CA2205959A1 - Continuous low-dose administration of parathyroid hormone or its agonist - Google Patents

Abstract

A method of promoting bone formation in a human patient, which includes the step of administering continuously to the patient parathyroid hormone or its agonist for a period of at least one month at a dosage between 10 and 400 units/24 hrs. Also disclosed are novel parathyroid hormone agonists.

Description

CA 02205959 l997-05-23 WO96/19246 PCT~S95/16554 ~ ~U~U~ LOW--DOSE ~nMTNTRTRATION
OF P~UY~ID ~ORMONE OR IT~ r~O~T8T

BACKGROUND OF THE Ihv~N~llON
Parathyroid hormone ("PTH") has been shown to effect a positive bone balance (reviewed in Dempster, D.W. et al., Endocrine Rev., 1993, 14, 690-709; Riggs, L., Amer. J. Med., 1991, 91 (Suppl 5B), 37S-41S). The mammalian parathyroid hormone is a polypeptide product of 10 the parathyroid glands. The mature circulating form of the hormone is comprised of 84 amino acid residues.
Parathyroid hormone-related protein ("PTHrP") is a 139 to 173 amino acid-protein with N-teL ; n~ 1 homology to PTH.
PTHrP shares many or the bioiogical e~rec~S OL PT~
15 including binding to a common PTH/PTHrP receptor (Schipani E., et al., Endocrinology, 1993, 132, 215?-2165; Broadus, A.E., Steward, A.F., Parathyroid hormone-related protein: In: The Parathyroids, Bilezikian, J.P., Levine, M.A., Marcus R. Eds, Raven Press, NY. 1994, 259-20 294).
The general efficacy of daily acute administrationof PTHi_84 and PTHl_34 has been demonstrated in young, adult, and aged rats of both sexes (Dempster, D.W. et al., Endocrine Rev., 1993, 14, 690-709; Gunness-Hey, M., 25 et al., Metab. Bone Dis. & Rel. Res., 1984, 5, 177-181), as well as in other animal models, both normal and osteoporotic (Liu, C.C. et al., J. Bone Miner. Res., 1990, 5, 973-981; Podbesek, R., et al., Endocrinology, 1983, 112, 1000-1006). The anabolic effect of 30 intermittently administered PTH is also observed in osteoporotic men (Slovik, D.M., et al., J. Bone Miner.
Res., 1986, 1, 377-381) and women (Reeve, J., et al., Br.
Med. J., 1990, 301, 314-318) and with concurrent antiresorptive therapy (Hesch, R-D., et al. Calcif Tissue CA 0220~9~9 1997-0~-23 WO 9611924fi PCT/US95/16554 Int, 1989, 176-180) suggesting that the process is not coupled to active resorption. PTH1_34 is a synthetic amino-terminal fragment of PTH1_84 (Tregear, G.W., et al., Endocrinology, 1973, 93, 1349-1353; Mosekilde, L., et 5 al., Endocrinology, 1991, 129, 421-428).
The me~hAn; for the anabolic effect of intermittent PTH is not entirely clear (Dempster, D.W. et al., Endocrine Rev., 1993, 14, 690-709). The effect is dose-dependent within a defined dose range. There is lo increase in net calcium absorption from the intestine, and an increase in both calcium balance and calcium accretion into bone, with a corresponding increase in bone mass. This effect is also evident in the increase in trabecular bone forming surfaces and osteoblast 15 number.
However, contrary to the demonstrated anabolic efficacy of daily subcutaneous injections of PTH, data from animal studies indicate that continuous administration (infusion) of the hormone either had no 20 effect on bone growth or resulted in bone loss in dogs (Podbesek, R., et al., Endrocrinology, 1983, 112, 1000-1006; Malluche, H.H., et al., Am. J. Physiol., 1982, 242, F197-F202). Continuous administration of PTH has also been shown to inhibit osteoblast activity in humans.
25 Simon, et al. (Simon, L.S., et al., J. Bone Miner. Res., 1988, 3, 241-246) ~ ined the effects of intravenously infused hPTH1_34 at a dose of 0.55 U/kg/hr (approximately 800 U over 24 hr) continuously for 24 hours in six osteoporotic subjects and two normal subjects. In all 30 cases, serum levels of collagen type I, produced principally by osteoblasts, decreased within 16 hours of infusion, which also correlated with an increase in blood levels of ionized calcium.

CA 0220~9~9 l997-0~-23 WO 96/19246 PCTlUSg5/16554 SUMMARY OF THE lN V~N~ oN
The invention features a method of promoting bone formation in a human patient (e.g., a patient who suffers from osteoporosis). The method includes the steps of 5 ~;n;~tering (e.g., transmucously, intravenously, transdermally, subcutaneously, via implantation, or via infusion) continuously to the patient PTH (mature form), PTHrP, or an agonist thereof for a period of at least one month (as long as the life time of the patient, if 10 necessary) at a dosage between 10 and 400 units/24 hrs.
PTH and PTHrP include, but are not limited to, human PTH (hPTH), human PTHrP (hPTHrP), bovine PTH
(bPTH), bovine PTHrP (bPTHrP), and rat PTH (rPTH). An agonist of PTH or PTHrP is a peptide which is a 15 structural analog or fragment (preferably, an N-terminal fragment containing 50 or fewer amino acids) of a naturally occurring PTH or PTHrP and, like PTH and PTHrP, also capable of binding to PTH receptor and, thereby, promoting bone formation. Examples of such an agonist 20 include, but are not limited to, hPTHl_34 NH2, hPTHl_38 2' hPTHl_44 NH2, hPTHl_68 NH2, [Nle8~18, Tyr34]hPTH1 3 NH2 ~ bPTHl_34 NH2 ~ [ Nle8 ~ 18, Tyr34]bPTH1_34, [Nle8,18 phe22,Tyr34]bPTHl-34 NH2, [ Nle8, 18 Argl9,Tyr34]bPTHl-34 NH2' [Nle3~18,Arg21,Tyr34]bPTHl_34 NH2, or [Nle8~18,Argl9~21 , Tyr34]bPTHl_34 NH2. The symbol NH2 denotes amidation of the carboxyl group (-CO OH) of the C-terminal amino acid to form -CO-NH2. The following publications disclose the sequences of PTH peptides: The Parathyroids Basic and 30 Clinical Concepts, ed. John P. 8ilezikian, 239-258 (Raven Press, NH 1994); Nissenson, R., et al., Structure &
Function of the Receptor for Parathyroid Hormone and Parathyroid Hormone-Releasing Hormone, 3 Receptor 193-202 1993; Bachem California 1993-1994 Catalog (Torrance, CA);
35 and Sigma Peptides and Amino Acids 1994 Catalog (St.

CA 0220~9~9 l997-0~-23 WO96/19246 PCT~S95/16554 Louis, MO). The following publications disclose the sequenc~s of PTHrP peptides: Yasuda, et al., 264 J.
Biol. Chem. 7720-7725 (1989); Schermer, D.T., Journal of Bone & Mineral Research 6:149-155 (1991) and Burtis, 5 W.J., 3~(11) Clinical Chemistry 2171-2183 (1992). More examples can be found in the following publications:
German Application 4203040 A1 (1993);
PCT Application 94/01460 (1994);
PCT Application 94/02510 (1994);
EP Application 477885 A2(1992);
EP Application 561412 A1 (1993);
PCT Application 93/20203 (1993);
U.S. Patent No. 4,771,124 (1988);
PCT Application 92/11286 (1992);
PCT Application 93/06846 (1993);
PCT Application 92/10515 (1992);
U.S. Patent No. 4,656,250 (1987);
EP Application 293158 A2 (1988);
PCT Application 94/03201 (1994);
EP Application 451,867 A1 (1991);
US Patent No. 5,229,489 (1993); and PCT Application 92/00753 (1992).
Note that partial PTH agonists can also be used to practice the method of this invention. Examples of 25 partial PTH agonists include, but are not limited to, N-terminal deletion analogs (e.g., [Tyr34]bPTH3_34 NH2; see U.S. Patent No. 4,771,124 (1988).
~ referred ranges of dosages include 10-300 units/24 hrs, 10-200 units/24 hrs, 10-100 units/24 hrs, 30 100-400 units/24 hrs, 200-400 units/24 hrs, and 300-400 units/24 hrs.
One unit of PTH, PTHrP, or an agonist thereof is defined by utilizing an in vitro cAMP accumulation assay with human SaOS-2 cells. Human SaOS-2 cells respond upon 35 exposure to PTH, PTHrP, or an agonist thereof with a CA 0220~9~9 1997-0~-23 dose-dependent stimulation of cAMP ac~~ lation. With ~ [Nle8~l8~Tyr34]hpTHl-34 NH2 as the reference stA~d analog (10,000 units/mg), a dose-response relationship ~ can be generated using st~n~d non-linear regression 5 analysis. The relative potency for various PTH analogs (in units/mg) can be deterr;ne~ from the ratio of the EC50 of the reference stAn~d analog to that of the PTH
analog. EC50 is defined as the dose that evokes a half ~ l response -- cAMP accumulation herein. The 10 detailed procedure for handling the cells, setting up the assay, as well as methods for cAMP quantitation, is described in Sistane, E ., et al., Pharmacopeial Forum 20(3), 7509-7520 (1994).
If the a~m;ni~tration is to be achieved via 15 infusion, an ambulatory (e.g., MINIMED~ 404-SP, M;n;Me~
Technologies, Sylmar, CA; Pharmacia Deltec CADD-MICRO
MODEL~ S900, Pharmacia Deltec Inc., St. Paul, MN; or Disetronic Medical System's PANOMAT~, Plymouth, MN) or an implantable pump (e.g., MEDTRONIC SYNCROMED~, Medtronic, 20 Inc., Minneapolis, MN) can be used.
Preferably, PTH, PTHrP, or an agonist thereof is administered as a sustained release formulation. As an example, the formulation may contain a homo- or a co-polymer prepared from lactic acid (D-isomer, L-isomer, or 25 a racemate), glycolide, glycolic acid, or caprolactone.
Examples of suitable sustained release formulations can be found in the following publications:
U.S. Patent No. 3,773,919 (1973);
U.S. Patent No. 5,187,1S0 (1993);
U.S. Patent No. 4,767,628 (1988);
U.S. Patent No. 4,675,189 (1987);
U.S. Patent No. 5,271,945 (1993);
U.S. Patent No. 4,917,893 (1990); and U.S. Patent No. 3,887,699 (1975).

CA 0220~9~9 1997-0~-23 WO96/19246 PCT~S95/16554 The sustained release formulations can be administered parenterally (e.g. subcutaneously, or intravenously) or by inhalation (e.g. using an aerosol delivery system; e.g., see WO93/00951 and WO94/07514).
In other embodiments, PTH, PTHrP, or an agonist thereof can be administered transmucously (e.g. nasal, vaginal, rectal) or transdermally (e.g. iontophoretic patch).
Also, if desired, a bone resorption inhibiting lo agent can also be administered during performance of the above-described method. Examples of a suitable bone resorption inhibiting agent include, but are not limited to, estrogen, calcitonin, bisphosphonate, tamoxifen, vitamin D, and calcium. Also, see U.S. Patent No.
15 5,118,6~67 (1992).
The preferred dose and duration for practicing the above-described method varies depending upon the manner of administration, the age and the body weight of the subject and the condition of the subject to be treated, 20 and ultimately will be decided by the attending physician. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active 25 ingredient(s) into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient (e.g., hPTH) with finely divided solid 30 carriers, and then, if necessary as in the case of tabletsy forming the product into the desired shape and size.
The invention also features novel PTH or PTHrP
agonists of the following formula:

CA 0220~9~9 1997-0~-23 WO 96/19246 PCT/US95/16~54 Rl Al-Val-Ser-Glu-Ile-Gln-A7-Nle-His-Asn-Leu-A12-Lys-His-Leu-Al6-ser-Nle-Alg-Asn-A2l-A22-A23 Leu Arg Lys- Lys-Leu-Gln-Asp-Val-His-Asn-A34-W

wherein:
A1 is Ser or Ala;
A7 is Leu or Phe;
A12 is Gly, Aib, Ala, or D-Ala;
A16 is Asn, Ser or Ala;
Alg is Glu, Arg, Lys, Asp, Ser, Thr, Gln, Asn, or Ala;
A21 is Val, Met, Arg, Lys, Glu, Asp, Ser, Thr, Gln, Asn, Leu, Ile, Nle, Ala, Phe, or p-X-Phe where X is OH, CH3, N02, or a halogen;
A22 is Glu, Asp, Phe, p-X-Phe where X is OH, CH3, NO2 or a halogen, Ser, Thr, Gln, Asn, Leu, Ile, Nle, Val, 20 Ala, or Met;
A23 is Trp, 1-Nal, or 2-Nal;
A34 is Phe, or p-X-Phe where X is OH, CH3, N02, or a halogen;
W is OH~ Cl-12 alkoxy, C7_20 phenylalkoxy~ Cl1 20 25 napthylalkoxy, or NR3R4; provided that when A12 is Gly, Alg is Glu, A21 is Val, and A22 is Gln, then A23 must be 1-Nal or 2-Nal; and each of R1, R2, R3, and R4 is, independently, H, Cl_l2 alkyl, C7_10 phenylalkyl, or CO E where E is C1_12 30 alkyl, C2_12 alkenyl, phenyl, naphthyl, or C7_20 phenylalkyl; or a pharmaceutically acceptable salt thereof.
In the above formula, the N-terminus is at the left and the C-terminus at the right in accordance with 35 the conventional representation of a polypeptide chain.

CA 0220~9~9 1997-0~-23 WO96/19~6 PCT~S95/16554 Also, The symbol Al, Val, A7, or the like in a peptide sequence stands for an amino acid residue, i.e., =N-CH(R)-CO- when it is at the N-terminus or -NH-CH(R)-CO-when it is not at the N-terminus, where R denotes the 5 side chain of that amino acid residue. Thus, R is -CH(CH3)2 for Val. Also, when the amino acid residue is optical]y active, it is the L-form configuration that is intendecl unless D-form is expressly designated. Further, the symbols Aib, 1-Nal, and 2-Nal herein are lO abbreviations for ~-aminoisobutyric acid, 3-(l-naphthyl) alanine, and 3-(2-naphthyl)alanine, respectively.
Preferred groups of compounds covered by the above formula include (i) those where Al is Ala; A7 is Phe; and A16 is Ser; (ii) those where A19 is Glu or Arg; and A21 is 15 Val or Arg; and (iii) those where A22 is p-X-Phe where X
is OH, ~H3, NO2, or a halogen. Below are particularly pre-ferred compounds: [Nle8~18,Arg19,Tyr34]bPTH1_34 NH2, tNle8~18 Arg2l~Tyr34]bPTH1_34 NH2, [Nle8~18 Argl9~2l~Tyr34]bpTHl-34 NH2~ and [Nle8~18,Phe22,Tyr34]bPTH1_34 NH2. Such compounds, as PTH
or PTHrP agonists, can be used to promote bone formation in a manner described above.
Other features and advantages of the present invention will be apparent from the following description 25 of the preferred embodiments, and also from the appending claims.

~ESCRIPTION OF PREFERRED EMBODIMENTS
The synthesis, selection and use of PTH, PTHrP or agonists thereof which are capabie of promoting bone 30 formation are within the ability of a person of ordinary skill in the art.

CA 0220~9~9 1997-0~-23 WO 96119246 PCT/US9~i/16~54 For example, well-known in vitro or in vivo assays can be used to determine the efficacy of various PTH/PTHrP agonists to promote bone formation in human patients. For in vitro binding assays, osteoblast-like 5 cells which are permanent cell lines with osteoblastic characteristics and possess receptors for PTH/PTHrP of either rat or human origin can be used. Suitable osteoblast-like cells include ROS 17/2 (Jouishomme, H., et al., Endocrinology, 1992, 130, 53-60), UMR 106 (Fujimori, A., et al., Endocrinology, 1992, 130, 29-60) and the human derived SaOS-2 (Fukuyama, S., et al., Endocrinology, 1992, 131, 1757-1769). The cell lines are available from American Type Culture Collection, Rockville, Maryland, and can be maintained in standard 15 specified growth media. Additionally, transfected human embryonic kidney cells (HEK 293) expressing the human PTC
receptor can also be utilized for in vitro binding assays. See Pines, et al., Endocrinology, 1994, 135, 1713-1716.
For in vitro functional assays, PTH and PTH-like agonist activities of peptide fragments or derivatives of parathyroid hormone or PTHrP can be tested by contacting a concentration range of the test compound with the cells in culture and assessing the stimulation of the PTH/PTHrP
25 receptors. Receptor stimulation is evidenced by the activation of second messenger molecules coupled to the receptors, for example, a stimulation of cyclic AMP
accumulation in the cell or an increase in enzymatic activity of protein kinase C, both of which are readily 30 monitored by conventional assays (Jouishomme, H., et al., Endocrinology, 1992, 130, 53-60; Abou-Samra, A.B., et al., Endocrinology, 1989, 125, 2594-2599; Fujimori, A., et al., Endocrinology, 1991, 128, 3032-3039; Fukayama, S., Et al., Endocrinology, 1994, 134, 1851-1858; Abou-35 Samra, A.B., et al., Endocrinology, 1991, 129, 2547-2554;

CA 0220~9~9 l997-0~-23 WO96/19246 PCT~S95116554 and Pines, et al., Endocrinology, 1994, 135, 1713-1716).
Other parameters of PTH action include increase in cytosolic calcium and phosphoinositols, and biosynthesis of collagen, osteocalcin, and alteration in alkaline 5 phosphatase activity.
PTH-like agonist activities of subfragments of PTH
have been successfully analyzed by contacting peptides with rat kidney cells in culture and assessing cyclic AMP
accumulation (Blind, E., et al., Clin. Endocrinol., 1993, lO 101, 150-155) and the stimulation of 1,25-dehydroxyvitamin D3 production (Janulis, M., et al., Endocrinology, 1993, 133, 713-719).
The following specific embodiments are, therefore, to be construed as merely illustrative, and not 15 limitative of the remainder of the disclosure in any way whatsoever. Furthermore, all of the references cited in this disclosure are incorporated by reference.

Example 1 The PTH or PTHrP agonists of the invention were 20 synthesized on an APPLIED BIOSYSTEMS~ 430A Automated Peptide Synthesizer (Applied Biosystems Inc., Foster City, CA) using version 1.40 of the software for NMP/HOBt Boc-based chemistry. The following side-chain protected amino acid derivatives were used in the course of the 25 synthesis: N-Boc-Arg(NG-Tosyl)-OH, N-Boc-Asp(cycHx)-OH, N-Boc-Glu(OBzl)-OH, N-Boc-His(Bom)-OH, N-Boc-Lys(Cl-Z)-OH, N-Boc-Ser(Bzl)-OH, N-Boc-Thr(Bzl)-OH, N-Boc-Tyr(Br-Z)-OH, and N-Boc-Trp(N'-For)-OH.
Cleavage from resin with concomitant removal of 30 the side-chain protecting groups of pMBHA-R-bound peptide was performed by liquid HF in the presence of 10% anisole ~20 mL/g resin-bound peptide) 1 hr at 0~C. Low-high HF
procedures shall be used for clevage from resin and deprotection for PTH agonists containing Trp. See Tam, CA 0220~9~9 1997-0~-23 WO 96/19246 PCT/US9~/16!554 et al., J. Am. Chem. Soc., 1983, 105, 6442. The resin-crude peptide mixture was washed with petroleum ether and ether. The dry resin-crude peptide mixture was extracted ~ consecutively with 5096 acetic acid and water. The 5 combined washes were lyophilized. The lyophilized crude peptides were subjected to preparative RP-HPLC
purification.
The full names for the abbreviations used above are as follows: Boc for t-butoxycarbonyl, For for 10 formayl, cycHx for cyclohexyl, Cl--Z for 2--chlorobenzyloxycarbonyl, OBzl is O-benzyl, BOM for benzyloxymethyl, Bzl for benzyl, Br-Z for 2-bromo-benzyloxycarbonyl, NG-Tosyl for tosyl at guanidyl site, and pMBHA-R for paramethoxybenzhydrylamine resin.
The crude peptides were purified on a WATERS DELTA
PREPTY 4000 (Waters, Milford, MA), preparative HPLC
system, connected to a PrepPack cartridge of Vydac~ C18 300A, 15-20 ,~Lm [47 x 300 mm] (Waters, Milford, MA) at a flow rate of 70 mL/min monitored at 220 nm. The - 20 analytical HPLC system included the following components:
Waters 600E multisolvent delivery system, 490E
programmable multiwavelength detector, 717 autosampler and a 747 data module. The samples were analyzed on a VYDAClY C18 218TP5415 (150 x 4.6 mm, 5 ,um), The Separation 25 Group (Hesperia, CA) at a flow rate of 1 mL/min monitored at 220 nm. The solvent mixtures for both the analytical and preparative HPLC were: A: 0.1% TFA in H2O, and B:
0.1% TFA in acetonitrile. The purity of the peptides and their derivatives exceeded 99% as determined from the 30 analytical RP--HPLC.
The following four bovine PTH agonists were synthesized:
[Nle8~18,Phe22,Tyr34]bPTHl_34 NH2 (Analog I);
tNle8~18~Argl9~Tyr34]bPTHl_34 NH2 (Analog II);
[Nle3~18,Arg21,Tyr34]bPTHl_34 NH2 (Analog III); and WO96/19~6 PCT~S95/16554 [Nle8~lg~Argls~2lrTyr34]bpTHl-34 NH2 (Analog IV)-These four analogs were analyzed using HPLC
(gradient of 30 min) and the results are shown in Table I
below. For each solvent gradient, only the intitial and final percentages of B of the solvent (which consists of both A and B -- see above for their compositions) are indicated.

ANALOG RETENTION TIME SOLVENT GRADIENT
(min.) (%) lo I 20.6 25 - 45 II 14.2 25 - 50 III 13.6 25 - 50 IV 17.1 20 - 50 The results of the amino acid analysis of the four 15 analogs are shown in Table II below, which lists both calculated and found values.

CA 02205959 l997-05-23 WO 96119246 PCrlUS95/16554 TABLE II

Cslcd. 3.00 3.00 3.00 3.00 Asx Found 3.23 3.09 3.16 3.22 Calcd. 3,00 3.00 3.00 3,00 Ser Found 2.75 2.54 2.48 2.86 Calcd. 4.00 4.00 S.00 4.00 GL~
Found 4.27 4.27 5.35 4.34 Calcd. 1.00 l.oo 1.00 1.00 Gly Found 1.03 1.08 1.01 1.07 Calcd. 1.00 1.00 1.00 1.00 Ala Found 1.04 1.03 0.97 0.95 Calcd. 3.00 3.00 2.00 2.00 Val Found 2.80 3.03 1.97 1.68 Csled. 1.00 1.00 1.00 1.00 lle l~ound 0.99 .096 0.91 0.81 Caled. 4.00 4.00 4.00 4.00 Leu Found 4.24 432 4.11 4.25 Calcd. 2.00 2.00 2.00 2.00 Nle Found 1.61 150 1.94 2.19 Calcd. 1.00 1.00 1.00 1.00 Tyr Found 1.15 1.06 1.01 1.10 Caled. 2.00 1.00 1.00 1.00 Phe Found 2.06 0.97 1.00 0.93 Calcd. 3.00 3.00 3.00 3.00 His Found 2.85 3.03 2.93 2.64 Calcd. 3.00 3.00 3 00 3 00 Lys Found 3.00 2.93 3.05 2.90 Calcd. 2.00 3.00 3.00 4.00 A~g Found 1.97 3.20 3.11 4.04 CA 0220~9~9 1997-0~-23 WO96/1924~ PCT~S95/16554 Table III demonstrates the FAB (fast-atom bombardment) mass spe~LL~l~ter analysis of the analogs.

TABLE III

ANALOG MEASURED PREDICTED
I 4106.8 4105.8 II 4114.7 4114.8 III 4146.4 4144.8 IV 4173.5 4172.8 Example 2 SaOS-2 B10 cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37~C in a humidified atmosphere of 5 C~2 in air. The medium was changed every three or four days, and the cells were subcultured every week by 15 trypsinization.
SaOs-2 B10 cells were maintained for four days after they had reached confluence. The medium was replaced with 5% FBS RPS/1640 medium and incubated for 2 hrs at room temperature with 10 x 104cpm mono-125~I-20 [Nle8~18,Tyr34(3-Il25)]bPTHl_34 NH2 in the presence or absence of a competing tested PTH agonist. The cells were washed four times with ice-cold PBS and lysed with 0.1 M NaOH, and the radioactivity associated with the cells was counted in a scintillation counter. Synthesis 25 of the radiolabelled [Nle8~18. Tyr34 (3-Il25)]bPTHl_34NH2 was carried out as described in Goldman ME et al., Endocrinology, 1988, 123, 1468-1475.
The binding assay was conducted on Analogs I-IV
and Analog V (i.e., [Nle8, Nle18, Tyr34]bPTHl_34 NH2). The 30 IC50's (half maximal inhibition of binding of mono-l25I-CA 0220~9~9 1997-0~-23 WO 96/19~46 PCT/US95/16554 [Nle8~18,Tyr34(3-I125)]bPTHl_34 NH2) for the five tested analogs were calculated and shown in Table IV below:
TABLE IV
., ANALOG IC50(nM) I 1.5 II 0.6 III 0.3 IV 0.3 V 1.5 10 Example 3 The adenylate cyclase activity induced by each of Analogs I-V was also measured in SaOS-2 B10 cells as described previously (Rodan et al. 1983 J. Clin. Invest.
72;1511; Goldman et al. 1988 Endocrinology 123, 1468).
15 Confluent SaOS-2 B10 cells in 24 wells plates were incubated 0.5 ~Ci[3H]adenine (26.9 Ci/mmol, New England Nuclear, Boston, MA) in fresh medium at 37~C for 2 hrs, and washed twice with Hank's solution. The cells were treated with 1 mM IBMX [isobutylmethylxanthine, Sigma, 20 St. Louis, MO] in fresh medium for 15 min, and a tested PTH analog was added to the medium to incubate for 5 min.
The reaction was stopped by the addition of 1.2 M TCA
followed by sample neutralization with 4 N KOH. Cyclic AMP was isolated by the two-column chromatographic method (see Salomon et al. 1974 Anal. Biochem. 58;541). The radioactivity was counted in a scintillation counter (Liguid scintillation counter 2200CA, PACKARD, Downers Grove, IL). The EC50's (half maximal stimulation of adenylate cyclase) were calculated for the five PTH
30 analogs and are shown below:

CA 0220~9~9 1997-0~-23 WO96/19~6 PCT~S95116554 TABLE ~

ANALOG EC50(nM) I 1.5 II 2.0 III 0.2 IV 0.5 V 2.0 Example 4 In vivo bone anabolic activities of PTH/PTHrP
10 agonists are tested by administering the peptide or a formulation containing the peptide into intact animals or an experimental animal model of osteopenia. The animal model can be osteoporosis in rats induced by ovariectomy (Hori, M., et al., Bone Miner., 1988, 3, 193-199; Geral, 15 et al., J. Bone Miner. Res., 1989, 4, Suppl. 1, S303; Liu C-C. & ]~alu, D.N., J. Bone Miner. Res., 1990, 5, 973-982;
Mosekilde, L., et al., Endocrinol., 1991, 129, 421-428;
Wronski, T.J., Yen C-F. Bone, 1994, 15, 51-58; Reviewed in Demster D.W., et al., Endocrine Rev., 1993, 14, 690-20 709) The bone anabolic effects of the compound aredetermined following 12 to 60 days of treatment by assessing the change in bone mineral density by dual energy x-ray absorptiometry or dry weight of femurs or 25 total ash weight (Hori, H., et al., Bone Miner., 1988, 3, 193-199; Hefti, E., et al., Clin. Sci., 1982, 62, 389-396). Increase in the rate of bone formation and mineralization are assessed using metabolic labels, e.g.
tetracycline (Tam, C.S., et al., Endocrinology, 1982, 30 110, 506-512). Qualitative and quantitative evaluations of changes in trabecular/cortical bone volume and CA 0220~9~9 1997-0~-23 WO96/19246 PCT~S9~116554 complexity are determined by standard hlstomorphometric methods (Wronskl, T.J., Yet C-F, Bone, 1994, 15, 51-58;
Tam C.S., et al., Endocrinology, 1982, 110, 506-612;
Podbesek, R., et al., Endocrinology 1983, 112, 1000-1006) 5 of bone samples from control (untreated) and treated s~n;r~ ~ls.

Example 5 The anabolic efficacy of the PTH/PTHrP agonists are tested in humans (Review in Dempster D.W., et al., lo Endocrine Rev., 1993, 14, 690-709). To determine if continuous administration of hPTHl_34 or its agonist is effective in promoting bone growth in humans, 30 post-menopausal women with established osteoporosis based on bone-density measurements are selected. A double-blind, 15 placebo-controlled, randomized experiment is conducted where two groups of 15 women each are either placed on placebo treatment or continuous infusion of a defined dose of hPTHl_34 (25 units to 400 units/24 hrs) using an infusion pump.
The patients prior to the study are subject to the following: complete health and physical GXAm;nation, evaluation of the nutritional status (particularly of calcium intake and serum calcium), full analysis of biomarkers for bone-turnover (Riis, B.J., Amer. J. Med.
25 1993, 95 [Suppl 5A], 17s-21s; Delmas, P.D., Amer. J.
- Med., 1951, 91 [Suppl 5B], 59s-63s), radiology, bone mass measurements of vertebral and axial sites (Gerant, H.K., et al., Amer. J. Med., 1991, 91 [Suppl 5B], 49s-53s;
Wasnich, R.D., Amer J Med, 1991, 91 [Suppl 5B], 54s-58s) 30 and bone biopsy, to establish the baseline parameters for each individual. After one and three months of treatment, the patients are reevaluated for changes in serum calcium and biomarkers of bone turnover to determine the outcome of continuous hPTHl_34 CA 0220~9~9 1997-05-23 WO96/19246 PCT~S95/16554 administration. When the biomarker analysis suggests an increase in osteoblast activity (e.g., increase in serum alkaline phosphatase and serum osteocalcin), the treatments are extended to twelve months, wherein bone 5 mass measurements and bone biopsy further provide clear indication of bone growth in the population treated with continuous infusion hPTHl_34.

OTHER EMBODIMENTS
From the above description, one skilled in the art lO can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims (20)

- 19 -

1. A method of promoting bone formation in a human patient, said method comprising administering continuously to said patient PTH, PTHrP, or an agonist thereof for a period of at least one month at a dosage between 10 and 400 units/24 hrs.

2. A method of claim 1, wherein said dosage is 10-300 units/24 hrs.

3. A method of claim 1, wherein said dosage is 10-200 units/24 hrs.

4. A method of claim 1, wherein said dosage is 10-100 units/24 hrs.

5. A method of claim 1, wherein said dosage is 100-400 units/24 hrs.

6. A method of claim 1, wherein said dosage is 200-400 units/24 hrs.

7. A method of claim 1, wherein said dosage is 300-400 units/24 hrs.

8. A method of claim 1, wherein said agonist is hPTH1-34 NH2 or [Nle8,18, Tyr34]hPTH1-34 NH2.

9. A method of claim 1, wherein said agonist is bPTH1-34 NH2, [Nle8-18,Tyr34]bPTH1-34 NH2, [Nle8,18,Phe22, Tyr34]bPTH1-34 NH2, [Nle8,18,Arg19,Tyr34]bPTH1-34 NH2, [Nie8,18,Arg21,Tyr34]bPTH1-34 NH2, or [Nle,8,l8,Arg19,21 , Tyr34]bPTH1-34 NH2.

10. A method of claim 1, wherein infusion is performed to administer said PTH, PTHrP, or an agonist thereof.

11. A method of claim 10, wherein said infusion is achieved with an ambulatory or implantable infusion pump.

12. A method of claim 1, wherein said PTH, PTHrP, or an agonist thereof is administered as a sustained release formulation.

13. A method of claim 12, wherein said sustained release formulation comprising a polymer prepared from lactic acid, caprolactone, glycolide, or glycolic acid.

14. A method of claim 1, further comprising the step of administering to said patient a bone resorption inhibiting agent.

15. A compound having the formula:

Lys-His-Leu-A16-ser-Nle-A19-Asn-A21-A22-A23-Leu-Arg-Lys- Lys-Leu-Gln-Asp-Val-His-Asn-A34-W

wherein:
A1 is Ser or Ala;
A7 is Leu or Phe;
A12 is Gly, Aib, Ala, or D-Ala;
A16 is Asn, Ser or Ala;

A19 is Glu, Arg, Lys, Asp, Ser, Thr, Gln, Asn, or Ala;
A21 is Val, Met, Arg, Lys, Glu, Asp, Ser, Thr, Gln, Asn, Leu, Ile, Nle, Ala, Phe, or p-X-Phe where X is OH, CH3,NO2, or a halogen;
A22 is Glu, Asp, Phe, p-X-Phe where X is OH, CH3, NO2 or a halogen, Ser, Thr, Gln, Asn, Leu, Ile, Nle, Val, Ala, or Met;
A23 is Trp, 1-Nal, or 2-Nal;
A34 is Phe, or p-X-Phe where X is OH, CH3, NO2, or a halogen;
W is OH, C1-12 alkoxy, C7-20 phenylalkoxy, C11-20 napthylalkoxy, or NR3R4; provided that when A12 is Gly, A19 is Glu, A21 is Val, and A22 is Gln, then A23 must be 1-Nal or 2-Nal; and each of R1, R2, R3, and R4 is, independently, H, C1-12 alkyl, C7-10 phenylalkyl, or CO~E where E is C1-12 alkyl, C2-12 alkenyl, phenyl, naphthyl, or C7-20 phenylalkyl; or a pharmaceutically acceptable salt thereof.

16. A compound of claim 15, wherein:
A1 is Ala;
A7 is Phe; and A16 is Ser; or a pharmaceutically acceptable salt thereof.

17. A compound of claim 15, wherein:
A19 is Glu or Arg; and A21 is Val or Arg, or a pharmaceutically acceptable salt thereof.

18. A compound of claim 15, wherein:
A22 is p-X-Phe where X is OH, CH3, NO2, or a halogen; or a pharmaceutically acceptable salt thereof.

19. A compound of claim 15, wherein said compound is [Nle8,18,Arg19,Tyr34]bPTH1-34 NH2, [Nle8,18,Arg21, Tyr34]bPTH1-34 NH2, or [Nle8,18,Arg19,21,Tyr34]bPTH1-34 NH2;
or a pharmaceutically acceptable salt thereof.

20. A compound of claim 15, wherein said compound is [Nle8,18,Phe22,Tyr34]bPTH1-34 NH2; or a pharmaceutically acceptable salt thereof.