HK1170414A - Parathyroid hormone peptides and parathyroid hormone-related protein peptides and methods of use - Google Patents

Description

Parathyroid hormone peptides and parathyroid hormone-related protein peptides and methods of use

Cross Reference to Related Applications

This application claims priority from U.S. provisional application No. 61/164,284, filed on 3/27/2009, the contents of which are incorporated herein by reference in their entirety.

Electronic filing material binding by reference

Incorporated by reference in its entirety are the computer-readable sequence listing filed concurrently herewith, which identifies the following: a 44.9 KB ASCII (text) file created on 26.3.2010-h, named "van067 fp409wo sequence listing.

Technical Field

The present invention is in the fields of biochemistry and medicine and relates to peptides or polypeptides that activate the parathyroid hormone receptor and methods of using these peptides or polypeptides to enhance bone growth (e.g., to treat osteoporosis) or to treat cancer.

Background

Parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules with distinct functions in bone biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) [ (1); for a review see (2, 3) ]. PTH is an 84 amino acid polypeptide endocrine hormone that is produced by the parathyroid gland and secreted into the circulation in response to low calcium levels [ for review see (4-6) ]. The typical effects of PTH are mediated by PTH1R expressed in bone and kidney tissues, including stimulation of osteoclastic bone resorption to maintain calcium homeostasis and stimulation of calcium resorption, synthesis of 1, 25-dihydroxy vitamin D3, and phosphate excretion in the kidney. Paradoxically, PTH also stimulates osteoblasts which lead to bone formation [ for a review see (7) ], providing a molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (8). Anabolic PTH therapy requires intermittent administration to avoid bone resorption that predominates with the continual rise of PTH in the circulation.

PTHrP is a 141 amino acid polypeptide, initially isolated as a factor responsible for malignant humoral hypercalcemia (9-12), and subsequently shown to be a crucial developmental factor regulating endochondral bone formation [ (13, 14); for a review see (15) ]. PTHrP is locally produced and acts in a paracrine/autocrine manner to activate PTH1R expressed on chondrocytes to regulate their proliferation and differentiation. PTHrP also has anabolic effects when administered to osteoporosis patients (16), but appears to be more purely anabolic than PTH produced by uncoupling bone resorption from bone formation (17).

PTH and PTHrP can activate several downstream signal transduction pathways through PTH1R to mediate their effects, but through G.alpha._sActivation of the coupled cAMP/PKA pathway predominates and is responsible for bone anabolism (18). A peptide fragment of PTH and PTHrP 34 residues N-terminal was sufficient to bind to and activate PTH1R to the same extent as the native molecule, and PTH- (1-34) and PTHrP- (1-34) were also effective in activating cAMP signal transduction (1). Its interaction with the receptor follows a "two-domain" model. Residues 1-14 interact with the 7-transmembrane (7-TM) helical domain embedded in the membrane, while residues 15-34 interact with the N-terminal extracellular domain (ECD) of the receptor (19, 20). The 1-14 fragments of PTH and PTHrP share 8 amino acid sequence identity, reflecting the crucial role this fragment plays in activating the receptor (20). The 15-34 fragment confers high affinity for receptor binding, but this portion of PTH and PTHrP is less conserved, with only 3 amino acids identity. PTH and PTHrP form similar α -helical structures in solution (21, 22).

While PTH and PTHrP have a common two-domain receptor binding mechanism, a common secondary structure and equal activation of signal transduction, their ability to bind to two pharmacologically distinct conformations of PTH1R, which are distinguished by the presence or absence of G-protein coupling, differs. Each peptide binds with similarly high affinity to G-protein coupled receptors (RG conformational state), but in the absence of G-protein coupling (R0 conformational state) PTHrP binding is significantly reduced, while PTH binding is only slightly reduced (23-25). Thus, PTHrP is more selective for RG than PTH. The different R0/RG-selective characteristics of PTH and PTHrP are associated with different temporal effects on cAMP signaling. PTH induces a longer cAMP signal than PTHrP after ligand washout (23). Divergent residue 5 (Ile in PTH, His in PTHrP) is a critical determinant of the R0/RG selectivity difference of the peptide (23, 25), but 15-34 fragments of PTH contribute to its strong R0 binding (23, 26), suggesting that interaction with ECD contributes to the persistence of cAMP signaling. Importantly, the time difference in cAMP signaling may have a significant role in vivo. In mice receiving daily injections, PTH analogs that exhibit increased R0 binding compared to wild-type PTH induce a sustained cAMP response in cells and result in increased trabecular bone volume and increased cortical bone resorption (26). These studies suggest that the R0/RG-selective characteristics of PTH and PTHrP contribute to the diverse physiological and therapeutic effects of peptides and underscore the importance of a detailed understanding of the structural basis of how PTH and PTHrP bind to receptors.

The present inventors previously developed methods (27) that enable them to determine the high resolution crystal structure of PTH1R ECD complexed with the C-terminally amidated 15-34 fragment of PTH [ PTH (15-34) NH2 ]. ECD is expressed in e.coli (e.coli) as a fusion protein linked to the C-terminus of bacterial Maltose Binding Protein (MBP). During the in vitro disulfide shuffling reaction required for the proper formation of 3 disulfide bonds, MBP solubilizes the ECD and facilitates crystallization of the fusion protein. PTH1R ECD forms a fold conserved in the class B GPCR ECD (28-30) and consists of an N-terminal alpha-helix followed by 2 antiparallel beta-folds and 1 short C-terminal alpha-helix, all held together by 3 disulfide bonds. PTH (15-34) NH2 forms an amphiphilic α -helix that binds to the hydrophobic groove at the interface of the secondary structural element.

Brief description of the invention

The present inventors developed a high resolution crystal structure of PTHrP that binds to the extracellular domain (ECD) of PTH1R and compared it to the PTH-PTH1R ECD complex. Like PTH, PTHrP forms an amphipathic α -helix that binds to the hydrophobic groove in the ECD, whereas PTH forms a continuous straight helix, whereas PTHrP helix bends slightly and "unfolds" at the C-terminus, causing a significantly different contact between the C-terminus of the peptide and the ECD. The receptor adapts to different binding modes by changing the conformation of the 2 residues of the peptide binding site. Guided by this structure, the present inventors designed hybrid peptides containing PTH and PTHrP residue exchanges at positions that determine different binding modes; hybrid peptides exhibiting reduced ECD-binding also have reduced affinity for the G protein uncoupled conformation of PTH1R in the cell membrane (R0), but retain normal binding to the G protein coupled conformation (RG). RG selective peptides stimulate cAMP accumulation in cells efficiently, but produce a shorter term response after ligand washout, suggesting that they may be of therapeutic value by providing an effective but pulsatile effect on the receptor.

The present inventors have demonstrated that PTH and PTHrP bind to ECD with similar affinity and propose a high resolution crystal structure of PTHrP [ PTHrP (12-34) NH2] C-terminally amidated 12-34 fragment complexed with MBP-PTH1R ECD fusion protein. Comparison of PTH binding structure and PTHrP binding structure revealed a significant difference in the ECD binding pattern of the peptide (mainly at the C-terminus of the peptide).

The present inventors used PTH-binding and PTHrP-binding structures as a guide to the design of PTH and PTHrP peptide analogs, some of which are hybrid PTH/PTHrP peptides containing residue exchanges at positions that determine different ECD binding patterns, enabling them to obtain RG-selective peptides that effectively stimulate cAMP signaling with a temporally shorter response. These analogs may have therapeutic potential as more pure anabolic PTH analogs lacking the hypercalcemic side effects by providing a potent but pulsatile effect at the receptor.

In accordance with the foregoing, the present invention provides PTH peptides comprising the amino acid sequence of wild-type PTH (SEQ ID NO: 9) having an amino acid substitution at a position within the C-terminal portion of the peptide (e.g., amino acids 15-34 of SEQ ID NO: 9). As first demonstrated herein, the PTH peptides of the present disclosure exhibit altered biological properties compared to wild-type PTH (e.g., reduced affinity for the G-protein uncoupled conformation of PTH1R (R0) compared to the affinity of wild-type PTH for the R0 conformation of PTH1R, while the PTH peptide retains binding to the G-protein coupled conformation (RG) of PTH1R and is capable of stimulating the accumulation of cAMP in cells; reduced binding to the ECD of PTH 1R). It is believed that this altered biological property enhances the therapeutic potential of these PTH peptides.

In some aspects, the PTH peptide comprises the amino acid sequence of wild-type PTH (SEQ ID NO: 9) having an amino acid substitution at one or more of amino acid positions 12, 14, 16, 17 and 27 or an analog thereof comprising a disulfide bond.

In some aspects, the PTH peptide comprises the amino acid sequence set forth in SEQ ID NO: 9 (amino acids 15-34 of SEQ id no: 9) with amino acid substitutions in one or more of positions 23, 27, 28 and 31 of wild type PTH. In some embodiments, the PTH peptide exhibits reduced affinity for the G protein uncoupled conformation (R0) of parathyroid hormone receptor (PTH1R), but remains bound to the G protein coupled conformation (RG) of PTH1R, and stimulates the accumulation of cAMP in the cell, compared to the affinity of wild-type PTH (SEQ ID NO: 9) for the R0 conformation of PTH 1R.

In other aspects, the PTH peptide comprises SEQ ID NO: 9, wherein there is an amino acid substitution at one or more of positions 20, 21, 23, 24, 27, 28, and 34. In particular embodiments, the PTH peptide exhibits reduced binding to the extracellular domain (ECD) of PTH1R compared to ECD binding of wild-type PTH to PTH 1R.

Also provided herein are PTHrP peptides comprising the amino acid sequence of wild-type PTHrP (SEQ ID NO: 79) having amino acid substitutions at positions within the C-terminal portion of the peptide (e.g., amino acids 12-34 of SEQ ID NO: 79). As demonstrated herein for the first time, the PTHrP peptides of the present disclosure exhibit altered biological properties compared to wild-type PTHrP (e.g., reduced affinity for the G-protein uncoupled conformation of PTH1R (R0) compared to the affinity of wild-type PTHrP for the R0 conformation of PTH1R, while the PTHrP peptides remain bound to the G-protein coupled conformation (RG) of PTH1R and are capable of stimulating the accumulation of cAMP in cells; reduced binding to the ECD of PTH 1R). It is believed that this altered biological property enhances the therapeutic potential of these PTHrP peptides.

In some aspects, the PTHrP peptide comprises the amino acid sequence set forth in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 (amino acids 12-34 of SEQ ID NO: 79). In particular aspects, the PTHrP peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R, but remains bound to the G-protein coupled conformation (RG) of PTH1R and stimulates the accumulation of cAMP in cells, as compared to the affinity of wild-type PTHrP (SEQ ID NO: 79) for the R0 conformation of PTH 1R.

In certain aspects, the PTHrP peptide comprises SEQ ID NO: 79 having amino acid substitutions at one or more of positions 20, 21, 23, 24, 27, 28, 32, and 33. In particular aspects, the PTHrP peptides exhibit reduced binding to the ECD of PTH1R compared to the binding of wild-type PTHrP to either ECD or PTH 1R.

In particular embodiments, the PTH peptide and PTHrP peptide (or polypeptide analog) described herein comprises SEQ ID NO: 1-101. Also provided herein are variants, fusion proteins, multimeric peptides, peptide mimetics (peptidomimetics), peptidomimetics, and chemical derivatives thereof. Such PTH or PTHrP peptide or polypeptide fusion proteins may comprise one of PTH or PTHrP peptide or polypeptide analogs, optionally a linker region and a second polypeptide linked to the PTH or PTHrP peptide or polypeptide analog or linked to the linker region, which second polypeptide is not naturally linked to the PTH or PTHrP peptide or polypeptide analog. Likewise, the PTH or PTHrP peptide or polypeptide analog may be linked to other peptides that facilitate entry into the cell.

In exemplary embodiments, the PTH peptide (or polypeptide analog) comprises SEQ ID NO: 1-78 and variants, fusion proteins, multimeric peptides, peptidomimetics, and chemical derivatives thereof. In particular embodiments, the PTH peptide comprises SEQ ID NO: 55.

In other exemplary embodiments, the PTHrP peptide (or polypeptide analog) comprises seq id NO: 79-101 and variants, fusion proteins, multimeric peptides, peptidomimetics, and chemical derivatives thereof. In a specific embodiment, the PTH peptide comprises seq id NO: 81, or a pharmaceutically acceptable salt thereof.

The invention also provides a kit for treating osteoporosis. In some aspects, the kit contains a peptide selected from the group consisting of a PTH peptide or analog described herein and a PTHrP peptide described herein, and instructions for administering the peptide to a subject having osteoporosis.

The PTH peptides, PTHrP peptides and analogs, chemical derivatives, fusion polypeptides, multimeric peptides described herein are useful in the preparation of medicaments for the treatment of osteoporosis. Thus, the present invention also provides compositions, e.g., pharmaceutical compositions, comprising a PTH or PTHrP peptide (or polypeptide analog, chemical derivative, fusion polypeptide, multimeric peptide, etc.) described herein and a pharmaceutically acceptable carrier or excipient.

Also included is a method of the invention for activating a PTH receptor in a cell, said method comprising introducing into a cell a PTH or PTHrP peptide or polypeptide analogue as described above, such that the peptide or polypeptide causes activation of said PTH receptor. Such a method may be performed in a living animal, the cells may be osteoblasts, and the introduction may be by oral delivery or injection.

Furthermore, the present invention includes a method for treating a mammalian subject having a disease or disorder associated with undesirable bone loss, said method comprising administering to a subject having undesirable bone loss an effective amount of the above-described pharmaceutical composition, thereby treating said subject. For this method, the subject may be a human and the disease may be osteoporosis.

The invention further provides methods of ameliorating a symptom associated with osteoporosis in a subject, methods of delaying progression of osteoporosis in a subject, and methods of regenerating bone in a subject, each comprising administering to the subject a pharmaceutical composition described herein in an amount effective to ameliorate the symptom associated with osteoporosis in the subject, delay progression of osteoporosis in the subject, or regenerate bone in the subject.

Other aspects and embodiments of the invention are described in more detail below.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings, certain preferred embodiments of the invention. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

FIGS. 1A-1D show PTH and PTHrP bound to PTH1R ECD. FIGS. 1A and 1B show the ability of the indicated PTH (FIG. 1A) or PTHrP (FIG. 1B) peptides to compete for association with the N-terminal biotinylated PTH (7-34) NH2(23nM) and MBP-PTH1R ECD fusion protein (23nM) evaluated using the AlphaScreen luminescent proximity assay (luminescent proximity assay). Data represent the average of duplicate samples. Fig. 1C and 1D show steady state analysis of real-time binding data obtained with the Octet Red system. Biotinylated PTH1R ECD was immobilized on streptavidin sensor contacts (tips) and binding of PTH (1-34) NH2 (FIG. 1C) and PTHrP (1-34) NH2 (FIG. 1D) was evaluated. Panels the curves in fig. 1C and 1D show the reaction versus peptide concentration curves obtained from the raw binding data (fig. 6). Kd values for PTH and PTHrP were 2.8. mu.M and 0.99. mu.M, respectively.

FIGS. 2A-2I show a comparison of the structure of PTH1R ECD complexed with PTHrP (12-34) and PTH1R ECD bound to PTH. FIG. 2A shows a close-up view of a PTHrP-ECD complex wherein ECD is slate blue and PTHrP is magenta. Residues 57-101 were not observed in the electron density map. For clarity, MBP is not shown. Fig. 2B shows the molecular surface of ECD, showing the hydrophobic channels contacted by F23 ', L24', L27 'and I28' of PTHrP. Surface coloring by atomic type: carbon is grey, nitrogen is blue and oxygen is red. Fig. 2C is an omitted electron density map of PTHrP. F_o-F_cThe omission figure is represented by a green grid line with a contour of 3 sigma,2F_o-F_cthe omitted plot is represented by the blue grid line with contour 1 σ. FIG. 2D shows a detailed view of the PTHrP-ECD interface. PTHrP is represented by red coils and the selected side chains by small bars. The red dashed line indicates hydrogen bonds and the red sphere is a water molecule. FIG. 2E is a close-up view of the hydrogen bonding network including residues 32-34 of PTHrP. FIG. 2F shows the structural alignment of PTHrP-ECD complex and PTH-ECD complex (PDB code 3C 4M). The PTHrP-ECD complex was colored yellow as above, and PTH-bound ECD was green. Selected side chains are represented by small bars. The hydrogen bonds of the PTHrP-ECD complex are indicated by red dashes and those of the PTH-ECD complex are indicated by green dashes. FIG. 2G is an amino acid sequence alignment of residues 1-34 of human PTH and PTHrP with α -helix lengths plotted above and below the sequence. FIG. 2H shows the results of a single point competition assay (single point competition assay) that evaluates the ability of PTH (15-34) NH2 alanine-scanning peptide (20 μ M) to compete with the interaction of biotin-PTH (23nM) and MBP-PTH1R-ECD (23 nM). Results are the average of duplicate samples. FIG. 21 shows the results of the same AlphaScreen assay as set forth in FIG. 2H, except for the PTHrP (12-34) NH2 alanine scanning peptide.

FIGS. 3A-3D show the binding of hybrid PTH/PTHrP peptides to MBP-PTH1R ECD protein. FIG. 3A shows the results of an AlphaScreen single-point competition assay that assesses the ability of hybrid peptides in PTH (15-34) NH2 scaffold (20 μ M) to compete with the interaction of biotin-PTH (23nM) and MBP-PTH1R-ECD (23 nM). Results are the average of duplicate samples. FIG. 3B shows the results of the same AlphaScreen assay as in FIG. 3A except for the hybrid peptide in the PTHrP (12-34) NH2 scaffold. Fig. 3C is a close-up view of van der waals contacts formed by W23 'and K27' in a PTH-ECD structure. FIG. 3D is a close-up view of Van der Waals contacts formed by F23 'and L27' in the PTHrP-ECD structure.

FIGS. 4A and 4B show the binding of the hybrid PTH/PTHrP (1-34) NH2 peptide to the R0 and RG states of PTH1R in cell membranes. FIG. 4A shows the binding of the indicated hybrid peptides in PTH (1-34) NH2 scaffold to R0 and RG receptor states. FIG. 4B shows the binding of the indicated hybrid peptides in the PTHrP (1-34) NH2 scaffold to R0 and RG receptor states.

FIGS. 5A and 5B show the signal transduction properties of RG-selective hybrid PTH/PTHrP (1-34) NH2 peptide. FIG. 5A shows the results of a dose-response assay for cAMP accumulation. COS cells transiently expressing PTH1R were stimulated with the indicated peptide at 37 ℃ for 30 minutes, after which the cells were lysed and assessed for cAMP content. Data are the average of duplicate samples and normalized to the highest cAMP level observed in the presence of wild type PTH (1-34) NH 2. Basal cAMP was 0.24 pmol/well and PTH induced maximal cAMP was 6.1 pmol/well. The EC50 values for PTH, PTH (W23 'F/V31' I), PTHrP and PTHrP (L27K) were 71.6pM, 41.5pM, 67.7pM and 44.4pM, respectively. Figure 5B shows the results of the ligand wash out assay. Cells were stimulated with the indicated peptides for 10 min at room temperature, ligand washed out, and cAMP accumulation was assessed at the indicated times after washing out. Results are the average of duplicate samples and normalized to cAMP levels induced during the initial stimulation. Basal cAMP was about 0.24 pmol/well and initial stimulation induced maximal cAMP was about 2.9 pmol/well.

Fig. 6A and 6B show Octet Red real-time analysis of PTH and PTHrP binding to PTH1R ECD. FIG. 6A shows immobilization of biotinylated PTH1R ECD on streptavidin sensor contacts and evaluation of PTH (1-34) NH at the following concentrations₂The combination of (1): 156.25nM, 312.5nM, 625nM, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M. FIG. 6B shows the evaluation of PTHrP (1-34) NH₂The same group diagram as fig. 6A except for the combination of (a).

Fig. 7A and 7B show the results of luciferase assay of PTH analogues. Each PTH analog has an identifier given in the form # # - ## - # -SBSL. In FIGS. 7A and 7B (0.1nM and 0.03nM), each PTH analog is labeled with # # # -SBSL (e.g., "07-12-18-40-SBSL" is denoted as "18-40").

Detailed Description

General rule

It is to be understood that the invention is not limited to the specific embodiments of the invention described below, as variations may be made in the specific embodiments and still fall within the scope of the appended claims. It is also to be understood that the terminology used is for the purpose of describing particular embodiments, and is not intended to be limiting. Rather, the scope of the invention should be determined by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry described below are those well known and commonly employed in the art. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are described below.

In this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Unless the context clearly dictates otherwise, it is understood that when a range of values is provided, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. If the stated range includes one or both of the limits, ranges that do not include either or both of those included limits are also included in the invention.

All references, patents, patent publications, papers, and databases mentioned in this application are herein incorporated by reference in their entirety as if each was specifically and individually indicated to be incorporated by reference. Such patents, patent publications, articles and databases are incorporated for the purpose of describing and disclosing the subject matter elements of the invention described in such patents, patent publications, articles and databases, which elements may be used in connection with the presently described invention. It is not an admission that the information provided herein is prior art to the present invention, but is provided solely to assist the understanding of the reader.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, embodiments and advantages of the invention will be apparent from the description and drawings, examples, sequence listing and claims. Preferred embodiments of the present invention may be understood more readily by reference to the following detailed description of specific embodiments, examples and sequence listing included herein.

For purposes of clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

PTH peptides and PTHrP peptides

PTH and PTHrP induce distinct biological effects by activating their common receptor, PTH 1R. Understanding the high resolution structure of how peptides bind to receptors is important for elucidating the biochemical mechanisms responsible for the regulation of mineral ion homeostasis and bone remodeling by PTH and bone development by PTHrP, and also has practical application to aid in rational design of peptide analogs for therapeutic purposes. The present inventors have analyzed the structure of PTHrP complexed with PTH1R ECD. In conjunction with the existing structure of the PTH-PTH1R ECD complex (27), the present inventors have taken an overview of the structural mechanisms adopted by PTH1RECD to recognize PTH and PTHrP. The peptides bind to the ECD as amphiphilic alpha-helices that contact the hydrophobic groove in the ECD. 2 of the 3 invariant residues on the 15-34 fragment, R20 'and L24', anchor the interaction. After residue L24', the peptide diverged (diverge) such that its C-terminus was in significantly different contact with ECD (fig. 2F), a result predicted by our ECD binding data that suggests that the C-terminal amide group of PTH, but not PTHrP, is important for ECD binding (fig. 1). The 3 rd invariant residue of fragment 15-34, H32', provides the receptor with a critical hydrogen bond in the PTHrP binding structure, but is exposed to solvents in the PTH binding structure. Interestingly, R20 'and L24' are conserved in TIP39, a peptide that activates PTH 2-type receptors (36), but H32 'is not, consistent with the present inventors' observation that the N-terminal portion of the 15-34 fragment provides an anchor point for peptide-ECD interactions, while the C-terminal portion varies in its interaction with the receptor.

The receptor was adapted to slightly different binding modes with relatively minor changes, including movement of Ile115 in response to PTHrP bending and the L41 rotamer toggle mechanism (toggle switch mechanism), to maintain van der waals contact with the ligand despite the different side chain volumes of the ligand at position 23 (fig. 3C and 3D).

With respect to ECD binding, PTHrP is generally more tolerant of changes than PTH. Only one of the 4 single crossover mutants of PTHrP remained normally associated with ECD, but 3 of the PTH single crossover mutants remained normally associated with ECD. PTH interactions may be more sensitive to conservative substitutions because of its higher surface complementarity to ECD and a larger buried surface area, which leaves less room for interface regulation in response to mutations. This suggests that PTHrP may provide a better scaffold to introduce changes therein for the development of therapeutic analogs.

The inventors originally suspected that different ECD binding patterns of PTH and PTHrP might contribute to their different R0/RG selectivity profiles. Although this does not seem to be the case, the availability of high resolution structures does lead them to rationally design changes in exchange that ultimately allow them to obtain peptides with high selectivity for G protein-coupled receptors. Unexpectedly, RG selectivity was increased by crossover mutations that attenuated ECD binding. However, R0/RG selectivity is more complex than ECD affinity alone. RG selectivity was greater for PTH (1-28) showing reduced ECD binding compared to PTH (1-34), while RG affinity was similarly reduced compared to PTH (1-34) (26). Clearly, residues 29-34 must contribute to the ability of the RG-selective hybrid PTH/PTHrP (1-34) peptide to maintain normal RG binding. Thus, there are at least two different approaches to alter the R0/RG selectivity profile of a given PTH or PTHrP peptide. RG selectivity can be increased by His at position 5 (presumably via interaction with the 7-TM domain of the receptor) and also by diminished ECD binding by the C-terminal portion of the peptide, but residues 29-34 are important for maintaining normal RG binding.

RG selective peptides efficiently stimulate cAMP accumulation but have a short signaling lifetime (fig. 5), suggesting that these peptides provide a pulsatile effect on the receptor. Although the underlying biochemical mechanisms responsible for the short duration of signal transduction are not understood, these peptides are pharmacologically useful as "biased" agents that induce anabolic effects on bone while limiting bone resorption by sustained stimulation of the receptor. Indeed, PTHrP appears to be a purer anabolic agent (anabolic agent) than PTH, lacking the deleterious hypercalcemic side effects (17). If this effect is contributed to by a decrease in the affinity of R0 of PTHrP, the RG-selective peptides provided herein, in particular PTHrP (1-34) [ L27K ], may have therapeutic potential as purer anabolic analogs for the treatment of osteoporosis.

In accordance with the foregoing, the present invention provides PTH peptides comprising the amino acid sequence of wild-type PTH (SEQ ID NO: 9) having an amino acid substitution at a position within the C-terminal portion of the peptide (e.g., amino acids 15-34 of SEQ ID NO: 9). As demonstrated herein for the first time, the PTH peptides of the present disclosure exhibit altered biological properties compared to wild-type PTH (e.g., reduced affinity for the G-protein uncoupled conformation of PTH1R (R0) compared to the affinity of wild-type PTH for the R0 conformation of PTH1R, while the PTH peptide remains bound to the G-protein coupled conformation (RG) of PTH1R and is capable of stimulating the accumulation of cAMP in cells; reduced binding to the ECD of PTH 1R). It is believed that this altered biological property enhances the therapeutic potential of these PTH peptides.

In some aspects, the PTH peptides provided herein comprise the amino acid sequence set forth in SEQ ID NO: 9 having an amino acid substitution on one or more of amino acid positions 12, 14, 16, 17 and 27 of wild type PTH (SEQ ID NO: 9) or an analogue thereof which comprises a disulfide bond.

In some embodiments, the nucleic acid sequence of SEQ ID NO: 9 is an amino acid substitution at one or more of the specific positions of SEQ ID NO: 9 is a conservative amino acid substitution of the natural amino acid. In particular embodiments, the conservative amino acid substitution is one that complies with the teachings set forth herein. See "variant peptides". In exemplary embodiments, a conservative amino acid substitution is the replacement of one amino acid with another amino acid in one of the following groups:

I. small aliphatic apolar or slightly polar residues:

Ala、Ser、Thr、Pro、Gly；

polar, negatively charged residues and their amides and esters:

asp, Asn, Glu, Gln, cysteamine and homocysteine;

polar positively charged residues:

his, Arg, Lys; ornithine (Orn);

large aliphatic apolar residues:

met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine;

large aromatic residues:

phe, Tyr, Trp, acetylphenylalanine.

In other embodiments, the amino acid substitution is not a conservative amino acid substitution, e.g., is a non-conservative amino acid substitution.

Thus, in a specific embodiment, the PTH peptide of the present disclosure comprises SEQ id no: 53, wherein the amino acid in position 12 is Gly, Val, Ala or conservative substitutions thereof, wherein the amino acid in position 14 is His, Phe, Leu or conservative substitutions thereof, wherein the amino acid in position 16 is Asn, Thr or conservative substitutions thereof, wherein the amino acid in position 17 is Ser, Asp, Asn or conservative substitutions thereof, and wherein the amino acid in position 27 is Lys, Leu or conservative substitutions thereof. In more specific embodiments, the PTH peptide of the present disclosure comprises SEQ id no: 1-10 and 13-20.

In some aspects, the PTH peptide of the present disclosure comprises the sequence set forth in SEQ ID NO: 9 (amino acids 15-34 of SEQ ID NO: 9) with amino acid substitutions in one or more of positions 23, 27, 28 and 31 of wild type PTH. In some embodiments, the PTH peptide exhibits reduced affinity for the G protein uncoupled conformation (R0) of parathyroid hormone receptor (PTH1R), but remains bound to the G protein coupled conformation (RG) of PTH1R, and stimulates the accumulation of cAMP in the cell, compared to the affinity of wild-type PTH (SEQ ID NO: 9) for the R0 conformation of PTH 1R. In some embodiments, the PTH peptide exhibits reduced binding to the extracellular domain (ECD) of PTH1R as compared to binding of wild-type PTH to the ECD of PTH 1R.

In specific embodiments, SEQ ID NO: 9 by one or more of SEQ ID NOs: 79 amino acid substitutions at the corresponding positions. For example, SEQ ID NO: 9 by the amino acid at position 23 of SEQ ID NO: 79 amino acid substitutions at the corresponding positions are Trp by SEQ ID NO: substitution of the amino acid (Phe) at position 23 of 79. Also, for example, SEQ ID NO: 9 by the amino acid at position 19 of SEQ ID NO: 79 amino acid substitution at the corresponding position of Glu by SEQ ID NO: substitution of the amino acid (Arg) at position 19 of 79.

In some embodiments, the PTH peptide is comprised in SEQ ID NO: 9, or 9 having an amino acid substitution at one or more of positions 23, 27, 28, and 31 of SEQ ID NO: 9 and further comprising amino acids 15-34 of SEQ ID NO: 9 amino acids 1-14. In such embodiments, the PTH peptide comprises the sequence set forth in SEQ ID NO: 9, or 9 having an amino acid substitution at one or more of positions 23, 27, 28, and 31 of SEQ ID NO: 9 amino acids 1-34. Thus, in some embodiments, the PTH peptide comprises SEQ ID NO: 57-66.

In some aspects, the PTH peptide comprises SEQ ID NO: 9 amino acids 15-34. In particular embodiments, the PTH peptide exhibits reduced binding to the extracellular domain (ECD) of PTH1R compared to the binding of wild-type PTH to the ECD of PTH 1R. In some embodiments, the PTH peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R compared to the affinity of wild-type PTH (SEQ ID NO: 9) for the R0 conformation of PTH1R, wherein the PTH peptide retains binding to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTH peptide stimulates the accumulation of cAMP in the cell. In some embodiments, the PTH peptide comprises an amino acid substitution at position 23 or 28, or at both positions 23 and 28. In some embodiments, the PTH peptide further comprises an amino acid substitution at one or more of positions 21, 27, and 34.

In some embodiments, the PTH peptide comprises SEQ ID NO: 9, wherein in SEQ ID NO: 9, and further comprising an amino acid substitution at one or more of positions 20, 21, 23, 24, 27, 28 and 34 of the PTH peptide, and further comprising SEQ ID NO: 9 amino acids 1-14. In such embodiments, the PTH peptide comprises SEQ ID NO: 9, wherein there is an amino acid substitution at one or more of positions 20, 21, 23, 24, 27, 28, and 34.

Amino acid substitutions at specified positions can be conservative amino acid substitutions or non-conservative amino acid substitutions as described herein. In a specific embodiment, the amino acid substitution is a replacement of the amino acid of the natural amino acid at the specified position with Ala. Thus, in particular embodiments, the PTH peptide comprises Ala at one or more of positions 20, 21, 23, 24, 27, 28 and 34. Thus, in some embodiments, the PTH peptide comprises SEQ ID NO: 68-71, 73, 74 and 78.

In addition to the PTH peptides described herein, the present invention also provides PTHrP peptides comprising the amino acid sequence of wild-type PTHrP (SEQ ID NO: 79) having amino acid substitutions at positions within the C-terminal portion of the peptide (e.g., amino acids 12-34 of SEQ ID NO: 79). As demonstrated herein for the first time, the PTHrP peptides of the present disclosure exhibit altered biological properties compared to wild-type PTHrP (e.g., reduced affinity for the G-protein uncoupled conformation of PTH1R (R0) compared to the affinity of wild-type PTHrP for the R0 conformation of PTH1R, while the PTHrP peptides retain binding to the G-protein coupled conformation (RG) of PTH1R and are capable of stimulating cAMP accumulation in cells; reduced binding to the ECD of PTH 1R). It is believed that this altered biological property enhances the therapeutic potential of these PTHrP peptides.

In some aspects, the PTHrP peptide comprises the amino acid sequence set forth in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 (amino acids 12-34 of SEQ ID NO: 79). In particular aspects, the PTHrP peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R, but remains bound to the G-protein coupled conformation (RG) of PTH1R and stimulates the accumulation of cAMP in cells, as compared to the affinity of wild-type PTHrP (SEQ ID NO: 79) for the R0 conformation of PTH 1R. In some embodiments, the PTHrP peptide exhibits reduced binding to the ECD of PTH1R as compared to the binding of wild-type PTHrP to the ECD or PTH 1R.

In a specific embodiment, in SEQ ID NO: 79 is substituted with one or more of SEQ ID NO: 9 amino acid substitution at the corresponding position. For example, SEQ ID NO: 79 is substituted with the amino acid at position 23 of SEQ ID NO: 9 is an amino acid substitution of Phe by SEQ ID NO: 9 by amino acid (Trp) at position 23. Also, for example, SEQ ID NO: 79 is substituted with the amino acid at position 5 of SEQ ID NO: 9 is a substitution of His with the amino acid at the corresponding position of SEQ ID NO: 9 (c) by substitution with an amino acid (He) at position 5.

In some embodiments, the PTHrP peptide is comprised in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 of SEQ ID NO: 79 and further comprises at the N-terminus of the PTHrP peptide the amino acids 12-34 of SEQ ID NO: 79 amino acids 1-11. In such embodiments, the PTHrP peptide is comprised in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 of SEQ ID NO: 79 amino acids 1-34. Thus, in some embodiments, the PTHrP peptide comprises SEQ ID NO: 80-90 in the presence of a protease.

In some aspects, the PTHrP peptide comprises SEQ ID NO: 79 having amino acid substitutions at one or more of positions 20, 21, 23, 24, 27, 28, 32, and 33. In particular aspects, the PTHrP peptides exhibit reduced binding to the ECD of PTH1R compared to the binding of wild-type PTHrP to either ECD or PTH 1R. In some embodiments, the PTH peptide exhibits a reduced affinity for the G protein uncoupled conformation (R0) of PTH1R compared to the affinity of wild-type PTHrP (SEQ ID NO: 79) for the R0 conformation of PTH1R, wherein the PTHrP peptide retains binding to the G protein coupled conformation (RG) of PTH1R, and wherein the PTHrP peptide stimulates the accumulation of cAMP in the cell. In some embodiments, the PTHrP peptide comprises an amino acid substitution at position 22 or 27, or at both positions 22 and 27.

In some embodiments, the PTHrP peptide comprises SEQ ID NO: 79, wherein there is an amino acid substitution at one or more of positions 20, 21, 23, 24, 27, 28, 32 and 33, and further comprising at the N-terminus of the PTHrP peptide the amino acid sequence of SEQ ID NO: 79 amino acids 1-11. In such embodiments, the PTHrP peptide is comprised in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 20, 21, 23, 24, 27, 28, 32 and 33 of SEQ ID NO: 79 amino acids 1-34.

Amino acid substitutions at specified positions can be conservative amino acid substitutions or non-conservative amino acid substitutions as described herein. In a specific embodiment, the amino acid substitution is a replacement of the amino acid of the natural amino acid at the specified position with Ala. Thus, in particular embodiments, the PTHrP peptide comprises Ala at one or more of positions 20, 21, 23, 24, 27, 28, 32 and 33. Thus, in some embodiments, the PTHrP peptide comprises SEQ ID NO: 93-98, 100 and 101.

Activity of PTH peptide and PTHrP peptide

In some aspects of the disclosure, PTH peptides, analogs thereof, and PTHrP peptides are potent agonists of PTH 1R. In exemplary embodiments, the PTH peptides, analogs thereof, and PTHrP peptides exhibit an EC50 for PTH1R that is at least the same or about the same as the EC50 of wild-type PTH or PTHrP for the PTH1R receptor. In some embodiments, the efficacy of a PTH peptide or PTHrP peptide of the present disclosure (calculated by dividing EC50 of the PTH peptide or PTHrP peptide of the present disclosure by EC50 of wild-type PTH or PTHrP to PTH1R, and then multiplying this number by 100%) is at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 100%, at least or about 200%, at least or about 500%, at least or about 1000%.

As described herein, activation of PTH1R by PTH and PTHrP results in the accumulation of cAMP in cells (e.g., osteoblasts). In some aspects of the disclosure, PTH peptides, analogs thereof, and PTHrP peptides are potent agonists of PTH1R and result in the accumulation of significant amounts of cAMP in cells. In a specific embodiment, the response to PTH1R stimulated by the PTH peptide and PTHrP peptide or analog thereof is a shorter cAMP response compared to the cAMP response of wild type PTH (SEQ ID NO: 9) or wild type PTHrP (SEQ ID NO: 79). In some specific embodiments, the PTH peptide comprises SEQ ID NO: 64. in some specific embodiments, the PTHrP peptide comprises SEQ ID NO: 81.

analogs of PTH peptides and PTHrP peptides

Also provided herein are analogs of PTH peptides described herein (e.g., PTH peptides comprising an amino acid sequence of wild-type PTH (SEQ ID NO: 9) having amino acid substitutions at one or more of amino acid positions 12, 14, 16, 17, and 27) comprising an intramolecular bridge connecting two non-contiguous amino acids. In a specific embodiment, the intramolecular bridge is a disulfide bridge. In some embodiments where the analog comprises a disulfide bond, the PTH peptide further comprises a sequence wherein the amino acid sequence of SEQ ID NO: 9 is an amino acid substitution in which a natural amino acid is substituted with a sulfur-containing amino acid (e.g., Cys (e.g., L-Cys, D-Cys), cysteic acid, homocysteine, etc.).

Thus, in some embodiments, the analog comprises SEQ ID NO: 54-56, wherein two of the amino acids at positions 19, 22 and 25 are linked by a disulfide bond. In a specific embodiment, a disulfide bond links the amino acid at position 19 to the amino acid at position 22. In other more specific embodiments, the amino acid at position 19 is D-Cys and the amino acid at position 22 is Cys. In other specific embodiments, the disulfide bond of the analog links the amino acid at position 22 to the amino acid at position 25, while in more specific embodiments, the amino acids at positions 22 and 25 are each Cys. As noted above, in certain embodiments, the analog comprises SEQ id no: 11. 12 and any one of 21-52.

Also provided herein are analogs of the PTHrP peptides described herein (e.g., PTHrP peptides comprising the amino acid sequence of wild-type PTHrP (SEQ ID NO: 79) with amino acid substitutions at one or more amino acids within the C-terminal portion of the PTHrP peptide (amino acids 12-34 of SEQ ID NO: 79), the analogs comprising an intramolecular bridge linking two non-contiguous amino acids.

Variant peptides

"variants" of a PTH or PTHrP peptide or polypeptide analogue refer to molecules which are substantially identical to said peptide or polypeptide analogue in which one or more amino acid residues have been substituted (substitution variants), or one or more residues have been deleted (deletion variants) or added (addition variants). Preferably, such substitutions, additions or deletions result in improved biological activity or improved clinical properties without significantly reducing the desired biological or biochemical effect. It is well within the skill of the art to assess whether a given substitution, addition or deletion of one or more residues will have a desired effect on the peptide. For a detailed description of the Protein chemistry and Structure, see Schulz, G.E., et al, Principles of Protein Structure, Springer-Verlag, New York, 1979 and Creighton, T.E., Proteins: structure and Molecular Principles, w.h.freeman & co, San Francisco, 1984, which are incorporated herein by reference. The type of substitution that can be made in the peptide molecules of the present invention is conservative substitution, defined herein as an exchange within one of the following groups: 1. small aliphatic apolar or slightly polar residues: such as Ala, Ser, Thr, Gly; 2. polar negatively charged residues and their amides: for example Asp, Asn, Glu, Gln; 3. polar positively charged residues: e.g., His, Arg, Lys; pro, due to its unusual geometry, tightly constrains the chain. By selecting less conservative substitutions, for example between the above mentioned groups (or two other groups of amino acids not shown above) rather than within a group, a considerable change in functional properties will be made, which will result in a more significant difference in the effect of maintaining: (a) the structure of the peptide backbone in the substitution region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Most substitutions of the present invention are substitutions that do not produce a radical change in the characteristics of the peptide molecule. Even if it is difficult to predict the exact effect of a substitution before doing so, one skilled in the art will appreciate that such effect can be assessed by conventional screening assays, preferably the biological and biochemical assays described herein. Improvements in peptide properties, including redox or thermostability, hydrophobicity, susceptibility to degradation by proteases, or tendency to aggregate with carriers or to aggregate into multimers, are determined by methods well known to those of ordinary skill in the art.

The variant may have 50% sequence identity to one of the PTH or PTHrP peptide or polypeptide analogs of the invention, or 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the PTH or PTHrP peptide or polypeptide analog.

Other modifications of PTH or PTHrP peptides

The invention also includes peptides and polypeptides in which one or more L-amino acids are substituted with one or more D-amino acids. In addition, modified amino acids or chemical derivatives of amino acids may also be provided such that the peptides contain other chemical moieties or modified amino acids that are not normally part of the native protein. Such derivatized moieties may improve solubility, absorption, biological half-life, and the like. Portions capable of mediating these effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16 th edition, Mack Publishing co., Easton, Pa, (1980). Chemical derivatives are further described herein. See "chemical derivatives".

The peptides described herein may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (by, for example, disulfide bridges) or converted to acid addition salts and/or optionally dimerized or multimerized or conjugated.

C-terminal amidation

As discussed further herein, in some embodiments, a PTH peptide, PTHrP peptide, or analog thereof of the present disclosure comprises a C-terminal amide group in place of a C-terminal carboxylic acid. In this case, the backbone of the C-terminal residue comprises N-C_α-CONH₂。

Salt (salt)

In some embodiments, the PTH peptide or PTHrP peptide (including analogs or variants thereof) is in the form of a salt, e.g., a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" as used herein, refers to salts of a compound that retain the biological activity of the parent compound, which are not biologically or otherwise undesirable. Such salts can be prepared in situ during the final isolation and purification of the analog, or separately by reacting the free base functionality with a suitable acid. Many of the compounds disclosed herein are capable of forming acid and/or base salts via the presence of amino and/or carboxyl groups or similar groups thereof.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-isethionate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate (palmitate), pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Salts derived from inorganic acids including: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids including: acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Examples of acids which may be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acids, and organic acids such as oxalic, maleic, succinic and citric acids.

Base addition salts can also be prepared in situ during the final isolation and purification of the salicylic acid source, or by reacting the carboxylic acid-containing moiety with a suitable base (e.g., a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation) or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations and aluminum salts based on alkali or alkaline earth metals (e.g., lithium, sodium, potassium, calcium, magnesium) and the like and non-toxic quaternary ammonium (quaternary ammmonia) and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, ethylammonium and the like. Other representative organic amines useful for forming base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like. Salts derived from organic bases include, but are not limited to, salts of primary, tertiary and secondary amines.

In addition, a basic nitrogen-containing group can be quaternized with the PTH or PTHrP peptides of the present disclosure to lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides); long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides); arylalkyl halides (e.g., benzyl and phenethyl bromides, etc.). Thereby obtaining a water-soluble or oil-soluble product or a dispersible product.

Chemical derivatives

"chemical derivative" of a PTH or PTHrP peptide or polypeptide analog refers to a molecule that contains other chemical moieties that are not normally part of the PTH or PTHrP protein. Covalent modifications of proteins are included within the scope of the invention. Such modifications may be introduced into the molecule by reacting the targeted amino acid residue with an organic derivatizing agent capable of reacting with the selected side chain or terminal residue. Such chemically derivatized moieties may improve the solubility, absorption, biological half-life, etc., of the protein. These changes can eliminate or attenuate the undesirable side effects of PTH or PTHrP peptide or polypeptide analogs in vivo. Portions capable of mediating these effects are disclosed, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, easton pa, (Gennaro 18 th edition, 1990). The following capping peptides are examples of preferred chemical derivatives of "native" uncapped peptides. Any combination of substitution, addition or deletion variants of the invention can be capped with any of the capping groups disclosed herein.

Chemical moieties

The chemical moiety that is part of a chemical derivative may be any chemical moiety, including naturally occurring or non-naturally occurring biomolecules, compounds, such as small molecular weight compounds, which in some embodiments are chemically inert to the PTH peptide or PTHrP peptide. Generally, a chemical moiety is a moiety that does not reduce the activity of the peptide. Alternatively, the chemical moiety is a chemical moiety that: which alter (e.g., improve or enhance) the properties of the PTH or PTHrP peptide, e.g., increase stability, solubility and/or potency of the peptide, decrease duration of action on PTH1R, increase selectivity for RG, decrease affinity for R0. In a particular embodiment, the chemical moiety is a polymer, carbohydrate, amino acid, peptide, or lipid, such as a fatty acid or steroid.

In some embodiments, the chemical moiety is an amino acid. The amino acid can be any of the 20 common amino acids known in the art (Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, Tyr) or any known non-naturally occurring amino acid. Suitable synthetic amino acids for the purposes herein include, but are not limited to, beta-alanine (beta-Ala), N-alpha-methyl-alanine (Me-Ala), aminobutyric acid (Abu), gamma-aminobutyric acid (gamma-Abu), aminocaproic acid (epsilon-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrolecarboxylic acid, aminopiperidinecarboxylic acid, aminoserine (Ams), aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, beta-aspartic acid (beta-Asp), azetidinecarboxylic acid, 3- (2-benzothiazolyl) alanine, alpha-tert-butylglycine, 2-amino-5-ureido-N-pentanoic acid (citrulline, Cit), beta-cyclohexylalanine (Cha), Acetamidomethyl-cysteine, diaminobutyric acid (Dab), diaminopropionic acid (Dpr), Dihydroxyphenylalanine (DOPA), Dimethylthiazolidine (DMTA), gamma-glutamic acid (gamma-Glu), homoserine (Hse), hydroxyproline (Hyp), isoleucine N-methoxy-N-methylamide, methyl-isoleucine (MeIle), hexahydroisonicotinic acid (Isn), methyl-leucine (MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine, methano-proline (methanoproline), methionine-sulfoxide (Met (O)), methionine-sulfone (Met (O)₂) Norleucine (Nle), methyl-norleucine (Me-Nle), norvaline (Nva), ornithine (Orn), p-aminobenzoic acid (PABA), penicillamine (Pen), methylphenylalanine (MePhe), 4-chlorophenylalanine (Phe (4-Cl)), 4-fluorophenylalanine (Phe (4-F)), 4-nitrophenylalanine (Phe (4-NO)), and mixtures thereof₂) 4-cyanophenylalanine ((Phe (4-CN)), phenylglycine (Phg), piperidinylalanine, piperidinylglycine, 3, 4-dihydroproline, pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec), O-benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta), 4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), 1, 2, 3, 4-tetrahydro-isoquinoline-3-carboxylic acid (Tic), tetrahydropyranylglycine, tetrahydroxyphenylalanine, dihydroproline, sarcosine, selenocysteine,thienylalanine (Thi), O-benzyl-phosphotyrosine, O-phosphotyrosine, methoxytyrosine, ethoxytyrosine, O- (bis-dimethylamino-phosphono) -tyrosine, tyrosine tetrabutylamine sulfate, methyl-valine (MeVal) and alkylated 3-mercaptopropionic acid. The amino acid may be the D-isomer or the L-isomer of the amino acid.

In some embodiments, the chemical moiety is a peptide, e.g., a peptide of 50 amino acids or less, e.g., a dipeptide, tripeptide, 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, 10-mer, 15-mer, 20-mer, 25-mer, 30-mer, 35-mer, 40-mer, 45-mer, 50-mer. The peptide may comprise any amino acid known in the art, for example any amino acid described herein.

The polymer may be any polymer. The polymer may be a derivatized polymer of any one of: a polyamide; a polycarbonate; polyalkylene and derivatives thereof including polyalkylene glycol, polyalkylene oxide, polyalkylene terephthalate; polymers of acrylates and methacrylates including poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate); polyvinyl polymers including polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyvinyl halide, poly (vinyl acetate), and polyvinyl pyrrolidone; polyglycolide; a polysiloxane; polyurethanes and their copolymers; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitro cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and cellulose sulfate; polypropylene; polyethylene, including poly (ethylene glycol), poly (ethylene oxide), and poly (ethylene terephthalate), and polystyrene.

The polymers can be biodegradable polymers including synthetic biodegradable polymers (e.g., polymers of lactic and glycolic acid, polyanhydrides, poly (ortho) esters, polyurethanes, poly (butyric acid), poly (valeric acid), and poly (lactide-caprolactone copolymers)) as well as natural biodegradable polymers (e.g., alginates and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitution, addition of chemical groups (e.g., alkyl, alkylene), hydroxylation, oxidation, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins (e.g., zein and other prolamins and hydrophobic proteins), and any copolymers or mixtures thereof, hi general, these materials degrade by enzymatic hydrolysis in vivo or exposure to water, by surface or bulk erosion.

The polymer may be a bioadhesive polymer, such as a bioerodible hydrogel disclosed in Macromolecules, 1993, 26, 581-587 (the teachings of which are incorporated herein) by h.s.sawhney, c.p.pathak and j.a.hubbell; poly hyaluronic acid, casein, gelatin, polyanhydride, polyacrylic acid, alginate, chitosan, poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), and poly (octadecyl acrylate).

In some embodiments, the polymer is a water soluble polymer. Suitable water-soluble polymers are known in the art and include, for example, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose, hydroxypropyl amylcellulose, methylcellulose, ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkyl and hydroxyalkyl celluloses, various cellulose ethers, cellulose acetate, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acid copolymers, polyhydroxyalkyl methacrylates, hydroxymethyl methacrylates, methacrylic acid copolymers, polymethacrylic acid, polymethyl methacrylate, maleic anhydride/methyl vinyl ether copolymers, polyvinyl alcohol, sodium and calcium polyacrylates, Polyacrylic acid, acid carboxyl polymers, carboxypolymethylene, carboxyvinyl polymers, polyoxyethylene polyoxypropylene copolymers, polymethylvinyl ether-maleic anhydride copolymers, carboxymethylamides, potassium methacrylate divinylbenzene copolymers, polyoxyethylene glycols, polyethylene oxide and derivatives, salts and combinations thereof.

The carbohydrate may be any carbohydrate. Carbohydrates may be, for example, monosaccharides (e.g. glucose, galactose, fructose), disaccharides (e.g. sucrose, lactose, maltose), oligosaccharides (e.g. raffinose, stachyose), polysaccharides (starch, amylose (amylose), amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, galactomannan.

The lipid may be any lipid, for example, a fatty acid (e.g., C4-C30 fatty acids, eicosanoids, prostaglandins, leukotrienes, thromboxanes, N-acylethanolamines), glycerolipids (e.g., mono-, di-, tri-substituted glycerols), glycerophospholipids (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipids (e.g., sphingosine, ceramide), sterol lipids (e.g., steroids, cholesterol), prenyl alcohol esters (prenyl lipid), glycolipids, polyketides, oils, waxes, cholesterol, sterols, fat soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids.

The peptide may be linked to the chemical moiety by a direct covalent bond by reacting targeted amino acid residues of the peptide with an organic derivatizing agent capable of reacting with selected side chains or the N-or C-terminal residues of these targeted amino acids. Reactive groups on a peptide or chemical moiety include, for example, aldehyde, amino, ester, thiol, α -haloacetyl, maleimide, or hydrazine groups. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugated through a cysteine residue), N-hydroxysuccinimide (conjugated through a lysine residue), glutaraldehyde, succinic anhydride, or other derivatizing agents known in the art.

Most commonly, the cysteinyl residue is reacted with an alpha-haloacetic acid (and corresponding amines) (e.g., chloroacetic acid, chloroacetamide) to give a carboxymethyl derivative or a carboxyamidomethyl derivative. Cysteinyl residues are also derivatized by reaction with: bromotrifluoroacetone, α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuriyl-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1, 3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because such derivatizing agents are relatively specific for histidyl side chains. P-bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1M sodium cacodylate at pH 6.0.

Lysyl residues and amino terminal residues are derivatized with succinic anhydride or other carboxylic acid anhydrides. Derivatization with cyclic carboxylic acid anhydrides has the effect of reversing the charge of the lysyl residue. Other suitable reagents for derivatizing the residue containing the quarteture amino group include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2, 4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

Arginyl residues are modified by reaction with one or more conventional reagents, among which are phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires reaction under alkaline conditions because of the high pKa of the guanidine functional group. In addition, these reagents can react with the groups of lysine as well as the arginine epsilon-amino group.

Specific modifications of tyrosyl residues can be carried out by reaction with aromatic diazo compounds or tetranitromethane, wherein the introduction of spectroscopic tags into tyrosyl residues is particularly advantageous. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively.

The pendant carboxyl groups aspartyl or glutamyl groups can be selectively modified by reaction with carbodiimides such as 1-cyclohexyl-3- (2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4, 4-dimethylpentyl) carbodiimide (R — N ═ C ═ N — R'), in addition aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonia.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the amino group of lysine (Creighton, supra, pages 79-86), acetylation of the N-terminal amine, and amidation of the C-terminal carboxyl group.

Another type of covalent modification involves chemically or enzymatically coupling the glycoside to the peptide. The sugar may be linked to: (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as cysteine, (d) free hydroxyl groups, such as serine, threonine or hydroxyproline, (e) aromatic residues, such as tyrosine or tryptophan, or (f) glutamine amide groups. These methods are described in WO87/05330 and Aplin and Wriston, CRC Crit. Rev. biochem., 259-306 (1981), published on 9/11/1987.

The chemical moiety may be indirectly attached to the peptide via a linker. As used herein, a "linker" is a chemical bond, molecule, or group of molecules that connects two separate entities to each other. The linker may provide the optimum spacing of the two entities, or may also provide an unstable bond that allows the two entities to be separated from each other. Labile bonds include photocleavable groups, acid labile moieties, base labile moieties, and enzyme cleavable groups. In some embodiments, the linker is an intermediate carrier, such as a polysaccharide or polypeptide carrier. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these and other amino acids (e.g., serine to provide the desired solubility properties to the resulting loaded carrier), and the like.

Fusion proteins

The present invention includes "fusion proteins" comprising a PTH or PTHrP peptide or polypeptide analog (variant or chemical derivative) fused to another peptide or polypeptide that confers useful properties to the fusion protein. For example, a PTH or PTHrP peptide or polypeptide analog can be fused to a polypeptide that promotes cellular uptake of PTH or PTHrP peptide or polypeptide analog.

In some embodiments, the PTH or PTHrP peptide or polypeptide analog (variant or chemical derivative) is fused directly to another peptide or polypeptide. In particular embodiments, the PTH or PTHrP peptide or polypeptide analog (variant or chemical derivative) is located N-terminal to the other peptide or polypeptide. In other embodiments, the PTH or PTHrP peptide or polypeptide analog (variant or chemical derivative) is located C-terminal to the other peptide or polypeptide.

In some embodiments, the PTH or PTHrP peptide or polypeptide analog (variant or chemical derivative) is fused to another peptide or polypeptide by a linker. Suitable linkers are known in the art and include, but are not limited to, amino acids (e.g., any of the amino acids described herein) and peptides (dipeptides, tripeptides, 4-mers, 5-mers, 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 15-mers, 20-mers, 25-mers, 30-mers, 35-mers, 40-mers, 45-mers, 50-mers). Suitable amino acids and peptides include those known in the art (e.g., any of the amino acids and peptides described in the "chemical section" herein).

The other peptide to which the PTH peptide or PTHrP (or an analogue or variant thereof) is fused may be a plasma protein, a secretion signal, a targeting moiety, a cytokine, a soluble factor or an immunoglobulin or part thereof (e.g. a variable region, CDR or Fc region). Known immunoglobulin (Ig) classes include IgG, IgA, IgE, IgD or IgM. The Fc region is the C-terminal region of the Ig heavy chain, which is responsible for binding to Fc receptors that perform activities such as recycling (which results in increased half-life), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

For example, according to certain definitions, the human IgG heavy chain Fc region extends from Cys226 to the C-terminus of the heavy chain. The "hinge region" typically extends from Glu216 to Pro230 of human IgG1 (the hinge region of other IgG isotypes can be aligned to the IgG1 sequence by aligning the cysteines involved in cysteine bonding). The Fc region of IgG comprises 2 constant domains CH2 and CH 3. The CH2 domain of the human IgGFc region typically extends from amino acid 231 to amino acid 341. The CH3 domain of the human IgG Fc region often extends from amino acids 342 to 447. The amino acid numbering of the mentioned immunoglobulins or immunoglobulin fragments or regions is based on Kabat et al, 1991, Sequences of Proteins of Immunological Interest, U.S. department of public Health, Bethesda, Md, in its entirety. In a related embodiment, the Fc region may comprise one or more natural or modified immunoglobulin heavy chain constant regions (excluding CH 1), such as the CH2 and CH3 regions of IgG and IgA or the CH3 and CH4 regions of IgE.

Suitable other peptides to which the PTH or PTHrP peptide is fused include portions of immunoglobulin sequences (which comprise an FcRn binding site). FcRn, a salvage receptor, is responsible for recycling immunoglobulins and returning them to circulation in the blood. According to X-ray crystallography, the region of the IgG Fc part that binds to the FcRn receptor is described (Burmeister et al 1994, Nature 372: 379). The main contact region of Fc to FcRn is near the junction of the CH2 and CH3 domains. The Fc-FcRn contacts are all within a single Ig heavy chain. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of the CH2 domain and amino acid residues 385-387, 428 and 433-436 of the CH3 domain.

And may or may not include an Fc γ R binding site. Fc γ R is responsible for ADCC and CDC. Examples of positions within the Fc region which are in direct contact with Fc γ R are the amino acids 234-. The lower hinge region of IgE is also involved in FcRI binding (Henry et al, Biochemistry 36, 15568-15578, 1997). Residues involved in IgA receptor binding are described by Lewis et al (J Immunol.175: 6694-701, 2005). Amino acid residues involved in IgE receptor binding are described in Sayers et al (J Biol chem.279 (34): 35320-5, 2004).

Amino acid modifications may be made to the Fc region of an immunoglobulin. Such variant Fc regions comprise at least one amino acid modification in the CH3 domain (residues 342-447) and/or at least one amino acid modification in the CH2 domain (residues 231-341) of the Fc region. Mutations thought to confer increased affinity for FcRn include T256A, T307A, E380A and N434A (Shields et al, 2001, j.biol.chem.276: 6591). Other mutations may reduce the binding of the Fc region to Fc γ RI, Fc γ RIIA, Fc γ RIIB, and/or Fc γ RIIIA without significantly reducing the affinity for FcRn. For example, substitution of the Asn at position 297 of the Fc region by Ala or another amino acid removes a highly conserved N-glycosylation site and may cause a reduction in immunogenicity with an increase in half-life of the Fc region, as well as a reduction in binding to Fc γ R (Routledge et al, 1995, Transplantation 60: 847; Friend et al 1999, Transplantation 68: 1632; Shields et al 1995, J.biol.Chem.276: 6591). Amino acids at position 233-236 of IgG1 have also been modified to reduce binding to Fc γ R (Ward and Ghetie 1995, therapeutic immunology 2: 77, and Armour et al 1999, Eur. J. immunol. 29: 2613). Some exemplary amino acid substitutions are described in U.S. patents 7,355,008 and 7,381,408, each of which is incorporated herein by reference in its entirety.

Multimer

The present invention also includes "multimeric peptides", i.e., longer peptides or polypeptides in which the basic peptide sequence of a PTH or PTHrP peptide or polypeptide analog repeats about 2 to about 100 times, with or without intervening spacers or linkers. In one embodiment, the peptide of SEQ ID NO: 10 (symbolically referred to in this section as "JJJ", where J, J and J do not represent a single amino acid) is represented by the following formulated example:

(JJJ-X_m)_n-JJJ, wherein m is 0 or 1, n is 1 to 100, X is a spacer, preferably C₁-C₂₀Alkyl radical, C₁-C₂₀Alkenyl radical, C₁-C₂₀Alkynyl, C containing up to 9 oxygen atoms₁-C₂₀Polyether or Glyz (z ═ 1-10).

It is understood that such multimers may be constructed from any of the peptides or polypeptides described herein. In addition, such multimers may comprise different combinations of peptide monomers and disclosed variants thereof. Such oligomeric or polymeric peptides can be prepared by chemical synthesis or by recombinant DNA techniques as described herein. If produced chemically, the oligomer preferably has 2 to 8 repeats of the basic peptide sequence. If produced recombinantly, the multimer may have as many repeats as the expression system allows, e.g., 2 to about 100 repeats.

Peptide mimetics

Another class of compounds useful in this regard are low molecular weight "peptidomimetic compounds" (which term also includes peptidomimetics). Such peptidomimetics can be identified by structural studies comparing the co-crystallization of PTH or one of the PTHrP peptides or polypeptide analogs with the PTH receptor in the presence or absence of a candidate peptidomimetic.

Peptidomimetics of PTH or PTHrP peptide or polypeptide analogs mimic the biological effects of one of PTH or PTHrP peptide or polypeptide analogs. A peptidomimetic may be a non-natural peptide or non-peptide substance that has the stereochemistry of a PTH or PTHrP peptide or polypeptide analog such that it has the binding activity or biological activity of a peptide. Thus, the invention includes compounds in which peptidomimetic compounds are coupled to other peptides.

A peptidomimetic may be a non-natural peptide or non-peptide substance that reproduces the steric properties of the binding element of a PTH or PTHrP peptide or polypeptide analog such that it has the binding activity or biological activity of a PTH or PTHrP peptide or polypeptide analog. Similar to the linear peptide corresponding to PTH or PTHrP peptide or polypeptide analog, the peptidomimetic may have a binding surface (which binds to P)TH receptor interaction) and non-binding surfaces. Furthermore, similar to the linear peptides of PTH or PTHrP peptides or polypeptide analogs, the non-binding surface of the peptidomimetic may contain functional groups that can be modified by various therapeutic moieties without modifying the binding surface of the peptidomimetic. One embodiment of the mimetic may contain aniline on the non-binding side of the molecule. The NH 2-group of the aniline has a pKa of about 4.5 and can therefore be modified by any NH2 selective agent without modifying any NH on the peptidomimetic binding surface₂A functional group. Other peptidomimetics may not have any NH at their binding surfaces₂Functional groups, therefore, not taking pK into account_aAny NH of₂May be present on the non-binding side as a conjugation site. In addition, other modifiable functional groups (e.g., - -SH and- -COOH) can be incorporated into the non-binding surface of the peptidomimetic as a conjugation site. The therapeutic moiety may also be incorporated directly during the synthesis of the peptidomimetic and preferably occurs on the non-binding surface of the molecule.

The invention also includes compounds that retain a partial peptide character. For example, any proteolytically labile bond within a peptide of the invention may be selectively replaced by a non-peptide element such as an isostere (N-methylation; D-amino acid at position S1) or a reducing peptide bond, while the remainder of the molecule retains its peptidic properties.

Peptidomimetics agonists, substrates, or inhibitors of a number of biologically active peptides, such as opioid peptides, VIP, thrombin, HIV protease, and the like, have been disclosed. Methods for designing and preparing peptidomimetic compounds are known in the art ((Hruby, V.J., Biopolymers 33: 1073-1082 (1993); Wiley, R.A. et al, Med. Res. Rev.13: 327-384 (1993); Moore et al, adv. in Pharmacol 33: 91-141 (1995); Gianis et al, adv. in Drug Res.29: 1-78 (1997)), which references are all incorporated by reference.) these methods are used to prepare peptidomimetics having at least the binding capacity and specificity of cyclic peptides and preferably also having biological activity The stereochemistry of the partner interaction allows for the rational design of such peptidomimetics.

Peptide mimetics

The present invention also includes "peptidomimetics" which are oligomers of N-substituted glycines (Simon R J et al, Proc Natl Acad Sci USA, 199289: 9367-. "peptide-like" approaches to design non-peptides, small molecule agonists and antagonists are also described in Horwell D C, Trends Biotechnol, 1995, 13: 132-134. Peptidomimetics of PTH or PTHrP peptides or polypeptide analogs include the substitution or addition of one or more of such N-substituted glycines. The substituent can be 4-aminophenol, isobutylamine, butanediamine (NH)₂(CH₂)₃NH₂) Cyclohexane methylamine, aminomethyl cyclopropane, benzylamine, methylamine, isopropylamine, R (+) - (L-methylbenzylamine), 5- (-1-. alpha. -methylbenzylamine, N-3-guanidinopropyl, and the like. Nguyen.J.T. et al (Science 1998, 282: 2088-2092) have previously demonstrated that such substituents lead to an increased biological activity of SH3 binding peptidomimetics. However, the substituent may be virtually any substituent substituted at the N-position of glycine, so long as the N-glycine product can be further coupled in the peptidomimetic.

PTH or PTHrP peptides or polypeptide analogs of the invention (e.g., the peptide of SEQ ID NO: 10) can be substituted at the amino-and carboxy-termini with, for example, an acetyl group ("Ac") and an amido group bound to the N-terminus (a- -NH group bound to the carboxy-terminus of the C-terminus), respectively₂("Am")) blocking or capping. Such peptides may be mentioned in the single letter codes for the blocking groups Ac and Am: Ac-SEQ ID NO: 10, -Am.

The N-terminal capping functional group is preferably linked to the terminal amino functional group and may be selected from: a formyl group; alkanoyl groups having 1 to 10 carbon atoms, such as acetyl, propionyl, butyryl; alkenoyl having 1 to 10 carbon atoms, such as hex-3-alkenoyl; alkynoyl having 1 to 10 carbon atoms, e.g.Hex-5-ynoyl; aroyl, such as benzoyl or 1-naphthoyl; heteroaroyl groups such as 3-pyrroloformyl or 4-quinolinoyl; alkylsulfonyl groups such as methanesulfonyl group; arylsulfonyl such as phenylsulfonyl or sulfonamido; heteroarylsulfonyl, such as pyridine-4-sulfonyl; substituted alkanoyl groups having 1 to 10 carbon atoms, such as 4-aminobutyryl; substituted alkenoyl groups having 1 to 10 carbon atoms, such as 6-hydroxy-hex-3-enoyl; substituted alkynoyl groups having 1 to 10 carbon atoms, such as 3-hydroxy-hex-5-ynoyl; substituted aroyl groups such as 4-chlorobenzoyl or 8-hydroxy-naphthalen-2-yl; substituted heteroaroyl groups, such as 2, 4-dioxo-1, 2, 3, 4-tetrahydro-3-methyl-quinazolin-6-yl; substituted alkylsulfonyl groups such as 2-aminoethanesulfonyl; substituted arylsulfonyl groups such as 5-dimethylamino-1-naphthalenesulfonyl; substituted heteroarylsulfonyl, such as 1-methoxy-6-isoquinolinesulfonyl; carbamoyl or thiocarbamoyl; substituted carbamoyl (R ' -NH-CO) or substituted thiocarbamoyl (R ' -NH-CS), wherein R ' is alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted aryl or substituted heteroaryl; substituted carbamoyl (R ' -NH-CO) or substituted thiocarbamoyl (R ' -NH-CS) wherein R ' is alkanoyl, alkenoyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl, substituted alkenoyl, substituted alkynoyl, substituted aroyl or substituted heteroaroyl, all as defined above; the C-terminal capping functional group may be located in an amide linkage with the terminal carboxyl group or in an ester linkage with the terminal carboxyl group. The capping function providing the amide bond is designated NR¹R²Wherein R is¹And R²Can be independently selected from the following groups: hydrogen; alkyl groups preferably having 1 to 10 carbon atoms, such as methyl, ethyl, isopropyl; alkenyl groups having preferably 1 to 10 carbon atoms, such as prop-2-enyl; alkynyl having preferably 1 to 10 carbon atoms, such as prop-2-ynyl; substituted alkyl groups having 1 to 10 carbon atoms, e.g. hydroxyalkyl, alkoxyalkyl, mercaptoalkyl, alkylthioalkyl, haloalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkanoylalkyl, carboxyalkyl, carbamoylalkyl(ii) a Substituted alkenyl groups having 1 to 10 carbon atoms such as hydroxyalkenyl, alkoxyalkenyl, mercaptoalkenyl, alkylthioalkenyl, haloalkenyl, cyanoalkenyl, aminoalkenyl, alkylaminoalkenyl, dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl; substituted alkynyl groups having 1 to 10 carbon atoms, such as hydroxyalkynyl, alkoxyalkynyl, mercaptoalkynyl, alkylthioalkynyl, haloalkynyl, cyanoalkynyl, aminoalkynyl, alkylaminoalkynyl, dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl; aroylalkyl having up to 10 carbon atoms, such as phenacyl or 2-benzoylethyl; aryl, such as phenyl or 1-naphthyl; heteroaryl, such as 4-quinolinyl; alkanoyl groups having 1 to 10 carbon atoms, such as acetyl or butyryl; aroyl, such as benzoyl; heteroaroyl groups such as 3-quinolinoyl; OR ' OR NR ' R ", wherein R ' and R" are independently hydrogen, alkyl, aryl, heteroaryl, acyl, aroyl, sulfonyl, sulfinyl; or SO 2-R ' or SO-R ', wherein R ' is a substituted or unsubstituted alkyl, aryl, heteroaryl, alkenyl, or alkynyl group.

All of the aforementioned variants, fusion proteins, multimeric peptides, peptidomimetics, and chemical derivatives of the PTH or PTHrP peptide or polypeptide analogs described herein must have the following biological or biochemical activity of the PTH or PTHrP peptide or polypeptide analog: at least about 20%, 30%, 40%, 50%, 60, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the activity of the PTH or PTHrP peptide or polypeptide analog as determined in the assay of the examples below. Alternatively or additionally, the aforementioned variants, fusion proteins, multimeric peptides, peptidomimetics and chemical derivatives of PTH or PTHrP peptide or polypeptide analogs should compete with the labeled PTH or PTHrP peptide or polypeptide analog for binding to the PTH receptor when tested in a binding assay with intact cells, cell fractions, isolated PTH receptor binding domain containing proteins or peptides or any other binding molecule.

Retro-inverso peptides (retroverterso peptide)

For each of the peptide and polypeptide sequences disclosed herein, the invention includes the corresponding retro-inverso sequences, wherein the orientation of the peptide chain is inverted, and wherein all of the amino acids are in the D-form. The entire range of N-terminal capping groups and the entire range of C-terminal capping groups specified for L-type peptides are also intended for D-type peptides.

Construction method

The peptides and polypeptides of the invention may be prepared using recombinant DNA techniques. Solid phase synthesis preparations may also be used, such as those outlined by Merrifield (J.Amer.chem.Soc, 85: 2149-54(1963)), but other equivalent chemical syntheses known in the art are also useful. Solid phase peptide synthesis can be initiated from the C-terminus of the peptide by coupling the protected α -amino acid to a suitable resin. Such starting materials can be prepared by linking an alpha-amino protected amino acid to a chloromethylated resin or to a hydroxymethyl resin via an ester bond, or to a BHA resin or MBHA resin via an amide bond.

Hydroxymethyl resins are prepared as described in Bodansky et al, chem.ind., 38: 1597-98(1966). The chloromethylated resins are commercially available from BioRad Laboratories, Richmond, Calif and from lab. Such resins are prepared as described by Stewart et al, "Solid Phase Peptide Synthesis" (Freeman & Co., san Francisco 1969), Chapter 1, 1-6. BHA and MBHA resin supports are commercially available and are generally used only when the desired polypeptide to be synthesized has an unsubstituted amide at the C-terminus.

Amino acids can be coupled to extended peptide chains using techniques well known in the art for peptide bond formation. For example, one method involves converting the amino acid into a derivative that renders the carboxyl group of the amino acid more susceptible to reaction with the free N-terminal amino group of the extended peptide chain. Specifically, the C-terminus of the protected amino acid can be converted into a mixed anhydride by reacting the C-terminus with an acid chloride such as ethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate, isobutyl chloroformate, or pivaloyl chloride. Alternatively, the C-terminus of the amino acid may be converted to an active ester, such as 2, 4, 5-trichlorophenyl ester, pentachlorophenyl ester, pentafluorophenyl ester, p-nitrophenyl ester, N-hydroxysuccinimide ester, or an ester formed from 1-hydroxybenzotriazole. Another coupling method involves the use of a suitable coupling agent, such as N, N '-dicyclohexylcarbodiimide or N, N' -diisopropylcarbodiimide. Other suitable coupling agents that will be apparent to those skilled in The art are disclosed in Gross et al, The Peptides: analysis, Structure, Biology, Vol.I, "Major Methods of Peptide BondFormat" (Academic Press 1979).

The alpha-amino group of each amino acid used for peptide synthesis must be protected during the coupling reaction to prevent side reactions involving its active alpha-amino functional group. Certain amino acids contain reactive side chain functional groups (e.g., sulfhydryl, amino, carboxyl, and hydroxyl), which must also be protected with suitable protecting groups to prevent chemical reactions at the following sites during both the initial and subsequent coupling steps: (1) an alpha-amino moiety, or (2) a reactive side chain moiety.

In selecting specific protecting groups for use in the synthesis of peptides, the following general rules are generally followed. Specifically, the α -amino protecting group (1) should render the α -amino functional group inert under the conditions used for the coupling reaction, (2) should be easily removed after the coupling reaction without removing the side chain protecting group and without changing the structure of the peptide fragment, and (3) should sufficiently reduce the possibility of racemization upon activation immediately before the coupling. On the other hand, side chain protecting groups should (1) render the side chain functional groups inert under the conditions used for the coupling reaction, (2) be stable under the conditions used to remove the α -amino protecting group, and (3) be readily removed from the desired fully assembled peptide under reaction conditions that do not alter the structure of the peptide chain.

It will be apparent to those skilled in the art that the protecting groups known for peptide synthesis differ in reactivity depending on the reagents used for their removal. For example, certain protecting groups, such as triphenylmethyl and 2- (p-biphenylyl) isopropoxycarbonyl, are very labile and can be cleaved under mild acid conditions. Other protecting groups such as t-Butoxycarbonyl (BOC), t-pentoxycarbonyl, adamantyloxycarbonyl, and p-methoxybenzyloxycarbonyl are less labile and require moderately strong acids for their removal, such as trifluoroacetic acid, hydrochloric acid, or boron trifluoride in acetic acid. Benzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl and isopropoxycarbonyl, among other protecting groups, are even less labile and require even stronger acids for removal, such as hydrogen fluoride, hydrogen bromide or boron trifluoroacetate in trifluoroacetic acid. Suitable protecting groups known in The art are described in Gross et al, The Peptides: analysis, Structure, Biology, volume 3: "Protection of Functional Groups in peptide Synthesis" (Academic Press 1981).

Some of the alpha-amino protecting groups are BOC and FMOC. For the side chain amino group present on Lys, it may be protected by any of the groups mentioned in (1) above, such as BOC, 2-chlorobenzyloxycarbonyl, and the like. For the guanidino group of Arg, protection may be provided by a nitro, tosyl, CBZ, adamantyloxycarbonyl, 2, 5, 7, 8-pentamethylbenzodihydropyran-6-sulfonyl, 2, 3, 6-trimethyl-4-methoxyphenylsulfonyl, or BOC group. For the hydroxyl group of Ser or Thr, the hydroxyl group can be replaced by, for example, tert-butyl; benzyl (BZL); or substituted BZL, such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl and 2, 6-dichlorobenzyl. For the carboxyl group of Asp or Glu, groups such as BZL, t-butyl, cyclohexyl, cyclopentyl and the like may be used, protected, for example, by esterification. For the imidazole nitrogen of His, a Benzyloxymethyl (BOM) or tosyl (tosyi) moiety is suitably used as a protecting group. As the phenolic hydroxyl group of Tyr, a protecting group such as tetrahydropyranyl group, t-butyl group, trityl group, BZL, chlorobenzyl group, 4-bromobenzyl group and 2, 6-dichlorobenzyl group is suitably used. A preferred protecting group is bromobenzyloxycarbonyl. For the side chain amino group of Asn or Gln, xanthenyl (Xan) is preferably used. For Met, it is preferred that the amino acid is unprotected. For the thiol group of Cys, p-methoxybenzyl is generally used.

Other standard α -amino deprotection reagents (e.g. HCl/dioxane) and conditions for removing a particular α -amino protecting group are within the skill of the art, such as those described in Lubke et al, Chemieund biochemide der Aminosauren, Peptide und Proteine I, Chapter II-1, 102-117(Georg Thieme Verlag Stuttgart 1975). After removal of the alpha-amino protecting group, the unprotected alpha-amino group, which is generally still side chain protected, can be coupled in a stepwise manner in the predetermined sequence.

An alternative to the stepwise approach is the fragment condensation method, in which preformed short length peptides (each representing part of the desired sequence) are coupled to an extended amino acid chain bound to a solid support. For this stepwise process, a particularly suitable coupling reagent is N, N' -dicyclohexylcarbodiimide or diisopropylcarbodiimide. Also, for the fragment method, the choice of coupling reagents and the fragmentation pattern required to couple the fragments of the desired nature and size are important for success and are known to those skilled in the art.

Each protected amino acid or amino acid sequence is generally introduced into the solid phase reactor in an amount in excess of the stoichiometric amount, and the coupling is suitably carried out in an organic solvent, such as Dimethylformamide (DMF), CH2Cl2, or a mixture thereof. If incomplete coupling occurs, the coupling step is generally repeated before the N-amino protecting group is removed in preparation for coupling with the next amino acid. After removal of the alpha-amino protecting group, the remaining alpha-amino groups and side chain protected amino acids can be coupled in a stepwise manner in the predetermined sequence. The success of the coupling reaction in each synthesis stage can be monitored. A preferred method of monitoring synthesis is by ninhydrin reaction, see Kaiser et al, anal. biochem., 34: 595(1970). The coupling reaction may also be automated using well known commercially available methods and apparatus, such as the Beckman 990 Peptide Synthesizer.

Upon completion of the desired peptide sequence, the protected peptide must be cleaved from the resin support and all protecting groups must be removed. Cleavage and deprotection of the protecting group is suitably carried out simultaneously or sequentially with the deprotection reaction. If the bond anchoring the peptide to the resin is an ester bond, it may be cleaved by any agent capable of breaking the ester bond and penetrating the resin matrix. One useful method is by treatment with liquid anhydrous hydrogen fluoride. Such reagents will generally not only cleave the peptide from the resin, but will also remove all acid labile protecting groups, and thus will directly provide a fully deprotected peptide. If other protecting groups are present, which are not acid labile, then additional deprotection steps must be performed. These steps may be performed before or after the hydrogen fluoride treatment described above, as the case may be, according to specific needs and circumstances.

If cleavage of the peptide is desired without removal of the protecting group, the protected peptide-resin can be methanolyzed, thus producing a protected peptide in which the C-terminal carboxyl group is methylated. This methyl ester can then be hydrolyzed under mildly basic conditions to give the free C-terminal carboxyl group. The peptide chain can then be deprotected by treatment with a strong acid, such as liquid hydrogen fluoride. One particularly useful technique for methanolysis is that in Moore et al, Peptides, Proc. Fifth Amer. Pept. Symp., 518-521 (edited by Goodman et al, 1977), the protected peptide-resin is treated with methanol and potassium cyanide in the presence of a crown ether.

If a chloromethylated resin is used, other methods for cleaving the protected peptide from the resin include (1) ammonolysis, and (2) hydrazinolysis. If desired, the resulting C-terminal amide or hydrazide can be hydrolyzed to the free C-terminal carboxyl moiety and the protecting group removed by conventional methods. The protecting group present on the N-terminal alpha-amino group can be cleaved off either before or after cleaving the protected peptide from the support. Purification of the peptides of the invention is typically achieved using chromatographic techniques such as preparative HPLC (including reverse phase HPLC), gel permeation chromatography, ion exchange chromatography, partition chromatography, affinity chromatography (including monoclonal antibody columns), and the like, or other conventional techniques such as reverse flow distribution methods, and the like.

Pharmaceutical compositions, formulations, routes and dosages

The pharmaceutical compositions of the invention comprise PTH or PTHrP peptide or polypeptide analogs (and variants, fusion proteins, multimeric peptides, peptidomimetics, and chemical derivatives thereof) in formulations known per se in the art. The pharmaceutical formulations of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, dissolving or lyophilizing procedures. Suitable excipients may include fillers, binders, disintegrants, adjuvants and stabilizers, all of which are known in the art. Suitable formulations for parenteral administration include aqueous solutions of the protein in water-soluble form (e.g., water-soluble salts). Additionally, suspensions of the active compounds may be administered as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil) or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension. It is absolutely necessary that the vehicle, carrier or excipient and the conditions under which the composition is formulated have to positively influence the biological or pharmaceutical activity of the PTH or PTHrP peptide or polypeptide analog.

The compositions may be in the form of lyophilized particulate material, sterile or aseptically produced solutions, tablets, ampoules, and the like. A vehicle, such as water (preferably buffered to a physiologically acceptable pH, e.g., in phosphate buffered saline) or other inert solid or liquid material, such as physiological saline or various buffers, may be present. The particular vehicle is not critical and one skilled in the art will recognize which vehicle may be used for any particular use described herein.

Other pharmaceutically acceptable carriers of the invention are liposomes, in which the pharmaceutical composition in which the active protein is contained is dispersed or otherwise present in the minibody, which consists of aqueous concentric layers attached to a lipid layer. The active protein is preferably present in the inner or outer aqueous and lipid layers or, in any case, in a heterogeneous system commonly referred to as a liposomal suspension.

The hydrophobic or lipid layer typically, but not exclusively, comprises phospholipids (such as lecithin and sphingomyelin), steroids (such as cholesterol), more or less ionic surface active substances (such as dicetyl phosphate, stearylamine or phosphatidic acid) and/or other materials with hydrophobic properties.

The systemically administered pharmaceutical formulations of the present invention may be formulated for enteral, parenteral or topical administration, and all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

In addition to pharmacologically active peptides or polypeptide analogs, in some aspects, the pharmaceutical compositions contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically, as is well known in the art. Suitable solutions for injection or oral administration may contain about 0.01-99% of the active compound together with excipients.

The expression "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions (e.g. gastric (gastric upset), dizziness, etc.) when administered to a human. Preferably, the term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the federal or a state government or registered in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The pharmaceutical compositions of the present invention may be administered by any method that achieves its intended purpose. The amount and regimen of administration can be readily determined by one of ordinary skill in the clinical art for treating any particular disease. Preferred amounts are as follows.

The methods of the invention include administration by parenteral routes, including subcutaneous (s.c.), intravenous (i.v.), intramuscular, intraperitoneal, intrathecal, transdermal, topical, or inhalation routes. The preferred route is by injection. Alternatively or simultaneously, administration may be by the oral route. The dose administered will depend on the age, health and weight of the recipient, the nature of concurrent treatment (if any), the frequency of treatment and the nature of the effect desired.

In the method of the invention, the composition may be administered once, but may also be administered 6-12 times (even more, as will be appreciated empirically by those skilled in the art). Treatment may be performed daily, but may also be performed every two or three days, or infrequently, such as once a week, depending on the beneficial and any toxic effects observed in the subject.

The pharmaceutical formulations disclosed herein may be designed to be short-acting, immediate-release, long-acting, or sustained-release as described below. Pharmaceutical formulations may also be formulated for immediate, controlled or slow release. The compositions of the invention may also include, for example, micelles or liposomes or some other microencapsulated form, or may be administered in a sustained release form to provide long-term storage and/or delivery effects. The pharmaceutical formulations of the present disclosure can be administered on any schedule, including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), every two days, every three days, every four days, every five days, every six days, every week, every two weeks, every three weeks, every month, or every two months.

According to some embodiments, there is provided a pharmaceutical composition comprising any PTH peptide, PTHrP peptide (or related analogue or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide), disclosed herein, preferably sterile and preferably having a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient. Such compositions may contain PTH peptide or PTHrP peptide in a concentration of at least A, wherein A is 0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, 10mg/ml, 11mg/ml, 12mg/ml, 13mg/ml, 14mg/ml, 15mg/ml, 16mg/ml, 17mg/ml, 18mg/ml, 19mg/ml, 20mg/ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml or more. In other embodiments, such compositions may contain PTH peptide or PTHrP peptide at a concentration of up to B, wherein B is 30mg/ml, 25mg/ml, 24mg/ml, 23mg/ml, 22mg/ml, 21mg/ml, 20mg/ml, 19mg/ml, 18mg/ml, 17mg/ml, 16mg/ml, 15mg/ml, 14mg/ml, 13mg/ml, 12mg/ml, 11mg/ml, 10mg/ml, 9mg/ml, 8mg/ml, 7mg/ml, 6mg/ml, 5mg/ml, 4mg/ml, 3mg/ml, 2mg/ml, 1mg/ml or 0.1 mg/ml. In some embodiments, the composition may contain PTH peptide or PTHrP peptide at a concentration ranging from A to B mg/ml, for example, 0.001 to 30.0 mg/ml. In one embodiment, the pharmaceutical composition comprises an aqueous solution that is sterilized and optionally stored in various containers. The PTH or PTHrP peptides of the present disclosure can be used in one embodiment to prepare a pre-formulated solution ready for injection. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may be further packaged as part of a kit as otherwise described herein, the kit comprising a disposable device for administering the composition to a patient. The container or kit may be indicated to be stored at ambient room temperature or at refrigerated temperatures.

The pharmaceutical composition may comprise any pharmaceutically acceptable ingredient including, for example, acidulants, additives, adsorbents, aerosol propellants, air release agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antibacterial agents, bases, binders, buffering agents, chelating agents, coating agents, colorants, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, solubilizing agents, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, flavoring agents, flavorants, flow promoters, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, inorganic bases, pastille bases, pigments, plasticizers, agents, preservatives, masking polishes, skin penetrating agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, Surface-active substances, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxic agents, viscosity increasing agents, water absorbing agents, water miscible co-solvents, water softening agents or wetting agents.

In some embodiments, the pharmaceutical composition comprises any one or a combination of the following components: acacia, acesulfame potassium, acetyl tributyl citrate, acetyl triethyl citrate, agar, albumin, alcohol, anhydrous alcohol, denatured alcohol, diluted alcohol, elaeostearic acid, alginic acid, aliphatic polyester, alumina, aluminum hydroxide, aluminum stearate, amylopectin, alpha-amylose, ascorbic acid, ascorbyl palmitate, aspartame, injectable bacteriostatic water, bentonite whey, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl chlorideAlcohols, benzyl benzoate, bronopol, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, sodium butylate, calcium alginate, calcium ascorbate, calcium carbonate, calcium cyclamate, anhydrous calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate (dibasic dehydrated calcium phosphate), tricalcium phosphate, calcium propionate, calcium silicate, calcium sorbate, calcium stearate, calcium sulfate hemihydrate, canola oil (canola oil), carbomer, carbon dioxide, carboxymethylcellulose calcium, carboxymethylcellulose sodium, beta-carotene, carrageenan, castor oil, hydrogenated castor oil, cationic emulsifying wax, cellulose acetate phthalate, ethylcellulose, microcrystalline cellulose, powdered cellulose, microcrystalline cellulose, sodium carboxymethylcellulose, cetostearyl alcohol, cetyltrimethylammonium bromide, cetyl alcohol, chlorhexidine, chlorobutanol, sodium benzoate, calcium hydrogen phosphate, calcium propionate, calcium silicate, calcium sorbate, carbomer, carbon dioxide, carboxymethylcellulose calcium, carboxymethylcellulose sodium, beta-carotene, carrageenan, castor oil, cationic emulsifying wax, cellulose acetate phthalate, ethyl cellulose, microcrystalline cellulose, powdered cellulose, silicified cellulose, microcrystalline cellulose, carboxymethylcellulose sodium, chlorocresol, cholesterol, chlorhexidine acetate, chlorhexidine gluconate, chlorhexidine hydrochloride, chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbon (CFC), chlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrous citric acid, citric acid monohydrate, cocoa butter, colorants, corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol, croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrin, dextrates, dextrin, glucose, anhydrous glucose, diazolidinyl urea (diazolidinylurea), dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate, difluoroethane (HFC), dimethyl- β -cyclodextrin, cyclodextrin-type compounds (e.g., Captisol)) Dimethyl ether, dimethyl phthalate, dipotassium edetate, disodium hydrogen phosphate, docusate calcium, potassium docusate, sodium docusate, dodecyl glycol gallate, dodecyl trimethyl ammonium bromide, disodium edetate, edetic acid (ethic acid), meglumine, ethanol, ethyl cellulose, ethyl gallate, ethyl laurate, ethyl maltol, ethyl oleate, ethylparaben, potassium ethylparaben, sodium ethylparaben, ethylvanillin, fructose, liquid fructoseMilled fructose, pyrogen-free fructose, powdered fructose, fumaric acid, gelatin, glucose, liquid glucose, glycerol ester mixture of saturated vegetable fatty acids, glycerol behenate, glycerol monooleate, glycerol monostearate, self-emulsifying glycerol monostearate, glycerol stearate citrate, glycine, glycols, glycofurol, guar gum, pentafluoropropane (HFC), cetyltrimethylammonium bromide, high fructose syrup, human serum albumin, Hydrocarbons (HC), dilute hydrochloric acid, hydrogenated plant type II, hydroxyethylcellulose, 2-hydroxyethyl-beta-cyclodextrin, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, 2-hydroxypropyl-beta-cyclodextrin, hydroxypropylmethylcellulose phthalate, imidurea, indigo carmine, ion exchanger, iron oxide, Isopropyl alcohol, isopropyl myristate, isopropyl palmitate, isotonic saline, kaolin, lactic acid, lactitol, lactose, lanolin alcohols, anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesium carbonate, normal magnesium carbonate, anhydrous magnesium carbonate, basic magnesium carbonate, magnesium hydroxide, magnesium lauryl sulfate, magnesium oxide, magnesium silicate, magnesium stearate, magnesium trisilicate, anhydrous magnesium trisilicate, malic acid, malt, maltitol solutions, maltodextrin, maltol, maltose, mannitol, medium chain triglycerides, meglumine, menthol, methyl cellulose, methyl methacrylate, methyl oleate, methyl hydroxybenzoate, potassium hydroxybenzoate, sodium hydroxybenzoate, microcrystalline cellulose and sodium carboxymethylcellulose, mineral oil, light mineral oil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine, montmorillonite, and mixtures thereof, Octyl gallate, oleic acid, palmitic acid, paraffin, peanut oil, petrolatum and lanolin alcohols, pharmaceutical glazes, phenol, liquefied phenol, phenoxyethanol, phenoxypropanol, phenylethanol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, polacrilin potassium, poloxamers, polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates, polyethylene-polyoxypropylene-block polymers, polymethacrylates, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates, polyethylene stearate estersAlcohols, polyvinylpyrrolidone, potassium alginate, potassium benzoate, potassium bicarbonate, potassium bisulfite, potassium chloride, potassium citrate, anhydrous potassium citrate, potassium hydrogen phosphate, potassium metabisulfite, potassium dihydrogen phosphate, potassium propionate, potassium sorbate, povidone, propanol, propionic acid, propylene carbonate, propylene glycol alginate, propyl gallate, propyl hydroxybenzoate, potassium propylhydroxybenzoate, sodium hydroxy phenylpropionate, protamine sulfate, rapeseed oil, ringer's solution, saccharin, ammonium saccharin, calcium saccharin, sodium saccharin, safflower oil, saponite, serum protein, sesame oil, colloidal silica, sodium alginate, sodium ascorbate, sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride, anhydrous sodium citrate, sodium citrate dihydrate (sodium citrate dihydrate), sodium chloride, sodium cyclamate, sodium edetate, sodium lauryl sulfate, Sodium lauryl sulfate, sodium metabisulfite, sodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium propionate anhydrous, sodium propionate, sodium sorbate, sodium starch glycolate, sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters (sorbitan fatty acids), sorbitol solution 70%, soybean oil, spermaceti, starch, corn starch, potato starch, pregelatinized starch, sterilizable corn starch, stearic acid, purified stearic acid, stearyl alcohol, sucrose, sugar, compressible sugar, confectioner's sugar, sugar spheres, invert sugar, sucrose-invert sugar polymers (Sugartab), Sunset Yellolfcf, synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane (HFC), cocoa butter, thimerosal, titanium dioxide, alpha tocopherol, vitamin E acetate, vitamin E alpha succinate, beta-tocopherol, Delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributyl citrate, triethanolamine, triethyl citrate, trimethyl-beta-cyclodextrin, trimethyltetradecylammonium bromide, tris (hydroxymethyl) aminomethane buffer, trisodium edetate, vanillin, hydrogenated vegetable oil type I, water, soft water, hard water, carbon dioxide-free water, pyrogen-free water, water for injection, sterile water for flushing, wax, anionic emulsifying wax, carnauba wax, cationic emulsifying wax, cetyl esters wax, microcrystalline wax, nonionic emulsifying wax, suppository wax, and the likeWhite wax, yellow wax, white petrolatum, lanolin, xanthan gum, xylitol, zein, zinc propionate, zinc salts, zinc stearate or any excipient of the Handbook of Pharmaceutical excipients, 3 rd edition, a.h. kibbe (Pharmaceutical Press, London, UK, 2000), all incorporated by reference. Remington's Pharmaceutical Sciences, 16 th edition, e.w. martin (Mack Publishing co., Easton, Pa., 1980), which is incorporated by reference in its entirety, discloses various components for formulating pharmaceutically acceptable compositions and known techniques for their preparation. Unless any conventional material is incompatible with the pharmaceutical composition, its use in pharmaceutical compositions is included. Supplementary active ingredients may also be incorporated into the compositions.

In some embodiments, the aforementioned components may be present in the pharmaceutical composition at any concentration, e.g., at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v, 1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60% w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the aforementioned components may be present in the pharmaceutical composition at any concentration, e.g., up to B, wherein B is 90% w/v, 80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v, 5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In other embodiments, the aforementioned components may be present in the pharmaceutical composition in any concentration range, for example, from about a to about B. In some embodiments, a is 0.0001% and B is 90%.

The pharmaceutical composition may be formulated to achieve a physiologically compatible pH. In some embodiments, the pH of the pharmaceutical composition may be at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or at least 10.5 up to and including pH 11, depending on the formulation and route of administration. In certain embodiments, the pharmaceutical composition may comprise a buffering agent to achieve a physiologically compatible pH. The buffer may include any compound capable of buffering at a desired pH, such as phosphate buffer (e.g., PBS), triethanolamine, Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES, and the like. In certain embodiments, the buffer has an intensity of at least 0.5mM, at least 1mM, at least 5mM, at least 10mM, at least 20mM, at least 30mM, at least 40mM, at least 50mM, at least 60mM, at least 70mM, at least 80mM, at least 90mM, at least 100mM, at least 120mM, at least 150mM, or at least 200 mM. In some embodiments, the buffer has an intensity of no more than 300mM (e.g., at most 200mM, at most 100mM, at most 90mM, at most 80mM, at most 70mM, at most 60mM, at most 50mM, at most 40mM, at most 30mM, at most 20mM, at most 10mM, at most 5mM, at most 1 mM).

Route of administration

The following discussion of routes of administration is provided merely to illustrate exemplary embodiments and should not be construed as limiting the scope in any way.

Formulations suitable for oral administration may include: (a) liquid solutions, such as an effective amount of an analog of the present disclosure dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, tablets, dragees and lozenges each containing a predetermined amount of the active ingredient as a solid or as granules; (c) powder; (d) suspensions in suitable liquids; and (e) a suitable emulsion. Liquid formulations may include diluents, such as water and alcohols, such as ethanol, benzyl alcohol and polyethylene glycol, with or without added pharmaceutically acceptable surfactants. Capsule forms can be the common hard or soft shell gelatin types containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch. The tablet form may comprise one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid and other excipients, colorants, diluents, buffering agents, disintegrating agents, wetting agents, preservatives, flavoring agents and other pharmacologically compatible excipients. In addition to containing such excipients as are known in the art, dragee forms can contain the analogs of the disclosure in a flavor, which is typically sucrose and acacia or tragacanth, and pastilles comprising the analogs of the disclosure in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like.

The PTH peptides, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-mimetics, retro-inverso peptides) of the present disclosure can be delivered by pulmonary administration, alone or in combination with other suitable components, and can be formulated as an aerosol for administration by inhalation. These aerosol formulations can be packaged in pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated for use in non-pressurized formulations (e.g., in a nebulizer or atomizer). Such spray formulations may also be used to spray mucous membranes. In some embodiments, the PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) is formulated as a powder mixture or as microparticles or nanoparticles. Suitable transpulmonary formulations are known in the art. See, e.g., Qian et al, Int J Pharm 366: 218-220 (2009); adjei and Garren, Pharmaceutical Research, 7 (6): 565 — 569 (1990); kawashima et al, JControled Release 62 (1-2): 279-287 (1999); liu et al, Pharm Res 10 (2): 228-; international patent application publication Nos. WO 2007/133747 and WO 2007/141411.

Formulations suitable for parenteral administration include aqueous and anhydrous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and anhydrous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The term "parenteral" means not through the alimentary canal but through some other route, such as subcutaneous, intramuscular, intraspinal, or intravenous. The PTH peptide, PTHrP peptide (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-mimetics, retro-inverso peptides) of the present disclosure can be administered with a physiologically acceptable diluent in a pharmaceutically acceptable carrier, such as a sterile liquid or liquid mixture, including water, saline, aqueous glucose and related sugar solutions, alcohols (such as ethanol or cetyl alcohol), glycols (such as propylene glycol or polyethylene glycol), dimethyl sulfoxide, glycerol, ketals (such as 2, 2-dimethyl-153-dioxolan-4-methanol), ethers, poly (ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without added pharmaceutically acceptable surfactants: such as soaps or detergents, suspending agents (e.g. pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose) or emulsifying agents and other pharmaceutically acceptable excipients.

Oils that may be used in parenteral formulations include petroleum, animal, vegetable or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, vaseline oil, and mineral oil. Suitable fatty acids for parenteral formulations include oleic acid, stearic acid and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for parenteral formulations include fatty alkali metal, ammonium and triethanolamine salts, and suitable detergents include (a) cationic detergents, such as dimethyl dialkyl ammonium halides and alkyl pyridinesHalide, (b) anionic detergents such as alkyl, aryl and alkene sulfonates, alkyl, alkene sulfates, ether sulfates and glycerol monosulfate salts and sulfosuccinates, (c) nonionic detergents such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as alkyl- β -aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

Parenteral formulations typically contain from about 0.5% to about 25% by weight of a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The amount of surfactant in such formulations is typically from about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. Parenteral formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient for injections, for example water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from various sterile powders, granules and tablets of the kind previously described.

The injectable formulations are in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art (see, e.g., pharmaceuticals and pharmaceutical practice, J.B. Lippincott Company, Philadelphia, PA, Bank and Chalmers, pp.238 and 250 (1982); and ASHP Handbook on InjectablDrugs, Toissel, 4 th edition, pp.622 and 630 (1986)).

In addition, PTH peptides, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-inverso peptides of the present disclosure) can be formulated into suppositories for rectal administration by mixing with various bases (e.g., an emulsifying base or a water-soluble base). Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

It will be appreciated by those skilled in the art that in addition to the pharmaceutical compositions described above, the PTH peptides, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-inverso peptides) of the present disclosure can be formulated into inclusion complexes (e.g., cyclodextrin inclusion complexes) or liposomes.

Dosage form

The pharmaceutical compositions within the scope of the present invention include all compositions wherein the peptide or polypeptide analog is contained in an amount sufficient to achieve the intended purpose. Although individual needs vary, determination of the optimal range of effective amounts of each component is within the skill of the art. Typical dosages include 0.1-100 mg/kg/body weight.

For purposes of the present disclosure, the amount or dose of a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure administered should be sufficient to achieve, e.g., a therapeutic or prophylactic response in a subject or animal within a reasonable time frame. For example, the PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure should be dosed at a dose sufficient to stimulate cellular secretion or accumulation of cAMP described herein in the cells or to activate the PTH receptor in the cells, treat a subject having a bone loss-related disease or disorder, ameliorate a symptom associated with osteoporosis, delay progression of osteoporosis, or regenerate bone within about 1-4 minutes, 1-4 hours, or 1-4 weeks or more (e.g., 5-20 or more weeks) from the time of administration. In certain embodiments, the time may be even longer. Dosages can be determined based on the efficacy of the particular PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure and the condition of the animal (e.g., human) to be treated as well as the weight of the animal (e.g., human).

Many assays for determining the dose administered are known in the art. For the purposes herein, the following assay can be used to determine the starting dose to be administered to a mammal: comprising comparing the extent of cAMP stimulated by cells when a prescribed dose of a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure is administered to a mammal in a group of mammals each administered a different dose of PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide). Methods of testing the extent of stimulation of cAMP production are known in the art and include the methods described in example 1 herein.

The dosage of the PTH peptide, PTHrP peptide (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-inverso peptides of the present disclosure) can also be determined by the presence, nature, and extent of any adverse side effects that may be associated with the administration of a particular analog of the present disclosure. The dosage of a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-retro peptide of the present disclosure) of the present disclosure for use in treating individual patients will typically be determined by the attending physician taking into account various factors such as age, weight, general health, diet, sex, PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-retro peptide) to be administered, route of administration and severity of the disease to be treated. By way of example and not intended to limit the invention, the dosage of a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure may be from about 0.0001 to about 1g/kg body weight/day, from about 0.0001 to about 0.001g/kg body weight/day, or from about 0.01mg to about 1g/kg body weight/day of the subject to be treated.

Targeting forms

It will be readily appreciated by one of ordinary skill in the art that the PTH peptides, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-inverso peptides) of the present disclosure can be modified in a number of ways such that the therapeutic or prophylactic efficacy of the PTH peptides, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, peptidomimetic, retro-inverso peptides of the present disclosure is enhanced by the modification. For example, a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure can be conjugated directly to a targeting moiety or indirectly through a linker. The manipulation of conjugating a compound, such as a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) described herein to a targeting moiety is known in the art. See, for example, Wadhwa et al, J Drug Targeting, 3, 111-. The term "targeting moiety" as used herein refers to any molecule or substance that specifically recognizes and binds to a cell surface receptor such that the targeting moiety directs the delivery of a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure to a population of cells on which the surface receptor is expressed. Targeting moieties include, but are not limited to, antibodies or fragments thereof, peptides, hormones, growth factors, cytokines, and any other natural or non-natural ligand that binds to a cell surface receptor (e.g., Epithelial Growth Factor Receptor (EGFR), T Cell Receptor (TCR), B Cell Receptor (BCR), CD28, platelet derived growth factor receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.). As used herein, a "linker" is a bond, molecule or group of molecules that binds two separate entities to each other. The linker may provide optimal spacing of the two entities, or may also provide an unstable bond that allows the two entities to be separated from each other. Labile bonds include photocleavable groups, acid labile moieties, base labile moieties, and enzyme cleavable groups. In some embodiments, the term "linker" refers to any substance or molecule that bridges a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure with a targeting moiety. One of ordinary skill in the art will recognize that sites on a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure that are not necessary for the function of an analog of the present disclosure are ideal sites for attachment to a linker and/or targeting moiety, provided that, once attached to a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic), retro-inverso peptide of the present disclosure, the linker and/or targeting moiety does not interfere with a PTH peptide of the present disclosure, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-inverso peptides) function to stimulate cAMP secretion from cells, treat osteoporosis or cancer.

Controlled release formulations

Alternatively, the PTH peptides, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-inverso peptides) described herein can be modified into depot forms such that the manner in which the PTH peptides, PTHrP peptides (or related analogs or variants or chemical derivatives, fusion polypeptides, multimeric polypeptides, peptidomimetics, retro-inverso peptides) of the present disclosure are released into the body to which they are administered is controlled with respect to time and location within the body (see, e.g., U.S. patent No. 4,450,150). The depot formulation form of a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-retro peptide) of the present disclosure can be, for example, an implantable composition comprising a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic) and porous or non-porous material (e.g., a polymer) of the present disclosure, wherein the PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-retro peptide) of the present disclosure is encapsulated by or diffused into the entire material and/or degraded non-porous material. The depot formulation is then implanted at a desired site in the body and the PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure is released from the implant at a predetermined rate.

In certain aspects, the pharmaceutical compositions are modified to have any type of in vivo release profile. In some aspects, the pharmaceutical composition is an immediate release, controlled release, sustained release, delayed release, or biphasic release formulation. Methods of formulating peptides for controlled release are known in the art. See, e.g., Qian et al, J Pharm 374: 46-52(209) and international patent application publication nos. WO2008/130158, WO2004/033036, WO2000/032218 and WO 1999/040942.

The compositions of the invention may also comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in a sustained release form to provide long term storage and/or delivery effects. The pharmaceutical formulations of the present disclosure can be administered according to any regimen, including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), every two days, every three days, every four days, every five days, every six days, weekly, biweekly, every three weeks, monthly, or every two months.

Combination of

The PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure can be used in combination with another active ingredient. For example, a PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) disclosed herein can be formulated with another anti-osteoporosis agent comprising any of the following: bisphosphonates such as alendronate (Fosamax), ibandronate (Boniva), risedronate (Actonel) and zoledronic acid (Reclast), which slow the rate of bone thinning and can lead to increased bone density; raloxifene (Evista), a Selective Estrogen Receptor Modulator (SERM) that slows down bone thinning and causes some increase in bone thickness; calcitonin (Calcimar or Miacalcin), a naturally occurring hormone that helps regulate calcium levels in the body and is part of the bone building process; parathyroid hormone (teriparatide [ Forteo ]), administered by injection; estrogens (e.g., with or without progestins); testosterone (injection, gel, patch);

in some embodiments, the PTH peptide, PTHrP peptide (or related analog or variant or chemical derivative, fusion polypeptide, multimeric polypeptide, peptidomimetic, retro-inverso peptide) of the present disclosure is formulated with an anti-cancer agent. Suitable anti-cancer drugs are known in the art and include any of the following chemotherapeutic agents: platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides.

In some embodiments, the platinum coordination compound is cis-diamminedianilinum (II) -ion; chloro (diethylenetriamine) -platinum (II) chloride; dichloro (ethylenediamine) -platinum (II), diammine (1, 1-cyclobutanedicarboxylato) platinum (II) (carboplatin); spiroplatinum; iproplatin; diammine (2-ethylmalonate) -platinum (II); ethylenediamine-platinum malonate (II); hydration of (1, 2-diaminocyclohexane (dyclohexane)) -platinum sulfate (II); (1, 2-diaminocyclohexane) malonic acid platinum (II); (4-carboxyphthalate) (1, 2-diaminocyclohexane) platinum (II); (1, 2-diaminocyclohexane) - (isocitrate) platinum (II); (1, 2-diaminocyclohexane) cis (pyruvate) platinum (II); (1, 2-diaminocyclohexane) platinum oxalate (II); ormaplatin and tetraplatin.

In some embodiments, cisplatin is a platinum coordination compound for use in the compositions and methods of the present invention. Cisplatin is known under the name placinol^TMCommercially available from Bristol Myers-Squibb corporation, below, as a powder for formulation with water, sterile saline, or other suitable vehicle. Other platinum coordination compounds suitable for use in the present invention are known and are commercially available and/or may be prepared by conventional techniques. Cisplatin, or cis-diamminedichloroplatinum II, has been successfully used as a chemotherapeutic agent in the treatment of various human solid malignancies for many years. Recently, in the treatment of various human solid malignanciesOther diamino-platinum complexes have also been shown to have efficacy as chemotherapeutic agents in neoplasms. Such diamino-platinum complexes include, but are not limited to, spiroplatinum and carboplatin. Although cisplatin and other diamino-platinum complexes are widely used in human chemotherapy drugs, they have to be delivered at high dose levels that can cause toxicity problems (e.g., kidney damage).

In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that are capable of altering the topology of DNA in eukaryotic cells. They are crucial for cell function and cell proliferation. Generally, there are two types of topoisomerases in eukaryotic cells, i.e., type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of about 100,000. The enzyme binds to DNA, introduces a transient single-stranded nick, unravels the double helix (or allows it to unravel), and then reseals the nick before dissociating from the DNA strand. Recent studies have shown that various topoisomerase inhibitors have clinical efficacy in the treatment of humans with ovarian, esophageal, or non-small cell lung cancer.

In some aspects, the topoisomerase inhibitor is a camptothecin or a camptothecin analog. Camptothecin is a water-insoluble cytotoxic alkaloid produced by Camptotheca acuminata (Camptotheca acuminata) of the hometown tree in china and pseudochaetomium fortunei (Nothapodytes foetida) of the hometown tree in india. Camptothecin exhibits tumor cell growth inhibitory activity against a variety of tumor cells. Compounds of the camptothecin analogue class are generally specific inhibitors of DNA topoisomerase I. The term "inhibitor of topoisomerase" refers to any tumor cell growth inhibiting compound structurally related to camptothecin. Compounds of the camptothecin analog class include, but are not limited to, topotecan, irinotecan, and 9-amino-camptothecin.

Use of

While not being bound by any particular theory, as first described herein, the PTH and PTHrP peptides described herein are expected to exhibit altered properties that confer improved therapeutic potential. In this aspect, the invention provides a method of treating a disease or disorder associated with undesirable bone loss. The method comprises administering to a subject in need thereof a pharmaceutical composition as described herein in an amount effective to treat the subject.

The present invention also provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein in an amount effective to treat the subject.

The term "treatment" refers to the administration of a pharmaceutical composition comprising a PTH or PTHrP peptide or polypeptide analog (variant or chemical derivative) to a subject. The term "treatment" as used herein does not necessarily mean 100% or complete cure. Rather, there are different degrees of treatment that one of ordinary skill in the art would consider to have a potential benefit or therapeutic effect. In this regard, the methods of the invention can provide any amount at any level to treat osteoporosis or cancer in a mammal. In addition, the treatment provided by the methods of the invention may comprise treating one or more conditions or symptoms of a disease or disorder (e.g., osteoporosis or cancer to be treated), and/or may comprise delaying the progression of a disease or disorder. For example, treating osteoporosis, including ameliorating symptoms associated with osteoporosis, delaying the progression of osteoporosis, and regenerating bone. Treatment includes administration of the drug to subjects at risk of developing osteoporosis or cancer prior to the appearance of signs of clinical disease, as well as subjects diagnosed with osteoporosis or cancer who have not been treated or have been treated by other methods. Therefore, the present invention can be used for preventing or inhibiting osteoporosis or cancer.

Accordingly, the present invention also provides methods of ameliorating osteoporosis-related symptoms. In some embodiments, the symptoms of osteoporosis are pain, back pain, spine, wrist or hip fracture, and loss of height. The method comprises administering to a subject in need thereof a pharmaceutical composition described herein in an amount effective to ameliorate osteoporosis-related symptoms in the subject.

The invention also provides methods of delaying the progression of osteoporosis. The method comprises administering to a subject in need thereof a pharmaceutical composition described herein in an amount effective to delay the progression of osteoporosis. The progression (or regression) of osteoporosis can be monitored by methods known in the art, including, for example, dual X-ray absorptiometry (DXA or DEXA), ultrasound, and Quantitative Computed Tomography (QCT).

The invention also provides methods of regenerating bone. The method comprises administering to a subject in need thereof a pharmaceutical composition described herein in an amount effective to regenerate bone. Bone regeneration can be monitored by methods known herein, including, for example, measuring bone mineral density of a subject.

An effective amount or dose of PTH or PTHrP peptide or polypeptide analog for treating bone loss (e.g., osteoporosis or cancer) ranges from about 0.1 to 100 mg/kg/body weight. Effective dosages can preferably be determined by injection of cells in vitro or injection of live animals in vivo, in order to determine the optimal dosage range using the various methods described herein. The dose administered will depend in part on the recipient's health and weight, the presence of other concurrent treatments (if any), the frequency of treatment, and the nature of the desired effect.

The expression "effective amount" or "effective for" as used herein means an amount sufficient for treatment or prevention, and preferably sufficient to reduce by at least about 25%, more preferably at least 50%, most preferably at least 90%, of a pathological feature such as elevated blood pressure, fever, or white blood cell count, which is a clinically significant change as may accompany its presence and activity. In reference to the present invention, the term may also refer to an amount sufficient to ameliorate or reverse bone loss or cancer.

In some embodiments, the undesired bone loss-related disease or disorder is osteopenia or osteoporosis.

For purposes herein, a cancer may be any cancer. The term "cancer" as used herein refers to any malignant growth or tumor caused by abnormal and uncontrolled cell division, which may spread to other parts of the body through the lymphatic system or the blood stream. The cancer may be any cancer, including any of the following: acute lymphocytic cancer, acute myelocytic leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, cancer of the intrahepatic bile duct, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myelogenous cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma (Hodgkin lymphoma), hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, cancer of the peritoneum, omentum, and mesenterium, pharynx cancer, prostate cancer, rectal cancer, kidney cancer (e.g., Renal Cell Carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and bladder cancer.

Subject of being treated

The term "subject" as used herein refers to any multicellular living organism. In some embodiments, the subject is a mammal. The mammal can be any mammal, including, but not limited to, mammals of the order Rodentia (Rodentia) such as mice and hamsters, and mammals of the order lagomorpha (Logomorpha) such as rabbits. In some embodiments, the mammal is derived from the order Carnivora (Carnivora), including felines (cats) and canines (dogs). In some embodiments, the mammal is derived from the order Artiodactyla, including Bovine (cattle) and Swine (Swine) or perissodactyla (perssodactylyla), including Equine (Equine) (horses). In some embodiments, the mammal is an animal of the order primates (primates), ceboids or simoids (monkeys) or apes (anthropoids) (humans and apes). Preferred animal subjects of the invention are mammals. The invention is particularly useful for treating human subjects.

In addition to the above-described methods of treatment, PTH peptides and PTHrP peptides (as well as variants, analogs, chemical derivatives, fusion or multimeric polypeptides, peptidomimetics, retro-inverso peptides) can be used to activate PTH receptors in cells. The methods comprise administering a PTH peptide or PTHrP peptide (or variant, analog, chemical derivative, fusion or multimeric polypeptide, peptidomimetic) thereof) to a cell such that a PTH receptor is activated.

In some embodiments, such methods are performed in vitro or ex vivo. In this regard, the methods can be used to monitor the reactivity of cells or tissues of a subject (e.g., a human) to PTH and PTHrP peptides (as well as variants, analogs, chemical derivatives, fusion or multimeric polypeptides, peptidomimetics, retro-inverso peptides). In some embodiments, the method is performed for basic research purposes or clinical research purposes.

In some embodiments, such methods are performed in vivo, such that the cells are living animal cells, and the PTH peptide and PTHrP peptide (as well as variants, analogs, chemical derivatives, fusion or multimeric polypeptides, peptidomimetics, retro-inverso peptides) are administered to a living animal, such as a human.

In some cases, the cell is an osteoblast cell. The method may be a therapeutic method when osteoblasts are within a living animal. In embodiments where the osteoblasts are not in a living animal, the method may be performed for basic research or clinical research purposes.

In some embodiments, the cell is in a living animal, and the cell is a lymphocyte or a leukocyte. Antibodies specific for PTH peptides and PTHrP peptides (as well as variants, analogs, chemical derivatives, fusion or multimeric polypeptides, peptidomimetics, retro-inverso peptides) can be produced by living animals in this manner. Antibodies against PTH peptides and PTHrP peptides (as well as variants, analogs, chemical derivatives, fusion or multimeric polypeptides, peptidomimetics, retro-inverso peptides) are contemplated herein.

Medicine box

The invention also provides kits comprising any of the PTH peptides, PTHrP peptides, variants, analogs, chemical derivatives, fusion polypeptides, or multimeric polypeptides described herein and instructions for use. In some embodiments, the kit includes instructions for administering a PTH peptide, PTHrP peptide, variant, analog, chemical derivative, fusion polypeptide, or multimeric polypeptide to a subject (e.g., a human). In some embodiments, the instructions include instructions for administration to a subject having osteoporosis or cancer. In some embodiments, the kit comprises a device, such as a syringe needle, pen (pen) device, jet injector, or other needle-free injector, for administering the PTH peptide, PTHrP peptide, variant, analog, chemical derivative, fusion polypeptide, or multimeric polypeptide to a subject. Alternatively or in addition, the kit may comprise one or more containers, such as vials, tubes, bottles, single or multi-chamber prefilled syringes, infusion pumps (external or implantable), jet injectors, prefilled pen devices, and the like, optionally prepackaged PTH peptides, PTHrP peptides, variants, analogs, chemical derivatives, fusion polypeptides, multimeric polypeptides in lyophilized form or in aqueous solution.

Having now generally described the invention, the same may be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless otherwise specified.

Examples

Example 1

Materials and methods of examples 2-5

Protein purification and peptide synthesis. Previously described was an MBP-PTH1R ECD- (His)6 fusion protein (27) containing residues 29-187 of PTH1R fused to the C-terminus of MBP. Isolated PTH1R ECD (residues 23-191) was prepared free of MBP and biotinylated at a single site on the N-terminal biotinylated tag as described in the supplementary methods. SynBioSci (Livermore, Calif.) was delegated to synthesize peptides for ECD binding studies and crystallization and HPLC purification. PTH/PTHrP (1-34) NH2 hybrid peptide was synthesized by the Massachusetts General Hospital Biopolymer Core organization and subjected to HPLC purification. All peptides were C-terminally amidated unless otherwise indicated.

ECD-peptide binding assay. By using AlphThe binding of the peptide to the MBP-PTH1R ECD-H6 fusion protein was evaluated in the aScreen assay (Perkin-Elmer). The reaction mixture was incubated at room temperature, containing 5. mu.g/ml each of streptavidin-coated donor beads and nickel chelate-coated acceptor beads, and 23nM each of N-terminally biotinylated PTH (7-34) NH2 and MBP-PTH1R ECD-H6 in 50mM MOPS (pH 7.4), 150mM NaCl and 7mg/ml BSA buffer. Biotinylated PTH and MBP-PTH1R ECD-H6 were pre-coupled with streptavidin beads and nickel chelate beads, respectively, for 1 hour alone. The pre-coupling reactions were mixed, unlabeled competitor peptide was added as indicated, and the reactions were incubated for 4-5 hours to reach equilibrium. Photon counts were recorded in 384-well optiplates using an Envision 2104 plate reader (Perkin-Elmer). Data were fitted to a fixed slope dose response inhibition equation used to determine IC50 values using Prism 5.0 software (graphpad software, San Diego). The binding of peptides to isolated PTH1R ECD was analyzed in real time using the Octet Red system (ForteBio) as described below.

Crystallization, data acquisition, structure analysis and refinement. MBP-PTH1R ECD-H6 in 10mM Tris-HCl (pH7.5), 50mM NaCl, 1mM maltose and 1mM EDTA was mixed with the synthesized PTHrP fragment (residues 12-34) in a molar ratio of 1: 1.1 (protein: peptide), incubated on ice for 30 minutes, and concentrated to 20mg/ml by centrifugation to crystallize. Bipyramidal crystals were grown by sitting drop (sitting drop) vapor diffusion at 20 ℃ using a 7.5% PEG2000, 13% PEG 400 stock solution. The crystals were transferred to a cryoprotectant solution of 10% PEG2000, 22% PEG 400, 50mM NaCl, 1mM EDTA by dialysis overnight and inserted into liquid nitrogen for snap freezing. At LS-CAT beamline 21-ID-F of Advanced Photon Source (Argonne, IL) toAnd a temperature of 100 ° K, a data set was collected from the single crystal. Data were processed using the HKL2000 software package (37) to convert the Scalepack intensity to a structure factor amplitude using the CCP4 kit (38). Using the search models of MBP and PTH1R ECD from PDB coordinate file 3C4M (27), molecular substitution was performed using Phaser (39)The structure was analyzed. Among the asymmetric units is an MBP-PTH1R ECD PTHrP (12-34) complex. The iterative loop of reconstruction in O (40) and refinement with the constraints of Refmac5(41) completes the structure. Also included was TLS refinement using two TLS groups corresponding to the MBP-maltose complex and the ECD-PTHrP complex (42). The structure verified by Procheck (43) showed that 93.6% of the residues were in the most favorable region of the laplace map, and no residues were located in the disallowed region. Data acquisition and refinement statistics are listed in table S1. Solvent accessible surface area and shape complementarity were analyzed in a CCP4 kit. The structure was plotted using PyMol (44).

A receptor binding assay. Conformational binding of peptides to G protein uncoupling receptor (R0) and G protein coupling Receptor (RG) in cell membranes was assessed as described previously (26).

Measurement of cAMP. COS-1 cells obtained from the American Type Culture Collection (Manassas, Va.) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 37 ℃/5% CO2 incubator. 500,000 cells were seeded in a 10cm dish and transfected the next morning by the DEAE-dextran method with 3. mu.g of the pcDNA3.1 vector encoding hPTHIR. Approximately 40,000 transfected cells were seeded in each well of a 96-well plate 24 hours after transfection. 48 hours after transfection, cells were washed twice with PBS (pH 7.4) and stimulated with peptide. For dose response assays, cells were stimulated with peptides at 37 ℃ for 30 minutes in Krebs-ringer-Hepes (KRH) buffer (25mM Hepes (pH 7.4), 104mM NaCl, 5mM KCl, 2mM CaCl2, 1mM KH2PO4, 1.2mM MgSO4) supplemented with 0.2% BSA, 0.01% soybean trypsin inhibitor, 0.1% bacitracin, and 2mM 3-isobutyl-1-methylxanthine (IBMX). The reaction was stopped with cold 6% perchloric acid and the cell lysate was neutralized with KHCO 3. cAMP was determined using the LANCE cAMP kit (Perkin-Elmer) according to the manufacturer's instructions. For the ligand wash assay, cells were stimulated with peptide (100nM) in KRH buffer supplemented as above but lacking IBMX for 10 min at room temperature. Cells were washed 3 times with buffer lacking IBMX and at the indicated time after ligand washout, the buffer was replaced with buffer containing IBMX, the reaction was stopped after 5 minutes, and cAMP content was assessed as above. Curve fitting was performed using Prism 5.0 software using a fixed slope dose response stimulation equation for dose response determination and a monophasic exponential decay equation for ligand washout determination.

And (4) constructing an expression plasmid. A DNA fragment encoding PTH1R residues 23-191 tagged with a C-terminal 6 histidine residue was PCR amplified from pcDNA3.1/PTH 1R. The fragment was digested with BamHI and NotI restriction endonucleases and ligated into pETDuet1 vector (Novagen) encoding Maltose Binding Protein (MBP) followed by thrombin cleavage site (Th) and biotinylation tag sequence (GGLNDIFEAQKIEWHEDT; biotinylation site in bold) in multiple cloning site 1 and BirA biotin ligase in multiple cloning site 2. The obtained vector co-expresses MBP-Th-biotin tag-PTHIR ECD-H6 fusion protein and BirA. The construct was confirmed by automated DNA sequencing of the coding region.

Expression and purification of MBP-Th-biotin tag-PTHIR ECD fusion protein. Coli Origami B (DE3) cells (Novagen) were transformed with the expression plasmid and cultured to mid-log phase at 37 ℃ in 12L LB medium. The temperature was lowered to 16 ℃, biotin was added to a final concentration of 5 μ M, and protein expression was induced with 0.4mM IPTG for a total induction time of about 19 hours. Cells were harvested and resuspended in 50mM Tris-HCl (pH7.5), 150mM NaCl, 10% glycerol and 25mM imidazole. Cells were lysed by homogenization at 10,000psi and the resulting lysate was clarified by centrifugation. The supernatant was applied directly to a 50ml Ni2+ ChelatingSepharose column (GE Healthcare). The column was washed with 600ml of 50mM Tris-HCl (pH7.5), 150mM NaCl, 10% glycerol and 72.5mM imidazole, and the fusion protein in the buffer was eluted with 262.5mM imidazole. The peak fractions were pooled and applied to a 50ml amylose high flow column (New England Biolabs) equilibrated in 50mM Tris-HCl (pH7.5), 150mM NaCl and 5% glycerol. The protein was eluted with a linear gradient of 0-10mM maltose. The peak fractions were pooled, diluted to 1mg/ml and the samples were treated with 1mM reduced glutathione, 1mM oxidised glutathione and a 1: 1 molar ratio of purified DsbC: MBP-Th-biotin tag-PTHRECD-H6 at 20 ℃ for 23 hours to allow the disulphide bridges to be shuffled. DsbC was purified as described previously (45). The disulfide shuffling reaction mixture was run on the above 50ml Ni2+ chelating sepharose column to remove DsbC. The eluted fusion protein was concentrated, applied to a 300ml Superdex200 column (GE Healthcare), and the peak fractions corresponding to the correctly folded protein were collected. Native gel electrophoresis of the purified samples indicated that the protein was approximately 50% biotinylated, as evidenced by two distinct bands of slightly different mobility (data not shown). The protein concentration was determined by the Bradford method (46).

Octet Red real-time analysis of peptide binding to biotinylated PTH1R ECD. The interaction between biotinylated PTH1RECD and PTH (1-34) NH2 and PTHrP (1-34) NH2 was analyzed by a biolayer interferometry technique using the OctetRed System (ForteBio). MBP was removed from MBP-Th-biotin tag-PTHIR ECD fusion protein by digestion with human alpha-thrombin (Haematologic Technologies Inc.) at a protease: fusion protein ratio of 1: 750 (wt./wt.) overnight at 4 ℃. Complete cleavage was confirmed by SDS-PAGE (data not shown). The high binding streptavidin sensor and in the supplemented with 2mg/ml BSA KRH buffer diluted to 2 u g/ml protein digestion reaction together temperature, followed by 10 u g/ml biocytin blocking, and in the supplemented with 2mg/ml BSA KRH buffer washing 3 times to wash out digestion mixture of non-biotinylated components, and establish the baseline. Peptides were diluted in KRH buffer with 2mg/ml BSA and each peptide association and dissociation was monitored for 20 min at 25 ℃. To account for non-specific binding, traces obtained with peptide and sensor contacts lacking PTH1R ECD were subtracted from traces obtained with peptide and immobilized PTH1R ECD. In addition, traces from sensor contacts with immobilized PTH1R ECD but no peptide were used to subtract baseline drift. Data were analyzed using Octet Red analysis software.

Example 2

PTH and PTHrP bind to PTH1R ECD

In vitro binding of PTH and PTHrP to MBP-PTH1R ECD-H6 fusion protein was evaluated using the AlphaScreen luminescent proximity assay (Perkin-Elmer). In this assay, N-terminally biotinylated PTH (7-34) NH2 was bound to streptavidin-coated donor beads and the fusion protein was bound to nickel chelate-coated acceptor beads via a (His)6 tag. The association of biotinylated PTH and fusion protein brings the beads into proximity, generating a luminescent signal. The present inventors previously demonstrated that PTH (15-34) NH2 competes for interaction with an IC50 value similar to a Kd value of about 1. mu.M determined by isothermal titration calorimetry (27). Both PTH (1-34) NH2 and PTH (12-34) NH2 compete for the AlphaScreen interaction with an IC50 value of approximately 1 μ M (FIG. 1A), consistent with previous observations by the present inventors. However, PTH (1-34) OH binds to the fusion protein with an affinity as low as about 1/10 compared to the C-terminally amidated peptide (fig. 1A). This result is consistent with the crystal structure of PTH1R ECD complexed with PTH (15-34) NH2, which suggests that the C-terminal amide group forms two hydrogen bonds with the ECD (27). In contrast, PTHrP (12-34) NH2 and PTHrP (12-34) OH compete for interaction at a similar IC50 value of about 2. mu.M (FIG. 1B), indicating that the C-terminal amide group of PTHrP is not important for ECD binding.

The inventors also investigated the interaction of peptides with isolated PTH1R ECD by employing the Octet Red system (ForteBio) which utilizes a bio-layer interferometry technique to monitor binding events in real time. Isolated PTH1R ECD biotinylated at a single residue of the biotinylated tag sequence at the N-terminus of the protein (residues 23-191) without MBP was prepared according to the procedure described in example 1. Biotinylated ECD was immobilized on the surface of the streptavidin sensor contact and binding of free PTH (1-34) NH2 and PTHrP (1-34) NH2 in solution was evaluated. Steady state analysis of the real-time binding curves (FIG. S1) indicated that the Kd values for PTH and PTHrP binding to the ECD were 2.8. mu.M and 0.99. mu.M, respectively (FIG. 1C and FIG. 1D). The Kd value of PTH is similar to that obtained by others using the Biacore technique (31). Summary binding data indicate that PTH and PTHrP bind with similar affinity to PTH1R ECD, but indicate differences in the underlying biochemical mechanism, as C-terminal amidation affects the ability of PTH, but not PTHrP, to bind to ECD.

Example 3

Structural basis for binding PTHrP to PTH1R ECD

To understand the structural basis under the difference in PTH and PTHrP binding mechanisms, the present inventors determined the crystal structure of MBP-PTH1R ECD-H6 fusion protein complexed with PTHrP (12-34) NH 2. The structure is analyzed by molecular replacement inAt resolution, refine to an R factor of 19.3% (free R factor 23.3%) (table 2).

TABLE 2

The electron density was observed for all PTH1R residues except for 29-30, 57-101, 176-187 and the C-terminal (His)6 tag (excluded from the final model). The conserved B-class GPCR ECD fold observed prior to PTH1R ECD formation (27), while PTHrP forms an amphipathic α -helix that binds to the hydrophobic groove of the ECD at the interface of the N-terminal α -helix, the 2 β -folds and the short C-terminal α -helix (fig. 2A and 2B). Clear electron densities of PTHrP residues 13 to 34 were observed (FIG. 2C). The interactions are mediated by hydrophobic interactions and extensive hydrogen bonding networks including PTHrP residues F23 ', L24', L27 'and I28' (fig. 2B, 2D and 2E). [ peptide residues are indicated with apostrophes to distinguish them from receptor residues ]. R19 ' forms an intermolecular salt bridge with E35, but this may be caused by interactions (not shown) that limit crystal packing of R19 ' mobility, since in native proteins R19 ' contacts the 7-TM domain of the receptor (32). Conserved R20 'forms intermolecular salt bridges with D137, intermolecular hydrogen bonds with the backbone carbonyl of M32, and intramolecular hydrogen bonds with the side chain of D17' that may stabilize the helical conformation of the peptide (fig. 2D). The PTHrP helix "unravels" at the C-terminus after residue I31 ' allows several additional intermolecular hydrogen bonds to form, including hydrogen bonds between the δ nitrogen of H32 ' and the backbone amide nitrogen of Y167, the side chain hydroxyl group of T33 ' and the backbone amide nitrogen of a165 and the backbone carbonyl group of T163, and the backbone carbonyl group of a165 and the backbone amide nitrogen of T33 ' and a34 ' (fig. 2E). Consistent with our binding data in fig. 1B, the C-terminal amide group of PTHrP did not form any interaction with the ECD.

Example 4:

PTH and PTHrP exhibit different ECD binding patterns.

Structural alignment of PTHrP-ECD complexes and the inventors' previously published PTH-ECD complexes highlighted similarities and differences in the binding mechanism of the two peptides (FIG. 2F). In both cases, the hydrophobic face of the amphiphilic peptide binds to the hydrophobic groove in the ECD and is anchored to interact by invariant residues R20 'and L24' (fig. 2G). The most significant difference in binding pattern occurred at the C-terminus of the peptide. PTH forms a continuous α -helix from L15 'to F34', with the C-terminal amide group forming an important hydrogen bond with the ECD. In contrast, the PTHrP helix extends from I15 'to I31', after which "unwinding", the C-terminal amide group is not involved in receptor binding. The PTHrP helix is slightly bent such that its helical axis deviates from that of PTH after residue L24 ', apparently due to residue differences at positions 23 ', 27 ', 28 ' and 31 ' of the peptide (fig. 2F and 2G). With PTHrP-ECD complexesIn contrast, the interfacial buried solvent accessible surface area of the PTH-ECD complex isThe peptide of PTH-ECD complex (33) measured by Sc value has slightly higher surface complementarity to the ECD (Sc ═ 0.782) compared to that of PTHrP-ECD complex (Sc ═ 0.726). Thus, PTH peptide is buried in slightly more surface area than PTHrP and adapts to ECD with a slightly "tighter" complementarity. However, both peptides showed similar affinity for ECD, probably formed by residues 32-34 of PTHrPResults of additional hydrogen bonding (fig. 2E).

Receptor adaptation accommodated the different binding patterns of the two peptides with subtle but significant changes. The ECD structures in both composites are very similar with an RMSD in the C-alpha position ofHowever, in both structures, the side chains of residues L41 and I115 exhibit different conformations (fig. 2F). There appears to be coupling between L41 and I115 of ECD and between positions 23 'and 27' of the peptide, respectively. In the PTHrP binding structure, L41 assumed the favorable χ 1 ═ asymmetric (+) rotamer and χ 2 ═ trans rotamer (34), so that there was van der waals contact between the δ 1 methyl group of L41 and the phenyl ring of F23'. In contrast, in PTH-bound structures, L41 adopted the less favorable χ 1 ═ trans rotamer and χ 2 ═ asymmetric (-) rotamer, enabling the receptor to accommodate the larger W23 'side chain while maintaining van der waals contact between the δ 2 methyl group of L41 and the indole ring of W23'. This L41 rotamer toggle switch mechanism was recently predicted in a remarkable study by Donnelly and coworkers (35). In both PTH-bound and PTHrP-bound structures, I115 assumes the same rotamer, but the side chains are significantly displaced inward toward the core of the ECD in the PTHrP-bound state, which is undoubtedly caused by bending of the PTHrP helix and displacement at residue 27'. This I115 conformation is incompatible with the PTH binding mode because the delta carbon of I115 is sterically conflicting with the gamma 2 carbon of V31'. Thus, the peptide binding site of ECD exhibits plasticity, adopting different binding patterns of PTH and PTHrP by altering the conformation of residues L41 and I115.

To validate the different binding patterns observed in the crystal structure, the inventors evaluated PTH (15-34) NH in the AlphaScreen assay₂And PTHrP (12-34) NH₂The ability of the alanine scanning mutants to compete for the interaction of biotinylated PTH with MBP-PTH1R ECD examined the contribution of specific PTH and PTHrP residues to ECD binding. As previously described (27), PTH residues R20 ', W23 ', L24 ' and L28 ' are most critical for ECD binding, and V21 ', K27 ' and F34 ' provideAdditional contribution (fig. 2H). PTHrP residues R20 ', R21', F23 ', L24', L27 ', I28', H32 'and T33' are all important for ECD binding, with R20 'and L24' providing the most critical contacts (figure 21). Clearly, for ECD binding, F23 'and I28' of PTHrP are less critical than W23 'and L28' of PTH. In contrast, for ECD binding, L27 'and H32' of PTHrP are more important than K27 'and H32' of PTH. These in vitro binding results are in full agreement with the binding pattern observed in the crystal structure.

Example 5

Binding of the hybrid PTH/PTHrP peptide to PTH1R ECD.

The inventors sought to determine exactly which residues are responsible for the different ECD binding patterns of PTH and PTHrP. The crystal structure indicates that residues at positions 23 ', 27', 28 'and 31' of the peptide largely determine its ECD binding mode. The inventors speculate that changing the residues at these positions of one peptide to the corresponding residues present in the other peptide may allow switching between the PTH binding mode and the PTHrP binding mode. This hypothesis was tested in the AlphaScreen assay by evaluating the binding of hybrid peptides containing PTH/PTHrP residue exchanges at these 4 positions to purified MBP-PTH1R ECD fusion protein. In PTH (15-34) NH₂And PTHrP (12-34) NH₂Hybrid peptides are prepared in scaffolds, including peptides that are altered by a single crossover at one of these 4 positions, a double crossover at two of these 4 positions, or a quadruple crossover at all 4 positions. Theoretically, the quadruple mutant should adopt another peptide binding conformation, maintain normal ECD binding affinity, and show sensitivity or insensitivity to C-terminal amidation depending on its binding mode.

PTH(15-34)NH₂The peptides were less tolerant of the corresponding PTHrP residues at positions 23 ', 31' and to a lesser extent 28 ', as shown by the diminished ECD binding of the W23' F, L28 'I and V31' I single mutant peptides (fig. 3A). In contrast, the K27 'L mutant maintained normal binding, and this alteration was able to rescue W23' F, L28 in the W23 'F/K27' L, K27 'L/L28' I and K27 'L/V31' I double mutant peptidesDefects in the 'I and VV 31' I mutations. The inclusion of all 4 crossover mutations together in the W23 'F/K27' L/L28 'I/V31' I peptide resulted in some attenuation of ECD-binding, suggesting that, at least for PTH, the difference in PTH and PTHrP binding patterns was not solely determined by these 4 positions. The combined effect of the exchanges at all 4 positions in the PTH scaffold may push the PTH towards the PTHrP binding mode, but N33 'and F34' of PTH may be incompatible with the PTHrP binding mode (fig. 2E).

PTHrP (12-34) NH when altered with the corresponding PTH residues at positions 23 ', 28' and 31₂Normal ECD binding was shown, but binding was reduced when altered with L27' K (fig. 3B). In the F23 'W/L27' K, L27 'K/I28' L and L27 'K/I31' V double mutant peptides, the defect of the L27 'K change is completely rescued by the F23' W change, and partially rescued by the I28 'L or I31' V change. The quadruple mutant PTHrP peptide showed normal binding to ECD, consistent with the possibility that this peptide adopted the PTH binding mode.

In fig. 3A and 3B, the most significant feature of the data is the coupling at positions 23 'and 27' that is evident for both peptides. In either scaffold, the combination of Phe at position 23 'and Lys at position 27' (F23 '/K27') resulted in a severe reduction in ECD binding, while the W23 '/K27', W23 '/L27', and F23 '/L27' combinations were not compromised. The simplest explanation is that the F23 '/K27' combination does not embed as much hydrophobic surface area as the other combinations (FIGS. 3C and 3D). The W23 'F change in PTH resulted in less hydrophobic contact with the receptor because the Phe side chain was smaller and this could be compensated by the additional van der waals contact available from Leu at position 27' (fig. 3C), which explains the rescue of the W23 'F defect by the K27' L change. Also, the L27' K change in PTHrP resulted in less hydrophobic contact with the receptor, since Lys, unlike Leu, was not branched at the gamma carbon (fig. 3D). The L27 'K defect can be compensated by an additional hydrophobic contact available from the Trp at position 23'.

Example 6

The hybrid PTH/PTHrP peptide is selective for G protein-coupled receptors.

Previous studies have shown that the ECD-binding portion of PTH contributes to its strong binding to the G-protein uncoupling receptor (R0) (23, 26). Since PTH and PTHrP exhibit similar affinity for ECD (fig. 1), the inventors wanted to know if different ECD binding patterns contribute to their R0/RG selectivity differences. To address this issue, the inventors incorporated exchange mutations into PTH (1-34) NH2 and PTHrP (1-34) NH2 scaffolds to facilitate analysis of peptide binding to the R0 and RG conformations of PTH1R in cell membranes. Experimentally, the R0 conformation was studied by including GTP γ S in the uncoupling reaction of the receptor and the G protein, while the RG conformation was studied by co-expressing the receptor with the dominant negative form of G α S (23, 25). If different ECD binding patterns contribute to R0/RG selectivity, the PTHrP (1-34) NH2[ F23 'W/L27' K/I28 'L/I31' V ] peptide exhibits reduced RG selectivity (higher affinity for R0) compared to wild-type PTHrP (1-34) because our ECD binding data are consistent with the idea that this hybrid peptide adopts the PTHECD binding pattern.

Consistent with previous studies, wild-type PTH and PTHrP peptides bind RG with similarly high affinity (IC50 of PTH about 0.3nM, IC50 of PTHrP about 0.1nM), PTHrP is more selective for RG, exhibits affinity as low as 1/245 for R0 (IC50 about 27nM), while PTH exhibits only affinity as low as about 1/14 for R0 (IC50 about 4nM) [ fig. 4A, fig. 4B and table 3 ].

TABLE 3

PTHrP (1-34) NH2[ F23 'W/L27' K/I28 'L/I31' V ] in comparison with wild-type PTHrP]The peptide (likely adopting the PTH ECD binding mode) showed a slightly improved affinity for R0 (higher RG selectivity), indicating that the different ECD binding modes do not contribute to R0/RG selectivity. Indeed, all crossover mutations that lead to diminished ECD binding also resulted in diminished R0 binding, but had little or no effect on RG binding, with the degree of decrease in R0 affinity being related to ECD binding capacity (fig. 3 and fig. 4 and table 3). PTH (1-34) NH₂[W23′F/V31′I]And PTHrP (1)-34)NH₂[L27′K]The most remarkable; both peptides exhibited poor ECD binding and significantly reduced R0 affinity, but maintained normal RG affinity. PTHrP (1-34) NH₂[L27′K]It is particularly RG selective, with reduced affinity for R0 of 1/7588(IC50 of about 1.2. mu.M) compared to RG (IC50 of about 0.16 nM). The attenuation of R0 binding by the exchange change that reduces ECD binding is rescued by other exchange changes that restore ECD binding (fig. 3 and 4). These results indicate that ECD affinity, rather than ECD binding mode, is a key determinant of R0/RG selectivity characteristics of PTH and PTHrP, and that RG selectivity can be increased by decreasing ECD affinity. The increased RG selectivity of wild-type PTHrP compared to wild-type PTH is probably determined mainly by divergent residue 5 (23, 25) because the peptides have similar affinity for ECD.

In COS cells transiently expressing PTH1R, the most RG selective PTH and PTHrP hybrid peptides were evaluated for their ability to stimulate cAMP accumulation. PTH (1-34) NH2[ W23 ' F/V31 ' I ] and PTHrP (1-34) NH2[ L27 ' K ] induced a cAMP response with essentially the same potency as the wild-type peptide, but approximately 80% of the maximum cAMP levels obtained with the wild-type peptide (FIG. 5A). Under these experimental conditions, the reduction in maximal response may reflect sub-stoichiometric G protein levels, such that a fraction of the receptors are always present in an uncoupled state and are not activated by the RG-selective peptide. To analyze the duration of cAMP signaling, we used a ligand wash out protocol (26). Cells were stimulated with peptide (100nM) for 10 min and cAMP signaling capacity was assessed at various times after ligand washout. The RG-selective PTH and PTHrP hybrid peptides showed a shorter response than the wild-type peptide (fig. 5B), indicating that the effect of the hybrid peptide on the receptor is more pulsatile.

Example 7

PTH analogue and luciferase assay.

The inventors designed a series of PTH analogues with better potency and selectivity (fig. 7A and 7B). Each PTH analog has an identifier given as: # - # # - # # - # # - ## # -SBSL. Such PTH peptides or polypeptides consist of PTH peptides (1-34) with amino acid substitutions at one or more of amino acid positions 12, 14, 16, 17 and 27; some analogs include a D-cysteine substitution at position 19 and a cysteine substitution disulfide at position 22, or cysteine substitution disulfide at positions 22 and 25 (see SEQ ID Nos. 1-52), as follows:

TABLE 4

Cell culture for PTH analogue luciferase assay:

AD-293 cells (Stratagene) were maintained at 37 ℃ in α -MEM supplemented with 10% FBS at 5% CO2 and 95% humidity.

PTH analogue luciferase assay:

AD293 cells (Stratagene) were seeded in 24-well plates for 24 hours and then co-transfected with CRE-luciferase (pGL4.29/CRE-luc2p, promega, 200 ng/well), pcDNA-PTH1R (10 ng/well) and pHRLTK (Renilla luciferase, 5 ng/well) using Lipofectamine2000(Invitrogen) according to the manufacturer's protocol. During transfection, the medium in the cell culture was changed to OPTI-MEM without FBS. Different concentrations of PTH analogue (0.1nM or 0.03nM) were then added to the medium 24 hours after transfection and luciferase activity in transfected cells was determined using a Dual-luciferase assay kit (Dual-luciferase assay kit, promega) over 4 hours. See fig. 7A and 7B.

While the foregoing specification has been described in terms of certain preferred embodiments, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that various modifications and other embodiments of the invention can be made, and that certain of the details described herein can be varied considerably without departing from the spirit and scope of the invention.

Reference to the literature

The following references are cited throughout this disclosure in accordance with the indicated numbers listed below.

Juppner, h., Abou-Samra, a.b., Freeman, m., Kong, x.f., schipinani, e., Richards, j., Kolakowski, l.f., jr., Hock, j., Potts, j.t., jr., Kronenberg, h.m., et al (1991) a G protein-linked receptor for a partial polypeptide-related peptide (G protein linked receptor for parathyroid and parathyroid hormone-related peptide) Science (New York, n.y 254, 1024-1026).

Gardella, T.J. and Juppner, H. (2000) Interaction of PTH and PTHrP with the same receptors Reviews in endo and metabolic disorders 1, 317- "329.

Gensure, R.C, Gardella, T.J. and Juppner, H. (2005) parathyroid hormone and parathyroid hormone-related peptide, and the peptide receptors thereof Biochemical and biophysical research communications 328, 666-.

Murray, t.m., Rao, l.g., Divieti, p. and Bringhurst, F.R, (2005) partial phosphor one session and action: evidence for discrete receptors in the carboxy-terminal region and related biological actions of the carboxy-terminal ligands, Endocrine reviews 26, 78-113.

Potts, j.t. (2005) Parathyroid hormone: past and present (review and present of parathyroid hormone.) The Journal of endocrinology 187, 311-325.

Potts, j.t. and Gardella, T.J. (2007) Progress, paramox, andpitotential: partial hormone research over live decapdes (progression, difficulty and potential: parathyroid hormone research over fifty years.) Annals of the New YorkAcadmayment of Sciences 1117, 196-208.

Jilkka, R.L. (2007) Molecular and cellular mechanisms of the anabolic effect of internittent PTH (Molecular and cellular mechanisms of intermittent PTH anabolic effect), Bone 40, 1434-.

New, R.M., Arnaud, C.D., Zanchetta, J.R., Prince, R., Gaich, G.A., register, J.Y., Hodsman, A.B., Eriksen, E.F., Ish-Shalom, S., Genant, H.K. et al (2001) Effect of partial hormone (1-34) on fresh and bone minor hormone in porous patent hormone with osteoporosis (Effect of parathyroid hormone (1-34) on bone fracture and bone mineral density in postmenopausal women with osteoporosis.) The New environmental and jrnal of medical hormone 344, 1434. 1441.

Burtis, W.J., Wu, T., Bunch, C, Wysolmerski, J.J., Insogna, K.L., Weir, E.C, Broadus, A.E., and Stewart, A.F, (1987) Identification of animal 17,000-dalton partial hormone-like kinase-stimulating protein from a tumor associated with malignant liquid hypercalcemia-related tumor Identification of parathyroid hormone-like adenylate cyclase stimulating protein of tumor tissue assisted with biological chemistry 262. journal of biological chemistry 262, 7151 7156.

Moseley, J.M., Kubota, M., Diefenbach-Jagger, H., Wettenhall, R.E., Kemp, B.E., Suva, L.J., Rodda, C.P., Ebeling, P.R., Hudson, P.J., Zajac, J.D. et al (1987) Parametric hormon-related protein purified from human lung cancer cell line, Proceedings of the National Academy of science of the United states of America 84, 5048-Bufonic acid 2.

Suva, l.j., Winslow, g.a., Wettenhall, r.e., hammons, r.g., Moseley, j.m., dieffenbach-Jagger, h., Rodda, c.p., Kemp, b.e., Rodriguez, h., Chen, e.y., et al (1987) a partial hormone-related protein expressed in a macromolecular hydrogel: cloning and expression (parathyroid hormone-related protein involved in malignant hypercalcemia: cloning and expression.) Science New York, N.Y 237, 893-896.

Klein, R.F., Strewler, G.J., Leung, S.C and Nissenson, R.A (1987) partial hormone-like acrylate cyclase-stimulating activity from a human cancer associated with bone resorption activity.

Karaplis, A.C, Luz, A., Glowacki, J., Bronson, R.T., Tybuliwicz, V.L., Kronenberg, H.M., and Mullgan, R.C. (1994) Lethalskeletal dysplassa from target division of the paragateway-related peptide gene (lethal skeletal dysplasia derived from a targeted disruption), Genes & depression 8, 277-.

Lanske, B., Karapis, A.C., Lee, K., Luz, A., Vortkamp, A., Pirro, A., Karperien, M., Defize, L.H., Ho, C, Mullgan, R.C. et al (1996) PTH/PTHrP receptor in early development and Indian hedge-regulated bone growth PTH/PTHrP receptor in early development, Science (New York, N.Y 273, 663 666).

Kronenberg, H.M, (2006) PTHrP and skeletal definition (PTHrP and skeletal development), Annals of the New York Academy of Sciences1068, 1-13.

Horwitz, M.J., Tedesco, M.B., Gundberg, C, Garcia-Ocana, A. and Stewart, A.F (2003) Short-term, high-dose partial-related protein as a skeletal anabolic agent for The treatment of postmenopausal osteoporosis Short-term high dose parathyroid hormone-related protein (Short-term high dose parathyroid hormone-related protein as a skeletal anabolic agent), The Journal of clinical cognitive and metabolic 88, 569-575.

Plotkin, h., Gundberg, C, Mitnick, m., and Stewart, A.F (1998) the association of bone formation from restriction during 2-week travel with human partial hormone-related peptides- (1-36) in humans: potential as an anabolic therapy for osteoporotosis (treatment with human parathyroid hormone-related peptide- (1-36) in humans for 2 cycles dissociating bone formation and bone resorption: potential for use as an anabolic therapy for osteoporosis.) The Journal of clinical endomycology and transmetabolism 83, 2786-.

Yang, D.D., Singh, R.D., Divieti, P.s., Guo, J.S., Bouxsein, M.L., and Bringhurst, F.R. (2007) controls of partial hormone (PTH)/PTH-related peptide receptor signaling pathway to the anabolic effect of PTH on Bone. Bone 40, 1453. 1461.

Bergwitz, C, Gardella, T.J., Flannery, M.R., Potts, J.T., Jr., Kronenberg, H.M., Goldring, S.R., and Juppner, H. (1996) Full activity of molecular receptors by antibodies beta protein receptor chemical hormone and polypeptide receptor evaluation for a common model of ligand-receptor interaction through a hybrid between parathyroid hormone and calcitonin, The Journal of biological chemistry 271, 26469-.

Luck, m.d., Carter, p.h., and Gardella, T.J (1999) The (1-14) fragment of partial hormone (PTH) activated domains and amino-terminated PTH-1 receptors (The (1-14) fragment of parathyroid hormone (PTH) activates The intact and amino-truncated PTH-1 receptor Molecular endo-phonologogy baltimore, Md 13, 670-.

Marx, U.C., Adermann, K., Bayer, P., Forssmann, W.G., and Rosch, P. (2000) Solution structures of human partial thermal effects hPTH (1-34) and hPTH (1-39) and bovine partial thermal effects bPTH (1-37) (Solution structures of human parathyroid hormone fragments hPTH (1-34) and hPTH (1-39) and bovine parathyroid hormone fragments bPTH (1-37). Biochemical and biomedical research communications 267, 213-.

Weidler, M., Marx, U.C., Seidel, G., Schafer, W., Hoffmann, E., Essswein, A. and Rosch, P. (1999) The structure of human parathyroid-related protein (1-34) in near-physiological solution FEBS letters 444, 239-.

Dean, t, Vilardaga, j.p., Potts, j.t., jr. and Gardella, T.J (2008) an alternative selection of partial hormone (PTH) and PTH-related protein (PTHrP) for differential interactions of the PTH/PTHrP receptor (selective change of parathyroid hormone (PTH) and PTH-related protein (PTHrP) to the unique conformation of the PTH/PTHrP receptor), Molecular endiannelogy pellet, Md 22, 156-.

Dean, t, lingrart, a, Mahon, m.j., base, m.p., Juppner, h.p., Potts, j.t., jr. and Gardella, T.J. (2006) Mechanisms of ligand binding to the Partial Telephone (PTH)/PTH-related protein receptor: selected of modified PTH (1-15) radioligand for GalphaS-coupled receptor ligands (mechanism of ligand binding to parathyroid hormone (PTH)/PTH related protein receptor: selectivity of modified PTH (1-15) radioligand for G.alpha.S-coupled receptor conformation). Molecular endicerology (Baltimore, Md 20, 931-943).

Horre, S.R., Gardella, T.J. and Usdin, T.B, (2001) evaluation of The signal transduction mechanism of The parathyroid hormone 1receptor, evaluation of The receptor-G-protein interaction on The ligand binding mechanism and receptor conformation, The Journal of biological chemistry 276, 7741-fold 7753.

Okazaki, m., Ferrandon, s., Vilardaga, j.p., Bouxsein, m.l., Potts, j.t., jr, and Gardella, T.J. (2008) Prologated signaling at the parathyroid hormone receptor by peptide ligands targeted to specific receptor conformations extends signal transduction to parathyroid hormone receptors. Proceedings of the National Academy of Sciences and United States of America 105, 16525 and 16530.

Pioszak, A.A. and Xu, H.E. (2008) Molecular recognition of partial hormomone by G protein-coupled receptor (Molecular recognition of parathyroid hormone by its G protein-coupled receptor.) Proceedings of the National academy Sciences of the United States of America 105, 5034-5039.

Parthier, C, Kleinschmidt, M., Neumann, P., Rudolph, R., Manhart, S., Schlenzg, D., fanghael, J., Rahfeld, J.U., Demuth, H.U., and Stubbs, M.T, (2007) crystalline structure of the secreted-bound extracellular domain of G protein-coupled receptors. Proceedings of the National academy Sciences of the cultured States of America 104, 13942 13947.

Pioszak, A.A., Parker, N.R., Suino-Powell, K.and Xu, H.E. (2008) Molecular recognition of corticotropin-releasing factor by its G-protein coupled receptor CRFR 1. The Journal of biological chemistry283, 32900-32912.

Run, S., Thogersen, H., Madsen, K., Lau, J., and Rudolph, R. (2008) Crystal structure of The ligand-bound glucose-like peptide-1receptor extracellular domain, The Journal of biological chemistry283, 11340-11347.

Grauschopf, u., Lilie, h., Honold, k., Wozny, m., Reusch, d., Esswein, a, Schafer, w., Rucknagel, k.p., and Rudolph, r. (2000) the N-terminal fragment of human parathyroid hormone receptor 1 constitutive host binding domain and derived a variant polypeptide pattern, Biochemistry 39, 8878 + 8887.

Gensure, R.C., Shimizu, N., Tscan, J. and Gardella, T.J (2003) Identification of a contact site for resistance 19 of partial hormone (PTH) and PTH-related protein analogs in a transmembrane domain of the PTH receptor type 1, recognition of the contact site of parathyroid hormone (PTH) and PTH-related protein analog residues 19. molecular diagnostics Baltimore, Md 17, 2647 and 2658.

Lawrence, M.C. and Colman, P.M. (1993) ShapeComplementarity at proteins/protein interfaces shape complementarity Journal of molecular biology 234, 946-.

Penel, S. and Doig, A.J, (2001) rotator strain energy in protein helices-quantification of major force against protein folding Journal of molecular biology 305, 961-968.

Mann, R., Wigglessorth, M.J., and Donnelly, D. (2008) Ligand-receptors at the partial hormone receptors: type binding selectivity is mediated via an interaction between the protein response 23 on the ligand and response 41 on the receptor (ligand-receptor interaction on parathyroid hormone receptor: subtype binding selectivity is mediated by interaction between ligand residue23 and receptor residue 41). Molecular pharmacology 74, 605-.

Horre, S.R., Clark, J.A. and Usdin, T.B, (2000) molecular modulators of molecular beacons of 39 responses (TIP39) selectivity for The partial hormone-2(PTH2) receptor, N-tertiary receptor of TIP39 reverse PTH2 receptor/PTH 1receptor binding selectivity for parathyroid hormone-2(PTH2) receptor of nodule funnel peptide of 39 residues (TIP 39). N-terminal PTH2 receptor/reverse PTH 1receptor binding selectivity of TIP 39. The Journal of biological chemistry 275, 74 27283.

Otwwinowski, Z and Minor, W. (1997) Processing of X-ray diffraction data collected in an oscillation mode. Methods in enzymology 276(Pt A), 307-326.

(1994) The CCP4 suite: programs for protein crystallography (CCP4 suite: protein crystallography program.) Acta crystallography 50, 760-.

McCoy, A.J., gross-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C and Read, R.J. (2007) phase crystalline Software, Journal of Applied crystalline 40, 658-

Kleywegt, G.J., and Jones, T.A, (1997) Model building and refinement practice, Methods in enzymology 277, 208-.

Murshudov, G.N., Vagin, A.A. and Dodson, E.J, (1997) reference of macromolecular structures by the maximum-likelihood method for the Refinement of macromolecular structures Actacrystallographics 53, 240-.

Wine, m.d., Isupov, m.n., and Murshudov, G.N, (2001) Use of TLS parameters to model anisotropic displacements in macromolecular refinement actarraycopic 57, 122-.

Laskowski, r.a., MacArthur, m.w., Moss, d.s., and Thornton, J.M, (1993) PROCHECK: a program to chemical the steric quality of the protein structure (PROCHECK: program for examining the stereochemical quality of the protein structure.) Journal of Applied Crystallography 26, 283-.

Delano, W. (2002) The PyMoI Molecular Graphics System (PyMol Molecular Graphics System.) Delano Scientific, Palo Alto, Calif., USA.

45.Pioszak, A.A. and Xu, H.E. (2008) Proceedings of the national academy of Sciences of the United States of America 105, 5034-5039.

46.Bradford，M.M.(1976)Analytical biochemistry 72，248-254。

The claims (modification according to treaty clause 19)

1. A parathyroid hormone (PTH) peptide comprising the amino acid substitution of SEQ ID NO: 9.

2. The PTH peptide or analog of claim 1 comprising SEQ ID NO: 53, wherein the amino acid at position 12 is Gly, Val, Ala or conservative substitution thereof, the amino acid at position 14 is His, Phe, Leu or conservative substitution thereof, the amino acid at position 16 is Asn, Thr or conservative substitution thereof, the amino acid at position 17 is Ser, Asp, Asn or conservative substitution thereof, and the amino acid at position 27 is Lys, Leu or conservative substitution thereof.

3. The PTH peptide or analog of claim 1 or 2, wherein the PTH peptide comprises SEQ ID NO: 1-10 and 13-20.

4. The PTH peptide or analog of claim 1, wherein the analog comprises the amino acid sequence of SEQ ID NO: 54-56, wherein two of the amino acids at positions 19, 22 and 25 are linked by a disulfide bond.

5. The PTH peptide or analog of claim 4, wherein the disulfide bond links amino acid 19 to amino acid 22.

6. The PTH peptide or analog of claim 5, wherein the amino acid at position 19 is D-Cys and the amino acid at position 22 is Cys.

7. The PTH peptide or analog of claim 4, wherein the disulfide bond links amino acid 22 to amino acid 25.

8. The PTH peptide or analog of claim 7, wherein the amino acids at positions 22 and 25 are each Cys.

9. The PTH peptide or analog of claim 4, wherein the analog comprises the amino acid sequence of SEQ ID NO: 11. 12 and any one of 21-52.

10. The PTH peptide or analog of any of the preceding claims, wherein the PTH peptide or analog thereof comprises a C-terminal amide group replacing the C-terminal carboxylic acid.

11. A PTH peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, or 9 having an amino acid substitution at one or more of positions 23, 27, 28, and 31 of SEQ ID NO: 9 amino acids 15-34.

12. The PTH peptide of claim 11, wherein the PTH peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of parathyroid hormone receptor (PTH1R) compared to the affinity of wild-type PTH (SEQ ID NO: 9) for the R0 conformation of PTH1R, wherein the PTH peptide remains bound to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTH peptide stimulates the accumulation of cAMP in cells.

13. The PTH peptide of claim 11, wherein the amino acid sequence set forth in SEQ ID NO: 9 by one or more of SEQ ID NOs: 79 amino acid substitutions at the corresponding positions.

14. The PTH peptide of claims 11-13, which comprises the sequence set forth in SEQ ID NO: 9, or 9 having an amino acid substitution at one or more of positions 23, 27, 28, and 31 of SEQ ID NO: 9 amino acids 1-34.

15. The PTH peptide of claim 14, comprising SEQ ID NO: 57-66.

16. The PTH peptide of any one of claims 11-15, wherein the PTH peptide exhibits a shorter cAMP response as compared to the cAMP response of wild type PTH (SEQ ID NO: 9).

17. The PTH peptide of claim 16, wherein said PTH peptide comprises SEQ ID NO: 64.

18. a PTH peptide comprising SEQ ID NO: 9 amino acids 15-34.

19. The PTH peptide of claim 18, wherein said PTH peptide exhibits reduced binding to the extracellular domain (ECD) of PTH1R as compared to the binding of wild-type PTH to the ECD of PTH 1R.

20. The PTH peptide of claim 18, wherein the PTH peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R as compared to the affinity of wild-type PTH (SEQ ID NO: 9) for the R0 conformation of PTH1R, wherein the PTH peptide remains bound to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTH peptide stimulates the accumulation of cAMP in cells.

21. The PTH peptide of claims 18-20, wherein said PTH peptide comprises an amino acid substitution at position 23 or 28 or at both positions.

22. The PTH peptide of claim 21, wherein said PTH peptide comprises an amino acid substitution at one or more of positions 21, 27 and 34.

23. The PTH peptide of any one of claims 18-22, comprising SEQ ID NO: 9, wherein there is an amino acid substitution at one or more of positions 20, 21, 23, 24, 27, 28, and 34.

24. The PTH peptide of claim 18, which comprises Ala at one or more of positions 20, 21, 23, 24, 27, 28 and 34.

25. The PTH peptide of claim 24, comprising SEQ ID NO: 68-71, 73, 74 and 78.

26. A PTHrP peptide comprising the amino acid sequence set forth in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 of SEQ ID NO: 79 amino acids 12-34.

27. The PTHrP peptide of claim 26, wherein the PTHrP peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R as compared to the affinity of wild-type PTHrP (SEQ ID NO: 79) for the R0 conformation of PTH1R, wherein the PTHrP peptide retains binding to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTHrP peptide stimulates the accumulation of cAMP in cells.

28. The PTHrP peptide of claim 27, wherein the peptide in SEQ ID NO: 79 is substituted with one or more of SEQ ID NO: 9 amino acid substitution at the corresponding position.

29. The PTHrP peptide of claims 26-28 which comprises the amino acid sequence set forth in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 of SEQ ID NO: 79 amino acids 1-34.

30. The PTHrP peptide of claim 29, comprising the amino acid sequence of SEQ ID NO: 80-90 in the presence of a protease.

31. The PTHrP peptide of any one of claims 26 to 30, wherein the PTHrP peptide exhibits a shorter cAMP response as compared to the cAMP response of wild-type PTHrP (SEQ ID NO: 79).

32. The PTHrP peptide of claim 31, wherein the PTHrP peptide comprises SEQ id no: 81.

33. a PTHrP peptide comprising SEQ ID NO: 79 amino acids 12-34.

34. The PTHrP peptide of claim 33 wherein said PTHrP peptide exhibits reduced binding to the ECD of PTH1R as compared to the binding of wild-type PTHrP to either ECD or PTH 1R.

35. The PTHrP peptide of claim 33, wherein the PTH peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R as compared to the affinity of wild-type PTHrP (SEQ ID NO: 79) for the R0 conformation of PTH1R, wherein the PTHrP peptide retains binding to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTHrP peptide stimulates the accumulation of cAMP in cells.

36. The PTHrP peptide of claims 33-35 wherein the PTHrP peptide comprises an amino acid substitution at position 22 or 27 or at both positions.

37. The PTHrP peptide of any one of claims 33 to 36, comprising the amino acid sequence of SEQ ID NO: 79 having amino acid substitutions at one or more of positions 20, 21, 23, 24, 27, 28, 32, and 33.

38. The PTHrP peptide of claim 37, which comprises Ala at one or more of positions 20, 21, 23, 24, 27, 28, 32 and 33.

39. The PTHrP peptide of claim 38 comprising the amino acid sequence of SEQ ID NO: 93-98, 100 and 101.

40. A chemical derivative comprising a PTH peptide or analogue or PTHrP peptide of any one of the preceding claims and a chemical moiety which is not part of wild-type PTH (SEQ ID NO: 9) or wild-type PTHrP (SEQ ID NO: 79).

41. A fusion polypeptide comprising (a) a PTH peptide or analog or PTHrP peptide of any one of the preceding claims, (b) optionally a linker region, and (c) a second polypeptide linked to the PTH peptide or analog or PTHrP peptide, or linked to said linker region, wherein said second polypeptide is not naturally linked to the PTH peptide or analog or PTHrP peptide.

42. A multimeric peptide comprising a base peptide sequence repeated from about 2 to about 100 times and optionally a spacer, wherein the base peptide sequence is the amino acid sequence of a PTH peptide or analogue or PTHrP peptide of any one of the preceding claims.

43. A pharmaceutical composition comprising a PTH peptide or analog or PTHrP peptide of any of the preceding claims, a chemical derivative of claim 40, the fusion polypeptide of claim 41 or the multimeric peptide of claim 42, and a pharmaceutically acceptable carrier or excipient.

44. A kit for treating osteoporosis comprising a peptide selected from the group consisting of a PTH peptide or analog of any one of claims 1-25 and a PTHrP peptide of any one of claims 26-39, and instructions for administering said peptide to a subject having osteoporosis.

45. Use of a peptide selected from the group consisting of a PTH peptide or analog of any one of claims 1-25 and a PTHrP peptide of any one of claims 26-39 in the manufacture of a medicament for the treatment of osteoporosis.

46. A method for activating a PTH receptor in a cell, the method comprising administering a PTH peptide or analogue or PTHrP peptide of any one of the preceding claims to a cell such that the PTH peptide or analogue or PTHrP peptide causes the PTH receptor to be activated.

47. The method of claim 46, wherein the cell is an osteoblast cell.

48. The method of claim 46 or 47, wherein the cell is in a living animal and the PTH peptide or analog or PTHrP peptide is administered to the living animal.

49. The method of claim 48, wherein said living animal is a human.

50. The method of claim 48 or 49, wherein said PTH peptide or analog or PTHrP peptide is administered by oral delivery or injection.

51. A method of treating a subject having a disease or disorder associated with undesired bone loss, the method comprising administering to the subject the pharmaceutical composition of claim 43 in an amount effective to treat the subject.

52. The method of claim 51, wherein the subject is a human.

53. The method of claim 51 or 52, wherein the disease or disorder is osteoporosis.

54. A method of ameliorating osteoporosis related symptoms in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 43 in an amount effective to ameliorate the osteoporosis related symptoms in the subject.

55. A method of delaying the progression of osteoporosis in a subject, the method comprising administering the pharmaceutical composition of claim 43 to the subject in an amount effective to delay the progression of osteoporosis in the subject.

56. A method of regenerating bone in a subject, the method comprising administering the pharmaceutical composition of claim 43 to the subject in an amount effective to regenerate bone in the subject.

Claims (56)

1. A parathyroid hormone (PTH) peptide comprising the amino acid substitution of SEQ ID NO: 9.
2. The PTH peptide or analog of claim 1 comprising SEQ ID NO: 53, wherein the amino acid at position 12 is Gly, Val, Ala or conservative substitution thereof, the amino acid at position 14 is His, Phe, Leu or conservative substitution thereof, the amino acid at position 16 is Asn, Thr or conservative substitution thereof, the amino acid at position 17 is Ser, Asp, Asn or conservative substitution thereof, and the amino acid at position 27 is Lys, Leu or conservative substitution thereof.
3. The PTH peptide or analog of claim 1 or 2, wherein the PTH peptide comprises SEQ ID NO: 1-10 and 13-20.
4. The PTH peptide or analog of claim 1, wherein the analog comprises the amino acid sequence of SEQ ID NO: 54-56, wherein two of the amino acids at positions 19, 22 and 25 are linked by a disulfide bond.
5. The PTH peptide or analog of claim 4, wherein the disulfide bond links amino acid 19 to amino acid 22.
6. The PTH peptide or analog of claim 5, wherein the amino acid at position 19 is D-Cys and the amino acid at position 22 is Cys.
7. The PTH peptide or analog of claim 4, wherein the disulfide bond links amino acid 22 to amino acid 25.
8. The PTH peptide or analog of claim 7, wherein the amino acids at positions 22 and 25 are each Cys.
9. The PTH peptide or analog of claim 4, wherein the analog comprises the amino acid sequence of SEQ ID NO: 11. 12 and any one of 21-52.
10. The PTH peptide or analog of any of the preceding claims, wherein the PTH peptide or analog thereof comprises a C-terminal amide group replacing the C-terminal carboxylic acid.
11. A PTH peptide comprising the amino acid sequence set forth in SEQ ID NO: 9, or 9 having an amino acid substitution at one or more of positions 23, 27, 28, and 31 of SEQ ID NO: 9 amino acids 15-34.
12. The PTH peptide of claim 11, wherein the PTH peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of parathyroid hormone receptor (PTH1R) compared to the affinity of wild-type PTH (SEQ ID NO: 9) for the R0 conformation of PTH1R, wherein the PTH peptide remains bound to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTH peptide stimulates the accumulation of cAMP in cells.
13. The PTH peptide of claim 11, wherein the amino acid sequence set forth in SEQ ID NO: 9 by one or more of SEQ ID NOs: 79 amino acid substitutions at the corresponding positions.
14. The PTH peptide of claims 11-13, which comprises the sequence set forth in SEQ ID NO: 9, or 9 having an amino acid substitution at one or more of positions 23, 27, 28, and 31 of SEQ ID NO: 9 amino acids 1-34.
15. The PTH peptide of claim 14, comprising SEQ ID NO: 57-66.
16. The PTH peptide of any one of claims 11-15, wherein the PTH peptide exhibits a shorter cAMP response as compared to the cAMP response of wild type PTH (SEQ ID NO: 9).
17. The PTH peptide of claim 16, wherein said PTH peptide comprises SEQ ID NO: 64.
18. a PTH peptide comprising SEQ ID NO: 9 amino acids 15-34.
19. The PTH peptide of claim 18, wherein said PTH peptide exhibits reduced binding to the extracellular domain (ECD) of PTH1R as compared to the binding of wild-type PTH to the ECD of PTH 1R.
20. The PTH peptide of claim 18, wherein the PTH peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R as compared to the affinity of wild-type PTH (SEQ ID NO: 9) for the R0 conformation of PTH1R, wherein the PTH peptide remains bound to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTH peptide stimulates the accumulation of cAMP in cells.
21. The PTH peptide of claims 18-20, wherein said PTH peptide comprises an amino acid substitution at position 23 or 28 or at both positions.
22. The PTH peptide of claim 21, wherein said PTH peptide comprises an amino acid substitution at one or more of positions 21, 27 and 34.
23. The PTH peptide of any one of claims 18-22, comprising SEQ ID NO: 9, wherein there is an amino acid substitution at one or more of positions 20, 21, 23, 24, 27, 28, and 34.
24. The PTH peptide of claim 18, which comprises Ala at one or more of positions 20, 21, 23, 24, 27, 28 and 34.
25. The PTH peptide of claim 24, comprising SEQ ID NO: 68-71, 73, 74 and 78.
26. A PTHrP peptide comprising the amino acid sequence set forth in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 of SEQ ID NO: 79 amino acids 12-34.
27. The PTHrP peptide of claim 26, wherein the PTHrP peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R as compared to the affinity of wild-type PTHrP (SEQ ID NO: 79) for the R0 conformation of PTH1R, wherein the PTHrP peptide retains binding to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTHrP peptide stimulates the accumulation of cAMP in cells.
28. The PTHrP peptide of claim 27, wherein the peptide in SEQ ID NO: 79 is substituted with one or more of SEQ ID NO: 9 amino acid substitution at the corresponding position.
29. The PTHrP peptide of claims 26-28 which comprises the amino acid sequence set forth in SEQ ID NO: 79 with amino acid substitutions at one or more of positions 23, 27, 28 and 31 of SEQ ID NO: 79 amino acids 1-34.
30. The PTHrP peptide of claim 29, comprising the amino acid sequence of SEQ ID NO: 80-90 in the presence of a protease.
31. The PTHrP peptide of any one of claims 26 to 30, wherein the PTHrP peptide exhibits a shorter cAMP response as compared to the cAMP response of wild-type PTHrP (SEQ ID NO: 79).
32. The PTHrP peptide of claim 31, wherein the PTHrP peptide comprises SEQ id no: 81.
33. a PTHrP peptide comprising SEQ ID NO: 79 amino acids 12-34.
34. The PTHrP peptide of claim 33 wherein said PTHrP peptide exhibits reduced binding to the ECD of PTH1R as compared to the binding of wild-type PTHrP to either ECD or PTH 1R.
35. The PTHrP peptide of claim 33, wherein the PTH peptide exhibits reduced affinity for the G-protein uncoupled conformation (R0) of PTH1R as compared to the affinity of wild-type PTHrP (SEQ ID NO: 79) for the R0 conformation of PTH1R, wherein the PTHrP peptide retains binding to the G-protein coupled conformation (RG) of PTH1R, and wherein the PTHrP peptide stimulates the accumulation of cAMP in cells.
36. The PTHrP peptide of claims 33-35 wherein the PTHrP peptide comprises an amino acid substitution at position 22 or 27 or at both positions.
37. The PTHrP peptide of any one of claims 33 to 36, comprising the amino acid sequence of SEQ ID NO: 79 having amino acid substitutions at one or more of positions 20, 21, 23, 24, 27, 28, 32, and 33.
38. The PTHrP peptide of claim 37, which comprises Ala at one or more of positions 20, 21, 23, 24, 27, 28, 32 and 33.
39. The PTHrP peptide of claim 38 comprising the amino acid sequence of SEQ ID NO: 93-98, 100 and 101.
40. A chemical derivative comprising a PTH peptide or analogue or PTHrP peptide of any one of the preceding claims and a chemical moiety which is not part of wild-type PTH (SEQ ID NO: 9) or wild-type PTHrP (SEQ ID NO: 79).
41. A fusion polypeptide comprising (a) a PTH peptide or analog or PTHrP peptide of any one of the preceding claims, (b) optionally a linker region, and (c) a second polypeptide linked to the PTH peptide or analog or PTHrP peptide, or linked to said linker region, wherein said second polypeptide is not naturally linked to the PTH peptide or analog or PTHrP peptide.
42. A multimeric peptide comprising a base peptide sequence repeated from about 2 to about 100 times and optionally a spacer, wherein the base peptide sequence is the amino acid sequence of a PTH peptide or analogue or PTHrP peptide of any one of the preceding claims.
43. A pharmaceutical composition comprising a PTH peptide or analog or PTHrP peptide of any of the preceding claims, a chemical derivative of claim 40, the fusion polypeptide of claim 41 or the multimeric peptide of claim 42, and a pharmaceutically acceptable carrier or excipient.
44. A kit for treating osteoporosis comprising a peptide selected from the group consisting of a PTH peptide or analog of any one of claims 1-25 and a PTHrP peptide of any one of claims 26-39, and instructions for administering said peptide to a subject having osteoporosis.
45. Use of a peptide selected from the group consisting of a PTH peptide or analog of any one of claims 1-25 and a PTHrP peptide of any one of claims 26-39 in the manufacture of a medicament for the treatment of osteoporosis.
46. A method for activating a PTH receptor in a cell, the method comprising administering a PTH peptide or analogue or PTHrP peptide of any one of the preceding claims to a cell such that the PTH peptide or analogue or PTHrP peptide causes the PTH receptor to be activated.
47. The method of claim 46, wherein the cell is an osteoblast cell.
48. The method of claim 46 or 47, wherein the cell is in a living animal and the PTH peptide or analog or PTHrP peptide is administered to the living animal.
49. The method of claim 48, wherein said living animal is a human.
50. The method of claim 48 or 49, wherein said PTH peptide or analog or PTHrP peptide is administered by oral delivery or injection.
51. A method of treating a subject having a disease or disorder associated with undesired bone loss, the method comprising administering to the subject the pharmaceutical composition of claim 43 in an amount effective to treat the subject.
52. The method of claim 51, wherein the subject is a human.
53. The method of claim 51 or 52, wherein the disease or disorder is osteoporosis.
54. A method of ameliorating osteoporosis related symptoms in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 43 in an amount effective to ameliorate the osteoporosis related symptoms in the subject.
55. A method of delaying the progression of osteoporosis in a subject, the method comprising administering the pharmaceutical composition of claim 43 to the subject in an amount effective to delay the progression of osteoporosis in the subject.
56. A method of regenerating bone in a subject, the method comprising administering the pharmaceutical composition of claim 43 to the subject in an amount effective to regenerate bone in the subject.