NOVEL PARATHYROID HORMONE-RELATED PEPTIDE ANALOGS

Abstract: The present invention relates to novel PTHrP analogs that have been converted into potent PTH-2 receptor agonists or antagonists by the substitution of one or more amino acid residues of PTHrP to the corresponding residue(s) of PTH. A method of treating various diseases states associated with altered function of the PTH-2 receptor, using these PTHrP analogs, is also described.

Description: Novel Parathyroid Hormone-Related Peptide Analogs Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government has certain rights in this invention. Background of the Invention Field of the Invention The present invention relates to novel parathyroid hormone-related peptide (PTHrP) analogs. In particular, the invention relates to PTHrP analogs having one or more amino acid substitutions that confer PTH-2 receptor antagonist or agonist properties to the analog. Description of Related Art Parathyroid hormone (PTH) is a major regulator of calcium homeostasis whose principal target cells occur in bone and kidney. Regulation of calcium concentration is necessary for the normal function of the gastrointestinal, skeletal, neurologic, neuromuscular, and cardiovascular systems. PTH synthesis and release are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH, in turn, maintains the serum calcium level by directly or indirectly promoting calcium entry into the blood at three sites of calcium exchange: gut, bone, and kidney. PTH contributes to net gastrointestinal absorption of calcium by favoring the renal synthesis of the active form of vitamin D. PTH promotes calcium resorption from bone by inhibiting osteoblasts and, indirectly, by stimulating differentiation of the bone-resorbing cells, osteoclasts. It also mediates at least three main effects on the kidney: stimulation of tubular calcium reabsorption, enhancement of phosphate clearance, and promotion of an increase in the enzyme that completes synthesis of the active form of vitamin D. PTH exerts these effects primarily through receptor-mediated activation of adenylate cyclase, although receptor-mediated activation of phospholipase C by PTH has also been reported (Hruska et al., J. Clin. Invest. 79:21,0 (1987)). Disruption of calcium homeostasis may produce many clinical disorders (e.g., severe bone disease, anemia, renal impairment, ulcers, myopathy, and neuropathy) and usually results from conditions that produce an alteration in the level of parathyroid hormone. Hypercalcemia is a condition that is characterized by an elevation in the serum calcium level. It is often associated with primary hyperparathyroidism in which an excess of PTH production occurs as a result of a lesion (e.g., adenoma, hyperplasia, or carcinoma) of the parathyroid glands. Another type of hypercalcemia, humoral hypercalcemia of malignancy (HHM) is the most common paraneoplastic syndrome. It appears to result in most instances from the production by tumors (e.g., squamous, renal, ovarian, or bladder carcinomas) of a novel class of protein hormone which shares amino acid homology with PTH. These PTH-related proteins (PTHrP) appear to mimic certain of the renal and skeletal actions of PTH and are believed to interact with the PTH receptor in these tissues. PTHrP is normally found at low levels in many tissues, including keratinocytes, brain, pituitary, parathyroid, adrenal cortex, medulla, fetal liver, osteoblast-like cells, and lactating mammary tissues. In many HHM malignancies, PTHrP is found in the circulatory system at high levels, thereby producing the elevated calcium levels associated with HHM. The pharmacological profiles of PTH and PTHrP are nearly identical in most in vitro assay systems, and elevated blood levels of PTH (i.e., primary hyperparathyroidism) or PTHrP (i.e., HHM) have comparable effects on mineral ion homeostasis (Broadus, A.E. & Stewart, A.F., "Parathyroid hormone-related protein: Structure, processing and physiological actions," in Basic and Clinical Concepts, Bilzikian, J.P. et al, eds., Raven Press, New York (1994), pp. 259-294; Kronenberg, H.M. et al., "Parathyroid hormone: Biosynthesis, secretion, chemistry and action," in Handbook of Experimental Pharmacology, Mundy, G.R. & Martin, T.J., eds., Springer- Verlag, Heidelberg (1993), pp. 185-201). The similarities in the biological activities of the two ligands can be explained by their interaction with a common receptor, the PTH/PTHrP receptor, which is expressed abundantly in bone and kidney (Urena, P. et al., Endocrinology 754:451-456 (1994)). The binding of either radiolabeled PTH-(l-34) or PTHrP-(l-36) to the PTH PTHrP receptor is competitively inhibited by either unlabeled ligand (Juppner, H. et al, J. Biol. Chem. 263:8557-8560 (1988); Nissenson, R.A. et al, J. Biol. Chem. 263: 12866-12871 (1988)). Thus, the recognition sites for the two ligands in the PTH/PTHrP receptor probably overlap. In both PTH and PTHrP, the 15-34 region contains the principal determinants for binding to the PTH/PTHrP receptor. Although these regions show only minimal sequence homology (only 3 amino acid identities), each 15-34 peptide can block the binding of either PTH-(l-34) or PTHrP-(l-34) to the PTH/PTHrP receptor (Nussbaum, S.R. et al, J. Biol. Chem. 255:10183-10187 (1980); Caulfield, M.P. etal, Endocrinology 127:83-87 (1990); Abou-Samra, A.-B. et al, Endocrinology 725:2215-2217 (1989)). Further, the amino terminal portion of each ligand is required for bioactivity, and these probably interact with the PTH/PTHrP receptor in similar ways, since 8 of 13 of these residues are identical in PTH and PTHrP. The PTH/PTHrP receptor is a member of a distinct family of G protein-coupled receptors (Juppner, H. et al, Science 254:1024-1026 (1991); Abou-Samra, A.B. et al, Proc. Natl. Acad. Sci (USA) 59:2732-2736 (1992)) that includes receptors for other peptide hormones such as secretin (Ishihara, T. et al, EMBOJ. 70:1635-1641 (1991)), calcitonin (Lin, H.Y. et al, Science 254:1022- 1024 (1991)) and glucagon (Jelinek, L.J. et al, Science 259:1614-1616 (1993)). Using degenerate oligonucleotides corresponding to conserved regions of the PTH/secretin/calcitonin receptor family, Usdin et al. has identified a new receptor cDNA derived from rat and human brain that was most closely related to the PTH/PTHrP receptor (51% overall amino acid sequence identity) (Usdin, T. et al, J. Biol. Chem. 270:15455-15458 (1995)). This new receptor, the PTH-2 receptor, responded efficiently, potently, and specifically to PTH-(l-34), but interestingly, did not respond at all to PTHrP. Id. This observation implied that structural differences in the PTH and PTHrP ligands determined selectivity for interaction with the PTH-2 receptor. The PTH-2 receptor was found predominantly in the brain and pancreas. Id. Since the PTH-2 receptor was found in brain and pancreas, it is likely to have a physiological role in the normal functioning of those organs. Diseases associated with brain and pancreatic dysfunction may in fact be explained by the altered action of the PTH-2 receptor. Such diseases could then be treated with a PTII-2 receptor selective antagonist. Other antagonists that are available are not selective and thus, are not desired because they would also antagonize the PTH/PTHrP receptor, which could have negative consequences in terms of calcium regulation and skeletal function. Accordingly, there is a need in the art for the development of PTH-2 receptor selective agonists and antagonists: (1) to assist in further elucidating the biological role of the PTH-2 receptor; (2) to map specific sites of ligand-receptor interaction; and (3) as potential new therapeutic compositions that can be used in the treatment of disorders associated with altered action or genetic mutation of the PTH-2 receptor. Summary of the Invention To begin to define residues involved in the ligand-specificity of the PTH-2 receptor, the inventors studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs, with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and He in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Tip in PTH) determined binding affinity. By changing these two residues of PTHrP (i.e., residue 5 and 23) to the corresponding residues of PTH, the inventors were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor, and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp 23 ]hPTHrP-(l-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Accordingly, the inventors concluded that residues 5 and 23 in PTH and PTHrP play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Thus, in accordance with one aspect of the present invention, there is provided a novel PTHrP analog that is a potent PTH-2 receptor agonist, as well as a potent PTH/PTHrP receptor agonist. In a preferred embodiment, the PTHrP analog is altered at residues 5 and 23 to the corresponding residues of PTH. Most preferably, the invention includes PTHrP analogs having an amino acid substitution of histidine for isoleucine at position 5 of PTHrP, as well as phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [He 5 , Trp 3 ]PTHrP-(l-36). In accordance with another aspect of the present invention, there is provided a novel PTHrP analog that is a potent PTH-2 receptor selective antagonist. In a preferred embodiment, the analog is altered at PTHrP residue 23 to the corresponding residue of PTH. Most preferably, the invention includes PTHrP analogs having a single amino acid substitution of phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Trp 23 ]PTHrP-(l-36). In accordance with yet a further aspect of the invention, there is provided a method for treating a medical disorder that results from altered or excessive action of the PTH-2 receptor, comprising administering to a patient a therapeutically efficient amount of a PTHrP analog, sufficient to inhibit activation of the PTH-2 receptor of said patient. In a preferred embodiment, the PTHrP analog used in the method has a single amino acid substitution of phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Trp 23 ]PTHrP-(l -36). In accordance with yet a further aspect of the invention, there is provided a method for treating osteoporosis, comprising administering to a patient a therapeutically efficient amount of a PTHrP analog, sufficient to activate the PTH/PTHrP receptor and PTH-2 receptor of said patient. In a preferred embodiment, the PTHrP analog used in the method has an amino acid substitution of histidine for isoleucine at position 5 of PTHrP, as well as phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [He 5 , Trp 23 ]PTHrP-(l-36). In addition, any other amino-acid substitutions at positions 5 and/or 23, which do not destroy the ability of the PTHrP analog to antagonize or agonize the PTH-2 receptor (as determined by assays known to the skilled artisan and discussed below), are included within the scope of the present invention. Brief Description of the Figures Figure I depicts ligand-binding and cAMP-signaling profiles of PTH/PTHrP and PTH-2 receptors. COS-7 cells transfected with human PTH/PTHrP receptors (Figures 1 A and 1 C) or human PTH-2 receptors (Figures IB and ID) were evaluated for inhibition of radioligand binding (Figures 1 A and I B), or ligand-induced cAMP accumulation (Figures 1C and ID). Binding studies were performed at room temperature (RT) for 2 hours. 125 I-[NleTyr 82l ,Tyr 34 ]rPTH-(l-34)NH 2 (100,000 CPM/well) was used as radioligand and varying amounts of unlabeled [Tyr 34 ]hPTH-(l-34)NH 2 (•) or [Tyr 36 ]hPTHrP-(l-36)NH 2 (■) were used as competitor ligands. Symbols are indicated in the figure key. The maximum amount of tracer that specifically bound to PTH/PTHrP and PTH-2 receptors in the absence of competitor (B 0 ) was -25% and -5% of total radioactivity added, respectively. For cAMP stimulation assays, cells were treated with ligand for 1 hour at room temperature in the presence of IBMX. Symbols in Figures 1 C and ID are the same as in Figures 1 A and IB. Data are the mean (± s.e.m.) of 3-10 experiments, each performed in duplicate. Figure 2 depicts a scatchard analysis of PTH-(l-34) binding to PTH/PTHrP or PTH-2 receptors. COS-7 cells transfected with plasmid DNA (200 ng/well) encoding either the human PTH/PTHrP receptor (Figure 2A) or human PTH-2 receptor (Figure 2B) were evaluated in competition-binding studies performed for six hours at 4°C. The radioligand was 125 I-[NleTyr 82, ,Tyr 3 ]rPTH-(l-34)amide (100,000 CPM/well), as a tracer , and the competitor ligand was the same unlabeled peptide. Data are the mean (± s.e.m.) of four experiments, each performed in duplicate. The insets show Scatchard plots of the individual data points from the linear portions of each experiment. The mean dissociation constants (kds) and B^ values, as receptors/cell, derived from these experiments are shown above the panel s. Statistical analysis indicated that the surface densities of the two receptors, but not the Kd values, differed significantly (p values = 0.0003, and 0.14, respectively, students t test). Figure 3 depicts the primary structures of PTH, PTHrP and hybrid ligands. The biologically active regions of PTH, PTHrP, and the PTH/PTHrP hybrid ligands used in this study are shown. Sequences corresponding to PTHrP are shaded. All peptides contained the carboxy-terminal modification of tyrosine-amide, and otherwise corresponded to the native human PTH or human PTHrP sequence. Figure 4 depicts the effects of substitutions at position 23 on the binding properties of PTH-( 15-34) and PTHrP-( 15-36) carboxy-terminal fragments. The binding properties of substituted and unsubstituted PTH-( 15-34) or PTHrP-( 15-36) to COS-7 cells expressing either PTH/PTHrP receptors (Figure 4A) or PTH-2 receptors (Figure 4B) are shown. Competition-binding studies were performed at room temperature for 2 hours with ,25 I-[NleTyr 8 ' 2, ,Tyr 34 ]rPTH-(l-34)NH 2 (100,000 CPM/well) as radioligand. The unlabeled competitor ligands used are indicated in the figure key, the structure of each was based on the human PTH or PTHrP sequence and contained tyrosine-amide as a carboxy-terminal residue. Data are the mean (± s.e.m.) of 3 or 4 experiments, each performed in duplicate. Figure 5 depicts the effects of substitutions at position 5 and 23 on the binding of PTH-( 1 -34) and PTHrP-( 1 -36) to PTH/PTHrP and PTH-2 receptors. The binding of substituted or unsubstituted PTH-(l-34) or PTHrP-(l-36) analogs to COS-7 cells expressing either PTH/PTHrP receptors (Figures 5A and 5C) or PTH-2 receptors (Figures 5B and 5D) are shown. Competition-binding studies (RT/2h) were performed with l25 I-[NleTyr 821 ,Tyr 34 ]rPTH-(l-34)amide (100,000 CPM/well) as radioligand. The unlabeled PTHrP (Figures 5A and 5B) and PTH (Figures 5C and 5D) analog ligands, used as competitors, are indicated in the figure keys; the structure of each was based on the human PTH or PTHrP sequence and contained tyrosine-amide as a carboxy-terminal residue. The binding curves of [Tyr 36 ]hPTHrP-(l-36)NH,, and [Tyr 34 ]hPTH-(l-34)NH 2 from Figures 1 A and B are shown again for comparison. Data are the mean (± s.e.m.) of 3-10 experiments, each performed in duplicate. Figure 6 depicts the effects of substitutions at positions 5 and 23 on cAMP signaling by PTΗ-(l-34) and PTHrP-(l-36) with PTH/PTHrP and PTH-2 receptors. The abilities of substituted and unsubstituted PTH-(l-34) or PTHrP-(l-36) peptide analogs to stimulate cAMP formation in COS-7 cells expressing either PTH/PTHrP receptors (Figures 6A and 6C) or PTH-2 receptors (Figures 6B and 6D) are shown. Cells were treated with the indicated amount of peptide for 1 hour at room temperature in the presence of IBMX. The PTHrP (Figures 6A and 6B) and PTH (Figures 6C and 6D) analog ligands used are indicated in the figure keys; each was based on the human PTH or PTHrP sequence and contained tyrosine-amide as a carboxy-terminal residue. The curves of [Tyr^hPTHrP-O^NH,, and [Tyr 34 ]hPTH-(l-34)NH 2 from Figures 1C and ID are shown again here for comparison. Data are the mean (± s.e.m.) of 3-5 experiments, each performed in duplicate. Figure 7 depicts the antagonism of PTH-(l-34)-induced cAMP formation in COS-7 cells expressing PTH/PTHrP or PTH-2 receptors. COS-7 cells expressing either PTH/PTHrP or PTH-2 receptors were treated with varying doses of [Leu",D-Tφ 12 ]hPTHrP(7-34)NH 2 or [Trp 23 ,Tyr 36 ]hPTHrP-(l-36) ) plus a near-maximal stimulatory dose of the agonist, [Tyr 34 ]hPTH-(l-34)NH 2 (1 nM), and then incubated in the presence of IBMX for 30 minutes at room temperature. The resulting intracellular cAMP levels are expressed as the percent of the cAMP levels in cells treated with [Tyr 34 ]hPTH-(l-34)NH 2 alone (1 nM), which were 167±17 and 70±8 pmoles/well for the PTH/PTHrP receptor and the PTH-2 receptor, respectively. The corresponding basal cAMP levels in cells not treated with peptide were 8.4 ±0.3 and 9.0 ±11.7 pmoles/well, respectively. Data are the mean (± s.e.m.) of two experiments, each performed in duplicate. It is of interest to note that these hybrid ligands, as well as [H5]PTH-(l-34), competed very poorly for binding to the PTH/PTHrP receptor, yet efficiently stimulated cAMP formation (Table 1 ). The inventors have found that radioiodinated hybrid-5 peptide binds efficiently to the PTH/PTHrP receptor in COS-7 cells (specific binding -8% of total radioactivity added. It thus appears that these altered ligands bind to the PTH/PTHrP receptor, but at a site that does not fully overlap with that used by 125 I-[NleTyr 82l ,Tyr 34 ]rPTH-(l-34)NH 2 . Table 1 Receptor-binding properties of PTH and PTHrP analogs COS-7 cells transfected with plasmids encoding the human PTH/PTHrP receptor or the human PTH-2 receptor were evaluated in competition binding studies that used [Nle 821 , Y 34 ]rPTH-(l-34)NH 2 as radioligand and the indicated unlabeled ligands as competitors. Assays were performed for 2 hours at room temperature. Data are the mean (± s.e.m.) of IC 50 values obtained from the number of experiments indicated (n). co x [Nle8, 21, Y34]rPTH-(l-34)NH2 58±4 5 1 1±2 5 m [Y34]hPTH-(l-34)NH2 39±4 7 16±4 7 31 c 10 [Y36]hPTHrP-(l-36)NH2 285±47 10 2,140±250 10 r- m ro Hybrid- 1 hPTHrP-(l - 14)/[Y34]hPTH-( 15-34)NH2 7,900±370 606±96 Binding IC50 (nM) PTH/PTHrP PTH-2 Ligand receptor receptor Hybrid-5 hPTHrP-(l - 18)/[Y34]hPTH-( 19-34)NH2 4,970±1,560 4 500±62 4 Hybrid-4 hPTHrP-(l-21)/[Y34]hPTH-(22-34)NH2 19±6 4 11±3 4 CO C ς • -> Hybrid -3 co H H hPTHrP-( 1 -23)/[Y34]hPTH-(24-34)NH2 1,000±471 4 6,050±990 4 C H m Hybrid-2 co x m hPTH-( 1 - 14)/[Y34]hPTHrP-( 15-34)NH2 497±144 4 750±210 4 m H 3 [Y36]hPTHrP-(l 5-36)NH2 > 10,000 4 4,970±970 4 r c- m 10 [W23, Y36]hPTHrP-(15-36)NH2 > 10,000 4 370±100 4 ro σ» [E22, W23, Y36]hPTHrP-(15-36)NH2 > 10,000 4 137±36 4 [Y34]hPTH-(15-34)NH2 > 10,000 624±1 16 Binding IC50 (nM) PTH/PTHrP PTH-2 Ligand receptor receptor [F23, Y34]hPTH-(15-34)NH2 > 10,000 > 10,000 [W23, Y36]hPTHrP-(l-36)NH2 47±8 5 30±7 5 [15, Y36]hPTHrP-(l-36)NH2 41±11 6 695±171 6 CO c 03 [15, W23, Y36]hPTHrP-(l-36)NH2 16±3 5 lO±l 5 CO H 3 [F23, Y34]hPTH-(l-34)NH2 95±11 4 453±53 4 H 5 m co [H5, Y34]hPTH-(l-34)NH2 5,100±1,000 4 249±18 4 x m m [H5, F23, Y34]hPTH-(l-34)NH2 >10,000 3 >10,000 3 3) c r- m ro Detailed Description of the Preferred Embodiments Previously, the present inventors had prepared a series of PTH/PTHrP hybrid ligands for the purpose of investigating whether homologous domains of PTH and PTHrP could be interchanged (Gardella, T . et al, J. Biol Chem. 270:6584-6588 (1995)). The present inventors have now used these hybrid ligands and derivative peptide analogs to map regions of PTH and PTHrP that contribute to the ligand selectivity of the PTH-2 receptor. These efforts have led to the identification of two residues in the ligands that play major roles in determining the efficiency of binding and signaling interactions with the PTH-2 receptor. The present invention provides a novel PTHrP analog that is a potent PTH-2 receptor agonist, as well as a potent PTH/PTHrP receptor agonist. In a preferred embodiment, the PTHrP analog is altered at residues 5 and 23 to the corresponding residues of PTH. Most preferably, the invention includes PTHrP analogs having an amino acid substitution of histidine for isoleucine at position 5 of PTHrP, as well as phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [He 5 , Trp 23 ]PTHrP-(l-36). In accordance with another aspect of the present invention, there is provided a novel PTHrP analog that is a potent PTH-2 receptor selective antagonist. In a preferred embodiment, the analog is altered at PTHrP residue 23 to the corresponding residue of PTH. Most preferably, the invention includes PTHrP analogs having a single amino acid substitution of phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Trp 23 ]PTHrP-(l-36). In addition, any other amino-acid substitutions of a nature, which do not destroy the ability of the PTHrP analog to antagonize or agonize the PTH-2 receptor (as determined by assays known to the skilled artisan and discussed below), are included in the scope of the present invention. The following Example is illustrative, and is not intended to be limiting. Materials and Methods Peptides and reagents The preparation and initial characterization of PTH/PTHrP hybrid ligands was described previously (Gardella, T.J. et al, J. Biol. Chem. 270:6584-6588 (1995)). [Nle 821 , Tyr 34 ]rat(r)PTH(l-34)NH 2 was purchased from Bachem Fine Chemicals (Torrance, CA). The antagonist, [Leu",D-Trp 12 ] hPTHrP(7-34)NH 2 , was purchased from Peninsula Laboratories (Belmont, CA). All other peptides were prepared by the biopolymer synthesis facility at Massachusetts General Hospital (Boston, MA) using solid-phase chemistry and F-moc protecting groups. ,25 I-[Nle 82, ,Tyr 34 ]rPTH-( 1 -34)NH 2 was prepared by chloramine-T iodination, and was HPLC-purified (Shigeno, C. et al, J. Biol. Chem. 265:3872-3878 (1988)). [ l25 I]-Na (2,000 Ci/mmol) was purchased from DuPont/New England Nuclear (Boston, MA). Dulbecco's modified Eagle's medium (DMEM), EGTA/trypsin and lOOx antibiotic mixture (10,000 units/ml penicillin G and 10 mg/ml streptomycin) was from GIBCO; fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT). DNA transfection of COS-7 cells The cDNAs encoding the human PTH/PTHrP receptor (Schipani, E. et al. , Endocrinology 752:2157-2165 (1993)) and the human PTH-2 receptor (Usdin, T. et al, J. Biol. Chem. 270:15455-15458 (1995)) were carried on the expression vectors pcDNA-1 and pcDNAI/Amp (InVitrogen, San Diego, CA), respectively. COS-7 cells were cultured and transfected as described previously (Lee, C. et al, Mol. Endocrinol. 9:1269-1278 (1995)). Cells were transfected in 24-well plates using plasmid DNA (200 ng/well) purified by cesium chloride/ethidium bromide gradient centrifugation. Ligand binding and cAMP accumulation assays were performed three days after transfection, by which time the cell density reached 500,000 ± 100,000 cells/well. Radioligand-receptor binding Binding reactions were performed as described previously. Id. Each well (final volume: 300 ml) contained 26 fmole of ,25 I-[Nle 82l ,Tyr 34 ]rPTH(l-34)NH 2 (100,000 CPM) and various amounts (0.4-300 pmole) of an unlabeled competitor ligand, both diluted in binding buffer (50 mM Tris-HCl, pH 7.7, 100 mM NaCl, 5 mM KC1, 2 mM CaCl, 5% heat-inactivated horse serum, 0.5% heat-inactivated fetal bovine serum). Incubations were at room temperature for 2 hours, except for experiments involving Scatchard analysis, which were performed at 4°C for 6 hours. At the end of the binding reaction, the cells were rinsed 3 times with 0.5 ml of binding buffer, lysed with 0.5 ml of 5M NaOH, and the entire lysate was counted. Nonspecific binding of tracer (NSB), determined in wells containing 1 μM of [NleTyr 821 ,Tyr 34 ]rPTH-(l-34)NH 2 , was -1% of total counts added. Maximum specific binding (B 0 ) was calculated as the total radioactivity bound to cells in the absence of unlabeled PTH ligand, minus NSB. IC 50 values (the dose of competing ligand that resulted in 50% inhibition of ,25 I-[NleTyr 8 - 21 ,Tyr 34 ]rPTH-(l-34)NH 2 binding) were determined from plots of log(B/B 0 -B) vs. log[competitor]. For Scatchard analyses, homologous competition binding studies were performed that used l25 I-[NleTyr 8 - 2, ,Tyr 34 ]rPTH-(l-34)NH 2 (26 fmole/well) and varying amounts (1.2-300 pmol) of the same unlabeled ligand. Estimates of the number of receptors/cell derived from these studies assumed a single class of binding sites and a transfection efficiency of 20% (Abou-Samra, A.B. et al, Proc. Natl. Acad. Sci (USA) 59:2732-2736 (1992)). Intracellular cyclic AMP Transfected COS-7 cells were rinsed with 500 ml of binding buffer. 200 μl of IBMX buffer (DMEM containing 2 mM IBMX, 1 mg/ml bovine serum albumin, 35 mM Hepes-NaOH, pH 7.4) and 100 ml of binding buffer or binding buffer containing various amounts of a PTH or PTHrP analog were then added. The plates were incubated for 60 minutes at room temperature. The buffer was then withdrawn and the cells lysed by placing the plates on powdered dry ice and adding 0.5 ml of 50 mM HC1. The acid lysate was diluted 1/30 in dH 2 O, and an aliquot (5-50 μl) analyzed for cAMP content by radioimmunoassay. EC 50 values (the dose of ligand that resulted in 50% of the maximum response (E max ) obtained for that ligand with the relevant receptor) were determined from plots of log(E/E max -E) vs. log[ligand], where E is the cAMP response measured at the corresponding dose of ligand. For antagonist studies, the inhibitor peptide [Leu n ,D-Trp 12 ]hPTHrP(7-34)NH 2 or [Trp 23 ,Tyr 36 ]hPTHrP-(l-36)NfJ wasapplied to COS-7 cells in 100 μl of binding buffer two minutes prior to the addition of IBMX buffer that contained a near-maximal stimulatory dose (1.5 nM) of [Tyr 34 )hPTH-(l-34)NH 2 agonist peptide. The cells were then incubated for 30 minutes at room temperature, and the resulting intracellular cAMP was measured by RIA. Data were analyzed using the AssayZap software (Elsevier Science Publishers BV, Cambridge, United Kingdom). Results The binding and cAMP-signaling responses obtained for PTH-(l-34) ([Tyr 34 ]hPTH-(l-34)NH 2 ) and PTHrP-(l-36) ([Tyr 36 ]hPTHrP-(l-36)NH ) in COS-7 cells expressing the human PTH/PTHrP and human PTH-2 receptors are shown in Figure 1. With the PTH PTHrP receptor, both PTH-(l-34) and PTHrP-(l-36) fully inhibited binding of the radioligand, ,25 I-[NleTyr 8 - 21 ,Tyr 34 ]rPTH-(l-34)NH 2 , although PTH-(l-34) was 4.8-fold more potent than PTHrP-(l-36) (IC 50 s = 39 and 285 nM, respectively, p<0.001 ; Figure 1A and Table 1). With cells expressing the PTH-2 receptor, PTH-(l-34) bound with high apparent affinity, while PTHrP-(l -36) bound poorly with a potency that was more than 100-fold weaker than that of PTH-( 1 -34) (Figure 1 B and Table 1 ). Both PTH-0-34) and PTHrP-(l-36) were full and potent agonists of cAMP production in COS-7 cells expressing the PTH/PTHrP receptor (Figure 1C and Table 2). In contrast, only PTH-(l-34) was a potent agonist with the PTH-2 receptor, while PTHrP-(l-36) was nearly inactive (Figure ID). These results confirm the markedly different cAMP-stimulating activities of PTH and PTHrP with the PTH-2 receptor (Usdin, T. et al, J. Biol. Chem. 270: 15455-15458 (1995)), and demonstrate that at least part of the signaling defect that PTHrP has with this receptor can be attributed to weak binding interactions. Scatchard analyses of competition binding data indicated that the PTH-2 receptor was expressed on the surface of COS-7 cells to levels that were 5-fold lower than the level of expression attained by the PTH/PTHrP receptor (Figures 2A and 2B). This difference in expression levels is likely to explain the lower cAMP responses compared to the PTH/PTHrP receptor (Figure 1 ). Although, the reason for the lower expression of the PTH-2 receptor is unclear at present, expression was fully adequate for comparing the interactions of different PTH and PTHrP analogs with this receptor. Table 2 Receptor-binding properties of PTH and PTHrP analogs COS-7 cells transfected with plasmids encoding the human PTH/PTHrP receptor or the human PTH-2 receptor were stimulated with the indicated ligand for lh at room temperature, and the resulting levels of intracellular cAMP were quantified by RIA. The maximum values indicate the highest level of cAMP that was attained for each ligand with each receptor. The mean basal cAMP values (not subtracted) were 4.6 ±0.2 and 5.4 ±0.3 pmoles/well for the PTH/PTHrP and PTH-2 receptors, respectively. Data are the mean (± s.e.m.) of values obtained from the number of experiments indicated (n). O c CD CO cAMP accumulation m PTH/PTHrP receptor PTH-2 receptor co X EC50 Maximum EC50 Maximum m m Ligand (nM) (pmoles/well) (nM) (pmoles well) H c (n) (n) r- m [Y34]hPTH-(l-34)NH2 0.46±0.18 1 13=1=13 5 0.6±0.1 81±5 5 10 [Y36]hPTHrP-(l-36)NH2 0.32±0.04 103±10 5 >1000 1 1=1=1 5 [15, Y36]hPTHrP-(l-36)NH2 0.26±0.08 1 12±9 3 7.0±2.1 80±5 3 cAMP accumulation PTH/PTHrP receptor PTH-2 receptor EC50 Maximum EC 50 Maximum Ligand (nM) (pmoles/well) (nM) (pmoles/well) [W23, Y36]hPTHrP-(l-36)NH2 0.26±0.05 123±14 4 >1000 12±1 4 [15, W23, Y36]hPTHrP-(l-36)NH2 0.21±0.06 109±10 4 0.5±0.3 65±7 4 CO c CD [H5, Y34]hPTH-(l-34)NH2 0.93±0.37 1 16±19 3 >1000 lO±l 3 CO H H [F23, Y34]hPTH-(l-34)NH2 0.23±0.02 104±16 4 7.6±2.2 50±8 4 C H m [H5, F23, Y34]hPTH-(l-34)NH2 1.18±0.29 110±19 3 >1000 7.1±0.9 3 co x m m H Hybrid- 1 3 c [ Y34]hPTHrP-( 1 - 14)/hPTH-( 15-34)NH2 0.23±0.04 140±17 3 >1000 5.3±0.6 r- m ro Hybrid-5 [ Y34]hPTHrP-( 1 - 18)/hPTH-( 19-34)NH2 0.43±0.16 133±14 3 >1000 6.2±0.4 10 Hybrid-4 [ Y34JhPTHrP-( 1 -21 ) hPTH-(22-34)NH2 0.27±0.06 107±17 4 >1000 6.7±0.6 Hybrid-3 cAMP accumulation PTH/PTHrP receptor PTH-2 receptor EC50 Maximum EC50 Maximum Ligand (nM) (pmoles/well) (nM) (pmoles/well) [Y34]hPTHrP-( 1 -23)/hPTH-(24-34)NH2 0.42±0.13 95±9 4 >1000 l l±l 4 Hybrid-2 O [Y34]hPTH-( 1 - 14)/hPTHrP-( 15-34)NH2 0.74±0.37 123±28 3 2.1±0.5 58±12 3 c CD CO H c H m co x m q 39 C m ro σ> PTH/PTHrP hybrid ligands To localize regions of PTH and PTHrP responsible for the difference in apparent binding affinities of the two ligands with the PTH-2 receptor, the inventors evaluated the series of PTH/PTHrP hybrid analogs shown in Figure 3. Some of the hybrid ligands (i.e., hybrids 1 and 5) were not highly informative, because unlike either parent ligand, they competed poorly for binding even to the PTH/PTHrP receptor, indicating that these analogs were unlike either parent ligand. Similarly weak binding potencies for these hybrid ligands were previously observed in studies with rat osteosarcoma cells (ROS 17/2.8). Those results were interpreted as alterations in ligand structure caused by incompatibility between residues in the amino-terminal 1-14 portion of PTHrP and the 15-34 region of PTH (Gardella, T.J. et al, J. Biol. Chem. 270:6584-6588 (1995)). For the current studies on the PTH-2 receptor, the most informative hybrid analogs were hybrid-3 and hybrid-4. Hybrid-4, PTHrP-(l-21)/PTH- (22-34) bound to the PTH-2 receptor with high potency (IC 50 = 1 1 nM), similar to PTH-(l-34) (Table 1). Extending the PTHrP sequence by two residues yielded hybrid-3, PTHrP-(l-23)/PTH-(24-34), which competed poorly for binding to the PTH/PTHrP receptor (IC 50 = 1 ,000 nM), and bound with even weaker potency to the PTH-2 receptor (IC50 ~ 6,000 nM). In this regard, the binding profile of hybrid-3 resembled that of PTHrP-(l-36). Substitutions at positions 22 and 23 Comparing the structures and binding properties of hybrids 3 and 4 led the inventors to hypothesize that the divergent residues at positions 22 and/or 23 contribute to PTH-2 receptor binding selectivity. To initially test the role of residues 22 and 23 in receptor binding, shorter-length PTHrP-(15-36) fragments were prepared in which Phe-22 and/or Phe-23 were replaced by the corresponding Glu and Tip residues of PTH. The unsubstituted parent peptide, PTHrP-(l 5-36), competed weakly for binding to the PTH-2 receptor (Figure 4B). The binding potency improved considerably with the combined Glu-22 and Trp-23 to Glu and Tφ substitution. Most of this enhancement could be accounted for by the single substitution of Tφ for Phe-23 (Table 1, Figure 4B). Binding of either unsubstituted or substituted PTHrP-(15-36) fragments to the PTH/PTHrP receptor could not be detected within these competition assays (Figure 4A). The Phe-23-to-Tφ modification was introduced into full- length PTHrP-(l-36), where it was found to enhance binding potency for the PTH-2 receptor by 71 -fold, as compared to the binding of PTHrP-(l -36) itself (Figure 5B; Table 1). The reciprocal change of Tφ-23-to-Phe in PTH-(l-34) led to a 28-fold reduction in binding potency for the PTH-2 receptor, as compared to unsubstituted PTH-(l-34) (Figure 5D and Table 1). Interestingly, the divergent residues at position 23 could also account for some of the difference in the apparent binding affinities that PTH and PTHrP exhibited for the PTH/PTHrP receptor. Thus, [Tφ 23 ]PTHrP-(l-36) bound to the PTH/PTHrP receptor with 6-fold stronger affinity than did PTHrP-(l-36) (Figure 5 A; Table 1), and [Phe 3 ]PTH-(l-34) bound to this receptor with 2-3 fold weaker affinity than did PTH-(l-34) (Figure 5C; Table 1). The effect of these position 23 changes on binding to the PTH/PTHrP receptor could be predicted from the binding properties that hybrid-3 and hybrid-4 displayed with this receptor (Table 1). Effects of residues 5 and 23 on cAMP signaling The above substitutions at position 23 in PTH or PTHrP had little or no effect on cAMP-signaling by the PTH-2 receptor; [Tφ 23 ]PTHrP-(l-36) was still inactive and [Phe 23 ]PTH-(l-34) was nearly a full agonist (Figures 6B and 6D; Table 2). The high potency of hybrid-2, PTH-(l-14)/PTHrP-( 15-34), in stimulating cAMP production with the PTH-2 receptor, compared with the weak activity of hybrid-1, PTHrP-(l-14)/PTH-(15-34) (Table 2), suggested that residues within the 1-14 sequence of PTH and PTHrP were involved in modulating the signaling properties of the two ligands with this receptor. Previous binding studies in ROS 17/2.8 cells revealed that position 5 (He in PTH and His in PTHrP) was one divergent site in the 1-14 region that could dramatically affect ligand-receptor interaction (Gardella, TJ. et al, J. Biol. Chem. 270:6584-6588 (1995)). Therefore, the effects of reciprocal position 5 substitutions on signaling interactions with the PTH-2 receptor were tested. Strikingly, [Ile 5 ]PTHrP-(l-36) became a full agonist with the PTH-2 receptor, promoting the same maximal cAMP response as that attained by PTH-(l-34) (Figure 6B and Table 2). However, the EC 50 of the [Ile 5 ]PTHrP-(l-36) response was still 12-fold higher than that of PTH-(l-34) (EC 50 s = 7 and 0.6 nM, respectively). The inventors, therefore, combined the Ile-5 substitution with the affinity-enhancing Tφ-23 modification. The resulting analog, [Ile 5 ,Tφ 23 ]PTHrP- (1-36), was as potent and efficacious as PTH-(l-34) in both binding and cAMP production with the PTH-2 receptor (Figure 6B, Tables 1 and 2). The observation that [Tφ 23 ]PTHrP-(l-36) (histidine at position 5) bound to the PTH-2 receptor without stimulating cAMP production, suggests that this analog could function as a PTH-2 receptor antagonist. Figure 7 shows that this analog was indeed at least as potent as [Leu",D-Tφ 1 ]hPTHrP(7-34)NH 2 , a highly potent antagonists of the PTH/PTHrP receptor antagonist (Nutt et al, Endocrinology 727:491-493 (1990)), in inhibiting PTH-(l-34)-induced activity on the PTH-2 receptor. With the PTH/PTHrP receptor, [Tφ 23 ]PTHrP-(l -36) was not an antagonist, but instead augmented the agonist response to PTH-(l-34). Discussion The studies discussed above demonstrate that the failure of PTHrP to efficiently bind to and activate the PTH-2 receptor can be attributed to two residues, phenylalanine at position 23 and histidine at position 5. In PTH, which is fully potent with the PTH-2 receptor, these residues are replaced by tryptophan and isoleucine, respectively. By exchanging residues 5 and 23 of PTHrP with the corresponding isoleucine and tryptophan residues of PTH, PTHrP could be converted into a full and potent PTH-2 receptor agonist. The histidine at position-5 in PTHrP blocked activation of the PTH-2 receptor. In previous competition binding studies in ROS 17/2.8 cells, residue 5 was identified as an important determinant for interacting with the PTH/PTHrP receptor (Gardella, T.J. et al, J. Biol. Chem. 270:6584-6588 (1995)). These earlier studies suggested that this residue was involved in putative intramolecular interactions between the 1-14 and with the 15-34 portions of the ligand. Evidence for this included the ability of the His-5-to-Ile-5 substitution in PTHrP to "cure" the deleterious effects that carboxy-terminal substitutions (i.e. at positions, 19, 21 and 24) had on binding potency. It now appears that the full role of residue 5 in the ligand in PTH and PTHrP is more complex, since the present data indicate that this residue strongly modulates signaling interactions with the PTH-2 receptor. The weak binding of PTHrP to the PTH-2 receptor can be attributed to the phenylalanine residue at position 23; replacing this residue by the corresponding tryptophan of PTH improved binding to the PTH-2 receptor by 71 -fold. The substitution of Tφ for Phe-23 could also explain the six-fold weaker binding that PTHrP-( 1 -36) exhibited for the PTH/PTHrP receptor, as compared to PTH-( 1 -34) (Table 1). Such weaker binding of PTHrP was observed in earlier studies with the cloned human PTH/PTHrP receptor expressed in COS-7 cells, although, nearly equivalent binding potencies were seen with the cloned rat PTH/PTHrP receptor (Schipani, E. et al, Endocrinology 732:2157-2165 (1993)). Earlier studies with the rat PTH/PTHrP receptor endogenous to ROS 17/2.8 cells also found comparable binding potencies for the two ligands (Juppner, H. et al, J. Biol. Chem. 265:8557-8560 (1988); Nissenson, R.A. et al, J. Biol. Chem. 265:12866-12871 (1988)). Therefore, it may be expected that the rat and human PTH/PTHrP receptors, and the human PTH-2 receptor, each differ at a site or sites that recognize residue 23 in the ligand. Thus, with both receptors, the tryptophan-23 Tφ modification in PTHrP enhanced the binding affinity of the analog to nearly that of PTH-(l-34). The effects on activity of a variety of modifications at position 23 in PTH have been investigated previously in PTH PTHrP receptors. Rosenblatt and coworkers found that the addition of a bulky ortho-nitrophenylsulfenyl group to the indole nitrogen of Tφ-23 had little or no effect on bioactivity (Rosenblatt, M. & Potts, J.T., Endo. Res. Comm. 4:115-133 (1977)), nor did a napthylalanine substitution at this site (Nakamoto, C. et al, Biochemistry 34:10546-10552 (1995)). However, methylation of the backbone nitrogen at Phe-23, or, the substitution of D-Tφ for Tφ-23, severely reduced receptor-binding affinity (Caulfield, M.P. & Rosenblatt, M., T.E.M. Jan/Fe 6:164-168 (1990)). Mutational analysis of residues 23-34 of PTH-(l -84) showed that cysteine substitution of Trp-23 reduced bioactivity by more than 10-fold, while leucine substitution reduced activity by 50% (Gardella, T.J. et al, "Scanning mutagenesis of the 23- 25 region of parathyroid hormone reveals important determinants of receptor binding," in Calcium Regulating Hormones and Bone Metabolism; Basic and Clinical Aspects, Vol. II, Cohn, D.V. et al, eds., Elsevier Science Publishers BV, Amsterdam (1992), pp. 218-222). NMR studies on PTH analogs (Barden, J.A. & Kemp, B.E., Biochemistry 32:7126-7132 (1993); Marx, U. et al, J. Biol. Chem. 270: 15194-15202 (1995); Klaus, W. et al, Biochemistry 50:6936-6942 (1991); Bundi, A. et al, Eur. J. Biochem. 97:201-208 (1978)) and PTHrP analogs (Barden, J.A. & Kemp, B.E., Eur. J. Biochem. 184:379-394 (1989); Ray, F.R. et al, Eur. J. Biochem. 277:205-211 (1993)) suggest that the aromatic residue at this position in either ligand is involved in intramolecular hydrophobic interactions. Residue 23 may, therefore, be important for maintaining ligand structure, as well as modulating receptor-binding interactions. Because the PTHrP-(l-36) analog containing the Tφ-23 modification exhibited high binding affinity for the PTH-2 receptor without having detectable agonist activity, we evaluated it as a potential PTH-2 receptor antagonist. This analog was at least as potent as [Leu 1 ',D-Tφ ,2 ]hPTHrP(7-34)NH 2 a previously characterized antagonist of the PTH/PTHrP receptor in inhibiting the agonist action of PTH-(l-34) on the PTH-2 receptor; however, unlike the amino- terminally truncated antagonist analog, however, [Tφ 23 ]PTHrP-(l-36) was a full and potent agonist with the PTH/PTHrP receptor (Figures 6A and 7A). Because this new analog appears to be a selective antagonist for the PTH-2 receptor, it could potentially be useful to in vivo studies aimed at further elucidating the physiological role of the PTH-2 receptor. PTHrP is expressed in many fetal and adult tissues and is a vital developmental moφhogen, a function that is likely to be mediated by the PTH/PTHrP receptor (Karaplis, A.C. et al, Genes & Dev. 5:277-289 ( 1994)). It is of interest that phenylalanine is preserved at position 23 in all native PTHrP ligands, despite the implications of the invention that tryptophan at this site, as is found in all native PTH ligands, is fully compatible with binding to the PTH/PTHrP receptor. It may be that phenylalanine has been selected for at position 23 in PTHrP, because of its ability to inhibit binding to the PTH-2 receptor. Likewise, the conserved histidine residue at position 5 in PTHrP may have been selected for because it blocks signaling interactions with the PTH-2 receptor. Together, these two conserved residues in PTHrP would thus ensure that productive interactions with the PTH-2 receptor do not occur. In summary, the present inventors have identified two sites in PTH and PTHrP that account for the ligand selectivity of the PTH-2 receptor. The inventors are now constructing chimeras between the PTH-2 and PTH/PTHrP receptors, as one approach towards identifying the cognate receptor sites involved in this ligand selectivities for the two ligands. The functional profiles of such receptor chimeras interacting with modified PTH and PTHrP ligands should help to refine and constrain models of the complexes formed between these peptide ligands and their G protein-coupled receptors. T erapeutic Use In accordance with yet a further aspect of the invention, there is provided a method for treating a medical disorder that results from altered or excessive action of the PTH-2 receptor, comprising administering to a patient a therapeutically efficient amount of a PTHrP analog, sufficient to inhibit activation of the PTH-2 receptor of said patient. In a preferred embodiment, the PTHrP analog used in the method has a single amino acid substitution of phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Tφ 23 ]PTHrP-(l-36). In this embodiment, a patient who is suspected of having a disorder resulting from altered action of the PTH-2 receptor may be treated using the peptide analogs of the invention shown to be a selective antagonist of the PTH-2 receptor. Such antagonists include the compounds of the invention which have been determined (by the assays described herein) to interfere with PTH-2 receptor-mediated cell activation or other analogs having similar properties. To administer the antagonist, the appropriate peptide is used in the manufacture of a medicament, generally by being formulated in an appropriate carrier or excipient such as, e.g., physiological saline, and administered intravenously, intramuscularly, subcutaneously, or orally, at a dosage that provides adequate inhibition of PTH binding to the PTH-2 receptor. Typical dosage would be 1 ng to 10 mg of the antibody or peptide per kg body weight per day. In accordance with yet a further aspect of the invention, there is provided a method for treating osteoporosis, comprising administering to a patient a therapeutically efficient amount of a PTHrP analog, sufficient to activate the PTH/PTHrP receptor and the PTH-2 receptor of said patient. In a preferred embodiment, the PTHrP analog used in the method has an amino acid substitution of histidine for isoleucine at position 5 of PTHrP, as well as phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [He 5 , Tφ 23 ]PTHrP-(l-36). Similar dosages and administration as described above for the PTHrP antagonist, may be used for administration of a PTHrP agonist, e.g., for treatment of conditions such as osteoporosis. By "agonist" is intended a ligand capable of enhancing or potentiating a cellular response mediated by the PTH-2 receptor. By "antagonist" is intended a ligand capable of inhibiting a cellular response mediated by the PTH -2 receptor. Whether any candidate "agonist" or "antagonist" of the present invention can enhance or inhibit such a cellular response can be determined using art-known protein ligand/receptor cellular response or binding assays, including those described elsewhere in this application. It will be appreciated to those skilled in the art that the invention can be performed within a wide range of equivalent parameters of composition, concentration, modes of administration, and conditions without departing from the spirit or scope of the invention or any embodiment thereof.

Claims: What Is Claimed Is: I . A PTHrP analog that is a PTH-2 receptor agonist, as well as a PTH/PTHrP receptor agonist. 2. The PTHrP analog of claim 1 , wherein the PTHrP analog is altered at amino acid residues 5 and 23 to the corresponding residues of PTH. 3. The PTHrP analog of claim 2, wherein said alteration at PTHrP residue 5 comprises an amino acid substitution of histidine for isoleucine, and said alteration at PTHrP residue 23 comprises an amino acid substitution of phenylalanine for tryptophan. 4. The PTHrP analog [He 5 , Tφ 3 ]PTHrP-(l-36). 5. A PTHrP analog that is a PTH-2 receptor selective antagonist. 6. The PTHrP analog of claim 5, wherein the analog is altered at PTHrP amino acid residue 23 to the corresponding residue of PTH. 7. The PTHrP analog of claim 6, wherein said alteration at PTHrP amino acid residue 23 comprises a substitution of phenylalanine for tryptophan. 8. The PTHrP analog [Tφ 23 ]PTHrP-(l-36). 9. A method for treating a medical disorder that results from altered or excessive action of the PTH-2 receptor, comprising administering to a patient a therapeutically efficient amount of the PTHrP analog of claim 6, sufficient to inhibit activation of the PTH-2 receptor of said patient. 10. A method for treating a medical disorder that results from altered or excessive action of the PTH-2 receptor, comprising administering to a patient a therapeutically efficient amount of the PTHrP analog of claim 7, sufficient to inhibit activation of the PTH-2 receptor of said patient. I I . A method for treating a medical disorder that results from altered or excessive action of the PTH-2 receptor, comprising administering to a patient a therapeutically efficient amount of the PTHrP analog of claim 8, sufficient to inhibit activation of the PTH-2 receptor of said patient. 12. A method for treating osteoporosis, comprising administering to a patient a therapeutically efficient amount of the PTHrP analog of claim 2, sufficient to activate the PTH/PTHrP receptor and PTH-2 receptor of said patient. 13. A method for treating osteoporosis, comprising administering to a patient a therapeutically efficient amount of the PTHrP analog of claim 3, sufficient to activate the PTH/PTHrP receptor and PTH-2 receptor of said patient. 14. A method for treating osteoporosis, comprising administering to a patient a therapeutically efficient amount of the PTHrP analog of claim 4, sufficient to activate the PTH/PTHrP receptor and PTH-2 receptor of said patient.

Inventors: GARDELLA THOMAS J, JUEPPNER HARALD, GARDELLA, THOMAS, J., JUEPPNER, HARALD
Applicants: GARDELLA THOMAS J, JUEPPNER HARALD, GARDELLA, THOMAS, J., JUEPPNER, HARALD
Application year: 1997
Main classification: C07K14
IPC codes: A61K38, A61P3, A61P19, A61P43, C07K14