drug.In order to assess the perviousness activity in this situation, the lycoremine of 40mg/ml lactic acid salt form and 25,50 and 100 μ M PN159 at solution, are made up among pH 5.0 and the osmolarity～270mOsm.Use external epithelium model to detect described combination, to detect the cytotoxicity of monitoring lycoremine infiltration, transepithelial electrical resistance (TER) and described preparation by LDH and MTT.

In described vitro tissue model, add PN159 and cause medicine to pass the remarkable increase of the infiltration of barrier cell.Particularly, at the P of 40mg/ml lycoremine _AppIn have 2.5-3.5 increase doubly.

In the presence of lycoremine, PN159 reduces TER, described in previous embodiment.

In the presence of the lactic acid lycoremine and PN159 of all detectable levels, the cell survival ability keeps higher (＞80%).On the contrary, as detecting by LDH, when having PN159 and lactic acid lycoremine, cytotoxicity is lower.These detections show that all PN159 does not have toxicity to epithelial membrane.

When not having PN159, the P of lycoremine _AppBe about 2.1 * 10 ^-6Cm/s.When existence 25,50 and 100mM PN159, P _AppBe respectively 5.1 * 10 ^-6, 6.2 * 10 ^-6And 7.2 * 10 ^-6Cm/s.Therefore, at the P of this model low-molecular-weight drug _AppIn, PN159 provides 2.4-to 3.4-increase doubly.

TJMP increases the epithelium infiltration as the lycoremine of model low-molecular-weight drug surprisingly.PN159 is joined the infiltration that remarkable enhancing lycoremine passes the epithelial cell individual layer in the lycoremine solution.Evidence shows that temporary transient minimizing of PN159 passes the TER of epithelial membrane, and does not damage the cell in the film, as measuring by high cell survival ability and low cytotoxicity.TJMP strengthens the bioavailability of lycoremine and other small-molecule drug in vivo, and it strengthens by the identical mechanism of using the external model proof in this article.Further predict TJMP and also strengthen the infiltration of lycoremine in higher concentration.

Embodiment 22

TJMP is to proteic infiltration enhancement

Having determined that PN159 is used for the purposes of striding the mucous membrane preparation of low-molecular weight compound, importantly is to distinguish whether these observations can be extrapolated to bigger molecule, for example therapeutic peptide and albumen.For this purpose,, the salmon calcitonin see calcimar as the mode treatment peptide is carried out vitro tissue research not having and exist 25,50 and during 100mM PN159.When not having PN159, for the P of thyrocalcitonin _AppBe about 1 * 10 ^-7Cm/s is lower than the P of lycoremine _AppAbout order of magnitude infers that this is because the difference of molecular weight.When having PN159, the remarkable increase of the bright thyrocalcitonin infiltration of data sheet is compared with the situation of having only thyrocalcitonin, at P _AppIn reach 23-and doubly increase (table 20) to 47-.

Table 20

The P that uses the vitro tissue model to detect _App

^aPH is 5.0.

In order to study the ubiquity of these discoveries, when not existing and having PN159, two kinds of other peptides of check, i.e. human parathyroid hormone 1-34 (PTH in described external model _1-34) and people's peptide YY3-36 (PYY _3-36) (P _AppData presentation is in table 20).When not having PN159, the P of these two kinds of peptides _AppP with thyrocalcitonin _AppConsistent.At PTH _1-34Situation in, PN159 exists in P _AppIn about 3-5 is provided increase doubly.When in the presence of PN159, preparing PYY _3-36The time, Papp increases about 12-to 17-doubly.Our discovery of these data acknowledgements, promptly TJMP strengthens small molecules and proteicly strides the ubiquity that the mucous membrane medicine is sent.

Embodiment 23

The chemical stability of TJMP

Under the relevant condition of storage of treatment, determine the chemical stability of PN159.Use the HPLC method of expression stability.Solution (50mM) is kept under different pH value (4.0,7.3 and 9.0) and temperature (5 ℃, 25 ℃, 35 ℃, 40 ℃ and the 50 ℃) condition.Sample in pH value 4 contains the 10mM citrate buffer.Sample in pH value 7.3 and 9.0 contains the 10mM phosphate buffered saline buffer.Stability in storage result's (comprising Arrhenius graphing method (Arrhenius plot)) shows, chemically is being the most stable at low temperature and pH value PN159.For example, at 5 ℃ and pH 4.0 or pH7.3, for 6 months storage, existing basically, 100% PN159 recovered.When storage temperature is elevated to 25 ℃, after 6 months, there is 7% and 26% natural PN159 loss respectively for sample at pH 4 or pH 7.In the temperature of pH 9 and/or rising, for example, 40-50 ℃, ensue the quick consumption of PN159.4.0-7.3 pH value scope and freezing temperature range to envrionment temperature be maximally related for preparation in the nose.Therefore, these data support that TJMP can keep chemical integrity under the condition of storage relevant with the IN preparation.

Embodiment 24

In rabbit, assess tight junction modulating peptide in vivo by intranasal administration

In rabbit, carry out pharmacokinetics (PK) research, send the peptide YY (PYY) that uses and the drug plasma dynamic metabolism feature of various tight junction modulating peptides (TJMPs) by (IN) in the nose with assessment.

Animal model

In this research, New Zealand white rabbit (Hra:(NZW) SPF) as test subject, to assess the drug plasma dynamic metabolism of MC-4RA by intranasal administration and intravenous infusion.The processing of animal is according to the regulations of listing in USDA animal welfare method (USDA Animal Welfare Act) (9CFR the 1st, 2 and 3 parts) and experimental animal feeding management and instruction manual (Guide for theCare and Use of Laboratory Animals, (ILAR publishes, 1996, national academy of sciences printing)) specified condition is carried out in.

Because the pharmacokinetics curve that obtains from the medicine that is administered to rabbit is similar to the PK curve of identical medicine among the people very much, so select the animal subjects of rabbit as this research.

Administration is used

Experimental design and dosage regimen about the TJMPs of 9 kinds of detections are summarised in the table 21.All experimental group are all passed through in the nose (IN) and are used and give with single TJMP or phosphate-buffered saline (PBS: Zu He 205 μ g/kg PYY (3-36) negative control).Use autospencer and disposable plastic suction nozzle, with every kind of preparation applied once in left nostril.With the perk of animal head back, and when sucking, animal uses described dosage, so that allow capillary action that solution is sucked the nostril.After IN used, control animal head about 15 seconds in the position of perk backward was with any loss of the dosage that prevents to use.In this step process, take the careful of extreme, with any potential tissue injury of avoiding causing by contacting with mucous membrane in the nose.

Table 21

Group	Size of animal	Approach	Tight junction modulating agent (concentration)	PYY3 (μg/kg)
					1	5M	In the nose	PBS	205
2	5M	In the nose	PN159(50μM)	205
					3	5M	In the nose	PN161(100μM)	205
4	5M	In the nose	PN202(100μM)	205
					5	5M	In the nose	PN27(250μM)	205
6	5M	In the nose	PN58(500μM)	205
					7	5M	In the nose	PN73(500μM)	205
8	5M	In the nose	PN228(500μM)	205
					9	5M	In the nose	PN183(1000μM)	205
10	5M	In the nose	PN556(1000μM)	205

PN556 has the primary sequence identical with PN283, but does not have maleimide amine-modified at the N of peptide end.

Blood and plasma sample are collected

After using by the IN administration, by directly carrying out venipuncture and collect the successive blood sample from every animal at auricular vein.Before administration, after the administration 5,10,15,20,30,45,60,90, collected blood sample in 120 and 180 minutes.Sample collection is to containing in the pipe of EDTA dipotassium as anti-coagulant.Pipe cooled off before centrifugal.All samples carry out centrifugal in collection within an hour.Collect blood plasma, and transfer in the plastic jar of mark in advance, freezing in dry ice/acetone batch, before carrying out the pharmacokinetics analysis, be kept at approximately-70 ℃ then.

Carry out clinical observation constantly at each blood sampling, and two nostrils of using all animals in the test set at IN were being checked in 1 hour before 5 minutes and behind the intranasal administration just.

Analytical procedure

By using ELISA, analyze PYY (3-36) level from the sample of every animal in all study group.With before the administration and the test sample after the administration on HPLC, move and carry out quality control.Blood plasma aliquots containig (0.1mL) is the albumen that acetonitrile precipitation is used in the mark back in adding bioanalysis.With the supernatant liquor nitrogen drying, reconstruct in the HPLC damping fluid, and be then injected in the HPLC system.Detect effluent by positively charged ion electro-spray ionization three utmost point quadrupole mass spectrometers (positive ion electrosprayionization tandem triple quadrupole mass spectrometer) of connecting.The PK data are analyzed by WinNonlin (method Saudi company (Pharsight Corp.), Mountain View (MountainView)).

The result

Average blood plasma PK parameter about each test set is summarised in the table 22.After using any preparation, do not observe disadvantageous clinical indication.Show without any rubescent there is not swelling behind the intranasal inspection in two nostrils of the animal by the IN administered formulation.PK research assessment C _Max(the maximum concentration of observing), T _MaxTerminal and the infinitely great limit (inf) of (moment of peak concentration) and AUC (area under curve).According to the horizontal 8 kinds of TJMPs of their body internal penetration, and be divided into 4 kinds of different performance grades, grade I comprises and has the most substantially TJMPs of internal penetration level, and each subsequent levels comprises the TJMPs with the body internal penetration level that reduces gradually.

Table 22

These data show for PN161 suitable with PN159 with the two observed body internal penetration of PN27; And remaining TJMPs in the concentration that is detected, obtains to be lower than the body internal penetration level of PN159.

Embodiment 25

Tight junction modulating peptide in external enhancing epithelium layer infiltration

Present embodiment is described the detection preparation (shown in the table 24) of screening representative peptide PN679 of the present invention and PN745 (shown in the table 23) and every kind of peptide, to determine the effective concentration scope of every kind of peptide about epithelial cell mono-layer osmotic enhancement.

Table 23

Tight junction modulating peptide

Peptide #	Aminoacid sequence	Molecular weight	Lot number	Purity (%)
					PN679	CNGRCGGKKKLKLLLKLL (SEQ ID NO：32)	1984.78	05-1882-758	94.01
PN745	LRKLRKRLLRLRKLRKRLLR-acid amides (SEQ ID NO:33)	2684.53	05-1882-761	99.29

Following table 24 is described the indivedual detection preparations that contain representative peptide of the present invention (" promoting agent " hurdle in the table 24), with the detection preparation as the contrast of positive and negative detection preparation, they detect by TER, LDH (cytotoxicity) and sample infiltration enhancement and test.Every kind of peptide is at 25 μ M, 100 μ M, and 250 μ M, 500 μ M and 1000 μ M concentration detect.PN159 herein (detecting preparation #11) is as the TJMP positive control, and previous having shown at 25 μ M effectively reduces TER and strengthen the ability that sample permeates.1% Triton X-100 ^TM(detecting preparation #14) reduces to detect the positive control of the two as cytotoxicity (LDH) detection and TER." special reinforced (specialsauce) " is (SS) herein as the small molecules penetration enhancers.DPBS++ is as negative control.Except the detection preparation #12 of purpose pH value with pH 5, every kind is detected preparation and has the final volume of 300 μ l and the purpose pH value of pH 7.1% Triton X-100 ^TMThe positive control that (detecting preparation #14) detected as cytotoxicity (LDH).

Detect in the preparation at every kind of 300 μ l cumulative volumes, only with 20 μ l sample application to the tracheae/bronchial epithelial cell (EpiAirway that derives from the people ^TMThe system of organize models), to assess every kind of effect that detects preparation to TER, LDH and sample infiltration.

Table 24

Detect preparation

SS=" special reinforced "

Embodiment 26

PN679 and PN745 are at external regulation and control tight junction protein

Present embodiment shows that representative peptide PN679 and PN745 effectively reduce TER in the dose dependent mode and significantly strengthen the infiltration of sample, and do not cause significant cytotoxicity, and this shows that these peptides are effective TJMPs.Table 25 has been summed up about TER, the LDH of the detection preparation of describing in the table 24 of embodiment 25 and sample infiltration (FD3) data.Detect twice about the detection preparation #1 of PN679 with about the detection preparation #6 of PN745.Other detected result about TER, LDH and sample infiltration is presented in the bracket.

Table 25

The summary of TER, LDH and sample infiltration enhancement data

SS=" special reinforced "

Comprise 100 μ M, 250 μ M, 500 μ M and 1000 μ M representative peptide PN679 of the present invention (detects preparation #1, #2, #3 and #4) or PN745 (detection preparation #6, #7, #8 and #9) any detection preparation TER is reduced to the degree that equates with " special reinforced ", and significantly be lower than the degree of definite TJMP contrast PN159.As estimating, the DPBS++ negative control does not reduce TER significantly.These two kinds of peptides reduce the ability of TER and their strengthen the ability height correlation of the infiltration of FD3 molecule.The PN679 of 100 μ M dosage (detecting preparation #4) shows the infiltration percentage ratio similar to PN159TJMP with PN745 (detecting preparation #9), but has lower cytotoxicity (lower %LDH discharges).Any peptide of greater concn causes the FD3 level of interpenetration that increases, the level of described horizontal exceeding PN159, but also increasing the emission levels of LDH, this shows the cytotoxicity of increase.As estimating, the DPBS++ contrast is not induced and can be discharged by detected LDH.Based on viewed TER minimizing, sample infiltration and cytotoxicity (LDH release), as if it is the most desirable that any representative peptide PN679 of 100 μ M dosage and PN745 are used for further analyzing these two kinds of TJMPs.

Aforementioned data shows unexpected discovery, that is, representational peptide PN679 and PN745 are at the external TER that reduces human epithelial cell's individual layer, and the infiltration of enhancing small molecules, and do not have significant toxicity.These data show, these tight junction modulating peptides (TMJP) are that the medicine that is used to pass mucous membrane surface is sent the fabulous material standed for that (IN) medicine is sent in the nose for example.

Embodiment 27

Tight junction modulating peptide permeates with observed enhanced permeated height is relevant in vivo in external enhancing

Carry out linear regression analysis, with determine in external EpitAirway epithelial cell model system observed TJMP penetration kinetics whether with for relevant with the observed drug disposition dynamic metabolism of a kind of TJMP data.In order to determine the body outer osmotic data whether as the good indication of effect in the body, area under curve-final (last) value (AUC-last) that will obtain from the drug disposition dynamic metabolism research of using PYY and TJMPs to carry out is at the epithelial cells in vitro mono-layer osmotic research drawing of using PYY and TJMPs to carry out.Body outer osmotic is expressed as percentage ratio, and AUC-last (AUC-is final) is expressed as a minute * pg/ml.Study in the external and body about 10 kinds of different TJMPs and draw, and carry out linear regression.Obtain 0.82 R ²Value (82% dependency), this shows the dependency that has height for the perviousness percentage ratio that derives from intravital AUC value and observation in vitro.Surprisingly, when between get rid of detecting during (inter-assay) mutability, obtain 0.996 R ²Value (basically 100%), this shows have direct dependency between the effect in body outer osmotic and body.Therefore, body outer osmotic can be used for effect in the predictor.

Embodiment 28

TJMP equals or exceeds the small molecules infiltration to infiltration enhancement in the body of peptide hormone therapeutical agent to be strengthened The enhancement of agent

The male New Zealand rabbit of 20 3-6 monthly ages and heavy 2.1-3.0kg is divided into 5 treatment group at random, every group of 4 animals.With test animal with 15 μ l/kg and by the suction pipe intranasal administration.The composition of 5 kinds of different administration groups of following table 26 expressions.

For administration group 1 (seeing Table 26), use the PYY clinical preparation that comprises the small molecules penetration enhancers.Small molecules toughener in these researchs comprises methyl-beta-cyclodextrin, didecyl acyl phospholipids phatidylcholine (DDPC) and/or EDTA.Administration group 2 accepts to be dissolved in the PYY in the phosphate-buffered saline (PBS).For administration group 3-5, the PN159 of different concns is added in the administration group 2, so that each group among the administration group 3-5 to be by PYY, PN159 and PBS form.

Table 26

The administration group

Group	Animal	Penetration enhancers	Administration concentration (mg/ml)	Administration volume (ml/kg)	PYY dosage (μ g/kg)
						1	4M	The small molecules penetration enhancers	13.67	0.015	205
2	4M	Do not have	13.67	0.015	205
						3	4M	25μMPN159	13.67	0.015	205
4	4M	50μMPN159	13.67	0.015	205
						5	4M	100μM PN159	13.67	0.015	205

Collect successive blood sample (every part of about 2ml) by directly carrying out venipuncture, collect and contain in the blood collection tube of EDTA as anti-coagulant at auricular vein.0,2.5,5,10,15,30,45,60 and 120 minutes collection blood samples after administration.Collect after the blood, pipe shakes several times gently, with anticoagulation, adds 50 μ l bovine pancreatic trypsin inhibitor solution then.About 4 ℃ with about 1,600xg centrifugal blood 15 minutes, and plasma sample is assigned in the duplicate aliquots containig, and in approximately-70 ℃ freezing preservation.

Average all 4 animals in a treatment group, detect the plasma concentration (table 27) of following PYY:

Table 27

The summary of test set PYY plasma concentration

	Group 1	Group 2	Group 3	Group 4	Group 5
						Time, minute	The small molecules penetration enhancers	No penetration enhancers	25μM PN159	50μM PN159	100μM PN159
0	183.825	257.3	228.675	424.4	294.225
						2.5	1280.7	242.8	526.375	749.975	1748.225
5	1449.425	273.675	1430.15	1293.4	3088.2
						10	8251.8	372.05	6521.7	12517.2	14486.6

15	13731.2	398.225	12563.075	34455.3	20882.725
						30	19537.55	476.475	15222.6	35294.375	25470.475
45	13036.075	340.7	9081.125	21582.225	16499.55
						60	7080.875	283.825	4843.15	9461.925	10676.625
120	1671.9	192.575	1224.2	2337.775	1891.275

Be presented at the following table 28 from the pharmacokinetic data of above-mentioned data computation:

Table 28

Pharmacokinetic data is summed up

Compare with group 2 (no toughener) preparation, determine following relative enhancing ratio (table 29):

Table 29

Relative enhancing ratio

Group	Preparation	Relative Cmax	Relative AUC last
				1	The small molecules penetration enhancers	38x	27x
3	PN159，25μm	30x	21x
				4	PN159，50μm	79x	46x
5	PN159，100μm	54x	38x

Aforementioned data shows, compares with the small molecules penetration enhancers, and TJMP will infiltration be strengthened to equal or bigger degree in the nose of people's hormone peptide therapeutics in vivo.Observe the maximum effect of described peptide in 50 μ M concentration.100 μ M concentration cause low slightly infiltration, although the two all causes the infiltration higher than small molecules penetration enhancers.

Embodiment 29

TJMP is to the infiltration enhancement of oligopeptides therapeutical agent

Present embodiment shows a kind of representative peptide of the present invention, and PN159 strengthens a kind of ring pentapeptide, melanocortin-4 receptor agonist (MC-4RA), the effect that a kind of epithelium that is used for the pattern oligopeptides agonist of mammalian cellular receptor permeates.In this embodiment, the combination of one or more perviousness peptides and MC-4RA is described.Useful in this case preparation can comprise the combination of oligopeptides therapeutical agent, perviousness peptide and one or more other penetration enhancers.Described preparation can also contain buffer reagent, tonicity agents, pH value conditioning agent and peptide/protein stabiliser such as amino acid, sugar or polyvalent alcohol, polymkeric substance and salt.

Assessment PN159 is to the effect of MC-4RA infiltration in this research.MC-4RA be a kind of have about 1, the methane sulfonates of 100Da molecular weight, the activity of its regulation and control MC-4 acceptor.The PN159 concentration of assessment is 5,25,50 and 100 μ M.45mg/ml M-β-CD is as the solubilizing agent of all preparations, to obtain the peptide concentration of 10mg/ml.Assessment PN159 self or with the effect of EDTA (1,2.5,5, or 10mg/ml) combination.The pH value of preparation is fixed on 4, and osmolarity is 220mOsm/kg.

The HPLC method

Analyze the concentration of MC-4RA in the basolateral part substratum by RP-HPLC, described RP-HPLC uses the C18RP chromatography, 25 ℃ of flow velocity 1mL/ minute and column temperatures.

Solvent orange 2 A: 0.1% TFA in water; Solvent B: 0.1% TFA in ACN

Volume injected: 50 μ L

Detect: 220nm

Working time: 15 minutes

MC-4RA and 5,25,50 and 100 μ M PN159 combination, and at pH value 4 and osmolarity～220mOsm/kg.Use external epithelium model to detect described combination, to detect and the cytotoxicity of monitoring PT H infiltration, transepithelial electrical resistance (TER) and described preparation by MTT and LDH.

The result of study of MC-4RA infiltration shows that except the mucous membrane infiltration that strengthens the peptide hormone therapeutical agent, TJMP also significantly strengthens the epithelium infiltration of oligopeptides therapeutical agent.

Embodiment 30

TJMP is to the infiltration enhancement of small-molecule drug

Present embodiment shows a kind of representative peptide of the present invention, and the PN159 enhancing is the effect that the epithelium of the small-molecule drug of example permeates with acetylcholinesterase (ACE) inhibitor lycoremine.The combination of one or more perviousness peptides and small-molecule drug is described in the present embodiment.Useful in this case preparation can comprise the combination of small-molecule drug, perviousness peptide and one or more other penetration enhancers.Described preparation can also comprise buffer reagent, tonicity agents, pH value conditioning agent, stablizer and/or sanitas.

The present invention is with lycoremine and PN159 combination, to strengthen the infiltration that lycoremine passes nasal mucosa.Because lycoremine is a kind of small molecules that can see through nasal epithelial membranes independently, so the increase of this drug osmotic is unexpected.Therefore, the lycoremine that mediates by the vehicle that adds the infiltration of enhancing peptide is surprising by the remarkable enhancement that epithelium permeates, and this is based on, and not expecting described vehicle significantly increases the infiltration that lycoremine passes the epithelium layer usually.Therefore, the present invention will promote the nose of lycoremine and other small-molecule drug to send by the bioavailability that increases them.

In this research, the lycoremine of 40mg/ml lactic acid salt form and 25,50 and 100 μ MPN159 make up among pH 5.0 and the osmolarity～270mOsm at solution.Use external epithelium model to detect described combination, to detect the cytotoxicity of monitoring lycoremine infiltration, transepithelial electrical resistance (TER) and described preparation by LDH mentioned above and MTT.The infiltration of lycoremine detects by standard HPLC analysis to be undertaken, as follows.

HPLC analyzes

Use no gradient LC (Waters Alliance (this associating of water)) method and UV and detect, determine in the preparation and the lycoremine concentration in substrate outside substratum (infiltration sample).

Post: this symmetrical posts of water (Waters Symmetry Shield), C18,5um, 25 * 0.46cm

Moving phase: the 5%ACN in the 50mM ammonium formiate, pH 3.0

Flow velocity: 1ml/ minute

Column temperature: 30 ℃

Working curve: 0-400 μ g/ml bromination lycoremine

Detect: in the ultraviolet ray (UV) of 285nm

Based on aforementioned research, PN159 improves the micromolecular mucosal delivery of striding.Select lycoremine as the model low-molecular-weight drug, and think that the result of this a part is the active prediction of perviousness peptide that is used for other small-molecule drug.In order to assess the perviousness activity in this situation, with the lycoremine of 40mg/ml lactic acid salt form and 25,50 and 100 μ M PN159 in solution, pH 5.0 and osmolarity～270mOsm combination.Use external epithelium model to detect described combination, to detect the cytotoxicity of monitoring lycoremine infiltration, transepithelial electrical resistance (TER) and described preparation by LDH and MTT.

In described vitro tissue model, add PN159 and cause medicine to pass the remarkable increase of the infiltration of barrier cell.Particularly, at the P of 40mg/ml lycoremine _AppIn have 2.5-3.5 increase doubly.

In the presence of lycoremine, PN159 reduces TER, described in previous embodiment.

In the presence of the lactic acid lycoremine and PN159 of all detectable levels, the cell survival ability keeps higher (＞80%).On the contrary, when detecting by LDH, when having PN159 and lactic acid lycoremine, cytotoxicity is low.These detections show that all PN159 does not have toxicity to epithelial membrane.

When not having PN159, the P of lycoremine _AppBe about 2.1 * 10 ^-6Cm/s.When existence 25,50 and 100mM PN159, P _AppBe respectively 5.1 * 10 ^-6, 6.2 * 10 ^-6And 7.2 * 10 ^-6Cm/s.Therefore, at the P of this model low-molecular-weight drug _AppIn, PN159 provides 2.4-to 3.4-increase doubly.

TJMP increases the epithelium infiltration as the lycoremine of model low-molecular-weight drug surprisingly.PN159 is joined the infiltration that remarkable enhancing lycoremine passes the epithelial cell individual layer in the lycoremine solution.Evidence shows that temporary transient minimizing of PN159 passes the TER of epithelial membrane, and does not damage the cell in the film, as measuring by high cell survival ability and low cytotoxicity.TJMP strengthens the bioavailability of lycoremine and other small-molecule drug in vivo, and it strengthens by the identical mechanism of using the external model proof in this article.Further predict TJMP and also strengthen the infiltration of lycoremine in higher concentration.

Embodiment 31

TJMP is to proteic infiltration enhancement

Having determined that PN159 is used for the purposes of striding the mucous membrane preparation of low-molecular weight compound, importantly is to distinguish whether these observations can be extrapolated to bigger molecule, for example therapeutic peptide and albumen.For this purpose,, the salmon calcitonin see calcimar as the mode treatment peptide is carried out vitro tissue research not having and exist 25,50 and during 100mM PN159.When not having PN159, for the P of thyrocalcitonin _AppBe about 1 * 10 ^-7Cm/s is lower than the P of lycoremine _AppAbout order of magnitude infers that this is because the difference of molecular weight causes.When having PN159, the remarkable increase of the bright thyrocalcitonin infiltration of data sheet is compared with the situation of having only thyrocalcitonin, at P _AppIn reach 23-and doubly increase (table 30) to 47-.

Table 30

The Papp that uses the vitro tissue model to detect

^aThe pH value is 5.0

In order to study the ubiquity of these discoveries, when not existing and having PN159, two kinds of other peptides of check, i.e. human parathyroid hormone 1-34 (PTH in described external model _1-34) and people's peptide YY3-36 (PYY _3-36) (P _AppData presentation is in table 30).When not having PN159, the P of these two kinds of peptides _AppP with thyrocalcitonin _AppConsistent.At PTH _1-34Situation in, PN159 exists in P _AppIn about 3-5 is provided increase doubly.When in the presence of PN159, preparing PYY _3-36The time, Papp increases about 12-to 17-doubly.Our discovery of these data acknowledgements, promptly TJMP strengthens small molecules and proteicly strides the ubiquity that the mucous membrane medicine is sent.

Embodiment 32

The chemical stability of TJMP

Under the relevant condition of storage of treatment, determine the chemical stability of PN159.Use the HPLC method of expression stability.Solution (50mM) is kept under different pH value (4.0,7.3 and 9.0) and temperature (5 ℃, 25 ℃, 35 ℃, 40 ℃ and the 50 ℃) condition.Sample in pH value 4 contains the 10mM citrate buffer.Sample in pH value 7.3 and 9.0 contains the 10mM phosphoric acid buffer.Stability in storage result's (comprising Arrhenius graphing method (Arrhenius plot)) shows, chemically is being the most stable at low temperature and pH value PN159.For example, at 5 ℃ and pH 4.0 or pH7.3, for 6 months storage, existing basically, 100% PN159 recovered.When storage temperature is elevated to 25 ℃, after 6 months, there is 7% and 26% natural PN159 loss respectively for sample at pH 4 or pH 7.In the temperature of pH 9 and/or rising, for example, 40-50 ℃, ensue the quick consumption of PN159.4.0-7.3 pH value scope and freezing temperature range to envrionment temperature be maximally related for preparation in the nose.Therefore, these data support that TJMP can keep chemical integrity under the condition of storage relevant with the IN preparation.

Embodiment 33

In rabbit, assess tight junction modulating peptide in vivo by intranasal administration

In rabbit, carry out pharmacokinetics (PK) research, send the peptide YY (PYY) that uses and the drug plasma dynamic metabolism feature of various tight junction modulating peptides (TJMPs) by (IN) in the nose with assessment.

Animal model

In this research, New Zealand white rabbit (Hra:(NZW) SPF) as test subject, to assess the drug plasma dynamic metabolism of MC-4RA by intranasal administration and intravenous infusion.The processing of animal is according to the regulations of listing in USDA animal welfare method (USDAAnimal Welfare Act) (9CFR the 1st, 2 and 3 parts) and experimental animal feeding management and instruction manual (ILAR publication, 1996, national academy of sciences printing) specified condition is carried out in.

Because the dynamic metabolism curve that obtains from the medicine that is administered to rabbit is similar to the PK curve of identical medicine among the people very much, so select the animal subjects of rabbit as this research.

Administration is used

Experimental design and dosage regimen about the TJMPs of 9 kinds of detections are summarised in the table 31.All experimental group are all passed through in the nose (IN) and are used and give with single TJMP or phosphate-buffered saline (PBS: Zu He 205 μ g/kg PYY (3-36) negative control).Use autospencer and disposable plastic suction nozzle, with every kind of preparation applied once in left nostril.With the perk of animal head back, and when sucking, animal uses described dosage, so that allow capillary action that solution is sucked the nostril.After IN used, control animal head about 15 seconds in the position of perk backward was with any loss of the dosage that prevents to use.In this step process, take the careful of extreme, with any potential tissue injury of avoiding causing by contacting with mucous membrane in the nose.

Table 31

Test set is summed up

Group	Size of animal	Approach	Tight junction modulating agent (concentration)	PYY3 (μg/kg)
					1	5M	In the nose	PBS	205
2	5M	In the nose	PN159(50μM)	205
					3	5M	In the nose	PN161(100μM)	205
4	5M	In the nose	PN202(100μM)	205
					5	5M	In the nose	PN27(250μM)	205
6	5M	In the nose	PN58(500μM)	205
					7	5M	In the nose	PN73(500μM)	205
8	5M	In the nose	PN228(500μM)	205
					9	5M	In the nose	PN183(1000μM)	205
10	5M	In the nose	PN556(1000μM)	205

PN556 has the primary sequence identical with PN283, but does not have maleimide amine-modified at the N of peptide end.

Blood and plasma sample are collected

After using by the IN administration, by directly carrying out venipuncture and collect the successive blood sample from every animal at auricular vein.Before administration, after the administration 5,10,15,20,30,45,60,90, collected blood sample in 120 and 180 minutes.Sample collection is to containing in the pipe of EDTA dipotassium as anti-coagulant.Pipe cooled off before centrifugal.All samples carry out centrifugal in collection within an hour.Collect blood plasma, and transfer in the plastic jar of mark in advance, freezing in dry ice/acetone batch, before carrying out the pharmacokinetics analysis, be kept at approximately-70 ℃ then.

Carry out clinical observation constantly at each blood sampling, and two nostrils of using all animals in the test set at IN were being checked in 1 hour before 5 minutes and behind the intranasal administration just.

Analytical procedure

By using ELISA, analyze PYY (3-36) level from the sample of every animal in all study group.With before the administration and the test sample after the administration on HPLC, move and carry out quality control.Blood plasma aliquots containig (0.1mL) is the albumen that acetonitrile precipitation is used in the mark back in adding bioanalysis.With the supernatant liquor nitrogen drying, reconstruct in the HPLC damping fluid, and be then injected in the HPLC system.Detect effluent by positively charged ion electro-spray ionization three utmost point quadrupole mass spectrometers of connecting.The PK data are analyzed by WinNonlin (method Saudi company (Pharsight Corp.), Mountain View (Mountain View)).

The result

To be summarised in the table 32 about the average blood plasma PK parameter of each test set.After using any preparation, do not observe disadvantageous clinical indication.Show without any rubescent also there is not swelling behind the intranasal inspection in two nostrils of the animal by the IN administered formulation.PK research assessment C _Max(the maximum concentration of observing), T _MaxTerminal and the infinitely great limit (inf) of (moment of peak concentration) and AUC (area under curve).According to the horizontal 8 kinds of TJMPs of their body internal penetration, and be divided into 4 kinds of different performance grades, grade I comprises and has the most substantially TJMPs of internal penetration level, and each subsequent levels comprises the TJMPs with the body internal penetration level that reduces gradually.

Table 32

Pharmacokinetic data is summed up

Group	Hierarchical arrangement in the body	T 1/2	T max(minute)	C max (pg/mL)	AUClast (minute * pg/mL)	AUCinf (minute * pg/mL)
							PBS		86.0	22.0	806	4.5×10 4	6.81×10 4
PN159	I	30.2	17.0	30200	1.52×10 6	1.55×10 6
							PN161	I	34.3	24.0	32100	1.62×10 6	1.65×10 6
PN27	I	29.9	33.0	29300	1.67×10 6	1.71×10 6
							PN228	II	30.4	31.0	21200	1.06×10 6	1.08×10 6
PN202	II	34.1	32.0	12700	7.35×10 5	7.63×10 5
							PN58	III	29.5	43.0	12800	8.3×10 5	8.71×10 5
PN73	IV	53.8	37.0	8220	3.46×10 5	3.55×10 5
							PN183	IV	33.7	22.0	5450	2.58×10 5	2.75×10 5
PN556	IV	51.2	22.0	4620	2.47×10 5	2.80×10 5

Embodiment 34

Purifying

The PN159 peptide (table 33) that has synthesized following PEGization:

Table 33

The PN0159 peptide tabulation of synthetic PEGization

PN526	(SEQ.ID NO 58) PEG1-KLALKLALKALKAALKLA-acid amides
		PN537	(SEQ.ID NO 59) PEG (5000Da)-KLALKLALKALKAALKLA-acid amides
PN570	(SEQ.ID NO 60) NH2-KLALKLALKALKAALKLA-PEG 1-acid amides
		PN571	(SEQ.ID NO 61) PEG1-KLALKLALKALKAALKLA-PEG1-acid amides
PN572	(SEQ.ID NO 62) PEG3-KLALKLALKALKAALKLA-acid amides

The thick peptide of 150mg amount is absorbed in the water that 15mL contains 0.1%TFA and 3mL acetate.After stirring and supersound process, mixture is transferred in the 1.5mL Eppendorf pipe, and centrifugal at 13000rpm.Collect supernatant liquor, and filter by Millex GV 0.22um injection filter.This solution is encircled with sample on 5mL/ minute the flow velocity to Zorbax 300SBC18 post (21.2mm ID * 250mm, 7um granular size) by the 5mL injection.Finish purifying by 0.2%B/ minute linear AB gradient of operation, wherein solvent orange 2 A is that 0.1%TFA in water and solvent B are the 0.1%TFA in acetonitrile.Under these conditions, peptide elutes in the scope of 15-17%B.

Embodiment 35

Cell

EpiAirway ^TMCell (with 96 well format (Air-196-HTS) or 24 independent hole insets (Air-100)), a kind of people's tracheae/bronchial tissue's model, available from Ma Te Tyke (MatTek) company (ashland, MA), with screening tight junction modulating peptide (TJMPs), screening is carried out transepithelial electrical resistance (TER) and infiltrative effect based on them.Cultured tissue derives from single donor, and is negative for HIV, hepatitis B, hepatitis C, mycoplasma, bacterium, yeast and fungi screening.

EpiAirway is organized in refrigerated transport on the substratum that replenishes sepharose.The EpiAirway tissue was recovered 24 hours at 37 ℃ with the substratum that the manufacturer provides.The perfect medium (Epi-CM) that is used for the EpiAirway model contains DMEM, EFG and other factor, gentamicin (5 μ g/ml), amphotericin B (0.25 μ g/ml) and phenol red as the pH indicator.

Embodiment 36

Determine TER

(world's precision instrument (World PrecessionInstrument) is company limited (WPI), and (Sarasota, Florida State) carried out TER to Air-196-HTS and detected in the tissue resistance system (REMS) of use automatization.In order to monitor the TER in 96 hole HTS forms, in incubator for tissue culture (hood), use Endhom-Multi (STX), to prevent pollution.When spending the night the recovery inset, 100 μ l substratum are used for end face, 250 μ l substratum are used for basal compartment.Background TER detects with blank inset (Mi Sibo (Millipore)), and deducts from organize inset.By with the reversing of described inset on paper handkerchief and substratum is inclined to.Then, inset is beaten on paper handkerchief gently, to guarantee the removing top substratum to greatest extent.For other TER point detection time, after processing immediately with inset with 150 μ l Epi-CM rinse three times gently, and before TER detects, drain fully.

Result (Fig. 8) shows, that tight junction modulating peptide PN159 of the present invention that detects on the simple epithelium cell and the PEGization form of PN159 all have is strong, reversible strengthens the infiltrative effect of epithelium.Use the two observed work to take place in order to predictable mode.In addition, the result shows that PEG-159 strengthens ion permeability (reducing TER) significantly than independent PN159.Maximum differential between the PEG-PN159 and 159 in TER is at 50 μ M PEG-PN159.

Embodiment 37

Perviousness detects

The dextrin (MW 3,000) of fluorescein isothiocyanate (FITC) mark is added in the treating mixture with 0.1-1mg/ml.Treating mixture is added to the side of roof, and with flat board the orbital oscillation device (new Eintracht Braunschweig science, Ai Dixun, in New Jersey (New Brunswick Scientific, Edison, NJ)) with 100rpm 37 ℃ of specified times of incubation.At the incubation end, the basal culture medium of triplicate 200 μ l is transferred to black wall fluorescence read on the flat board.(VT) detection is in the fluorescence intensity of 470nm wavelength for rich Tyke difficult to understand (BIO-TEK) Instr Ltd., Winooski to read instrument FLx800 by microtest plate fluorescence.The serial dilution of standard substance is used for obtaining typical curve and calculating concentration.Perviousness detects in two ways, as the ratio of donor quality (top chamber) or as the ratio of acceptor quality (basal compartment), represents with percentage ratio.

When having PN159 of the present invention and PEG-PN159, observe the remarkable increase (Fig. 9) of PTH infiltration.Use the two observedly to act on some concentration dependent between 10 μ M and the 100 μ M.In addition, the result shows that PEG-PN159 is than the remarkable enhancing molecule infiltration of PN159.

When the perviousness of PEG-PN159 increases with PN159 relatively (drawing as the ratio between two values in Figure 10), the maximum differential of infiltration increase is in 50 μ M concentration.

Embodiment 38

Cytotoxicity detects

LDH detects the cytotoxicity that is used for assessing described processing.The LDH level detects (Pu Luomaige (Promega), Madison, the state of Wisconsin) by the "dead" cytotoxicity of CytoTox96 and determines according to manufacturer's method.For base side LDH level, the basal culture medium of triplicate 50 μ l is used for determining the LDH level.For top LDH level, by in the chamber, top, adding 150 μ l Epi-CM, and remove the top sample of the dilution of 150 μ l, substratum is mixed by pressure-vaccum up and down, and remove 150 μ l substratum and dilute 2 times (final 8 times of dilutions), be used for triplicate detection with 50 μ l.Total LDH level is determined by lysing cell in the Triton-X100 of final concentration 0.9%.LDH in every kind of sample is expressed as the percentage ratio of Triton-X100 lysis.Result (Figure 11) shows that PEG-PN159 has the cytotoxicity lower than PN159.

Embodiment 39

Pharmacokinetic data in the rabbit

With 25 male New Zealand rabbits, at about 3 monthly ages, be used for this research.Rabbit is accepted single intranasal administration, tight connection (TJ) peptide of nostril one dosage and PYY3-36 group, and this uses autospencer and disposable plastic suction nozzle to carry out.Rabbit is according to the TH peptide and the control group administration that show in table 34.TJ peptide (PN407, PN408, PN526 (PEG-PN159), and PN159) is all in having the 0.75x DPBS of calcium and magnesium.Negative control is the 0.75x DPBS (PBS) that only contains calcium and magnesium.Be not contained in the DDPC, the EDTA that comprise the TJ peptide in the citrate buffer and the positive PYY3-36 control formulation of MbCD and be used for comparison (PDF).

When sending described dosage, with the head perk a little backward of animal.After the administration, control animal head about 15 seconds in the position of perk backward.Collect successive blood sample (every part of about 1.5mL) by directly carrying out venipuncture, collect and contain in the blood collection tube of EDTA as anti-coagulant at auricular vein.0 (before the administration) after the group administration in nose, 5,10,15,30,45,60,120 and 240 minutes collection blood samples.After the collection, pipe reverses several times gently, with anticoagulation, adds 50 μ l bovine pancreatic trypsin inhibitors then in collection tube, and still thoroughly mixes gently.About 4 ℃ with about 1, before centrifugal 15 minutes of the 600xg, the blended sample is placed in the frozen-pack.Plasma sample is divided into duplicate aliquots containig (every part of about 0.35mL), and is kept at approximately-70 ℃ then.

Table 34

The administration group that is used for the research of rabbit pharmacokinetics

Use is purchased ELISA test kit (" active total peptide YY (PYY) ELISA ", catalog number (Cat.No.) .DSL-10-33600, diagnositc system experiment (the Diagnostic Systems Laboratories of company limited, Inc.), Robert Webster (Webster), Texas) the PYY3-36 bioanalysis of carrying out in rabbit plasma detects.Described detection is a kind of enzymatic amplification " step " sandwich-like immunodetection.In described detection, using anti--PYY antibody incubation in the microtiter well with another kind of resisting-PYY antibody sandwich with proofreading and correct thing, contrast and unknown sample.After incubation and the washing, hole luminous substrate, tetramethyl benzidine incubation.Add acid stop bath then, and by 450 and the dual wavelength absorbance detection of 620nm determine the enzymatic degree of conversion of substrate.The concentration of the absorbancy of measuring and the PYY of existence is proportional.

The logical data simplified method of 5 parameters (five-parameter logistic data reductionmethod) is used to proofread and correct the result, to produce the calibration curve about every kind of detection.Calibration curve is used for according to their absorbancy result and the PYY concentration value of interior insertion unknown sample.The test kit composition is used for all detection steps, except following exception: PYY _3-36Reference substance is used for producing correction thing and contrast; Proofread and correct thing and contrast the rabbit plasma that merges with wash-out (strip) (C18 solid-phase extraction column) and prepare as thinner; And if desired, unknown sample is diluted in the rabbit plasma of wash-out merging.Will be at the antibody Combinatorial Optimization in this test kit, to detect complete people PYY _1-36, and with mouse PYY _1-36With people PYY _3-36Fully cross reaction.

Average pharmacokinetics (PK) data and standard deviation (SD) about contrast (PBS and PDF) and TJ peptide (PN159, PN407, PN408, and PN526) preparation are presented in the table 35.Relative bioavailability (%BA) about every kind of tight junction modulating agent and contrast is presented in the table 36.Variation factor percentage ratio about the pharmacokinetics variable is presented in the table 37.

Table 35

About the PYY in rabbit _3-36Mean P K parameter and standard deviation (SD)

Preparation	T max(minute)	C max (pg/mL)	AUC last(minute * pg/mL)	AUC inf(minute * pg/mL)	T1/2 (minute)	Kel (1/ minute)
							PBS	33.75	2646.25	118438.13	147625.18	83.12	0.009
SD	18.87	1381.06	23611.86	42331.68	22.53	0.003
							PDF	30.00	19004.40	1289219.50	1319034.73	38.56	0.019
SD	10.61	8174.32	589127.80	612688.59	11.12	0.005
							PN159	27.00	18346.60	973038.80	985572.89	34.43	0.021
SD	19.56	9671.72	549668.76	546060.77	7.20	0.005
							PN407	21.00	13980.20	725950.50	753080.86	47.46	0.016
SD	8.22	7124.99	388368.38	397975.49	14.51	0.004
							PN408	15.00	15420.00	721601.50	758951.24	44.23	0.016
SD	0.00	7644.40	361013.89	360247.20	8.23	0.003
							PN526	27.00	36066.20	1786973.50	1819888.30	41.04	0.018
SD	6.71	22447.13	1065867.60	1084222.74	9.66	0.005

Table 36

The % bioavailability of tight junction modulating agent

Preparation	AUC last(minute * pg/mL)	％F
			PBS	118438.13	9.19
PDF	1289219.50
			PN159	973038.80	75.48
PN407	725950.50	56.31
			PN408	721601.50	55.97
PN526	1786973.50	138.61

Table 37

Variation % coefficient about pharmacokinetic parameter

Preparation	T max(minute)	C max (pg/mL)	AUC last(minute * pg/mL)	AUC inf(minute * pg/mL)
					PBS	55.9	52.2	19.9	28.7
PDF	35.4	43.0	45.7	46.4
					PN159	72.4	52.7	56.5	55.4
PN407	39.1	51.0	53.5	52.8
					PN408	0.0	49.6	50.0	47.5
PN526	24.8	62.2	59.6	59.6

Quantized lower limit (LLOQ) is thought 15.8pg/mL.The raw value of any＜NUMBER is made as 7.9pg/mL to be used for analyzing.Nose is used back mean P YY _3-36Plasma concentration shows with linear graph in Figure 12, shows with logarithm-linear graph in Figure 13.Use the mean P YY of the animal of nose dosage _3-36Serum-concentration is represented all group peak concentration (T between 15-34 after the administration minute _Max).For nose PBS with the dosage level of 205 μ g/kg; PDF; PN159; PN407; The average C of PN408 and PN526 _MaxBe respectively 2,646.25; 19,004.40; 18,346.60; 13,980.20; 15,420.00 and 36,066.20pg/mL.For nose PBS; PDF; PN159; PN407; The average A UC of PN408 and PN526 _LastBe respectively 118,438.13; 1,289,219.50; 973,038.80; 725,950.50; 721,601.50 and 1,786,973.50 minutes * pg/mL.For nose PBS; PDF; PN159; PN407; The average A UC of PN408 and PN526 _InfBe respectively 147,625.18; 1,319,034.73; 985,572.89; 753,080.86; 758,951.24 and 1,819,888.30 minutes * pg/mL.For all nasal preparation t1/2 is about 35-48 minute; Yet PBS is 83 minutes.Referring to the complete list of table 35 about all pharmacokinetic parameters of comprising standard deviation.For PN159, PN407, PN408 and PN526 are based on the AUC about the relative PDF preparation of tight junction modulating agent _Last%BA be respectively 75,56,56 and 139%.Compare with PDF, the % bioavailability of PBS only is 9%.Also compared the variation coefficient (table 37).When comparative drug dynamic metabolism parameters C between preparation _MaxAnd during AUC, all tight junction modulating agent have similar variation.Use the pharmacokinetics between all 5 kinds of preparation groups of one-way analysis of variances model analysis to change, and find about C _Max, AUC _LastAnd AUC _InfThe PBS preparation significantly is lower than PN526.(T _max：p＝0.27；C _max：p＝0.009；AUC _last：p＝0.008；AUC _inf：p＝0.0097)。

Compare C _Max, the tight junction modulating agent PN526 of PEGization is higher than 1.9 times of PDF, and be higher than respectively PBS, PN407 and PN40813.6,2.6 and 2.3 times.Compare AUC _Last, the tight junction modulating agent PN526 of PEGization is higher than 1.4 times of PDF, and be higher than respectively PBS, PN407 and PN40815.1,2.5 and 2.5 times.Except PBS 80 minutes, for all about 40 minutes of t1/2 of group.

As comparative drug dynamic metabolism parameter, C _MaxDuring with AUC, between PN526 and PBS preparation, there is significant difference; Yet, between the tight junction modulating agent, do not have significance.

Compare with all other tight junction modulating agent, use the PN526 bioavailability to increase, and compare with the PBS control formulation, pharmacokinetic parameter is a significance,statistical.These data show, the peptide formulations of PEGization, and PN526 has the %BA of increase, and it is higher than the preparation of the peptide that does not have PEGization, PN159, PN407, PN408, and PBS.In addition, the %BA of PN526 also is higher than the positive control PDF of the peptide that does not have PEGization.

The embodiment that this paper provides is presented for purposes of illustration, and is not intended to limit the present invention's scope as claimed in claim.Although use concrete term and value in this article, it is representational that described term and value are construed as, and does not limit the scope of the invention.

For all purposes, all publications quoted in this disclosure and reference are incorporated into this by reference fully.

Claims (according to the modification of the 19th of treaty)

1. compound or pharmaceutically acceptable salt thereof that contains peptide, it has the activity that the mucosal epithelium of enhanced activity agent is transported by the perviousness of the described mucous membrane of regulation and control in the Mammals mucous membrane, wherein said peptide has the molecular weight less than 10 kilodaltons, and wherein said peptide comprises the aminoacid sequence of the SEQ ID NO:34 that prolongs one or more amino-acid residues.

2. the compound of claim 1, wherein said peptide comprises the aminoacid sequence that is selected from the group of being made up of SEQ ID NOS:41-43.

3. compound or pharmaceutically acceptable salt thereof that contains peptide, it has the activity that the mucosal epithelium of enhanced activity agent is transported by the perviousness of the described mucous membrane of regulation and control in the Mammals mucous membrane, wherein said peptide has the molecular weight less than 10 kilodaltons, and comprises the aminoacid sequence of SEQ ID NO:35.

4. the compound of claim 3, wherein said peptide is selected from the group of being made up of SEQ ID NO:35.

5. compound or pharmaceutically acceptable salt thereof that contains peptide, it has the activity that the mucosal epithelium of enhanced activity agent is transported by the perviousness of the described mucous membrane of regulation and control in the Mammals mucous membrane, wherein said peptide has the molecular weight less than 10 kilodaltons, and comprises the aminoacid sequence of SEQ ID NO:38.

6. the compound of claim 5, wherein said peptide is selected from the group of being made up of SEQ ID NO:38.

7. compound or pharmaceutically acceptable salt thereof that contains peptide, it has the activity that the mucosal epithelium of enhanced activity agent is transported by the perviousness of the described mucous membrane of regulation and control in the Mammals mucous membrane, wherein said peptide has the molecular weight less than 10 kilodaltons, and comprises the aminoacid sequence of the SEQ ID NO:34 that is rich at least 60% Methionin, leucine and/or L-Ala.

8. the compound of claim 7, wherein said peptide comprise and are selected from the NOS:32 by SEQ ID, the aminoacid sequence of 33,36 and 50 groups of forming.

9. each compound among the claim 1-8 wherein strengthens in perviousness described in the mucous membrane, and keeps the cell survival ability.

10. each compound among the claim 1-8, wherein said compound and water-soluble chain are covalently bound.

11. the compound of claim 10, wherein said compound are poly-(oxirane) chains.

12. the compound of claim 11, wherein said poly-(oxirane) chain are that side chain arranged or unbranched.

13. the compound of claim 12, wherein said poly-(oxirane) chain is polyoxyethylene glycol (PEG) chain.

14. the compound of claim 13, wherein said PEG have about 0.2 and about 200 kilodaltons (kDa) between molecular size.

15. the compound of claim 13, wherein said PEG has the size that is lower than 40kDa, and preferably wherein said PEG has the size that is lower than 20kDa, and more preferably wherein said PEG has the size that is lower than 10kDa.

16. the compound of claim 13, wherein said PEG has the size that is lower than 5kDa, and preferably wherein said PEG has the size that is lower than 2kDa.

17. the compound of claim 13, wherein said poly-(oxirane) has the polydispersity value (Mw/Mn) less than 2.00.

18. the compound of claim 13, wherein said poly-(oxirane) has the polydispersity value (Mw/Mn) less than 1.20.

19. a pharmaceutical preparation, it comprises among the claim 1-18 that strengthens mucosal epithelium transhipment significant quantity each compound or comprises the compound of aminoacid sequence of described SEQ ID NO:34 and the promoting agent of treatment significant quantity.

20. the preparation of claim 19, wherein said preparation reduces to pass the resistance of mucous membrane tissue barrier.

21. the preparation of claim 20, wherein said resistance be reduced at least 80%.

22. the preparation of claim 21, wherein said preparation increases the perviousness that described promoting agent passes the mucous membrane tissue barrier with respect to the compound that does not comprise among the claim 1-33 each or the similar preparation of compound that contains the aminoacid sequence of SEQ ID NO:34.

23. the preparation of claim 22, the wherein infiltrative twice at least that increases to.

24. the preparation of claim 22, wherein said perviousness is a parietal cell.

25. the preparation of claim 22, the perviousness of wherein said increase are closely connected by regulation and control and cause.

26. the preparation of claim 22, wherein said perviousness are transcellular or stride cell and the mixing of parietal cell.

27. the preparation of claim 22, wherein said mucous membrane tissue barrier is an epithelium layer.

28. the preparation of claim 27, wherein said epithelial cell is selected from by tracheal cell, segmental bronchus cell, alveolar cell, nose cell, pneumonocyte, gastrointestinal cell, the group that epidermic cell and Stomatocyte are formed.

29. the preparation of claim 28, wherein said epithelial cell are the nose cells.

30. the preparation of claim 19, wherein said promoting agent are peptide, albumen or nucleic acid.

31. the preparation of claim 30, wherein said peptide or albumen comprise 2-1000 amino acid.

32. the preparation of claim 30, wherein said peptide or albumen comprise 2-50 amino acid.

33. the preparation of claim 30, wherein said peptide or albumen are annular.

34. the preparation of claim 30, wherein said peptide or albumen are dimer or oligomer.

35. the preparation of claim 30, wherein said peptide or albumen are selected from the group of being made up of the inhibitor of GLP-1, PYY3-36, PTH1-34 and glucagon-like peptide-4.

36. the preparation of claim 30, wherein said albumen are selected from the group of being made up of beta-interferon, alpha-interferon, Regular Insulin, erythropoietin, G-CSF, GM-CSF, tethelin and their analogue.

37. comprise the formulation of each preparation among the claim 19-36, wherein said formulation is a liquid.

38. the formulation of claim 37, wherein said liquid is droplet form.

39. the formulation of claim 37, wherein said liquid is aerosol form.

40. comprise the formulation of each preparation among the claim 19-36, wherein said formulation is a solid.

41. the formulation of claim 40, wherein said solid is reconstruct in liquid before using.

42. the formulation of claim 40, wherein said solid is used as powder.

43. the formulation of claim 40, wherein said solid are capsule, tablet or gel form.

44. use the method for molecule to animal for one kind, it comprises provides as each preparation among the claim 19-36, and described preparation is contacted with the mucous membrane surface of described animal.

45. the method for claim 44, wherein said mucous membrane surface is in the nose.

46. the method for the bioavailability of a promoting agent that increases intranasal administration in Mammals, it comprises provides as each preparation among the claim 19-36, and described preparation is administered to described Mammals.

47. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is siRNA.

48. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is dsDNA.

49. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a hematopoietic; Anti-infective; The dementia resisting medicine; Antiviral agent, antitumour drug, febrifugee, anodyne, antiphlogistic drug, anti-ulcerative drug, anti-allergic agent, thymoleptic, antipsychotic medicine, cardiotonic drug, anti-arrhythmic, vasodilator, antihypertensive drug, hypotensor diuretic(s), antidiabetic drug, anti-coagulant, pravastatin is used for the therapeutical agent of osteoporosis, hormone, microbiotic, or vaccine.

50. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a cytokine, peptide hormone, somatomedin, the cardiovascular factor, cell adhesion factor, the central or peripheral nervous system factor, the body fluid ionogen factor, the blood organic substance, bone growth factor, the gastro-intestinal system factor, the kidney factor, the reticular tissue factor, the sensory organ factor, the immunity system factor, the respiratory system factor, or the reproductive organ factor.

51. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is male sex hormone, oestrogenic hormon, prostaglandin(PG), tethelin, gonad-stimulating hormone, interleukin, steroid or cytokine.

52. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is the vaccine that is used for hepatitis, influenza, respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, canary pox, varicella, measles, mumps, rubella, pneumonia or Human Immunodeficiency Virus (HIV).

53. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is the bacterial toxoid of diphtheria, tetanus, pseudomonas (pseudomonas) or mycobacterium tuberculosis (mycobactriumtuberculosis).

54. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a r-hirudin, hirulos or leech element.

55. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is monoclonal antibody, polyclonal antibody, humanized antibody, antibody fragment or immunoglobulin (Ig).

56. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a morphine, hydromorphone, oxymorphone, Lip river cut down coffee promise, levallorphan, morphine monomethyl ether, Nalmefene, nalorphine, naloxone, TREXUPONT; buprenorphine, butorphanol, or nalbuphine.

57. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a cortisone, hydrocortisone, fluohydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, Betamethasone Valerate, paramethasone, or fluocinolone acetonide.

58. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a colchicine, paracetamol, acetylsalicylic acid, Ibuprofen BP/EP, Ketoprofen, indomethacin, Naproxen Base, meloxicam, or piroxicam.

59. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18; wherein said promoting agent is an acyclovir; ribavirin; trifluorothymidine, Ara-A (ARA-A), acyl group guanosine; Nordeoxyguanosine; Zidovodine, DIDEOXYADENOSINE, or zalcitabine.

60. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a spironolactone, testosterone, estradiol, Progesterone, gonad-stimulating hormone, oestrogenic hormon or progesterone.

61. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is a Papaverine, pannonit, vasoactive intestinal peptide, the gene peptide that thyrocalcitonin is relevant, Cyproheptadine, doxepin, imipramine, Cimitidine Type A/AB, Dextromethorphane Hbr, Clozaril, superoxide dismutase, neural enkephalinase, amphotericin B, grisovin, miconazole, KETOKONAZOL, tioconazole, itraconazole, fluconazole, cynnematin, tsiklomitsin, aminoglycoside, erythromycin, gentamicin, PXB, 5 FU 5 fluorouracil, bleomycin, Rheumatrex, and hydroxyurea, didanosine, floxuridine, Ismipur, Dx, daunorubicin, idarubicin, safe plain, taxol, vitamin-E, Quinidine, Prazosin, verapamil, nifedipine, or diltiazem

62. each formulation among each preparation or the claim 37-43 among each compound or the claim 19-36 among the claim 1-18, wherein said promoting agent is to organize profibr(in)olysin activation factor (TPA), Urogastron (EGF), fibroblast growth factor (the acid or alkalescence of FGF-), Thr6 PDGF BB (PDGF), transforming growth factor (TGF-α or β), vasoactive intestinal peptide, tumour necrosis factor (TNF), hypothalamic releasing factor, prolactin antagonist, thyrotropic hormone (TSH), thyroliberin (ACTH), Rat parathyroid hormone 1-34 (PTH), follicle stimulating hormone (FSF), gonadotropin releasing hormone (LHRH), endorphin, hyperglycemic-glycogenolytic factor, thyrocalcitonin, pitocin, carbetocin, aldoetecone, enkephalin, somatostin, tethelin, somatomedin, α-melanotropin, lignocaine, sufentanil, terbutaline, droperidol, Scopolamine, gonadorelin, ciclopirox, buspirone, thyrocalcitonin, sodium cromoglycate or midazolam, ciclosporin, lisinopril, captopril, delapril, Ranitidine HCL, famotidine, superoxide dismutase, asparaginase, arginase, the arginine desaminase, the adenosine deaminase rnase, trypsinase, chemical trypsinase, papoid, bombesin, P material, beta-hypophamine, alpha-globulin, transferrin, factor I, beta lipoprotein, betaglobulin, prothrombin, ceruloplasmin, α 2-glycoprotein, α 2-sphaeroprotein, Pp63 glycophosphoproteins, A1LP, α 1-sphaeroprotein, albumin, or prealbumin.

63. medicament production, it comprise among the compound that contains among the claim 1-18 each or the claim 19-36 each preparation or claim 37-43 in each formulation solution and be used in mucous membrane, the nose or the setter of lung's spraying.

Claims (78)

1. compound that contains peptide, it has in the Mammals mucous membrane activity of the mucosal epithelium transhipment of enhanced activity agent by the perviousness of regulation and control mucous membrane, or its pharmaceutical salts.

2. the compound of claim 1, wherein said peptide has the molecular weight less than 10 kilodaltons.

3. the compound of claim 1, wherein said peptide is a tight junction modulating peptide.

4. the compound of claim 1, wherein said compound contains nexin transduction domain, DNA-binding domains or fusion structure territory.

5. the compound of claim 1, wherein said compound contains Zinc finger domain.

6. the compound of claim 1, wherein said compound contains at least 60% Methionin, leucine and/or alanine residue.

7. the compound of claim 1, wherein said compound formation alpha-helix.

8. the compound of claim 1, wherein said perviousness is external or intravital.

9. the compound of claim 1, the reversible enhancing of wherein said perviousness.

10. the compound of claim 1, wherein said perviousness is basically less than about 90 minutes time cycle.

11. the compound of claim 1, wherein said perviousness are enhanced being less than in about 60 minutes time cycle basically.

12. the compound of claim 1 wherein is enhanced in perviousness described in the described mucous membrane, and does not induce basic cytolysis.

13. the compound of claim 1 wherein is enhanced in perviousness described in the described mucous membrane, and keeps the cell survival ability.

14. the compound of claim 1, the mucous membrane lipid is mobile to be strengthened wherein said perviousness by increasing.

15. the compound of claim 1, wherein said compound are peptide PN159.

16. the compound of claim 1, wherein said compound are analogue, conjugate, derivative, variant, fragment, stand-in, fusion molecule or the mixture of PN159.

17. the compound of claim 1, wherein said peptide are selected from the group of being made up of SEQ.ID NOs 1-72.

18. the compound of claim 1, wherein said peptide are selected from the 32-35 by SEQ.ID NOs, and 38,46-49,53 and 55 groups of forming.

19. each compound among the claim 1-18, wherein said compound and water-soluble chain are covalently bound.

20. the compound of claim 19, wherein said compound has the molecular weight less than about 300 kilodaltons.

21. the compound of claim 19, wherein said compound has the molecular weight less than about 200 kilodaltons.

22. each compound among the claim 1-21, wherein said compound is covalently bound with poly-(oxirane) chain.

23. the compound of claim 22, wherein said poly-(oxirane) chain are that side chain arranged or unbranched.

24. the compound of claim 22, wherein said poly-(oxirane) chain is polyoxyethylene glycol (PEG) chain.

25. the compound of claim 24, wherein said PEG have about 0.2 and about 200 kilodaltons (kDa) between molecular size.

26. the compound of claim 24, wherein said PEG has the size that is lower than 40kDa.

27. the compound of claim 24, wherein said PEG has the size that is lower than 20kDa.

28. the compound of claim 24, wherein said PEG has the size that is lower than 10kDa.

29. the compound of claim 24, wherein said PEG has the size that is lower than 5kDa.

30. the compound of claim 24, wherein said PEG has the size that is lower than 2kDa.

31. the compound of claim 22, wherein said poly-(oxirane) has the polydispersity value (Mw/Mn) less than 2.00.

32. the compound of claim 22, wherein said poly-(oxirane) has the polydispersity value (Mw/Mn) less than 1.20.

33. the compound of claim 22, wherein said compound and poly-(oxirane) are puted together by compartment.

34. a pharmaceutical preparation, it comprises each compound and the promoting agent of treatment significant quantity among the claim 1-33 that strengthens mucosal epithelium transhipment significant quantity.

35. the preparation of claim 34, wherein said preparation reduces to pass the resistance of mucous membrane tissue barrier.

36. the preparation of claim 35, wherein said resistance be reduced at least 80%.

37. the preparation of claim 34, wherein said preparation increases the perviousness that described promoting agent passes the mucous membrane tissue barrier with respect to the similar preparation that does not comprise each compound among the claim 1-33.

38. the preparation of claim 37, the wherein infiltrative twice at least that increases to.

39. the preparation of claim 37, wherein said perviousness is a parietal cell.

40. the preparation of claim 37, the perviousness of wherein said increase are closely connected by regulation and control and cause.

41. the preparation of claim 37, wherein said perviousness are transcellular or stride cell and the mixing of parietal cell.

42. the preparation of claim 37, wherein said mucous membrane tissue barrier is an epithelium layer.

43. the preparation of claim 42, wherein said epithelial cell is selected from by tracheal cell, segmental bronchus cell, alveolar cell, nose cell, pneumonocyte, gastrointestinal cell, the group that epidermic cell and Stomatocyte are formed.

44. the preparation of claim 42, wherein said epithelial cell are the nose cells.

45. the preparation of claim 34, wherein said promoting agent are peptide, albumen or nucleic acid.

46. the preparation of claim 45, wherein said peptide or albumen comprise 2-1000 amino acid.

47. the preparation of claim 45, wherein said peptide or egg comprise 2-50 amino acid.

48. the preparation of claim 45, wherein said peptide or albumen are annular.

49. the preparation of claim 45, wherein said peptide or albumen are dimer or oligomer.

50. the preparation of claim 45, wherein said peptide or albumen are selected from the group of being made up of the inhibitor of GLP-1, PYY3-36, PTH1-34 and glucagon-like peptide-4.

51. the preparation of claim 45, wherein said albumen are selected from the group of being made up of beta-interferon, alpha-interferon, Regular Insulin, erythropoietin, G-CSF, GM-CSF, tethelin and their analogue.

52. comprise the formulation of each preparation among the claim 34-51, wherein said formulation is a liquid.

53. the formulation of claim 52, wherein said liquid is droplet form.

54. the formulation of claim 52, wherein said liquid is aerosol form.

55. comprise the formulation of each preparation among the claim 34-51, wherein said formulation is a solid.

56. the formulation of claim 55, wherein said solid is reconstruct in liquid before using.

57. the formulation of claim 55, wherein said solid is used as powder.

58. the formulation of claim 55, wherein said solid are capsule, tablet or gel form.

59. use the method for molecule to animal for one kind, it comprises provides as each preparation among the claim 34-58, and described preparation is contacted with the mucous membrane surface of described animal.

60. the method for claim 59, wherein said mucous membrane surface is in the nose.

61. the method for the bioavailability of a promoting agent that increases intranasal administration in Mammals, it comprises provides as each preparation among the claim 34-58, and described preparation is administered to described Mammals.

62. the compound of claim 1, wherein said promoting agent is siRNA.

63. the compound of claim 1, wherein said promoting agent is dsDNA.

64. the compound of claim 1, wherein said promoting agent is a hematopoietic; Anti-infective; The dementia resisting medicine; Antiviral agent, antitumour drug, febrifugee, anodyne, antiphlogistic drug, anti-ulcerative drug, anti-allergic agent, thymoleptic, antipsychotic medicine, cardiotonic drug, anti-arrhythmic, vasodilator, antihypertensive drug, hypotensor diuretic(s), antidiabetic drug, anti-coagulant, pravastatin is used for the therapeutical agent of osteoporosis, hormone, microbiotic, or vaccine.

65. the compound of claim 1, wherein said promoting agent is a cytokine, peptide hormone, somatomedin, the cardiovascular factor, cell adhesion factor, the central or peripheral nervous system factor, the body fluid ionogen factor, blood organic substance, bone growth factor, the gastro-intestinal system factor, the kidney factor, the reticular tissue factor, the sensory organ factor, the immunity system factor, the respiratory system factor, or the reproductive organ factor.

66. the compound of claim 1, wherein said promoting agent are male sex hormone, oestrogenic hormon, prostaglandin(PG), tethelin, gonad-stimulating hormone, interleukin, steroid or cytokine.

67. the compound of claim 1, wherein said promoting agent are the vaccines that is used for hepatitis, influenza, respiratory syncytial virus (RSV), parainfluenza virus (PIV), tuberculosis, canary pox, varicella, measles, mumps, rubella, pneumonia or Human Immunodeficiency Virus (HIV).

68. the compound of claim 1, wherein said promoting agent are the bacterial toxoids of diphtheria, tetanus, pseudomonas (pseudomonas) or mycobacterium tuberculosis (mycobactrium tuberculosis).

69. the compound of claim 1, wherein said promoting agent is a r-hirudin, hirulos or leech element.

70. the compound of claim 1, wherein said promoting agent are monoclonal antibody, polyclonal antibody, humanized antibody, antibody fragment or immunoglobulin (Ig).

71. the compound of claim 1, wherein said promoting agent is a morphine, hydromorphone, and oxymorphone, coffee promise (lovorphanol), levallorphan, morphine monomethyl ether, Nalmefene, nalorphine, naloxone, TREXUPONT, buprenorphine, butorphanol, or nalbuphine are cut down in the Lip river.

72. the compound of claim 1, wherein said promoting agent is a cortisone, hydrocortisone, fluohydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, Betamethasone Valerate, paramethasone, or fluocinolone acetonide.

73. the compound of claim 1, wherein said promoting agent is a colchicine, paracetamol, acetylsalicylic acid, Ibuprofen BP/EP, Ketoprofen, indomethacin, Naproxen Base, meloxicam, or piroxicam.

74. the compound of claim 1, wherein said promoting agent is an acyclovir, ribavirin, trifluorothymidine, Ara-A (ARA-A), acyl group guanosine, Nordeoxyguanosine, Zidovodine, DIDEOXYADENOSINE, or zalcitabine.

75. the compound of claim 1, wherein said promoting agent is a spironolactone, testosterone, estradiol, Progesterone, gonad-stimulating hormone, oestrogenic hormon or progesterone.

76. the compound of claim 1, wherein said promoting agent is a Papaverine, pannonit, vasoactive intestinal peptide, the gene peptide that thyrocalcitonin is relevant, Cyproheptadine, doxepin, imipramine, Cimitidine Type A/AB, Dextromethorphane Hbr, Clozaril, superoxide dismutase, neural enkephalinase, amphotericin B, grisovin, miconazole, KETOKONAZOL, tioconazole, itraconazole, fluconazole, cynnematin, tsiklomitsin, aminoglycoside, erythromycin, gentamicin, PXB, 5 FU 5 fluorouracil, bleomycin, Rheumatrex, and hydroxyurea, didanosine, floxuridine, Ismipur, Dx, daunorubicin, idarubicin, safe plain, taxol, vitamin-E, Quinidine, Prazosin, verapamil, nifedipine, or diltiazem

77. the compound of claim 1, wherein said promoting agent are to organize profibr(in)olysin activation factor (TPA), Urogastron (EGF), fibroblast growth factor (the acid or alkalescence of FGF-), Thr6 PDGF BB (PDGF), transforming growth factor (TGF-α or β), vasoactive intestinal peptide, tumour necrosis factor (TNF), hypothalamic releasing factor, prolactin antagonist, thyrotropic hormone (TSH), thyroliberin (ACTH), Rat parathyroid hormone 1-34 (PTH), follicle stimulating hormone (FSF), gonadotropin releasing hormone (LHRH), endorphin, hyperglycemic-glycogenolytic factor, thyrocalcitonin, pitocin, carbetocin, aldoetecone, enkephalin, somatostin, tethelin, somatomedin, α-melanotropin, lignocaine, sufentanil, terbutaline, droperidol, Scopolamine, gonadorelin, ciclopirox, buspirone, thyrocalcitonin, sodium cromoglycate or midazolam, ciclosporin, lisinopril, captopril, delapril, Ranitidine HCL, famotidine, superoxide dismutase, asparaginase, arginase, the arginine desaminase, the adenosine deaminase rnase, trypsinase, chemical trypsinase, papoid, bombesin, P material, beta-hypophamine, alpha-globulin, transferrin, factor I, beta lipoprotein, betaglobulin, prothrombin, ceruloplasmin, α 2-glycoprotein, α 2-sphaeroprotein, Pp63 glycophosphoproteins, A1LP, α 1-sphaeroprotein, albumin, or prealbumin.

78. a medicament production, it comprises the solution of the compound that contains claim 1 and is used in the mucous membrane, nose or the setter of lung's spraying.

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