CN101262888A - Formulations for enhanced mucosal delivery of PYY - Google Patents
Formulations for enhanced mucosal delivery of PYY Download PDFInfo
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- CN101262888A CN101262888A CNA2006800332373A CN200680033237A CN101262888A CN 101262888 A CN101262888 A CN 101262888A CN A2006800332373 A CNA2006800332373 A CN A2006800332373A CN 200680033237 A CN200680033237 A CN 200680033237A CN 101262888 A CN101262888 A CN 101262888A
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Abstract
Pharmaceutical formulations are described for enhancing mucosal delivery of peptide YY (PYY) to a mammal. A PYY dosage form is described that is suitable for multi-use administration. The PYY dosage form comprises a bottle containing an aqueous pharmaceutical formulation and an actuator effective intranasal administration of the formulation. The formulation comprises a therapeutically effective amount of PYY, a buffer to control pH, a water-miscible polar organic solvent and a chelating agent for cations. The PYY dosage form exhibits at least 90% PYY recovery after storage as used for greater than about five days.
Description
Background of invention
Obesity and associated conditions thereof all are general and very serious public health problem in the U.S. and the whole world.Shown when to the mammal administered peripherally some during in conjunction with the peptide of Y2 receptor, it induces weight loss.Described Y2 receptor-binding peptides is the neuropeptide in conjunction with the Y2 receptor.These Y2 receptor-binding peptides belong to the peptide family that comprises peptide YY (PYY), neuropeptide tyrosine (NPY) and pancreatic polypeptide (PP).
These are about 36 amino acid whose peptides and have the helical structure closely that comprises " PP-is folding " at the middle part of this peptide.Concrete feature comprises poly proline spiral, the βZhuan Jiao that is positioned at residue 9 to 14, the α spiral that is positioned at residue 15 to 30 that is positioned at residue 1 to 8, outwards outstanding C-terminal and the carboxyl terminal amide that is positioned at residue 30 to 36, and this carboxyl terminal amide is seemingly crucial for biological activity.Shown when by injection by peripherally administered when individual, be called peptide YY (1-36) [PYY (1-36)] [YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY, (SEQ ID NO:1)] 36 amino acid peptides therefore cause weight loss and can be used as the medicine of the fat and relevant disease of treatment, Morley, J., Neuropsychobiology 21:22-30 (1989).Be found to be afterwards and produced this effect PYY and Y2 receptors bind, and the decline that causes carbohydrate, protein and meal amount to absorb of Y2 agonist and combining of Y2 receptor, Leibowitz, S.F.et al., Peptides 12:1251-1260 (1991).Another molecular forms of PYY is PYY (3-36) IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (the residue 3-36 of SEQ ID NO:1), Eberlein, Eysselein et al., Peptides 10:797-803,1989).Hereinafter term PYY refers to total length PYY and in conjunction with any PYY fragment of Y2 receptor.
Knownly can give PYY with the fatal hypotension (U.S. Patent number 4 that in the shock that particularly endotoxin causes of suffering a shock, runs into of treatment by venoclysis or injection, 839,343), by perfusion, parenteral, vein or subcutaneous administration and by implanting to suppress the propagation (U.S. Patent number 5 of pancreas tumor in the mammal, 574,010), and treatment fat (Morley (1989) and Application No. 20020141985).Also statement can give PYY to domestic animal or people with the amount of the weight increase of the described individuality of effective raising by parenteral, oral, nasal cavity, rectum and local approach, this raising is by increasing the nutraceutical gastrointestinal absorption (U.S. Patent number 5 that cotransports that sodium relies on, 912,227).Yet, for treatment obesity and relevant disease, comprising diabetes, administering mode is restricted to vein IV infusion, does not optimize effective preparation of other administration that is used for PYY.The instruction of these prior aries does not all provide to contain with design and is used for strengthening the PYY of the mixed with excipients that mucosa (being nasal cavity, buccal, oral) sends or the preparation of PYY (3-36), they also instruction do not contain the value of endotoxic Y2 receptors bind peptide formulations for non-infusion administration.
In the past, the preparation that is used for the PYY intranasal administration is described to some extent in patent application, comprise patent application open WO040563142, US2004/0115135, US2004/0157777, US2004/0209807 and US2005/0002927, incorporate this paper into by the mode of reference.These applications disclose and have been adapted at containing among the 0.1ml 20 μ g to 200 μ g dosage, the i.e. preparations of 0.2mg/ml to 2.0mg/ml PYY concentration.In the preparation that comprises 10mM citrate and 100mM NaCl, 40 ℃ continue 5 days, under different pH value, tested the stability of the dosage form of 0.3mg/ml PYY.Find that optimal pH is 4.9, wherein after hatching 5 days, surpass 80% peptide reservation.Yet, find that subsequently PYY stability is subjected to the appreciable impact of PYY concentration: under higher concentration, the PYY stability decreases.In addition, the preparation that is used for intranasal administration of describing in the past and testing comprises the excipient such as methyl-p-cyclodextrin and L-α-DDPC, and they are not generally regarded as safe (GRAS) excipient.Therefore, in order to have the dosage form and the preparation of general effectiveness under the usual conditions that are provided at medicine, need the alternative preparation compositions that exploitation has the stability of raising badly.In addition, need exploitation badly and comprise the preparation of GRAS (generally recognized as safe) excipient and dosage form as the different choice of using non-pharmacopeia excipient.
Brief Description Of Drawings
Fig. 1: the PYY3-36 of the preparation of test infiltration in embodiment 1.
Fig. 2: the PYY3-36 of the preparation of test infiltration in embodiment 2.
Fig. 3: the PYY3-36 of the preparation of test infiltration in embodiment 3.
Fig. 4: the PYY3-36 of the preparation of test infiltration in embodiment 4.
Fig. 5: the peptide of relative time reclaims: (A) 25 ℃ of storages; (B) 40 ℃ of storages; (C) 50 ℃ of storages.
Fig. 6: the peptide of relative time reclaims, 40 ℃ of storages: (A) based on the preparation of citrate; (B) based on the preparation of acetate; (C) based on the preparation of glutamate, Glu; (D) not buffered preparation.
Fig. 7: the PYY3-36 stability of the sample of under the atomizing stress of the temperature that raises and 3 sprayings every day, in embodiment 8, testing.
Fig. 8: the PYY3-36 stability of the sample of under the atomizing stress of the temperature that raises and 3 sprayings every day, in embodiment 9, testing.
Fig. 9: the PYY3-36 stability under the atomizing stress of the temperature that raises and 3 sprayings every day, test in embodiment 10: (A) sample 5-1,5-2 and 5-3; (B) sample 5-4,5-5,5-6 and 5-7; (C) sample 5-8,5-9,5-10 and 5-11; (D) sample 5-12,5-13 and 5-14.
The detailed description of invention
In order to understand the present invention better, provide to give a definition and to describe in detail:
The Y2 receptor-binding peptides
The Y2 receptor-binding peptides that is used for mucosa preparation of the present invention comprises 3 naturally occurring biologically active peptide families, PP, NPY and PYY.Example of Y2 receptor-binding peptides and uses thereof is in U.S. Patent number 5,026,685; U.S. Patent number 5,574,010; U.S. Patent number 5,604,203; U.S. Patent number 5,696,093; U.S. Patent number 6,046,167; Gehlert et al., Proc.Soc.Exp.Biol.Med.218:7-22 (1998); Sheikh et al., Am.J.Physiol.261:701-15 (1991); Fournier et al., Mol.Pharmacol.45:93-101 (1994); Kirby et al., J.Med.Chem.38:4579-4586 (1995); Rist et al., Eur.J.Biochem.247:1019-1028 (1997); Kirby et al., J.Med.Chem.36:3802-3808 (1993); Grundemar et al., Regulatory Peptides 62:131-136 (1996); U.S. Patent number 5,696 has description in 093 (example of PYY agonist), the U.S. Patent number 6,046,167.According to the present invention; the Y2 receptor-binding peptides comprises: free alkali; acid-addition salts or slaine; for example potassium salt or sodium salt, or peptide are by the process (U.S. Patent number 6 of for example amidatioon, glycosylation, acidylate, sulphation, phosphorylation, acetylation and cyclisation; 093; 692, U.S. Patent number 6,225,445 and Pegylation) adorned Y2 receptor-binding peptides.
Peptide YY agonist
As used herein, " PYY " refers to the PYY (1-36) (SEQ IDNO:1) of native sequences or variant form, and PYY derivant, fragment and the analog in any source, no matter be natural, synthetic or reorganization.This PYY is made up of last at least 15 amino acid residue PYY (22-36) or its analog of PYY sequence.Other operable PYY peptide is PYY (1-36) (SEQID NO:1), PYY (3-36), PYY (4-36), PYY (5-36), PYY (6-36), PYY (7-36), PYY (8-36), PYY (9-36), PYY (10-36), PYY (11-36), PYY (12-36), PYY (13-36), PYY (14-36), PYY (15-36), PYY (16-36), PYY (17-36), PYY (18-36), PYY (19-36), PYY (20-36) and PYY (21-36).These peptides are usually in conjunction with Y receptor, especially Y2 and/or the Y5 receptor in brain and other places.Usually, these peptides are synthetic not contain endotoxin or pyrogen-free form, although always this is unessential.
Other PYY peptide comprises those PYY peptides that wherein carried out the conservative amino acid residues change, and for example the sudden change of the site-specific of PYY peptide comprises [Asp
15] PYY (15-36) (SEQ ID NO:2), [Thr
13] PYY (13-36) (SEQ ID NO:3), [Val
12] PYY (12-36) (SEQ ID NO:4), [Glu
11] PYY (11-36) (SEQ ID NO:5), [Asp
10] PYY (10-36) (SEQ ID NO:6), [Val
7] PYY (7-36) (SEQ ID NO:7), [Asp
6] PYY (6-36) (SEQ ID NO:8), [Gln
4] PYY (4-36) (SEQ ID NO:9), [Arg
4] PYY (4-36) (SEQ ID NO:10), [Asn
4] PYY (4-36) (SEQ ID NO:11), [Val
3] PYY (3-36) (SEQ ID NO:12) and [Leu
3] PYY (3-36) (SEQ ID NO:13).Other PYY peptide comprises those PYY peptides that wherein carried out at least two conservative amino acid residues changes, comprises [Asp
10, Asp
15] PYY (10-36) (SEQ IDNO:14), [Asp
6, Thr
13] PYY (6-36) (SEQ ID NO:15), [Asn
4, Asp
15] PYY (4-36) (SEQ ID NO:16) and [Leu
3, Asp
10] PYY (3-36) (SEQ ID NO:17).
The analog that also comprises PYY, for example U.S. Patent number 5,604, those disclosed in 203 and number 5,574,010.These comprise following peptide:
General formula 1A
For general formula 1A, can create following PYY (22-36) peptide analogues, wherein:
X is Cys or disappearance; R
1And R
2In each all be bonded on the amino acid whose alpha-amino nitrogen-atoms of N-terminal; R
1Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; R
2Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
8Aralkyl, or C
7-C
18Alkaryl; A
22Be ArAA, Ala, Aib, Anb, N-Me-Ala or disappearance; A
23Be Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala, D-Trp, or disappearance; A
24Be Leu, Gly, Ile, Val, Trp, Nle, Nva, Aib, Anb, N-Me-Leu, or disappearance; A
25Be Arg, Lys, homology (homo)-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
26Be Ala, His, Thr, 3-Me-His, 1-Me-His, β-pyrazolyl alanine, N-Me-His, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
27Be Nal, Bip, Pcp, Tic, Trp, Bth, Thi, or Dip; A
28Be Leu, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A
29Be Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A
30Be Leu, Ile, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A
31Be Val, Leu, Ile, Trp, Nle, Nva, Aib, Anb, or N-Me-Val; A
32Be Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp; A
33Be Cys, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or C
6-C
18Or Orn aryl); A
34Be Cys, Gln, Asn, Ala, Gly, N-Me-Gin, Aib, or Anb; A
35Be Cys, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or C
6-C
18Or Orn aryl); A
36Be ArAA, or Cys; R
3Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; R
4Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl, or its pharmaceutically-acceptable salts.
The example of PYY (22-36) general formula 1A peptide analogues comprises:
N-α-Ac-Ala-Ser-Leu-Arg-His-Trp-Leu-Asn-Leu-Val-Thr-Arg-Gln-A rg-Tyr-NH
2(SEQ ID NO:18); N-α-Ac-Ala-Ser-Leu-Arg-His-Thi-Leu-Asn-Leu-Val-Thr-Arg-Gln-A rg-Tyr-NH
2(SEQ ID NO:19), or its pharmaceutically-acceptable salts.
Operable other general formula 1A analog comprises:
Residue A wherein
28And A
29, A
29And A
30, A
30And A
31, A
31And A
32, A
33And A
34, A
34And A
35Or A
35And A
36Between--the CO--NH--key is by CH
2--NH, CH
2--S, CH
2--CH
2Or CH
2-O replaces, perhaps residue A wherein
35And A
36Between the CO--NH key by CH
2--NH replaces.
For general formula 1B, can create following PYY (22-36) peptide analogues, wherein:
X is Cys or disappearance; R
1And R
2Be bonded to N-terminal aminoacid; R
1Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; R
2Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; A
22Be ArAA or disappearance; A
23Be Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, Me-Ala, D-Trp, or disappearance; A
24Be Leu, Gly, Ile, Val, Trp, Nle, Nva, Aib, Anb, N-Me-Leu, or disappearance; A
25Be Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
26Be Ala, His, Thr, 3-Me-His, 1-Me-His, β-pyrazolyl alanine, N-Me-His, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or aryl), or Orn; A
27It is the ArAA except that Tyr; A
28Be Leu, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A
29Be Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A
30Be Leu, Ile, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A
31Be Val, Leu, Ile, Trp, Nle, Nva, Aib, Anb, or N-Me-Val; A
32Be Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp; A
33Be Cys, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or C
6-C
18Or Orn aryl); A
34Be Cys, Gln, Asn, Ala, Gly, N-Me-Gin, Aib, or Anb; A
35Be Cys, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or C
6-C
18Or Orn aryl); A
36Be ArAA, or Cys; R
3Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; R
4Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl, or its pharmaceutically-acceptable salts.
The example of PYY (22-36) general formula 1B peptide analogues comprises:
N-α-Ac-Tyr-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-A rg-Tyr-NH
2(SEQ ID NO:20), or its pharmaceutically-acceptable salts.
Can use other general formula 1B analog, wherein A
27Be Phe, Nal, Bip, Pcp, Tic, Trp, Bth, Thi, or Dip.
Another example of PYY (22-36) general formula 1B peptide analogues comprises: N-α-Ac-Phe-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-A rg-Tyr-NH
2(SEQ ID NO:21).
For general formula 2, can create following PYY (25-36) peptide analogues, wherein:
R
1And R
2Be bonded on the amino acid whose alpha-amino nitrogen-atoms of N-terminal; R
1Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; R
2Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; A
25Be Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
26Be Ala, His, Thr, 3-Me-His, β-pyrazolyl alanine, N-Me-His, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or aryl), or Orn; A
27It is ArAA; A
28Be Leu, Val, Trp, Nle, Nva, Aib, Aib, Anb, or N-Me-Leu; A
29Be Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A
30Be Leu, Ile, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A
31Be Val, Leu, Ile, Trp, Nle, Nva, Aib, Anb, or N-Me-Val; A
32Be Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp; A
33Be Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or C
6-C
18Aryl), Cys, or Orn; A
34Be Cys, Gln, Asn, Ala, Gly, N-Me-Gin, Aib, or Anb; A
35Be Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH--R, side chain or straight chain C
1-C
10Alkyl, or C
6-C
18Aryl), Cys, or Orn; A
36Be ArAA, or Cys; R
3Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; And R
4Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl, or its pharmaceutically-acceptable salts.
Can use other analog, the A of its formula of 2
27Be Phe, Nal, Bip, Pcp, Tic, Trp, Bth, Thi, or Dip.
The example of PYY (22-36) general formula 2 analog comprises:
N-α-Ac-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-TYr-NH
2Or its pharmaceutically-acceptable salts (SEQ.ID.NO:22).
Operable other general formula 2 analog comprise:
Residue A wherein
28And A
29, A
29And A
30, A
30And A
31, A
31And A
32, A
33And A
34, A
34And A
35Or A
35And A
36Between--the CO--NH--key is by CH
2--NH, CH
2--S, CH
2--CH
2Or CH
2-O replaces.
In addition, analog can comprise the dimer chemical compound, this dimer chemical compound comprises two peptides of two peptides, general formula 1B of general formula 1A or two peptides of general formula 2, perhaps 1 peptide of 1 peptide of 1 peptide of 1 peptide of 1 peptide of 1 of general formula 1A peptide and general formula 1B or general formula 1A and general formula 2 or general formula 1B and general formula 2; Wherein said dimer forms by amido link between described two peptides or disulphide bridges.
Abbreviation comprises the Asp=D=aspartic acid; The Ala=A=alanine; The Arg=R=arginine; The Asn=N=agedoite; The Cys=C=cysteine; The Gly=G=glycine; Glu=E=glutamic acid; The Gln=Q=glutamine; The His=H=histidine; The Ile=I=isoleucine; The Leu=L=leucine; Lys=K=lysine; The Met=M=methionine; The Phe=F=phenylalanine; The Pro=P=proline; The Ser=S=serine; The Thr=T=threonine; The Trp=W=tryptophan; Tyr=Y=tyrosine; The Val=V=valine; The Orn=ornithine; Nal=2-naphthyl alanine; The Nva=norvaline; The Nle=nor-leucine; The Thi=2-thienylalanine; The Pcp=4-chlorophenylalanine; Bth=3-benzothiophene alanine; Bip=4,4 '-the diphenylprop propylhomoserin; Tic=tetrahydroisoquinoline-3-carboxylic acid; The Aib=aminoisobutyric acid; Anb=. the amino n-butyric acie of α .-; Dip=2,2-diphenylprop propylhomoserin; And Thz=4-thiazole alanine (U.S. Patent number 5,604,203).
United States Patent (USP) 5,574, the analog of describing in No. 010 comprises following:
General formula 3
The analog of general formula 3, wherein X is a 0-5 amino acid whose chain, comprises the aminoacid of its N-terminal and R
1And R
2Bonding; Y is a 0-4 amino acid whose chain, comprises the aminoacid of its C-terminal and R
3And R
4Bonding; R
1Be H, C
1-C
12Alkyl (for example methyl), C
6-C
18Aryl (for example phenyl, naphthalene acetyl group), C
1-C
12Acyl group (for example formoxyl, acetyl group and myristoyl), C
7-C
18Aralkyl (for example benzyl), or C
7-C
18Alkaryl (for example p-aminomethyl phenyl); R
2Be H, C
1-C
12Alkyl (for example methyl), C
6-C
18Aryl (for example phenyl, naphthalene acetyl group), C
1-C
12Acyl group (for example formoxyl, acetyl group and myristoyl), C
7-C
18Aralkyl (for example benzyl), or C
7-C
18Alkaryl (for example p-aminomethyl phenyl); A
22Be ArAA, Ala, Aib, Anb, N-Me-Ala or disappearance; A
23Be Ser, Thr, Ala, N-Me-Ser, N-Me-Thr, N-Me-Ala, or disappearance; A
24Be Leu, lie, Vat, Trp, Gly, Aib, Anb, N-Me-Leu, or disappearance; A
25Be Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH-R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
26Be His, Thr, 3-Me-His, 1-Me-His, β-pyrazolyl alanine, N-Me-His, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH-R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
27It is the ArAA except that Tyr; A
28Be Leu, Ile, Vat, Trp, Aib, Aib, Anb, or N-Me-Leu; A
29Be Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A
30Be Leu, Ile, Val, Trp, Aib, Anb, or N-Me-Leu; A
31Be Vat, Ile, Trp, Aib, Anb, or N-Me-Val; A
32Be Thr, Ser, N-Me-Set, or N-Me-Thr; R
3Be H, C
1-C
12Alkyl (for example methyl), C
6-C
18Aryl (for example phenyl, naphthalene acetyl group), C
1-C
12Acyl group (for example formoxyl, acetyl group and myristoyl), C
7-C
18Aralkyl (for example benzyl), or C
7-C
18Alkaryl (for example p-aminomethyl phenyl); R
4Be H, C
1-C
12Alkyl (for example methyl), C
6-C
18Aryl (for example phenyl, naphthalene acetyl group), C
1-C
12Acyl group (for example formoxyl, acetyl group and myristoyl), C
7-C
18Aralkyl (for example benzyl), or C
7-C
18Alkaryl (for example p-aminomethyl phenyl), or its pharmaceutically-acceptable salts.
The particularly preferred analog of general formula 3 comprises:
N-.α.-Ala-Ser-Leu-Arg-His-Trp-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH
2(SEQ.ID.NO：23)。
Another peptide YY analog is a general formula 4, wherein N-terminal aminoacid and R
1And R
2Bonding; Y is a 0-4 amino acid whose chain, comprises the aminoacid of its C-terminal and R
3And R
4Bonding; R
1Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; R
2Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; A
25Be Arg, Lys, homology-Arg, diethyl-homology Arg, (wherein R is H to Lys-∈-NH-R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
26Be Ala, His, Thr, 3-Me-His, 1-Me-His, β-pyrazolyl alanine, N-Me-His, Arg, Lys, homology-Arg, diethyl-homology-Arg, (wherein R is H to Lys-∈-NH-R, side chain or straight chain C
1-C
10Alkyl, or aryl), Orn, or disappearance; A
27It is ArAA; A
28Be Leu, Ile, Val, Trp, Aib, Anb, or N-Me-Leu; A
29Be Asn, Ala, Gln, Gly, Trp, or N-Me-Asn; A
30Be Leu, Ile, Val, Trp, Aib, Anb, or N-Me-Leu; A
31Be Val, Ile, Trp, Aib, Anb, or N-Me-Val; A
32Be Thr, Set, N-Me-Set, or N-Me-Thr or D-Trp; R
3Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl; And R
4Be H, C
1-C
12Alkyl, C
6-C
18Aryl, C
1-C
12Acyl group, C
7-C
18Aralkyl, or C
7-C
18Alkaryl, or its pharmaceutically-acceptable salts.
It should be noted that except as otherwise noted, for all peptide YY agonist described herein, each amino acid residue, for example Leu and A
1, represent the structure of NH--C (R) H--CO--, wherein R is a side chain.Line representative between the amino acid residue connects amino acid whose peptide bond.Equally, when amino acid residue when being optically active, be intended to refer to L type conformation, unless clearly indicate the D type.
Abbreviation: Aib=aminoisobutyric acid; Anb=. the amino n-butyric acie of α .-; Bip=4,4 '-the diphenylprop propylhomoserin; Bth=3-benzothiophene alanine; Dip=2,2-diphenylprop propylhomoserin; Nat=-naphthyl alanine; The Orn=ornithine; The Pcp=4-chlorophenylalanine; The Thi=2-thienylalanine; Tic=tetrahydroisoquinoline-3-carboxylic acid.(U.S. Patent number 5,574,010).
The example of other PYY synthetic analogues comprises:
[im-DNP-His
26] PYY:YPAKPEAPGEDASPEELSRYYASLR[im-DNP-His
26] YLNLVTRQRY--NH
2(SEQ.ID No.24); [Ala
32] PYY:ASLRHYLNLV[Ala] RQRY-NH
2(SEQ.ID No.25); [Ala
23,32] PYY:A[Ala] LRHYLNLV[Ala] RQRY-NN
2(SEQ.ID No.26); [Glu
28] PYY (22-36): ASLRHY[Glu] NLVTRQRY--NH
2(SEQ.IDNo.27); N-α-Ac-PYY (22-36): N-α-Ac-ASLRHYLNLVTRQRY--NH
2(SEQ.ID No.28); N-α-Ac[p.CL.Phe.sup.26] PYY:N-α-Ac-ASLR[p.Cl.Phe
26] YLNLVTRQR (SEQ.ID No.29); N-α-Ac[Glu
28] PYY:N-α-Ac-ASLRHY[GIu] NLVTRQRY-NH
2(SEQ.ID No.30); N-α-Ac[Phe
27] PYY:N-α-Ac-ASLRH[Phe] ENLVTRQR[N-Me-Tyr]-NH
2(SEQ.ID No.31); N-α-Ac] 8N-Me-Tyr] PYY:N-α-Ac-ASLRHYENLVTRQR[N--Me--Tyr]--NH
2(SEQ.ID No.32); N-α-myristoyl-PYY (22-36): N-α-myristoyl-ASLRHYLNLVTRQRY--NH
2(SEQ.ID No.33); N-α-naphthalene acetyl group-PYY (22-36): N-α-naphthalene acetyl group-ASLRHYLNLVTRQR (SEQ.ID No.34); N-α-Ac[Phe
27] PYY:N-α-Ac-ASLRH[Phe] ENLVTRQR[N--Me--Tyr]--NH
2(SEQ.ID No.35); N-α-Ac-PYY (22-36): N-α-Ac-ASLRHYLNLVTRQRY--NH
2(SEQ.ID No.36); N-α-Ac-[Bth
27] PYY (22-36): N-α-Ac-ASLRH[Bth] LNLVTRQRY--NH
2(SEQ.ID No.37); N-α-Ac-[Bip
27] PYY (22-36): N-α-Ac-ASLRH[Bip] LNLVTRQRY--NH
2(SEQ.ID No.38); N-α-Ac-[Nal
27] PYY (22-36): N-α-Ac-ASLRH[NaL] LNLVTRQRY--NH
2(SEQ.ID No.39); N-α-Ac-[Trp
27] PYY (22-36): N-α-Ac-ASLRH[Trp] LNLVTRQRY--NH
2(SEQ.ID No.40); N-α-Ac-[Thi
27] PYY (22-36): N-α-Ac-ASLRN[Thi] LNLVTRQRY-NH
2(SEQ.ID No.41); N-α-Ac-[Tic
27] PYY (22-36): N-α-Ac-ASLRH[Tic] LNLVTRQRY--NH
2(SEQ.ID No.42); N-α-Ac-[Phe
27] PYY (25-36): N-α-Ac-H[Phe] LNLVTRQRY--NH
2(SEQ.ID No.43); N-α-Ac-[Phe
27, Thi
36] PYY (22-36): N-α-Ac-ASLRH (phel LNLVTRQR[Thi]-NH
2(SEQ.ID No.44); N-α-Ac-[Thz
26, Phe
27] PYY (22-36): N-α-Ac-ASLR[Thz] [Phe] LNLVTRqRY--NH
2(SEQ.ID No.45); N-α-Ac.[Pcp
27] PYY (22-36): N-α-Ac-ASLRH[Pcp] LNLVTRQRY--NH
2(SEQ.ID No.46); N-α-Ac-[Ph
22,27] PYY (22-36): N-α-Ac-[Phe] SLRN[Phe] LNLVTRQRY--NH
2(SEQ.ID No.47); N-α-Ac-[Tyr
22, Phe
27] PYY (22-36): N-α-Ac-[Tyr] SLRH[Phe] LNLVTRQRY--NH
2(SEQ.ID No.48); N-α-Ac-[Trp
28] PYY (22-36): N-α-Ac-ASLRHY[Trp] NLVTRQRY--NH
2(SEQ.ID No.49); N-α-Ac-[Trp
28] PYY (22-36): N-α-Ac-ASLRHYLN[Trp] VTRQRY--NH
2(SEQ.ID No.50); N-α-Ac-[Ala.sup.26, Phe
27] PYY (22-36): N-α-Ac-ASLR[Ala] [Phe] LNLVTRQRY--NH
2(SEQ.ID No.51); N-α-Ac-[Bth
27] PYY (22-36): N-α-Ac-ASLRH[Bth] LNLVTRQRY--NH
2(SEQ.ID No.52); N-α-Ac-[Phe
27] PYY (22-36): N-α-Ac-ASLRH[Phe] LNLVTRQRY--NH
2(SEQ.ID No.53); N-α-Ac-[Phe
27,36] PYY (22-36): N-α-Ac-ASLRH[Phe] LNLVTRQR[Phe]-NH
2(SEQ.ID No.54); N-α-Ac-[Phe
27, D-Trp
32] PYV (22-36): N-α-Ac-ASLRH[Phe] LNLV[D-Trp] RQRY--NH
2(SEQ.ID No.55).
Other analog is described in Balasubramaniam, et al., Peptide Research 1:32 (1988); Japanese patent application 2,225,497 (1990); Balasubramaniam, et al., Peptides14:1011,1993; Grandt, et al., Reg.Peptides 51:151 (1994); The PCT international application discloses in 94/03380.
Balasubramaniam, et al. have described analog PYY (1-28), PYY (1-22), PYY (22-28), PYY (22-36) and PYY (27-36).Grandt, et al. have discussed PYY (1-36) (SEQ ID NO:1) and PYY (3-36).Above-mentioned peptide is usually in conjunction with Y receptor, especially Y2 and/or the Y5 receptor in brain and other places.Usually, these peptides are synthetic not contain endotoxin or pyrogen-free form, although always this is unessential.
The PYY agonist comprises P of Rats YY:Tyr Pro Ala Lys Pro Glu Ala Pro Gly GluAsp Ala Ser Pro Glu Glu Leu Ser Arg Tyr Tyr Ala Ser Leu Arg His Tyr LeuAsn Leu Val Thr Arg Gln Arg Tyr (SEQ ID NO:56) and the amino terminal clipped form corresponding with the people; Pig PYY:Tyr Pro Ala Lys Pro Glu Ala Pro Gly Glu Asp Ala SerPro Glu Glu Leu Ser Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu ValThr Arg Gln Arg Tyr (SEQ ID NO:57) and the amino terminal clipped form corresponding with the people; And Cavia porcellus PYY:Tyr Pro Ser Lys Pro Glu Ala Pro Gly Ser Asp Ala Ser ProGlu Glu Leu Ala Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val ThrArg Gln Arg Tyr (SEQ ID NO:58) and the amino terminal clipped form corresponding with the people.
According to the present invention; the PYY peptide also comprises free alkali; acid-addition salts or slaine, for example potassium salt of this peptide or sodium salt, or process and the adorned PYY peptide of other known covalent modification method by for example amidatioon, glycosylation, acidylate, sulphation, phosphorylation, acetylation and cyclisation.These peptides are usually in conjunction with Y receptor, especially Y2 and/or the Y5 receptor in brain and other places.Usually, these peptides are synthetic not contain endotoxin or pyrogen-free form, although always this is unessential.
Neuropeptide Y agaonists
NPY is another kind of Y2 receptor-binding peptides.The NPY peptide comprises total length people NPY (1-36): the fragment of TyrPro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala ArgTyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr (SEQID NO:59) and NPY (1-36), its at amino terminal by truncate.In conjunction with the Y2 receptor, the NPY agonist should have last 11 aminoacid of carboxyl terminal at least, promptly comprises NPY (26-36) for effectively.Other example in conjunction with the NPY agonist of Y2 receptor is NPY (3-36), NPY (4-36), NPY (5-36), NPY (6-36), NPY (7-36), NPY (8-36), NPY (9-36), NPY (10-36), NPY (11-36), NPY (12-36), NPY (13-36), NPY (14-36), NPY (15-36), NPY (16-36), NPY (17-36), NPY (18-36), NPY (19-36), NPY (20-36), NPY (21-36), NPY (22-36), NPY (23-36), NPY (24-36) and NPY (25-36).
Other NPY agonist comprises rat NPY:Tyr Pro Ser Lys Pro Asp Asn Pro GlyGlu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His TyrIle Asn Leu Ile Thr Arg Gln Arg Tyr (SEQ ID NO:60) and as the amino terminal clipped form from NPY (3-36) to NPY (26-36) in people's form; Rabbit NPY:Tyr Pro Ser LysPro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr SerAla Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr (SEQ ID NO:61) and as the amino terminal clipped form in people's form from NPY (3-36) to NPY (26-36); Dog NPY:Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu AspMet Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg GlnArg Tyr (SEQ ID NO:62) and as the amino terminal clipped form in people's form from NPY (3-36) to NPY (26-36); Pig NPY:Tyr Pro Ser Lys Pro Asp Asn Pro Gly GluAsp Ala Pro Ala Glu Asp Leu Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr IleAsn Leu Ile Thr Arg Gln Arg Tyr (SEQ ID NO:63) and as the amino terminal clipped form in people's form from NPY (3-36) to NPY (26-36); Cattle NPY:Tyr Pro Ser LysPro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Leu Ala Arg Tyr Tyr SerAla Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr (SEQ ID NO:64) and as the amino terminal clipped form in people's form from NPY (3-36) to NPY (26-36); Sheep NPY:Tyr Pro Ser Lys Pro Asp Asn Pro Gly Asp Asp Ala Pro Ala Glu AspLeu Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg GlnArg Tyr (SEQ ID NO:65) and as the amino terminal clipped form in people's form from NPY (3-36) to NPY (26-36); And Cavia porcellus NPY:Tyr Pro Ser Lys Pro Asp Asn Pro GlyGlu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His TyrIle Asn Leu Ile Thr Arg Gln Arg Tyr (SEQ ID NO:66) and as the amino terminal clipped form in people's form from NPY (3-36) to NPY (26-36).According to the present invention; the PYY peptide also comprises free alkali; acid-addition salts or slaine, for example potassium salt of this peptide or sodium salt, and the process and the adorned NPY peptide of other known covalent modification method that pass through for example amidatioon, glycosylation, acidylate, sulphation, phosphorylation, acetylation and cyclisation.These peptides are usually in conjunction with Y receptor, especially Y2 and/or the Y5 receptor in brain and other places.Usually, these peptides are synthetic not contain endotoxin or pyrogen-free form, although always this is unessential.
Pancreatic polypeptide
Pancreatic polypeptide (PP) and PP agonist are also in conjunction with the Y2 receptor.The example of PP agonist is total length people PP (1-36): Ala Ser Leu Glu Pro Glu Tyr Pro Gly Asp Asn Ala Thr Pro GluGln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr ArgPro Arg Tyr (SEQ ID NO:67) and many PP fragments, it is in the amino terminal truncate.For in conjunction with the Y2 receptor, the PP agonist must have last 11 amino acid residues of carboxyl terminal, PP (26-36).Example in conjunction with other PP of Y2 receptor is PP (3-36), PP (4-36), PP (5-36), PP (6-36), PP (7-36), PP (8-36), PP (9-36), PP (10-36), PP (11-36), PP (12-36), PP (13-36), PP (14-36), PP (15-36), PP (16-36), PP (17-36), PP (18-36), PP (19-36), PP (20-36), PP (21-36), PP (22-36), PP (23-36), PP (24-36) and PP (25-36).
Other PP agonist comprises sheep PP:Ala Pro Leu Glu Pro Val Tyr Pro Gly AspAsn Ala Thr Pro Glu Gln Met Ala Gln Tyr Ala Ala Asp Leu Arg Arg Tyr IleAsn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO:68) and as the amino terminal clipped form from PP (3-36) to PP (26-36) in people's form; Pig PP:Ala Pro Leu Glu Pro ValTyr Pro Gly Asp Asp Ala Thr Pro Glu Met Ala Gln Tyr Ala Ala Glu Leu ArgArg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO:69) and as the amino terminal clipped form in people's form from PP (3-36) to PP (26-36); Dog PP:Ala Pro LeuGlu Pro Val Tyr Pro Gly Asp Asp Ala Thr Pro Glu Gln Met Ala Gln Tyr AlaAla Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO:70) and as the amino terminal clipped form in people's form from PP (3-36) to PP (26-36); Cat PP:Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln Met AlaGln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu Thr Arg Pro Arg Tyr (SEQ ID NO:71) and as the amino terminal clipped form in people's form from PP (3-36) to PP (26-36); Cattle PP:Ala Pro Leu Glu Pro Glu Tyr Pro Gly Asp Asp Ala Thr ProGlu Gln Met Ala Gln Tyr Ala Ala Glu Leu Arg Arg Tyr Ile Asn Met Leu ThrArg Pro Arg Tyr (SEQ ID NO:72) and as the amino terminal clipped form in people's form from PP (3-36) to PP (26-36); P of Rats P:Ala Pro Leu Glu Pro Met Tyr ProGly Asp Tyr Ala Thr His Glu Gln Arg Ala Gln Tyr Glu Thr Gln Leu Arg ArgTyr Ile Asn Thr Leu Thr Arg Pro Arg Tyr (SEQ ID NO:73) and as the amino terminal clipped form in people's form from PP (3-36) to PP (26-36); Mice PP:Ala Pro Leu GluPro Met Tyr Pro Gly Asp Tyr Ala Thr His Glu Gln Arg Ala Gln Tyr Glu ThrGln Leu Arg Arg Tyr Ile Asn Thr Leu Thr Arg Pro Arg Tyr (SEQ ID NO:74) and as the amino terminal clipped form in people's form from PP (3-36) to PP (26-36); And Cavia porcellus PP:Ala Pro Leu Glu Pro Met Tyr Pro Gly Asp Tyr Ala Thr Pro Glu GlnMet Ala Gln Tyr Glu Thr Gln Leu Arg Arg Tyr Ile Asn Thr Leu Thr Arg ProArg Tyr (SEQ ID NO:75) and as the amino terminal clipped form in people's form from PP (3-36) to PP (26-36).
According to the present invention; the PP peptide also comprises free alkali; acid-addition salts or slaine, for example potassium salt of this peptide or sodium salt, and the process and the adorned PP peptide of other known covalent modification method that pass through for example amidatioon, glycosylation, acidylate, sulphation, phosphorylation, acetylation and cyclisation.These peptides are usually in conjunction with Y receptor, especially Y2 and/or the Y5 receptor in brain and other places.Usually, these peptides are synthetic not contain endotoxin or pyrogen-free form, although always this is unessential.
Peptide and albumen analog and analogies
Be included in derivant or the salt that are used for biologically active peptide of the present invention and proteic definition for peptide (comprising 2 or more a plurality of covalently bound aminoacid), albumen, peptide or protein fragments, peptide or albumen analog and the bioactive peptide or the proteic chemical modification of natural or synthetic, therapeutic activity or prophylactic activity.The multiple useful analog and the analogies of expection Y2 receptor-binding peptides are used for the present invention, and can produce and test its biological activity according to known method.Often, be used for peptide or albumen or other biologically active peptide or the mutant protein (mutein) of albumen for obtaining easily of Y2 receptor-binding peptides of the present invention, this obtains substituting, adding or disappearance by the amino acid whose part in naturally occurring or natural (for example wild type, naturally occurring mutant or allelic variant) peptide or the protein sequence.In addition, comprise native peptides or proteic bioactive fragment.Such mutant derivative and fragment keep native peptides or proteic required biological activity basically.Have under the situation of carbohydrate chain at peptide or albumen, be also included within the present invention by the biological activity variant of the change flag of these carbohydrate species.
As used herein, term " conserved amino acid substitute " refers to have the common interchangeability of the amino acid residue of similar side chain.For example, one group of common interchangeable aminoacid with aliphatic lateral chain is alanine, valine, leucine and isoleucine; One group of aminoacid with aliphatic-hydroxyl side chain is serine and threonine; One group of aminoacid with amide containing side chain is agedoite and glutamine; One group of aminoacid with aromatic side chain is phenylalanine, tyrosine and tryptophan; One group of aminoacid with basic side chain is lysine, arginine and histidine; And one group of aminoacid with sulfur-containing side chain is cysteine and methionine.Conservative alternate example comprises such as isoleucine, valine, and nonpolar (hydrophobic) residue of leucine or methionine substitutes another.Equally, the substituting of desired polarity of the present invention (hydrophilic) residue, for example between arginine and the lysine, between glutamine and the agedoite and between threonine and the serine.In addition, also expect to substitute another, or substitute another such as the acidic amino acid of aspartic acid or glutamic acid such as the alkaline residue of lysine, arginine or histidine.The example of conserved amino acid alternate sets is: Val-Leu-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and agedoite-glutamine.By peptide or albumen analog are compared with corresponding native peptides or albumen the best, and determine selected bioactive suitable mensuration by using, for example attachment proteins or receptors bind are measured, and can easily identify the feasible peptide and the albumen analog that are used for the inventive method and compositions.Feasible peptide and albumen analog antibody specific immune common and at corresponding native peptides or albumen generation reacts.
Pharmacokinetics (PK) parameter
As used herein, the " peak serum concentration (C of Y2 receptor-binding peptides
Max) ", " the plasma concentration time graph (AUC) of Y2 receptor-binding peptides down area ", " in blood plasma, reach the time (t of the maximal plasma concentration of Y2 receptor-binding peptides
Max) " be pharmacokinetic parameter well known by persons skilled in the art.Laursen et al.，Eur.J.Endocrinology 135：309-315，1996。" Cot curve " measured after individuality being given Y2 receptor-binding peptides dosage by intranasal, intramuscular, subcutaneous or other parenteral approach, should the serum of individuality with respect to the time in the concentration of Y2 receptor-binding peptides." C
Max" be behind the Y2 receptor-binding peptides that individuality is given single dose, the Cmax of Y2 receptor-binding peptides in this individual serum." t
Max" be behind the Y2 receptor-binding peptides that individuality is given single dose, in this individual serum, reach the time of the Cmax of Y2 receptor-binding peptides.
As used herein, add then that according to linear trapezoid method residual area calculates " the plasma concentration time graph (AUC) of Y2 receptor-binding peptides is area down ".Use 90% probability (error of the second kind B=10%) can detect 23% minimizing or 30% increase.By reaching Cmax (C
Max) and time (t
Max) contrast, estimate " delivery rate " or " absorption rate ".C
MaxAnd t
MaxAll use the nonparametric technique analysis.Relatively being undertaken of the pharmacokinetics of intramuscular, subcutaneous, vein and the administration of intranasal Y2 receptor-binding peptides by variance analysis (ANOVA).For comparing in twos, use the Bonferroni-Holmes sequential grammar to estimate significance.By the dose-effect relationship between 3 nasal-cavity administrations of regression analysis assessment.It is remarkable that P＜0.05 is considered to.The result with meansigma methods+/-standard error provides.
Can change by different mucosal delivery reinforcing agents according to the present invention although absorb the mechanism that promotes, useful reagent does not influence mucosal tissue basically nocuously and will select according to the physical chemical characteristics of specific Y2 receptor-binding peptides or other activity or delivery enhancer in the case.In the case, improve mucosal tissue penetrate or infiltrative delivery enhancer will often cause some change of the protectiveness permeability barrier of mucosa.In order to make such delivery enhancer valuable, be reversible in the time limit that any remarkable change of expecting mucosal permeability usually was fit in the expectation persistent period of sending for medicine to the present invention.In addition, the toxicity that life-time service should not have significantly, accumulates is not induced permanent harmful change of mucosal barrier characteristic yet.
Stability
A kind of method of stablizing solid protein preparation of the present invention is the physical stability that improves such as freeze dried purifying protein.This will suppress the gathering by hydrophobic interaction and covalency approach, and this gathering increases along with the albumen unfolding.Stabilization formulations often comprises the preparation based on polymer in the case, for example biodegradable aqueogel/delivery system.As mentioned above, the pivotal role of known water in protein structure, function and stability.Usually, albumen remove under the solid state of big water gaging relatively stable.Yet, solid treatment protein formulation may be because under high humility, store hydration or from hydration between slow releasing composition or device delivery period.Proteic stability descends along with the increase of hydration usually.Water can play remarkable effect in solid protein is assembled, for example, by increasing the accessibility that the albumen flexibility causes the increase of reactive group, by removable phase is provided for reactant, and by remove such as β and some destructive processes of hydrolysis in as reactant.
The protein formulation that contains 6% to 28% water of having an appointment is least stable.Be lower than this level, the activeness of bonded water and proteic internal motion are low.Be higher than this level, those of water activeness and the approaching hydration fully of albumen motion.When height is put to certain, in some systems, observed along with hydration increases and the solid phase gathering susceptibility of increase.Yet, when high moisture content more, because diluting effect is observed less gathering.
According to these principles, the solid-state accumulative stabilized peptide of antagonism and the proteic effective ways that are used for mucosal delivery are to control the moisture of solid preparation and the water activity in the preparation is remained on optimum level.This level depends on proteic character, but usually, the albumen that remains under its " monolayer " water covering shows higher solid-state stability.
In the present invention, provide multiple effective control moisture to improve additive, diluent, substrate and the delivery vector of protein stability.Effectively these reagent and the carrier material as anti-aggregating agent prepared therefrom comprises in this meaning, for example, has multiple functional polymer, for example Polyethylene Glycol, dextran, diethylin ethyl glucosan and carboxymethyl cellulose, it significantly improves stability and reduces and its mixing or peptide that is connected and the gathering of proteic solid phase.
Some additive is also given such as the significant physical stability of freeze dried desiccation protein.These additives also can be used in the present invention not only also to protect described albumen antagonism to assemble between the storage life in drying regime during the lyophilizing.
This paper provides multiple other preparation component and method and concrete formulation additives, its production is used to have the peptide of assembling tendency and the preparation of proteic mucosal delivery, wherein use stabilizing agent with described peptide or protein stabilized basically pure, not in the aggregated forms.Relate to a series of components and additive and be used for these method and formulations.The example of these stabilizing agents is cyclodextrin (CDs), the hydrophobic side chain of its selective binding polypeptide.Have been found that these CDs are with the protein-bonded hydrophobic flakes of the accumulative mode of remarkable inhibition.This inhibition all is optionally for CD that relates to and albumen.Such selectivity of protein aggregation is suppressed in the intranasal delivery method and composition of the present invention other advantage is provided.The other reagent that is used for this situation comprises different geometric CD dimer, trimer and the tetramer that has by the junctional complex control of special prevention peptide and protein aggregation.But the stabilizing agent and the method that are included in the present invention comprise that use peptide and peptide mimics selectivity stop protein-protein interaction.On the one hand, the peptide and the extended albumen that arrives of peptide mimics that the specific bond hydrophobic side chain of CD polymer report are stoped similarly protein aggregation by use.Can obtain being used to be included in the compositions and methods of the invention interior multiple suitable method and anti-aggregating agent prepared therefrom.
Protease inhibitor
Another excipient that can be included in the mucosa preparation is the degradability enzyme inhibitor.The exemplary mucosal adhesive polymer-enzyme inhibitor complex that can be used for mucosa preparation of the present invention and method includes but not limited to: carboxymethyl cellulose-pepstatin (having the antipepsin activity), poly-(acrylic acid)-Bowman-Birk inhibitor (chymotrypsin inhibitor), poly-(acrylic acid)-chymotrypsin inhibitor (chymostatin) (chymotrypsin inhibitor), poly-(acrylic acid)-elastase inhibitor (elastatinal) (elastase inhibitor), carboxymethyl cellulose-elastase inhibitor (elastase inhibitor), polycarbophil-elastase inhibitor (elastase inhibitor), chitosan-protease inhibitor (antipain) (antitrypsin), poly-(acrylic acid)-bacitracin (anti-Aminopeptidase N), chitosan-EDTA (anti-Aminopeptidase N, anti-Carboxypeptidase A), chitosan-EDTA-protease inhibitor (antitrypsin, chymotrypsin inhibitor, elastase inhibitor).As hereinafter in greater detail, certain embodiments of the present invention will randomly comprise the chemical modification form of new chitosan derivatives or chitosan.A kind of be used for so new derivant of the present invention be represented as β-[1 → 4]-2-guanidine radicals-2-deoxy-D-glucose polymer (poly--GuD).
Any inhibitory enzyme activity all can be used for the compositions and methods of the invention with the inhibitor of protection bioactivator.Can be used for protecting the enzyme inhibitor of biological activity protein and peptide to comprise; for example, soybean trypsin inhibitor, pancreas trypsin inhibitor, from Rhizoma Solani tuber osi (solanum tuberosum L.) the isolating chymotrypsin inhibitor of tuber and trypsin and chymotrypsin inhibitor.Can use the combination or the mixture of inhibitor.Being used for other proteinase inhibitor of the present invention comprises ovomucoid-enzyme, gabexate mesilate, alpha1-antitrypsin, presses down enzyme peptide, amastatin, ubenimex, puromycin, bacitracin, leupeptin, alpha2-macroglobulin, pepstatin and egg white or soybean trypsin inhibitor.These and other inhibitor can separately or be united use.Described one or more inhibitor can be merged in or be attached in the carrier of hydrophilic polymer for example, this carrier is coated on the surface with the dosage form of contact nasal membrane, or the surface that is incorporated into described surface mutually in, with the bioactivator associating or in administration respectively (for example pre-administration) preparation.
To change according to following factors such as the optional amount of incorporating the described inhibitor of the proteinase inhibitor in the present composition into: (a) characteristic of concrete inhibitor, (b) number of the functional group that exists in the molecule (can with its reaction to induce the required ethylene type desaturation of monomeric copolymerization that forms hydrogel), and (c) be present in number in the inhibitor molecules such as the lectin group of glucosides class.It can also depend on concrete therapeutic agent to be administered.Generally speaking, the consumption that has of enzyme inhibitor is that about 0.1mg/ml of preparation (the protease inhibitor preparation that promptly separates or contain inhibitor and the combination formulations of bioactivator) is to about 50mg/ml, often from about 0.2mg/ml to about 25mg/ml, and more frequent from about 0.5mg/ml to 5mg/ml.
Under the situation that trypsin suppresses, the inhibitor that is fit to can be selected from and for example press down enzyme peptide, BBI, soybean trypsin inhibitor, the sticking egg of ovum gallinaceum class, avian ovomucoid inhibitor, HPTIN, Camostat mesilate (camostat mesilate), flavone compound inhibitor, protease inhibitor, leupeptin, p-aminophenyl amidine salt, AEBSF, TLCK (tosyllysine chloromethylketone), APMSF, DFP, PMSF and polyacrylic acid ester derivant.Under the situation that chymase suppresses, the inhibitor that is fit to can be selected from, and for example presses down enzyme peptide, BBI, soybean trypsin inhibitor, chymotrypsin inhibitor, benzyloxycarbonyl group-Pro-Phe-CHO, FK-448, avian ovomucoid inhibitor, sugared biphenyl boric acid complex, DFP, PMSF, beta-phenylpropionic acid ester and polyacrylic acid ester derivant.Under the situation that elastoser suppresses, the inhibitor that is fit to for example can be selected from elastase inhibitor, methoxyl group succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (SEQID NO.76) (MPCMK), BBI, soybean trypsin inhibitor, avian ovomucoid inhibitor, DFP and PMSF.
Be used for other enzyme inhibitor of the present invention and be selected from different non-widely protein inhibitor on usefulness and toxic level.As hereinafter in greater detail, can easily implement these auxiliary reagents are fixed on substrate or other delivery vector, or the analog of exploitation chemical modification, when running into them, to reduce or even elimination toxic action.Be used for candidate's enzyme inhibitor of the present invention this widely the group organophosphor inhibitor is arranged, for example diisopropylphosphofluoridate (DFP) and Phenylmethanesulfonyl fluoride (PMSF), it is effective, the irreversible inhibitor of serine protease (for example trypsin and chymase).These chemical compounds make them uncontrolled sending high toxicity be arranged in the setting to the extra inhibition of acetylcholinesterase.Another candidate inhibitor, 4-(2-aminoethyl)-benzene sulfonyl fluorine (AEBSF) have the inhibition activity suitable with PMSF with DFP, but toxicity is significantly lower.(4-aminophenyl)-sulfonyl methane villiaumite hydrochlorate (APMSF) is another kind of effectively trypsin inhibitor, but poisonous in uncontrolled setting.Opposite with these inhibitor, 4-(4-isopropyl Fructus Piperis absorption carbonyl (isopropylpiperadinocarbonyl) phenyl 1,2,3,4 ,-tetrahydrochysene-1-naphthoic acid methanesulfonates (FK448) is the hypotoxicity material, represents effective and special chymotrypsin inhibitor.Other representative of the non-albumen cohort of this class of inhibitor material standed for and also show the hypotoxicity risk for Camostat mesilate (N, N '-dimethylamino formyl methyl-p-(p ' guanidine radicals-benzoyloxy) phenylacetic acid methanesulfonates).
The enzyme inhibitor that is used in another type in the method and composition of the present invention is to disturb the aminoacid of enzymatic degradation of concrete therapeutic compound and modified aminoacid.Be to use in this case, aminoacid and modified aminoacid are nontoxic on substantially and can be with low-cost production.Yet because its low molecular size and fine solubility, they can easily be diluted and be absorbed in the mucosa environment.Yet under proper condition, aminoacid can be as the reversibly-competitive inhibitor of protease.Some modified aminoacid can show much better than inhibition activity.Qi Wang modified aminoacid is called as " transition state " inhibitor in the case.The strong inhibitory activity of these chemical compounds based on they in its transition state geometry arrangement with the structural similarity of substrate, and they selectedly usually have the affinity more much higher than substrate itself with the avtive spot for enzyme.The transition state inhibitor is reversible, competitive inhibitor.The example of this class inhibitor is the alpha-amino boronic acid derivant, for example boron-leucine, boron-valine and boron-alanine.
Boron atom in these derivants can form tetrahedron borate (boronate) ion, and it has been considered to simulate the transition state of peptide during it is by the aminopeptidase hydrolysis.These amino acid derivativges are effective and reversible amastatins, and have reported that in enzyme suppresses boron-leucine surpasses 100 times and surpass 1000 times than puromycin is more effective than ubenimex is more effective.Another modified aminoacid that its strong protease inhibiting activity has been in the news is N-acetylcystein, and it suppresses the enzymatic activity of Aminopeptidase N.This auxiliary reagent also shows mucolytics characteristic, and this characteristic can be used in the method and composition of the present invention to reduce the effect of mucus diffusion barrier.
Other useful enzyme inhibitor that is used for medication arranged side by side of the present invention and compositions can be selected from peptide and modified peptidase inhibitors.The important representative of this class inhibitor is the ring dodecapeptide that obtains from Bacillus licheniformis (Bacillus licheniformis), bacitracin.Except the peptide of these types, some dipeptides and tripeptides show weak, the nonspecific inhibition activity to some protease.Analogize according to aminoacid, can improve their inhibition activity by chemical modification.For example, the phosphonic acids dipeptide analog also has strong inhibitory activity " transition state " inhibitor to aminopeptidase.Reported the bright deltorphin delta that it is used to stablize nasal-cavity administration.Another example of transition state analogs is modified pentapeptide pepstatin (pepstatin), and it is very effective pepsin inhibitor.By testing the inhibition activity of some synthetic analogues, the structural analysis of pepstatin has illustrated is responsible for the active molecule primary structure-functional character of described inhibition.The modified peptide of another specific type is included in to have in its structure and is positioned at the terminal functional inhibitor of aldehyde.For example, satisfy the effective reversible inhibitor that sequence benzyloxycarbonyl group-Pro-Phe-CHO that the known main and less important specificity of chymase requires is found to be this target protease.Other has the chemical constitution that is positioned at the terminal functional inhibitor of aldehyde also is known in the art, for example protease inhibitor, leupeptin, chymotrypsin inhibitor and elastase inhibitor, as the structure of other known reversible modified inhibitor peptides, for example phosphoramidon, ubenimex, puromycin and amastatin.
Because their commeasurable high molecular are sent for concentrating in medicine-carrier matrix, the smaller chemical compound of polypeptide protein enzyme inhibitor is easier to control.The other reagent that is used for the protease inhibition in preparation of the present invention and method comprises the use chelating agent.These reagent suppress by the intranasal environment mediation enzyme of depriving (or preparation or therapeutic combination) bivalent cation, and this bivalent cation is the cofactor of many protease.For example, the auxiliary reagent of chelating agent EDTA that gives side by side and DTPA or associating prescription is enough to the protease that suppresses to select under debita spissitudo, strengthen the intranasal delivery of bioactivator of the present invention thus.Other of this class inhibitor is represented as EGTA, 1,10-phenanthroline and hydroxyquinoline (hydroxychinoline).In addition, because the tendency of its chelating bivalent cation, these and other chelating agent can be used as direct absorption enhancer in the present invention.
As other parts herein in greater detail, also expection use various polymer especially the mucosal adhesive polymer as administration arranged side by side of the present invention, repeatedly handle and/or unite enzyme inhibitor in the prescription method and composition.For example, the activity of multiple protein enzyme be can influence, trypsin, chymase comprised such as the polyacrylic acid ester derivant of polyacrylic acid and polycarbophil.The inhibitory action of these polymer also can be based on such as Ca
2+And Zn
2+The complexation of bivalent cation.As indicated abovely expect that also these polymer can be used as the coupling partner or the carrier of other enzyme inhibitor.For example, developed chitosan-EDTA conjugate and can be used for the present invention, it shows the strong inhibitory action to the enzymatic activity of zinc dependence protein enzyme.Be expected in the case in the mucoadhesive properties of the covalently bound post polymerization thing of other enzyme inhibitor and significantly do not damaged, expect that also these polymer are not detracted as the general service of the delivery vector of bioactivator among the present invention.On the contrary, the delivery vector and the distance between the mucomembranous surface of being given by mucosal adhesive mechanism reduce and will minimize the presystemic metabolism of activating agent, and covalently bound enzyme inhibitor keeps concentrating on the medicine site of delivery, toxicity and other side effect of minimizing the diluting effect of the inhibitor of not expecting and causing thus.In this way, the effective dose of the enzyme inhibitor of administration can be reduced owing to get rid of diluting effect side by side.
The exemplary mucosal adhesive polymer-enzyme inhibitor complex that can be used for mucosa preparation of the present invention and method includes but not limited to: carboxymethyl cellulose-pepstatin (having the antipepsin activity), poly-(acrylic acid)-Bowman-Birk inhibitor (chymotrypsin inhibitor), poly-(acrylic acid)-chymotrypsin inhibitor (chymotrypsin inhibitor), poly-(acrylic acid)-elastase inhibitor (elastase inhibitor), carboxymethyl cellulose-elastase inhibitor (elastase inhibitor), polycarbophil-elastase inhibitor (elastase inhibitor), chitosan-protease inhibitor (antitrypsin), polyacrylic acid-bacitracin (anti-Aminopeptidase N), chitosan-EDTA (anti-Aminopeptidase N, anti-Carboxypeptidase A), chitosan-EDTA-protease inhibitor (antitrypsin, chymotrypsin inhibitor, elastase inhibitor).
Mucosa is passed contrary
Mucosal delivery preparation of the present invention comprises Y2 receptor-binding peptides, analog and analogies, and they make up with one or more pharmaceutically acceptable carriers and optional other treatment component usually.Described carrier must be " pharmaceutically acceptable ", and the meaning is with other component compatibility of described preparation and does not cause curee's unacceptable illeffects.Described carrier is above described to some extent at this paper, perhaps is that the technical staff of area of pharmacology is known.Desirably, described preparation should not comprise the material such as enzyme or oxidant, knownly treats that the bioactivator of administration is incompatible with these materials.Described preparation can prepare by the known any method in pharmaceutics field.
In the compositions and methods of the invention, Y2 receptors bind peptide protein disclosed herein, analog and analogies and other biological activating agent can deliver medicine to the experimenter by multiple mucosa delivery mode, comprise by in oral cavity, rectum, vagina, intranasal, the lung or transdermal delivery, or by local delivery to eyes, ear, skin or other mucomembranous surfaces.Randomly, Y2 receptors bind peptide protein disclosed herein, analog and analogies and other biological activating agent can be by non-mucosal route synergistically or the administration of auxiliary ground, comprise by in intramuscular, subcutaneous, intravenous, the atrium, intraarticular, intraperitoneal or parenteral approach.In other optional embodiments, described bioactivator can be used as the component of the vitro tissue that for example contains described bioactivator in suitable liquid or solid carrier or organ treatment preparation, carries out treated in vitro by directly being exposed to the cell, tissue or the organ that come from mammalian subject.
Usually the liquid solution with nose or lung spray gives the present composition, can make up a prescription with spray form by several different methods well known by persons skilled in the art.The 4th, 511, No. 069 U.S. Patent Publication the liquid that makes up a prescription of preferred nasal spray.Described preparation can provide in multi-dose container, for example in the sealing dispensing system of the 4th, 511, No. 069 U.S. Patent Publication.Other aerosol drug delivery form comprises for example compressed air sprayer, jet nebulizer, ultrasonic nebulizer and piezo jet day with fog, and it is sent dissolving or is suspended in for example bioactivator in water, ethanol or their mixture of drug solvent.
Nose of the present invention and lung spray solution comprise described medicine or medicine to be sent usually, and it is randomly prepared with surfactant and one or more buffer such as nonionic surfactant (for example Polyoxyethylene Sorbitan Monooleate).In some a little embodiment of the present invention, described nose spray solution also comprises propellant.The pH of described nose spray solution chooses wantonly at about pH 3.0 to 6.0, preferred 4.5 ± 0.5.The suitable buffer that is used for these compositionss as mentioned above or or be known in the art.Other components be can add to strengthen or to keep chemical stability, antiseptic, surfactant, dispersant or gas comprised.The antiseptic that is fit to includes but not limited to phenol, methyl parahydroxybenzoate, p-Hydroxybenzoate, metacresol, thimerosal, methaform, benzalkonium chloride (benzylalkonimumchloride) etc.The surfactant that is fit to comprises but is not limited to oleic acid, sorbitan trioleate, poly-mountain alcohol ester, lecithin, phosphatidylcholine, and various long-chain diglyceride and phospholipid.The dispersant that is fit to includes but not limited to editic acid etc.The gas that is fit to includes but not limited to nitrogen, helium, Chlorofluorocarbons (CFCs), hydrogen fluorine carbon (HFCs), carbon dioxide, air etc.
In optional embodiment, the mucosa preparation is as the dry powder formulations administration that is used for intranasal administration, described dry powder formulations comprise have suitable particle size or the drying in the suitable particle size scope, the bioactivator of lyophilized form usually.Be suitable for sedimentary minimum particle size Chang Weiyue 0.5 μ mass median equivalent air kinetic diameter (MMEAD) in nose or pulmonary passageway, common about 1 μ MMEAD, more typical about 2 μ MMEAD.Be suitable for that sedimentary maximum particle size is generally about 10 μ MMEAD in nasal meatus, about 8 μ MMEAD usually, more typical about 4 μ MMEAD.The absorbable powder of intranasal in these magnitude range can produce by multiple routine techniques, and for example fluid energy mill, spray drying, solvent deposition, supercritical liq are condensing etc.The dry powder of these suitable MMEAD can deliver medicine to the patient through conventional Diskus (DPI), and described Diskus depends on lung or the fashionable patient's of snuffing breathing and powder is dispersed into the aerosol amount.Alternatively, dry powder can help the administration of formula device through gas, and described device uses extra power that powder is dispersed into the aerosol amount, for example piston pump.
In order to prepare the compositions that is used for mucosal delivery among the present invention, can be with described bioactivator and various pharmaceutically acceptable additive and be used to disperse the substrate of described activating agent or carrier combinations together.Desired additives includes but not limited to the pH controlling agent, for example arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid etc.In addition, can comprise local anesthetic (for example benzyl alcohol), isotonic agent (for example sodium chloride, mannitol, Sorbitol), absorption inhibitor (for example tween 80 (Tween 80)), solubilizing agent (for example cyclodextrin and derivant thereof), stabilizing agent (for example serum albumin) and Reducing agent (for example glutathione).When the compositions that is used for mucosal delivery is liquid, usually the tension force of preparation (as measured with reference to the tension force as 0.9% (w/v) normal saline solution of unit) is adjusted in the nasal mucosa medicine-feeding part and does not induce numerical value remarkable, irreversible disorganization.Usually, with the tension adjustment of the solution numerical value to about 1/3 to 3, more typical 1/2 to 2 and the most frequently used 3/4 to 1.7.
Described bioactivator can be scattered in substrate or the vehicle, and described substrate or vehicle can comprise the hydrophilic compounds with the ability of disperseing described activating agent and any desired additives.The optional suitable carriers of described substrate from wide region, include but not limited to, the copolymer of polycarboxylic acid or its salt, carboxylic acid anhydrides (for example maleic anhydride) and other monomers (for example methacrylic acid (first) ester, acrylic acid etc.), the hydrophilic ethylene polymer is polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone for example, cellulose derivative is hydroxy methocel, hydroxypropyl cellulose etc. for example, and natural polymer for example chitosan, collagen, sodium alginate, gelatin, hyaluronic acid and avirulence slaine thereof.Usually, biodegradable polymer is selected as substrate or carrier, for example polylactic acid, poly-(lactic-co-glycolic acid) copolymer, poly hydroxybutyric acid, poly-(hydroxybutyric acid-hydroxyacetic acid) copolymer and composition thereof.Alternatively or in addition, synthetic fatty acid ester for example polyglyceryl fatty acid ester, sucrose fatty acid ester etc. can be used as carrier.Hydrophilic polymer and other carriers can be used alone or in combination, and can be by partially crystallizable, ionic bonding, crosslinked or the like give carrier enhanced structural intergrity.Carrier can provide in a variety of forms, comprises, is used to directly apply to liquid or viscosity solution, gel, paste, powder, microsphere and the film of nasal mucosa.The use of selected carrier can cause promoting the absorption of described bioactivator among the present invention.
Compositions of the present invention can contain alternatively near the required pharmaceutically acceptable carrier mass of physiological condition, for example pH regulator agent and buffer, tension regulator, wetting agent or the like.For example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, Span 20, Emulphor FM etc.For solid composite, can use the conventional pharmaceutically acceptable carrier of avirulence, it comprises for example pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, Talcum, magnesium carbonate or the like.
The therapeutic combination that is used to carry out described bioactivator administration also can be mixed with solution, microemulsion or other the orderly structures that is suitable for the high concentration active component.Described carrier can be solvent or disperse medium, comprises for example water, ethanol, polyhydric alcohol (for example glycerol, propylene glycol, liquid macrogol etc.) and suitable mixture thereof.Can by for example use encrusting substance for example lecithin, by maintaining dispersible formulation agent situation desired particle size and by using surfactant to keep the adequate liquidity of solution.
In certain embodiments of the invention, described bioactivator carries out administration in the time delivery formulations, for example in the compositions that comprises slow release polymers.Described activating agent can be prepared with the carrier that prevents rapid release, for example as the drug-supplying system of polymer, microcapsule embedding or the controlled release vehicles thing of bioadhesive gel.The prolongation of the described activating agent in the various compositionss of the present invention is sent can be by comprising that in composite preparation for example aluminum monostearate hydrogel and gelatin are realized for the reagent that postpone to absorb.When the controlled release preparation of the described bioactivator of needs, be applicable to that controlled release bonding agent of the present invention comprises that for described activating agent be inertia and any biocompatible controlled-release material that can mix described bioactivator.The such material of many kinds is known in the art.Useful controlled release bonding agent be after its intranasal delivery under physiological condition (for example after the mucosal on nasal mucosa surface or under the situation that body fluid exists) slow metabolic material.The bonding agent that is fit to includes but not limited to before be used in the art the biocompatible polymer and the copolymer of extended release preparation.These biocompatible compounds are to the surrounding tissue avirulence and have inertia, and do not cause significant adverse side effect, for example nose stimulation, immunoreation, inflammation etc.They are metabolised to and also are bio-compatible and the easy metabolite of eliminating from body.
Sterile solution can prepare by in suitable solvent the described reactive compound of requirement being blended together with the component of above enumerating a kind of or combination, as needs, and filtration sterilization afterwards.Usually, dispersant contains basic dispersion medium and prepares from the aseptic vehicle of required other components of listed those above by described reactive compound is mixed.In the situation of sterilized powder, preparation method comprises vacuum drying and lyophilization, and it produces described active component and adds powder from any other required component of its previous aseptic filtration solution.The prevention of microbial action can realize by various antibacterial agents and antifungal, for example metagin, methaform, phenol, sorbic acid, thimerosal etc.
Allow patient's effective automedication to treat according to mucosa delivery of the present invention, condition is to be provided with enough safety measures with control and monitoring dosage and side effect.Mucosa delivery has also overcome some shortcoming of other form of medication, for example injection, and they make us pain and make the patient be exposed to the problem that possible infection also can bring drug bioavailability.Send for nose and lung, it is known being used to the system of controlled release aerosol dispensing of the treatment liquid of spray.In one embodiment, the activating agent of dosing is sent (U.S. Patent number 4,511,069) by the mode of the mechanical pump valve of special structure.
Dosage
For prevention and therapeutic purposes, bioactivator disclosed herein can deliver medicine to experimenter's (for example by per hour, every day or repeat administration scheme weekly) with the quick administration of single or with the repeat administration scheme.In the application's context, the treatment effective dose of PYY can be included in multiple dosage in the prevention of prolongation or the therapeutic scheme, and it produces alleviates one or more symptoms that are associated with target disease mentioned above or state or the clinical remarkable result of detectable state.The definite of effective dose is the human clinical trial based on Research of Animal Model for Study usually afterwards in the application's context, and by determining that significantly alleviating experimenter's the target disease symptom or the generation of state or the effective dose and the dosage regimen of seriousness is instructed.Comprise for example mice, rat, pig, felid, non-human primate and other acceptable animal model objects known in the art at the suitable model aspect this.Alternatively, effective dose can use external model to determine (for example immunity and histopathology are measured).Use these models, usually only need general calculating and regulate to determine the described bioactivator that suitable concentration and dosage treats effective dose (intranasal that for example causes required reaction is effective, percutaneous is effective, intravenous is effective or intramuscular is effectively measured).
The actual dose of bioactivator is certainly according to following factor and difference: disease indication and experimenter's particular case (for example degree of experimenter's age, size, symptom, predisposing factor etc.) for example, administration time and approach, other drug that gives simultaneously or treatment, and the concrete pharmacology who is used to cause the described bioactivator of experimenter's required activity or biological respinse.Can adjust dosage regimen so that best prevention or therapeutic response to be provided.Still a kind of like this amount of treatment effective dose: the amount that promptly surpasses any toxicity or illeffects at the treatment beneficial effect of the described bioactivator of clinicing aspect.The non-limiting scope of the treatment effective dose in the inventive method and the preparation is that 0.7 μ g/kg is to about 25 μ g/kg.In order to promote to lose weight, intranasal dose is with up to being enough to promote satietion but be low to moderate the dosed administration that the side effect that is enough to can not to induce any non-expectation is for example felt sick.The preferred intranasal dose of PYY3-36 is about 1 μ g-10 μ g/kg patient weight, and most preferably from about 1.5 μ g/kg are to about 6 μ g/kg patient weight.In standard dose, the patient accepts 40 μ g to 2000 μ g, more preferably from about between 50 μ g to the 600 μ g, and 100 μ g to 400 μ g most preferably.Alternatively, in the inventive method and the preparation the non-limiting scope of the treatment effective dose of bioactivator at the about 0.001pmol of every kg body weight to about 100pmol, the about 0.01pmol of every kg body weight is to about 10pmol, the about 0.1pmol of every kg body weight is to about 5pmol, or the about 0.5pmol of every kg body weight is to about 1.0pmol.Dosage in this scope can obtain by the single or multiple administration, for example comprises multiple dosing every day, once a day or weekly administration.According to about 0.1 to 24 hour, the preferably arrangement of time between 0.5 and 24.0 hour between the administration between the administration, the danger that the repeated intranasal administration of preparation of the present invention is kept normally, the Y2 receptor-binding peptides of continued treatment level minimizes over-exposure and side effect with the maximization clinical benefit.But give this dosage every day several times to promote satietion, preferably maybe when producing hunger, give this dosage half an hour before the meal.Purpose is a kind of like this amount of mucosal delivery Y2 receptor-binding peptides: promptly be enough to improve the amount of the concentration of the Y2 receptor-binding peptides in individual blood plasma with the concentration of the Y2 receptor-binding peptides that (promptly after individuality is finished feed) produces after the meal under the simulation normal condition.
If the oneself carries out the administration of nonprescription drugs dosage form keeping the desired concn of target site, the Y2 agonist for example dosage of PYY can be according to attending doctor or patient and difference.
In optional embodiment, the invention provides the compositions and the method for the intranasal administration that is used for the Y2 receptor-binding peptides, wherein said Y2 receptors bind peptide compounds is by the effective dosage regimen repeat administration of intranasal, and this dosage regimen relates in every day or the Y2 receptor-binding peptides repeatedly given in the time pulsation level that treatment that the experimenter keeps the Y2 receptor-binding peptides in the administration process that is prolonging is effectively increased or reduced weekly.Described compositions and method provide such Y2 receptors bind peptide compounds: promptly its by the experimenter between every day 1 and 6 times with the pulsation level that increase and reduce of nasal preparation automedication with Y2 receptor-binding peptides during maintaining 8 hours to 24 hours the administration of prolongation.
The present invention also comprises and contains aforementioned pharmaceutical compositions, active component that is useful on prevention and the treatment disease of mammalian subject or other diseases and/or test kit, packing and a plurality of container unit that carries out the method for aforementioned pharmaceutical compositions, active component administration.In brief, these test kits comprise such container or preparation: promptly it contains pharmaceutical preparation and one or more Y2 receptors bind peptide proteins, analog or analogies and/or the other biological activating agent combination of mucosa delivery reinforcing agent that is used for mucosa delivery that be mixed with disclosed by the invention.
Intranasal preparation of the present invention can use any spray bottle or syringe to carry out administration.An example of nasal spray bottle is " the nose atomizing pump with safety clamp " (Pfeiffer SAP #60548), the length of tube that soaks that it is sent the dosage of 0.1ml/ spray and has 36.05mm.It can be available from the Pfeiffer of N.J. Princeton.For example the Y2 receptor-binding peptides intranasal dose of PYY can be 0.1 μ g/kg to about 1500 μ g/kg.When as the intranasal spray administration, the granularity of preferred spray is 10-100 μ m (micron) size, preferred 20-100 μ m size.
In order to promote to lose weight, Y2 receptor-binding peptides PYY is with up to being enough to promote satietion but be low to moderate and be enough to can not induce the side effect dosage such as the non-expectation of feeling sick to carry out administration.The preferred intranasal dose of Y2 receptor-binding peptides such as PYY (3-36) is about 3 μ g-10 μ g/kg weight in patients, most preferably from about 6 μ g/kg weight in patients.At standard dose, the patient accepts 50 μ g to 800 μ g, 100 μ g to 400 μ g more preferably from about, and most preferably 150 μ g are to about 200 μ g.Such as the Y2 receptor-binding peptides of PYY (3-36) preferably on the feed before at least 10 minutes to 1 hour but be no more than the feed precontract and carried out administration in 12 to 24 hours and feel sick preventing.At least once whenever deliver medicine to the patient preferred every day before the meal, alleviates the body weight of aequum up to the patient.The weekly at least preferred maintenance dose once a day of patient's acceptance loses weight to keep then.
As shown in,, find that PYY (3-36) reduces appetite when using Y2 receptors bind peptide formulations intranasal administration of the present invention in man-hour from the data of following embodiment.Embodiment has shown also and can use PY2 receptors bind peptide formulations of the present invention to reach the physiological level after the meal first of PYY peptide by the intranasal administration approach that wherein PYY (3-36) is the Y2 receptor-binding peptides.
The aerosol nasal administration of PYY
We find that the PYY in above-mentioned preparation can use nasal spray or aerosol to carry out intranasal administration.This is surprising, because numerous protein and peptide show that in the art, following definitions is useful because the mechanical force of actuator generation is sheared or degeneration in the process of preparation spray or aerosol.
Aerosol---under pressure, pack and contain the goods of the therapeutic activity agent that when the valve system that is fit to activates, discharges.
Metered aerosol---comprise the pressurization dosage form of metered dose valve, it allows to carry out sending of homogeneous amount spray when each the activation.
Powder aerosol---under pressure, pack and contain the goods of the therapeutic activity agent of the powder type that when the valve system that is fit to activates, discharges.
Atomizing aerosol---utilize Compressed Gas to provide the aerosol goods of discharge as the required power of the goods of wet spray agent as propellant, it is usually applicable to the solution of the medicament in aqueous solvent.
Spray---spray and the liquid of refinement by air or air-flow.The treatment active component that nose spray Tetramune contains dissolving in excipient solution in non-pressurised dispersant or the mixture or suspends.
Metering spray agent---non-pressurised dosage form, it comprises that permission distributes the valve of specified quantitative spray when each the activation.
The suspension spray---contain and be scattered in the liquid vehicle and with droplet form or as the liquid preparation of the solid solid particle of refinement.
The hydrodynamics feature of the spray that spurts as drug delivery device (" DDD ") by metering nose atomizing pump.Spray feature is an indispensable part of the research and development to novel and existing nose atomizing pump, quality assurance and the necessary laws and regulations requirement of Detection of Stability rules of Food and Drug Administration (" FDA ") approval.
Found that geometric all features of spray are the best indications of nose atomizing pump overall performance.Especially, find: when the measurement of the dispersion angle (plume (plume) geometry) of spray during from the ejection of described device; The cross section ellipticity of spray, the uniformity and particles/droplets distribution (spray pattern (spray pattern)); And develop spraying time progress be the most representative performance component in the feature of atomizing pump.In the detection of quality assurance and stability, plume geometry and spray pattern measurements are to be used to confirm identify with the key of the concordance of the approval data standard of nose atomizing pump and compatibility identify.The character that following definition is used to spray.
The plume height---from the actuator top to the plume angle because the destruction of linear flow and the measured value of the nonlinear point that becomes.Based on range estimation, and, define the height of 30mm for this research in order to set up the width measure point consistent with the measurement point farthest of spray pattern to digital picture.
The longest band of main shaft-can in crossing the match spray pattern of COMw, draw with ultimate unit (mm).
The minimum band of minor axis-can in crossing the match spray pattern of COMw, draw with ultimate unit (mm).
Ellipticity-main shaft is to the ratio of minor axis.
D
10The total liquid volume of-sample 10% by the droplet diameter of forming than the droplet of minor diameter (μ m).
D
50The total liquid volume of-sample 50% by the droplet diameter of forming than the droplet of minor diameter (μ m), be also referred to as mass median diameter.
D
90The total liquid volume of-sample 90% by the droplet diameter of forming than the droplet of minor diameter (μ m).
The measurement of span-dispersion of distribution is worth more for a short time, and it is narrow more to distribute.Following calculating span:
%RSD-relative standard deviation percent (standard deviation is divided by serial mean and multiply by 100) is also referred to as %CV.
The nose sprayer unit comprises bottle and the actuator of placing the PYY preparation, and described causing led device orders about PYY when activating or work spraying plume and sprayed from aerosol container by described actuator.Described bottle can be the smooth glass bottle that comprises I type Pyrex.Described bottle can have threaded top and concave bottom.Valve protection cap can be the polypropylene of SANYE stricture of vagina (trifoil-lined).SANYE (Tri-Foil) WP comprise by 0.00067 " white LDPE is bonded to 0.0035 " aluminium foil then in conjunction with LDPE film/foam/film coextrusion thing 0.0005 " clear polymer.The all components of this liner is GRAS.Valve protection cap can comprise polypropylene and be used for required bottle with suitable screw thread.
Embodiment
Embodiment 1:
The vitro tissue model evaluation of various PYY3-36 preparations
There is PYY under the situation in evaluation at various excipient (EDTA, polysorbate80 (Tween 80), oleic acid, Sorbitol and ethanol)
3-36Body outer osmotic.Use 10mM citrate buffer (citric acid/sodium citrate) that preparation is adjusted to pH4.The several formulations that is detected is shown in table 1.All samples is transparent and colourless.
The description of the preparation that is detected among table 1 embodiment 1
With vitro tissue model evaluation sample.Employed cell line is trachea/bronchial epithelial cell (EpiAirway normal, that come from the mankind
TMOrganize models, MatTek Corporation).Cell is as providing at the insert that comprises confluent growth on the Millipore Millicell-CM filter of transparent hydrophilic polyflon (PTFE).When collecting, before use with thin film in 1ml basal medium (reactive phenol Da Erbaikeshi red and no hydrocortisone improves Yi Geershi culture medium (DMEM)) in 37C/5%CO
2Cultivated 24-48 hour.Provide nutrient to carry out the collection of every day to insert.
Each hole of 1ml MatTek basal medium will respectively be organized insert to put into to contain.At the end face of insert, use the detection preparation of 50 μ l according to the research design in the table 1, and sample is put in 37 ℃ of vibrators (～100rpm) last 1 hour.Following media samples is stored in 4 ℃ reaches 48 hours to carry out (enzyme immunoassay (EIA) (EIA) evaluation of lactic acid dehydrogenase (LDH, cytotoxicity) and sample permeability.Before 1 hour incubation and measure afterwards through epithelium resistance (TER).After incubation, through the cell viability of mitochondrion dehydrogenase (MDH) determination and analysis cell insert.
1. stride the resistance of monolayer
Use utilizes electrode cable to be connected in to have EVOM epithelium voltohmyst, and (Endohm-12 tissue resistance FL) is measured the measurement that TER is implemented in the chamber for World PrecisionInstruments, Sarasota.Before checking calibration in the MatTek culture medium of outage the blank insert of counter electrode and tissue culture at least 20 minutes.The 1.5ml culture medium and the 300 μ l culture medium in the blank insert that are used in the Endohm tissue cavity are measured background resistance.Adjust top electrodes make its near but do not contact the end face that embeds film.The background resistance of blank insert is about 5-20 ohm.Measure for each TER, 300 μ l MatTek culture medium are added into insert, then put into the Endohm chamber.Ohmmeter is shown (measured resistance-blank) X0.6cm
2
The result shows that negative control and culture medium expose any significant change that does not show TER afterwards to impinging upon 1 hour.The Triton contrast shows that TER reduces basically fully.Contain EDTA and alcoholic acid sample performance TER and reduce, this is with close-connected open consistent.
2. cell viability
Use MTT to measure (MTT-100, MatTek test kit) and estimate cell viability.The MTT concentrated solution that melts and dilute is moved to 24 well culture plates with pipet (300 μ l).Gently drying is organized insert, put it into to cultivate in the plate hole, and in 37 ℃ of incubations 3 hours.After incubation, take out each insert from culture plate, the soft suction printed, and puts into 24 holes and extract culture plate.Then cell is trained in the extractant solution that insert is dipped in every hole 2.0ml (covering sample fully).Cover and seal and extract culture plate to alleviate the evaporation of extractant.Spend the night in the room temperature dark after the incubation, the liquid in each insert is refunded to the hole of therefrom taking out it, and abandon insert.Extractant solution (200 μ l, with double) is at least blankly moved to 96 hole microtitration plates with pipet together with extracting.On plate reading machine in the optical density of 550nm measuring samples.
All samples and negative control and the acceptable cell viability of culture medium contrast performance promptly compare at least 80% with the culture medium contrast.As expection, Triton X contrast significantly reduces cell viability.
3. cytotoxicity
(Promega Corp., Madison WT) measure lactic acid dehydrogenase (LDH) is measured cell death from the forfeiture of cell quantity (cytotoxicity) by using CytoTox 96 cytotoxic assay test kits.
The LDH that estimates the top culture medium analyzes.Consider initial sample load volume, the culture medium of appropriate amount is added into end face in proper order to 300 μ L altogether.Vibration insert 5 minutes takes out 150 μ L top culture medium then, and it is dispensed in the eppendorf pipe, and at 10000rpm centrifugal 3 minutes.Take out the supernatant of 2 μ L volumes, and it is added in 96 well culture plates.The culture medium of 48 μ L volumes is used to dilute supernatant with preparation 25x diluent.LDH for substrate outside culture medium analyzes, and 50 μ L samples, 96 holes of packing into are measured in the culture plate.Fresh acellular culture medium is as blank.50 μ L matrix solutions are added into each hole and incubation culture plate 30 minutes in the room temperature dark.After incubation, 50 μ L stop buffers are added into each hole and read plate in 490nm on the optical density plate reading machine.LDH measures by the substrate outside, and all samples and negative control and culture medium contrast all have lower cytotoxicity, promptly compares with the culture medium contrast and is no more than 20%.As expection, Triton X contrast has higher cytotoxicity.
4. cell permeability
PYY
3-36The EIA test kit is available from Phoenix Pharmaceuticals, and (Belmont CA), and measures according to the description that is provided Inc..
Permeability results (Fig. 1) shows that 10mM EDTA or 1-2% ethanol provide significant % permeability, i.e. the medicine of the about 1.5-2% of infiltration after 1 hour.By contrast, observe the extremely low % permeability of negative control (isoosmotic, impermeability reinforcing agent), for example＜0.3%.Observe sample 11 (1mg/mL EDTA, 1% ethanol) and 12 (10mg/mL EDTA, 2% ethanol) and have maximum % permeability.Combination provides and close-connected reduction and the increase PYY that opens consistent TER EDTA with ethanol
3-36Permeability has acceptable low cytotoxicity and high cell viability.
The in-vitro evaluation of various PYY3-36 preparations
Purpose is in order further to detect ethanol, EDTA and Tween 80 as PYY
3-36The effect of permeability reinforcing agent.Use 10mM citrate buffer (citric acid/sodium citrate) that preparation is adjusted to pH4.The various preparations that detect among the embodiment 2 are shown in table 2.Except these samples, comprise that also feminine gender etc. oozes contrast, cell culture medium contrast and Triton-X contrast.
The description of the preparation that detects among table 2: the embodiment 2
| Preparation | Describe |
| Current | 45mg/niL M β CD, 1mg/mL DDPC, 1mg/mL EDTA, 10mM citrate buffer (pH 5.0), 25mM lactose, 100mM Sorbitol, 0.5% |
| GRAS# | |
| 1 | 1mg/mL EDTA, 1%EtOH, 10mM acetate buffer (pH 4.0), 0.02% |
| GRAS# | |
| 2 | 10mg/mL EDTA, 2%EtOH, 10mM acetate buffer (pH 4.0), 0.02%BZK |
| GRAS#3 | 10mg/mL EDTA, 10mM second salt buffer (pH 4.0), 0.02%BZK |
| Saline | Isotonic saline solution |
TER, MTT, LDH and the PYY of each sample in the table 2 have been detected
3-36Permeability is as the various assay methods of enforcement as described in the embodiment 1.
After 1 hour exposure, negative control and culture medium contrast do not show any significant change of TER.The Triton contrast shows that TER reduces basically fully.The sample of all detections shows that TER reduces.The compositions of these data declarations EDTA, ethanol, Tween 80 and these excipient reduces TER, and this is with close-connected open consistent.
LDH measures by the substrate outside, and all samples and negative control and culture medium contrast have lower cytotoxicity, promptly compares with the culture medium contrast and is no more than 20% toxicity.Triton X contrast has higher cytotoxicity.
The infiltrative data of percentage ratio are shown in Fig. 2.Negative control has extremely low permeability, and this is consistent with embodiment 1.These three kinds of GRAS samples all show remarkable high permeability under the testing conditions.These data confirm that also the compositions of EDTA, ethanol, Tween 80 and these excipient reduces TER and improves the PYY3-36 permeability, has acceptable low cytotoxicity.
Embodiment 3:
The vitro tissue model evaluation of various PYY3-36 preparations
The purpose of this research is in order further to detect ethanol, EDTA and Tween 80 as PYY
3-36The effect of potential permeability reinforcing agent.
There is PYY under the situation in evaluation at various excipient (EDTA, ethanol, Tween 80, DDPC and methyl-beta-schardinger dextrin-)
3-36Body outer osmotic.Use 10mM citrate buffer (citric acid/sodium citrate) that preparation is adjusted to pH4.2-4.3.The various preparations that detect among the embodiment 3 are shown in table 3.Except these samples, comprise that also feminine gender etc. oozes contrast, cell culture medium contrast and Triton-X contrast.Sample 3-1 contains methyl-beta-schardinger dextrin-, and (compositions of M-β-CD), DDPC and EDTA had shown before that said composition provided PYY
3-36Enhanced permeability (the 10/768th, No. 288 U.S. Patent application [publication No. 20040209807]).
TER, MTT, LDH and the PYY of each sample in the table 3 have been detected
3-36Permeability is as each method for measuring of enforcement as described in the embodiment 1.
The description of the preparation that is detected among table 3: the embodiment 3
All samples is transparent and colourless.After 1 hour exposure, culture medium and negative control contrast do not show any significant change of TER.Sample 3-1 to 3-13 all shows the remarkable reduction with respect to the TER of culture medium contrast, shows that the compositions of EDTA, ethanol, Tween 80 and these excipient reduces TER.Data show that also the preparation that contains methyl-beta-schardinger dextrin-, EDTA and DDPC reduces TER.
The infiltrative data of percentage ratio (Fig. 3) show the higher % permeability of all samples performance of 3-1 to 3-13.Negative control has extremely low % permeability, and this is consistent with embodiment 1.These data confirm that further EDTA, ethanol, Tween 80 and compositions thereof provide the reduction of TER, with close-connected open consistent and increase PYY
3-36Permeability.These preparations have obtained to be equivalent to or to be better than the permeability (the 10/768th, No. 288 U.S. Patent application) of the preparation of the previous described EDTA of containing, DDPC and methyl-beta-schardinger dextrin-.
Embodiment 4:
The vitro tissue model evaluation of various PYY3-36 preparations
The purpose of this research is in order further to detect ethanol, EDTA and Tween 80 as PYY
3-36The effect of potential permeability reinforcing agent.In this embodiment, to different buffer (citrate buffer, acetate buffer and glutamate, Glu buffer), and different antiseptic (methaform and benzalkonium chloride) is tested.The data of all preparations are compared with the preparation that contains methyl-beta-schardinger dextrin-, DDPC and EDTA.
There is PYY under the situation in evaluation at various excipient (EDTA, ethanol, Tween 80, DDPC and methyl-beta-schardinger dextrin-)
3-36Body outer osmotic.Use 10mM citrate buffer (citric acid/sodium citrate) that preparation is adjusted to pH4.The various preparations that detected are shown in table 4.Except these samples, comprise that also feminine gender etc. oozes contrast, cell culture medium contrast and Triton-X contrast.Sample 4-1 comprises methyl-beta-schardinger dextrin-, and (preparation of M-β-CD), DDPC and EDTA, the previous demonstration provides PYY
3-36Enhanced permeability (No. the 10/768th, 288, U.S. Patent application).
The description of the preparation that is detected among table 4: the embodiment 4
TER, MTT, LDH and the PYY of the various samples in the table 4 have been detected
3-36Permeability is as each method for measuring of enforcement as described in the embodiment 1.In table 4, " BZK "=0.2mg/mL benzalkonium chloride, " CB "=5mg/mL methaform.
Triton X contrast shows that TER reduces basically completely.Negative control and culture medium contrast show the TER no change.All test sample performances are with respect to the remarkable reduction of the TER of negative control.Data show is at PYY
3-36Acetate and glutamate, Glu buffer can replace citrate buffer in the preparation.Data are also supported can for example benzalkonium chloride and methaform be added into PYY with antiseptic
3-36Preparation.
Permeability data (Fig. 4) shows the higher % permeability of all samples, and 4-4,4-6 and the highest % permeability of 4-9 performance.These data further confirm the permeability potentiation of EDTA, ethanol, Tween 80 and compositions thereof.Acetate and glutamate, Glu buffer replace citrate buffer and do not lose permeability.In addition, benzalkonium chloride and methaform are successfully added as antiseptic.
These data acknowledgements EDTA, ethanol and Tween 80 preparation can realize being equivalent to or being better than the permeability of the preparation of the previous described EDTA of containing, DDPC and methyl-beta-schardinger dextrin-.At PYY
3-36In the preparation, acetate and glutamate, Glu buffer can replace citrate buffer, and can for example benzalkonium chloride and methaform are added into PYY with antiseptic
3-36In the preparation.
The intranasal PYY that comprises acetate buffer, ethanol and EDTA
3-36
The pharmacokinetics test of preparation
In mammal, carry out the PYY of different intranasal preparations
3-36Pharmacokinetics test.Described preparation comprises as the EDTA of penetration enhancers and ethanol (acetate buffer; PH 4.0).For comparing, (this combination that had before shown excipient causes PYY also to comprise the preparation of methyl-beta-schardinger dextrin-, DDPC and EDTA as penetration enhancers at intranasal administration
3-36The enhancing (Application No. 10/768,288) of infiltration.In addition, administration does not contain the preparation of reinforcing agent, so that illustrate the usefulness that penetration enhancers improves drug conveying.
The different preparations that detect among the embodiment 5 are described in table 5.Sample 5-1 comprise as reinforcing agent methyl-beta-schardinger dextrin-, DDPC and EDTA and as the CB of antiseptic, intranasal administration 5-1 is used for contrast.Sample 5-2,5-3 and 5-4 comprise 1 or 10mg/mL EDTA and 0,10 or 20mg/mL ethanol, and comprise BZK as antiseptic.Preparation sample 5-5 does not contain penetration enhancers in buffer.
Detect the description of preparation among table 5: the embodiment 5
| Sample number | Dosage (μ g/kg) | PYY 3-36Concentration (mg/mL) | Preparation |
| 5-1 | 205 | 13.6 | 45mg/mL M β CD, 1mg/mL DDPC, 1mg/mL EDTA, 10mM citrate (pH 5.0), 25mM lactose, 100mM Sorbitol, 0.5%CB |
| 5-2 | 205 | 13.6 | 1mg/mL EDTA, 1%EtOH, 10mM acetate (pH 4.0), 0.02%BZK |
| 5-3 | 205 | 13.6 | 10mg/mL EDTA, 2%EtOH, 10mM acetate (pH 4.0), 0.02%BZK |
| 5-4 | 205 | 13.6 | 10mg/mL EDTA, 10mM acetate buffer (pH 4.0), 0.02%BZK |
| 5-5 | 205 | 13.6 | Isotonic saline solution |
The pharmacokinetics of carrying out in the New Zealand white rabbit (PK) is identified and is observed management of laboratory animal principle (NIH publishes 86-23, revision in 1985).Before administration and behind the intranasal administration 2.5,5,10,15,30,45,60 and 120 minutes, extract blood sample from the edge ear vein.Measure PYY in the blood plasma by EIA
3-36Concentration (Foerder C. waits the people, Quantitative Determination of PeptideYY3-36 in Plasma by Radioimmunoassay, AAPS 2004 NationalBiotechnology Conference, Boston, MA, May 2004).(Mountain View CA) carries out PK and calculates for Pharsight Corporation, 4.0 versions to adopt non-compartment model method to utilize WinNonlin software.Data are expressed as mean+/-standard error.
PK result is summarized in table 6.Concerning all samples, the T of intranasal approach
MaxApproximately between the 26-43.Sample 5-1 has the highest C
MaxAnd AUC, with respect to the situation that does not have reinforcing agent (sample 5-5), the latter increases by 27 times.5-1 compares with sample, the C of sample 5-2 and 5-3
MaxShow lower standard deviation.And 5-1 compares with sample, the Auc of sample 5-2
LastShow lower standard deviation.Sample 5-2 shows and sample 5-1 Auc much at one
Last
Detect the PK general introduction of preparation among table 6: the embodiment 5
| Sample | Tmax (min) | Cmax (pg/mL) | Cmax SD(％) | Auclast (min ＊pg/mL) | AUC SD (％) | The multiple that AUC increases ＊ |
| 5-1 | 29 | 20713 | 46 | 1002914 | 43 | 27 |
| 5-2 | 38 | 14271 | 23 | 929365 | 31 | 25 |
| 5-3 | 43 | 13954 | 22 | 783633 | 44 | 21 |
| 5-4 | 40 | 8902 | 75 | 722448 | 79 | 20 |
| 5-5 | 26 | 517 | n/d | 36476 | n/ |
1 |
N/d=does not detect;
*With respect to no reinforcing agent (8-5)
Compare with no reinforcing agent, all groups that comprise EDTA and/or ethanol combination (sample 5-2,5-3 and 5-4) show enhanced basically infiltration, reach about 20-to 25-doubly.
Embodiment 6:
Under the condition of not having any other excipient existence, different buffer are to PYY
3-36
Thermal stress stability
Influence
Press the listed preparation preparation of table 7.Tested buffer comprises citrate, tartrate, acetate and glutamate, Glu.In all cases, PYY
3-36Be 1mg/mL, pH is 5.0.The brown non-silanization bottle of 1-cc is filled preparation to be measured, every bottled 1mL, bottle SANYE stricture of vagina (trifoil-lined) cap seal mouth.Bottle available from SGD Glass Inc. (New York, NY).These bottles have screw lid and concave bottom (U-shape structure).Bottle is made up of I type Pyrex.(Union NJ), is made up of polypropylene lid, the bottle that is used to be scheduled to by suitable screw threadization available from O ' BerkCompany.Lid is the SANYE stricture of vagina.Tri-Foil WP is by 0.0005, and " transparent polyester is formed, and described polyester is attached to LDPE film/foam/film co-extrusion thing then by 0.00067 " white LDPE is attached to 0.0035 " aluminium foil.The assembly of all liners all is GRAS (generally recognized as safe).
Bottle is stored in 25,40 and 50 ℃, detects chemical stability in different time points by HPLC, shows as the peptide response rate % peptide of data determination (with respect to the t=0 time).At 45 ℃, and HPLC method use 5-μ m C18 post (Supelco, BIO Wide-pore, 250 * 4.6mm), using the mobile phase component is 0.1% trifluoroacetic acid (A) and 0.08% trifluoroacetic acid (B) that is dissolved in acetonitrile, isocratic elution in 27%A/73%B.Utilize UV to detect at 210nm.Undertaken quantitatively by external standard method.
The preparation of estimating among table 7: the embodiment 6
| Group # | PYY 3-36 (mg/mL) | Citrate buffer (mM) | Tartrate buffer (mM) | Acetate buffer (mM) | Glutamate, Glu buffer (mM) | pH |
| 1-1 | 1 | 10 | - | - | - | 5.0 |
| 1-2 | 1 | - | 10 | - | - | 5.0 |
| 1-3 | 1 | - | - | 10 | - | 5.0 |
| 1-4 | 1 | - | - | - | 10 | 5.0 |
The HPLC data show utilizes acetate and glutamate, Glu buffer to reach optimum stabilization (the highest response rate after the different condition preservation).The peptide response rate the results are shown in Fig. 5 A, 5B and 5C, corresponds respectively to 25,40 and 50 ℃.
This result of experiment is revelatory, because those preserve PYY
3-36The best buffer of stability is the monovalence buffer, and those do not improve PYY
3-36Stable is the multivalence buffer.The monovalence buffer may strengthen PYY under thermal stress conditions
3-36Stability.
Embodiment 7:
Under the sorbitol existence condition as tonicity agents (Tonicifier), buffer type and pH should to heat
The influence of power stability
Press the listed preparation preparation of table 8.Tested buffer is citrate, acetate and glutamate, Glu.In all cases, PYY
3-36Be 2mg/mL, the sorbitol of existence provides the osmolality of 225mOsm.PH from 3.5 to 5.0.The brown non-silanization bottle of 1-cc of then these preparations being packed into, every bottled 1mL, bottle is with SANYE stricture of vagina cap seal mouth.Preserve sample, press the embodiment 6 described mensuration response rate.
The preparation of estimating among table 8: the embodiment 7
| Group # | PYY (mg/mL) | Citrate buffer (mM) | Acetate buffer (mM) | Glutamate, Glu buffer (mM) | Sorbitol (mM) | pH |
| 2-1 | 2 | 10 | 0 | 0 | 210 | 5.0 |
| 2-2 | 2 | 10 | 0 | 0 | 210 | 4.5 |
| 2-3 | 2 | 10 | 0 | 0 | 210 | 4.0 |
| 2-4 | 2 | 10 | 0 | 0 | 210 | 3.5 |
| 2-5 | 2 | 0 | 10 | 0 | 210 | 5.0 |
| 2-6 | 2 | 0 | 10 | 0 | 210 | 4.5 |
| 2-7 | 2 | 0 | 10 | 0 | 210 | 4.0 |
| 2-8 | 2 | 0 | 10 | 0 | 210 | 3.5 |
| 2-9 | 2 | 0 | 0 | 10 | 210 | 5.0 |
| 2-10 | 2 | 0 | 0 | 10 | 210 | 4.5 |
| 2-11 | 2 | 0 | 0 | 10 | 210 | 4.0 |
| 2-12 | 2 | 0 | 0 | 10 | 210 | 3.5 |
| 2-13 | 2 | 0 | 0 | 0 | 0 | 5.0 |
| 2-14 | 2 | 0 | 0 | 0 | 0 | 4.5 |
| 2-15 | 2 | 0 | 0 | 0 | 0 | 4.0 |
| 2-16 | 2 | 0 | 0 | 0 | 0 | 3.5 |
The preparation for temperature optimal heat stability that the HPLC data show is identified is acetate and glutamate, Glu buffer and the preparation that do not have buffer.Peptide response rate result such as Fig. 6 A, 6B, shown in 6C and the 6D, its buffer is respectively citrate, acetate, glutamate, Glu or does not have buffer.
Identical with embodiment 6, the optimum performance preparation is that those comprise monovalence buffer (that is, acetate or glutamate, Glu) or do not comprise pH scope that surpass to identify and the preparation of the buffer of temperature.Those preparations that comprise multivalence buffer (that is citrate) do not reach optimum performance.In addition, keep PYY3-36 the pH of suitable stability show as pH 3.5-pH 4.5, irrelevant with the buffer that uses.
Embodiment 8:
Different PYY
3-36
The heat stability of preparation and atomizing stress stability
The target of this research is check PYY
3-36The stability of the heat of preparation and atomizing stress, described PYY
3-36Preparation comprises as PYY
3-36The ethanol of potential penetration enhancers, EDTA and Tween-80 (Tween 80).In this embodiment, add different buffer (acetate buffer and glutamate, Glu buffer), and the antiseptic (benzalkonium chloride) that is used for repeatedly using preparation.
At the non-silanization Brown Glass Brown glass bottles and jars only of the 3mL about 3.9mL of solution that packs into.Bottle adjunction 100 μ L actuators.Actuator available from Pfeiffer of America (Princeton, NJ).Bottle causes up to realizing for the first time spraying fully by activating.After observing for the first time spraying fully, carry out other twice actuating to confirm initiation completely.After initiation is finished, spray the first time that is construed to this research next time and spray.All actuatings all are manually to carry out.
Be stored in 30 ℃/65% relative humidity (by day and night) at whole spray booth bottles.After the container taking-up, bottle sprays (in 5 minutes), puts back to container then.Whole spray booths minimum 1 hour at interval.Bottle sprays (TID) 3 times, totally 10 days every day.
By the embodiment 1 described HPLC method of carrying out.The data of stabilized peptide are expressed as percentage ratio and reclaim the concentration of native peptides (with respect to the initial t=0 time) and percentage ratio purity (peak area of native peptides is divided by the relevant peak area of all peptides).At the 0th day, 5 days and 10 days of the atomizing stress that is exposed to 3 sprayings of elevated temperature and every day, measure the HPLC data of peptide content.
The different preparations that detect among the embodiment 8 are described in table 9.All samples comprises 6mg/mLPYY
3-36Sample comprises 10mg/mL EDTA, 20mg/mL ethanol, benzalkonium chloride (BZK) (as being used for nonexpondable representative antiseptic) and 0 or 1mg/mL Tween 80.Sample 3-1 and 3-2 comprise the 10mM acetate buffer (acetic acid/sodium acetate buffer system) of pH 4.3.Sample 3-3 comprises the 10mM glutamate, Glu buffer (glutamic acid/sodium glutamate buffer system) of pH 4.3.
The description of tested preparation among table 9: the embodiment 8
| Sample # | EDTA(mg/mL) | EtOH(mg/mL) | Tween 80 (mg/mL) | Buffer (10mM) |
| 3-1 | 10 | 20 | 1 | Acetate |
| 3-2 | 10 | 20 | 0 | Acetate |
| 3-3 | 10 | 20 | 0 | Glutamate, Glu |
In the HPLC data show of the peptide content of the 0th day, 5 days of the atomizing stress that is exposed to elevated temperature and every day 3 times spraying and 10 days in Fig. 7.
Data show is in containing the alcoholic acid preparation of 10mg/mL EDTA and 20mg/mL, and the existence of 1mg/mL Tween-80 (3-1, black triangle) has stabilizing effect with respect to the same preparation that does not contain Tween-80 (3-2, open squares).With respect to acetate buffer (3-2, open squares), glutamate, Glu buffer (3-3, open diamonds) provides higher stability.PYY
3-36Residual main kind has and natural PYY in the data show solution of purity
3-36Identical HPLC retention time is consistent with the peptide loss that gathering causes.Precipitation is mainly by PYY
3-36Monomer is formed, and shows the PYY under heat and the atomizing stress
3-36Loss comes from hydrophobicity (for example, non-covalent) gathering.
When being exposed to the combination of the stress that sprays high temperature and every day 3 times, PYY
3-36Preparation may suffer hydrophobic, and is for example non-covalent, assembles.Under certain condition, the existence of Tween-80 improves this situation.Simultaneously, data show is for stability, and glutamate, Glu may be preferred buffer system with respect to acetate.
Embodiment 9:
Different PYY
3-36
The heat stability of preparation and atomizing stress stability
The target of this research is check PYY
3-36The stability of preparation heat resistanceheat resistant and atomizing stress, described PYY
3-36Preparation comprises as PYY
3-36The ethanol of potential penetration enhancers, EDTA and Tween 80.In this embodiment, detect the acetate buffer from pH 3.8 to 4.4, methaform is as nonexpondable antiseptic.
The method of using among this embodiment is described identical with top embodiment 8.Identical with embodiment 8, bottle was stored in 30 ℃/65% relative humidity (reaching night by day) between all sprayed herein, bottle is sprayed 3 times (TID) every day, and totally 10 days, at the PYY that used in the definite spraying of HPLC (the last injection on the same day) in the 0th, 5 and 10 day of spraying
3-36Content.
The different preparations that detect among this embodiment are described in table 10.All samples comprises 6mg/mLPYY
3-36, 10mM acetate buffer (acetic acid/sodium acetate buffer system) and 5mg/mL chlorobutanol (CB).Sample 4-1 comprises 10mg/mL EDTA to 4-8,20mg/mL ethanol and 0 or 1mg/mL Tween 80, and pH is between 3.8 to 4.4.For comparing, last sample, 4-9 comprises 45mg/mL methyl-beta-schardinger dextrin-, 1mg/mL DDPC and 1mg/mL EDTA, pH 4.0.The back one preparation before on the books (US number of patent application 10/768288, people such as Quay. " Compositions and methods for enhanced mucosal delivery of Y2receptor-binding peptides and methods for treating and preventing obesity ").
The description of tested preparation among table 10: the embodiment 9
| Group # | Me-β-CD (mg/mL) | DDPC (mg/mL) | EDTA (mg/mL) | Ethanol (mg/mL) | Tween 80 (mg/mL) | pH |
| 4-1 | 0 | 0 | 10 | 20 | 1 | 3.8 |
| 4-2 | 0 | 0 | 10 | 20 | 0 | 3.8 |
| 4-3 | 0 | 0 | 10 | 20 | 1 | 4.0 |
| 4-4 | 0 | 0 | 10 | 20 | 0 | 4.0 |
| 4-5 | 0 | 0 | 10 | 20 | 1 | 4.2 |
| 4-6 | 0 | 0 | 10 | 20 | 0 | 4.2 |
| 4-7 | 0 | 0 | 10 | 20 | 1 | 4.4 |
| 4-8 | 0 | 0 | 10 | 20 | 0 | 4.4 |
| 4-9 | 45 | 1 | 1 | 0 | 0 | 4.0 |
Abbreviation:
Me-β-CD=methyl-beta-schardinger dextrin-
The EDTA=disodiumedetate
DDPC=L-α-two caprinoyl phosphatidylcholines (L-α-phosphatidylcholine didecanoyl).
In the HPLC data show of the peptide content that is exposed to the 0th day, 5 days of the atomizing stress that sprays elevated temperature and every day 3 times and 10 days in Fig. 8.
Data show is after the heat and atomizing stress of combination, and the existence of 1mg/mL Tween-80 improves PYY
3-36Stability (comparative sample 4-1 and 4-2 (being respectively solid and open diamonds); Comparative sample 4-3 and 4-4 (being respectively solid and open squares); Comparative sample 4-5 and 4-6 (being respectively solid and open circles); With comparative sample 4-7 and 4-8 (being respectively solid and hollow triangle)).When pH when 4.4 are reduced to 3.8, also have stable trend of rising.The sample that comprises 1mg/mLTween-80 (4-1) of pH 3.8 shows and sample 4-9 stability much at one.
The existence of 1mg/mL Tween-80 provides PYY
3-36Preparation is for the stability of heat and spraying stress, described PYY
3-36Preparation comprises 10mg/mL EDTA and 20mg/mL ethanol.When pH when 4.4 are reduced to 3.8, stability raises.
Embodiment 10:
PYY
3-36
The heat stability of preparation and atomizing stress stability
The target of this research is check PYY
3-36The stability of preparation heat resistanceheat resistant and atomizing stress, described PYY
3-36Preparation comprises as PYY
3-36The ethanol of potential penetration enhancers, EDTA and Tween 80.In this embodiment, buffer is acetate or glutamate, Glu, and pH is 4.0, and the level of Tween-80 from 0 to 50mg/mL.Add methaform as antiseptic.The method that this embodiment uses is described in embodiment 8.Bottle is stored in 30 ℃/65% relative humidity between all sprayings, bottle spraying TID, and totally 10 days, at the PYY that used in the definite spraying of HPLC (the last spraying on the same day) in the 0th, 5 and 10 day of spraying
3-36Content.The different preparations that detect among this embodiment are described in table 11.All samples comprises 6mg/mL PYY
3-36With 5mg/mL methaform (CB).Sample 5-1 comprises 1-10mg/mL EDTA to 5-13,10-20mg/mL ethanol, and 0-50mg/mL Tween-80 also comprises 10mM acetate buffer/pH 5 or 10mM glutamate, Glu buffer/pH 4.For comparing, last sample, 5-14 comprises 45mg/mL methyl-beta-schardinger dextrin-, 1mg/mL DDPC and 1mg/mL EDTA, pH 4.0.
The description of tested preparation among table 11: the embodiment 10
Fig. 9 A show sample 5-1, the stability data of 5-2 and 5-3.The interpolation that relatively shows 1mg/mL Tween 80 of 5-1 and 5-2 does not have improved effect, because under experimental condition (for example, 1mg/mL EDTA and 10mg/mL ethanol, pH 4), two samples all obtain advantages of excellent stability.The relative 5-3 of sample 5-2 relatively is presented under the experimental condition, observes the lower a little stability of the relative acetate buffer of glutamate, Glu buffer.
Fig. 9 B shows 5-4,5-5, the stability data of 5-6 and 5-7.All these samples comprise 10mg/mL EDTA, 20mg/mL ethanol and 10mM acetate buffer/pH 4.0.This series of samples has the Tween 80 of varying level, promptly from 0 to 50mg/mL.Usually, compare with the sample among Fig. 9 A, observed stability is lower a little under Fig. 9 B condition.Sample 5-7 observes the highest stability, and described sample 5-7 comprises the Tween 80 (50mg/mL) of top level in this series.
Fig. 9 C shows heat and atomizing stress to sample 5-4,5-5, the influence of 5-6 and 5-7.All these samples comprise 10mg/mL EDTA, 20mg/mL ethanol and 10mM glutamate, Glu buffer/pH 4.0.This series of samples has the Tween 80 of varying level, promptly from 0 to 50mg/mL.Usually, compare with the sample among Fig. 9 B with Fig. 9 A, observed stability is lower a little under Fig. 9 C condition.Sample 5-11 observes the highest stability, and described sample 5-11 comprises the Tween 80 (50mg/mL) of top level in this series.
At last, sample 5-12, the stability data of 5-13 and 5-14 is shown in 9D.All 3 samples all show advantages of excellent stability,, are being exposed to heat and atomizing stress does not have the variation of the peptide response rate basically after 10 days that is.
Claims (93)
1. be used to strengthen the pharmaceutical preparation to mammal mucosal delivery PYY, wherein said preparation comprises the PYY that treats effective dose, polar organic solvent and cation chelating agent that can be miscible with water.
2. preparation according to claim 1, wherein PYY is PYY (3-36), described can be that ethanol and described cation chelating agent are EDTA with the miscible polar organic solvent of water.
3. preparation according to claim 2, wherein alcoholic acid formula concentration are about 1% (v/v) or higher.
4. preparation according to claim 2, wherein alcoholic acid formula concentration are about 2% (v/v) or higher.
5. preparation according to claim 2, wherein alcoholic acid formula concentration are about 10% (v/v) or higher.
6. preparation according to claim 2, wherein the concentration of EDTA in described preparation is at least about 1mg/mL.
7. preparation according to claim 2, wherein the concentration of EDTA in described preparation is at least about 2mg/mL.
8. preparation according to claim 2, wherein the concentration of EDTA in described preparation is at least about 10mg/mL.
9. preparation according to claim 2, it also comprises surfactant.
10. preparation according to claim 9, wherein said surfactant are tween 80.
11. preparation according to claim 10, wherein tween 80 in described preparation with 50mg/mL or lower the existence.
12. preparation according to claim 10, wherein tween 80 in described preparation with 10mg/mL or lower the existence.
13. preparation according to claim 10, wherein tween 80 in described preparation with 1mg/mL or lower the existence.
14. preparation according to claim 2, it also comprises buffer salt.
15. preparation according to claim 9, it also comprises buffer salt.
16. preparation according to claim 14, wherein said buffer salt are acetate or glutamate, Glu.
17. preparation according to claim 15, wherein said buffer salt are acetate or glutamate, Glu.
18. preparation according to claim 16, wherein said buffer salt is a glutamate, Glu.
19. preparation according to claim 17, wherein said buffer salt is a glutamate, Glu.
20. preparation according to claim 2, wherein said pH are about 5.0 or lower.
21. preparation according to claim 2, wherein said pH are about 4.4 or lower.
22. preparation according to claim 2, wherein said pH are about 4.0 or lower.
23. preparation according to claim 2, wherein said pH are about 3.8 or lower.
24. preparation according to claim 2, it also comprises antiseptic.
25. preparation according to claim 24, wherein said antiseptic are methaform or benzalkonium chloride.
26. preparation according to claim 2 wherein gives the P that described preparation causes and measures by contact monolayer mucomembranous cell in the isosmotic solution that does not contain penetration enhancers
AppCompare the P of measurement
AppBe about its 2 times or higher.
27. preparation according to claim 2 wherein gives the P that described preparation causes and measures by contact monolayer mucomembranous cell in the isosmotic solution that does not contain penetration enhancers
AppCompare the P of measurement
AppBe about its 5 times or higher.
28. preparation according to claim 2 wherein gives the P that described preparation causes and measures by contact monolayer mucomembranous cell in the isosmotic solution that does not contain penetration enhancers
AppCompare the P of measurement
AppBe about its 10 times or higher.
29. preparation according to claim 2 wherein gives the P that described preparation causes and measures by contact monolayer mucomembranous cell in the isosmotic solution that does not contain penetration enhancers
AppCompare measured P
AppBe about its 10 times or higher.
30. according to claim 26,27,28 or 29 described preparations, wherein said mucomembranous cell is a bronchial epithelial cell.
31. preparation according to claim 2, wherein intranasal gives described preparation and causes and give not contain the measured AUC of normal isotonic saline solution of penetration enhancers for intranasal in mammal
LastCompare the AUC of measurement
LastBe about its 2 times or higher.
32. preparation according to claim 2, wherein intranasal gives described preparation and causes and give not contain the measured AUCl of normal isotonic saline solution of penetration enhancers for intranasal in mammal
AstCompare measured AUC
LastBe about its 5 times or higher.
33. preparation according to claim 2, wherein intranasal gives described preparation and causes and give not contain the measured AUC of normal isotonic saline solution of penetration enhancers for intranasal in mammal
LastCompare the AUC of measurement
LastBe about its 10 times or higher.
34. preparation according to claim 2, wherein intranasal gives described preparation and causes and give not contain the measured AUC of normal isotonic saline solution of penetration enhancers for intranasal in mammal
LastCompare the AUC of measurement
LastBe about its 20 times or higher.
35. be used to strengthen the pharmaceutical preparation to mammal mucosal delivery PYY, wherein said preparation comprises the PYY that treats effective dose, about 2% (v/v) ethanol, about 10mM EDTA, about 1% tween 80, and pH about 4.0.
36. preparation according to claim 35, it also comprises antiseptic, and wherein said antiseptic is methaform or benzalkonium chloride.
37. preparation according to claim 36, it also comprises buffer salt, and wherein said buffer salt is acetate or glutamate, Glu.
38. according to the described preparation of claim 37, it also comprises buffer salt, wherein said buffer salt is a glutamate, Glu.
39. be suitable for nonexpondable PYY dosage form, comprise the sealing bottle that contains aqueous pharmaceutical preparations, wherein said preparation comprises the PYY that treats effective dose, polar organic solvent and cation chelating agent that can be miscible with water, and wherein such PYY dosage form shows at least 90% the PYY response rate after storing at least 10 days under 5 ℃.
40., descend storage to have after at least 6 months at 5 ℃ and be higher than 90% the PYY response rate according to the described PYY dosage form of claim 39.
41., descend storage to have after at least 1 year at 5 ℃ and be higher than 90% the PYY response rate according to the described PYY dosage form of claim 39.
42., descend storage to have after at least 2 years at 5 ℃ and be higher than 90% the PYY response rate according to the described PYY dosage form of claim 39.
43. be fit to nonexpondable PYY dosage form, its comprise the bottle that contains aqueous pharmaceutical preparations and effectively intranasal give the actuator of described preparation, wherein said preparation comprises the PYY that treats effective dose, polar organic solvent and cation chelating agent that can be miscible with water, and wherein such dosage form shows at least 90% the PYY response rate after storage is longer than about 5 days.
44. according to the described PYY dosage form of claim 43, wherein said administration is every day 3 times.
45., between all sprayings, under 30 ℃/65% relative humidity, have and be higher than about 90% the PYY response rate according to the described PYY dosage form of claim 44.
46. according to claim 39 and 43 described PYY dosage forms, it also comprises the pH buffer agent with clean single ionizing part, described ionizing partly has the interior pKa of 2 pH units of the pH of described preparation.
47. according to the described PYY dosage form of claim 46, wherein said buffer agent has the ionizing part of the pKa in 1 pH unit of clean single pH with described preparation.
48. according to the described PYY dosage form of claim 47, wherein said preparation is selected from glutamate, Glu, acetate, glycine, histidine, arginine, lysine, methionine, lactate, formates and glycollate.
49. according to the described PYY dosage form of claim 48, it also comprises glutamate, Glu or acetate buffer.
50. according to the described PYY dosage form of claim 48, wherein said pH is about 5.0 or lower.
51. according to the described PYY dosage form of claim 48, wherein said pH is about 4.4 or lower.
52. according to the described PYY dosage form of claim 48, wherein said pH is about 4.0 or lower.
53. according to the described PYY dosage form of claim 48, wherein said pH is about 3.8 or lower.
54. according to claim 40 and 45 described PYY dosage forms, wherein PYY is PYY (3-36).
55. according to the described PYY dosage form of claim 54, wherein the concentration of PYY is at least about 20 μ g/ml.
56. according to the described PYY dosage form of claim 54, wherein the concentration of PYY is at least about 100 μ g/ml.
57. according to the described PYY dosage form of claim 54, wherein the concentration of PYY is at least about 200 μ g/ml.
58. according to the described PYY dosage form of claim 54, wherein the concentration of PYY is at least about 1mg/ml or higher.
59. according to the described PYY dosage form of claim 54, wherein the concentration of PYY is at least about 2mg/ml or higher.
60. according to the described PYY dosage form of claim 54, wherein the concentration of PYY is at least about 6mg/ml or higher.
61. according to the described PYY dosage form of claim 54, wherein the concentration of PYY is at least about 10mg/ml or higher.
62. according to the described PYY dosage form of claim 54, wherein said dosage form is fit to intranasal administration to reach the dosage of about 2 μ g to the described PYY of about 1000 μ g.
63. according to the described PYY dosage form of claim 54, wherein said dosage form is fit to intranasal administration to reach the dosage of about 100 μ g to the described PYY of about 600 μ g.
64. according to the described PYY dosage form of claim 54, wherein said can be that ethanol and described cation chelating agent are EDTA with the miscible polar organic solvent of water.
65. according to the described PYY dosage form of claim 64, wherein alcoholic acid formula concentration is at least about 0.1% (v/v).
66. according to the described PYY dosage form of claim 64, wherein alcoholic acid formula concentration is at least about 1% (v/v).
67. according to the described PYY dosage form of claim 64, wherein alcoholic acid formula concentration is at least about 10% (v/v).
68. according to the described PYY dosage form of claim 64, the concentration of EDTA in described preparation is at least about 1mg/mL.
69. according to the described PYY dosage form of claim 64, the concentration of EDTA in described preparation is at least about 10mg/mL.
70. according to the described PYY dosage form of claim 64, the concentration of EDTA in described preparation is at least about 50mg/mL.
71. according to the described PYY dosage form of claim 64, it also comprises surfactant.
72. according to the described PYY dosage form of claim 71, wherein said surfactant is a tween 80.
73. according to the described PYY dosage form of claim 72, wherein tween 80 in described preparation to exist at least about 1mg/mL.
74. according to the described PYY dosage form of claim 72, wherein tween 80 in described preparation to exist at least about 10mg/mL.
75. according to the described PYY dosage form of claim 72, wherein tween 80 in described preparation to exist at least about 50mg/mL.
76. according to the described PYY dosage form of claim 64, it also comprises antiseptic.
77. according to the described PYY dosage form of claim 76, wherein said antiseptic is methaform or benzalkonium chloride.
78. be used to strengthen the pharmaceutical preparation to mammiferous Y2 receptor-binding peptides mucosal delivery, wherein said preparation comprises the described peptide for the treatment of effective dose, polar organic solvent and cation chelating agent that can be miscible with water.
79. be used to strengthen the pharmaceutical preparation to mammiferous PYY functional analogue mucosal delivery, wherein said preparation comprises the described analog for the treatment of effective dose, polar organic solvent and cation chelating agent that can be miscible with water.
80. according to claim 78 or 79 described preparations, wherein said can be that ethanol and described cation chelating agent are EDTA with the miscible polar organic solvent of water.
81. 0 described preparation according to Claim 8, wherein alcoholic acid formula concentration is about 1% (v/v) or higher.
82. 0 described preparation according to Claim 8, the concentration of EDTA in described preparation is at least about 1mg/mL.
83. 0 described preparation according to Claim 8, it also comprises surfactant.
84. 3 described preparations according to Claim 8, wherein said surfactant is a tween 80.
85. 4 described preparations according to Claim 8, wherein tween 80 in described preparation with 50mg/mL or lower the existence.
86. 0 described preparation according to Claim 8, it also comprises buffer salt.
87. 0 described preparation according to Claim 8, wherein said pH is about 5.0 or lower.
88. 0 described preparation according to Claim 8, it also comprises antiseptic.
89. 8 described preparations according to Claim 8, wherein said antiseptic is methaform or benzalkonium chloride.
90. be used to strengthen the pharmaceutical preparation to mammiferous Y2 receptor-binding peptides mucosal delivery, wherein said preparation comprises the described peptide for the treatment of effective dose, about 2% (v/v) ethanol, about 10mMEDTA, about 1% tween 80, and pH about 4.0.
91. be used to strengthen the pharmaceutical preparation to mammiferous PYY functional analogue mucosal delivery, wherein said preparation comprises the described analog for the treatment of effective dose, about 2% (v/v) ethanol, about 10mM EDTA, about 1% tween 80, and pH about 4.0.
92. according to claim 90 or 91 described preparations, it also comprises antiseptic, wherein said antiseptic is methaform or benzalkonium chloride.
93. according to the described preparation of claim 92, it also comprises buffer salt, wherein said buffer salt is acetate or glutamate, Glu.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US69805205P | 2005-07-11 | 2005-07-11 | |
| US60/698,052 | 2005-07-11 | ||
| US60/699,294 | 2005-07-13 |
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| Publication Number | Publication Date |
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| CN101262888A true CN101262888A (en) | 2008-09-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800332373A Pending CN101262888A (en) | 2005-07-11 | 2006-07-10 | Formulations for enhanced mucosal delivery of PYY |
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| CN (1) | CN101262888A (en) |
| ZA (1) | ZA200800285B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104371005A (en) * | 2014-11-14 | 2015-02-25 | 中山大学 | Polypeptide PP3 capable of promoting growth hormone of tilapia mossambica to express and application of polypeptide PP3 |
-
2006
- 2006-07-10 CN CNA2006800332373A patent/CN101262888A/en active Pending
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- 2008-01-09 ZA ZA200800285A patent/ZA200800285B/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104371005A (en) * | 2014-11-14 | 2015-02-25 | 中山大学 | Polypeptide PP3 capable of promoting growth hormone of tilapia mossambica to express and application of polypeptide PP3 |
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| ZA200800285B (en) | 2009-08-26 |
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