Parathyroid hormone analogues for the treatment of osteoporosis

Details Parathyroid hormone analogues for the treatment of osteoporosis Parathyroid hormone analogues for the treatment of osteoporosis

2000-03-28

2003-04-01

Russel, Jeffrey E. (Department: 1654)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200, C530S317000, C530S324000

Reexamination Certificate

active

06541450

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to analogues of human parathyroid hormone, which have been found to be effective in the treatment of osteoporosis.
BACKGROUND OF THE INVENTION
Osteoporosis is a leading cause of disability in the elderly, particularly elderly women. It has recently been realized that human parathyroid hormone (hPTH) and certain analogues are stimulators of bone growth that are useful in the treatment of osteoporosis. Osteoporosis is a progressive disease which results in the reduction of total bone mass and increased bone fragility. This often results in spontaneous fractures of load-bearing bones and the physical and mental deterioration characteristic of immobilizing injuries. Postmenopausal osteoporosis is caused by the disappearance of estrogens which trigger a decade-long acceleration of bone turnover with an increased imbalance between resorption of old bone and formation of new bone. This results in thinning, increased porosity, and trabecular depletion of load-bearing bones. Osteoporosis is also associated with hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and the use of certain steroidal drugs. Remedies historically have involved increase in dietary calcium, estrogen therapy, and increased doses of vitamin D, but mainly with agents such as antiresorptives that inhibit bone resportion by osteoclasts.
Parathyroid hormone (PTH) is produced by the parathyroid gland and is a major regulator of blood calcium levels. PTH is a polypeptide and synthetic polypeptides may be prepared by the method disclosed by Erickson and Merrifield,
The Proteins
, Neurath et al, Eds., Academic Press, New York, 1976, page 257, and as modified by the method of Hodges et al (1988),
Peptide Research
1, 19, or by Atherton, E. and Sheppard, R. C.,
Solid Phase Peptide Synthesis
, IRL Press, Oxford, 1989.
When serum calcium is reduced to below a normal level, the parathyroid gland releases PTH and the calcium level is increased by resorption of bone calcium, by increased absorption of calcium from the intestine, and by increased renal reabsorption of calcium from nascent urine in the kidney tubules. Although continuously infused low levels of PTH can remove calcium from the bone, the same low doses, when intermittently injected can actually promote bone growth.
Tregear, U.S. Pat. No. 4,086,196, described human PTH analogues and claimed that the first 27 to 34 amino acids are the most effective in terms of the stimulation of adenylyl cyclase in an in vitro cell assay. Rosenblatt, U.S. Pat. No. 4,771,124, disclosed the property of hPTH analogues wherein Trp
23
is substituted by amino acids phenylalanine, leucine, norleucine, valine, tyrosine, &bgr;-naphthylalanine, or &agr;-naphthylalanine as a PTH antagonist. These modified hPTH analogues also have the 2 and 6 amino terminal acids removed, resulting in loss of most agonist activities when used to treat osteoporosis. These analogues were designed as inhibitors or PTH and PTH-related peptide. The analogues were claimed as possibly useful in the treatment of hypercalcemia associated with some tumors,
Pang et al, WO93/06845, published Apr. 15, 1993, described analogues of hPTH which involve substitutions of Arg
25
, Lys
26
, Lys
27
with numerous amino acids, including alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. These are claimed, with no supporting data from animal or human trials, to be effective in the treatment of osteoporosis with minimal effects on blood pressure and smooth muscle.
PTH operates through activation of two second messenger systems, G
s
-protein activated adenylyl cyclase (AC) and G
q
-protein activated phospholipase C
&bgr;
. The latter results in a stimulation of membrane-bound protein kinase Cs (PKC) activity. The PKC activity has been shown to require PTH residues 29 to 32 (Jouishomme et al (1994)
J. Bone Mineral Res.
9, (1179-1189). It has been established that the increase in bone growth, i.e. that effect which is useful in the treatment of osteoporosis, is coupled to the ability of the peptide sequence to increase AC activity. The native PTH sequence has been shown to have all of these activities. The hPTH-(1-34) sequence is typically shown as (A):
Ser Val Ser Glu lie Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys LeuGln Asp Val His Asn Phe-OH (SEQ ID NO:22)A
The following linear analogue, hPTH-(1-31)-NH
2
, for which data is included in Table 1, below, has only AC-stimulating activity and has been shown to be fully active in the restoration of bone loss in the ovariectomized rat model (Rixon, R. H. et al (1994)
J. Bone Miner. Res.
9, 1179-1189; Whitfield et al (1996),
Calcified Tissue Int.
58, 81-87; Willick et al, U.S. Pat. No. 5,556,940 issued Sep. 17, 1996):
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val-NH
2
B
The above molecule, B SEQ ID NO:32, and its counterpart with a Leu
27
substitution SEQ ID NO:2 may have a free carboxyl ending instead of the amide ending illustrated.
It is an object of the present invention to produce new PTH analogues with greater metabolic stability, increased bone restoration activity, increased AC activity, and minimal clinical side effects.
BRIEF SUMMARY OF THE INVENTION
According to one aspect of the invention, human parathyroid hormone hPTH and pharmaceutically acceptable salts thereof are provided, having the amino acid sequence
R-NH-R1-Val-Ser-Glu-Ile-Gln-Leu-R2-His-Asn-Leu-Gly-Lys-R3-R4-R5-R6-R7-Glu-Arg-Val-R8--Trp-Leu-R9--R10--R11-Leu-R12-Asp--Y (SEQ ID NO:23)
wherein,
R=hydrogen or any linear or branched chain alkyl, acyl or aryl group,
R1=Ser, Ala or Aib,
R2=Met, or a naturally occurring hydrophobic amino acid,
R3=His or a water soluble amino acid,
R4=Leu or a water soluble amino acid,
R5=Asn or a water soluble amino acid,
R6=Ser or a water soluble amino acid,
R7=Met, or a naturally occurring hydrophobic amino acid,
R8=Glu, Lys or Asp,
R9=Arg, Cys, Lys, Orn, or His
R10=Arg, Lys, Orn, Gln, Glu or Asp
R11=a naturally occurring hydrophobic or polar amino acid,
R12=Gln, Arg, Glu, Asp, Lys or Orn,
Cterm=OH, NH2, and
Y=Cterm, Val-Cterm, Val-His-Cterm, Val-His-Asn-Cterm, Val-His-Asn-Phe-Cterm, Val-His-Asn-Phe-Val-Cterm, Val-His-Asn-Phe, Val-Ala-Cterm and Val-His-Asn-Phe,Val-Ala-Leu-Cterm, (SEQ ID NOS:24-27)
cyclized as between one or two amino acid pairs 22 and 26, 26 and 30, 27 and 30, and 25 and 29 are lactams when R9 is Lys or Orn and R12 is Glu or Asp, excluding cyclo(Lys
26
-Asp
30
)(Leu
27
)-hPTH-(1-34)-NH
2
, cyclo(Lys
27
-Asp
30
)-h-PTH-(1-34)-NH
2
and cyclo(Lys
26
-Asp
30
)-(Leu
27
)-hPTH-(1-34)-OH.
Examples of the salts include salts of inorganic acids, salts of organic acids such as formic acid, acetic acid, tartaric acid and citric acid, salts of inorganic bases such as sodium and ammonium and salts of organic bases such as triethylamine, ethylamine and methylamine.
According to another feature of the present invention, cyclisation is effected by the formation of lactams, involving the coupling of the side-chains of the selected amino acid pairs such as between natural residues 22 and 26, or 26 and 30. Other types of cyclisations, such as the formation of a disulfide bridge e.g., between Cys containing analogues Cys
22
-Cys
26
and Cys
26
-Cys
30
are also contemplated.
Substitutions of various amino acids have also been found to be effective. Lys
27
may be replaced by a Leu or by various other naturally occurring hydrophobic or polar residues. Another factor is how well the residue fits to the receptor. Ala is not as hydrophobic as Leu. Lys and Tyr are generally considered to be polar, but nonetheless have hydrophobic interactions with the receptor. Lys, for example, can fold so that the hydrophobic part interacts with other hydrophobic residues in t

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