MXPA98001630A - Pharmaceutical compositions of nasal administration of useful compounds for the treatment of osteoporo - Google Patents
Pharmaceutical compositions of nasal administration of useful compounds for the treatment of osteoporoInfo
- Publication number
- MXPA98001630A MXPA98001630A MXPA/A/1998/001630A MX9801630A MXPA98001630A MX PA98001630 A MXPA98001630 A MX PA98001630A MX 9801630 A MX9801630 A MX 9801630A MX PA98001630 A MXPA98001630 A MX PA98001630A
- Authority
- MX
- Mexico
- Prior art keywords
- leu
- arg
- glu
- lys
- seq
- Prior art date
Links
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- ILMRJRBKQSSXGY-UHFFFAOYSA-N tert-butyl(dimethyl)silicon Chemical group C[Si](C)C(C)(C)C ILMRJRBKQSSXGY-UHFFFAOYSA-N 0.000 description 1
- 125000004187 tetrahydropyran-2-yl group Chemical group [H]C1([H])OC([H])(*)C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- QEMXHQIAXOOASZ-UHFFFAOYSA-N tetramethylammonium Chemical compound C[N+](C)(C)C QEMXHQIAXOOASZ-UHFFFAOYSA-N 0.000 description 1
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- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 101710042243 trpE(G) Proteins 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
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Abstract
The present invention relates to a pharmaceutical composition for the nasal delivery of compounds useful for treating osteoporosis, comprising an effective amount of a truncated physiologically active analog of PTH or PTHrp, or salt thereof, in which the amino acid residues (22- 31) form an amphipathic alpha-helix, said residues (22-31) selected from (SEQ ID NOS 85, 86, 26, 27, 28, 29, and 30), an absorption enhancer selected from the group consisting of dimethyl -beta-cyclodextrin and biliary acid surfactants; and
Description
PHARMACEUTICAL COMPOSITIONS FOR THE NASAL DELIVERY OF USEFUL COMPOUNDS FOR THE TREATMENT OF OSTEOPOROSIS
BACKGROUND OF THE INVENTION a) Field of the Invention This invention relates to pharmaceutical compositions for the treatment of osteoporosis via nasal administration of the therapeutically effective amounts of certain novel analogs of the parathyroid hormone and the parathyroid hormone-related peptide. bl Description of the Related Art Osteoporosis is the most common form of metabolic bone disease and can be considered as the symptomatic fracture stage of bone loss (osteopenia). Although osteoporosis can occur secondarily to a number of preceding diseases, 90% of all cases appear to be idiopathic. Postmenopausal women are particularly at risk for idiopathic osteoporosis (postmenopausal osteoporosis or Type I). Another group of high risk for idiopathic osteoporosis is the elderly of either sex (senile osteoporosis or Type I I). Osteoporosis has also been linked to the use of corticosteroids, immobilization or prolonged bed rest, alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa, primary and secondary amenorrhea, and oophorectomy. In the various forms of osteoporosis, bone fractures, which are the result of bone loss that has reached the point of mechanical failure, frequently occur. Postmenopausal osteoporosis is characterized by fractures of the wrist and spine, while femoral fractures of the neck appear to be the dominant feature of senile osteoporosis. The mechanism by which bone is lost in people with osteoporosis is thought to involve an imbalance in the process by which the skeleton renews itself. This process has been called bone remodeling. This occurs in a series of discrete receptacles of activity. These receptacles spontaneously appear within the bone matrix on a given bone surface as a site of bone resorption. Osteoclasts (resorbing cells or bone solvents) are responsible for the resorption of a bone portion of generally constant dimension. This process of resorption is followed by the appearance of osteoblasts (bone-forming cells), which fill the cavity left by the osteoclasts with new bone. In a healthy adult, the rate at which osteoclasts and osteoblasts are formed is such that bone formation and bone resorption are in balance. However, in people with osteoporosis an imbalance develops in the process of bone remodeling, which results in bones that are lost at a faster rate than the bones that are made. Although this imbalance occurs to some degree in most individuals according to their age, it is much more severe and occurs at an early age in people with postmenopausal osteoporosis or after oophorectomy.
Adachi, et al. in Seminars in Arthritis and Rheumatism, 22: 6, 375-84 (June 1993), report that despite many conflicting data regarding the pathophysiology of osteoporosis induced by corticosteroids, it is generally agreed that there is a relative decrease in bone formation and a relative increase in bone resorption. The loss of bones with resulting fractures and osteonecrosis is a frequent consequence of corticosteroid therapy. There is evidence that bone loss occurs rapidly within the first 6 to 12 months of corticosteroid therapy; there also seems to be a close relationship between the rate of bone loss and corticosteroid dose. Men are equally susceptible to the effects of corticosteroids. The estimated incidence of fractures and osteonecrosis vary from 30 to 50%. There have been many attempts to treat osteoporisis with the goal of either decreasing additional bone loss or, more conveniently, produce a net gain in bone mass. Certain agents, such as estrogen and bisphosphonates, appear to decrease additional bone loss in people with osteoporosis. Agents that decrease bone loss, due to the different durations of resorption and bone formation, may appear to increase bone mass (in the order of 3 to 7%). However, this apparent increase is limited in time, not progressive, and is due to a decrease in the "remodeling space". In addition, due to the close coupling between resorption and formation, treatments that prevent bone resorption also ultimately prevent bone formation. It has been suggested that treatment with parathyroid hormone (PTH) would lead to both increased bone production and a positive calcium balance. However, human clinical trials have shown that any increase in trabecular bone is compensated for by a decrease in cortical bone, so that there is no net increase in the total bone. Hefti, et al. in Clinical Science 62, 389-396 (1982) have reported that daily subcutaneous doses of either bPTH (1-84) or hPTH (1-34) increased whole body calcium and weight of individual bone ash in both female rats adult osteoporotic and normal. Liu, et al. in J. Bone Miner. Res. 6: 10, 1071-80 (1991) have noted that ovariectomy of adult female rats induced a 47% loss in the percentage of trabecular bone in the proximal tibial metaphysis, accompanied by a significant increase in the number of osteoblasts and trabecular osteoclasts. Daily subcutaneous injections of hPTH (1-34) completely reversed the loss of trabecular bone and resulted in trabecular bone amounts exceeding those of the controls operated by susbtitutos. The number of osteoblasts increased and the number of osteoclasts decreased. Hock et al. in J. Bone Min. Res. 7: 1, 65-71 (1992) have reported that daily subcutaneous injections of hPTH (1-34) for healthy adult male rats for 12 days increased cortical and trabecular bone calcium and weight dry. Total bone mass, trabecular bone volume, trabecular number and thickness, and osteoblastic surfaces were increased. Mammalian parathyroid hormones, e.g., human (hPTH), bovine (bPTH), and porcine (pPTH), are simple polypeptide chains of 84 amino acid residues, with molecular weights of about 9500. Biological activity is associated with the N-terminal portion, with residues (1-34) apparently the minimum required. The N-terminal segment of human PTH differs from the N-terminal segment of the bovine and porcine hormones by only three and two amino acid residues, respectively: hPTH 1 - 34} : Ser Val Ser Glu l ie Gln Leu Met Hi s A = r. Le _ _ -, Lys -5 Asn Ser Met Glu Arg Val .u JTU i = D 20 Val His Asn Phe (SEQ _D NO: 1);
bPTH (l-34): Ala Val Ser Glu lie Glr. Phe Met: His Asn Leu X; > Lys -i = Leu
1 5? O "'~ i5
Being Ser Met Glu Arg Val Glu Trc Leu Are Lys Lvs: -u Si-. Asp 20 25"''" 3c "
Val His Asn Phe 'SEQ ID NO: 2);
pPTH (l-34): Ser Val Ser Glu lie Gln Leu Met Fis s-. Le "G ~ - -> Lys" J- ^ leu
1 5: ¿'' * ~~ 15
Being Ser Leu Glu Arg Val Glu Trp Leu Arg Lvs Lys Leu Gin Asp 20 25"* * 30 *
Val His Asn Phe (SEQ ID NO: 3).
The primary function of PTH is to produce the adaptation changes that serve to maintain a constant Ca2 + concentration in the extracellular fluid. PTH acts on the kidneys to increase the tubular reabsorption of Ca2 + in the urine, as well as to stimulate the conversion of calcifediol to calcitriol, which is responsible for the absorption of Ca2 + from the intestines. A prominent effect is to promote the mobilization of Ca2 + from the bone. PTH acts on the bone to increase the rate of Ca2 + and phosphate resorption. PTH stimulates the rate of bone resorption by osteoclasts, increases the rate of differentiation of mesenchymal cells to osteoclasts, and prolongs the half-life of these latter cells. With prolonged action of PTH, the number of bone-forming osteoblasts is also increased; In this way, the speed of bone production and remodeling is intensified. However, individual osteoblasts appear to be less active than normal osteoblasts. Rosenblatt, et al. in U.S. Patent Nos. 4,423,037, 4,968,669 and 5,001, 223 have described PTH antagonists obtained by the deletion of the N-terminal amino acids (1-6) and the selective replacement of Phe7, Met8, 18, and Gly12. Reportedly, Tyr34-N H2 increased the activity and stability of these compounds. The parathyroid hormone-related peptide (PTHrp), a protein of 140+ amino acids, and fragments thereof, reproduces the main biological actions of PTH. PTHrp is produced by a number of human and animal tumors and other tissues and may play a role in hypercalcemia of malignancy. The sequence of hPTHrp (1-34) is as follows: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser lie 1 5 10 15
Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu l ie Wing Glu 20 25 30
He His Thr Ala (SEQ ID NO: 4). The homology of the sequence between hPTH and hPTHrp is greatly limited to the 13 N-terminal residues, 8 of which are identical; only 1 of 10 amino acids in the receptor binding region (25-34) of hPTH is conserved in h PTHrp The conformational similarity can sustain the common activity. Cohen, et al in J. Biol Chem 266 3, 1997-2004 (1991) have suggested that much of the sequence of PTH (1 -34) and PTHrp (1 -34), in particular regions (5-1 8) and (21 -34), assume an a-helical configuration, while noting that there is some question of whether this configuration prevails for the carboxy terminal end under physiological conditions Such a secondary structure may be important for lipid interaction, receptor interaction, and / or structural stabilization We have synthesized analogs of PTH and PTHrp with the aim of developing improved therapeutic agents for bone mass restoration in mammalian subjects, including those afflicted by osteoporosis Delivery of these agents in the most efficient way possible is highly desirable, both in view of the convenience for the patient and the expected high cost of the peptide. It seems possible that the compounds, analogs to PTH, will be delivered very effectively in a Pulsatile Although subcutaneous injection is compatible with this objective, there are decided disadvantages for this mode of delivery, not the least of which is the pain associated with daily injections. Oral delivery has unfortunately not been particularly useful for the delivery of peptide drugs, mostly due to degradation in the gastrointestinal tract and low bioavailability (<; 1%) resulting from the ingested medication. Non-traditional, alternative modes of delivery, such as transdermal, pulmonary and nasal can prove to be more attractive for the delivery of these labile agents. The present invention encompasses the discovery and development of liquid compositions with particular utility for nasal delivery. Nasal delivery of peptides has found some limited commercial success, such as with Synarel® Nasal Solution (Syntex Corporation, Palo Alto, CA), a potent decapeptide (nafarelin) indicated for the treatment of endometriosis, for which a bioavailability up to about 3%. Intensified nasal formulations of LH-RH analogs, including nafarelin, with significantly higher bioavailabilities, have been reported by Anik in US Pat. Nos. 4,476, 1 16 and 5, 16, 817. Merkus et al. have reported in PCT Patent Publication No. 92/01440 compositions for the nasal administration of peptides, particularly insulin, including dimethyl-β-cyclodextrin as an absorption enhancer. Aliverti et al. in US Patent No. 5, 183,802 discloses a calcitonin pharmaceutical composition and a glycyrrhizinate absorption enhancer for nasal administration in the treatment of osteoporosis. However, none of the nasal compositions developed to date is completely satisfactory in the delivery of peptide compounds with acceptable levels of bioavailability.
SUMMARY OF THE INVENTION This invention provides a pharmaceutical composition for the nasal delivery of compounds useful for treating osteoporosis, comprising an effective amount of a truncated physiologically active analog of PTH or PTHrp, or salt thereof, in which residues of amino acids (22-31) form an amphipathic a-helix, said residues (22-31) selected from (SEQ ID NOS: 85, 86, 26, 27, 28, 29 and 30); an absorption enhancer selected from the group consisting of dimethyl-β-cyclodextrin and bile acid surfactants; and water.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and definitions The abbreviations of one and three letters for the various amino acids and bases of common nucleotides are as recommended in Puré Appl. Chem. 31, 639-645 (1972) and 40, 277-290 (1974) and the Commission on Biochemical Nomenclature I UPAC-I U B and comply with 37 CFR § 1 .822 (55 FR 18245, May 1, 1990). The abbreviations of one and three letters are as follows:
Abbreviations of amino acids Amino acids Three-letter symbol One-letter symbol
Alanine Wing A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Asn Asp Asp Asx B Cysteine Cys C Glutamine Gln Q Glutamic Acid Glu E Gl + Glu Glx Z Glycine Gly G Histidine Hist H Isoleucine He I Leucine Leu L Lysine Lys K Methazine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr And Valine Val V Other amino acid Xaa X Abbreviations represent L-amino acids unless otherwise designated as D- or D, L-. Certain amino acids, both natural and non-natural, are achiral, for example glycine. All peptide sequences are presented with the N-terminal amino acid on the left and the C-terminal amino acid on the right. Additional abbreviations for other amino acids and compounds used herein are: hSer homoserine hSerlac homoserin lactone Nle norleucine PEG2 diethylene glycol methyl ether radical, a. k.a. methoxydi (ethyleneoxy), CH3O (CH2CH2O) 2-, (MW = 1 19) PEG5000 radical of poly (ethylene glycol methyl ether), a. k. to. methoxypoly (ethyleneoxy), CH3O (C H2CH2O) n0-, (average MW = 5000) PEGX radical of poly (ethylene glycol methyl ether), CH3O (CH2CH2O) "-, n = 2 - 225, (average MW = 100 to 10,000 )
"Hydrophilic amino acid (Haa)" refers to an amino acid having at least one hydrophilic functional group in addition to those required for bone formation with peptide, such as arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine , serine, threonine and their counterparts.
"Lipophilic amino acid (Laa)" refers to an aromatic or aliphatic amino acid, uncharged, such as isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine and their homologs. For the purposes of this invention, alanine is classified as "amphiphilic" that is, capable of acting either as hydrophilic or lipophilic. "Physiologically active truncated analog or homolog of PTH or PTHrp" refers to a polypeptide having a sequence that comprises less than the full complement of amino acids found in PTH or PTHrp which, however, produces a similar physiological response. PTH or truncated PTHrp does not need to be completely homologous with PTH or PTHrp to produce a similar physiological response. PTH (1-34) and PTHrp (1-34) are preferred, but not exclusive, representatives of this group. "The amphipathic a-helix refers to the secondary structure exhibited by certain polypeptides in which the amino acids assume an a-helical configuration having opposite polar and polar faces oriented along the long axis of the helix. α-helical in the polypeptide of interest can be explored to some degree by the construction of a "Schiffer-Edmundson wheel" (M. Schiffer and AB Edmundson, Biophys. J. 7, 121 (1967)), of appropriate inclination and noting the segregation of the hydrophilic and lipophilic residues on opposite sides of the cylinder circumscribing the helix., empirical evidence, such as circular dichroism or x-ray diffraction data, may be available indicating the presence of an a-helical region in a given polypeptide. An ideal a-helix has 3.6 amino acid residues per turn with adjacent side chains separated by 100 ° arc. Eisenberg et al. in Nature 299: 371-374 (1982) and Proc. Nat. Acad. Sci. USA 81: 140-144 (1984) have combined a hydrophobicity scale with the helical wheel to quantify the concept of amphipathic helices. The main hydrophobic moment is defined as the sum of vectors of the hydrophobicities of the component amino acids that make the helix. The following hydrophobicities for amino acids are those reported by Eisenberg (1984) as the "consensus" scale: lie 0.73; Phe 0.61; Val 0.54; Leu 0.53; Trp 0.37; Met 0.26; Wing 0.25; Gly 0.16; Cys 0.04; Tyr 0.02; Pro -0.07; Thr -0.18; Ser -0.26; His -0.40; Glu -0.62; Asn -0.64; Gln -0.69; Asp -0.72; Lys -1. 10; Arg -1 .76. The hydrophobic moment, μH, for an a-helix having 3.6 residues per turn (or an arc of 100 ° (= 360 * 73.6) between side chains), can be calculated from: μH = [(? HNsind (N- 1)) 2 + (S HNcosd (N-1)) 2] 1/2, where HN is the hydrophobicity value of the Nβth amino acid and the sums are taken on the N amino acids in the sequence with periodicity d = 100 °. The hydrophobic moment can be expressed as the main hydrophobic moment per residue by dividing μH by N to obtain < μH. A value of < μH > at 100 ° ± 20 ° of about 0.20 or greater is suggestive of amphipathic helix formation. The values < μH > at 100 ° for hPTHrp (22-31) and hPTH (22-31) are 0.19 and 0.37, respectively.
Cornett, et al. , in J. Mol. Biol., 195: 659-685 (1987) have further extended the study of amphipathic a-helices by introducing the "amphipathic index" as a prediction of amphipathicity. They concluded that approximately half of all known a-helices are amphipathic, and that the dominant frequency is 97.5 ° instead of 100 °, the number of residues per turn being closer to 3.7 than to 3.6. Although such refinements are scientifically interesting, the basic approach of Eisenberg, et al. it is sufficient to classify a given sequence as amphipathic, particularly when one is designing an ab initio sequence to form an amphipathic a-helix. A substitute amphipathic a-helical amino acid sequence may lack homology to the sequence of a given segment of a naturally occurring polypeptide but produces a similar secondary structure, i.e., an a-helix having opposite polar and non-polar faces , in the physiological environment. The replacement of the amino acid sequence that occurs naturally with an alternative sequence can beneficially affect the physiological activity, stability or other properties of the altered parent polypeptide. The guide in order to design and select such sequences is provided in J. L. Krstenansky, et al. , FEBS Letters 242: 2, 409-413 (1989), and J. P. Segrest, et al. Proteins: Structure, Function, and Genetics 8: 103-1 17 (1990) among others. The amphipathic a-helix of ten amino acids of this invention has the formula: Haa (Laa Laa Haa Haa) 2 Laa wherein the Haa are selected from the group of hydrophilic amino acids and the Laa are selected from the group of lipophilic amino acids, as defined above . Assuming an idealized a-helix, residues 1, 4, 5, 8 and 9 are distributed along one face (A) of the helix within about 140 ° of each other, while residues 2, 3, 6, 7 and 10 occupy an opposite 140 ° arc on the other face (B) of the propeller. Preferably, all the residues on one face are of the same polarity while all those on the other face are of the opposite polarity, that is, if the entire A face is hydrophilic, the whole B face is lipophilic and vice versa. The skilled person will recognize that the helices of this invention are described by Haa (Laa Laa Haa Haa) 2 Laa the reverse sequence, Laa (Haa Haa Laa Laa) 2 Haa will also find the waste distribution criteria and is an equivalent description of the propellers of this invention. Alanine can be substituted by either hydrophilic or lipophilic amino acids, since Ala can easily reside on either side of an amphipathic a-helix, although Ala10 does not form an amphipathic a-helix. In general, proline, cysteine and tyrosine are not used; however, their presence and other random errors in the sequence can be tolerated, for example, a hydrophilic residue on the lipophilic side, as long as the remaining amino acids in the segment substantially conform to the lipophilic hydrophilic face splitting. A convenient method for determining whether a sequence is amphipathic enough to be a sequence of this invention is to calculate the main hydrophobic moment, as defined above. If the peak peak moment per residue at 100 ° ± 20 ° exceeds about 0.20, then the sequence will form an amphipathic helix and is a sequence of this invention. For example, the main hydrophobic moment per residue at 100 ° for (SEQ ID NO: 26), Xaa = Glu, is calculated as follows:
A.A. H_N d (N-1) H without dN-1) H eos d (N-1)
E -.62 0 0 -.62 L .53 100 .52 -.17 L .53 200 -.18 -.50 E -.62 300 .34 -.31 K -1.1 400 -.70 -.85 L. 53 500 .34.-41 L .53 600 -.46 -.27 E -.62 700 .21 -.58 K -1.1 800 -1.08 -.19 L .53 900 0 -.53? = 0.81 S = - 4.43
μH = [(0.81) 2 + (-4.43) 2] 1/2 = 4.50 < μH > = 4.50 / 10 = 0.45 For this sequence, the peak main hydrophobic moment occurs at 92 ° and has a value of 0.48. By applying this concept to the parathyroid hormone and the peptide related to parathyroid hormone, a hypothesis was formulated that either or both regions (7-16) and (22-31) may exhibit a secondary helical structure and may be replaced with a non-homologous sequence that has similar structural tendencies, without loss of biological activity or induction of immunoreaction.
Preferred Modes In one aspect, this invention provides analogs of PTH, PTHrp and physiologically active truncated homologs and analogues of PTH and PTHrp, or salts thereof, in which the amino acid residues (22-31) form an a-helix amphipathic, the sequence of said residues (22-31) selected from: a) Xaa1 Xaa2 Leu Xaa4 Xaa5 Leu Xaa7 Xaa8 Xaa9 Xaa10 wherein Xaa1 and 1 5 Xaa4 are independently Glu, Glu (OCH3), His or Phe; Xaa2 is Leu or Phe; Xaa5 is Lys or His; Xaa7 and Xaa10 are independently Leu or He; Xaa8 is Ala, Arg, or Glu; and Xaa9 is Lys or Glu (SEQ ID NO: 85); preferably Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu wherein 1 5 10 Xaa is Glu or Arg (SEQ ID NO: 26);
b) Xaa1 Xaa2 Leu Xaa4 Arg Leu Leu Xaa8 Arg Leu wherein 1 5 10 Xaa1 and Xaa4 are independently Glu, Glu (OCH3), His, or Phe; Xaa2 is Leu or Phe; Xaa8 is Glu, Lys, or Lys (COCH2PEGX) and PEGX is a poly- (ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO: 86); preferably, Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu wherein 1 5 10 Xaa is Glu, Lys, or Lys (COCH2PEGX) and PEGX is a radical? oli- (ethylene glycol methyl ether) of molecular weight from 100 to 10,000 (SEQ ID NO: 27); c) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu (SEQ ID NO: 28); 1 5 10 d) Be Leu Leu Be Ser Leu Leu Be Ser Leu (SEQ I D NO: 29); 1 5 10 e) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu (SEQ ID NO: 30). 1 5 10 In another aspect, this invention provides analogs of PTH,
PTHrp and physiologically active homologous and truncated analogues of
PTH and PTHrp, or salts thereof, of the formula: Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Leu His Xaa10 Xaa1 1 Gly Xaa13 Ser He Gln Xaa17 Leu Xaa19 Xaa20 Xaa21 Xaa22"31 Xaa32 Xaa33 Xaa35 Xaa36 Xaa37
Xaa38 Term, where: Xaa1 is absent or is Ala; Xaa2 is absent or is Val; Xaa3 is absent or is Ser; Xaa4 is absent or is Glu or Glu (OCH3);
Xaa5 is absent or is His or Ala; Xaa6 is absent or is Gln; Xaa7 is absent or is Leu; Xaa10 and Xaa17 are independently Asp or Asp (OCH3); Xaa1 1 is Lys, Arg, or Leu; Xaa13 is Lys, Arg, Tyr, Cys, Leu, Cys (CH2CONH (CH2) 2NH (biotinyl)), Lys (7-dimethylamino-2-oxo-2H-1-benoxopyran-4-acetyl), or
Lys (dihydrocinnamoyl); Xaa20 is Arg or Leu; Xaa19 and Xaa21 are independently Lys, Ala or Arg; Xaa22'31 is selected from (SEQ ID NO: 26, 27, 28, 29, or 30); Xaa32 is His, Pro, or Lys; Xaa33 is absent, or is Pro, Thr, Glu, or Ala; Xaa34 is absent, or is Pro, Arg, Met, Ala, hSer, hSer lactone, Tyr, Leu or 1,4-diaminobutyryl lactam; Xaa35 is absent or is Pro, glu, Ser, Ala, or Gly; Xaa36 is absent or is Ala, Arg, or lie; Xaa37 is absent or is Arg, Trp, or 3 - (- 2-naphthyl) -L-alanine; Xaa38 is absent or is Ala or hSer or Xaa38"42 is Thr Arg Ser Ala Trp, and the Term is OR or N H2 where each R is independently H,
(C? -C4) alkyl or phenyl (C? -C) alkyl; and the pharmaceutically acceptable salts thereof. In still another aspect, this invention includes polypeptide analogs of the physiologically active truncated homologue hPTHrp (1-34), as shown in Formula (I):
Ala Val Ser Glu Xaas Gln Leu Leu His Asp Xaa11 Gly Xaa13 Ser He Gln Asp Leu Xaa19 Arg Xaa21 Xaa22"31 Xaa32 Xaa33 Xaa34 Term, where: Xaa5 is His or Ala, Xaa1 1 and Xaa13 are independently Lys, Arg, or Leu Xaa19 and Xaa21 are independently Ala or Arg; Xaa22'31 is selected from: a) Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu where 1 5 10 Xaa is Glu or Arg (SEQ ID NO: 26); Leu Leu Glu Arg Leu Leu Xaa Arg Leu where 1 5 10 Xaa is Glu, Lys, or Lys (COCH2PEGX) and PEGX is a poly- (ethylene glycol methyl ether) radical of molecular weight from 100 to 10,000 (SEQ ID NO: 27); c) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu (SEQ ID NO: 28); 1 5 10 d) Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu (SEQ ID NO: 29); 1 5 10 e ) Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu (SEQ ID NO: 30); 1 5 10 Xaa32 is His or Lys; Xaa33 is Thr, Glu, or Ala; Xaa34 is ala, hSer, Tyr, or Leu; is Gly Arg Arg, lactone, OH or N R2, where each R is H or (C? -C4) alkyl and its pharmaceutically acceptable salts. to I).
A more specific aspect of the invention includes those polypeptides of Formula (I), wherein Xaa22"31 is (SEQ ID NO: 26), for which < μH > at 100 ° exceeds 0.45. Still a more specific aspect of the invention includes those polypeptides of Formula (I) wherein Xaa22'31 is (SEQ ID NO: 26), Xaa1 1 and Xaa13 are both Lys, and Xaa19 and Xaa21 are both
Arg. Representative polypeptides include, but are not limited to
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arg Lys 20 25 30
Leu H s Thr Wing OH 'S? Q:.: Í.O 5., Wing Val Ser 3lu His Gin e- Le "His As Lys G_ Lvs Ser lie 1 - n" "'"
Gln Asp Leu Arg Arg Arg _ *! _ L = _ Le ~ - - »- • - * _ • _: .- - -. -ys 20 2 5 3 0
Leu His Thr Ala OH [SEO. 12 NO -, Ala Val Ser Glu His Glr. i_eu Le- H: A = p Lys G l y l ys o l e 1 5 10 *] 5
Gln Asp Leu Arg Arg Arg G_- e- Leu Glu Lys Leu Leu Glu Lys 20 25"30
Leu H s Thr Wing NK, 'S? 0 TO: Z "X
Ala Val Ser Giu His G ~ r. You. > .sp? yí,? y.-y? Se: 1 5 .0 * 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Giu Lys 20 25 30
Leu His Thr hSer NH, 'SEO ID NO
Ala Val Ser Glu His Gln Le _ Leu Ais .As ._ / s G "! \ Lye Ser lie 1 5 i:" 15
Gln Asp Leu Arg Arg Arg 31 _ Le_ Leu 0-1- Lys Lea Leu G * .- Lys 20 25 20
Leu His Tnr hSerlac 'SEQ 10 NC 91;
Wing Val Ser Glu His Gin Leu Leu His Aso Lys Gly Lys Ser He
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Giu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala Gly Arg Arg OH (SEQ ID NO: 10); 35 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Giu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu Lys Glu Leu NK: (SEQ ID NO: 11). Yet another aspect of this invention includes those polypeptides of Formula (I) wherein Xaa22-31 is (SEQ ID NO: 26); Xaa11 and Xaa13 are both Lys; and one of Xaa19 and Xaa21 is Arg and the other is Ala. Representative polypeptides of this subgenus include, but are not limited to: Ala Val Ser Giu His Glr. leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Wing Arg Arg Glu Leu Leu Giu Lys L u Leu Glu Lys 20 30
Leu His Thr Ala NH, (SEQ TD NO: 12 ^
Ala Val Ser .u His r-i je His Asp Lys Gly Lys Ser lie
Gln Asp Leu Arg Arg Ala Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala NH, (SEQ ID NO: 13).
In another aspect, this invention includes those polypeptides of Formula (I) wherein Xaa22"31 is (SEQ ID NO: 26), one of Xaa11 and Xaa13 is Leu and the other is Lys; and Xaa19 and Xaa21 are both Arg. Representative polypeptides of this subgenus include, but are not limited to:
Wing Val Ser Glu Wing Gin Leu Leu His Ase Leu Gly'Lys Ser He 10 15 Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Ala Leu OH (SEQ ID NO - 14). In another aspect, this invention includes those polypeptides of Formula (I) wherein Xaa22"31 is (SEQ ID NO: 27), for which < μH > a
100 ° exceeds 0.50. A further aspect of this invention includes those polypeptides of Formula (I) wherein Xaa22-31 is (SEQ ID NO: 27); Xaa1 1 and
Xaa13 are both Lys or both Arg; and Xaa19 and Xaa21 are both Arg. Representative polypeptides of this subgenus include, but are not limited to: Al a Val Ser Giu H s Gl r. e L u -'1 * - j - V Lys Ser i i-2 1 5 10 15
Gln Asp Leu Arg Arg Arg Gl _ L-? - - *:, r 7, »^ L u 11 _ Are 2:" Z 5 30 ~
Leu His Tnr Ala OH = ?; TI NI I;
Ala Val Ser Gl ms .-. Rzz. ' and ie
1 0 15
Gln Asp Leu Arg Arg Glu Leu Leu Glu Ar: Leu J6U Arg 20 30
Leu His Thr Ala OH! SL .. *. • < v.- ': _ o
Ala Val Ser Giu His Glr. Le_ Leu His Asp Arg Giy Arg Ser He 1 5 lo "15
Gln Asp Leu Arg Arg Arg Gl- Leu Leu Giu Arg Leu Leu Lys Arg 2? "" 25 30
Leu His Thr Ala OH li-v - -
Ala Val Ser Glu His Glr. Le_ Le .. H s Aso Arg Gly Arg Ser II «1 5 l" "'" 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
Lys (COCH2PEG2) Arg Leu 'Ai s Thr Wing CH (SEQ ID NO: LSI; 30 Wing Val Ser Glu His Gir. Leu Leu His Asp Arg Gly Arg Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu 20"25 Lys (COCH, PEG5000) Arg Leu H_s Thr Wing OH (SEQ ID NO: 19). In another aspect, this invention includes polypeptides of Formula (I) wherein Xaa22"31 is (SEQ ID NO: 28), for which < μH > at 100 ° is approximately 0.25. Representative polypeptides of this subgenus include, but are not limited to, Ala Val Ser Glu His Gin Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Without Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala Glu Ala 20 25 30
Leu His Thr Ala NH2 (SEQ ID NO 2!
In another aspect, this invention includes polypeptides of Formula (I) wherein Xaa22"31 is (SEQ ID NO 29), for which < μH > at 100 ° is approximately 028 Representative polypeptides of this subgenus include, but are not limited to, are limited to
Ala Val Ser Gl? His Gln Leu Leu His Asp Lys Giy Lys Ser He 1 5 10 * 15
Gin Asp Leu Arg Arg Arg Ser Leu Leu £ ~ r Ser Leu Leu Ser Ser 20 25 30 Leu His Thr Wing NH2 (SSQ ID NO: 21).
In another aspect, this invention includes polypeptides of Formula (I) wherein Xaa22"31 is (SEQ ID NO 30), for which < μH > at 100 ° is approximately 029 Representative polypeptides of this subgenus include, but are not limited to, are limited to Ala Val Ser Glu His Gln Leu Leu His Aso Lys Gly Lys Ser He 1 5 10 * "15
Gln Asp Leu Arg Arg Arg Wing Phe Tyr ASD Lys Val Wing Glu Lys 20 25 30
Leu His Thr Ala NH2 (SEQ ID NO: 22).
Yet another aspect of this invention includes polypeptide analogues of the physiologically active truncated homolog bPTH (1-34), as shown in Formula (II): Xaa1 Val Ser Glu lie Gln Xaa7 Xaa8 His Asn Leu Gly Lys His Leu Xaa16 Ser Xaa18 Xaa19 Arg Xaa21 Xaa22"31 His Asn Xaa34 Term, where: Xaa is Ser or ala, Xaa7 is Leu or Phe, Xaa8 is Met or Nle, Xaa 16 is Asn or Ser, Xaa18 is Leu, Met or N le; Xaa19 is Glu or Arg; Xaa21 is Val or Arg; Xaa22 is selected from (SEQ ID NO: 26, 27. 28, 29 and 30), Xaa34 is Phe or Tyr, Term is OH or N R2, where each R is H or (dC) alkyl, and the pharmaceutically acceptable salts thereof (Formula II) Representative polypeptides include, but are not limited to: Ala Va l Ser Giu He Gin Phe ie H s A = r Leu Gl and Lys His Leu I 5 10 15
Being Ser Nle Glu Arg Val Giu Leu Leu Glu _eu -eu Glu Lys 20"25 30
Leu iis Asn Tyr NH, (SEQ ID NO: 23) Wing Val Ser Glu He Gln Phe Nle His Asn Leu Gly Lys His Leu 1 5 i 10 15
Be Being Nle Arg Arg Arg Glu Leju Leu Glu Lys Leu Leu Glu Lys 20 I 25 30
Leu His Asn Tyr NH, (SEQ ID NO: 241
In yet another aspect of this invention, it has surprisingly been found that homologs and analogue; of PTH and PTHrp that have less than 34 amino acids are also potent bone remodeling agents. These compounds are of the general formula: Ala Val Ser Glu Xaa5 Gln Leu Leu His Asp Xaa11 Gly Xaa13 Ser lie Gln Asp
Leu Xaa .1l9s Arg Xaa'1 Xaa, 2"2--3511 Xaa, 3J2 'Xa a33 Xaa34 Term
Representative polypeptides include, but are not limited to: Compound 41: AVSEHQLLHD KGKSIQ DLRR RELLEKLLEK LHP-NH2 (SEQ ID NO: 55)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser lie 5 10 15
Gln Asp Leu Arq Arg Ara Glu Leu Leu Glu Lys Leu Leu Giu Lys 20 25 3: Leu His Pro NH2 (SEQ ID NO: 55)
Physical data p.f. 142.8-166.1 ° C [a -5380 (c 0.38, H20) FAB (C173H295Nss? 49): [M + H] + 3929 AAA: Asx 2.0 (2) Glx 5.7 (6) Ser 1 8 (2) His 3.0 ( 3) Gly 1.1 (1) Wing 09 (1) Arg 2.8 (3) Val 1.2 (1) Me 0.9 (1) Leu 7.4 (8) Lys 4.4 (4) Pro 09 (1)
Compound 42: AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH2 (SEQ ID NO: 56)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys, Ser He
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Olu Lys 20 25 30
Leu Pro NK, (SEQ ID NO: 56).
Physical data mp 161 0-1770 ° C [a] D25-61 97 (c 0 19, H20) FAB (C167H288N52? 48) [M + H] + 37920 AAA Asx 22 (2) Glx 59 (6) Ser 1 8 (2) His 21 (2) Gly 1 1 (1) Wing 1 0 (1) Arg 30 (3) Val 1 1 (1) lie 1 0 (1) Leu 79 (8) Lys 43 (4) Pro 09 ( 1)
The skilled person will appreciate that numerous permutations of the polypeptide analogs can be synthesized, which will possess the desirable attributes of those described herein as long as an amino acid sequence having a major hydrophobic moment per residue at 100 ° ± 20 ° greater than approximately 0.20 is inserted in the positions (22-31).
Classical Synthesis of the Polypeptides The polypeptides of the present invention can be synthesized by methods such as those set forth in J. M Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed. , Pierce Chemical Co., Rockford, Illinois (1984) and J. Meienhofer, Hormone Proteins and Peptides, vol. 2, Academic Press, New York, (1973) for solid-phase synthesis and E. Schroeder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for synthesis in solution. In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain. Normally, either the carboxyl or amino group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either bound to an inert solid support, or used in solution, and the next amino acid in the sequence, also suitably protected, is added under receptive conditions for amide bond formation. After all the desired amino acids have been linked in the proper sequence, the protecting groups and any solid support are removed to provide the crude polypeptide. The polypeptide is desalted and purified, preferably by chromatography, to produce the final product. A preferred method for preparing analogues of physiologically active truncated polypeptides, having less than about forty amino acids, involves peptide synthesis in solid phase. In this method, a-amino (Na) functions and any reactive side chain is protected by acidic or base-sensitive groups. The protecting group must be stable to the conditions of peptide bond formation, while it must be easily removed without affecting the existing polypeptide chain. The a-amino protecting groups include, but are not limited to t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc), isobornyloxycarbonyl, a, a-dimethyl-3,5 -dimethoxybenzyloxycarbonyl, -nitrophenylsulphenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and the like, preferably t-butoxycarbonyl (Boc). Suitable side chain protecting groups include, but are not limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), g-bromobenzyloxycarbonyl, t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z), 2, 6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos), trimethylsilyl, and trityl. In solid phase synthesis, the C-terminal amino acid is first attached to a suitable resin support. Suitable resin supports are those materials which are inert to the reactants and reaction conditions of the gradual deprotection and condensation reactions, as well as being insoluble in the medium used. Examples of commercially available resins include styrene / divinylbenzene resins modified with a reactive group, for example, chloromethylated cco-poly- (styrene-divinylbenzene), co-poly (styrene-divinylbenzene) hydroxymethylated, and the like. Phenylacetamidomethyl hydroxymethylated, benzylated (PAM) resin is preferred. When the C-terminal of the compound is an amide, a preferred resin is the p-methyl-benzylhydrylamino-co-poly (styrene-divinyl-benzene) resin. The binding to the PAM resin can be achieved by the reaction of the N -protected amino acid, preferably the amino acid-Boc, such as its ammonium, cesium, triethylammonium, 1,5-diazabicyclo- [5.4.0) undec-5-ene, tetramethylammonium or similar salt in ethanol, acetonitrile, N, N-dimethylformamide (DMF), and the like, preferably the cesium salt in DMF, with the resin at an elevated temperature, for example between about 40 ° and 60 ° C, preferably about 50 ° C, for from about 12 to 72 hours, preferably about 48 hours. The Na-Boc-amino acid can be bound to the benzhydrylamine resin by means of, for example, an N.N'-diisopropylcarbodumide (DIC) / 1-hydroxybenzotriazole (HOBt) measured the coupling for from about 2 to about 24 hours, preferably about 2 hours at a temperature between 10 ° and 50 ° C, preferably 25 ° C in a solvent such as dichloromethane or dimethylformamide, preferably dichloromethane. The successive coupling of protected amino acids can be accomplished by methods well known in the art, typically in an automatic peptide synthesizer. Following neutralization with triethylamine or similar base, each protected amino acid is preferably introduced at approximately 1.5 to 2.5 times the molar excess and the coupling is carried out in a polar, non-aqueous, inert solvent such as dichloromethane, DMF, or mixtures thereof. same, preferably in dichloromethane at room temperature. Representative coupling agents are N.N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropyl-carbodiimide (DIC) or other carbodiimide, either alone or in the presence of 1-hydroxybenzotriazole (HOBt), O-acyl ureas , benzotriazol-1-yl-oxitris (pyrrolidino) phosphonium, hexafluorophosphate (PyBop), N-hydroxysuccinimide, other N-hydroximides, or oximes. Alternatively, active esters of protected amino acids (e.g., p-nitrophenyl, pentafluorophenyl and the like) or symmetric anhydrides can be used. At the end of the solid phase synthesis, the fully protected peptide is removed from the resin. When the bond to the resin support is of the benzyl ester type, the cut can be effected by means of aminolysis with an alkylamine or fluoroalkylamine for peptides with a C-terminal alkylamide, or by aminolysis with, for example, ammonia / methanol or ammonia. ethanol for peptides with an unsubstituted C-terminal amide, at a temperature between about -10 ° C and 50 ° C, preferably about 25 ° C, for between about 12 and 24 hours, preferably about 18 hours. Peptides with a C-terminal hydroxy can be cut by HF or other strongly acid deprotection regime or by saponification. Alternatively, the peptide can be removed from the resin by transesterification, for example, with methanol, followed by aminolysis or saponification. The protected peptide can be purified by silica gel chromatography.
The side chain protecting groups can be removed from the peptide by treating the aminolysis product with, for example, anhydrous liquid hydrogen fluoride in the presence of anisole or other carbonium ion cleaner, treatment with hydrogen fluoride / pyridine complex , treatment with trifluoroacetic acid and tris (trifluoroacetyl) boron, by reduction with hydrogen and palladium on carbon or polyvinylpyrrolidone, or by reduction with sodium in liquid ammonia, preferably with liquid hydrogen fluoride and anisole at a temperature between about -10 ° and + 10 ° C, preferably at about 0 ° C, for between about 15 minutes and 2 hours, preferably about 1.5 hours. For peptides in the benzydrylamine resin, the steps of deprotection and resin cutting can be combined in a single step using liquid hydrogen fluoride and anisole as described above. The solution can be desalted (for example with BioRad AG-3® ion exchange resin) and the peptide purified by a sequence of chromatographic steps employing any or all of the following types: exchange of ions in a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on co-poly (styrene-divinylbenzene) without derivatization, for example Amberlite® XAD; silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, for example in Sephadex® G-25; countercurrent distribution; or high performance liquid chromatography (HPLC), especially reverse phase HPLC in octyl- or octadecylsilylsilicate (ODS) bound phase column packing.
Thus, another aspect of the present invention relates to the processes for preparing the polypeptides and pharmaceutically acceptable salts thereof, which processes comprise sequentially condensing protected amino acids in a suitable resin support, removing the protective groups and resin support, and purifying the product, to produce analogs of the truncated physiologically active analogs and homologues of PTH and PTHrp, preferably PTH (1-34) and PTHrp (1-34), in which the amino acids at the positions (22-31) ) form an amphipathic α-helical peptide sequence, as defined above.
Recombinant synthesis of the polypeptides Alternatively, the polypeptides of this invention can be prepared by cloning and expressing a gene encoding the desired polypeptide. In this process, a plasmid containing the desired DNA sequence is prepared and inserted into an appropriate host microorganism, typically a bacterium, such as £. coli, or a yeast, such as Saccharomyces cerevisiae, inducing the host microorganism to produce multiple copies of the plasmid, and thus of the cDNA encoding the polypeptide analogs of this invention. First, a synthetic gene encoding the selected PTH or PTHrp analog is designed with convenient restriction enzyme cleavage sites to facilitate subsequent alterations. The polymerase chain reaction (PCR), as shown by Mullis in U.S. Patent Nos. 4,683, 195 and 4,683,202, can be used to amplify the sequence. The amplified synthetic gene can be isolated and ligated to a suitable plasmid, such as a Trp LE plasmid, in which four copies of the gene can be inserted in tandem. The preparation of the Trp LE plasmids is described in U.S. Patent No. 4,738,921 and European Patent Publication No. 0.212532. The Trp LE plasmids generally produce 8-10 times more protein than the Trp E plasmids. The multi-copy gene can then be expressed in an appropriate host, such as E. coli or S. Cervisiae. The specific expression vector used herein was Trp LE 18 Prot (lie3, Pro5) containing the following elements: a fragment pBR322 (EcoRI-BamH I) containing the ampicillin-resistant gene and the origin of replication plasmid; an EcoRI-Sacll fragment containing the trp promoter and the trpE gene; a fragment of HIV protease gene (He3, Pro5) (Sacl l-Hindl I); a gene fragment bGRF (HindII I-BamH I); and a transcription terminator of the E. coli rpoc gene. The bGRF and portease HIV gene fragments are not critical and can be replaced with other coding sequences, if desired. The expressed multimeric fusion proteins accumulate intracellularly in stable inclusion bodies and can be separated by centrifugation of the rest of the cellular protein. The isolated fusion protein is converted to the PTH analog or monomeric PTHrp and can be purified by reverse phase cation exchange and / or H PLC.
Alternative methods of cloning, amplification, expression and purification will be apparent to the skilled person. Representative methods are described in Maniatis, et al., Molecular Cloning, a Laboratory Manual, 2 * ed., Cold Spring Harbor Laboratory (1989).
Utility and administration The polypeptides of this invention are useful for the prevention and treatment of a variety of mammalian conditions manifested by loss of bone mass. In particular, the compounds of this invention are indicated for the prophylaxis and therapeutic treatment of osteoporosis and osteopenia in humans. In general, the polypeptides of this invention, or salts thereof, are administered in amounts between about 0.002 and 10 μg / kg of body weight per day, preferably from about 0.04 to about 0.2 μg / kg of body weight per day. For a 50 kg woman, the daily dose of active ingredient is from about 0.1 to about 500 μg, preferably from about 2.0 to about 100 μg. In other mammals, such as horses, dogs and cattle, higher doses may be required. This dose may be delivered in a conventional pharmaceutical composition by a single administration, by multiple applications, or by controlled release, as necessary to achieve the most effective results, preferably one or more times per day by injection.
The selection of the exact composition and dose and the most appropriate delivery regimen will be influenced by, inter alia, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient. In the treatment of corticosteroid-induced osteopenia, it is expected that the required dose of polypeptide will be higher for higher doses of corticosteroids. Representative delivery regimens include oral, parenteral (including subcutaneous, intramuscular, and intravenous), rectal, buccal (including sublingual), pulmonary, transdermal, and intranasal. The pharmaceutically acceptable salts retain the desired biological activity of the parent polypeptide without side toxic effects.
Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids, such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, acids naphthalenedisulfonic acids, polygalacturonic acid and the like; (b) base addition salts foraminated with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed of N, N'-dibenzylethylenediamine or ethylenediamine; or
(c) combinations of (a) and (b), for example, a zinc tannate salt and the like.
A further aspect of the present invention relates to pharmaceutical compositions comprising as an active ingredient a polypeptide of the present invention, or a pharmaceutically acceptable salt thereof, in admixture with a non-toxic, pharmaceutically acceptable carrier. As mentioned above, such compositions can be prepared for parenteral administration (subcutaneous, intramuscular or intravenous), particularly in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; for pulmonary or intranasal administration, particularly in the form of powders, nasal drops or aerosols; and for rectal or transdermal administration. The compositions can be conveniently administered in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences17th ed. , Mach Publishing Compnay, Easton, PA. , (1985). Formulations for parenteral administration may contain as excipients saline or sterile water, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. For oral administration, the formulation can be enhanced by the addition of bile salts or acylcarnitines. Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be oily or aqueous solutions for use in the form of measured nasal drops or spray. For buccal administration, typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like. When formulated for nasal administration, absorption through the nasal mucous membrane can be enhanced by surfactant acids, such as, for example, glycocholic acid, cholic acid, taurocholic acid, ethocolic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, acid glycodeoxycholic, cyclodextrins and the like in an amount in the range between about 0.2 and 0.15 percent by weight, preferably between about 0.5 and 4 percent by weight, most preferably about 2 percent by weight. Delivery of the compounds of the present invention to the subject for prolonged periods of time, for example, for periods of one week to one year, can be achieved by simple administration of a controlled release system containing sufficient active ingredient for the period of desired release. Various controlled release systems, such as reservoir or monolithic microcapsules, reservoir implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, onotrophoretic devices and alternative injectable dosage forms can be used for this purpose. The location at the site to which the active ingredient enters is desired, in an additional feature of some controlled release devices, which may prove beneficial in the treatment of certain disorders. A form of controlled release formulation contains the polypeptide or its salt dispersed or encapsulated in a non-antigenic, non-toxic, slowly degradable polymer, such as copoly (lactic / glycolic acid), as described in the pioneering work of Kent, Lewis, Sanders and Tice, U .S. 4,675, 189. The compounds or, preferably, their relatively insoluble salts, can also be formulated into cholesterol or other lipid matrix pellets, or silastomer matrix implants. Injectable formulations or depot implant, further slow release will be apparent to the expert. See, for example, Sustained and Controlled Relay Drug Delivery Systems, J. R. Robinson ED. , Marcel Dekker, Inc., New York, 1978, and R. W. Baker, Controlled Relay of Biologically Active Agents, John Wiley & amp;; Sons, New York, 1987.
Nasal delivery systems Commercially available bottles and atomizer pumps were used to deliver nasal solutions for evaluation in male cynomolgous monkeys. The atomizer pumps with delivery volumes of 50 and 100 μl were obtained from Valois. For initial studies, adult-sized spray tips were used. For later studies, pediatric-sized spray tips were used. The size of the tips did not affect the performance of the atomizer pumps. The samples from each batch of atomizer pumps were tested to determine the number of actions required to load the pumps, the accuracy of the delivery volumes and the consistency in their spray patterns.
The monkeys were anesthetized before dosing and reclined on their backs when the dosage was applied. To deliver a 4 mg dose of peptide, an atomization of 100 μl of 100 mg / ml solution was dosed in each nostril. To deliver a dose of 1 mg of peptide, an atomization of 50 μl of 20 mg / ml solution was dosed into a nostril. Each monkey was dosed subcutaneously with 50 μg of peptide in a subsequent study to serve as its own control. The nasal bioavailability was calculated in relation to the subcutaneous injection. Blood samples were extracted at 1, 0.25, 0.5, 1, 2, 4, 6 and 8 hours post-dosing and were evaluated for the peptide by RIA. The pharmacokinetic parameters were calculated from the plasma profiles. The area under the curve (AUC) of the plasma concentration profile was calculated by the trapezoidal rule, and the bioavailability was determined in relation to the average AUC dosing s.c. for all animals. The average bioavailabilities close to 50% were obtained for the compositions of this invention. The Tmax for the nasal formulations was between 0.3 and 0.7 hours, similar to the Tmax measured for s.c. administration (0.6 hours). Among the bile acid surfactants useful in this invention are glycolic acid, cholic acid, taurocholic acid, colonic acid, ethocolic acid, deoxycholic acid, chenodeoxycholic acid, and pharmaceutically acceptable salts thereof, including sodium, potassium, ammonium salts, calcium and magnesium, as well as other inorganic and non-toxic organic cations. Preferably, the bile acid surfactant is an alkali metal salt of glycocholic or taurocholic acid, most preferably the sodium salt. Other materials, such as condoms, salts to achieve isotonicity, buffers and the like, can be added to nasal formulations. Typically, the peptide is present in an amount of about 1 mg / ml to about 100 mg / ml and the concentration of absorption enhancer is from about 0.5 mg / ml to about 50 mg / ml. The following specific Examples are intended to illustrate the synthesis and testing of the representative compounds of the invention, and should not be construed as limiting the scope of the claims. In the Examples, "p.f." is the melting point, "[a] D25" is the optical activity at 25 ° C at the given concentration in the indicated solvent, "FAB" is fast atom bombardment mass spectrometry, and "AAA" is the analysis of amino acids, with the expected values in parentheses that follow the observed values. The amino acid analyzes were conducted on a Hewlett-Packard AminoQuant Analyzer following the protocols recommended by the manufacturer. The primary amino acids were derived with o-phthalaldehyde; the secondary amino acids with Fmoc. The fluorescent detection of the derived amino acids was used for quantification. The protected amino acids were obtained from Applied Biosystems Inc. (Foster City, CA).
EXAMPLE I Compound 1 (SEQ ID NO: 7) was prepared on a 0.5 mmol scale by the solid phase method on 4-methylbezhydrylamine resin, using an automatic Applied Biosystems Model 430a peptide synthesizer. The a-amino groups were protected with t-butoxycarbonyl (Boc). The side chain protecting groups were: benzyl (Bzl) for Asp, Glu, and Ser; tosyl (Cough) for Arg; benzyloxymethyl (Bom) for His; and 2-chlorobenzyl (Cl-z) for Lys, The amino acids were sequentially coupled using N, N-dicyclohexylcarbodiim ida / 1-hydroxybenzotriazole (DCC / HOBt) following Stewart and Young (supra). After coupling of each amino acid, the peptide was acetylated using a mixture of acetic anhydride and diisopropylethylamine in N-methylpyrrolidone. The completed peptide was cut from the resin with simultaneous deprotection of the side chain protecting groups using anhydrous HF (25 ml) in the presence of anisole (2.5 ml) at -10 ° C for 30 minutes and at 0 ° C for 60 minutes. After evaporation of the HF in vacuo, the residue was washed with anhydrous ether, and the crude peptide was extracted with 10% acetic acid. Lyophilization of the 10% acetic acid extract gave 900 mg of crude product. The peptide was purified by medium pressure ODS reverse phase column chromatography using a gradient of 22-45% CH3CN in 0.1% TFA. The product was extracted in three fractions, which were concentrated and lyophilized to give 130 mg of white solid of purity > 98%.
Compound 1:
AVSEHOLLHDKGKSIODLRRRELLEKLLEKLHTA-NH7 (SEO ID NO: 7) Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala NH2. { SEQ ID NO: 7)
Physical data: p.f. 150-159 ° C [a] ß25 -34.88 ° (c 0.16, H, 0)
FAB (CpíHjooNjßOjí): [M + H] + 4005.5 AAA: Asp, 1.9 (2); Glu, 5.6 (6); Ser, 1.6 (2); His, 2.7 (3); Gly,
1. 0 (1); Thr, 0.9 (1); Ala, 1.9 (2); Arg, 2.8 (3); Val, 1.0 (1); He, 0.9 (1); Leu, 7.3 (8); Lys, 4.0 (4).
Similarly, compounds 2, 5-18, 21-27, 29-36, 38-48, 50-54, 58-64 and 66-70 were prepared and characterized, replacing the PAM resin as was necessary for the synthesis of hydroxy-terminal polypeptides.
Compound 2:
AVS? HQLLHDKGKSIQDLRRR? LLEKLL? KLHTA-OH =? Q ID NO: 6]
Wing Val Ser Glu His Gin Leu Leu His Aso Lys Gly Lvs Ser 'le 1 5 10 * 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lvs Leu Leu Glu "vs 20 25 'or Leu His Thr Wing OH (SEQ ID NO: 6) Physical data: Mp 154-170 ° C [a] D ~ -49.35 ° ( c 0 46 H, 0¡
FAB (C17SH3OINS7OJO): [M + H] * 4005.0 '"
AAA: Asp, 2.1 (2); Glu, 5.9 (6); Ser, 1.7 (2); His, 2.9 (3); Gly 1.1 (1); Thr, 1.0 (1); Ala, 1.9 (2); Arg, 3.0 (3); Val, 1.2 (1); He, 1.0 (1); Leu, 7.3 (8), Lys, 4.2 (4).
Compound 5:
AVSEHOLLHDKGKSIQDLRRRELLERLLERLHTA-OH (SEO ID NO: 15) Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 * 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg 20 25 30
Leu Kis Thr Ala OH (SEQ ID NO: 15) Physical data: p.f. 147-165 ° C íc] D :! -49.17 ° (c 0.66, H20)
FAB (C175K299N59052): [M + H] '4061 AAA: Asp, 2.1 (2); Gly, 6.1 (6); Ser, 1.3 (2); His, 3.1 (3); Gly,
1. 1 (1); Thr, 1.0 (1); Ala, 2.0 (2), Ars, 5.0 (5); Val, 1.0 (1); He, 0.9 (1); Leu, 1.K3X Lys, 1.9 (2).
Compound 6:
AV.?EHOLLHDZ, PQTQnLRRRELLERLLE LHTA ^ OH (SEQ ID NO: 16; Ala Val Ser Glu His Gln Leu Leu His As? Ara Gly Arg Ser lie
! 5 - Gln Aso Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg '20 25 - 0
Leu His Thr Ala OH (SEQ ID NO -.16 ''
Physical data: p.f. 150-170 ° C [a] t¡: i -46.65 = < r 0 5- H, 0-FAB (C175H299N6305,): [M + H] '4118.0""' '
AAA: Asp, 2.1 (2); Glu, 6.1 (6); Ser, 1.6 (2-; His 3 2 (3) - Glv 1.2 (1); Thr, 1.0 (1); Wing 2.0 (2!; Ar, ¿) Val 'Y'
1. 0 (1); He, 1.0 (1), - Leu, 7.3 (8).
Compound 7:
AVSEHOLLHDRGRSIODLRRRELLERLLKRLHTA-OH (SEO ID NO: 17) Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Lys Arg 20 25 30
Leu His Thr Ala OH (SEQ ID NO: 17) Physical data: P.f. 177-182 ° C ra] D ~ -46.17 ° (c 0.14, H, 0¡
FAB (CpßHjwNwOso): [M + H] ^ 4117 AAA: Asp, 2.0 (2); Glu, 4.8 (5); Ser. 1.8 (2); His, 3.2 (3); Gly,
1. 1 (1); Thr, 0.9 (1); Ala, 1.9 (2); Arg, 6.7 (7); Val, 1.0 (1); He, 1.0 (1); Lys, 7.7 (8); Lys, 0.9 (1).
Compound 8:
AVSEHOLLHDKGKSIODLRRRELLEKLLRKLHTA-OH (SEO ID NO: 5) Ala Val Ser Glu His Glr. Leu Leu His Asp Lys Giy Lys Ser He i 5 l? ' fifteen
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arq Lvs 20 25"30
Leu His Thr Ala OH (SEQ ID NO:) Physical data: p.f. 147-165 ° C [a] Du -49.17 ° (c 0.66.H-, 0;
FAB (C176H3OJNS9049): [M + H] + 4033.0 AAA: Asp, 2.0 (2); Glu, 4.3 (5); Ser, 1.8 (2); His, 2.7 (3); Gly,
1. 1 (1); Thr, 0.9 (1); Ala, 2.0 (2); Arg, 3.9 (4); Val, 1.0 (1); He, 1.0 (1); Leu, 7.9 (8); Lvs, 4.0 (4).
Compound 9:
AVSEHOLLHDKGKSIODLRRRELLEKLLEKLHTAGRR-QH (SEO ID NO: 10)
Wing Val Ser Glu His Gln Leu Leu His Aep Lys Gly Lys Ser He
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala Gly Arg Arg OH (SEQ ID NO: 10/35
PHYSICAL DATA: p.f. L? 3 -1DO ° L ¡zt) X 44. "^ ?: C. I H, 0)
FAB (C, S9K326? 05, [M] "4375 C AAA: Asp, 2 0 (2), Glu, = 9 \ C, Ser, 1.7 '2, .3 ¿-, J Gxy,
2. 3 (2 /, Thr, 1.0 (1), Wing, 1.9 (2), Arg, 5.C '- 1.2: i); He, 1.0 (1,; Leu, 7 8'8 <; Lvs, 4 3,
Compound 10
AVSEAOLLHDLGKSIODLRRRELL? KLLEKLHAL-0H (SEO ID NO -14 '
Ala Val Ser Glu Ala Glr. Leu Leu His Asp Leu Gly Lys Ser I ", e 1 5 10" '"15
Glr. Asp Leu Arg Arg Arg GH Leu Leu Glu Lys Le '. I read. Gla L _? Yy i 20 25 30
Leu His Ala Leu OH (S? Q ID NO.14 / Physical data: p.f.70-75 ° C.] D- -31.59 °; c 0.54, H, C;
FAB (C174H3CON52051): [M + H]. 3936.0 AAA: Asp, 2.0 (2); Glu, 6.0 (6 /; Ser, 1.8 (2); His, 2.0 (2 '; Glv, 1.2 (1); Wing, 3.0 (3); Arg, 2.8 (3); Val, 1 i'l); lie, 1.0 (1); Leu, 9.9 (10); Lys, 3.0 (3).
Compound 11:
AVSEHOLLKDKGKSIODLRRRELLEKLLELLKEL-NH-, (SEO ID NO: 11)
Wing Val Ser Glu His Gln Leu Leu His ASO Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20"25 30
Leu Lye Glu Leu NH2 (SEQ ID NO: 11) Physical data: p.f. 172-174 ° C [a] -} 25 -43.29 °! C: .2, H: 0)
FAB (C179H3nNS50S2): [M + Hj * 4065.3 AAA: Asp, 2.2 (2); Glu, 7.7 (7); Ser, 1.7 (2); His, 2. C (2.).; Giy,
1. 0 (1;; Ala, 1.0 (1); Arg, 3.0 (3); Val, 1.1 (1); lie. 1.0 (1;; Leu, 9.3 (9); Lys, 5.1 (5).
Compound 12:
AVSEIQFXHNLGKHLSSXERVELLEKLLEKLHNY-NIL fX = Nle. SEO ID NO: 23)
Ala Val Ser Glu He Gln Phe ie His Asn Leu Giy Lys His Leu i 5 10 '* 15
Being Ser Nle Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Giu Lye 20 25 '30
Leu His A ?? Tyr NK,; SEQ ID NO: 23;
Physical data: p.f. 178 ° C [a] c ~ -35.83 ° '.c 0.4, H-, 0)
FAB (Clg2H295N50Osl): ÍM + Kj * 4001.6 AAA: Asp, "2.1 (2), Giu, 6.5 (6); Ser, 2.7 (35; His, 3.1 (3;; Gly,
1. 1 (15; Ala, 1.0 (1); Ar, 1.0 (1); Ty, 0.3 (1); Val, 2.0 (2); Phe, 1.0 (1); He, 0.9 (1); Leu + Nle, 8.5 (7-2); Lys 3.1 (3).
Compound 13:
AVSEIQFXHNLGKHLSSXRRRELLEKLLEKLHNY-NH, (X = Nle, SEO ID NO: 24)
Wing Val Ser Glu He Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Being Ser Arg Arg Arg Glu Leu Leu Glu Lys Leu Glu Lys 20 25 30
Leu His Asn Tyr NH, (SEQ ID NO: 24)
,
Compound 14:
AVS? HOLLHDKGKSIODLRRRALAEALAEALHTA-NH-, (SEO ID NO: 20)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He
1 5 10 15
Gln Asp Leu Arg Arg Arg Ala Leu Ala Giu Ala Leu Ala Glu Ala 2? "25 30
Leu His Thr Ala NH2 (SEQ ID NO -.20) Physical data: p.f. I90-225 ° C [a] D ^ -56.58 ° (c 0.36, H2G)
FAB (C151H272N5.049): [M + H] * 3747.0 AAA: Asp, 2.1 (2); Glu, 4.9 (5); Ser, 1.7 (2); His, 2.6 (3); Gly,
1. 1 (1); Thr, 1.0 (1); Ala, 7.6 (7); Arg, 2.8 (3); Val, 1.2 (1); He, 1.0 (1); Leu, 6.6 (6); Lys, 1.9 (2).
Compound 15:
AVSEHOLLHDKGKSIODLARRELLEKLLEKLHTA-NH, (SEO ID NO: 12)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He! 5 10 5
Gln ASD Leu Ala Arg Arg Hu Leu Leu Glu Lys Leu Leu Giu Lys 20 25 30 Leu His Thr Wing NH, (SEQ ID NO: 12) Physical data: p.f. 170-18Q ° C [a] D ~ s -48.13 ° (c 0.2, H, C?
FAB (CmH293N530): [M + H] * 3919.0 AAA .: Asp, 2.1 (2); Glu, 6.1 (6); Ser, 1.7 (2 /; H s, 3.1 (3;; Gly
1. 1 (1); Thr, 1.0 (1), Wing, 3.0 (3); Arg, 2.1 (2); Val, 1.1 (1); He, 1.0 (1); Leu 8.0 (8); Lys, 4.4 (4).
Compound 16:
AVSEHOLLHDKG SICDLP.RAELLEKLLEKLHTA-NH, 'SEO 10 NO: 13
Ala Val Ser Glu His Gln Leu Leu His Asr > Lys Giv Lye Ser He 1 5 10 * Gln Aep Leu Arg Arg Wing Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 'Leu His Thr Wing NH2 (S? Q ID NO: 13) Physical data: p.f. 190-I95 ° c [a] 0-50.50 ° ic .4, H, 0)
FAB (C? 7: H293N5305i): [M + H] '3919.0 AAA: Asp, 2.1 (2); Glu, 6.0 (6); Ser, 1.8 (2); His, 3.1 (3): Giv
1. 1 (1); Thr, 1.0 (1); Ala, 3.0 (3): Arg, 2.1 (2); Val. 1.1 (1); He, 1.0 (1); Leu, 7.5 (8); Lys, 1.2 í).
Compound 17:
& VfiBHOT, T-HDKGKSTOnt.RRRS LSST.LSSLHTA-Nfí, (SEO ID NO: 21) Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser lie 10 Gln Asp Leu Arg Arg Arg Ser Leu Leu Ser Ser Leu Leu Se: 5 20 2 ¿0
Leu His Thr Ala NH, (SEQ ID NO: 21)
Physical data: p.f. 195-204 ° C Mo25 -67.11 ° (c 0.3 .0)
FAB (C163H280N54Oso): [M + H] + 3796.0 AAA: Asp, 2.1 (2); Glu, 2.9 (3); Ser, 6.8 (7), Ais, 3. ^ -y.
1. 2 (1); Thr, 1.0 (1); Ala, 2.0 3. C (3) 1.0 (1); He, 1.0 (1); Leu, 8.2 (8) Lvs, 2. C (2)
Compound 18:
AVS? HOLLHDKG SIODLERRAFYDKV.AEKLHTA-NHL (SFQ ~ ^ NO- ?? ',
Ala Val Ser Glu His Gir. Leu Leu His Aso Lys Glv Lvs Ser lie 1 5? O "15
Gin Asp Leu Arg Arg Arg Wing Phe Tyr Asp Lys Val Wing Giu Lvs 20 25 Leu His Thr Wing NH, (SEQ ID 130.22: Physical data: mp 200-207 ° c [«] o25 -60.26 ° (c 0.6, H, 0)
FAB (Cn4H284N56Os0): M + H] ~ 3960.0 AAA: Asp, 2.9 (3), Glu, 3.5 (4); Ser, 1.4 (2); His, 2.6 (3); Glv, 0.9 (1); Thr, 1.0 (1); Wing, 4.0 (4); Ara, 3.0 (3); Tyr, 0.9 (1); Val, 1.9 (2); Phe, 1.1 (1); He, 0.9 (1); Leu, 3.6 (4); Lys, 4.1 (4).
Compound 21:
AVSEIOFLHN LGKHLSSLRR RELLEKLLEK LHNY-NH, (SEO ID NO: 35)
Ala Val Ser Glu He Gln Phe Leu His Asn Leu Gly Lys His Leu 1 5 10 5
Being Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Asn Tyr NH, (SEQ ID NO: 35) Physical data: p.f. 143--155 ° C [a] ^ -45.97 (c 0.26, H20)
F? B (C iwliS60A9): [M + H] ~ 4084 AAA: Asx, 2.1 (2); Gix, 5.0 (5); Ser, 2.7 (3); His, 3.0 (3); Gly,
1. 0 (1); Ala, 0.9 (1); Arg, 3.1 (3); Tyr, 0.9 (1); Val, 1.0 (1); Phe, 0.9 (1); He, 0.9 (1); Leu 9.3 (9); Lye, 3.2 (3)
Compound 24:
AVSEHOLLHD KGKSIODLKL K? LLEKLLE LHTA-NH, (SEO ID NO: 38)
Wing Val Ser Glu His Gin Leu Leu His Asp Lvs Gly Lys Ser He 1 5 lo '' '15
Gln Asp Leu Lys Leu Lys Giu Leu Leu Giu Lys Leu Leu Glu Lys 20 25 3
Leu His Thr Ala NH, (S? Q ID NO: 38)
Physical data: p.f. 175-182 ° C; a¡D- -49.99 (c 0.47, H, 0) FAB (C17SH299N49051) = [M + H] * 3906.5 AAA: Asx, 2.1 (2); Glx, 6.5 (6); Ser, 1.8 (2); His, 3.1 (3); Gly,
1. 1 (1); Thr, 1.0 (1); Ala, 2.1 (2); Val, 1.1 (1); He, 1.0 (1); Leu, 9.1 (9); Lys, 6.5 (6). Compound 25:
AVSEHOLLHD KGKSIODLRR RELLERLLER LHTA-NH, (SEO ID NO -.39)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Giu Are 20 25 '30
Leu His Thr Ala-NH, (SEQ ID NO: 39) Physical data: p.f. 136.5-153.5 ° C [¡X -3L.57 (c 0.13, 11.0)
FABtCpsHjooNßoOj!): [M + H] + 4060.8 AAA: Asx, 2.2 (2); Glx, 6.2 (6); Ser, 1.8 (2); Hie, 3.2 (3): Gly, 1.1 (1); Thr, 1.0 (1); Ala, 2.1 (2); Arg, 5.2 (5); Val, 1.1 (1); He, 1.1 (1); Leu, 6.4 (6 ', - Lys. 2.2 (2).
Compound 26:
AVSEHOLLHD KGKSIODLRR R? LLERLL? R LHTAP-OH (SEO ID NO: 40)
Wing Val Ser Glu His Gln Leu Leu His Aso Lvs Gly Lys Ser He
1 5 10 * * 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Giu Arg Leu Leu Glu Arg 20 25 30
Leu His Thr Ala Pro OH (SEQ ID NO: 40: Physical data: p.f 125.8-127.2 ° C .IX -54.62 (c 0.23, H, 0 >
FAB (Clg0H3O6N60O53): [M + H] + 4158.0 AAA: Asx, 2.1 (2); Glx, 6.2 (6); Ser, 1.8 (2); His, 2.9 (3); Gly, 1.1 (1); Thr, 1.0 (1); Ala, 2.0 (2) - Arg, 5.1 (5); Val, 1.0 (1); He, 1.0 (1); Leu, 8.0 (8). Lys, 2.1 (2); Pro, 1.1 (1).
Compound 27:
AVSEHOLLHD KGKSIODLRR RELLERLLER LHTAGRR-OH (SEO ID NO: 1)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15 Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg 20"25 30
Leu His Thr Ala Gly Arg Arg OH (SEQ ID NO: 41) 35
Physical data: p.f. 1C5-L37.3 ° C] ^ -39 55, c 0.6", K-, 0)
FAB (Clg9H326N6gG < 5): [M + H] * 4430.5 AAA: Asx, 2. -. (2); Gix, 5 9 (6;, - Ser, 1.5 (2); His, 2.7 (3), Giy 2.2 (2); Thr, 1.0 (1); Ala, 1.8 (2); Arq, 7.3 (7 /; Val, 0.8 (1), He, 1.01J, Leu, 8.1 (8), Ly s, 2.1 (2).
Compound 29:
AVSEHCLLHD KGKSIODLRR RELL? XLLEK LHTY-NH, (SEO ID NO: 3)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lvs 20 25 30
Leu His Thr Tyr NH2 (SEQ ID NO: 43) Physical data: p.f. 160-172 ° C [a]? V > -49.85. { c 0.34, H, 0)
FAB (C? GlH3 < wN56052): [M + H] '4096.9 AAA: Asx, 2.0 (2); Glx, 5.6 (6); Ser, 1.7 (2); His, 3.1 (3); Gly, 1.1 (1); Thr, 0.9 (1); Ala, 0.9 (1) .- Arg, 3.0 (3); Tyr, 0.9 (1); Val, 1.0 (1); He, 1.0 (1); Leu, 7.7 (8); Lv =, 4.4 (4).
Compound 30:
AVSEHOLLHD KGYSIODLRR RELLEKLLEK LHTA-NH, (SEO ID NO: 4)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Tyr Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala NH, (S? Q ID NO: 44) Physical data: p.f. 130-171 ° C [at o25 -40.65! C 0.34, H, 0)
FA3 (C, 7gH, r,; N, 5052): [M + H] '4039.4 AAA: Asx, 2.0 (2); Glx, 5.5 (6); Ser, 1.8 (2); His, 3.4 (3;, - Gly 1.1 (1); Thr, 1.0 (1); Ala, 2.0 (2); Arq, 2.9Í3); Tyr, 0.8 (1); Val, 1.0 (1); He, 0.9 (1), Leu, 7.9 (8); Lvs, 3.4 (3).Compound 31
AVSEHOLLHD KGC5I0IL ?? RELL? KLLEK LHTA-NH- SEO ID NO: 45
Ala Va¿ be Glu his G.r. Leu Leu His Asp Lys Gly Cys Ser He
1 5 10 15
Glp Aso Leu Arg Arg Aro- Glu Leu Leu Glu Lvs Leu Leu Glu Lvs 20 25":
Leu His Thr Wing NK, ID NO: 45) Physical data: p.f 140-160 ° C [a] o15 -44.48 (c 0.25,? 20)
FAB (C172H293N55051S,): [M + H] '3979 AAA: Asx + Cys, 3.0 (2 + 1); Glx, 5.6 (6); Ser, 1.7 (2); His, 3.0 (3) Gly, 1.0 (1); Thr, 0 9 (1); Ala, 1.9 (2); Arg, 2.5 (3); Val, 1.0 (1); He, 0.9, 1); Leu, 7.5 (8); Lys, 3.3 (3).
Compound 32:
AVSEHOLLHD KGXSIODLRR RELLEKLLEK LHTA-NH, (SEO ID NO: 46) (X = Cvs (CH? CONH (CH?), NH (biotinyl))) Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Xaa Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30 Leu His Thr Wing NH2, (Xaa = Cys (CH, CONK (CH2) 2NH (biotinyl))), (SEQ ID NO: 46)
Physical data: p.f. (not determined) [a] D25 (not determined) FAE (Cl? 6H3le 59Ow3,): [M-H] * 4306.6 AAA: Asx, 2.2 (2), - Glx, 6.1 (6); Ser, 1.8 (2): His, 3.3 (3 ',; Glv, 1.0 (1 /; Thr, 1.0 (1); Wing, 2.0 (2); Arg, 3.U3); Val, l.Hl; He, 0.9 (1); Leu, S .3 v); Lys, 2.3 '2). Compound 33:
AVSEHOLLHD KGXSIQDLR RELLEKLLEK LHTA-I ?. 'SEO ID NO -47) (X = Lys (7-dimethylamino-2-oxo-2H-1-benoxopyran-4-acetyl))
Ín Asp Leu Arg Arg Ar; ! lu Leu Leu Glu -JGI I and Í Giu Lys 20 25 30
Leu His Thr Ala NK,,
(Xaa = Lys (7-dimethylamino-2-oxo-2H-1-benxop? Ran-4-acet? L), (SEQ ID NO: 47)
Physical data p.f 135-205 ° C [a] D25 -2692 (c 0.104, 50% aqueous HOAc)
FAB, ClgliH3l, i; 57C; 4): [M + HJ '4233 AAA: Asx, 1.9 (2;; Glx, 6.3: 6;; Ser. 1.7 (2); His, 3.2 (3'.; Giy 1.0;; I '; Thr, 1.1 (1); Wing, 2.3 (2); Arg, 3.2 (3); Val, 1.1 (1); lie, 0.9 (1), Leu, 3.2 (S); Lys, 4 6 (4) .
Compound 34:
AVSEHOLLHD KGKSIQDLRR RELLEKLLEK LHTAG-OH (SEO ID NO: 8)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 lo "15
Gln Asp Leu Arg Arg Arg Glu Leu Leu C-lu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala Gly OH. { SEQ ID NO: 8) 35 Physical data: p.f. 92.1-146.6 ° C [cric25 -40.7 (c C.34, H,
FAB (C, 7H302N56O53): [M + H] + 4062.0 AAA: Asx, 2.0 (2); Glx, 5.7 (6); Ser, 1.3 (2); His, 3.0 (3); Gly 2.2 (2); Thr, 0.9 (1); Ala, 1.9 (2); Arg, 2.3 (3); Val, He, 0.9 (1); Leu, 7.5 (8 '., - Lys, 4.2 (4).
Compound 35: AVSX.HOLLHX, KGKSIOX, LRR RX | LLX, KLLX, K LHA-OH (SEQ ID NO: 49 i (X, = Glu (OCH,); X, = ep (OCH,)) Ala Ser Ser Xaa , Hie Gln Leu Leu His Xaa, Lys Gly Lye Ser He
1 5 20"15
Gln Xaa, Leu Arg Arg Arg Xaa, Leu Leu Xaa, Lys Leu Leu Xaa, Lys 20"25 * '3'
Leu His Ala OH IXaa, = Glu (OCH3); Xaa, = AspíOCH3)) (SEQ ID NO: 49)
Physical data: p.f. (not determined) [s.] D25 -21.96 (c 0.132, H20)
FAB íC; 8! H3pN550l2; : [M -'- K] 4089.0 AAA; Asx, 2.1 (2); Glx, 6.3 (6); Ser, 1.8 (2); His, 3.3 (3), - 'lv, 1.1 (1); Thr, 1.0 (1); Ala, 2.0 (2); Arg, 3.1 (3); Val i 1.1 (1); He, 0.9 (1); Leu, 8.0 (8); Lys, 4 2 (4).
Compound 36:
AVSX.HQLLHX, KGKSIOX, LRR RX, LLX, KLLX, K LHA.-OCH, (SEO ID NO: 50 ^ (X, = Glu (Oc ?,); 'X, = ASP (OCH,)) Ala Val Ser Xaa, His Gln Leu Leu His Xaa, Lye Gly Lys Ser He 1 5 10"15
Gln Xaa, Leu Arg Arg Arg Xaa, Leu Leu Xaa, Lys Leu Leu Xaa, Lys "20 '25 30
Leu His Ala OCH, (Xaa, = Glu (OCH3); Xaa, = ASD (OCH3)) (SEQ ID NO: 50) Physical data: p.f. (not determined) ía] D25 -46.80 (c 0.07,
H, 0) FAB (C18, H3l3N55O2): [M + H] "4103 AAA: Asx, 2.1 (2); Glx -3.2 (6 ',; 3er, 1.4 (2); Hie, 3 0 (3;, - Gly,
1. 1 (1 ', Thr, 1.1, 1; A1 a, 1 7.2); Arg, ¿2 (3); Go 1, 0.6 (1); He, 0.9 (lj; Leu, 3.0 (8) Lys, 4.1 (4).
Compound 38
AVSEHOLLHD KGKSIODLRR RELLEKLLEK LHT7-.P-OH (SEO ID NO: 52)
Wing Val Ser Glu His Gln Leu Leu Kis Asp Lys Gly Lye Ser He 1 5 10 l =
Gin Asp Leu Arg Arg Arg Giu Leu Leu Glu Lvs Leu Leu Glu Lys 20"25 30
Leu His Thr Ala Pro OH (SEQ ID NO -52) 35 Physical data p.f 15:? AL 33 FAB (C18oH306ÍJ56O53): [M + H] * 4102.6 AAA: Asx, 2.0 (2); Glx, 5.6 (5 '; Ser, 1.7 (2,; Kis 2 9 (3) 1.1 (1); Thr, 0.9 (1); Ala, 1.9 (2), Arg,? .9' '; Val 1.2 ( 1); He, 1.0 (1); Leu, 7.7: 3,; L s, 4.3 (4,.
Compound 39:
AVSEHOLLHD KGKSIODLRR RELLEKLLEK LHTP-OH (SEO ID NO: 53)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Pro OH i SEQ ID NO: 53)
Physical data: p.f. 120 .43.2 ° C] D- -52.78 (c 0 47
H, 0) FAB (C, -7H30, N < 5C5,). [M + H] '4031 AAA- Asx, 2 0 (2;; Gix, (2)?, 2 -V, u _ / Tr.r, 1.0' 1 9 '3, 2 1, He, 0 9, 1. "£ 6 (3) Pro,
Compound 40
AV ?? HOLLHD XGKSIQDLRR RSLL? KLLEK LHTP-NH- (SEQ ID NO: 5 '
Ala Val Ser Gl i Kis Glr. Leu Leu Kis Asp Lys Gly Lys Ser He 1 5 l "15
Gln Asp Leu Arg Arg Arg Glu Read Leu 3lu Lye Leu Leu Giu Lys 2? "25 30 Leu His Thr Prc NH, (SEQ ID NO: 54) Physical data: pf 133 9- 155? ° c XXDZ5 -54.22 X 0.37,;;,and;
FAB (C177H30, N56O5,). [M + H] * 4030 ~? AAA- Asx, 2.0 (2); Gix, 5.6 (6); Se-, 1 9 (2), His, 2.9 (3); Gly 1.1 (1); Thr, 0.9 (1), Ala, 0.9 (1), Arg, 2.8 (3); Val, 1.2 (1;, He, 1.1 (1); Leu, 7.8 (8, Lys, 4.2 (4); Pro, 0 S (1)
Compound 41 AVSEHOLLHD KGKSIODLRR R? LLEKLLEK LHP-NH, (SEO ID NO: 55)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He
1 5 10 15 Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Pro NH2 (SEQ ID NO: 55)
Physical data: p.f. 142.8-166.1 ° C [a] ^ 5 -53.80 (c 0.38, H, 0)
FAB (C173H, 95N55049): [M + H] * 3929 AAA .: Asx, 2.0 (2); Glx, 5.7 (6); Ser, 1.8 (2); His, 3.0 (3); Gly, 1.1 (1); Ala, 0.9 (1); Arg, 2.8 (3); Val, 1.2 (1); He, 0.9 (1); Leu, 7.4 (8); Lys, 4.4 (4); Pro, 0.9 (1).
Compound 42
AVSEHQLLHD KOKSIQDLRR RELLEKLLEK LP-NH, (SEO ID NO: 56 'Ala Val Ser C-lu His Glr. Leu Leu His Asp Lys Giy Lys Ser He
iU Are uj.-. _eu? _e, i ^
~ - > - ~ w_ IL NO:
Physical data: p.f 161.0- 1 ~ 7.0 ° C -61.97 • 'c 0.19, K, 0)
FA3'.ClS7H: 88N5? C48-: [M + H] * 3 ^ 52.0 _ _ AAA. Asx, 2.2 (2 ^, Glx, 5.9 (6; ber. _.S: 2i; is, 2.1.2,; _-_ *, 1.1 (1;; Ala, 1.0 (1); Arg, 3.0 (3; Val, i.l), He, 1.0 (1), Leu, 7.9 (8), Lys, 4.3 (4, Pro, 0.3 (1).
Compound 43
AVSEHOLLHD KGKSIQDLRR RELLEKLLE _LHT Aj ^ -Oj_ (SEO ID NO: 57)
Wing Val Ser Glu Kis Gln Leu Leu Hi e Asp Lye Gly Lys Ser H «1 5? O * l '~ Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 3o
Leu His Thr Arg Ser Ala Trp OH (SEQ ID NO: 57) 35 Physical data: p.f. 181-202 ° C [a] D "-45.14 (c 0.19, H, 0) FAB (C195H326N62056): [M + H] + 4435.2 AAA: Asx, 2.0 (2); Glx, 5.8 (6); Ser, 2.8 (3); His, 2.8 (3); Gly 1.1 (1); Thr, 0.9 (1); Ala, 1.9 (2); Arg, 3.7 (4); He, 0.9 (1); Leu, 7.5 ( 8); Lys, 4.3 (4); Trp, 0.9 (1).
Compound 44:
AVSEHOLLHD RGRSIODLRR RELLERLLER LHTAGRRTRSAW-GH (SEO ID NO: 58) Jla Val Ser Glu Hxs Gln Leu Leu His Asp Arg Gly Arg Ser He 5 10 * 15
Gin Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg L "? Leu Glu Arg 20 25 30
Leu H e Thr Arg Glv Arg Arg Thr Arg Ser Wing Trp CK iSEQ ID NO: 58) 35 41 Physical data p f 130-132.2 ° C ia! ^ -46.65 (c 0.195, H, 0,
FAB (C ,, 6K365N81062): [M + H] "5C83.8 AAA, Asx, 2.2 (2), Glx, 6.0 (61; be, 2 7 (3); Hie, 3.0 (3), Gly, 2.2 (2), Thr, 2.1 (2), Wing, 3.0 '3 Arg 10.5 (10), Val, 0. H), He, 1.0 (1), Leu, 8.2 (8) • Trp, 1.0 (1).
Compound 45
Wing Val Str Clu His Cin Le. Leu • -'- s -lsp Arg l 1 5 10 * -.n Asp Leu Arg Ax - ^ Gi e- l-? H,. \ ~. * > . ^ 20;? * "'^'" "" - "'Leu H s Thr Arg Gly Arg Ara Tr.r Arg Se- a - w- 35 40 Physical data: pf 158-174 ° C [a] Da - -4433 .. 5577 ((cc 00..5533 ,, HH, 200))
FAB (C, 16H366Ng206l): [M + H] 5087 4 AAA: Asx, 1.9 (2); Glx, 5.6 (6), Ser, 2.6 (3); • His, 3.3 (3); Gly, 2.1 (2); Thr, 2.0 (2); Ala, 2.9 (3); Arg, 10.1 (10); Val, 0.9 (1); He, 1.0 (1); Leu, 8.3 (8); Trp 1-1 (1).
Compound 46: AVSEHOLLHD RGXSIQDLRR RELL? RLLER LHTAGRRTRSAW-OH (SEO ID NO: 60) (X = Lys (di idrocinamoil) Ala Val Ser Glu His Gln Leu Leu Hrs Asp Arg Gly Xaa Ser He
1 5 10 * 15
Gln Asp Leu Arg Arg Arg C-iu Leu Ara -, rg 20 25 i J
Leu His Thr Arg Gly Arg Arg Thr Wing Tr: .Xaa - 35 40 Lys (d? H? Dro? Namo? L), (SEQ ID NO: 60) Physical data, p.f 165.4-175. ° ^ ~ cA 2 ^ i - 4¿ 0 '0.43 0 K, G
FABtCüsHjwNjoOa): [M + H] * 5191 AAA: Asx, 2.1 (2), Glx, 6.3 (6), Ser, 2.3 ¡3); His, 3 2 (3), G 2.1 (2), Thr, 2.0 (2); Ala, 3.2 (3); Arg, 9.9 -.9 j Val, 1.0 (1), He, 0.9 (1); Leu, 8.6 (3); Lys, 1.1 i 1) Trp, 1.1 (1).
Compound 47 AVS? IOFXHN XT? 5AWLRKKLCL VK.'Y-NH "D NO: 61 (X = norleucine)
r.r rii '^ i T or Leu Are: -v? _eu 20' Val Kis Asa Tyr NK, 'SEQ ID NO: 61 Physical data: p.f. 140 -160 ° C [cr] 2Í -56.88 (c 0.16, H, 0)
FABÍCuoHjnNssOjg): [M + H] * 3989. AAA: Asx, 3.0 (3); Glx, 2.9 (3); Ser, 3 7 (4); His, 2.8 (3); Gly,
1. 1 (1), - Thr, 0.9 (1); Wing, 1.9 (2) Arg, 2.0 (2); Tyr, 1.0 (1); Val, 1 7 (2); Phe, 0.9 (1) He, 0.9 (1); Leu + Nle 5.8 (2 + 4); Lys, 3.4 (3); Trp, 1.1 (:).
Compound 48:
AVSEHOLLKD KGKSIQDLRR RELLEKLLEK LHTMA-NH, (SEO ID NO: 62)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Giu Lys Leu Leu Gl? Lys 20 u
, eu His Thr Met Ala NK, 'SEQ ID NO: 6! 35
Physical data, pf i40-2i: u2 X.-c ~ '- ^ 7 73' c 0.173, FA3 (C¡goH30oN57? 5, S,): [M + Hj '4135.0 AAA- Asx, 2.3 * (2) , Glx. 6.6 (5); Ser, 1 4 (2); Kis, 3.2 '3); 1.1 (1); Thr, 1.0 (1); Ala, 2.0 (2, Ara, 3.1, 3) • Val, 0.9 (1); Met, 1.1 (1); lie, 1.0 (1), Leu, 8.8 (8); Lye, 4.4 (4)
Compound 50
AVSEHOLLHD KGKSIQDLRR RFFLEKLL? K LHTA-NH. SEO ID NO: 64
Wing Val Ser Glu His Gln Leu Leu His ASD L / S G.V Lys Ser I i *
1 5 11 *: 5
Glr, Asp Leu Are Arg Arg Phe Phe Leu 01 Lyt,. u Leu Olu Ly: 20 25 30
Leu His Thr Wing NH 'SEQ ID NO: 64) 35"Physical data: p.f. 136.5-156. > 3 ° C [a] ^ -49.89 (c 0.24 H, 0)
FAB (C182Hj00NS6O49): [M + H] * 4056..0 AAA: ASX, 2.2 (2); Glx, 5 .0 (5); • Being, 1.9 (2), - His, 3 • 3 (3) Gly,
1. 0 (1); Thr, 1.0 (1); Ala, 2.1 (2); Arg, 3.1 (3); Val 1.0 (1); Phe, 2.0 (2); He, 0.9 (1); Leu, 7.2 (7),; Lys 3.5 (4).
Compound 51
AVSEHOLLHD KGKSIODLRR RELLHKLLEK LHTA-NH-, (SEO ID NO: 65)
Ala Val Ser Giu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 * "'15 (1 Gin Aso Leu Arg Arg Arg Glu Leu Leu His Lys Leu Leu Giu Lys 20 25 30
Leu Hi = Thr Ala NH: (SEQ ID NO: 65) Physical data, p.f 80.7-141.0 ° C [a] o25 -55.38 (c 0.23, H, 0,
FA3 (C176H300N5gOi ?;: [M + H] * 4012.8 AAA: Asx, 2.2 (2); Glx, 4.9 (5-Ser, 1.8 (2); K.is, 4.3 (4); G. and, 1.1 ( 1); Thr, 1.0 (1); Ala, 2.0 (2); Arg, 3.H3); Val, llii); He, 1.0 (1); Leu, 8.1 (8); Lye, 3.9 (4).
Compound 52
AVSEHOLLHD KGXSIQDLRR RELL? HLLEK LHTA-NH, (SEO ID NO.66 '
Wing Val Ser Glu Hie Gln Leu Leu His Asp Lys Glv Lys Ser He 0 1 5 10 * 15
Gln Aep Leu Arg Leu His Thr Wing NH, (SEQ ID NO: 6)
Physical data: pf134.3 -157.9 ° C [ajD: 5 -50.72 (c 0.45, H, 0] FAB (Cl7sH295N57OSI): [M + H] + 4012.8 AAA: Asx, 2.1 (2); Glx, 5.9 (6 ); Ser, 1.8 (2); His, 4.2 (4); Gly,
1. 1 (1); Thr, 1.0 (1); Ala, 2.0 (2); Arg, 3.0 (3); Val, 1.1 (1); He, 0.9 (1); Leu, 8.1 (8); Lys, 3.1 (3). Compound 53:
AVSEHOLLHD KGKSIQDLRR RELLEKLIAK LKTA-NH, (SEO ID NO: 67)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
C- r. Asp Leu Arg Arg Ara Glu Leu Leu Glu Lys Leu He Wing Lv = 20 25 3Ó W, (SEQ ID NO -.67)
Physical data: p.f. 7-159 ?? j (c C [M- j, 94 Asx, 2.2 (2); Gl, 4.9 5 5;; Ser, 1. £: 2,; Kis,: 1.1 (1); Thr. 1.0 ( 1,; Ala, 3.1 (3); Arg, 3.1 (3 1.0 (1); He, 1.9 (2); Leu, 7.0 (7); Lys, i.3 (4.
Compound 54
AVSEHCLLKD KGKSIODLRR R? LLEKLLEE IKTA-NK, (SEO ID NC -_S_31
Ala Val Ser Giu His Glr. Leu Leu His Asp Lys Gly Lye Ser __e 5 10 13
Glr. Asp Leu Arg Arg Arq Glu Leu Leu Giu Lys Leu Leu Glu C-lu 20 * "25 3
He His Thr Ala NH, (= EQ ID NO: 68) Physical Data: pp. 138-185 ° C [X X -50.17 (c 0.14, H, 0:
FAB (C, 74H295N550S3): [M + H] + 4005 AAA: Asx, 2.2 (2); Glx, 7.1 (7); Ser, 1.7 (2); His, 2.8 (3); Giv 1.0 (1); Thr, 1.0 (1); Ala, 2.1 (2); Arg, 3.1 (3 / .- Val, 1.1 (1); He, 1.7 (2!; Leu, 7.1 (7;, - Lys, 2.7 (3).
Compound 58:
AVSEHOLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-NH, (SEO ID NO: 72
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He! 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 3G
Leu His Thr Arg Ser Wing Trp NH2 (SEQ ID NO: 72) 35 Physical data: P.f. 158-163 ° C [a] Q25 -46.06 (c 0.17, H-O '
F B (C, «H, j, NHOs,). [M * H] * 4434.8 AAA: Asx, 2.0 (2); Glx, 5.5 (6); Ser, 2.7 (3); Kis, 3 1 (3) - H 1.0 (1); Ala, 1.8 (2); Arg, 4.0 (4); Thr, 0.9 (1; Val, 0.9 (1), - He, 0.9 (1); Leu, 7.5 (6;, - Lye, 3.9 '4', - Trp,
Compound 59: AVSEKQG LHI GK, LI¿.L_LR P.? LL? KLL? K LKTPSAX-OK
(X = Nal (2) = 3- (2-naphthyl) -L-alanine
.TJ, s Gln .eu e y-, o, Lyjos j? V _, ys ber __. Í 1 15
Leu His Thr Arg Ser Ala 3- (2-naphthyl) -L-alanine-OH (SEQ ID NO. 73) 35 Physical data: p.f. 156-162ÜC [a] D '-44.44 X. C.18Í
FAB (C, 9, K, 2iiN ,, p ,,: il'-K] + 4445. d AAA- Asx, 2.1 (2), Glx, 5.5, 6;, - S r, 2.3 (3;; His; , 2.9 (3); Gly,
); Ala, 2.0 (2) Arg, 4.0 ', 4:, Thr. 0.911 -: Val, (ll; He, 0 -, I r, "ys (4 /, - Nal Compound 60:
AVSEHOLLHD KGKSIODLRR RELLEKLLEK LHTASAW-OH (SEO ID NO: 74)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala Ser Ala Trp OH (SEQ ID NO: 74) 35 Physical data: p.f. 159-164 ° C [] D * -50.94 (c 0.29, H, 0)
FAB (C mHjjoNßoOss): [M + H] + 4349.0 .AAA: Asx, 2.0 (2); Glx .. 5.6 (6); Ser, 2.7 (3); His, 2.2 (3); Gly,
1. 0 (1); Wing, 3.1 (3 /, - Arg. 2.8 (3); Thr, 1.0 (1); Val, 1.1 (1); He, 0.9 (1); Leu, 7.6 (3); Lys, 4.0 (4. Tr,
Compound 61
AVSFHQLLKD KGKS1QCLRR RELLEKLLEK LHTAEIRA-QH • SEP ID N: '75
Wing Val Ser Giu His Gln Leu Leu Kis ASD Lve Gly Lvs Ser He 1 5 10 * 15
Gln Aso Leu Arg Arg Arg Glu Leu Leu Giu Lys Leu Leu Glu Lvs ~ 20 25 3 * 0 .1IS: r Wing to Artr A_.a JA LS: 1
Physical data: p.f. 155-210 ° C [za - - 6.15 'r ~ 12.? AB'. C? 95H3, N6: 058, l M + K] "4475.8 AAA: Asx 2.2 (2), Glx, 6.9 '7,. Ser, .7? ..: Kis, 1 _ 3, 1.1: 1); Wing 3.1 (3,; Arg, 4.014;, Tr.r, 3.3, 1, Val, li: i • I le, 1 9 (2!, Le, 3.1 (8 Lvs, -i .1; '1
Compound 62
AVSEHOLLHD KGKSIODLRR RELLEKLLEK LHTAEIR-OH (SEO ID NO: 76)
Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr Ala Glu He Arg OH (SEQ ID NO: 76) 35 [a] o25 -52.73 (c 0.265, H, 0) Ser, 1.9 (2); His, 3.4 (3); Gly, 3.3 (4); Thr, 1.0 (1); Val, 7.9 (8); Lys, 4.0 (4).
Compound 63:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHT.A? I-OH (SEO ID NO: 77)
Ala Val Ser Glu His Glr. Leu Leu Kis Asp Lys Giv Lys Ser lie 1 5 10 * "15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Giu Lys Leu Leu Olu Lvs 20 25 3C
, eu His Thr Ala Glu He OH (? EQ NO: 35 Physical data: p.f. 169-205 ° C íCtl D: o.te C. O. K, 0)
FAB (C186H317N5705): [M + H] '4243.0 AAA: Asx, 2.2 (2); Glx, 6.8 (7); Ser, 1. I His, 3.3 (3) 1.0 (1); Ala, 2.0 (2); Arg 3.0 (3) 1.0 (1); Val 1.0 (1); He, 1.8, 2); Leu, 7.8 (8) Lys 3.5 (4:.
Compound 64. AVSEHOLLKD KGKSIPDLPP RELL? KLL? K LKTA? -OK 'SEO LD NO - 7? Ala Val Ser Glu His Glr. Leu Leu Kis Asp Lys Gly Lys Ser He 1 5 10 * 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Giu Lys 20 ~ 25 30
Leu His Thr Ala Gli OH (SEQ ID NO: 78! 35
Physical data: p.f. 199- 205 ° C [ajr, 25 • 52.47 (c 0.41, H, 0)
FAB (CIgoH306N56O55): [M + H] ^ 4135.0 AAA: Asx, 2.0 (2); Glx, 6.6 (7); Ser, 1.9 (2); His, 3 3 (3); Gly,
Id) Ala,; Arg, 2.9 (3); Thr, 1.0 (1) Val, ??: He,, Le, 3.2 (8); Lys, 3.8 (4)
Compound 66:
SEHOLLHD KGKSIC'LRR -? LLE LLEK LHTA-NH, 'SEO ID NO: 80'
Ser Glu His Gln Leu Leu Kis? Sp Lys Gly Lys Ser He Gln Asp 1 5 10 15
Leu Arg Arg Arg Giu Leu Le ^ H Lys Leu Leu Giu Lys Leu His 20 '25 30
Thr Ala NH, (SEQ ID NO: 80 / Physical data 'P.f. 134.2 ° C [a] 5 -48.12 (c 0.36, K-0
FAB (C167HM6NS4049): [M ~ H] '3834.4 AAA: Asx, 2.0 (2); Glx, 3.7 (6,; Ser, 1.7 (2); HlS, 2.9 (3); Gly, 1.0 (1); Thr, 0.9 (1 /, Wing, 1.0 (1), Arg, 2.8 (3); 0.9 (1); Le u, 7.4 (3;, Lys, 4.3 (4).
Compound 67 L KD KGKSIQDLPR R? LL? - "L? K LFT -NH, iSEQ Leu Leu His Asp Lys Giy Lys Ser Lie Gln Asp Leu Arg Arg Arg 1 5 10 Id Glu Leu Leu Glu Lys Leu Leu C-lu Lvs Leu His Thr Ala NH, (SEQ ID NO: 81) "2 20 25
Physical data: p.f. 128.5-184.5 ° C Lor j -6.53 (c 0.69, .Me
FAB •; C.igH259 47041J: [+ KF 3353 AAA: Asx, 2 V I; Gix, 4.1 (4); Ser, 0 9 (1); His, 2 1 (2) 1.0 (1) T'pv 0.9 (1); Wing 1.0 (1) Arg, 3.0 (3) He 1.0 (1); Leu 8.H8); Lys, 4 2 (4)
Compound 68 '
.HD KGKSIODLER RELLE- Leu H s Asp Lys Gly Lys Ser Lie Gln Asp e Arg Arg Arg 21- D .-, U ^
Leu Leu Glu Lys Leu Leu Giu Lys Leu Kis Thr A__a NH2 (S? Q ID NO: 82) 20 25
Physical data: p.f 165-210 ° C IX D25 - ^ G.05 (c 0.12, H-D) AAA: Asx, 2.0 (2); Glx, 3.9 (4): Ser, 3 9 (1); His, 1.3 (2); 01"1.0 (1); Thr, 1.0 (1); Wing, 1.0 (1); Arg, 2.9 (3); He. 0.9 (1); Leu, 6.8 (7); Lys, 4.2 (4).
Compound 69
S? HPLLKD RGRSIQDLP? R? LLE LLE? IIIAORRTRSA - ^ H. _1GE -Q -_ -, - - \ V •
Ser Giu His Gin Leu Leu His Asp Arg Giy Arg Ser He Glr, Asp 1 5 10 15 Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu H-i s 20 25 30
Leu His Arg Gly Arg Arg Thr Arg Ser Wing Trp OH (SEQ ID NO- 83 35 40
Physical data: p.f. 150-210 ° C [a] or -18.0 (c 0.64, H, 0)
FAB (CiogH ^ N ^ Ofio): [M + H] * 4918.6 AAA: Asx, 2.2 (2); Glx, 6.2 (6); Ser, 2.3 (3); His, 3.1 (3); Giy, 2.2 (2); Thr, 2.2 (2); Ala, 2.2 (2); Arg, 10.4 (10); He, 1.0 (1); Leu, 8.0 (8); Trp, 1.1 (1).
Compound 70
.KD RGRSIQDLRR RSLL? RLLER LHAGRRTRSA -OH (SEO ID NO: 34 ', eu H s Asi Arg Glv Arg Se: He Asp Leu Arg Arg Arg 5 10
2 r, ^ r.
Physical data: p.f. 15C-21C ° C (a '~:' -i 7- > (- - ~ .c • - c
- AB? Clí9H3: 4 7, Os: /. ÍM + H] '4437.14' "" '
AAA: Asx, 2.1 (2 ': Gix, 4.1 (4); Ser "< > R-. S - Q < -, 2.1 (2); Thr, 2.0 (2,, - Ala, 2.1 ( .2.; ~ * -q '9 r,.' '-' ^ '0, 9 H), - Leu, "' .4 (3," - >, -,
The side-chain cyclized analogue (Compound 57) was synthesized as before except that N'-Boc-N-Fmoc-Lys and N'-Boc-N-Fmoc-Asp were placed at positions 13 and 17, respectively
Upon completion of the synthesis of the amino acid Boc, the resin was treated with 20% piperidine in DMF at room temperature for 30 minutes. The residue was filtered and washed with DMF, MeOH and CH2Cl2. The resin (1.1 g) was suspended in 10 ml of DMF containing 250 mg of PyBOP. The pH was adjusted to 8-9 with DIEA and the resin was stirred for 1 hour. The resin was filtered, washed with DMF and CH 2 Cl 2 > then it was re-suspended in DMF. The coupling was repeated using 125 mg of PyBOP. The resin was filtered, washed with DMF, MeOH, and CH2Cl2 and dried. The resin was then treated with HF and the peptide was purified as mentioned above.
Compound 57:
Ala Val Ser Giu Gln Leu Leu Kie As Lys C-Iy Lys Ser He 3"1 Glp Asp Leu Arg Arg Arg Giu Leu Leu Glu Lye Leu Leu <- Ly = 20 -5 30
Leu His Thr Ala -NK, (SEQ ID NO: 71) Physical data: p f. 142.5 - 163.5 ° C [a i,? : 4.31 ic 0.1 ~ \ H, 0,
FAB (C, 7, H298N56Ow) [MrH] * 3986.4 AAA .: Asx, 1.9 (2;; Glx, 5.9 (6); Ser, 1.8 (2); His, 3.2 (3); Giy 1.1 (1); Ala, 2-0 (2); Arg, 3.0 (3); Thr, 1.0 (1); Val, 1 1 (1); He, 0.9 (1); Leu, 8.0 (8); Lye, 4.0 (4) -.
EXAMPLE II [Met34, Ala35] Compound 1, AVSEHQLLHDKGKSIQDLRRRELLEK-LLEKLHTMA-NH2, (SEQ ID N025), was prepared and purified followed by the procedures of Example I This popeptide was converted to homoserine lactone as follows. The purified peptide (160 mg) was dissolved in 44% formic acid (4 ml). This solution was combined with a premixed solution of cyanogen bromide (700 mg) and phenol (16 mg) in 44% formic acid (4 ml). ) at 0 ° C The solution was stirred at 0 ° C for 2 h and at room temperature for 2 h. The formation of the product was monitored by HPLC (Vydac® C-18, 300 A, 46 x 250 mm, flow of 1.2 ml / min. , gradient 25-45% of acetonitplo in 0.1% of TFA over 10 min). The reaction was completed within 4 h Half of the sample was concentrated and purified by preparative RP-HPLC (Vydac® C-18, gradient 25-45% acetonitoplo in 01% TFA) The homosepna lactone peptide fractions were extracted and lyophilized to give 28 mg of white solid of purity > 95%, Compound 4
Compound 4
VSEKQLLHDKGKSIQDIPRPELL? GLI EKLHTX 'v = hSerlac, SL- / •: o
Ala Val Ser Giu His Glr. Leu Leu His As? Lys Gly Lys ber He
1 5 10 15
Glr. Ast Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Giu Lys 20 25 30
Leu His Thr hSerlac (SEQ ID NO 9;
Physical data
mp 138-142 ° C aJa23 - "66 ° (c 0.1, K, C FAB ÍC" 6H2WN3,052) ÍMrH] * 401"'61 AAA? sp, 2.1 (~ 2 > Giu, 6 1'6,, Ser, "8 (2), His, 3.0 '3), Thi 1 1 ¡1X Ma, 1 1 (1), Arg, 2 1 (3), Val, 1.0.H, He, 1 3 (1), Leu, 8.2 (8), Lye, 3 8 '4,, Gly 1 09 (1); hSer, 1.09 f 1) Similarly, Compound 65 was prepared according to this procedure.
Compound 65: AVSEIOFX, HN KGKH SSX.ER VE LRKXLOD YHNX, (SEO ID NQ_1_ ^ (Xi = L-norleucine; X? = Homoserine lactone)
Ala Val Ser Glu He Gln Phe His Asn Lys Gly Lys Kis 1 5 10 Ser Ser Nie Glu Arg Val Glu Tro u u Arg ^ ys _ys JTU Gln Asi 20 25 ~ J Val His Asp hSerlac (SEG ID NO: 79)
PHYSICAL DATOS P f, r - -? - co r - "71 16 - 1 / b C [ü J D- - 52 22 'c 0 ~ 5 -t ^'
FAB ÍCigoH, 8sN54O50; : [M-Hj '4003 -: -. '' "" ";
AAA: Asx, 3.1 (31.- Glx, 4.3 (5, Se-, 2 - ">? C.? O - - -. -,. 3.1Í1); Wing, 1 1 (1);? rg, 2.0 (2,, Vaí. 2? (3 '""' - > h «'"' + .0H); He, 1.0 (1,; Le _ - Nie 5.5, .2 í, ys i ' - '3
EXAMPLE III To prepare the homosepna amide, the crude analogue hSerlactone compound 4 was concentrated and treated with 25 ml of saturated NH3 in methanol. The solution was stirred at 0 ° C for 2 h and at room temperature for 16 h. The reaction was then boosted by HPLC (Vidac® C-18.
Á, 46 x 250 mm, flow of 1 2 ml / min, gradient 20-45% of acetonitoplo in
01% TFA) and it was completed in 18 h The solution was concentrated and purified by preparative RP-HPLC (Vydac® C-18, gradient of 25-45% acetonitoplo in 0.1% TFA) The homosepne peptide fractions Amide was extracted and lyophilized to give 30 mg of white solid of purity > 98%, Compound 3.
Compound 3
AVSEHOLLHDKGKSIODLRRRELLEKLLEKLHTX-NH, (X = hSer. SEO ID NO: 8)
Wing Val Ser Glu His Gln Leu Leu His Asp Lye Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Giu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr hSer NH2 (SEQ ID NO: 3).
Physical data p f 33- 42 °? .-._3 -r6"3J2 ^ 5o '-' 5 AAA Asp, - > ii 'i2 I-lu:" S Sir 1 5 (2 - Hrs, 2 8', 3? .9, f- ÍD; 1. 1 or 2 Va_, - 3 9 '4;
Similarly, Compounds 22, 23 and 28 were prepared following this procedure
Compound 22 AVSEIQFLHN LGKKLSSLRR F? LL? LLEK LKTC1-NH-. SEO ID NO: 36) (X = homosepna) Ala Val Ser Ha He 0_r. Phe Leu Kis Asr. Leu Gly Lys Hrs L., 1 5 10 15
Being Ser Leu ^ rg Arg Ar Glu Le i Leu Gl. Lvs Leu Leu Giu Lvs 20"25 '30
Leu His Asn hSer NK, 'SEQ ID NO: 36) 3.93 (c 0.15 H20) His, 2 .8 (3) Gly, 1.0 (1); Phe 3.4 (3)
Compound 23: AVSEIOFLHN KGKHLSSLRR RELLEKLLEK LHNX-NH, (SEO ID NO: 37) (X = homoserine)
Ala Val Ser Glu? I¿ Glr. Phe Leu His Asn Lys Gly Lys His 1 5 10 Ser Ser Leu Ar u Ly e _EU: 1U -Jí Leu His Asn hSe: 'SEQ ID NO: 37) Physical data: p.f 87.1-142. i ° C [ex] o 25 ?? B (Cll9timK510? ti): [M + H] * 4038 AAA: Asx, 2.1 (2); Glx, 4.9 (5); Ser, 2.7 (3,; Kis, 2.3 (3); Gl "1.0 (1); hSer, 1.0 (1); Wing, 1.0 (1); Arg 3.0 (3;, - Val, 1.1 (1); Phe , 0.9 (1); He, 0.9 (1); Leu, 7.9 (8); Lys, 3.7 (4).
Compound 28 AVSEHOLLHD KG SI 2DLRR RELLEKLLER LKTAGRR - NHL (SEO ID NO (X = homoserine) Wing Val Ser S j Kis Glr. Le z Leu Kis As;; Lvs Glv Lvs Ser II
E - r Asp Leu Lr Aig? Rg IX. Leu eí J,. ^. "i- 20
Leu Hie Thr Al Gly Arg Arg hSer NH-, (SEQ ID NO: 4:
Physical data: p.f. 80 ° C] 25 -48.64 (c 0.09, H20) FAB (C ^ HJ ^ NTOO *): [M + H] + 4530.0 AAA: Asx, 2.2 (2); Glx, 6.1 (6); Ser, 1.7 (2); His, 3.0 (3); Gly, 1.9 (2), hSer, 1.0 (1); Thr, 1.0 (1), Wing, 2.1 (2); Arg, 7.2 (7); Val, 0.8 (1); He, 1.0 (1); Leu, 8.4 (8); Lys, 2.1 (2).
The homoserine alkylamides were similarly prepared from the homosepna lactone by dissolving it in DMF containing an excess of the corresponding alkylamine. After stirring at room temperature for several days (the reaction was monitored by analytical HPLC), the mixture was evaporated to the dryness and the peptide purified by preparative HPLC The homosepna representative alkylamides are Compounds 55 and 56
Compound 55 AVS? KOLLHD KG SIQDL? R RELLEKLLSK LHT \ -NHCH, CH, (SEO I NO 6 c (X = homosepna) Wing Val Ser Glu Kis Gln Leu Leu His Aep Lys Giy Lys Ser He 1 5 0 15
;? n Aep Leu Are Arg Arg Glu Leu Leu Giu jys Leu Leu G ^ u "2 25 30 .eu Kis Thr hSer NHCK, CH, SEQ ID NO 6:
Physical data pf (not determined) I] D25 (not determined) F.AB (C "BH30N56o;;,) [M + H] - 4063 0 AAA Asx, 2.1 (21, GJx, D 8 'D,, Ser, 1.7 (2), HxS, 3.1, 3), Gly, 0.9 (1;; Tftr, 1.0, 1); Wing, 0.9 '1, Arg, 3.0 (3), Val, 1.1 (1), He, 1.0 (1), Leu, 3.4 (8), Lys, 3.7 (4); hSer, 0 9 (1).
Compound 56. AVSEHOLLHD KGKSIODT.RR RELI.FKTT.py H ^ Y- ^ - "yr" Í OG tr NQ .- 7 o) "ii ^ ti - ^? ^^ 2i = ^ ¿^ is L EQ ID ( X = homosepna)
Ala Val Ser Glu His Gln Leu Leu His Aso Lvs Glv Lys Ser He 1 5 10"" '' 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Giu Lys 20 25 30
Leu His Thr hSer NHCHjOLCéH, > SEQ ID NO: 70 '.
Physical data pf (not determined) [a] D (not determined) AB (C1MH310N56OS2. '[MTHG 4133 8 AAA, Asx, 2.0-2), Glx, 5.9 (6) Ser, 1 2. - ^ _v 0 9 '1); Thr,: 0 (1); Ala, C.S ti ', "> í 3 1.0' 1;, II -: -, 0 9 (1; Leu, 6. J '8" i? 0 9? I;
EXAMPLE IV The cesium salt of acid N -Boc-N'-Fmoc-L-2,4-d? A? Nobutyr? Co was bound to the Merpfield resin (DMF, 50 ° C, 48 h) and used in a solid phase synthesis instead of the Boc-Ala resin as in Example I Upon completion of the synthesis the peptide was treated with 20% pipepdine in DMF at room temperature for 30 minutes to remove the side chain Fmoc protecting group The protected peptide spontaneously cyclized to the lactam, thereby cutting itself off the ream. The solution was filtered from the resin and evaporated in vacuo to leave an oil. The residue was treated with liquid HF as in Example I to produce the peptide. unprotected crude The peptide was treated and purified as in Example I Compound 49: AVSEHOLLHD KGKSIQDLRR RSLLEKLLEK LHTX (SEQ ID NO: 63) (X = L-2,4-diaminobutyryl lactam) A Gln Asp Leu Arg Arg Arg Glu Leu Leu Giu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr L-2, 4-diaminobutyryl lactam (S? Q ID NO: 63)
Physical data: p.f. R i ^ "* 8 FAB.'C¡16H3l) 0NS6? 5i) - 4015. AA: Asx, 2.1 (2) 2 (6;; 2;; Kis, 3.3 (3;; Ji 1 n Ala,. , 2.? X i; Val, U. (1); Zv '=,. .6 í 4;
EXAMPLE V An aqueous solution of homoserin lactone analog of Example
II was treated with pork liver esterase (Sigma Chemical Company, St. Louis, MO). Hydrolysis of the lactone to the C-terminal homoserin was monitored by analytical HPLC. When the hydrolysis was considered complete, the material was purified by preparative HPLC as in Example I.
Compound 37 AVSEKQLLKD KGKSIQDLR R? LE ^ .J - - rv .ilJH-l SEQl ^ _ NO • =. '.
(X = homoserine) Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He 1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
Leu His Thr hSer OH (SEQ ID NO: 51) Physical data: p.f (not determined) [a] o25 (not determined) FAB (C176H301N55OS3): [M + H] + 4035.1. AAA: Asx, 2.1 (2); Glx, 5.9 (6); Ser, 2.0 (2); His, 3.1 (3); Gly, 0.8 (1); hSer, 0.8 (1); Thr, 1.0 (1); Ala, 1.0 (1); Arg, 3.0 (3); Val, 1.3 (1); He, 1.0 (1); Leu, 8.1 (8); Lys, 3.8 (4).
EXAMPLE VI Following example I, the protected peptide resin BocAVS (Bzl) E (Obz) H (Bom) QLLHD (Obzl) R (Ts) GR (Ts) S (Bzl) IQD (Obz) -LR (Ts) R (Ts) E (Obz) LLE (Obzl) R (Ts) LLK (Fmoc) R (Ts) LH (Bom) T (Bzl) A- O-PAM was synthesized on a scale of 035 mmol All Nl groups were protected with t-butoxycarbonyl (Boc), the side chain protecting groups were as indicated. Upon completion of the synthesis, the peptide resin was treated with 50 ml of 20% pipepdine in dimethylformamide (DMF) at room temperature for 30 minutes to remove the fluorenylmethoxycarbonyl (Fmoc) protecting group in lysine The resin was washed successively with DMF, MeOH, CH 2 Cl 2 and dried to give 16 g of partially protected peptide 08 g (0175 mmol) of the partially protected peptide acylated in lysine with 044 g (03 mmol) of methox? D? (Et? Lenox?) Acet? Co [PEG (2) CH2COOH] in the presence of 016 g (03 mmol) of benzotr? Azole? Ox? -tr? S hexafluorophosphate ( pyrrolidone) phosphono (PyBop) 'and 0067 g (0525 mmol) of dnsopropylethyl amine (DIEA) in 20 ml of DMF at room temperature for 5 h. After 5 h, the resin was filtered and washed successively with DMF, MeOH and CH2Cl2. The acylation step was repeated twice until a negative ninhydrin result was obtained in the resin. The final peptide was cut from the resin with removal of the side chain protecting groups and purification as in Example I; 100 mg of Compound 19 were obtained.
Compound 19 AVSEHOLLHDRGRSIODLRRRELL? RLLKRLHTA-OH (SEO ID NO: 18!
CH30 (CH: CH, 0), CH, C = 0 Ala Val Ser Gl? Kis Glr: Leu Leu Kis Aep Arg Glv Arg Ser Lie 1 5 10"'15
Gir. Aep Leu Arg? Rg Arg Giu Leu Leu Glu Are Leu Leu - o o c
Lys (COCH; PEG2, Arg Leu Kis Tr.r Wing CHE? Q LD NO: 13) 30 Physical data:
p.f 145- 195 ° C íct z > 15 -44.6C '(c 0.2, H20) FAB (Clg3H3l6NM0) -. [M + HG 4276.2 AAA: Asp, 2.1 (2 ''; Ci, 5.0 (5,, Ser, 1.6 (2); Kis 2.9 (3); L - "_ v 0.9 (1); Thr, lL (2:; Arg, 7.1 (7;. Val, 1.1 (1); lio, 1.0 (1); Leu, 3.0 .. S), - Lys, 0.9 (1).
EXAMVII The peptide was synthesized, cut and purified in the same manner as in ExamIV, except that 2-methoxy-poly (ethylene-oxy) acid, co [PEG (5000) CH2CO2H] was used as the acetylated 08 g (0.175 mmol) of partially protected peptide yielded 300 mg of pure Compound 20 Compound 20
AVSEHOLLHDRGRSIODLRRRELLERLLKRLHT -OK (SEO CH30 (CH, CH, 0) p0CH, C = O Ala Val Ser Glu Kis Glp Leu Leu His Asp Arg Giy Arg Ser 1 5 10 G "in A.sp Leu Arg Ars Arg Glu Leu Leu Glu Arg Leu Leu 20 25 Lys (COCH, P? G5000) Arg Leu His Thr Wing OH (SEQ ID NO: 15- .30 Physical Data: pf 105 ° C [ar] Da -22.95 ° (c 0.11, 50% aq. )
.AAA: Asp, 2.0 (2); Glu, 4.8 '5); Ser, 1.6 (2); His, 2.6 • '3'; 1.1 (1); Thr, 1.1 (1); Arg, 7.3 (7); Val, 0.8'i); l s 0.9 (1); Leu, 8.3 (8); Lys, 1.1 (1); Ala, 1.8 (2).
EXAMVIII Analog gene synthesis hPTHrp (1-34) A synthetic gene encoding the hPTHrp analogue (1-34) Compound 4 (SEQ ID NO: 9) was designed. The required oligodeoxynucleotides were prepared with a DNA synthesizer (Milligen / Biosearch) using the phosphoramidite process of Sinha, et al., Nucleic Acid Research 12, 4539-4557 (1984). After deprotection, the crude oligonucleotides were purified by gel electrophoresis in 15% pohacrylamide gels. Preparations Oligonucleotides were placed with UV, cut out of the gel, desalted on Waters c18 Sep-pak'5 'cartridges and lyophilized.
Amplification via polymerase chain reaction (PCR) was performed in a Perkin-Elmer Cetus thermal cycle former, with 25 cycles of: 94 ° C for 1 minute, 50 ° C for 2 minutes, and 72 ° C for 3 minutes , using reagents, including Taq polymerase, from the "GeneAmp" DNA amplification set (Perkin-Elmer Cetus). Two overlapping oligonucleotides, one 88mer (2μg), PTH3,
(SEQ ID NO: 31): CCTCTAGATC TCCGCGGCGC TAGC ATG GCT GTT TCT GAA CAT CAG 45 Met Wing Val Ser Glu His Gin i 5 CTG CTT CAT GAC AAA GCT AAA TCG ATT CAA GAT CTG AGA CGT C 88 Leu Leu Hie Aep Lys Giy Lys Ser He Glr. Asp Leu Arg Aro 10 * 15 * 20
and an anti-sense 90mer, 2 μg, PTH4 (SEQ ID NO 32): CCTCGAA.GCT TATGCATCAT TATCTAGA CAT AGT ATG CAG CTT TTC 46 30 AAG CAG TTT CTC CAG CAG CTC GCG ACG TCT CAG ATC TTG AAT 88 Leu Leu Lys Glu Leu Leu Glu Arg Arg r Leu Ase 31r. He 2 5 2 0"" 15 CG 90,
were prepared as the template DNA sequence for the hPTH analog gene rp (1-34). Using the two flanking initiators. PTH PCR 1: CCTCTAGATC TCCGCGGCGC TAG (SEQ I D NO 33) and PTH PCR2: CCTCGAAGCT TATGCATCAT TATC (SEQ I D NO 34), the whole gene was amplified by PCR. The amplified DNA products were purified by gel electrophoresis on NuSieve® 4% agarose gel. The band containing the synthetic hPTH rp (1-34) synthetic gene, approximately 1 50 bases in length, was cut from the gel and approximately 200 ng of DNA were isolated by DNA extraction on Elu-Quik® glass gel (Schleicher &Schuell, Keene, NH).
EXAMIX Molecular Cloning of an Analog Aene hPTHrp (1-34) 1 To subclone the analogous gene hPTHrp (1-34) of ExamVI, 200 ng of the amplified DNA was isolated and corroded by the restriction enzymes HinD II and Sac I I The DNA was ligated to 2 μg of plasmid Tr1 8 Prot (lie3, Pro5) previously cut with Hind III and Sac I I. The resulting plasmid, Tr18 hPTHrp (1-34) 1, containing a copy of the analogous gene hPTHrp (1-34) was then transformed into competent E. coli HB 101 cells (CLONTECH, Palo Alto, CA). The transformants were subjected to PCR analysis to verify the insertion.
Colonies of transformed cells were selected and boiled in
200 μl of water for 5 minutes; 2 μl were subjected to PCR with two primers flanking the insertion. The PCR product was then analyzed on a 1% agarose gel to confirm the presence of a copy of the hPTHrp / 1 -34 gene insert. The TrpLE 18 hPTHrp (1-34) 1 construct was then verified by DNA sequencing in an automatic DNA sequencer (Applied Biosystems Model 373A, Foster
City, CA) using the vendor's Dye Deoxi Terminator Sequencing set.
EXAMPLE X Construction of a Trp LE 18 vector containing multiple copies of the analogous gene hPTHrp (1-34) The unique Nhe I and Xba I restriction sites are located near the start and end of the analog gene sequence hPTHrp (1-34) ). These two sites, which recognize different sequences, but produce identical single filament cohesive terms, allow the construction of the hPTHrp (1-34) genes of multiple copies within the Trp LE 18 vector. In separate reactions, 5 μg of plasmid Trp LE 18 hPTHrp (1-34) 1 containing a single copy of the gene was cut with Bam Hl + Nhe I and Xba I + Bam H l. Of each digestion, about 300 ng of the fragment containing the analogous gene hPTHrp (1-34) were isolated. These two fragments were mixed and ligated to form the plasmid Trp LE 18 h PTHRrp (1-34). This plasmid was used to transform competent B-cell 101 of E. coli. The size of the PCR products transformed on 1% agarose gel was used to determine the presence of two insert copies of the hPTHrp gene (1-34). TrpLE 18 hPTHrp (1-34) 2 containing two copies of the gene was then confirmed by DNA sequencing. The correct fusion of the two hPTHrp genes (1-34) resulted in the elimination of the Nhe I and Xba I sites at the junction. This makes the remaining Xba I and Nhe I sites flanking the unique tandem genes. By repeating this process, the final plasmid Trp Le 18 hPTHrp (1-34) 4 containing four copies of the hPTHrp gene (1-34) is constructed. The sequence of Trp LE 18 hPTHrp (1 -34) 4 was found to be correct by DNA sequence analysis.
EXAMPLE XI Expression and purification of Trp LE 18 hPTHrp 1 -34) 4 Induction of Trp LE 18 hPTHrp (1-34) 4. A starting culture of 50 ml of the culture medium LB, J. H. , "Experiments in Molecular Genetics," p.431 (1972), incorporated herein by reference, containing 50 μg / ml ampicillin and 100 μg / ml tryptophan, was inoculated with E. coli cells containing the plasmid Trp LE hPTHrp (1-34) 4, was grown overnight at 37 ° C with vigorous stirring at an AS50 of about 6. Two liters of LB culture medium for production were pre-warmed to 37 ° C and plated with 20 ml of the starter culture to give an A55o of approximately 0.06. The culture was then grown with vigorous agitation at an A550 of between 0.6 and 0.8, wherein 2 ml of a solution of 10 mg / ml indole acrylic acid (IAA) was added. The growth was continued with good aeration for about 16 h for a final As50 of about 6 (typically, between 4 and 10). The cells were concentrated by centrifugation and resuspended in 500 ml of 50 mM Tris-HCl, pH 7.5, 0. 1 mM EDTA buffer (Tris buffer). The suspension was sonic using a Model 220F sonicator from Heat Systems-Ultrasonics, Inc. (equipped with a 1 .9 cm cornet) operated at 50% of the total capacity to avoid overheating. To determine the degree of induction, whole cells were analyzed by SDS-PAGE. The gene products derived from the TrpLE 1 8 hPTHrp (1-34) 4 construct were viewed as a major band of the predicted molecular weight of approximately 17,000. This is considered as much as 10% of the total cellular protein.
Isolation of the fusion protein. The cell lysate was centrifuged for 15 min. to approximately
3600 x g to form the pellet of the fusion protein Trp LE 18 hPTHrp (1-34) 4; the supernatant was discarded. The pellet was resuspended in 200 ml of Tris buffer, (typically 40-80 A5so / ml).
EXAMPLE XII Processing of the fusion protein and purification of peptide hPTHrp (1-34) of homo-Serlactone The cleavage of the methionine residues flanking the multimeric fusion protein hPTHrp (1-34) with CN Br releases the hPTH polypeptide rp (1-34) of desired homo-Serlactone, which was purified as described below.
CNBr treatment of the fusion protein. The washed pellet of TrpLE 18 hPTHrp (1 -34) 4 fusion protein was resuspended by gently swirling in 60 ml of 70% formic acid (approximately 20 mg / ml total protein, usually material of 1000 A55 or units is dissolved of cells in 3 ml). A few drops of octanol were added and N2 was bubbled through the solution for 20 minutes before adding 5.5 g of CN Br. This reaction was allowed to proceed for 6 hours at 25 ° C before an equal volume of 50.50 MeOH H2O will be mixed with the sample and subsequently removed by rotary evaporation. After 2 to 4 repetitions of this process, the aggregate of formic acid and CNBr were essentially removed. The sample was then evaporated to dryness, redissolved in 200 ml of water and it was lyophilized for storage
Purification of hPTHrp (1-34) of homo-Serlactone. The cut-off CNBr supernatant was dialyzed against 50 mM KH2PO4 pH 6 5 for 24 hours with multiple changes During dialysis, the pH was maintained at 6 5 After dialysis, the precipitates were removed by high speed centrifugation. The supernatant was clarified through a 0 4μ Gelman filter device (Acrodisc 4184)
Chromatography of cation exchange. The initial purification was completed by cation exchange chromatography on a column of H PLC Bio-Gel TSK-SP-5PW
(21 5 x 150 mm) The chromatographic conditions for a flow rate 3 ml / min and a production of approximately 12 mg of peptide hPTHRrp (1-34) of highly purified homo-Serlactone were 1 column equilibrium in 50 mM KH2PO4 , pH 6 5 2 Clarified supernatant of 10 ml of loading (approximately 1 5 l of culture broth or 2 4 g of inclusion) 3. Wash column with 50 mM of KH2PO4 pH 6.5 containing 50 mM of NaCl until the line of base stabilizes. 4. Extraction column with 50 mM KH2PO4 pH 6.5 containing 90 mM NaCl. Fractions collected for approximately 45 minutes. 5. Analyze the 90 mM NaCl fractions for the hPTHrp (1-34) content of homo-Serlactone by C18 H PLC before extracting and storing.
HPLC reverse phase chromatography. A column of 4.6 x 100 mm Poros R / H reverse phase (Perseptive biosystems, Cambridge, MA) was used for the final purification step. The chromatographic conditions were as follows: Mobile phase A: 0. 1% trifluoroacetic acid (TFA) / water B: 0.1% trifluoroacetic acid (TFA) / CH3CN
TI EMPO FLOW% B 0 min 4 ml / min 15 5.0 min 4 ml / min 40 5.2 min 4 ml / min 100 6.8 min 4 ml / min 100 7.0 min 4 ml / min 1 5
Retention time of hPTHrp (1-34) of homo-Serlactone, Compound 4, was approximately 2943 minutes. The purified peptide was approximately 98% pure as determined by mass spectroscopy.
EXAMPLE XIII The compounds of this invention were evaluated for their effect on bone mass in ovariectomized rats, generally in accordance with the procedures of Gunness-Hey and Hock, Metab. Bone Dis. 5: 177-181 (1984). Adult female Sprague-Dawley rats were acclimatized, grouped by weight (n = 9, 10 or 12), and underwent bilateral ovariectomy (OVX) or surrogate surgery. Dosing was started 17 days after surgery and continued for 20 days. The test compound was administered subcutaneously once a day in 2% rat serum / saline vehicle. After 20 days of dosing, the rats were sacrificed and the right femurs excised. The femurs were cut in half and the femurs of the distal half (DH F) were further separated into trabecular bone (TB) and cortical bone (CB) when perforating the trabecula. Calcium was extracted and measured by Calcette calcium analyzer and expressed as Ca of the main bone in mg / DHF / 100 g of body weight. The two sample t tests were used to compare the OVX and substitute groups. A one-way ANOVA was used to compare OVX groups, followed by multiple comparison of Fisher's LSD to compare each treatment group with the vehicle.
Ovariectomy induced loss of substantial total bone, mainly from trabecular bone. Total bone calcium was 47 to 54% lower than for the substitute operation controls. bPTH (1-34) and hPTHrp (1-34) at 80 μg / kg / day provided statistically significant increases in total bone calcium for treated OVX rats, ranging from 53 to 95% and 18 to 40%, respectively; however, there was no significant increase in cortical bone calcium. The compounds of this invention, dosed at 80 μg / kg / day, increased total bone calcium by from 66 to 138% and trabecular calcium by from 87 to 128%. Calcium from cortical bone, trabecular thickness, and bone volume also increased significantly over untreated OVX controls. In this test, the following compounds were tested:
Number of n Calcium of bone Compound bone calcium (# of tests) trabecular (% of total (% increase over increase over OVX) OVX) Compound 1 (SEQ ID NO: 7) 6 101 -128% 88-138%
Compound 2 (SEQ ID NO: 6) 3 87-128% 66- 1 14%
Compound 4 (SEQ I D NO: 9) 3 - 88-1 14%
In similar studies, ovariectomized rats were dosed druante 5, 10 and 20 days, at 40 ug / kg / day, with the following results:
Number of n # days (d) of Composite bone calcium (# of tests) total dosage (% increase on OVX) Compound 1 (SEQ ID NO: 7) 3 20d 73-109%
Compound 4 (SEQ ID NO: 9) 5 20d 79-105%
Compound 4 (SEQ ID NO: 9) 1 10d 79% Compound 49 (SEQ ID NO: 63) 1 10d 93% Compound 4 (SEQ ID NO: 9) 1 5d 55% Compound 42 (SEQ ID NO: 56) 1 5d 60%
EXAMPLE XIV Mature white New Zealand female rabbits (Hazelton Research Products, l n., 3.6 kg, n = 9-1 1 / treatment group) were injected daily with vehicle or 0.15 mg / kg of prednisone for months. Bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry (Hologic Model QDR-1500W,
Hologic, Inc., Waltham, MA) and biochemical markers of metabolism were followed. After 9 months, the subgroups also received
0. 05, 0.15 or 0.5 ug / kg / day is Compound 1 (Seq ID No. 7). Two months later, the doses were elevated to 0.5, 3 and 10 ug / kg / day. The prednisone treatment resulted in a rapid initial loss (2-4 months) followed by stable BMD for the remainder of the study. The final BMD losses of the baseline of 12.4 + 1.3%, 1 8. 1 ± 1.9% and 11.8 + 2.5% were observed in the A / P view of the lumbar spine and the distal femur . The smallest decrease
(7.8 ± 2.2%) was observed in the femoral diaphysis. The early, smaller doses did not change these figures. After 30-60 days, 10 ug / kg of Compound 1 in animals continuing in BMD recovered from prednisone at all sites at values not significantly different from those seen in control rabbits treated with the vehicle alone for 12 months. Serum osteocalcin fell 6 4 times from a baseline of 50 + 8 ng / ml in rabbits treated with prednisone, but was returned to normal by 10 ug / kg concomitantly with Compound 1
EXAMPLE XV Nasal formulations of a peptide of this invention Compound 1 (SEQ ID NO 7), were prepared and evaluated for efficacy to deliver the compound with high bioavailability Solutions containing 20 mg / ml Compound 1, 2 mg / ml of citric acid, 2 6 mg / ml of sodium citrate, 0 2 mg / ml of benzalkonium chloride, and varying amounts of sorbitol and different intensifiers were prepared in sterile water and the pH was adjusted to 6 0 with 1 N NaOH. Solutions were administered nasally to mono cynomolgus and and the pharmacokinetics were evaluated as described above. The composition of the formulations tested and the results of the tn vivo tests are shown in Table 1, where it can be seen that the formulations of this invention provide bioavailabilities that they are close to 50% of that obtainable by subcutaneous injection and, thus, they can be suitable for clinical use in the treatment of osteoporosis Table 1. PTHrp analogous nasal formulations (SEQ ID NO: 7) Formulation A B C D E F G
Compound SE ID NO 7 20 20 20 20 20 20 20
Citric acid 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Sodium Citrate 2.6 2.6 2.6 2.6 2.6 2.6 2.6
Benzalkonium Chloride 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Sorbitol 30 30 12 - 35 40 35
Methylcellulose 1500 - 2.5 - Propylene Glycol - - 10 50 Dimethyl-β-cyclodextrin - - - - 50 Taurocholate of sodium 10
Sodium glycocolate - - - - - - 1 0
Dosage N Tmax Cmax AUC avg. Percentage of (mg) (hours) (ng / ml) (ng / ml h) average bioavailability
B 4 3 0.3 2.7 1 .7 ± 0.6 0.6 ± 0.2
C 4 6 0.3 2.8 2.7 ± 2.8 1 .0 + 1 .0
D 4 3 0.3 4.0 3.3 + 2.6 1 .2 ± 1 .0
D 1 6 0.4 0.5 1. 1 +2. 1 1 .7 ± 3.0
E 4 8 0.3 40 36 ± 40 13 ± 1 5 F 4 6 0.6 1 19 128 + 48 47 + 18 G 4 6 0.3 1 57 133 + 77 49 ± 29 Subcutaneous injection 0.05 23 0.6 2.0 3.4 + 2.8 100 ± 82 LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Syntex (USA) Inc. (ii) TITLE OF THE INVENTION: PHARMACEUTICAL COMPOSITIONS FOR COMPOSITE NASAL DELIVERY USEFUL FOR THE
TREATMENT OF OSTEOPOROSIS (iii) SEQUENCE NUMBER: 86 (iv) ADDRESS FOR CORRESPONDENCE: (A) DESTINY: Heller Ehrman White & McAuliffe (B) STREET: 525 University Ave. (C) CITY: Palo Alto (D) STATE: CA (D) COUNTRY: EU (E) POSTAL CODE: 94301 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEANS : FLEXIBLE DISK (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PACKAGE: Patent in release # 1.0, version # 1.25 (vi) CURRENT REQUEST DATA: (A) NUMBER REQUEST: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Chow, Y. Ping (B) REGISTRATION NUMBER: 30,740 (C) REFERENCE NUMBER / CASE : 27610-04 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 415-324-7078 (B) TELEFAX: 415-324-0638
(2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL : NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Ser Val Ser Giu lie Gln Leu Met His Asn Leu Gly Lys His Leu
Asn 10
Being Met Giu Arg Val Glu Trp Leu Arg Lys Lys Leu Glr. Asp Val ns 20 25 3o
Asn Phe (2) INFORMATION FOR SEQ ID NO: 2. (0 CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) Linear TOPOLOGY
(?? TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-TERMAL FRAGMENT (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 2 Ala Val SP: l e Gln Phe Met pis Asn Leu Glv Lys His ser I 5 10 LL
Be Met G? ? rg Val Giu Trp Leu Arg Lys Lys Leu Glr. Ase Val 20 25 30
Asn Phe (2) INFORMATION FOR SEQ ID NO 3 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) ) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 Ser Val Glu Lie Gin Leu Met His Asn Leu Giy Lys His Leu
Ser 1 10 15
Being Leu Giu Arg Val Giu Trp Leu Arg Lys Lys Leu Gln Asp Val
Hi 20 25 30
Asn Phe (2) INFORMATION FOR SEQ ID N04 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE amino acid (D) TOPOLOGY linear (??) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE N-terminal FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 4 Ala Val Ser Giu His Gir. 3U Leu Hrs Asp Lys Giy Lys Ser
C-ln i
Asp Leu Arg Arg Arg Pr.e Ppe Leu His His Leu lie Ala His 20 25
Thr Ala (2) INFORMATION FOR SEQ ID NO 5 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) ) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5 Wing Val Ser Glu His Glp Leu Leu His Asp Lys Gly Lys Ser Lie
Gln 1 5 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arg Lys Leu
His 20 25 3C
Thr Ala (2) INFORMATION FOR SEQ ID N06 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-terminal FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 6 Ala Val Ser Glu His Gln Leu Leu His Aso Lys Glv Lvs; m 1
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu L-; His 20 25
Tnr Ala (2) INFORMATION FOR SEQ ID NO 7 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL NO (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7 Wing Val Ser Giu His Gin Leu Leu His ASD Lys Gly Lys Ser lie
Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His 20 25 30
Thr Ala (2) INFORMATION FOR SEQ ID N08 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-terminal FRAGMENT (ix) FEATURE (A) NAME / KEY Modified site (B) LOCATION 34 (D) OTHER INFORMATION / note = "Xaa34 = homosepna" (xi) SEQUENCE DESCRIPTION SEQ ID NO 8 Ala Val Ser Glu His Gin Leu Leu His Ase Lvs Gly Lys Ser lie Gip 1 5 10 15
Asp Leu ^ g Arg Aia Glu Leu Le_ Glu Lvr- Leu Leu 51 _ Cvs Le-His 20 25 30 Thr Xaa
(2) INFORMATION FOR SEQ ID NO 9 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (vi) CHARACTERISTIC: (A) NAME / KEY: modified site (B) LOCATION: 34 (D) OTHER INFORMATION: / note = "Xaa34 = homoserin lactone"! (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO.9 Wing Ser Giu His ST- -. V: Se: Gm
Asp, eu Are .rg GIU_eu _eu Giu úeu ,, eu Glu ivs _ev
Thr Xaa (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) ) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10 Ala Val Ser Giu His Gln Leu Leu Hi -.?z _ys i and Lys? ß.? E
G 1 n Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu is 20 25 30 Thr Wing Gly Arg Arg 35 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (li) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL- NO (v) TYPE OF N-TERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 11 Wing Val Ser Giu His Gin Leu Leu Hie Asp Lys Gly Lys 3rd Lie
J 5 10 15
Ast. Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Read Gl_ I vs Leu 2 c 25; :
Giu Leu (2) INFORMATION FOR SEQ ID NO 12 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-TERMAL FRAGMENT (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 12 Wing Val Se J, a His Gin Leu L- His Asi G ia 13 Asp Leu Wing Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His 20 25 30 Thr Ala (2) INFORMATION FOR SEQ ID NO: 13 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE, peptide (iii) HYPOTHETICAL- NO (v) N-terminal FRAGMENT TYPE (xi) SEQUENCE DESCRIPTION. SEQ ID NO 13 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Se- Lie
ASD eu M: g Arg A? Glu I read, eu Giu Ly: Leu Leu Gl? 'S _e?
'and s 20 25
Thr Ala (2) INFORMATION FOR SEQ ID NO 14 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE amino acid (D) TOPOLOGY linear (li) TYPE OF MOLECULE peptide (lii) HYPOTHETICAL NO (v) TYPE OF N-TERMAL FRAGMENT (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 14 Ala Val Ser Gl_: Ala Gln Leu Leu His Aso Leu Gl G 1 n 1 5 1D Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu 20 25 30
Ala Leu (2) INFORMATION FOR SEQ ID NO.15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) ) HYPOTHETIC- NO (v) N-terminal FRAGMENT TYPE (xi) SEQUENCE DESCRIPTION SEQ ID NO 15 Wing Val Ser Glu His Without Leu Leu H ^ s Aso L Gl "Lv? Gln
Asp Leu? Rg Arg Arg Clu Leu Leu Glu Arg Leu Leu Cl; Arg Le
His ¿J ¿O _} 'J Thr Ala (2) INFORMATION FOR SEQ ID NO 16 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (n) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) ) TYPE OF N-TERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 16 Ala Val Ser Glu His Gm Leu Leu His Aso Arq i. - - Arq S ^ - "Gln '_ - Asp Leu Arg Arg Arg Glu Leu Leu Giu Arg Leu Leu Glu Arg Leu His 20 25 30 Thr Wing (2) INFORMATION FOR SEQ ID NO.17: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY, linear (ii) TYPE OF MOLECULE- peptide (iu) HYPOTHETICAL NO (v) TYPE OF N-TERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 17 Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arr Ser Lie G "¡-
As-3 Leu. Arg Arg Arg Glu Leu Lea Glu Arg Leu Leu H 3 20 25 3L Thr Ala (2) INFORMATION FOR SEQ ID NO 18 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid ( D) linear TOPOLOGY (n) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-TERMAL FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 29 (D) OTHER INFORMATION / note = "Xaa29 = l? S? Na- (OCCH2 (OCH2CH2) 2OCH3)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser He
Glr. 1 5 10 15 Asp Leu Arg Arg Arg Giu Leu Leu Glu. Arg Leu Leu Xaa Arg Leu His 20 25 30
Thr Ala
(2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH- 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY: linear (? ¡) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO ( v) N-terminal FRAGMENT TYPE (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 29 (D) OTHER INFORMATION / note = "Xaa29 = sina- (OCCH2 (OCH2CH2) 110OCH3" (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 19 Wing Val Ser Glu Kis Cir. Leu Leu Hrs Ase Ari Gln Ser Le 1C
Asp Leu Arg Arg Arg. U L6'J íl .Ar His Leu Xaa .10
Thr Ala (2) INFORMATION FOR SEQ ID NO 20 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) ) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE "SEQ ID NO 20 Wing Val Ser Glu His Gln Leu Leu His ASO Lys Giy Lys Yes - _e
Gln
Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala Gi His 20
Thr Ala (2) INFORMATION FOR SEQ ID NO 21 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-THERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 21 Ala Val c, ez Ciu His Glr. Leu Leu His A.sp L s Gly L / s Gln 1 5 10
Asp Leu Arg Arg. Arg Ser Leu Le er be Ser Leu Leu =; His 20 25
Thr Ala (2) INFORMATION FOR SEQ ID NO 22 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) ) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22 Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lye Ser He Gln 1 5 10 15
Asp Leu Arg Arg Arg Wing Phe Tyr Asp Lys Val Wing Glu Lys Leu His 20 25 30
Thr Ala (2) INFORMATION FOR SEQ ID NO 23 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-TERMAL FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION (D) OTHER INFORMATION / note = "Xaa8 = norleucma" (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION (D) OTHER INFORMATION / note = "Xaa18 = norleucma" (xi) SEQUENCE DESCRIPTION SEQ ID NO 23 Ala Val Ser Glu Lie Gin? Pe Xaa His Asr Leu Gly Lys H s ._ =.
Se 1 5 10 L
Ser Xaa Glu Arg Val Clu Leu Leu Ciu Lys Leu Leu Clu Lys Le
His 23 25 30 Asn Tyr (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE. (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) N-terminal TYPE OF FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 8 (D) OTHER INFORMATION / note = "Xaa8 = norleucma" (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 18 (D) OTHER INFORMATION / note = "Xaa18 = norleucine '(xi) SEQUENCE DESCRIPTION SEQ ID NO 24 Aa Val 5c - llu He Glr.Phe Xaa His As :. Leu 31 and Lye FE be 1 5 10
Being Xaa .Arg A ^ Arg Glu Leu Lau Glu Lys Leu .eu Gir
His 20 25 30
Asn Tyr (2) INFORMATION FOR SEQ ID NO 25 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 35 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF N-THERMAL FRAGMENT (xi) DESCRIPTION OF SEQUENCE: SEQ ID N0.25 Wing Val Ser Giu His Gin Leu Leu Hrs Asp Lys Gly Lys Ser He Gin 1 5 10 15
Asp Leu Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His 20 25 30
Thr Met Ala 35 (2) INFORMATION FOR SEQ ID N0: 26: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 10 amino acids (B) TYPE of amino acid (D) helical TOPOLOGY (II) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF INTERNAL FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY region (B) LOCATION 8 (D) OTHER INFORMATION / note = Xaa8 = glutamic acid or arginine "(x) FEATURE (A) NAME / KEY modified site (B) LOCATION 1 10 (D) OTHER INFORMATION / note = "Sequence 26 is embedded in positions 22 to 31 of sequences 5, 6, 7, 8, 9, 10, 11 12 13 and 14" (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 26 Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO 27 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 10 amino acids (B) TYPE of amino acid (D) helical TOPOLOGY (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) TYPE FRAGMENT: internal (ix) CHARACTERISTICS: (A) NAME / KEY: modified site (B) LOCATION: 8 (D) OTHER INFORMATION: / note = "Xaa8 = glutamic acid, lysine, or lysine- (OCCH2PEGX)" (ix) ) CHARACTERISTICS: (A) NAME / KEY: region (B) LOCATION: 1..10 (D) OTHER INFORMATION / note = "Sequence 27 is embedded in positions 22 to 31 of sequences 15, 16, 17, 18 and 19"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 27
Glu Leu Leu Glu Arg Leu Leu Xaa Arq Leu 5?
(2) INFORMATION FOR SEQ ID NO 28 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 10 amino acids (B) TYPE of amino acid (D) helical TOPOLOGY (II) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF FRAGMENT internal (ix) CHARACTERISTICS (A) NAME / KEY peptide (B) LOCATION 1 10 (D) OTHER INFORMATION / note = "Sequence 28 is embedded in positions 22 to 31 of sequence 20 (xi) SEQUENCE DESCRIPTION SEQ ID NO 28
Ala Leu Ala Ciu Ala Leu Ala Clu Ala Leu - 5 _
(2) INFORMATION FOR SEQ ID NO 29 (i) CHARACTERISTICS OF THE SEQUENCE. (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: helical (li) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: internal (ix) CHARACTERISTIC: (A) NAME / KEY: peptide (B) LOCATION: 1..10 (D) OTHER INFORMATION "/ note =" Sequence 29 is embedded in positions 22 to 31 of sequence 21 (xi) SEQUENCE DESCRIPTION. SEQ ID NO 29
SP- > - Leu Leu Ser Ser Leu Leu Ser Ser Leu X "5 10
(2) INFORMATION FOR SEQ ID NO 30 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 10 amino acids (B) TYPE of amino acid (D) helical TOPOLOGY (n) TYPE OF MOLECULE peptide (m) HYPOTHETICAL NO (v) TYPE OF FRAGMENT internal (ix) CHARACTERISTICS (A) NAME / KEY peptide (B) LOCATION 1 10 (D) OTHER INFORMATION. / note = "Sequence 30 is embedded in positions 22 to 31 of sequence 22 (xi) SEQUENCE DESCRIPTION SEQ ID NO 30 Ala Ph-Tyr Asp Lys Val Ala L_u Lys Le1 '
(2) INFORMATION FOR SEQ ID NO 31 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 88 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE-cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION SEQ ID N031
CCTCTAGATC TCCGCGGCGC TACC.ATCC: 5AAC iTCAGCTGCT TC.ATGACAAA 60 GGTAAATCGA TTCAAGATCT GAC.ACGTC
(2) INFORMATION FOR SEQ ID NO 32
(i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 90 base pairs (B) TYPE nucleic acid (C) FILAMENTO simple (D) TOPOLOGÍA lineal
(H) TYPE OF MOLECULE cDNA (ni) HYPOTHETICAL NO (iv) ANTI-SENSE YES (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 32
CCTCGAAGCT TATGCATCAT TAT 60 GCAGCTCGCG ACGTCTCAGA 90
(2) INFORMATION FOR SEQ ID NO 33 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 23 base pairs (B) TYPE nucleic acid (C) FILAMENTO simple (D) TOPOLOGY linear (ii) TYPE OF MOLECULE: cDNA (iii) ) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xí) SEQUENCE DESCRIPTION. SEQ ID NO 33
CCTCTAGATC TCCGGGCGCT AGC 23
(2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH - 24 base pairs j0 (B) TYPE nucleic acid (C) FILAMENTO simple (D) Linear TOPOLOGY (n) TYPE OF MOLECULE cDNA (ni) HYPOTHETICAL NO. 15 (iv) ANTI-SENSE YES (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 34
CCTCGAAGCT I'ATGCATCAT TATC 24
(2) INFORMATION FOR SEQ ID NO 35 0 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein 5 (ni) HYPOTHETICAL NO (v) TYPE OF N-TERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 35 Ala Val Ser Giu He Gir. Pne Leu His Asn "eu Gl Lv Ser Ser Leu Arg Arg Arg Gl? Leu Leu Glu Lys Leu Leu Glu Lys Leu
His 20 25 30 Asn Tyr (2) INFORMATION FOR SEQ ID NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (ix) CHARACTERISTIC: (A) NAME / KEY: modified site (B) LOCATION: 34 (D) OTHER INFORMATION: / note = "Xaa is homoserin "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36 Ala Val Ser Glu He Cir. Phe J ~ z. Hrs As- Leu Glv Lys Hrs Le Ser
Ser Leu Arg Ar Arg Giu 's Leu Leu Glu Lvs
H i s 20 25
Asn Xaa (2) INFORMATION FOR SEQ ID N037: (i) CHARACTERISTICS OF THE SEQUENCE. (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (ix) CHARACTERISTIC: (A) ) NAME / KEY: modified site (B) LOCATION: 34 (D) OTHER INFORMATION: / note = "Xaa is homosepna" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37 Ala Val Ser Glu He Gln Phe Leu His Asr. Lys Gly Lys His Leu
1 5 0 15
^ Be Leu Arg Arg Giu Leu Leu GH Lys Leu Leu Giu Lys Leu 20 25 30
Asn Xaa
(2) INFORMATION FOR SEQ ID NO'38 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO (v) TYPE OF N-thermal FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 38 Ala Val Ser Glu His Gir. Leu Leu Krs Asp Lys Gly Lys Ser He
Gln 10 15
Ase Leu LVÍ _, and? Leu Gl L \ .JG.? U YES. ~ eu 20
rnr Ala (2) INFORMATION FOR SEQ ID NO 39 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (i?) HYPOTHETICAL. NO (v) TYPE OF FRAGMENT: N-terminal Wing Val Ser Giu His Gln Leu Leu His Aso Lvs Giy Lys Se- ~ e Gln 1 5 10 - =
Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Are Leu
His 20 25 30
Thr Ala
(2) INFORMATION FOR SEQ ID NO 40 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 35 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO (v) TYPE OF FRAGMENT N-thermal (xi) SEQUENCE DESCRIPTION SEQ ID NO 40 Ala Val Ser Gia His Gln Leu Leu His ASP Lys Giy Gln i 5 10
Asp Leu Arg Arg Arg Clu Leu Leu Gl i Arg Le-: eu His 20 25 Thr Ala Pro 35 (2) INFORMATION FOR SEQ ID NO 41 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 37 amino acids (B) TYPE of amino acid ( D) Linear TOPOLOGY (n) TYPE OF MOLECULE protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He Gln 1 5 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu
His 20 25 30
Thr Ala Gly Arg Arg 35 (2) INFORMATION FOR SEQ ID N042 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 38 amino acids (B) TYPE 'amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO (v) TYPE OF N-THERMAL FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 38 (D) OTHER INFORMATION / note = 'Xaa is homosepna "(xi) SEQUENCE DESCRIPTION SEQ ID NO 42 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Gln 1 5 10 i:
Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu GH Arg. 'μ ilS 20 25 30
Thr Ala Gly Arg Arg Xaa 35 (2) INFORMATION FOR SEQ ID NO 43 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43 Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He Gln 1 5 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Giu Lvs Leu
His 20 25 30
Thr Tyr (2) INFORMATION FOR SEQ ID NO 44 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO (v) TYPE OF N-THERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 44 Ala Val Ser Glu H s Gln Leu Leu His Asp Lvs Gly Gln 1 5 10
Asp Leu Arg Arg Ara Clu Leu Leu Gi Lvs Leu Leu Giu Lvs Le _ His 20 25 3C
Thr Ala (2) INFORMATION FOR SEQ ID NO 45 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID N045 Wing Val Ser Glu His Gln Leu Leu His Asp Lys Gly Cys Ser He
Gln 1 5 10 15
Asp Leu Arg Arq Arg Giu Leu Leu Glu Lys Leu Leu Glu Lys His 20 25 30
Thr Ala (2) INFORMATION FOR SEQ ID NO 46 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO (v) TYPE OF N-THERMAL FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 13 (D) OTHER INFORMATION / note = "Xaa is Cys (CH-2CONH (CH-2) -2NH (b? Ot? n? l)) "(xi) SEQUENCE DESCRIPTION SEQ ID NO 46 Ala Val Ser Giu His Gin Leu Leu His Aep Lys Gly Xaa Ser 10
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu G__ Ly:
His 20 25 30 Thr Ala (2) INFORMATION FOR SEQ ID NO: 47: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (¡i¡) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (¡x) CHARACTERISTIC: (A) NAME / KEY: modified site (B) LOCATION: 13 (D) OTHER INFORMATION: / note = "Xaa is Lys (7-dimethylamino-2-oxo-2H-1-benoxopyran-4-acetyl) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47 Wing Val Ser Glu His Gln Leu Le z His Asu Lvs Glv Xaa Ser
G:
Asp Leu Arg Arg Arg Gi? Leu L-? C _? hi:
Thr Ala
(2) INFORMATION FOR SEQ ID NO: 48 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL- NO (v) TYPE OF N-TERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO: 48 Val Val Ser Clu His Gln Leu Leu H: .s Asp Lys 3iy Ly.
Gln 1 5 10 Asp Leu Arg Arg Arg C-lu Leu Leu Ciu Lys Leu Leu Giu Lv
His V. eu 20 25
Thr Ala Gly 35 (2) INFORMATION FOR SEQ ID NO: 49"(i) CHARACTERISTICS OF THE SEQUENCE '(A) LENGTH- 33 amino acids (B) TYPE: amino acid (D) TOPOLOGY" linear (ii) TYPE OF MOLECULE- protein (iii) HYPOTHETICAL- NO (v) N-terminal FRAGMENT TYPE (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 4 (D) OTHER INFORMATION / note = "Xaa4 is Glu (OCH-3)" (ix) CHARACTERISTICS (A) NAME / KEY modified site (B) LOCATION 10 (D) OTHER INFORMATION / note = "Xaa10 is Asp (OCH-3)" (ix) FEATURE (A) NAME / KEY modified site (B) LOCATION 17 (D) OTHER INFORMATION / note = "Xaa17 is Asp (OCH-3)" (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 22 (D) OTHER INFORMATION / note = "Xaa22 is Glu ( OCH-3) "(ix) CHARACTERISTICS (A) NAME / KEY modified site (B) LOCATION 25 (D) OTHER INFORMATION / note =" Xaa25 is Glu (OCH-3) "(ix) FEATURE (A) NAME / KEY modified site (B) LOCATION 29 (D) OTHER INFORMATION / note = "Xaa2 9 is Glu (OCH-3) "(xi) SEQUENCE DESCRIPTION: SEQ ID NO.49 Ala Val Ser Xaa His Gln Leu Leu His Xaa Lys Gly Lys Ser: le
Gln 1 5 10 li
Xaa Leu Arg Arg Arg Xaa Leu Leu Xaa Lys Leu Leu Xaa Lys Leu
His 20 25 30
Wing (2) INFORMATION FOR SEQ ID NO: 50: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 33 amino acids (B) TYPE amino acid (D) TOPOLOGY linear (n) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO (v) TYPE OF N-THERMAL FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 4 (D) OTHER INFORMATION / note = "Xaa4 is Glu (OCH-3)" (ix) FEATURE (A) NAME / KEY modified site (B) LOCATION 10 (D) OTHER INFORMATION / note = "Xaa10 is Asp (OCH-3)" (ix) CHARACTERISTICS (A) NAME / KEY modified site (B) LOCATION 17 (D) OTHER INFORMATION / note = "Xaa17 is Asp (OCH-3)" (ix) CHARACTERISTICS (A) NAME / KEY modified site (B) LOCATION 22 (D) OTHER INFORMATION / note = 'Xaa22 is Glu (OCH-3) "(ix) FEATURE (A) NAME / KEY modified site (B) LOCATION 25 (D) OTHER INFORMATION: / note = "Xaa25 is Glu (OCH-3)" (ix) FEATURE: (A) NAME / KEY: modified site (B) LOCATION : 29 (D) OTHER INFORMATION: / note = "Xaa29 is Glu (OC) H-3) "(xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 50 Wing Val Ser Xaa His Gln Leu Leu His Xaa Lys Gly Lys Ser He inn 10
25 30
Wing (2) INFORMATION FOR SEQ ID N051 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO (v) TYPE OF FRAGMENT N-thermal (ix) CHARACTERISTICS (A) NAME / KEY modified site (B) LOCATION 34 (D) OTHER INFORMATION / note = "Xaa is homosepna" (xi) SEQUENCE DESCRIPTION SEQ ID NO 51 Ala Val Ser Giu His Gln Leu Leu His Aso Lvs Giv Lys Ser _ i < _
1 5 1
As: ßu Arg Arg Are L-Í: i-.e. Glu Lys Leu Leu G HlJ 25
Thr Xaa (2) INFORMATION FOR SEQ ID NO 52 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52 Wing Val Ser Glu His Gin Leu Leu His Asp Lys Gly Lys Ser He Gin 1 5 10 15
Asp Leu Arg Arg Arg Lu Leu Leu Glu Lys Le jeu Gl _eu nis 20 25
Thr Ala Pro 35 (2) INFORMATION FOR SEQ ID NO: 53 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY, linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL NO (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE SEQ ID NO: 53 Wing Val Ser Gi.? Kis Glr. Leu Leu His Asp Lys Ciy Gln
Asp Leu Arg Arg Ar Giu Leu i.o Ciu Lys _- _ ^ u Kis z > n 25
Thr Pro (2) INFORMATION FOR SEQ ID NO: 54: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i¡) TYPE OF MOLECULE: protein ( iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54 Wing Val Ser Glu His Gln Leu Leu His Asp Lys Glv Lys Ser He Gln 1 5 10 15
Asp Leu Arg Ars Ara Giu Leu Leu Glu Lys Leu Leu Glu Lys Leu His 20 25 30 Thr Pro (2) INFORMATION FOR SEQ ID NO: 55 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 33 amino acids (B) TYPE: amino acid (D) TOPOLOGY- linear (ii) TYPE OF MOLECULE- protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT- N-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID N055 Wing Val Ser Giu His Gln Leu Leu Hie Asp Lys Gly Lys Ser He
Gln 10 15
Asp Leu Arg Arg Arg Glu LÍ e Giu Lys Leu Leu Giu His 20
Pro (2) INFORMATION FOR SEQ ID NO. 56: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL : NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser H e Gln 1 5 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu Pro 20 25 30
(2) IN FORMATION FOR SEQ ID NO 57 (i) CHARACTERISTICS OF THE SECU ENCIA (A) LENGTH 37 amino acids (B) TI PO amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (ni) HI POTÉTICA NO (v) ) N-terminal FRAGMENT TYPE (xi) SEQUENCE DESCRIPTION SEQ IDNO 57 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lye Ser He
Gln 10 15
Asp Leu Arg Arg Arg Giu Leu Leu Gl u Lys Leu Leu Gi u Lys Le \
His 20 25 3 0
Thr Arg Ser Ala Trp 35 (2) IN FORMAC TION FOR SEQ ID NO 58 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : protein (iií) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58 Ala Val. Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser He
Gln 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu
His 20 25 30
Thr Ala Gly Arg Arg Thr Arg Ser Ala Tro 35 40 (2) INFORMATION FOR SEQ ID N0.59: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59 Wing Val Ser Glu Kis Gln Leu Leu His Asp Arg Giy Arg Ser I have
Gln 10
Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu His 20 25 30 Thr Arg Gly Arg Arg Thr rg Ser Wing Trp 35"" (2) INFORMATION FOR SEQ ID NO: 60: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 42 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (ix) CHARACTERISTIC: ( A) NAME / KEY: modified site (B) LOCATION: 13 (D) OTHER INFORMATION: / note = "Xaa13 is Lys (dihydrocinnamoyl)" (xi) SEQUENCE DESCRITION: SEQ ID NO 60 Ala Val Ser Glu His Gin Leu Leu His ASD Arg Gly Xaa Ser He
Gln 10
Asp Leu Arg Arg Arg Glu Leu Leu Ciu Aig Leu Leu His 20 25
Thr Arg Gly Arg Arg Thr Arg Ser Wing 35 40 (2) IN FORMATION FOR SEQ ID NO 61 (i) CHARACTERISTICS OF THE SEQUENCE (A) LONGITU D. 34 amino acids (B) TI PO: amino acid (D) Linear TOPOLOGY ( li) MOLECY TYPE THE PROTEIN (iii) HYPOTHETICAL: NO (v) N-THERMAL FRAGMENT TI PO (ix) CHARACTERISTIC (A) NAME / KEY- modified site (B) U BICAC ION 8 (D) ANOTHER IN TRAINING / note = "Leu8 is Norleucma" (ix) STIC CHARACTERISTIC. (A) NAME / KEY modified site (B) LOCATION. 18 (D) OTHER INFORMATION: / note = "Leu18 is Norleucine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO.61 Wing Val Ser Glu He Gln Phe Leu His Asn Leu Gly Lys His Leu
Ser 10 15
Being Leu Thr Arg Being Wing Trp Leu Arg Lys Lys Leu Glñ Asp Val
His 20 25 30
Asn Tyr (2) INFORMATION FOR SEQ ID NO: 62:
(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(i¡) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62 Wing Val Ser Glu His Glr. Leu Leu Kis Asr Lvs ivs Ser ^ _e Gln
Asp Leu Arg Arg Arg Glu Leu Glu Lys Leu
His 20 or 30
Thr Met Ala 35 (2) INFORMATION FOR SEQ ID NO: 63: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (ix) CHARACTERISTIC: (A) NAME / KEY: modified site (B) LOCATION: 33 (D) OTHER INFORMATION: / note = "Xaa is Thr 1 , 4-diaminobutyryl lactam "(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He
Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu
His 20 25 30
Xaa
(2) INFORMATION FOR SEQ ID NO: 64: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL : NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64 Wing Val Ser Glu His Gln Leu Leu His Asp Lys Giy Lys Ser He
Glr. 1 5 10 - = Asp Leu Arg Arg Arg Phe Phe Leu Glu Lys Leu Leu Clu Lys Le \
His 20 25 30
Thr Ala
(2) INFORMATION FOR SEQ ID NO: 65: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL : NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65 Wing Val Ser Glu His Gln Leu Leu His Aso Lys Gly Lys Ser He Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu His Lys Leu Leu Glu Lys Leu His 20 25 30
Thr Ala
(2) INFORMATION FOR SEQ ID NO: 66: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (li) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL- NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66 Wing Val Ser Ciu His Cln Leu Leu K_.s Ase Lys Gly Lvs S ^ Lie Gin 1 5 10 11 Asr Leu Arg Ar :? rg .Lu Leu Leu C_ H-¿L u Lee i_ Lvs Le. is 20? - 30
T.lr
(2) INFORMATION FOR SEQ ID NO: 67 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 34 amino acids (B) TYPE, amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67 Wing Val Ser Giu His Gln Leu Leu His Asp Lys Gly Lys Ser lie
Gln 1 5 10 J-3 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu He Ala Lys?, Eu
Thr Ala
(2) INFORMATION FOR SEQ ID NO: 68: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ii) HYPOTHETICAL : NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION SEQ ID NO 68 Ala Val Ser Glu His Gin Leu Leu His Asp Lys Gly Lys Ser He
Gln 1 5 10 Asp Leu Arg. Arg Arg His
Thr Ala
(2) INFORMATION FOR SEQ ID NO.69 '(i) CHARACTERISTICS OF THE SEQUENCE. (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He
Gln 1 5 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu
His 20 25 30
Thr Xaa (2) INFORMATION FOR SEQ ID NO: 70: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULA protein (lii) HYPOTHETICAL . NO (v) TYPE OF FRAGMENT: N-terminal (ix) CHARACTERISTIC: (A) NAME / KEY: modified site (B) LOCATION: 34 (D) OTHER INFORMATION: / note = "Xaa is omosepna" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 70 Ala Val Ser Clu Kis Clr. Leu Leu His Asp ~ ys Giy Lye Gln
"&?,
Thr Xaa (2) INFORMATION FOR SEQ ID N0: 71: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: both (ii) TYPE OF MOLECULE: protein (! ii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO.71 Wing Val Ser Ciu His Gin Leu Leu His Asp Lys Gly Lys Se:
Gln 1 5 10 1:
Asp Leu Arg Gl \ J ^ J.Lv LVS .eu
T r Ala (2) INFORMATION FOR SEQ ID NO: 72: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 amino acids (B) TYPE: amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE, protein (iii) ) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 72 Wing Val Ser Glu Hr Cir. Leu Leu K's Asp Lys Giy 10
Asp Leu Arg Arg. Arg Glu Leu Leu Glu Lys Leu Leu "> r 75
Thr Arg Ser (2) INFORMATION FOR SEQ ID NO: 73: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (¡x) CHARACTERISTIC: (A) NAME / KEY- modified site (B) LOCATION: 36 (D) OTHER INFORMATION- / note = "Xaa is Ala 3- (2-naphthyl) -L-alanine "(xi) SEQUENCE DESCRIPTION SEQ ID NO 73 Ala Val Ser Güu His Gln Leu Leu His Asp Lys Gly Lys Ser m
sp Leu .Arg Í-. r j .K. His
Thr Arg Ser Xaa 35 (2) INFORMATION FOR SEQ ID NO 74 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 37 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (II) TYPE OF MOLECULE protein (ni) HYPOTHETICAL NO ( v) TYPE OF N-THERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 74 Wing Val Ser Glu His Gin Leu Leu ihs fisv Lvs Gl-- Lv.
Gln 1 5 10 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu is 20 25 30 Thr Wing Being Wing Trp 35 (2) INFORMATION FOR SEQ ID NO: 75: (i) CHARACTERISTICS OF THE SEQUENCE: (A ) LENGTH: 38 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF SEQUENCE SEQ ID NO 75 Ala Val Ser Clu His Cln Leu Leu His Ase Lvs Giy Lys -Ler Lie Cin
Asp Leu Arg Arg Ar! ^ U JT Lvs Leu Leu Gie Lvs Le
.iis 3? z
Thr Ala Giu He Ara Ala 35 (2) INFORMATION FOR SEQ ID NO 76. (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 37 amino acids (B) TYPE: amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE: protein ( iii) HYPOTHETICAL: NO (v) TYPE OF N-THERMAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO 76 To Val Ser G_ u Hrs Gin Leu Leu His Ase: / s G] - "Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu
His 20 25 30
Thr Ala Glu He Arg 35 (2) INFORMATION FOR SEQ ID NO: 77: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77 Wing Val Ser Giu His Sin Leu Leu Kis Asp Lys Gly Lys Ser Glr. 1 5 10:
Asp Leu Arg Arg Arg Glu Leu Leu Ciu Lys Leu Leu Ciu Lys Leu His 20 25 30 go to the Gi r Lie 35 (2) INFORMATION FOR SEQ ID NO: 78: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH : 35 amino acids (B) TYPE: amino acid (D) TOPOLOGY, linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: N-terminal (xi) SEQUENCE DESCRIPTION SEQ ID NO: 78 Ala Val Ser Glu His Gln Lee Leu His Asp Lys Giy Lys Ser He
Gin i S i r. 15 Asp Leu Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu is 20 25 30
Thr Ala Glu 35 (2) INFORMATION FOR SEQ ID NO: 97 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (? ¡) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL- NO (v) TYPE OF FRAGMENT: N-terminal (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 8 (D) OTHER INFORMATION / note = "Leu8 is Norleucma" (ix) FEATURE (A) NAME / KEY- modified site (B) LOCATION 18 (D) OTHER INFORMATION / note = "Leu18 is Norleucma" (ix) FEATURE (A) NAME / KEY modified site (B) LOCATION 34 (D) OTHER INFORMATION / note = "Xaa is homosepna lactone" (xi) SEQUENCE DESCRIPTION SEQ ID NO 79 Ala Va. Ser Clu He 3 Go. Phe Leu Hie Asn Lys Gi-- Lys Ser 1 5 1C
er Leu Glu Arg Va] Glu Trp Leu Arg Lys Lys Leu Gln .Asr V;
Kr s' ^ 25 3 Asn Xaa
(2) INFORMATION FOR SEQ ID NO 80 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) ) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80 Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser He Gln Asp
Le 1 5 10 15
Arg Arg Arg Glu Leu Leu Glu Lye Leu Leu Glu Lys Leu Kis Thr
Wing 20 25
(2) INFORMATION FOR SEQ ID NO: 81 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO.81 Leu Leu His SO Lys Giy Lvs Ser He Glr. Aszz. Leu Aruj .-. R;
Lr / s Leu Leu Glu Lys Leu His Thr Aid 20"25
(2) INFORMATION FOR SEQ ID NO 82: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82 Leu His Asp Lys Gly Lys Ser He Gln Asp Leu Arg Arg Arg Glu
Leu 1 5 10 15
Leu Glu Lys Leu Leu Glu Lys Leu His Thr Wing 20 25
(2) INFORMATION FOR SEQ ID NO: 83: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH 41 amino acids (B) TYPE: amino acid (D) TOPOLOGY linear (n) TYPE OF MOLECULE 'protein (in) HYPOTHETICAL: NO (v) TYPE OF INTERNAL FRAGMENT (xi) SEQUENCE DESCRIPTION SEQ ID NO. 83 Ser Giu His Glr. Leu Leu His Asp Arg Gly Arg Be He Glr. Asp Le 5 10 Arg Arg Arg Glu Leu Leu Ax Leu e .Are- Leu 20 3
Arg Gly Arg Arg Thr Arg Ser Wing Trp
(2) INFORMATION FOR SEQ ID NO 84 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 35 amino acids (B) TYPE of amino acid (D) TOPOLOGY linear (ii) TYPE OF MOLECULE- protein (i? I) HYPOTHETICAL: NO (v) ) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84 Leu Leu His Asp Arg Gly Arg Ser He Gln Asp Leu Arg Arg Arg
Glu 10 15
Leu Leu Glu Arg Leu Leu Glu Arg Leu His Wing Gly Arg Arg T:
Arg 20 25 30
Ser Ala Trp 35 '(2) INFORMATION FOR SbU ID NO 85 (1) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 10 amino acids (B) TYPE of amino acid (D) helical TOPOLOGY (ii) TYPE OF MOLECULE peptide (111) HYPOTHETICAL NO ( v) TYPE OF INTERNAL FRAGMENT (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 1 and 4 (D) OTHER INFORMATION / note = "Xaa1 and Xaa4 = Glu, Glu (OCH3), His, or Phe" (ix) CHARACTERISTICS (A) NAME / KEY modified site (B) LOCATION 2 (D) OTHER INFORMATION / note = "Xaa2 = Leu or Phe" (ix) FEATURE (A) NAME / KEY modified site (B) LOCATION 5 ( D) OTHER INFORMATION / note = 'Xaa5 = Lys or His "(? X) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 7 and 10 (D) OTHER INFORMATION / note =" Xaa7 and Xaa10 = Leu or lie "(ix) FEATURE: (A) NAME / KEY, modified site (B) LOCATION: 8 (D) OTHER INFORMATION: / note =" Xaa8 = Wing, Arg or Glu "
(ix) FEATURE: (A) NAME / KEY: modified site (B) LOCATION: 9 (D) OTHER INFORMATION: / note = "Xaa9 = Lys or Glu"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85
Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO 86 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH 10 amino acids (B) TYPE of amino acid (D) helical TOPOLOGY (n) TYPE OF MOLECULE peptide (ni) HYPOTHETICAL NO (v) TYPE OF FRAGMENT internal (ix) CHARACTERISTICS (A) NAME / KEY modified site (B) LOCATION 1 and 4 (D) OTHER INFORMATION / note = "Xaa1 and Xaa4 = Glu, Glu (OCH3), His, or Phe" (ix) FEATURE ( A) NAME / KEY modified site (B) LOCATION 2 (D) OTHER INFORMATION / note = "Xaa2 = Leu or Phe" (ix) CHARACTERISTIC (A) NAME / KEY modified site (B) LOCATION 8 (D) OTHER INFORMATION / note = "Xaad = Glu, Lys or Lys (COCH2PEGX)" (xi) SEQUENCE DESCRIPTION SEQ ID NO 86 Xaa Xaa Leu Xaa Arg Leu Leu Xaa Arg Leu 1 5 10
Claims (10)
1 . A pharmaceutical composition for the nasal delivery of compounds useful for treating osteoporosis, comprising an effective amount of a truncated physiologically active analog of PTH or PTHrp, or salt thereof, in which the amino acid residues (22-31) form a amphipathic a-helix, said residues (22-31) selected from (SEQ ID NOS: 85, 86, 26, 27, 28, 29 and 30); an absorption enhancer selected from the group consisting of dimethyl-β-cyclodextrin and bile acid surfactants; and water.
2. A composition of claim 1, wherein the bile acid surfactant is selected from the salts of glycolic acid and taurocholic acid.
3. A composition of claim 2, wherein the bile acid surfactant is sodium taurocholate.
4. A composition of claim 2, wherein the bile acid surfactant is sodium glycocholate.
5. A composition of claim 1, wherein the concentration of PTH analog or PTHrp is from 1 mg / ml to about 100 mg / ml.
6. A composition of claim 1, wherein the concentration of absorption enhancer is from about 0.5 to about 50 mg / ml.
7. A composition of claim 1, wherein the PTH analog rp is the compound of (SEQ ID NO: 7).
8. A composition of claim 7, wherein the absorption enhancer is dimethyl-β-cyclodextrin.
9. A composition of claim 7, wherein the absorption enhancer is sodium taurocholate.
10. A composition of claim 7, wherein the absorption enhancer is sodium glycollate. eleven . A pharmaceutical composition comprising from about 1 to about 100 mg / ml of a compound of (SEQ ID NO: 7), from about 0.5 to about 50 mg / ml of an absorption enhancer selected from the group consisting of dimethyl-β-cycle -dextrin, sodium glycocholate, and sodium taurocholate, and water.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/521,097 US5977070A (en) | 1992-07-14 | 1995-08-29 | Pharmaceutical compositions for the nasal delivery of compounds useful for the treatment of osteoporosis |
US08521097 | 1995-08-29 |
Publications (2)
Publication Number | Publication Date |
---|---|
MX9801630A MX9801630A (en) | 1998-08-30 |
MXPA98001630A true MXPA98001630A (en) | 1998-11-12 |
Family
ID=
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