CA2120946A1 - Parathyroid hormone analogues substituted at aa 25.26.27 and use in osteoporosis treatment - Google Patents
Parathyroid hormone analogues substituted at aa 25.26.27 and use in osteoporosis treatmentInfo
- Publication number
- CA2120946A1 CA2120946A1 CA002120946A CA2120946A CA2120946A1 CA 2120946 A1 CA2120946 A1 CA 2120946A1 CA 002120946 A CA002120946 A CA 002120946A CA 2120946 A CA2120946 A CA 2120946A CA 2120946 A1 CA2120946 A1 CA 2120946A1
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- Prior art keywords
- seq
- ala
- ser
- leu
- parathyroid hormone
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
Analogues of bovine and human parathyroid hormone, wherein twenty-fifth, twenty-sixth and twenty-seventh positions of the natural hormone, Arg-Lys-Lys- each have been substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val have been found to retain bone cell effect with minimal effects on blood pressure and smooth muscle, including cardiac muscle. It has further been found that this effect can be obtained by using a synthetic PTH containing only the first 34 amino acids of PTH, with substitution at the twenty-fifth, twenty-sixth and twenty-seventh amino acids as described. These analogues of PTH also are effective in the treatment of osteoporosis and other bone diseases.
Description
~093/06~ PCT/~S92/0~77 P~RATHYROID ~ORMONB ANAL0~UE8 ~U~8TITUTED AT
a25~ 26, 27 AND U9E IN O~TEOPOR0~I8 TREATMENT
21209~6 FIELD OF T~IE INVENTION
This invention relates to analogues of parathyroid hormone which, by substitution at the twenty-fifth, twenty-six and twenty-seventh positions of natural parathyroid hormone, have bee~l foun~ to affect calcium chan~e in bone cells without ~ro~ucillg tlle typical effects of paratl~yroi~ hormone O~l systolic at~d diastolic blood pressure, the effects on smooth muscle relaxation, vascular smooth muscle calcium c11allge as well as positive chronotropic and inotropic effects on t}le heart.
BACKGROUND OF T~IE INVENTION
Parathyroid hormone ~hereinafter, PT~ produced by the parathyroid gland and is involved in tlle control of calcium levels in blood. It is a hypercalcemic hoFmone, elevatillg blood calcium levels. PT~I is a polypeptide and the amino acid sequences of bovine and human PT~ are closely related. Only the residues at l.ocations olle, seven and sixteen differ between t~le two. Synthetic polypeptides containillg t~e first thirty-~our residues of PT~I may be prepared using the method disclose~
~3~ Erickson and Merri.field, l'he ~rotei~s, ~eurat~ et al~, E~ls., ~cademic Press, New York, 1976, page 257, preferably as modi~ied by tile met~lod of l~odg2s et al ., PePtide ~esearcl~, 1 , 19 (lgB8).
Wllen serum calcium is re~uced to below a "normal" level, the parathyroid gland releases PT~3 and re~orptio~l of ~one calcium and increased absorption of calcium from the inte.sti.~le, as well as renal reabsorption o~ calcium, occur.
The antagonist of PTH is calcitonin, which acts to reduce tlle level of circulating calcium. PTII is known to stimulate osteoclasts and its activity requires the presence of W093/06~S 2 1 2 0 9 4 ~ PCT/US92/~77 derivatives of vitamin D3, especially 1,25-dihydroxyc}-olecalciferol.
Intracellular calcium, particularly in the cells of the vascular system, has been shown to affect changes in vascular tension, as can be measured by changes in blood pressure. U.S.
Patent Application 603,745 describes one method which has been discovered to regulate calcium uptake in vascular cells.
Osteoporosis is a progressive disease whicll is particularly characteristic of postmenopausal women, and results in the reduction of total bone mass. T}~e seq~elae frequently involve fractures of load-bearing boneç and the physical degenerations characteristic of immobilizing injuries.
Osteoporosis is associated wit~l }1yperthyroidism, hyperparathyroidism, Cushings syndrome and the use of certain steroidal drugs. Remedies historically have involved increase in dietary calcium, estrogen therapy and increased doses o~
vitamin D.
- PTH has been used to treat osteoporosis. ~iowever, while tll~ use o~ Pl'l~ is ef~ective in the treatment of osteoporosis by diminishing the loss of bone mass, PT~ may exl-ibit otller undesired pharmalogical effects, such as hypotension and smooth m~lscle rel~xation (e.g. relaxation of gastrointestin~l org~ns, u~erus, tracheal and vas deferens) as well as pos3tive chronotropic and inotropic e~`ects on the heart. The 2S relaxation effects of PT~ on smooth muscle as well as positive chronotropic and inotropic effects of PTH are described in Pang et al, I`ren~s in Pharmacoloaical Sciences, Vol. 7, ~o. 9, pp.
3~0-3~1 (September 1986).
U.S. Patent No. 4,771,124 discloses the property of bovine and human M`l~ analogues wherein Trp23 is substituted by amino acids phenylalanine, leucine, norleucine, valine, tyrosine, beta-naphtylalanine and alpha-naphtylalanine as a Pl`H
alltagollist. While it was suggested that these analogues migllt be useful in the treatment of osteoporosis, it was based on the allalogues antagonistic action to PTII. Furthermore, there was no data to in~icate the effectiveness these analogues on bone or other tissue. In addition, analogues with substituted at W093/~ ~ PCT/US92~0~77 Trp23 with leucine, phenylalanine or tyrosine would produce undesired secondary effects of smootll muscle relaxation, vascular smooth muscle calcium change as well as positive chronotropic and inotropic effects on the heart.
Because PTI~ is a peptide, topical administration would be the preferred method of administration. However, topical application of PTH or the aforementioned analogues which exllibit vasoactivity would likely produce an undesired local vascular reaction. This reaction could be potentially detrimental if, for example, nasal administration is employe~.
It is one object of this invention to ameliorate bone loss while preventing smooth muscle relaxation as well as positive chronotropic and inotropic effects 011 the ~leart and without significantly changing blood pressure. It is another object of this invention to identify that portion of PT~3 which is responsible for calcium regulation and that portion which appears to be primarily related to control of blood pressure and smooth-muscle action.
BRIEF SUM~U~RY OF THE INVENTION
..
Modification of either bovine or human PTII at each o~ the twenty-fifth, twenty-sixth and twenty-seventh amino ~cid positions to substitute for -arginine-lysine-lysine- either alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycinej histidine, isoleucine, leucine, 2S methionil-e, phenylalanine, proline, serine, t~lreonine, tryptophan, tyrosine or valine produces substantially no change in systolic and diastolic blood pressure, substantially no change in muscle tension and substantially no change in the rate of contraction and the force of contraction of the heart as compared to native PTH. It also has been observed that th~
P'l'llanalogue containing only the first thirty-four amino acids, with substitution at the twenty-fifth, twenty-sixth and twenty-se~enth positions, is equally effective in the '`osteo effectn without changillg blood pressure or causing muscle relaxation or positive chrono-ropic and inotropic effects on the heart.
:;
W093l0~5 2 1 2 0 9 4 6 PCT/~S9210~, The analogues of the present invention should be effective in ameliorating bone loss while preventing smooth muscle relaxation as well as positive chronotropic and inotropic effects on the heart and without significantly changing blood pressure.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la shows the structure of natural bovine PTH (SEQ ID
~10: 1) . ' Fig. lb shows the structure of natural human PTII (SEQ ID ;
N0:2).
Fig. 2 shows the structure of bPTH (1-34) with each of ~ositions 2s, 26 and 27 substituted with Xaa (SEQ ID N0:3).
Fig. 3 shows the structure of bPTH (1-34) with each o~
positions 25, 26 and 27 substituted with ~la (SEQ ID N0:4).
Fig. ~ shows the structure of hPTI~ (1-34) with each oE
positions 25, 26 and 27 substituted Xaa (SEQ ID N0:5).
Fig. 5 shows the structure of hPTH (1-34) with each of positions 25, 26 and 27 substituted Ala (SEQ ID N0:6).
Fiq. 6 shows the structure of bPTII with each of positions 25, 26 and 27 substituted with Xaa (SEQ ID ~lo:7).
Fig. 7 shows the structure of bPT~ with each of positions 25, 26 and 27 substituted with Ala (SEQ ID N0:8).
Fig. 8 shows the structure of hPT~ with each of positions 25, 26 and 27 substituted with Xaa (SEQ ID N0:9).
a25~ 26, 27 AND U9E IN O~TEOPOR0~I8 TREATMENT
21209~6 FIELD OF T~IE INVENTION
This invention relates to analogues of parathyroid hormone which, by substitution at the twenty-fifth, twenty-six and twenty-seventh positions of natural parathyroid hormone, have bee~l foun~ to affect calcium chan~e in bone cells without ~ro~ucillg tlle typical effects of paratl~yroi~ hormone O~l systolic at~d diastolic blood pressure, the effects on smooth muscle relaxation, vascular smooth muscle calcium c11allge as well as positive chronotropic and inotropic effects on t}le heart.
BACKGROUND OF T~IE INVENTION
Parathyroid hormone ~hereinafter, PT~ produced by the parathyroid gland and is involved in tlle control of calcium levels in blood. It is a hypercalcemic hoFmone, elevatillg blood calcium levels. PT~I is a polypeptide and the amino acid sequences of bovine and human PT~ are closely related. Only the residues at l.ocations olle, seven and sixteen differ between t~le two. Synthetic polypeptides containillg t~e first thirty-~our residues of PT~I may be prepared using the method disclose~
~3~ Erickson and Merri.field, l'he ~rotei~s, ~eurat~ et al~, E~ls., ~cademic Press, New York, 1976, page 257, preferably as modi~ied by tile met~lod of l~odg2s et al ., PePtide ~esearcl~, 1 , 19 (lgB8).
Wllen serum calcium is re~uced to below a "normal" level, the parathyroid gland releases PT~3 and re~orptio~l of ~one calcium and increased absorption of calcium from the inte.sti.~le, as well as renal reabsorption o~ calcium, occur.
The antagonist of PTH is calcitonin, which acts to reduce tlle level of circulating calcium. PTII is known to stimulate osteoclasts and its activity requires the presence of W093/06~S 2 1 2 0 9 4 ~ PCT/US92/~77 derivatives of vitamin D3, especially 1,25-dihydroxyc}-olecalciferol.
Intracellular calcium, particularly in the cells of the vascular system, has been shown to affect changes in vascular tension, as can be measured by changes in blood pressure. U.S.
Patent Application 603,745 describes one method which has been discovered to regulate calcium uptake in vascular cells.
Osteoporosis is a progressive disease whicll is particularly characteristic of postmenopausal women, and results in the reduction of total bone mass. T}~e seq~elae frequently involve fractures of load-bearing boneç and the physical degenerations characteristic of immobilizing injuries.
Osteoporosis is associated wit~l }1yperthyroidism, hyperparathyroidism, Cushings syndrome and the use of certain steroidal drugs. Remedies historically have involved increase in dietary calcium, estrogen therapy and increased doses o~
vitamin D.
- PTH has been used to treat osteoporosis. ~iowever, while tll~ use o~ Pl'l~ is ef~ective in the treatment of osteoporosis by diminishing the loss of bone mass, PT~ may exl-ibit otller undesired pharmalogical effects, such as hypotension and smooth m~lscle rel~xation (e.g. relaxation of gastrointestin~l org~ns, u~erus, tracheal and vas deferens) as well as pos3tive chronotropic and inotropic e~`ects on the heart. The 2S relaxation effects of PT~ on smooth muscle as well as positive chronotropic and inotropic effects of PTH are described in Pang et al, I`ren~s in Pharmacoloaical Sciences, Vol. 7, ~o. 9, pp.
3~0-3~1 (September 1986).
U.S. Patent No. 4,771,124 discloses the property of bovine and human M`l~ analogues wherein Trp23 is substituted by amino acids phenylalanine, leucine, norleucine, valine, tyrosine, beta-naphtylalanine and alpha-naphtylalanine as a Pl`H
alltagollist. While it was suggested that these analogues migllt be useful in the treatment of osteoporosis, it was based on the allalogues antagonistic action to PTII. Furthermore, there was no data to in~icate the effectiveness these analogues on bone or other tissue. In addition, analogues with substituted at W093/~ ~ PCT/US92~0~77 Trp23 with leucine, phenylalanine or tyrosine would produce undesired secondary effects of smootll muscle relaxation, vascular smooth muscle calcium change as well as positive chronotropic and inotropic effects on the heart.
Because PTI~ is a peptide, topical administration would be the preferred method of administration. However, topical application of PTH or the aforementioned analogues which exllibit vasoactivity would likely produce an undesired local vascular reaction. This reaction could be potentially detrimental if, for example, nasal administration is employe~.
It is one object of this invention to ameliorate bone loss while preventing smooth muscle relaxation as well as positive chronotropic and inotropic effects 011 the ~leart and without significantly changing blood pressure. It is another object of this invention to identify that portion of PT~3 which is responsible for calcium regulation and that portion which appears to be primarily related to control of blood pressure and smooth-muscle action.
BRIEF SUM~U~RY OF THE INVENTION
..
Modification of either bovine or human PTII at each o~ the twenty-fifth, twenty-sixth and twenty-seventh amino ~cid positions to substitute for -arginine-lysine-lysine- either alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycinej histidine, isoleucine, leucine, 2S methionil-e, phenylalanine, proline, serine, t~lreonine, tryptophan, tyrosine or valine produces substantially no change in systolic and diastolic blood pressure, substantially no change in muscle tension and substantially no change in the rate of contraction and the force of contraction of the heart as compared to native PTH. It also has been observed that th~
P'l'llanalogue containing only the first thirty-four amino acids, with substitution at the twenty-fifth, twenty-sixth and twenty-se~enth positions, is equally effective in the '`osteo effectn without changillg blood pressure or causing muscle relaxation or positive chrono-ropic and inotropic effects on the heart.
:;
W093l0~5 2 1 2 0 9 4 6 PCT/~S9210~, The analogues of the present invention should be effective in ameliorating bone loss while preventing smooth muscle relaxation as well as positive chronotropic and inotropic effects on the heart and without significantly changing blood pressure.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la shows the structure of natural bovine PTH (SEQ ID
~10: 1) . ' Fig. lb shows the structure of natural human PTII (SEQ ID ;
N0:2).
Fig. 2 shows the structure of bPTH (1-34) with each of ~ositions 2s, 26 and 27 substituted with Xaa (SEQ ID N0:3).
Fig. 3 shows the structure of bPTH (1-34) with each o~
positions 25, 26 and 27 substituted with ~la (SEQ ID N0:4).
Fig. ~ shows the structure of hPTI~ (1-34) with each oE
positions 25, 26 and 27 substituted Xaa (SEQ ID N0:5).
Fig. 5 shows the structure of hPTH (1-34) with each of positions 25, 26 and 27 substituted Ala (SEQ ID N0:6).
Fiq. 6 shows the structure of bPTII with each of positions 25, 26 and 27 substituted with Xaa (SEQ ID ~lo:7).
Fig. 7 shows the structure of bPT~ with each of positions 25, 26 and 27 substituted with Ala (SEQ ID N0:8).
Fig. 8 shows the structure of hPT~ with each of positions 25, 26 and 27 substituted with Xaa (SEQ ID N0:9).
2~ Fig. 9 shows the structure of hPT~i with each of positions 25, 2G and 27 substituted w~th ~la (SEQ ID No:lo~.
Fig. lO shows the effect of bPTH-(1-34) and its analogues on diastolic blood pressure of anesthetized Sprague-Dawley rats.
Fig. 11 shows the effect of bPTH-(1-34) and its analogues on systolic blood pressure of anesthetized Sprague-Dawley rats.
Fig. 12 shows the va50relaxing ef~ect of bPTl1-(1-3~) an~
its analogues on rat tail artery helical strip in vitro.
Fig. 13 shows the depolarizing concentrations of KCl which increased calcium ion levels in cultured osteoblasts. ~r~g 7 is an an~ti-osteoporotic agent which inhibits the KCl effect.
W093/06~5 2 1 2 0 9 ~ ~ PCT/US92/08477 Figs. 14 a-d show tlle depolarizin~ concentrations of KCl which increased calcium levels in cultured osteoblasts.
Addition of bPTH-(1-34) inhibits the KCl effect.
Fig. 15 shows the effect of Cs88 ~bPTH~ 34)~ on the mean 5arterial blood pressure of anesthetized Sprague-Dawley rats.
Fig. 16 shows the dose-response relationship between Cs88 [bPTH-(1-34)] and the tension of rat tail artery helical strips precontracted with KCl, norepinephrine and AVP.
Fig. 17 shows the effect of Cs88 on [Ca2+]i in cultured lOU~ osteoblast cells.
Fig. 18 shows the effect of Cs88 on [Ca2+]i in cultured UMR cells.
Fig. 19 shows a comparison of the effect of Cs221 and cSsg and bPTII on the mean arterial blood pressure of anesthetized 1~Sprague-Dawley rats.
Fig. 20 s~-ows the relation between the relaxation curves of Sprague-Dawley rat tail artery helical strips, precontracted with AVP when treated with CslOO, Cs99, Cs88, Csll7 and Cs221.
Fig. 21 shows ~ com~arison between the effect of Cs221 ~n~
20hPT~I on the intracellular calcium uptake in the presence of KCl in UMR cells in culture.
Fig. 22 shows the effect of Cs221 on the intracellular calcium uptake in tl-e presence of KCl in UMR cells in culture.
Fig. 23 shows the effect of CslO01 on the intracellular 25calcium uptake in the presence of KCl in UMR cells in culture.
Fig. 24 shows the effect of Cs221 on the contractility an~
contractioll rate of right atrial tissue of Sprague-Dawley rats.
Fig. 25 shows the effect of Cs2001 and CslOOl Oll the contractility and contraction rate of right atrial tissue of 30Sprague-Dawley rats.
DETAILE~ DESCRI~TION OE T~E INVSNTION
There are at least two known catagories of functions for PT~I. PTI~ is involved in calcium balance in the blood stream and controls both the amount of calcium uptake from the W093/0~5 PCT~US92/0~77 21209~6 gastrointestinal tract and the deposition and removal of calcium from bone. Calcium also has been found to be effective in the maintenance of blood pressure. Cox, J. Cardiovascu~
Pharmacoloqy, Vol. 8 (1986), Supp. 8 S48. Control of calcium in the walls of blood vessels is a useful therapeutic regimen for controlling hypertension and calcium channel blockers, which prevent the introduction of calcium into cell walls, is a convelltional therapy for hypertensioll. Needleman et al. in Goodman and Gilman's The Pharmacoloaical Basis of Therapeutics, M~cMillan, New York, (1985), page ~lG ff.
Administration of therapeutic doses of PTtl has been found to be effective for the control of osteoporosis, particularly in individuals who have been subjected to thyroidectomies~
parathyroidectomies. Therapeutic dosages of PTH will, in some individuals, result in unacceptable diminution of bloo~
pressure and may result in relaxation of smooth muscles such as gastrointestinal, uterus, tracheal, vas deferens as well as exhibit positive chronotropic and inotropic effects on the ~leart. To avoid hypotensive effects, smooth muscle relaxation effects and positive chronotropic and inotropic ef~ects Oll the heart, it was envisaged that the structure of PTI~ could be modi~ie~ to decouple the hypotensive, smootl- muscle relaxation and positive chronotropic and inotropic function from the bond calcium and bone ~deposition functioll. lt llas now beell discovered that a critical site exi-sts at amino acid twenty-five, twenty-six and twenty-seven, which is -Arg-Lys-Lys- in both bovine and human PTI~. Substitution at ttle -~rg-Lys-Lys-site with -~la-~la-Ala- diminishes the hypotensive as well as smooth muscle relaxation an~ positive chronotropic and inotropic effects without denigrating from the osteo effect.
These results suggest that substitution at the -Arg-Lys-Lys-site with amino acids other than basic amino acids arginine and lysine would also diminish the hypotensive, smooth muscle relaxation and positive chronotropic and inotropic effects 3s without denigrating from the osteo effect.
l~he procedure of Erickson and Merrifield, as modified by llodqes et al., as described above, may ~e used to synthesize , ~ ' W093/06~ PCT/US92/0~77 ~120946 .
synthetic PTil or fragments thereof. The procedure enables substitution for the nàturally occurring PTI~ at substantially every location and it is possible to prepare both bovine and human synthetic PTH at full length or in the sequence of the first thirty-four amino acids, which is more facilely performed. Such substitution can also be accomplished by genetic engineering.
Substitution at position twenty-five, twenty-six and twenty-seven invariably alters the observed hypotensive, smooth muscle relaxation and positive chronotropic an~ inotropic effects, whether the full length PTH or the 1-34 fragment is administered. Substitution of -Ala-Ala-Ala- for -Arg-Lys-Lys-at position twenty-~ive, twenty-six and twenty-seven is particularly preferred because the change in blood pressure, smooth muscle relaxation and positive chronotropic and inotropic effects from this substitution are minimal and calcium uptake, as measured in osteoblasts, mimics the results from the administration of native PT~. The 1-34 PTE{ fragment with -~la25-~la26-~la27- i~ particularly preferred because the pharmacological properties are those which are desired and the difficulty of synthesis is minimized. Synthesis of the compoun~s used in the development of this invention was performed at U berta Peptide Institute (~PI) and t~e cooperation o~ API is gratefully acknowledged.
The structure of bovine parathyroid hormone (bPTI~) and human parathyroid hormone (hPTH) are shown in Figs. la (SEQ ID
No:l) and lb (SEQ ID ~10:2). Representative synthetic analogues are described in Table l and are further shown in Figs. 2-9 and SEQ ID N0:3-SEQ ID N0:10. The hypotensive effects of these analogues is shown in Figs. 10, ll, 15 and l9. All o~ the analogues produce either no or less diminution of blood pressure than does native PTH. With only one amino acid at either the 25, 26 or 27 position substituted, the analogue shows less e~fect than native PTH. With all three positions substituted, it provides almost no change. ~t the level o 5 /kg of r'lll, the blood pressure in Sprague-Dawley rats is 5UCl that they are essentially moribund.
.
`::
W093/~ 2 1 2 U 9 4 6 PCT/US92/0~77 We have developed a method for modeling the hypotensive effects of natural and synthetic chemical compounds using helically cut tail arteries from Sprague-Dawley rats in a Sawyer-8artlestone chamber, measuring the change in tension with a force displacement transducer. This method and the effect of bovine PTH-(1-34) in this system is described in Blood Vessels. 22, 57 (1985). It is demonstrated in this papar - that bPTH-(1-34) produces dose-dependent relaxation of helical strïps of rat tail artery which have been previously contracted by ~rginine-vasopressin (~VP). Figs. 12, 16 and 20 illustrate the effect of the PTI~ analogues of this invention as measured using this in vitro technique. Alternatively, the strips may be precontracted using other pressor ~ubstances such as norepinephrine (NE) or KCl.
We have also developed a method of modeling the chronotropic effects of natural and synthetic chemicals using the right atrium from Sprague-Dawley rats and measuring the change in the force and rate of atrium contraction. lhis met~lod and the effects of bovine PTI~ 34) in this system ~re described in Tenner et al, ~he Canadian Journal of Ph~sioloqv and Pharmacoloqv, Volume 61, No. 10 (1983) pp, 1162-1167. It is demonstrated in this paper that bPTI~ (1-34) produces significant dose-dependent chronotropic effects on rat cardiac pacemaker tissue. Figs. 24-25 illustrate the effect of the PTil analogues of this invention as measured using this in vitro technique.
Decause osteoporosis i5 a progressive syndrome, a model is required and the use of cultured osteoblasts of the UMR-106 rat osteosarcoma cells, ATCC CRL 1661 have been used as the model.
Uptake of calcium in these cells has been monitored using the ~URA-2 method, wherein a fluorescent dye which is spe~ific for calcium is used as a marker for calcium change into the cells.
Cells are incubated Wit)l 1-10 IlN of the acetomethoxy ester of FUR~-2 for 30-60 minutes. Vpon uptake, the ester is hydrolyzed to release free FVRA-2, which selectively binds free ca2~.
~UR~-2 has a characteristic fluorescence spectrum, which wavelength is shifted when the dye binds to free Ca2~.
~093/0~s 2 1 2 0 9 ~ ~ PCT/US92/0~77 ~ccording to the method, Ca2+ which is present in the cell can be quantified by exciting the dye at two different wavelengths, 340 and 380 nm. The emission fluorescence i~ measured at 510 nm. The calcium concentration is proportional to the ratio of the fluorescent emission when excited at 340 nm to the emission at 380 nm. It is conventional to report the concentration of calcium within the cell in terms of the fluorescence ratio, the 340/380 ratio. This technique is described in Grynkiewicz et al., J. Biol. Chem.. 260, 3440 (1985) and Pang et al., P. N. A.
S. (US~ , 623 (199~).
Figs. 13, 14 a-d, 17, 18, 21, 22 and 23 illustrate t}~e results of the above-described measurements when inhibitors sucll as an anti-osteoporotic agent (788) or bPTll~ 34) or Csll4 were used in the presence of KCl.
~s call be readily seen from the figures, the PTII
analogues, whether full length or 1-34, which contain anomalous amino acids at positions twenty-five, twenty-six and twenty-seven (most particularly those which contain Ala2S-Ala26-~la27), do no~ effect a hypotensive and smooth muscle relaxation response, including positive chronotropic effects, but do inhibit calcium uptake as stimulated by KCl in osteoblasts, which indicates that these compounds would have the same effect on bone cells as PTIl and would be useful in the treatment of osteoporosis in mammals and, particularly, in man, without the aformentioned deleterious side effects in~the elderly. -While not being bound by any theory, it is suggested that substitution ~rg25-Lys26-Lys27 by other amino acids in 1-84 PT~
alld in the 1-34 analogues removes the vasodepressor, smooth muscle relaxation and positive chronotropic and inotropic effects of either bPTH or hPTH. The effect on KCl induced calcium uptake in osteoblasts, however, is essentially unchanged for 1-84 or 1-34 PTII. In otller words, the effect on bone cells i5 unchanged from PTII.
lhe physiological significance of an inhibiting effect on tl)e l`~CL induced calcium uptake in bone cells i~ not yet understood. One hypothesis is that the analogues interact ' `' W093/0~ PCT/US92/0~77 212094(;
fully with bone cell receptor activity. The fact that the same e~fect is seen for both PTII and the analogues disclosed herein suggests that the site of interaction with the osteoblast cell receptor is unchanged by the substitution.
The analogues of the present invention can be used in the treatment of osteoporosis and other bone related diseases and disorders involving bone cell calcium regulation.
The analogues of the present invention may be administered to a warm-blooded mammalian in need thereof, particularly a lluman, by parental, topical, rectal administration or by inhalation. The analogues may be conventionally formulated in a parenteral dosage form compounding about 1 to about 300 mg per unit o~ dosage with a conventional vehicle, excipient, binder, preservative, stabilizer, color, agent or the like as called for by accepted pharmaceutical pra~tice.
For p~rental administration, a 1 to 10 ml intravenous, intramuscular or sub~utaneous injection would be given one to four times daily. The injection would contain an analogue of the present invention in an aqueous isotonic sterile solution or suspension optionally with a preservative such as phenol or a solubilizing agent such as ethylenediamin~tetraacetic acid (EDT~ mong the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic s~dlum chloride solutiol~. In addition, sterile, fixed oils are 2~ conventionally employed as a solvent or suspending medium.
Synthetic monoglycerides, diglycerides, fatty acids (such as oleic acid~ find use as fixed oil in the preparation of injectables.
For rectal administration, the analogues of the present invention can be prepared in the form of suppositories by mixing with a suitable non-irritating excipient such as cocoa butter or polyethylene glycols.
For topical use, the analogues of tlle present inventio can be prepared in the form of ointments, jellies, solutions, - 35 suspensions or dermal adhesive patches.
In a powdered aerosol, analogues of the present invention may be` administered by a spinhaler turbo-inhaler device ~` .
.
~W093/~ ~ 2 1 2 0 9 4 G PCT/US92/0~ ~
obtained from Fisons Corporation of Bedford, Massachusetts, at a rate of about 0.1 to 50 mg per capsule! 1 to 8 capsules being administered daily for an average human. In a liquid aerosol, the compounds of the present invention are administered at the rate of about 100 to 1000 micrograms per "puff" or activated release of a standard volume of propellant. The liquid aerosol would be given at the rate of 1 to 8 "puffs" per day with variation in dosages due to the severity of the conditions being t'reated, the weight of the patient and the particle size distributio11 of t~e aerosol. ~ fluorinated hydrocarbon or isobutane find use as propellants for liquid aerosols.
Daily doses are in the range of about 0.01 to about 200 mg per kg o body weight, depending on the activity of the - specific compound, the age, weight, sex and conditions of t~
subject to be treated, the type and severity of the disease, the fre~uency and route of administration. As would be w~l~
known, the amount of active ingredient that may be combined with the carried materials to produce a single dosage will vary ~epe~ing UpO~l the host treate~ and the particular mo~e of administration.
The following examples demonstrate ~he utility o~
applicants' invention. The examples are not limiting, but are illustrative only, and modifications which wQuld be apparen~ to those skilled in the art are included within the scope of t~is disclosure.
Exam~le 1 In Vlvo Blood Pressure Measurement.
Sprague-Dawley (S-D) rats were anaesthetized with pentobarbital and a cannula was inserted into the carotid artery. The rats were kept sedated durin~ the procedure ~nd were injected with PTH peptides only when the blood pressure ~
tlle rats were stable~ Peptides were injected tllrough a cannula in the jugular vein, in amounts of 1, 3 and 5 or more ~g/kg and the mean systolic and diastolic blood pressure was monitore~
co~ltinuously t~roughout the procedure. ~e~ults are reported with comparison to bPTH-(1-34).
.
W093/0~S 2 1 2 0 ~ 4 1; 12 PCT/~S92/0~77 ExamPle 2 In Vitro Rat Tail ~rtery Helical Strip Tension Assay The assay was performed according to Pang et al., Bloo~
Vessels. 22, 57 (1985). Sprague-Dawley rats were anaesthetized with pentobarbital and the tail artery excised and placed in ice-cold Krebs-}~anseleit solution (KHS) ~xygenated with 95%`02, 5% C2 Each artery was cut helically and strips of approximately 1.5 cm were secured in a Sawyer-Bartlestone chamber containing KHS. The force generated by the strips was measured with a Grass FT03 force displacement transducer and recorded on a polygraph. Isolated tail artery helical strips were equilibrated for 1 hour prior to use.
One to two minutes prior to addition of a peptide, the strips were contracted by addition of either arginine vasopressin (~VP~, potassium chloride (KCl) or norepinephrine E) to the bath. The peptide was-then added to the bath and - the degree of relaxation measured. Bovine serum albumin was used as a control. Results are reported as percent decrease in tension for each drug and dose used. Drug dose is calculated on the basis of the final concentration in the bath solution.
: . .
~; ExamPle 3 In Vitro atrial contractility and contraction rate measurement q`he assay .was performed according to Tenner et al., Canadian Journal of Ph~sioloaY and PharmacoloqY, Vol. 61, No.
1~ (1983) pp. 1162-1167. Sprague-Dawley rats weighing between 100 and 250 g were treated with heparin (500 IU, i.p.) 15 minutes prior to decapitation. Thoracotomies were performed and the heart rapidly excised and placed in a cold physiological salt solution (PSS) having the following composition (in millimolar): NaCl, 120; KCl, 5.63: CaC12, 2.0 MgC12, 2.1: NaHC03, 25.0; dextrose, 9.7. The solution was continously aerated by a gas mlxture of 95% 2-5% C02. l~i-e right atrium was isolated and suspended in a tissue chamber co~ltaining 20 mL of PSS at 370C, p~ 7.~. ~tria were allowed to equilibrate for 1 hr under a restlng tension of 1 g.
The atrial rate and force were determined from W093~ PCT/US92/0~7~ ~
2120~6 contractions recorded by a Grass FT.03 force-displacement transducer and a Grass model 79 polygrapll. The Basial atrial rate for control atria (as determined by counting the frequency of contractions) was 258 ~ 7 bpm (n=29). Basal developed force of the spontaneously beating right atria was 0.33 ~ 0.06 g (n=10). Dose-response curves for the peptides were obtained by cumulative addition of the respective peptides. Drug dose is calculated on the basis of the final concentration in the batll solution.
ExamDle 4 Measurement of Intracellular Free Calcium Concentration In vitro Intracellular free calcium concentration was measured using the fluorescent dye FURA-2 according to the method of Grynkiewicz et al., J. Biol. Chem... 260, 3440 (1985) and Pang et al., P. ~. A. S. (USA~. 87, 623 (1990). UMR-106 ra~
osteosarcoma cells (ATCC CRL-1661) are incubated in 1-10 FUR~-2 AM (Sigma Chemical Co., St. Louis), the acetomethoxy ester of FUR~-2. Upon hydrolysis within the cell, FURA-2 is ;~ 20 released which sèlectively binds to free Ca2~ inding to Ca2+
shifts tl~e fluorescent spectrum of FUR~-2. Quantitation is obtained by exciting the dye at two different wavelengths, prePerably 340 and 380 nm alld measuring the fluorescent emission at 510 nm. The concentration of calcium is proportional to the ratio of the fluorescence emitted at 340 n~
to tllat at 380 nm.
KCl is used ln the medium to stimulate ~Ca2~i increase.
~fter the intracellular ~Ca2+]i llad been measured, the cells we~e washed with the original medium and the analogues added and the intracellular ~Ca2~]i measured again. KCl was then added without washing to measure the effect of the analogue on KCl induced ~Ca2+]i cl-anges. ~fter measurement, the cells were washed Witll the medium 3-4 times and KCl again added to determine the recovery of the cells after removal of t~le allalogue. ~esults are s}lown by actual traces and ~; histograms summarizing the results. As can be seen from Figs.
, w093/~W5 2 1 2 0 9 4 6 PCT/US92/~'7 14 a-d, PTH inhibits intracellar ~Ca2+]i increases as stimulated by KCl and measured by the method. Figs. 18, 21, 22 and 23 illustrate comparable results for the aa25~26~27 analogues.
-5 The comparability of the analogues and PT~ itself is considered to indicate that the analogues would be as use~ul as PTi~ for the treatment of osteoporosis.
W093/06~ 2 1 2 0 9 4 G PCT/US9~/08~77 Table I -~esiqnation Lenath Source Substitution Site . , Cs88 1-34 bovine none Cs99 1-34 bovine Ala 25 CslOO 1-34 bovine Ala 26 .
csll? 1-34 bovine Ala 27 cs 221 1-34 human Ala25,26,27 CslOOl 1-34 human none Cs2001 1-84 humall none WQ93/06~ PCT/VS92/0~77 2120~6 , ,, SEQUENCE LISTING
(1) GENERAL INFORMATION:
~i) APPLICANT: PANG, Peter K.T.
JIE, Shan (ii) TITLE OF INVENTION: PARATHYROID HORMONE ANALOGUES
SUBSTITUTED AT AA 25~ 26~ 27 AND USE IN OSTEOPOROSIS
TREATMENT
(iii) NUMBER OF SEQUENCES: 10 - (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Nikaido, Marmelstein, Murray & Oram (B) STREET: 655 Fifteenth Street N.W., Suite 330 (C) CITY: Washington D.C.
(D) STATE:
(E) COUNTRY: United States of America (F) ZIP: 2000S-5701 (v) COMPUTER READABLE ~ORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release ~1.0, Version ~1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C~ CLASSIFICATION:
.
(vii) PRIOR APPLICATION DATA:
: ~ . (A) APPLI~ATION NUMBER: US 773,098 (B) FILING DATE: 10-Oct-91 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORM~TION:
(A) NAME: Murray, Robert Bo (B) REGISTRATION NUMBER: 22,890 (C) REFERENCE/DOCKET NUMBER: ~610-2002 (ix) T~LECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 638-5000 (B) TELEFAX: (202) 638-4810 (2) INFO~MATION FOR SEQ ID NO:1:
: (i) SEQUENCE CHARAC~ERISTICS:
(A) LENGTH: 84 amino acids .
(B) TYPE: amino acid ,~
SUBS ~ ITUTE ~SHFFT
W093/~5 2 1 2 0 9 ~ 6 PCT/US92/0~7 :~
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Val Ser Glu Ile Gln Phe Met ~is Asn Leu Gly Lys His . Leu Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala - 30 35 ~
Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp : - 45 50 55 Asn Val Leu Val Glu Ser His Gl~ Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INFORMATION FOR SEQ ID NO:2:
: (i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 84 amino acids (B) TYPE: amino acid (~) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: . ~:
Ser Val Ser Glu Ile Gln Leu Met ~is Asn Leu Gly Lys ~is ~;
Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys 1ys Leu SU~STITU7E SH~
W093/0684 2 1 2 0 9 4 6 PCT/US~2/0~77 Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INF0RMATION FOR SEQ ID NO:3:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser Ser Net Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu 15 .20 25 Gln Asp Val His Asn Phe (2) INFORMATION FOR SEQ ID NO:4: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~l JR~TITUTr SHEET
. ~093/06~; 2 1 2 0 9 ~1 ~ PCT/US92/0~477 (xi) SEQUENCE DESC~IPTION: SEQ ID NO:4:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu Gln Asp Val His Asn Phe (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ser Val Ser Glu lle Gln Leu Met His Asn Leu Gly Lys His .
Leu Asn Ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu ~.
15 20 25 ~.
Gln Asp Val His Asn Phe ' (2) INFORMATION FOR SÆQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (~3) TYPE: amino acid (~) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His c l IR~TITI I~F ~ FT
W093/0~ PCT/US92/0~77 Leu Asn Ser Net Glu Arg Val Glu Trp Leu Ala Ala Ala Leu Gln Asp Val His Asn Phe (2) INFORMATION FOR SEQ ID NO:7:
.(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His 1 - 5 10 , ~:~ Leu Ser Ser Net Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile AIa Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp ' Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2)`INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 84 amino acids (B) TYPE: amino acid ' SUBSTI~Ul ~ E~
::
WO 93/06845 P~/US92/08477 21 21209~1~
( D) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His 1 5 1~
Leu Ser Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu ~5 20 25 Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys L~s Glu Asp ` - 45 50 55 Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala .~sp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 84 amino acids (~) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE ~ESCRIPTION: SEQ ID NO: 9:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu ~UBSTITI~E SHEE~
WO 93/0684~; 21 2 0 9 ~ G PCI'/USg2/1)8~77 ~ln Asp Val Hls Asn Phe Val Ala Leu Gly Ala Ser Ile Ala Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala . 60 65 70 Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INFORMATION FOR SEQ ID NO:10: .
(i) SEQUENCE CHAR~CTERISTICS: ~;
(A) LENGTH: 84 amino acids (B) TYPE: am'ino acid (D) TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:10:
Ser Val Ser ~lu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu 25~
Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala 3~ 40 Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln SUBSTITI~TE SHEET
Fig. lO shows the effect of bPTH-(1-34) and its analogues on diastolic blood pressure of anesthetized Sprague-Dawley rats.
Fig. 11 shows the effect of bPTH-(1-34) and its analogues on systolic blood pressure of anesthetized Sprague-Dawley rats.
Fig. 12 shows the va50relaxing ef~ect of bPTl1-(1-3~) an~
its analogues on rat tail artery helical strip in vitro.
Fig. 13 shows the depolarizing concentrations of KCl which increased calcium ion levels in cultured osteoblasts. ~r~g 7 is an an~ti-osteoporotic agent which inhibits the KCl effect.
W093/06~5 2 1 2 0 9 ~ ~ PCT/US92/08477 Figs. 14 a-d show tlle depolarizin~ concentrations of KCl which increased calcium levels in cultured osteoblasts.
Addition of bPTH-(1-34) inhibits the KCl effect.
Fig. 15 shows the effect of Cs88 ~bPTH~ 34)~ on the mean 5arterial blood pressure of anesthetized Sprague-Dawley rats.
Fig. 16 shows the dose-response relationship between Cs88 [bPTH-(1-34)] and the tension of rat tail artery helical strips precontracted with KCl, norepinephrine and AVP.
Fig. 17 shows the effect of Cs88 on [Ca2+]i in cultured lOU~ osteoblast cells.
Fig. 18 shows the effect of Cs88 on [Ca2+]i in cultured UMR cells.
Fig. 19 shows a comparison of the effect of Cs221 and cSsg and bPTII on the mean arterial blood pressure of anesthetized 1~Sprague-Dawley rats.
Fig. 20 s~-ows the relation between the relaxation curves of Sprague-Dawley rat tail artery helical strips, precontracted with AVP when treated with CslOO, Cs99, Cs88, Csll7 and Cs221.
Fig. 21 shows ~ com~arison between the effect of Cs221 ~n~
20hPT~I on the intracellular calcium uptake in the presence of KCl in UMR cells in culture.
Fig. 22 shows the effect of Cs221 on the intracellular calcium uptake in tl-e presence of KCl in UMR cells in culture.
Fig. 23 shows the effect of CslO01 on the intracellular 25calcium uptake in the presence of KCl in UMR cells in culture.
Fig. 24 shows the effect of Cs221 on the contractility an~
contractioll rate of right atrial tissue of Sprague-Dawley rats.
Fig. 25 shows the effect of Cs2001 and CslOOl Oll the contractility and contraction rate of right atrial tissue of 30Sprague-Dawley rats.
DETAILE~ DESCRI~TION OE T~E INVSNTION
There are at least two known catagories of functions for PT~I. PTI~ is involved in calcium balance in the blood stream and controls both the amount of calcium uptake from the W093/0~5 PCT~US92/0~77 21209~6 gastrointestinal tract and the deposition and removal of calcium from bone. Calcium also has been found to be effective in the maintenance of blood pressure. Cox, J. Cardiovascu~
Pharmacoloqy, Vol. 8 (1986), Supp. 8 S48. Control of calcium in the walls of blood vessels is a useful therapeutic regimen for controlling hypertension and calcium channel blockers, which prevent the introduction of calcium into cell walls, is a convelltional therapy for hypertensioll. Needleman et al. in Goodman and Gilman's The Pharmacoloaical Basis of Therapeutics, M~cMillan, New York, (1985), page ~lG ff.
Administration of therapeutic doses of PTtl has been found to be effective for the control of osteoporosis, particularly in individuals who have been subjected to thyroidectomies~
parathyroidectomies. Therapeutic dosages of PTH will, in some individuals, result in unacceptable diminution of bloo~
pressure and may result in relaxation of smooth muscles such as gastrointestinal, uterus, tracheal, vas deferens as well as exhibit positive chronotropic and inotropic effects on the ~leart. To avoid hypotensive effects, smooth muscle relaxation effects and positive chronotropic and inotropic ef~ects Oll the heart, it was envisaged that the structure of PTI~ could be modi~ie~ to decouple the hypotensive, smootl- muscle relaxation and positive chronotropic and inotropic function from the bond calcium and bone ~deposition functioll. lt llas now beell discovered that a critical site exi-sts at amino acid twenty-five, twenty-six and twenty-seven, which is -Arg-Lys-Lys- in both bovine and human PTI~. Substitution at ttle -~rg-Lys-Lys-site with -~la-~la-Ala- diminishes the hypotensive as well as smooth muscle relaxation an~ positive chronotropic and inotropic effects without denigrating from the osteo effect.
These results suggest that substitution at the -Arg-Lys-Lys-site with amino acids other than basic amino acids arginine and lysine would also diminish the hypotensive, smooth muscle relaxation and positive chronotropic and inotropic effects 3s without denigrating from the osteo effect.
l~he procedure of Erickson and Merrifield, as modified by llodqes et al., as described above, may ~e used to synthesize , ~ ' W093/06~ PCT/US92/0~77 ~120946 .
synthetic PTil or fragments thereof. The procedure enables substitution for the nàturally occurring PTI~ at substantially every location and it is possible to prepare both bovine and human synthetic PTH at full length or in the sequence of the first thirty-four amino acids, which is more facilely performed. Such substitution can also be accomplished by genetic engineering.
Substitution at position twenty-five, twenty-six and twenty-seven invariably alters the observed hypotensive, smooth muscle relaxation and positive chronotropic an~ inotropic effects, whether the full length PTH or the 1-34 fragment is administered. Substitution of -Ala-Ala-Ala- for -Arg-Lys-Lys-at position twenty-~ive, twenty-six and twenty-seven is particularly preferred because the change in blood pressure, smooth muscle relaxation and positive chronotropic and inotropic effects from this substitution are minimal and calcium uptake, as measured in osteoblasts, mimics the results from the administration of native PT~. The 1-34 PTE{ fragment with -~la25-~la26-~la27- i~ particularly preferred because the pharmacological properties are those which are desired and the difficulty of synthesis is minimized. Synthesis of the compoun~s used in the development of this invention was performed at U berta Peptide Institute (~PI) and t~e cooperation o~ API is gratefully acknowledged.
The structure of bovine parathyroid hormone (bPTI~) and human parathyroid hormone (hPTH) are shown in Figs. la (SEQ ID
No:l) and lb (SEQ ID ~10:2). Representative synthetic analogues are described in Table l and are further shown in Figs. 2-9 and SEQ ID N0:3-SEQ ID N0:10. The hypotensive effects of these analogues is shown in Figs. 10, ll, 15 and l9. All o~ the analogues produce either no or less diminution of blood pressure than does native PTH. With only one amino acid at either the 25, 26 or 27 position substituted, the analogue shows less e~fect than native PTH. With all three positions substituted, it provides almost no change. ~t the level o 5 /kg of r'lll, the blood pressure in Sprague-Dawley rats is 5UCl that they are essentially moribund.
.
`::
W093/~ 2 1 2 U 9 4 6 PCT/US92/0~77 We have developed a method for modeling the hypotensive effects of natural and synthetic chemical compounds using helically cut tail arteries from Sprague-Dawley rats in a Sawyer-8artlestone chamber, measuring the change in tension with a force displacement transducer. This method and the effect of bovine PTH-(1-34) in this system is described in Blood Vessels. 22, 57 (1985). It is demonstrated in this papar - that bPTH-(1-34) produces dose-dependent relaxation of helical strïps of rat tail artery which have been previously contracted by ~rginine-vasopressin (~VP). Figs. 12, 16 and 20 illustrate the effect of the PTI~ analogues of this invention as measured using this in vitro technique. Alternatively, the strips may be precontracted using other pressor ~ubstances such as norepinephrine (NE) or KCl.
We have also developed a method of modeling the chronotropic effects of natural and synthetic chemicals using the right atrium from Sprague-Dawley rats and measuring the change in the force and rate of atrium contraction. lhis met~lod and the effects of bovine PTI~ 34) in this system ~re described in Tenner et al, ~he Canadian Journal of Ph~sioloqv and Pharmacoloqv, Volume 61, No. 10 (1983) pp, 1162-1167. It is demonstrated in this paper that bPTI~ (1-34) produces significant dose-dependent chronotropic effects on rat cardiac pacemaker tissue. Figs. 24-25 illustrate the effect of the PTil analogues of this invention as measured using this in vitro technique.
Decause osteoporosis i5 a progressive syndrome, a model is required and the use of cultured osteoblasts of the UMR-106 rat osteosarcoma cells, ATCC CRL 1661 have been used as the model.
Uptake of calcium in these cells has been monitored using the ~URA-2 method, wherein a fluorescent dye which is spe~ific for calcium is used as a marker for calcium change into the cells.
Cells are incubated Wit)l 1-10 IlN of the acetomethoxy ester of FUR~-2 for 30-60 minutes. Vpon uptake, the ester is hydrolyzed to release free FVRA-2, which selectively binds free ca2~.
~UR~-2 has a characteristic fluorescence spectrum, which wavelength is shifted when the dye binds to free Ca2~.
~093/0~s 2 1 2 0 9 ~ ~ PCT/US92/0~77 ~ccording to the method, Ca2+ which is present in the cell can be quantified by exciting the dye at two different wavelengths, 340 and 380 nm. The emission fluorescence i~ measured at 510 nm. The calcium concentration is proportional to the ratio of the fluorescent emission when excited at 340 nm to the emission at 380 nm. It is conventional to report the concentration of calcium within the cell in terms of the fluorescence ratio, the 340/380 ratio. This technique is described in Grynkiewicz et al., J. Biol. Chem.. 260, 3440 (1985) and Pang et al., P. N. A.
S. (US~ , 623 (199~).
Figs. 13, 14 a-d, 17, 18, 21, 22 and 23 illustrate t}~e results of the above-described measurements when inhibitors sucll as an anti-osteoporotic agent (788) or bPTll~ 34) or Csll4 were used in the presence of KCl.
~s call be readily seen from the figures, the PTII
analogues, whether full length or 1-34, which contain anomalous amino acids at positions twenty-five, twenty-six and twenty-seven (most particularly those which contain Ala2S-Ala26-~la27), do no~ effect a hypotensive and smooth muscle relaxation response, including positive chronotropic effects, but do inhibit calcium uptake as stimulated by KCl in osteoblasts, which indicates that these compounds would have the same effect on bone cells as PTIl and would be useful in the treatment of osteoporosis in mammals and, particularly, in man, without the aformentioned deleterious side effects in~the elderly. -While not being bound by any theory, it is suggested that substitution ~rg25-Lys26-Lys27 by other amino acids in 1-84 PT~
alld in the 1-34 analogues removes the vasodepressor, smooth muscle relaxation and positive chronotropic and inotropic effects of either bPTH or hPTH. The effect on KCl induced calcium uptake in osteoblasts, however, is essentially unchanged for 1-84 or 1-34 PTII. In otller words, the effect on bone cells i5 unchanged from PTII.
lhe physiological significance of an inhibiting effect on tl)e l`~CL induced calcium uptake in bone cells i~ not yet understood. One hypothesis is that the analogues interact ' `' W093/0~ PCT/US92/0~77 212094(;
fully with bone cell receptor activity. The fact that the same e~fect is seen for both PTII and the analogues disclosed herein suggests that the site of interaction with the osteoblast cell receptor is unchanged by the substitution.
The analogues of the present invention can be used in the treatment of osteoporosis and other bone related diseases and disorders involving bone cell calcium regulation.
The analogues of the present invention may be administered to a warm-blooded mammalian in need thereof, particularly a lluman, by parental, topical, rectal administration or by inhalation. The analogues may be conventionally formulated in a parenteral dosage form compounding about 1 to about 300 mg per unit o~ dosage with a conventional vehicle, excipient, binder, preservative, stabilizer, color, agent or the like as called for by accepted pharmaceutical pra~tice.
For p~rental administration, a 1 to 10 ml intravenous, intramuscular or sub~utaneous injection would be given one to four times daily. The injection would contain an analogue of the present invention in an aqueous isotonic sterile solution or suspension optionally with a preservative such as phenol or a solubilizing agent such as ethylenediamin~tetraacetic acid (EDT~ mong the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic s~dlum chloride solutiol~. In addition, sterile, fixed oils are 2~ conventionally employed as a solvent or suspending medium.
Synthetic monoglycerides, diglycerides, fatty acids (such as oleic acid~ find use as fixed oil in the preparation of injectables.
For rectal administration, the analogues of the present invention can be prepared in the form of suppositories by mixing with a suitable non-irritating excipient such as cocoa butter or polyethylene glycols.
For topical use, the analogues of tlle present inventio can be prepared in the form of ointments, jellies, solutions, - 35 suspensions or dermal adhesive patches.
In a powdered aerosol, analogues of the present invention may be` administered by a spinhaler turbo-inhaler device ~` .
.
~W093/~ ~ 2 1 2 0 9 4 G PCT/US92/0~ ~
obtained from Fisons Corporation of Bedford, Massachusetts, at a rate of about 0.1 to 50 mg per capsule! 1 to 8 capsules being administered daily for an average human. In a liquid aerosol, the compounds of the present invention are administered at the rate of about 100 to 1000 micrograms per "puff" or activated release of a standard volume of propellant. The liquid aerosol would be given at the rate of 1 to 8 "puffs" per day with variation in dosages due to the severity of the conditions being t'reated, the weight of the patient and the particle size distributio11 of t~e aerosol. ~ fluorinated hydrocarbon or isobutane find use as propellants for liquid aerosols.
Daily doses are in the range of about 0.01 to about 200 mg per kg o body weight, depending on the activity of the - specific compound, the age, weight, sex and conditions of t~
subject to be treated, the type and severity of the disease, the fre~uency and route of administration. As would be w~l~
known, the amount of active ingredient that may be combined with the carried materials to produce a single dosage will vary ~epe~ing UpO~l the host treate~ and the particular mo~e of administration.
The following examples demonstrate ~he utility o~
applicants' invention. The examples are not limiting, but are illustrative only, and modifications which wQuld be apparen~ to those skilled in the art are included within the scope of t~is disclosure.
Exam~le 1 In Vlvo Blood Pressure Measurement.
Sprague-Dawley (S-D) rats were anaesthetized with pentobarbital and a cannula was inserted into the carotid artery. The rats were kept sedated durin~ the procedure ~nd were injected with PTH peptides only when the blood pressure ~
tlle rats were stable~ Peptides were injected tllrough a cannula in the jugular vein, in amounts of 1, 3 and 5 or more ~g/kg and the mean systolic and diastolic blood pressure was monitore~
co~ltinuously t~roughout the procedure. ~e~ults are reported with comparison to bPTH-(1-34).
.
W093/0~S 2 1 2 0 ~ 4 1; 12 PCT/~S92/0~77 ExamPle 2 In Vitro Rat Tail ~rtery Helical Strip Tension Assay The assay was performed according to Pang et al., Bloo~
Vessels. 22, 57 (1985). Sprague-Dawley rats were anaesthetized with pentobarbital and the tail artery excised and placed in ice-cold Krebs-}~anseleit solution (KHS) ~xygenated with 95%`02, 5% C2 Each artery was cut helically and strips of approximately 1.5 cm were secured in a Sawyer-Bartlestone chamber containing KHS. The force generated by the strips was measured with a Grass FT03 force displacement transducer and recorded on a polygraph. Isolated tail artery helical strips were equilibrated for 1 hour prior to use.
One to two minutes prior to addition of a peptide, the strips were contracted by addition of either arginine vasopressin (~VP~, potassium chloride (KCl) or norepinephrine E) to the bath. The peptide was-then added to the bath and - the degree of relaxation measured. Bovine serum albumin was used as a control. Results are reported as percent decrease in tension for each drug and dose used. Drug dose is calculated on the basis of the final concentration in the bath solution.
: . .
~; ExamPle 3 In Vitro atrial contractility and contraction rate measurement q`he assay .was performed according to Tenner et al., Canadian Journal of Ph~sioloaY and PharmacoloqY, Vol. 61, No.
1~ (1983) pp. 1162-1167. Sprague-Dawley rats weighing between 100 and 250 g were treated with heparin (500 IU, i.p.) 15 minutes prior to decapitation. Thoracotomies were performed and the heart rapidly excised and placed in a cold physiological salt solution (PSS) having the following composition (in millimolar): NaCl, 120; KCl, 5.63: CaC12, 2.0 MgC12, 2.1: NaHC03, 25.0; dextrose, 9.7. The solution was continously aerated by a gas mlxture of 95% 2-5% C02. l~i-e right atrium was isolated and suspended in a tissue chamber co~ltaining 20 mL of PSS at 370C, p~ 7.~. ~tria were allowed to equilibrate for 1 hr under a restlng tension of 1 g.
The atrial rate and force were determined from W093~ PCT/US92/0~7~ ~
2120~6 contractions recorded by a Grass FT.03 force-displacement transducer and a Grass model 79 polygrapll. The Basial atrial rate for control atria (as determined by counting the frequency of contractions) was 258 ~ 7 bpm (n=29). Basal developed force of the spontaneously beating right atria was 0.33 ~ 0.06 g (n=10). Dose-response curves for the peptides were obtained by cumulative addition of the respective peptides. Drug dose is calculated on the basis of the final concentration in the batll solution.
ExamDle 4 Measurement of Intracellular Free Calcium Concentration In vitro Intracellular free calcium concentration was measured using the fluorescent dye FURA-2 according to the method of Grynkiewicz et al., J. Biol. Chem... 260, 3440 (1985) and Pang et al., P. ~. A. S. (USA~. 87, 623 (1990). UMR-106 ra~
osteosarcoma cells (ATCC CRL-1661) are incubated in 1-10 FUR~-2 AM (Sigma Chemical Co., St. Louis), the acetomethoxy ester of FUR~-2. Upon hydrolysis within the cell, FURA-2 is ;~ 20 released which sèlectively binds to free Ca2~ inding to Ca2+
shifts tl~e fluorescent spectrum of FUR~-2. Quantitation is obtained by exciting the dye at two different wavelengths, prePerably 340 and 380 nm alld measuring the fluorescent emission at 510 nm. The concentration of calcium is proportional to the ratio of the fluorescence emitted at 340 n~
to tllat at 380 nm.
KCl is used ln the medium to stimulate ~Ca2~i increase.
~fter the intracellular ~Ca2+]i llad been measured, the cells we~e washed with the original medium and the analogues added and the intracellular ~Ca2~]i measured again. KCl was then added without washing to measure the effect of the analogue on KCl induced ~Ca2+]i cl-anges. ~fter measurement, the cells were washed Witll the medium 3-4 times and KCl again added to determine the recovery of the cells after removal of t~le allalogue. ~esults are s}lown by actual traces and ~; histograms summarizing the results. As can be seen from Figs.
, w093/~W5 2 1 2 0 9 4 6 PCT/US92/~'7 14 a-d, PTH inhibits intracellar ~Ca2+]i increases as stimulated by KCl and measured by the method. Figs. 18, 21, 22 and 23 illustrate comparable results for the aa25~26~27 analogues.
-5 The comparability of the analogues and PT~ itself is considered to indicate that the analogues would be as use~ul as PTi~ for the treatment of osteoporosis.
W093/06~ 2 1 2 0 9 4 G PCT/US9~/08~77 Table I -~esiqnation Lenath Source Substitution Site . , Cs88 1-34 bovine none Cs99 1-34 bovine Ala 25 CslOO 1-34 bovine Ala 26 .
csll? 1-34 bovine Ala 27 cs 221 1-34 human Ala25,26,27 CslOOl 1-34 human none Cs2001 1-84 humall none WQ93/06~ PCT/VS92/0~77 2120~6 , ,, SEQUENCE LISTING
(1) GENERAL INFORMATION:
~i) APPLICANT: PANG, Peter K.T.
JIE, Shan (ii) TITLE OF INVENTION: PARATHYROID HORMONE ANALOGUES
SUBSTITUTED AT AA 25~ 26~ 27 AND USE IN OSTEOPOROSIS
TREATMENT
(iii) NUMBER OF SEQUENCES: 10 - (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Nikaido, Marmelstein, Murray & Oram (B) STREET: 655 Fifteenth Street N.W., Suite 330 (C) CITY: Washington D.C.
(D) STATE:
(E) COUNTRY: United States of America (F) ZIP: 2000S-5701 (v) COMPUTER READABLE ~ORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release ~1.0, Version ~1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C~ CLASSIFICATION:
.
(vii) PRIOR APPLICATION DATA:
: ~ . (A) APPLI~ATION NUMBER: US 773,098 (B) FILING DATE: 10-Oct-91 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORM~TION:
(A) NAME: Murray, Robert Bo (B) REGISTRATION NUMBER: 22,890 (C) REFERENCE/DOCKET NUMBER: ~610-2002 (ix) T~LECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 638-5000 (B) TELEFAX: (202) 638-4810 (2) INFO~MATION FOR SEQ ID NO:1:
: (i) SEQUENCE CHARAC~ERISTICS:
(A) LENGTH: 84 amino acids .
(B) TYPE: amino acid ,~
SUBS ~ ITUTE ~SHFFT
W093/~5 2 1 2 0 9 ~ 6 PCT/US92/0~7 :~
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Val Ser Glu Ile Gln Phe Met ~is Asn Leu Gly Lys His . Leu Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala - 30 35 ~
Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp : - 45 50 55 Asn Val Leu Val Glu Ser His Gl~ Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INFORMATION FOR SEQ ID NO:2:
: (i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 84 amino acids (B) TYPE: amino acid (~) TOPOLOGY: linear ( ii ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: . ~:
Ser Val Ser Glu Ile Gln Leu Met ~is Asn Leu Gly Lys ~is ~;
Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys 1ys Leu SU~STITU7E SH~
W093/0684 2 1 2 0 9 4 6 PCT/US~2/0~77 Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INF0RMATION FOR SEQ ID NO:3:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser Ser Net Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu 15 .20 25 Gln Asp Val His Asn Phe (2) INFORMATION FOR SEQ ID NO:4: -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~l JR~TITUTr SHEET
. ~093/06~; 2 1 2 0 9 ~1 ~ PCT/US92/0~477 (xi) SEQUENCE DESC~IPTION: SEQ ID NO:4:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu Gln Asp Val His Asn Phe (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ser Val Ser Glu lle Gln Leu Met His Asn Leu Gly Lys His .
Leu Asn Ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu ~.
15 20 25 ~.
Gln Asp Val His Asn Phe ' (2) INFORMATION FOR SÆQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (~3) TYPE: amino acid (~) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His c l IR~TITI I~F ~ FT
W093/0~ PCT/US92/0~77 Leu Asn Ser Net Glu Arg Val Glu Trp Leu Ala Ala Ala Leu Gln Asp Val His Asn Phe (2) INFORMATION FOR SEQ ID NO:7:
.(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His 1 - 5 10 , ~:~ Leu Ser Ser Net Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile AIa Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp ' Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2)`INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 84 amino acids (B) TYPE: amino acid ' SUBSTI~Ul ~ E~
::
WO 93/06845 P~/US92/08477 21 21209~1~
( D) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His 1 5 1~
Leu Ser Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu ~5 20 25 Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys L~s Glu Asp ` - 45 50 55 Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala .~sp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 84 amino acids (~) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE ~ESCRIPTION: SEQ ID NO: 9:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Xaa Xaa Xaa Leu ~UBSTITI~E SHEE~
WO 93/0684~; 21 2 0 9 ~ G PCI'/USg2/1)8~77 ~ln Asp Val Hls Asn Phe Val Ala Leu Gly Ala Ser Ile Ala Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala . 60 65 70 Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln (2) INFORMATION FOR SEQ ID NO:10: .
(i) SEQUENCE CHAR~CTERISTICS: ~;
(A) LENGTH: 84 amino acids (B) TYPE: am'ino acid (D) TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:10:
Ser Val Ser ~lu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Ala Ala Ala Leu 25~
Gln Asp Val His Asn Phe Val Ala Leu Gly Ala Ser Ile Ala 3~ 40 Tyr Arg Asp Gly Ser Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Gln Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln SUBSTITI~TE SHEET
Claims (10)
1. A bovine parathyroid hormone analogue comprising the structure shown in SEQ ID NO:3, wherein each of Xaa25, Xaa26 and Xaa27 is Alanine (Ala), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamine (Gln), Glutamic acid (Glu), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Methionine (Met), Phenyalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) or Valine (Val).
2. A bovine parathyroid hormone analogue having the structure shown in SEQ ID NO:4.
3. A human parathyroid hormone analogue comprising the structure shown in SEQ ID NO:5, wherein each of Xaa25, Xaa26 and Xaa27 is Alanine (Ala), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamine (Gln), Glutamic acid (Glu), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Methionine (Met), Phenyalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) or Valine (Val).
4. The human parathyroid hormone analogue having the structure shown in SEQ ID NO:6.
5. A bovine parathyroid hormone analogue comprising the structure shown in SEQ ID NO:7, wherein each of Xaa25, Xaa26 and Xaa27 is Alanine (Ala), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamine (Gln), Glutamic acid (Glu), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Methionine (Met), Phenyalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) or Valine (Val).
6. The bovine parathyroid hormone analogue having the structure shown in SEQ ID NO:8.
7. A human parathyroid hormone analogue comprising the structure shown in SEQ ID NO:9, wherein each of Xaa25, Xaa26 and Xaa27 is Alanine (Ala), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamine (Gln), Glutamic acid (Glu), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Methionine (Met), Phenyalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) or Valine (Val).
8. The human parathyroid hormone analogue having the structure shown in SEQ ID NO:10.
9. A pharmaceutical composition comprising a PTII
analogue according to any one of claims 1-8 and a pharmaceutically acceptable carrier.
analogue according to any one of claims 1-8 and a pharmaceutically acceptable carrier.
10. A method of treatment of osteoporosis in a patient in need of such treatment without causing substantial induction of hypotension, smooth muscle relaxation and cardiac inotropic and chronotropic action, said method comprising administering an osteoporotic effective amount of a PTII analogue according to any one of claims 1-8.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/773,098 US5317010A (en) | 1991-10-10 | 1991-10-10 | Parathyroid hormone analogues substituted at AA 25, 26, 27, and use in osteoporosis treatment |
| US773,098 | 1991-10-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2120946A1 true CA2120946A1 (en) | 1993-04-15 |
Family
ID=25097201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002120946A Abandoned CA2120946A1 (en) | 1991-10-10 | 1992-10-09 | Parathyroid hormone analogues substituted at aa 25.26.27 and use in osteoporosis treatment |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US5317010A (en) |
| EP (1) | EP0607325A4 (en) |
| JP (1) | JPH07502024A (en) |
| AU (1) | AU665587B2 (en) |
| CA (1) | CA2120946A1 (en) |
| WO (1) | WO1993006845A1 (en) |
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|---|---|---|---|---|
| CN102399285A (en) * | 2011-05-31 | 2012-04-04 | 成都金凯生物技术有限公司 | Parathyroid hormone (PTH) derivative |
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-
1991
- 1991-10-10 US US07/773,098 patent/US5317010A/en not_active Expired - Lifetime
-
1992
- 1992-10-09 AU AU28712/92A patent/AU665587B2/en not_active Ceased
- 1992-10-09 WO PCT/US1992/008477 patent/WO1993006845A1/en not_active Application Discontinuation
- 1992-10-09 JP JP5507124A patent/JPH07502024A/en active Pending
- 1992-10-09 EP EP92922071A patent/EP0607325A4/en not_active Ceased
- 1992-10-09 CA CA002120946A patent/CA2120946A1/en not_active Abandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399285A (en) * | 2011-05-31 | 2012-04-04 | 成都金凯生物技术有限公司 | Parathyroid hormone (PTH) derivative |
| CN102399285B (en) * | 2011-05-31 | 2014-06-04 | 成都金凯生物技术有限公司 | Parathyroid hormone (PTH) derivative |
Also Published As
| Publication number | Publication date |
|---|---|
| US5317010A (en) | 1994-05-31 |
| JPH07502024A (en) | 1995-03-02 |
| WO1993006845A1 (en) | 1993-04-15 |
| EP0607325A1 (en) | 1994-07-27 |
| AU665587B2 (en) | 1996-01-11 |
| EP0607325A4 (en) | 1996-11-06 |
| AU2871292A (en) | 1993-05-03 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |