Patent Publication Number: US-2007099828-A1

Title: Biomarkers for the efficacy of calcitonin and parathyroid hormone treatment

Description:
FIELD OF THE INVENTION  
      This invention relates generally to the analytical testing of tissue samples in vitro, and more particularly to aspects of gene expression profiling concerning calcium regulation.  
     BACKGROUND OF THE INVENTION  
      Calcium is essential for many cellular processes in the body and especially important for bone metabolism. The level of calcium in the body is carefully maintained by an endocrine control system. Two of the hormones in this endocrine control system are calcitonin and parathyroid hormone.  
      Calcitonins, which are polypeptide hormones of about 32 amino acids, are endogenous regulator of calcium homeostasis and can be used as anti resorptive agents for the treatment of hypocalcaemia-associated disorders. Calcitonin is produced in the parafollicular cells (C cells) of the thyroid gland. Various calcitonins, including e.g. salmon and eel calcitonin, are commercially available and are commonly employed in the treatment of e.g. Paget&#39;s disease of bone, malignant hypocalcaemia and post-menopausal osteoporosis. Pondel M,  Intl. J. Exp. Pathol . 81(6): 405-22 (2000). A version of calcitonin (Miacalcin®) is available as a nasal spray.  
      Parathyroid hormone (PTH) is a polypeptide of 84 amino acids. Parathyroid hormone regulates bone remodelling and Ca 2+  homeostasis. Parathyroid hormone is also a known paracrine activator of osteoclast differentiation and activity. PTS893 [SDZ PTS 893; Leu8, Asp10, Lys11, Ala16, Gln18, Thr33, Ala34 human PTH 1-34 [hPTH(1-34)]]is a 34 amino acid parathyroid analogue that enhances bone mass and biomechanical properties. Kneissel M et al.,  Bone  28: 237-50 (March 2001); Stewart A F et al.,  J. Bone. Miner. Res . 15(8): 1517-25 (August 2000); Thomsen J S et al.,  Bone  25(5):561-9 (November 1999).  
      Calcitonin and parathyroid hormone are known to interact in a complex and interdependent manner, but the understanding of how calcitonin and parathyroid hormone interact has been incomplete. Calcitonin inhibitory effects on osteoclast resorptive activity, and renal tubular calcium resorption have been well documented. However, potential calcitonin effects on osteoblasts and interactions with any other skeletal-metabolism-related factors have remained controversial.  
      Multi-organ gene profiling analysis would provide a better picture of the changes induced by a compound on the whole organism and also give a new perspective to the understanding of the pharmacology of hormones. Genomics technologies are a source of the new hypothesis-generating capabilities that are now empowering biomedical researchers. In the context of drug development, they provide with a new perspective to the understanding of the pharmacology of drugs. Accordingly, there is a need in the art for an organism-wide understanding of the activity of calcitonin and parathyroid hormone.  
     SUMMARY OF THE INVENTION  
      The invention provides a response to the need in the art. Multi-organ gene profiling analysis provides with a complete picture of the changes induced by a compound on the whole organism, and gives a new perspective to the understanding of the pharmacology of drugs. In one aspect, the invention provides the first description of the molecular mechanisms of action of hormonal-mediated bone remodelling by salmon calcitonin by gene profiling analysis. The known mechanisms of action of calcitonin as anti-resorptive agent could be reconstructed at the molecular level. Effects on effectors and pathways linked to bone remodelling activities—BMPs, IGFs, extracellular matrix components and VEGF—were also observed. These results support the role of calcitonin as an anabolic agent. In another aspect, the invention provides the first reconstruction of the molecular mechanisms of action of a pharmacological agent on one of its target tissues in an intact primate animal model, by evaluating the gene expression changes induced by salmon calcitonin or the parathyroid hormone analogue PTS893 on bone in cynomolgus monkeys, to elucidate the molecular mechanisms of action mediating their effects. Gene profiling analysis allowed the reconstruction of the pathways involved in calcitonin signal transduction, triggered by protein-G-linked-receptor stimulation and their influence on cell cycle, as indicated by the changes observed in yclins. In vivo gene-profiling expression studies allow the identification of the molecular mechanisms underlying a pharmacological effect.  
      In one embodiment, the invention provides for the use of calcitonin in the manufacture of a medicament for the treatment of a condition for which treatment with an anabolic agent is indicated. In one embodiment, the condition is atherosclerosis.  
      The invention also provides for the use of calcitonin in the manufacture of a medicament for the treatment of disorders of calcium metabolism in a selected patient population, where the patient population is selected on the basis of the gene expression profile indicative of calcitonin efficacy by the patient to whom calcitonin is administered. In one embodiment, the calcitonin is salmon calcitonin. The invention fuirther provides for the use of a parathyroid hormone or parathyroid hormone analogue in the manufacture of a medicament for the treatment of disorders of calcium metabolism in a selected patient population, where the patient population is selected on the basis of the gene expression profile indicative of parathyroid hormone or parathyroid hormone analogue efficacy by the patient to whom parathyroid hormone or parathyroid hormone analogue is administered. In one embodiment, the hormone analogue is PTS893. In one embodiment, the medicament is administered in a therapeutic dose prior to determining the gene expression profile by the patient. In another embodiment, the medicament is administered in a sub-therapeutic dose prior to determining the gene expression profile by the patient.  
      The invention also provides a method for treating a condition in a subject, wherein the condition is one for which administration of a calcitonin, parathyroid hormone, a parathyroid hormone analogue or a combination thereof is indicated. The method involves, first administering a compound of interest to the subject (e.g., a primate subject) and then obtaining the gene expression profile of the subject following administration of the compound. The gene expression profile of the subject is compared to a biomarker gene expression profile. The biomarker gene expression profile is indicative of efficacy of treatment by a calcitonin, parathyroid hormone, a parathyroid hormone analogue or a combination thereof In one embodiment, the biomarker gene expression profile is the baseline gene expression profile of the subject before administration of the compound. In another embodiment, the biomarker gene expression profile is the gene expression profile or average of gene expression profiles of a vertebrate to whom calcitonin (e.g., salmon calcitonin) or parathyroid hormone or a parathyroid hormone analogue (e.g., PTS893) has been administered. A similarity in the gene expression profile of the subject to whom the compound was administered to the biomarker gene expression profile is indicative of efficacy of treatment with the compound.  
      Accordingly, the invention provides biomarkers for the efficacy of treatment of a condition for which calcitonin, parathyroid hormone or a combination thereof is indicated. Among the biomarkers are the expression profiles of the genes for Y-box binding protein, bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), vascular endothelial growth factor (VEGF), α-2-HS glycoprotein (AHSG), osteoclast stimulating factor (OSF), nuclear receptors (steroid/thyroid family) and others.  
      The invention provides methods for determining a subject for inclusion in a clinical trial, based upon an analysis of biomarkers expressed in the subject to be treated. The compound to be tested is administered to the subject. In one embodiment, the compound to be tested is administered in a sub-therapeutic dose. Then, the gene expression profile of the subject following administration of the compound is obtained. The subject may be included in the clinical trial when the gene expression profile of the subject to whom the compound was administered is similar to a biomarker gene expression profile indicative of efficacy of treatment by a calcitonin, parathyroid hormone, a parathyroid hormone analogue or a combination thereof. The subject may be excluded from the clinical trial when the gene expression profile of the subject is dissimilar to the biomarker gene expression profile indicative of efficacy of treatment. Such similarities or dissimilarities are observable to those of skill in the art.  
      The invention also provides clinical assays, kits and reagents for determining treatment efficacy of a condition for which administration of a calcitonin, parathyroid hormone or a parathyroid hormone analogue is indicated. In one embodiment, the kits contain reagents for determining the gene expression of biomarker genes, by hybridization. In another embodiment, the kits contain reagents for determining the gene expression of biomarker genes, by the polymerase chain reaction. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
      This invention is based upon an understanding of the effects of administration to a subject of calcitonin (e.g., salmon calcitonin; SEQ ID NO:1) or parathyroid hormone (SEQ ID NO:2) or an analogue thereof (e.g., PTS893; SEQ ID NO:3). A multi-organ gene profiling analysis of the results of an administration to a subject of salmon calcitonin or a parathyroid hormone analogue provides biomarkers of calcitonin treatment efficacy and parathyroid hormone or parathyroid hormone analogue treatment efficacy. As used herein, a subject is a vertebrate. In one embodiment, the vertebrate is a mammal. In a more particular embodiment, the subject is a primate, e.g., a cynomolgus money or a human.  
      The analysis provided here globally describes the molecular mechanisms of action of salmon calcitonin and the PTS893 in changing the ribonucleic acid (RNA) content in different organs by multi-organ gene profiling analysis in primates. The RNA content of the cell, the “transcriptome” is a reflection of the cell functions and status. Inside an individual cell or an organ, the expressions of the different elements of a transcriptome are not independent. The change in expression level can trigger a series of events that will lead finally to another modification of the transcriptome. These interdependent events are described in terms of pathways. Because the changes in the different functions inside a cell are tightly interconnected, the changes in different organs inside the organism are linked. Applying gene profiling to different organs submitted to the same treatment gives an improved overview of the effects and the modifications of the physiological status. As shown herein, this is particularly the situation when multi-organ profiling analysis of pleiotropic compounds, such as calcitonin, is to be performed. Indeed, the global signature described for calcitonin is reflected not only in the main target organ (i.e., bone) but also in the other organs analyzed herein.  
      In this multi-organ gene profiling analysis, the known mechanisms of action of calcitonin as an anti-resorptive agent and the parathyroid hormone PTS893 as a paracrine activator of osteoclast differentiation and activity could be reconstructed at the molecular level. The calcitonin inhibitory effect on osteoclasts could be reconstructed, with changes affecting, among others, genes for PU.1 (SPI1; SpiB; SEQ ID NO:4), colony stimulating factor (CSF-1 (SEQ ID NO:6); differentiation and survival) carbonic anhydrase (SEQ ID NO:8), H + -ATPases, cathepsin K (resorptive activity) tubulins, PAK4 (motility). Effects on effectors and pathways linked to bone remodelling activities (bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), extracellular matrix components, steroid hormones, vascular endothelial growth factor (VEGF) and α-2-HS glycoprotein (AHSG)) were also observed, shared in many cases by both salmon calcitonin and PTS893. Interestingly, salmon calcitonin also regulates the expression of the gene codifying for osteoclast stimulating factor (OSF), and cystatin. Also interestingly, PTS893 also regulates the genes implicated in osteoclast differentiation and survival (SPI1, CSF-1, monocyte to macrophage differentiation-associated protein (1)). PTS893 also produced a strong up-regulation on nuclear receptors (steroid/thyroid family). Accordingly, these results support the role of calcitonin as anabolic agent.  
      Calcitonin is presently used in the treatment of systemic skeletal diseases characterized by high bone mass which are a consequence of imbalance between bone formation (anabolic) and resorption of bone, with the former predominating. Calcitonin promotes the synthesis of bone morphogenetic protein-2 (BMP-2), which is known to be a potent anabolic agent. The evidence is strong that when calcitonin gets to bone cells, they can have an anabolic effect by increasing production of BMP-2. Thus calcitonin can be used in a method of treating an individual to adjust a subject&#39;s bone minneral density.  
      This the first approach to characterise in an in vivo model the effects of calcitonin on bone metabolism by gene expression profiling. The calcitonin inhibitory effect on osteoclasts could be reconstructed, with changes affecting genes as carbonic anhydrase, H + -AThases and cathepsin K. Salmon calcitonin also seemed to regulate the expression of the gene codifying for cystatin, being this effect described here for the first time. Salmon calcitonin has also modulating effects on genes affecting the direct, autocrine, paracrine and endocrine regulation of the mesenchymal cell functions such as pleiothropin, periostin, fibroblast growth factor, transforming growth factor betas (TGF-betas), insulin-like growth factors/binding proteins (IGFs/IGFBPs), bone morphogenetic proteins (BMPs), Vascular Endothelial Growth Factor (VEGF), Tumour Necrosis Factor (TNF), neurochondrin, follistatin-like 3, or parathyroid hormone receptor. It also regulates the synthesis and degradation of extracellular matrix components (collagens, osteopontin, osteocalcin, dermatopontin, chondroadherin, glypican or syndecan) and enzymes. Salmon calcitonin also influenced some aspects of bone mineralization, since changes in dentin were observed.  
      As provided herein, calcitonin can also be used as an anabolic agent in the treatment of other conditions where anabolism or tissue growth is therapeutically desirable. Such a condition is atherosclerosis, an atheromatous disease in which the atheromatous plaque is complicated by fibrosis and calcification.  
      Moreover, the invention provides biomarkers of the efficacy of calcitonin or parathyroid hormone treatment. As used herein, a gene expression profile is diagnostic for determining the efficacy of treatment when the increased or decreased gene expression is an increase or decrease (e.g., at least a 1.5-fold difference) over the baseline gene expression following administration of the compound (i.e., the biomarker gene expression profile is the baseline gene expression profile of the subject before administration of the compound). Alternatively or in addition, the gene expression profile is diagnostic for determining the efficacy of treatment as compared with treatment of calcitonin (e.g., satmon calcitonin) or parathyroid hormone or parathyroid hormone analogues (e.g., PTS893) when the gene expression profile of the treated subject is comparable to a standard biomarker gene expression profile. In one embodiment, the standard biomarker gene expression profile is the gene expression profile or average of gene expression profiles of a vertebrate to whom a calcitonin, parathyroid hormone, a parathyroid hormone analogue of a combination thereof has been administered, this profile or profile being the standard to which the results from the subject following administration is compared. Such an approach, which contains aspects of therapeutics and diagnostics, is termed “theranostic” by many of those of skill in the art.  
      In one embodiment, the subject is a vertebrate. In a particular embodiment, the vertebrate is a mammal. In a more particular embodiment, the mammal is a primate, such as a cynomolgus monkey or a human. As used herein, the administration of an agent or drug to a subject or patient includes self-administration and the administration by another.  
      As used herein, a gene expression profile is diagnostic of the efficacy of calcitonin or parathyroid hormone treatment when the increased or decreased gene expression is an increase or decrease (e.g., at least a 1.5-fold difference) over the baseline gene expression following administration of a calcitonin or of parathyroid hormone or an analogue. As used herein, a gene expression pattern is “higher than normal” when the gene expression (e.g., in a sample from a treated subject) shows a 1.5-fold difference (i.e., higher) in the level of expression compared to the baseline samples. A gene expression pattern is “lower than normal” when the gene expression (e.g., in a sample from a treated subject) shows a 1.5-fold difference (i.e., lower) in the level of expression compared to the baseline samples.  
      Techniques for the detection of gene expression of the genes described by this invention include, but are not limited to northern blots, RT-PCT, real time PCR, primer extension, RNase protection, RNA expression profiling and related techniques. Techniques for the detection of gene expression by detection of the protein products encoded by the genes described by this invention include, but are not limited to, antibodies recognizing the protein products, western blots, immunofluorescence, immunoprecipitation, ELISAs and related techniques. These techniques are well known to those of skill in the art. Sambrook J et al.,  Molecular Cloning: A Laboratory Manual, Third Edition  (Cold Spring Harbor Press, Cold Spring Harbor, 2000). In one embodiment, the technique for detecting gene expression includes the use of a gene chip. The construction and use of gene chips are well known in the art. See, U.S. Pat Nos. 5,202,231; 5,445,934; 5,525,464; 5,695,940; 5,744,305; 5,795,716 and 5,800,992. See also, Johnston, M.  Curr Biol  8:R171-174 (1998); Iyer VR et al.,  Science  283:83-87 (1999) and Elias P, “New human genome ‘chip’ is a revolution in the offing”  Los Anigeles Daily News  (Oct. 3, 2003).  
      The gene expression profile may include one or more genes selected from the group of acid phosphatase 1 isoform a; activin A receptor type II like 1; activin A type IIB receptor precursor; activin beta C chain; alpha 2 HS glycoprotein; amelogenin; annexin V; arylsulfatase E precursor; ATPase H(+) vacuolar; ATPase H(+) vacuolar subunit; AITase, H+ transport, lysosomal; ATPase, H+ transporting, lysosomal; ATPase, H+ transporting, lysosomal; biglycan; bone morphogenetic protein 1; bone morphogenetic protein 10; bone morphogenetic protein 2A; bone morphogenetic protein 5; bone morphogenetic protein 6 precursor; calcium binding protein 1 (calbrain); calcium/caimodulin dependent protein kinase (CaM Idnase) II gamma; calreticulin; cAMP responsive element modulator (CREM); carbonic anhydrase I; carbonic anhydrase II; cartilage oligomeric matrix protein precursor; cathepsin K; cathepsin W; CDC like kinase 1; CDC like kinase 2 isoform hclk2/139; chondroitin sulphate proteoglycan 2 (versican); chondroitin sulphate proteoglycan 3 (neurocan); chorionic somatomammotropin hormone 1; chymotrypsin C (caldecrin); collagen type 1 and PDGFB fusion transcript; collagen type II alpha 1; collagen type III alpha 1; collagen type IV alpha 2; collagen type IX alphal; collagen type VI alpha 1; collagen type VI alpha 2 (AA 570 998); collagen type XI alpha 1; collagen type XI alpha2; collagen type XI alpha2; collagen, type I, alpha 2; collagen, type IV, alpha 1; collagen, type IX, alpha 2; collagen, type V, alpha 2; collagen, type VI, alpha 1; collagen, type VI, alpha 1 precursor; collagen, type XVI, alpha 1; collagen, type XVI alpha 1; collagenase 3 (matrix metalloproteinase 13); connective tissue growth factor; cyclin A2; cyclin B1; cyclin D2; cyclin E2; cyclin dependent kinase 5; cyclin dependent kinase 5, regulatory subunit 1 (p35); cyclin dependent linase 6; cyclin dependent kinase inhibitor 1A (p21, Cip1); cystatin B (stefin B); cytokine inducible kinase; death associated protein kinase 1; death associated protein kinase 3; dentin matrix acidic phosphoprotein 1 (DMP); dual specificity phosphatase 9; dystrophia myotonica protein kinase; ectonucleotide pyrophosphatase/phosphodiesterase 1; ectonucleotide pyrophosphatase/phosphodiesterase 1; endothelial differentiation, G protein coupled receptor 6 precursor; oestrogen receptor; oestrogen receptor; oestrogen receptor related protein; oestrogen responsive B box protein (EBBP); fibroblast activation protein; fibroblast growth factor 1 (acidic); fibroblast growth factor 18; fibroblast growth factor 4; fibroblast growth factor receptor; follistatin like 1; follistatin like 1; glutamate receptor, metabotropic 1; GPI1 N acetylglucosaminyl transferase component Gpi1; granulocyte macrophage colony stimulating factor (CSF1); growth arrest and DNA damage inducible, alpha; growth factor receptor bound protein 10; heparan sulphate proteoglycan 2 (perlecan); inositol 1,4,5 triphosphate receptor, type 1; inositol 1,4,5 triphosphate receptor, type 1; inositol 1,4,5 triphosphate receptor, type 2; inositol 1,4,5 trisphosphate 3 kinase isoenzyme; inositol polyphosphate 4 phosphatase type I beta; inositol polyphosphate 5 phosphatase; inositol(myo) 1(or 4) monophosphatase 1; inositol(myo) 1(or 4) monophosphatase 2; insulin like growth factor (IGF II); insulin like growth factor 2 (somatomedin A); insulin like growth factor binding protein; insulin like growth factor binding protein 2; insulin like growth factor binding protein 3; insulin like growth factor binding protein 5; insulin like growth factor binding protein 2; insulin like growth factor II precursor; insulin like growth factor II precursor; integrin alpha 10 subunit; interleukin 1 receptor associated kinase; Janus kinase 3; LIM protein (similar to rat protein kinase C binding enigma); lysyl oxidase like protein; MAD, mothers against decapentaplegic homolog 3; MAGUKs (membrane associated guanylate kinase homologues; MAP kinase kinase kinase (MTK1); MAPK13: mitogen activated protein kinase 13; MAPK8IP1: mitogen activated protein kinase 8 interacting protein 1; MEK kinase; metalloproteinase; mitogen activated protein kinase 1; mitogen activated protein kinase 8; mitogen activated protein kinase kinase 1; mitogen activated protein kinase kinase kinase kinase 4; mitogen activated protein kinase activated protein kinase 2; mitogen activated protein kinase activated protein kinase 3; MMD: monocyte to macrophage differentiation associated; neurochondrin; nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1; OS 4 protein (OS 4); OSF 2os osteoblast specific factor 2 (periostin); osteoclast stimulating factor (OSF); PAK4; PDGF associated protein; phosphatidylinositol 4 kinase, catalytic, beta polypeptide; phosphatidylinositol glycan, class L; phosphatidylinositol polyphosphate 5 phosphatase, isoform b; phosphatidylinositol 4 phosphate 5 kinase isoform C (1); phosphatidylinositol 4 phosphate 5 kinase, type I, beta; phosphatidylinositol 4 phosphate 5 kinase, type II, beta; phosphatidylinositol glycan class C (PIG C); phosphodiesterase 4A, cAMP specific; phosphodiesterase 4D, cAMP specific (dunce ( Drosophila ) homolog phosphodiesterase E3); phosphodiesterase B, calmodulin dependent; phosphoinositide 3 kinase; phosphoinositide 3 kinase, catalytic, gamma polypeptide; phosphoinositide 3 kinase, class 3; phospholipase C b3; phospholipase C, beta 4; phospholipase D; phosphotidylinositol transfer protein; PKD2 Protein kinase D2; preprocollagen type I alpha 2; preprocollagen type I alphal; procollagen alpha 1 type II; pro collagen lysine 5 dioxygenase; procollagen proline, 2 oxoglutarate 4 dioxygenase (proline 4 hydroxylase), alpha polypeptide I; progestagen associated endometrial protein (placental protein 14, pregnancy associated endometrial alpha 2 globulin, alpha uterine protein); prolidase (imidodipeptidase) PEPD; proliferating cell nuclear antigen; prolyl 4 hydroxylase beta; protease, serine, 11 I(GF binding); proteasome (prosome, macropain) subunit, beta type, 10; protein inhibitor of activated STAT X; protein kinase 1 PCTAIRE; protein kinase C substrate 80K H; protein kinase C, alpha; protein kinase, cAMP dependent, catalytic, gamma; protein kinase, cAMP dependent, regulatory, type I, beta; protein kinase, cAMP dependent, regulatory, type II, alpha; purinergic receptor P2Y, G protein coupled, 11; RAC2 Ras related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2); receptor tyrosine kinase DDR; retinoid X receptor gamma; ribosomal protein S6 kinase; ribosomal protein S6 kinase, 90 kD, polypeptide 3; SCAMP1: secretory carrier membrane protein 1 (vesicular transport); secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T lymphocyte activation 1); serine (or cysteine) proteinase inhibitor, clade H (heat shock protein 47), member 2; serine/threonine kinase 38; serine/threonine protein kinase; SF 1; Steroidogenic factor 1; signal transducer and activator of transcription 1; signal transducer and activator of transcription 2, 113 kD; signal transducer and activator of transcription 5A; signal transducer and activator of transcription 5A; signal transducer and activator of transcription 6 (STAT6); Smad 3; Smad anchor for receptor activation, isoform 1; Smad5; SMAD6 (inhibits BMP/Smad1 (MADH1); SNF 1 related kinase; SpiB transcription factor (SPI1/PU.1 related); Stat5b (stat5b); Ste20 related serine/threonine kinase; TEIG; TGFB inducible early growth response; TGFB inducible early growth response; TIEG; TGFB1 induced anti apoptotic factor 1; TGF beta induced apoptosis protein 12; TGF beta precursor; TGF beta superfamily protein; Tob; tousled like kinase 1; transforming growth factor, beta receptor III (betaglycan, 300 kD); transforming growth factor beta 3 (TGF beta 3); TRIO: triple functional domain (PTPRF interacting); tubulin alpha 1; tubulin alpha 3; tubulin alpha isotype H2 alpha; tubulin beta 2; tubulin beta 3; tubulin beta 4; tubulin beta, cofactor D; type VI collagen alpha 2 chain precursor; ubiquitin carrier protein E2 C; vascular endothelial growth factor; vascular endothelial growth factor; vascular endothelial growth factor B; and Y box binding protein 1.  
      As used herein, the administration of an agent or drug to a subject or patient includes self-administration and the administration by another.  
      Calcitonin. The term “calcitonin” includes not only the naturally occurring calcitonins, but also their pharmaceutically active derivatives and analogues, e.g. in which one or more of the peptide residues present in the naturally occurring product is replaced, or in which the N- or C-terminal is modified. Preferred calcitonins for use in accordance with the invention are salmon, human and porcine calcitonins and Elcatonin. All of these compounds are commercially available and have been extensively described, together with their pharmaceutical properties, in the literature. See, U.S. Pat. Nos. 5,733,569 and 5,759,565, the contents of which are incorporated by reference.  
      The amount of calcitonin to be administered in accordance with the method of the invention and hence the amount of active ingredient in the composition of the invention depends on the particular calcitonin chosen, the condition to be treated, the desired frequency of administration and the effect desired.  
      The bioavailability for calcitonins, in particular sahnon calcitonin, as determined in terms of blood plasma concentration following nasal administration is high, generally of the order of ca. 50% of levels achieved on intra-muscular injection. Accordingly administration in accordance with the invention will appropriately be effected so as to give a dosage rate of the order of two times or more, e.g. from about two to four times the dosage rate required for treatment via intra-parietal, e.g. intra-muscular, administration. Information regarding the administration of Miacalcin® (calcitonin-salmon) nasal spray is available in the Miacalcin® Prescribing Iniformation (Novartis, November 2002).  
      For intra-muscular injection, individual dosages of ca. 50 to 100 MRC units are applied at a rate of from ca. one time daily to ca. three times weekly. For nasal administration in accordance with the present invention, treatment will therefore suitably comprise administration of dosages of from about 50 to about 400 MRC units, more preferably from about 100 to about 200 MRC units at a frequency of from about one time daily to about three times weekly. Conveniently dosages as aforesaid will be administered in a single application, i.e. treatment will comprise administration of single nasal dosages comprising about 50 to about 400 MRC units, preferably about 100 to about 200 MRC units, calcitonin. Alternatively such dosages may be split over a series of e.g. two to four applications taken at intervals during the day, the dosage at each application then comprising about 10 to about 200 MRC units, preferably about 25 to about 100 MRC units.  
      The total composition quantity administered at each nasal application suitably comprises from about 0.05 to 0.15 ml, typically about 0.1 ml, e.g. 0.09 ml. Compositions for use accordingly suitably comprise from about 150 to about 8,000, preferably from about 500 to about 4,000, more preferably from about 500 to about 2,500, and most preferably from about 1,000 to about 2,000 MRC units calcitonin, e.g. salmon calcitonin, per ml.  
      The term “calcitonin” also encompasses active peptide analogues and mimetics, such as described for example, in U.S. Pat. Nos. 5,719,122, 5,175,146, and 5,698,6721. See, U.S. Pat. Appln. 2003015815. The “calcitonin superfamily” consists of calcitonin, calcitonin gene-related peptide (CGRP), and amylin. Calcitonin and CGRP derive from the CT/CGRP gene, in humans. Alternative splicing of the primary RNA transcript leads to the translation of CGRP and CT peptides in a tissue-specific manner. CGRP (a 37-amino-acid neuropeptide) and its receptors are widely distributed in the body. Amylin (a 37-amino-acid peptide) is generated from a gene located on chromosome 12 (thought to be an evolutionary duplication of chromosome 11) and shares 46% amino acid sequence homology with CGRP and 20% with human calcitonin. The term “calcitonin gene-related peptide” or “CGRP” includes native CGRP, preferably human CGRP, and its active analogues. CGRP is known to have a variety of roles in bone formation. The term “amylin” includes native amylin, typically from a human source, and its pharmaceutically active analogues. The hormone is known to induce bone-mass formation through a variety of mechanisms. “Calcitonin-like agents” include “calcitonin,” “CGRP,” and “amylin.” See, U.S. Pat. Appln. 003015815.  
      Paratltyroid hormone. The term “parathyroid hormone” refers to parathyroid hormone, fragments or metabolites thereof and structural analogues thereof which can stimulate bone formation and increase bone mass. Also included are parathyroid hormone related peptides and active fragments and analogues of parathyroid related peptides. See, U.S. Pat. Nos. 4,086,196, 5,001,223, 6,541,450 and 6,649,657 and published PCT patent applications WO 94/01460 and WO 93/06845. Parathyroid hormone functional activity is readily determined by those skilled in the art according to standard assays. A variety of these compounds are described and referenced below, however, other parathyroid hormones will be known to those skilled in the art. Exemplary parathyroid hormones are disclosed in the references cited in U.S. Pat. Nos. 6,541,450 and 6,649,657, the entire contents of which are incorporated by reference. The utility of parathyroid hormones as medical agents in the treatment of conditions which present with low bone mass (e.g., osteoporosis) in mammals is demonstrated by the activity of the parathyroid hormones in conventional assays, including in vivo assays, receptor binding assay, cyclic AMP assays and fracture healing assays.  
      PTS893 is an analogue of the endogenous parathyroid hormone, in which certain sites of chemical instability are eliminated within N-terminal parathyroid hormone fragments by making appropriate amino acid substitutions at particular residues which results in stable and biologically active human parathyroid hormone fragments. N-terminal fragments of human parathyroid hormones include hPTH(1-34)OH muteins and hPTH(1-38)OH muteins. PTS893 comprises at least the first 27 N-terminal amino acid units of parathyroid hormone. Preferred parathyroid hormone derivatives are those comprising at least one amino acid unit replaced in one or more of the following positions of the parathyroid hormone sequence: 8-11, 13, 16-19, 21, 22, 29 to 34, particularly 8-11, 16-19, 33 and/or 34. These compounds exhibit desirable bone-forming properties both in vivo and in vitro which are equal to or above the level of natural PTH and its N-terminal fragments. See, European patent EP 0 672 057; published PCT patent application WO 94/02510; Kneissel M et al.,  Bone  28: 237-50 (March 2001); Stewart A F et al.,  J Bone Miner Res  15(8): 1517-25 (August 2000); Thomsen J S et al.,  Bone  25(5):561-9 November 1999).  
      Kits. The kits of the invention may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, e.g., to determine whether a patient is responding effectively or can respond effectively to a compound for use in treating a condition for which calcitonin, parathyroid hormone, a parathyroid hormone analogue or a combination thereof is indicated. In several embodiments, the use of the reagents can be according to the methods of the invention. In one embodiment, the reagent is a gene chip for determining the gene expression of relevant genes.  
      The following EXAMPLE is presented in order to more fully illustrate the preferred embodiments of the invention. This EXAMPLE should in no way be construed as limiting the scope of the invention, as defined by the appended claims.  
     EXAMPLE  
     Salmon Calcitonin and PTS893, Pharmacogenomics Exploratory Study in Monkeys; Microarray Gene Expression Analysis  
      Introduction and summary. The purpose of this EXAMPLE was to evaluate the gene expression changes in cynomolgus monkeys following a two-week subcutaneous treatment with salmon calcitonin (sCT) at 50 μg/animal/day and PTS893 at 5 μg/animal/day to elucidate the mechanisms of action mediating their effects as well as the identification of biomarkers of therapeutic indications. This EXAMPLE is believed to be the first analysis that globally describes the molecular mechanisms of action of salmon calcitonin and a parathyroid hormone analogue by multi-organ-gene-profiling analysis in primates. This is also believed to be the first gene profiling analysis which describes the molecular mechanisms of action of hormonal-mediated bone remodelling by salmon calcitonin and PTS893.  
      In this EXAMPLE, salmon calcitonin and PTS893 were both found to have modulating effects on genes affecting the direct, autocrine, paracrine and endocrine regulation of the mesenchymal cell functions such as transforming growth factor betas (TGF-βs), insulin-like growth factors (IGFs), bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF). Both compounds also regulate the synthesis and degradation of extracellular matrix components. Salmon calcitonin also regulates oestrogen receptor and steroidogenic factor, whereas PTS893 produced a strong up-regulation on nuclear receptors of the steroid/thyroid receptor family. These data therefore support the role of calcitonin as an anabolic agent.  
      In addition, salmon calcitonin and PTS893 also influenced some aspects of the mineralization of the extracellular matrix, since changes in amelogenin, dentin and ectonucleotide pyrophosphatases were observed.  
      In addition, PTS893 showed an effect on mediating the paracrine activation of osteoclast differentiation and activity, through cytokine and RANK ligand.  
      No significant differences in gene expression profiling were attributable to the fact of administering salmon calcitonin and PTS893 in combination, with respect to the single therapy.  
      Thus, gene profiling analysis in this EXAMPLE allowed the reconstruction of the pathways involved in calcitonin and parathyroid hormone signal transduction, triggered by protein-G-linked-receptor stimulation and their influence on cell cycle, as indicated by the changes observed in cyclins.  
      Animals. A two-week subcutaneous treatment was carried out with salmon calcitonin (sCT), PTS893 or a combination of the two, each of which were dissolved in phosphate buffered saline (PBS) containing 9% autologous serum. Solvent was used as vehicle for the control group.  
      The animals used in this analysis were cynomolgus monkeys ( Macaca fascicularis ), supplied by Centre de Recherches Primatologiques, Port Louis, Mauritius. Two animals were used per group and sex. At the beginning of the treatment period, the animals were at least 24 months old, with a body weight of approximately 3 kg. Animals were kept under standard conditions for animal welfare. Animals were examined daily for mortality, food consumption and clinical observations. Body weight was recorded once per week. The dosages were 0 μg/animal/day (as the control), 50 μg/animal/day of salmon calcitonin and 5 μg/animal/day of PTS893.  
      As shown below, clinical observations and analysis, as well as the histopathological examinations performed in this EXAMPLE, showed that salmon calcitonin administered subcutaneously at a dose of 50 μg/animal/day was well tolerated by the cynomolgus monkeys.  
      In vivo examinations. No significant histopathological changes were observed. No relevant changes were observed other than a body weight decrease ranging from 8 to 12% in the salmon calcitonin group. A decrease in food consumption was also observed, although not always consistent with the decrease in body weight.  
               TABLE 1                       Food Consumption - Males                  Control                         Day                                                                 −6   −5   −4   −3   −2   −1   1   2   3   4   5               Animal no. W62501   50   100   100   100   100   100   100   100   100   100   100       Animal no. W62502   50   100   100   100   100   100   100   100   100   100    25                                 Day                                                             6   7   8   9   10   11   12   13   14   Avg.               Animal no. W62501    75   100   100   100   100   100   100    25       91.7       Animal no. W62502   100    75    75   100   100   100    75   100   50   91.7       Both animals                                       91.7                         Salmon Calcitonin                         Day                                                                 −6   −5   −4   −3   −2   −1   1   2   3   4   5               Animal no. W62503   50   75   50   75   100   75   75   25    50   100   100       Animal no. W62504   50   75   75   75   100   75   50   25   100    75   100                                 Day                                                             6   7   8   9   10   11   12   13   14   Avg.               Animal no. W62503   75   100   100   100   100   75   75   25       70.8       Animal no. W62504   75    75    75   100   100   75   75   25   75   75.0       Both animals                                       72.9                         PTS893                         Day                                                                 −6   −5   −4   −3   −2   −1   1   2   3   4   5               Animal no. W62505   50   100   100   100    75   100   100   100   100   100   100       Animal no. W62506   50   100   100   100   100   100   100   100   100   100   100                                 Day                                                             6   7   8   9   10   11   12   13   14   Avg.               Animal no. W62505   100   100   100   100   100   100   100    50    75   87.5       Animal no. W62506   100   100   100   100   100   100   100   100   100   91.7       Both animals                                       89.6                  
 
      The animals to whom salmon calcitonin was administered presented with a decrease in body weight ranging between 8 to 12%, which can be attributed to a decrease in food consumption. An anorectic effect had previously been described for salmon calcitonin acting through amylin receptors Eiden S et al.,  J. Physiol . 541(pt3): 1041-1048 (2002); Lutz T A et al.,  Peptides  21 (2): 233-8 (2000). However, no signs of toxicity were observed here. Hormonal and lipid changes observed in this EXAMPLE are most probably related to a consequent metabolic adaptation.  
      No relevant changes in electrocardiograms (ECG) or blood pressure were observed.  
               TABLE 2                          Blood Pressure                                     Animal       Compound   Week-1   Week 2   Difference       number   Sex   administered   (mmHg)   (mmHg)   (mmHg)                                             W62501   Male   Control   121   98   −23       W62501   Male   Control   90   29   −61       W62502   Male   Control   86   107   21       W62502   Male   Control   26   34   8       W62503   Male   Salmon   135   99   −36               Calcitonin       W62503   Male   Salmon   61   40   −21               Calcitonin       W62504   Male   Salmon   102   79   −23               Calcitonin       W62504   Male   Salmon   56   35   −21               Calcitonin       W62505   Male   PTS893   76   87   11       W62505   Male   PTS893   18   22   4       W62506   Male   PTS893   106   101   −5       W62506   Male   PTS893   53   33   −20       W62551   Female   Control   96   76   −20       W62551   Female   Control   27   26   −1       W62552   Female   Control   102   93   −9       W62552   Female   Control   26   36   10       W62553   Female   Salmon   98   82   −16               Calcitonin       W62553   Female   Salmon   50   25   −25               Calcitonin       W62554   Female   Salmon   92   44   −48               Calcitonin       W62554   Female   Salmon   26   30   4               Calcitonin       W62555   Female   PTS893   92   70   −22       W62555   Female   PTS893   43   42   −1       W62556   Female   PTS893   78   87   9       W62556   Female   PTS893   24   28   4                  
 
      Blood sampling. Animals were fasted overnight before blood collection but had free access to water. Blood samples were taken from a peripheral vein. Standard haematology and clinical chemistry analysis were performed once during pretest and at the end of the treatment period. Blood samples were collected from each animal at the same intervals as described for the clinical chemistry investigations. The serum samples were deep-frozen (approximately −80° C.) until analyses for hormone determination.  
      Clinical chemistry and hormone determinations. A slight anaemia was observed in all animals of the study, including the controls. This was attributed to the repeated blood sampling and not considered to be relevant.  
               TABLE 3                       Haematology - Males                  Control                         Animal no.   W62501   W62502                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               WBC   G/l   10.0   11.1   12.9   6.1   11.2   6.3       RBC   T/l   7.3   6.5   6.4   6.8   6.5   6.2       HB   g/dl   12.9   11.9   11.7   13.1   12.3   11.9       PCV   l/l   0.44   0.40   0.44   0.42   0.41   0.41       MCV   fl   60   61   68   61   63   66       MCH   pg   17.8   18.2   18.1   19.3   19.0   19.0       MCHC   g/dl   29.8   29.6   26.8   31.5   30.1   28.9       PLAT   G/l   316   371   266   458   500   547       N   G/l   6.46   4.93   3.65   2.09   6.77   1.24       E   G/l   0.01   0.14   0.20   0.10   0.10   0.10       B   G/l   0.02   0.03   0.06   0.02   0.02   0.00       L   G/l   3.05   5.45   8.44   3.60   3.65   4.51       M   G/l   0.46   0.51   0.54   0.33   0.64   0.46                         Salmon Calcitonin                         Animal no.   W62503   W62504                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               WBC   G/l   7.7   11.8   8.0   11.5   9.5   8.8       RBC   T/l   6.3   5.9   5.6   6.9   6.0   5.4       HB   g/dl   12.6   11.7   11.2   13.6   11.5   10.3       PCV   l/l   0.40   0.39   0.39   0.43   0.37   0.36       MCV   fl   64   66   70   62   62   67       MCH   pg   20.2   19.9   20.2   19.7   19.2   19.2       MCHC   g/dl   31.4   30.3   29.0   32.0   31.3   28.7       PLAT   G/l   351   396   302   247   330   389       N   G/l   3.36   4.11   1.90   3.93   3.31   3.04       E   G/l   0.02   0.10   0.13   0.16   0.09   0.01       B   G/l   0.02   0.04   0.03   0.08   0.04   0.03       L   G/l   4.00   6.79   5.38   6.55   5.57   4.92       M   G/l   0.30   0.73   0.57   0.76   0.45   0.76                         PTS893                         Animal no.   W62505   W62506                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               WBC   G/l   10.4   8.4   8.8   9.1   15.0   11.9       RBC   T/l   7.6   6.4   6.8   6.5   5.9   5.8       HB   g/dl   13.6   11.3   11.7   13.2   11.9   11.8       PCV   l/l   0.43   0.38   0.43   0.40   0.40   0.41       MCV   fl   57   60   63   62   67   70       MCH   pg   18.0   17.7   17.3   20.4   20.2   20.3       MCHC   g/dl   31.5   29.3   27.5   33.1   30.2   29.2       PLAT   G/l   325   456   330   459   589   452       N   G/l   4.45   1.77   2.88   4.80   8.73   6.51       E   G/l   0.21   0.30   0.19   0.03   0.08   0.07       B   G/l   0.00   0.02   0.04   0.02   0.03   0.03       L   G/l   5.07   5.91   5.37   3.99   5.30   4.86       M   G/l   0.62   0.39   0.27   0.27   0.83   0.46                 d −6, d 7 and d 13 indicate day −6, day 7 and day 13 relative to the starting day of dosing             
 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
               
               
                 Haematology - Females 
               
               
                   
               
             
            
               
                 Control 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62551 
                 W62552 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 WBC 
                 pg/ml 
                 8.2 
                 13.7 
                 10.0 
                 10.1 
                 9.1 
                 10.4 
               
               
                 RBC 
                 nmol/ 
                 6.5 
                 6.2 
                 5.8 
                 6.7 
                 6.2 
                 5.8 
               
               
                   
                 l 
               
               
                 HB 
                 pg/ml 
                 12.8 
                 11.8 
                 11.3 
                 13.1 
                 11.7 
                 11.4 
               
               
                 PCV 
                 mU/l 
                 0.42 
                 0.43 
                 0.41 
                 0.42 
                 0.42 
                 0.41 
               
               
                 MCV 
                 pg/ml 
                 64 
                 69 
                 71 
                 63 
                 68 
                 70 
               
               
                 MCH 
                 ng/ml 
                 19.7 
                 19.1 
                 19.4 
                 19.5 
                 18.9 
                 19.5 
               
               
                 MCHC 
                 pg/ml 
                 30.6 
                 27.7 
                 27.4 
                 30.9 
                 27.7 
                 27.9 
               
               
                 PLAT 
                 nmol/ 
                 463 
                 445 
                 468 
                 286 
                 292 
                 275 
               
               
                   
                 l 
               
               
                 N 
                 nmol/ 
                 4.45 
                 5.86 
                 3.53 
                 6.69 
                 3.13 
                 4.23 
               
               
                   
                 l 
               
               
                 E 
                 mUI/l 
                 0.03 
                 0.13 
                 0.12 
                 0.01 
                 0.15 
                 0.19 
               
               
                 B 
                 pg/ml 
                 0.03 
                 0.07 
                 0.04 
                 0.02 
                 0.03 
                 0.03 
               
               
                 L 
                 pg/ml 
                 3.40 
                 7.09 
                 5.91 
                 3.14 
                 5.39 
                 5.34 
               
               
                 M 
                 nmol/ 
                 0.27 
                 0.51 
                 0.39 
                 0.25 
                 0.39 
                 0.59 
               
               
                   
                 l 
               
               
                   
               
            
           
           
               
            
               
                 Salmon Calcitonin 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62553 
                 W62554 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 WBC 
                 pg/ml 
                 7.0 
                 9.5 
                 12.0 
                 8.3 
                 17.0 
                 13.3 
               
               
                 RBC 
                 nmol/ 
                 6.5 
                 6.2 
                 5.2 
                 7.0 
                 6.6 
                 5.7 
               
               
                   
                 l 
               
               
                 HB 
                 pg/ml 
                 12.3 
                 11.5 
                 10.1 
                 13.8 
                 12.7 
                 11.0 
               
               
                 PCV 
                 mU/l 
                 0.40 
                 0.40 
                 0.33 
                 0.45 
                 0.44 
                 0.37 
               
               
                 MCV 
                 pg/ml 
                 61 
                 64 
                 64 
                 65 
                 68 
                 65 
               
               
                 MCH 
                 ng/ml 
                 19.1 
                 18.6 
                 19.5 
                 19.8 
                 19.4 
                 19.5 
               
               
                 MCHC 
                 pg/ml 
                 31.2 
                 29.0 
                 30.3 
                 30.6 
                 28.7 
                 29.9 
               
               
                 PLAT 
                 nmol/ 
                 549 
                 594 
                 451 
                 304 
                 356 
                 229 
               
               
                   
                 l 
               
               
                 N 
                 nmol/ 
                 3.45 
                 3.83 
                 5.41 
                 3.13 
                 9.82 
                 6.16 
               
               
                   
                 l 
               
               
                 E 
                 mUI/l 
                 0.03 
                 0.36 
                 0.73 
                 0.03 
                 0.04 
                 0.06 
               
               
                 B 
                 pg/ml 
                 0.02 
                 0.03 
                 0.03 
                 0.01 
                 0.07 
                 0.05 
               
               
                 L 
                 pg/ml 
                 3.26 
                 4.61 
                 5.18 
                 4.79 
                 6.21 
                 6.58 
               
               
                 M 
                 nmol/ 
                 0.25 
                 0.63 
                 0.69 
                 0.30 
                 0.82 
                 0.39 
               
               
                   
                 l 
               
               
                   
               
            
           
           
               
            
               
                 PTS893 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62555 
                 W62556 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 WBC 
                 pg/ml 
                 10.1 
                 18.4 
                 13.2 
                 14.3 
                 12.3 
                 10.1 
               
               
                 RBC 
                 nmol/ 
                 6.9 
                 6.2 
                 5.9 
                 6.7 
                 6.4 
                 5.9 
               
               
                   
                 l 
               
               
                 HB 
                 pg/ml 
                 13.4 
                 11.7 
                 11.3 
                 12.9 
                 12.1 
                 11.3 
               
               
                 PCV 
                 mU/l 
                 0.44 
                 0.41 
                 0.40 
                 0.43 
                 0.43 
                 0.39 
               
               
                 MCV 
                 pg/ml 
                 63 
                 67 
                 67 
                 64 
                 68 
                 66 
               
               
                 MCH 
                 ng/ml 
                 19.3 
                 18.9 
                 19.3 
                 19.3 
                 19.0 
                 19.2 
               
               
                 MCHC 
                 pg/ml 
                 30.6 
                 28.2 
                 28.6 
                 30.2 
                 28.1 
                 29.2 
               
               
                 PLAT 
                 nmol/ 
                 501 
                 525 
                 496 
                 213 
                 382 
                 309 
               
               
                   
                 l 
               
               
                 N 
                 nmol/ 
                 5.34 
                 10.8 
                 6.36 
                 9.05 
                 5.49 
                 4.18 
               
               
                   
                 l 
               
               
                 E 
                 mUI/l 
                 0.00 
                 0.12 
                 0.21 
                 0.26 
                 0.49 
                 0.29 
               
               
                 B 
                 pg/ml 
                 0.00 
                 0.06 
                 0.03 
                 0.03 
                 0.04 
                 0.04 
               
               
                 L 
                 pg/ml 
                 3.92 
                 6.29 
                 5.81 
                 4.40 
                 5.87 
                 5.21 
               
               
                 M 
                 nmol/ 
                 0.80 
                 1.12 
                 0.82 
                 0.54 
                 0.44 
                 0.37 
               
               
                   
                 l 
               
               
                   
               
               
                   d −8, d 7 and d 13 indicate day −8, day 7 and day 13 relative to the starting day of dosing    
               
            
           
         
       
     
      Among the standard clinical chemistry tests performed, slight to moderate decreases in phosphorus and/or magnesium and a moderate to marked decrease in triglycerides were seen in the groups administered salmon calcitonin and PTS893.  
               TABLE 5                       Clinical Chemistry - Males                  Control                         Animal no.   W62501   W62502                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               Na+   mmol/l   154   151   153   152   153   148       K+   mmol/l   4.05   5.31   4.26   4.09   4.05   4.51       Cl−   mmol/l   109   113   108   107   110   111       Ca++   mmol/l   2.57   2.47   2.69   2.72   2.52   2.75       I.PHOS   mmol/l   2.21   1.93   2.76   1.88   1.69   1.99       Mg++   mmol/l   1.09   0.91   0.95   0.88   0.79   1.14       GLUC   mmol/l   3.85   4.51   4.68   3.44   5.30   6.13       UREA   mmol/l   9.7   4.9   5.0   7.6   6.1   5.2       CREAT   μmol/l   85   60   75   65   55   57       TOT.BIL.   μmol/l   6.0   2.0   2.0   7.0   3.0   4.0       PROT   g/l   89   80   88   90   83   85       A/G       1.89   1.57   1.45   1.62   1.53   1.50       CHOL   mmol/l   3.30   3.20   3.50   3.30   3.40   3.10       HDL-CHOL   mmol/l   1.49   1.45   1.70   1.54   1.45   1.49       LDL-CHOL   mmol/l   1.63   1.62   1.84   1.56   1.93   1.49       TRIG   mmol/l   0.94   0.36   0.43   0.65   0.36   0.45       ALP   IU/l   1559   1241   1313   1463   1423   1493       BAP-E   IU/l   543   439   457   452   476   464       ASAT   IU/l   22   22   25   30   26   26       ALAT   IU/l   22   32   30   29   41   37       CK   IU/l   150   45   127   74   67   102       LDH   IU/l   392   585   549   421   518   592       GGT   IU/l   128   92   111   89   71   75       ALB   %   65   61   59   62   61   60       A1-GLOB   %   1.90   2.70   2.50   1.90   2.10   2.30       A2-GLOB   %   7.60   8.30   7.90   8.20   8.90   8.50       B-GLOB   %   16   18   19   18   19   19       G-GLOB   %   9.2   9.9   10.9   9.6   9.3   10.2       ALB   g/l   58   49   52   56   50   51       A1-GLOB   g/l   1.70   2.20   2.20   1.70   1.70   2.00       A2-GLOB   g/l   6.80   6.60   7.00   7.40   7.40   7.20       B-GLOB   g/l   14   14   17   17   16   16       G-GLOB   g/l   8.2   7.9   9.6   8.6   7.7   8.7                         Salmon Calcitonin                         Animal no.   W62503   W62504                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               Na+   mmol/l   151   145   148   154   142   144       K+   mmol/l   4.24   4.90   4.34   4.85   5.15   4.48       Cl−   mmol/l   107   104   104   113   106   101       Ca++   mmol/l   2.66   2.68   2.91   2.71   2.54   2.73       I.PHOS   mmol/l   2.05   1.67   2.06   2.10   1.73   1.94       Mg++   mmol/l   0.97   0.68   0.73   0.99   0.71   0.72       GLUC   mmol/l   3.57   3.58   4.29   3.70   4.98   6.19       UREA   mmol/l   7.9   1.3   2.9   6.6   3.3   2.9       CREAT   μmol/l   78   57   62   64   50   56       TOT.BIL.   μmol/l   5.0   2.0   1.0   3.0   2.0   2.0       PROT   g/l   87   82   87   91   83   89       A/G       1.76   1.68   1.42   1.42   1.26   1.05       CHOL   mmol/l   3.30   3.60   3.70   3.80   3.90   3.40       HDL-CHOL   mmol/l   1.49   2.09   2.44   1.46   1.48   1.39       LDL-CHOL   mmol/l   1.21   1.28   1.26   1.87   2.51   1.83       TRIG   mmol/l   0.96   0.24   0.27   0.92   0.22   0.68       ALP   IU/l   1488   1023   1226   857   587   626       BAP-E   IU/l   508   363   302   311   188   180       ASAT   IU/l   28   31   28   24   17   24       ALAT   IU/l   38   39   43   48   24   31       CK   IU/l   124   56   119   75   45   173       LDH   IU/l   439   400   427   356   384   519       GGT   IU/l   105   80   75   121   75   69       ALB   %   64   63   59   59   56   51       A1-GLOB   %   1.60   2.00   2.40   1.90   2.80   3.60       A2-GLOB   %   8.00   8.80   8.80   8.70   8.70   7.80       B-GLOB   %   18   18   20   19   21   24       G-GLOB   %   8.3   8.5   9.7   12.0   12.1   13.6       ALB   g/l   56   51   51   54   46   46       A1-GLOB   g/l   1.40   1.60   2.10   1.70   2.30   3.20       A2-GLOB   g/l   7.00   7.20   7.70   7.90   7.20   6.90       B-GLOB   g/l   16   15   18   17   17   21       G-GLOB   g/l   7.2   7.0   8.4   10.9   10.0   12.1                         PTS893                         Animal no.   W62505   W62506                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               Na+   mmol/l   151   151   152   151   149   149       K+   mmol/l   5.13   4.00   4.27   4.72   4.76   4.12       Cl−   mmol/l   110   107   110   112   106   106       Ca++   mmol/l   2.81   2.39   2.59   2.64   2.45   2.51       I.PHOS   mmol/l   2.59   1.68   2.22   2.12   1.12   1.77       Mg++   mmol/l   1.04   0.71   0.77   0.97   0.70   0.76       GLUC   mmol/l   5.09   4.76   5.42   3.88   5.26   4.96       UREA   mmol/l   11.6   3.7   6.4   15.0   4.9   5.8       CREAT   μmol/l   86   66   79   77   63   70       TOT.BIL.   μmol/l   5.0   2.0   1.0   7.0   2.0   1.0       PROT   g/l   81   74   81   88   86   89       A/G       1.89   1.70   1.76   1.58   1.28   1.40       CHOL   mmol/l   3.20   3.30   3.10   2.50   2.50   2.60       HDL-CHOL   mmol/l   1.49   1.49   1.61   1.24   1.25   1.38       LDL-CHOL   mmol/l   1.39   1.73   1.51   1.27   1.22   1.38       TRIG   mmol/l   0.96   0.30   0.63   0.49   0.39   0.35       ALP   IU/l   1703   1494   1768   1414   1363   1486       BAP-E   IU/l   523   532   564   445   423   497       ASAT   IU/l   24   18   24   25   27   29       ALAT   IU/l   32   30   27   23   19   20       CK   IU/l   111   82   148   86   73   125       LDH   IU/l   367   400   528   354   432   464       GGT   IU/l   133   99   105   112   85   91       ALB   %   66   63   64   61   56   59       A1-GLOB   %   2.20   2.80   2.60   2.40   3.60   2.80       A2-GLOB   %   8.80   8.90   8.70   7.30   8.30   7.50       B-GLOB   %   17   18   19   19   22   20       G-GLOB   %   6.9   6.9   6.3   9.8   10.5   10.9       ALB   g/l   53   47   52   54   48   52       A1-GLOB   g/l   1.80   2.10   2.10   2.10   3.10   2.50       A2-GLOB   g/l   7.10   6.60   7.10   6.40   7.10   6.70       B-GLOB   g/l   14   14   15   17   19   18       G-GLOB   g/l   5.6   5.1   5.1   8.6   9.0   9.7                 d −6, d 7 and d 13 indicate day −6, day 7 and day 13 relative to the starting day of dosing             
 
     
       
         
           
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
               
               
                 Clinical Chemistry - Females 
               
               
                   
               
             
            
               
                 Control 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62551 
                 W62552 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 Na+ 
                 mmol/l 
                 152 
                 148 
                 155 
                 148 
                 150 
                 148 
               
               
                 K+ 
                 mmol/l 
                 4.16 
                 4.23 
                 4.92 
                 3.82 
                 4.11 
                 5.27 
               
               
                 Cl− 
                 mmol/l 
                 110 
                 105 
                 111 
                 109 
                 106 
                 108 
               
               
                 Ca++ 
                 mmol/l 
                 2.64 
                 2.61 
                 2.61 
                 2.48 
                 2.44 
                 1.80 
               
               
                 I.PHOS 
                 mmol/l 
                 1.98 
                 2.61 
                 2.28 
                 1.84 
                 1.98 
                 1.84 
               
               
                 Mg++ 
                 mmol/l 
                 1.00 
                 0.97 
                 1.03 
                 0.88 
                 0.84 
                 0.31 
               
               
                 GLUC 
                 mmol/l 
                 3.65 
                 8.39 
                 3.86 
                 2.79 
                 3.86 
                 3.60 
               
               
                 UREA 
                 mmol/l 
                 11.0 
                 8.3 
                 8.2 
                 11.3 
                 6.9 
                 6.3 
               
               
                 CREAT 
                 μmol/l 
                 73 
                 77 
                 62 
                 67 
                 60 
                 50 
               
               
                 TOT.BIL. 
                 μmol/l 
                 4.00 
                 2.00 
                 3.00 
                 5.00 
                 1.00 
                 2.00 
               
               
                 PROT 
                 g/l 
                 85 
                 80 
                 80 
                 83 
                 83 
                 77 
               
               
                 A/G 
                   
                 1.77 
                 1.67 
                 1.55 
                 1.68 
                 1.39 
                 1.27 
               
               
                 CHOL 
                 mmol/l 
                 3.20 
                 2.80 
                 3.00 
                 3.70 
                 3.40 
                 3.50 
               
               
                 HDL-CHOL 
                 mmol/l 
                 1.63 
                 1.44 
                 1.49 
                 1.75 
                 1.82 
                 1.80 
               
               
                 LDL-CHOL 
                 mmol/l 
                 1.55 
                 1.25 
                 1.90 
                 1.57 
                 1.28 
                 1.66 
               
               
                 TRIG 
                 mmol/l 
                 0.64 
                 0.54 
                 0.57 
                 0.83 
                 0.48 
                 0.50 
               
               
                 ALP 
                 IU/l 
                 1037 
                 1088 
                 1187 
                 1332 
                 1298 
                 1182 
               
               
                 BAP-E 
                 IU/l 
                 310 
                 369 
                 346 
                 432 
                 419 
                 379 
               
               
                 ASAT 
                 IU/l 
                 27 
                 33 
                 31 
                 21 
                 22 
                 23 
               
               
                 ALAT 
                 IU/l 
                 44 
                 52 
                 46 
                 16 
                 19 
                 20 
               
               
                 CK 
                 IU/l 
                 69 
                 169 
                 81 
                 83 
                 68 
                 87 
               
               
                 LDH 
                 IU/l 
                 420 
                 520 
                 481 
                 474 
                 471 
                 516 
               
               
                 GGT 
                 IU/l 
                 104 
                 95 
                 102 
                 84 
                 67 
                 66 
               
               
                 ALB 
                 % 
                 64 
                 63 
                 61 
                 63 
                 58 
                 56 
               
               
                 A1-GLOB 
                 % 
                 1.90 
                 2.60 
                 3.40 
                 2.00 
                 2.60 
                 3.50 
               
               
                 A2-GLOB 
                 % 
                 8.00 
                 7.60 
                 7.70 
                 7.00 
                 8.10 
                 7.70 
               
               
                 B-GLOB 
                 % 
                 17 
                 18 
                 18 
                 15 
                 18 
                 18 
               
               
                 G-GLOB 
                 % 
                 9.4 
                 9.2 
                 9.9 
                 12.9 
                 13.2 
                 14.8 
               
               
                 ALB 
                 g/l 
                 54 
                 50 
                 49 
                 52 
                 48 
                 43 
               
               
                 A1-GLOB 
                 g/l 
                 1.60 
                 2.10 
                 2.70 
                 1.70 
                 2.20 
                 2.70 
               
               
                 A2-GLOB 
                 g/l 
                 6.80 
                 6.10 
                 6.20 
                 5.80 
                 6.70 
                 5.90 
               
               
                 B-GLOB 
                 g/l 
                 14 
                 14 
                 15 
                 13 
                 15 
                 14 
               
               
                 G-GLOB 
                 g/l 
                 8.0 
                 7.4 
                 7.9 
                 10.7 
                 11.0 
                 11.4 
               
               
                   
               
            
           
           
               
            
               
                 Salmon Calcitonin 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62553 
                 W62554 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 Na+ 
                 mmol/l 
                 145 
                 147 
                 147 
                 145 
                 143 
                 147 
               
               
                 K+ 
                 mmol/l 
                 3.51 
                 3.73 
                 4.62 
                 3.89 
                 4.07 
                 4.95 
               
               
                 Cl− 
                 mmol/l 
                 106 
                 104 
                 107 
                 100 
                 96 
                 107 
               
               
                 Ca++ 
                 mmol/l 
                 2.62 
                 2.77 
                 2.57 
                 2.73 
                 2.91 
                 2.68 
               
               
                 I.PHOS 
                 mmol/l 
                 1.62 
                 1.48 
                 1.81 
                 1.97 
                 1.75 
                 1.83 
               
               
                 Mg++ 
                 mmol/l 
                 0.87 
                 0.63 
                 0.76 
                 0.91 
                 0.77 
                 0.80 
               
               
                 GLUC 
                 mmol/l 
                 3.84 
                 4.88 
                 4.98 
                 4.11 
                 5.31 
                 4.04 
               
               
                 UREA 
                 mmol/l 
                 10.3 
                 6.6 
                 5.0 
                 10.0 
                 6.3 
                 5.9 
               
               
                 CREAT 
                 μmol/l 
                 81 
                 71 
                 61 
                 88 
                 77 
                 65 
               
               
                 TOT.BIL. 
                 μmol/l 
                 3.00 
                 2.00 
                 2.00 
                 6.00 
                 5.00 
                 2.00 
               
               
                 PROT 
                 g/l 
                 88 
                 90 
                 80 
                 91 
                 95 
                 83 
               
               
                 A/G 
                   
                 1.46 
                 1.45 
                 1.30 
                 1.48 
                 1.42 
                 1.26 
               
               
                 CHOL 
                 mmol/l 
                 2.70 
                 2.80 
                 2.20 
                 3.30 
                 4.00 
                 3.00 
               
               
                 HDL-CHOL 
                 mmol/l 
                 1.04 
                 1.11 
                 0.96 
                 1.46 
                 1.99 
                 1.66 
               
               
                 LDL-CHOL 
                 mmol/l 
                 1.61 
                 1.51 
                 1.46 
                 1.13 
                 1.93 
                 1.42 
               
               
                 TRIG 
                 mmol/l 
                 0.79 
                 0.25 
                 0.39 
                 0.88 
                 0.30 
                 0.38 
               
               
                 ALP 
                 IU/l 
                 1197 
                 965 
                 842 
                 1132 
                 877 
                 890 
               
               
                 BAP-E 
                 IU/l 
                 416 
                 326 
                 304 
                 344 
                 325 
                 294 
               
               
                 ASAT 
                 IU/l 
                 24 
                 21 
                 25 
                 20 
                 18 
                 20 
               
               
                 ALAT 
                 IU/l 
                 21 
                 24 
                 19 
                 19 
                 14 
                 19 
               
               
                 CK 
                 IU/l 
                 99 
                 72 
                 107 
                 76 
                 64 
                 77 
               
               
                 LDH 
                 IU/l 
                 286 
                 423 
                 429 
                 319 
                 372 
                 363 
               
               
                 GGT 
                 IU/l 
                 88 
                 63 
                 54 
                 82 
                 72 
                 62 
               
               
                 ALB 
                 % 
                 59 
                 59 
                 57 
                 60 
                 59 
                 56 
               
               
                 A1-GLOB 
                 % 
                 2.70 
                 2.70 
                 3.10 
                 2.20 
                 2.20 
                 3.10 
               
               
                 A2-GLOB 
                 % 
                 6.50 
                 6.10 
                 6.80 
                 8.00 
                 7.70 
                 7.80 
               
               
                 B-GLOB 
                 % 
                 21 
                 23 
                 21 
                 15 
                 17 
                 17 
               
               
                 G-GLOB 
                 % 
                 10.8 
                 8.6 
                 12.4 
                 14.9 
                 14.6 
                 16.3 
               
               
                 ALB 
                 g/l 
                 52 
                 54 
                 45 
                 54 
                 56 
                 46 
               
               
                 A1-GLOB 
                 g/l 
                 2.40 
                 2.40 
                 2.50 
                 2.00 
                 2.10 
                 2.60 
               
               
                 A2-GLOB 
                 g/l 
                 5.70 
                 5.50 
                 5.40 
                 7.30 
                 7.30 
                 6.50 
               
               
                 B-GLOB 
                 g/l 
                 18 
                 21 
                 17 
                 14 
                 16 
                 14 
               
               
                 G-GLOB 
                 g/l 
                 9.5 
                 7.7 
                 9.9 
                 13.6 
                 13.9 
                 13.5 
               
               
                   
               
            
           
           
               
            
               
                 PTS893 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62555 
                 W62556 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 Na+ 
                 mmol/l 
                 153 
                 151 
                 152 
                 150 
                 148 
                 149 
               
               
                 K+ 
                 mmol/l 
                 4.82 
                 4.54 
                 4.63 
                 3.85 
                 3.81 
                 4.31 
               
               
                 Cl− 
                 mmol/l 
                 107 
                 109 
                 111 
                 108 
                 107 
                 114 
               
               
                 Ca++ 
                 mmol/l 
                 2.77 
                 2.61 
                 2.20 
                 2.64 
                 2.62 
                 2.35 
               
               
                 I.PHOS 
                 mmol/l 
                 2.11 
                 1.31 
                 1.51 
                 2.10 
                 1.60 
                 1.50 
               
               
                 Mg++ 
                 mmol/l 
                 0.96 
                 0.65 
                 0.59 
                 0.90 
                 0.74 
                 0.66 
               
               
                 GLUC 
                 mmol/l 
                 3.57 
                 4.18 
                 3.59 
                 3.22 
                 4.45 
                 3.52 
               
               
                 UREA 
                 mmol/l 
                 8.2 
                 8.7 
                 6.3 
                 8.4 
                 6.6 
                 6.8 
               
               
                 CREAT 
                 μmol/l 
                 77 
                 62 
                 58 
                 68 
                 63 
                 58 
               
               
                 TOT.BIL. 
                 μmol/l 
                 5.00 
                 1.00 
                 2.00 
                 5.00 
                 2.00 
                 2.00 
               
               
                 PROT 
                 g/l 
                 89 
                 87 
                 78 
                 84 
                 83 
                 76 
               
               
                 A/G 
                   
                 1.64 
                 1.62 
                 1.65 
                 1.84 
                 1.78 
                 1.50 
               
               
                 CHOL 
                 mmol/l 
                 2.90 
                 2.70 
                 2.80 
                 2.70 
                 2.40 
                 2.70 
               
               
                 HDL-CHOL 
                 mmol/l 
                 1.31 
                 1.48 
                 1.51 
                 1.12 
                 0.99 
                 1.25 
               
               
                 LDL-CHOL 
                 mmol/l 
                 1.69 
                 1.12 
                 1.71 
                 1.62 
                 1.28 
                 1.58 
               
               
                 TRIG 
                 mmol/l 
                 0.59 
                 0.27 
                 0.25 
                 0.67 
                 0.34 
                 0.47 
               
               
                 ALP 
                 IU/l 
                 1535 
                 1223 
                 1332 
                 1638 
                 1307 
                 1313 
               
               
                 BAP-E 
                 IU/l 
                 457 
                 350 
                 426 
                 456 
                 390 
                 400 
               
               
                 ASAT 
                 IU/l 
                 23 
                 18 
                 25 
                 24 
                 20 
                 25 
               
               
                 ALAT 
                 IU/l 
                 35 
                 25 
                 32 
                 33 
                 19 
                 21 
               
               
                 CK 
                 IU/l 
                 84 
                 65 
                 175 
                 63 
                 144 
                 172 
               
               
                 LDH 
                 IU/l 
                 468 
                 465 
                 557 
                 309 
                 313 
                 358 
               
               
                 GGT 
                 IU/l 
                 85 
                 71 
                 70 
                 103 
                 85 
                 83 
               
               
                 ALB 
                 % 
                 62 
                 62 
                 62 
                 65 
                 64 
                 60 
               
               
                 A1-GLOB 
                 % 
                 2.30 
                 2.50 
                 2.50 
                 1.90 
                 2.10 
                 2.70 
               
               
                 A2-GLOB 
                 % 
                 7.50 
                 8.00 
                 8.30 
                 7.50 
                 7.50 
                 8.10 
               
               
                 B-GLOB 
                 % 
                 18 
                 19 
                 18 
                 17 
                 17 
                 20 
               
               
                 G-GLOB 
                 % 
                 9.7 
                 8.4 
                 8.7 
                 8.8 
                 9.1 
                 8.7 
               
               
                 ALB 
                 g/l 
                 55 
                 54 
                 49 
                 55 
                 53 
                 46 
               
               
                 A1-GLOB 
                 g/l 
                 2.10 
                 2.20 
                 2.00 
                 1.60 
                 1.70 
                 2.10 
               
               
                 A2-GLOB 
                 g/l 
                 6.70 
                 7.00 
                 6.50 
                 6.30 
                 6.20 
                 6.20 
               
               
                 B-GLOB 
                 g/l 
                 16 
                 17 
                 14 
                 14 
                 14 
                 16 
               
               
                 G-GLOB 
                 g/l 
                 8.6 
                 7.3 
                 6.8 
                 7.4 
                 7.6 
                 6.6 
               
               
                   
               
               
                   d −8, d 7 and d 13 indicate day −8, day 7 and day 13 relative to the starting day of dosing    
               
            
           
         
       
     
      No relevant changes were observed in the standard urinalysis tests performed.  
               TABLE 7                       Urinary analysis - Males                  Control                         Animal no.   W62501   W62502                                             Test   Units   −6   −5   13   −6   −5   13               VOLUME   ml   15   10   77   22   130   30       CREAT   μmol/l   18000   17000   5460   7920   2480   5160       NTx   nM BCE   —   9954   3425   —   11979   3167       CTx   μg/l   —   21592   6810   —   27169   5323       D-PYR   nmol/l   —   2345   1110   —   2904   1461       LDH   IU/L   6.0       nd   8.0       8.0       NAG   IU/l   3.5       1.5   3.2       1.6       Na+   mmol/l   163       43   87       77       K+   mmol/l   258       67   125       75       Cl−   mmol/l   132       43   52       59       Ca2+   mmol/l   5.15       16.80   15.95       15.50       I.PHOS   mmol/l   11.10       1.05   11.30       8.90       Mg2+   mmol/l   2.75       7.50   7.85       6.25       Na/Crea   mM/mM   9.10       7.90   11.00       14.90       K/Crea   mM/mM   14.30       12.20   15.80       14.50       Cl/Crea   mM/mM   7.40       7.90   6.50       11.40       Ca/Crea   mM/mM   0.29       3.08   2.01       3.00       Pho/Crea   mM/mM   0.62       0.19   1.43       1.73       Mg/Crea   mM/mM   0.20       1.40   1.00       1.20       LDH/crea   IU/mM   0.33       nd   1.01       1.55       NAG/crea   IU/mM   0.19       0.28   0.40       0.31       NTx/Crea   nME/mM       586   627       4830   614       CTx/Crea   μg/μm.       1270   1247       10955   1032       Pyr/Crea   nM/mM       138   203       1171   283                         Salmon Calcitonin                         Animal no.   W62503   W62504                                             Test   Units   −6   −5   13   −6   −5   13               VOLUME   ml   62   38   68   37   10   54       CREAT   μmol/l   4300   7840   4620   13600   17360   4400       NTx   nM BCE   —   6023   5186   —   16067   3790       CTx   μg/l   —   11618   10088   —   26370   6130       D-PYR   nmol/l   —   1733   1083   —   5113   1476       LDH   IU/L   9.0       7.0   13.0       17.0       NAG   IU/l   2.7       1.4   4.2       7.2       Na+   mmol/l   22       14   119       15       K+   mmol/l   65       78   134       76       Cl−   mmol/l   10       55   64       68       Ca2+   mmol/l   0.90       18.25   3.70       23.40       I.PHOS   mmol/l   4.35       2.50   5.33       3.00       Mg2+   mmol/l   1.40       7.05   7.55       9.80       Na/Crea   mM/mM   5.20       3.10   8.70       3.40       K/Crea   mM/mM   15.10       16.90   9.90       17.20       Cl/Crea   mM/mM   2.20       11.80   4.70       15.30       Ca/Crea   mM/mM   0.21       3.95   0.27       5.32       Pho/Crea   mM/mM   1.01       0.54   0.39       0.68       Mg/Crea   mM/mM   0.30       1.50   0.60       2.20       LDH/crea   IU/mM   2.09       1.52   0.96       3.86       NAG/crea   IU/mM   0.63       0.30   0.31       1.64       NTx/Crea   nME/mM       768   1123       926   861       CTx/Crea   μg/μm.       1482   2184       1519   1393       Pyr/Crea   nM/mM       221   234       295   336                         PTS893                         Animal no.   W62505   W62506                                             Test   Units   −6   −5   13   −6   −5   13               VOLUME   ml   14   14   48   58   34   130       CREAT   μmol/l   16160   16160   7840   9940   16120   3840       NTx   nM BCE   —   5403   4871   —   8757   2102       CTx   μg/l   —   11865   9365   —   20108   3705       D-PYR   nmol/l   —   1660   1676   —   2278   782       LDH   IU/L   7.0       14.0   9.0       19.0       NAG   IU/l   23.4       2.9   7.1       2.6       Na+   mmol/l   174       111   59       35       K+   mmol/l   86       107   125       69       Cl−   mmol/l   22       117   50       48       Ca2+   mmol/l   5.10       7.55   3.50       13.10       I.PHOS   mmol/l   74.40       0.10   3.86       0.17       Mg2+   mmol/l   11.25       8.70   2.95       5.25       Na/Crea   mM/mM   10.80       14.10   6.00       9.10       K/Crea   mM/mM   5.30       13.60   12.60       17.90       Cl/Crea   mM/mM   1.40       15.00   5.00       12.60       Ca/Crea   mM/mM   0.32       0.96   0.35       3.41       Pho/Crea   mM/mM   4.60       0.01   0.39       0.04       Mg/Crea   mM/mM   0.70       1.10   0.30       1.40       LDH/crea   IU/mM   0.43       1.79   0.91       4.95       NAG/crea   IU/mM   1.45       0.37   0.71       0.68       NTx/Crea   nME/mM       334   621       543   547       CTx/Crea   μg/μm.       734   1195       1247   965       Pyr/Crea   nM/mM       103   214       141   204                 d −6, d −5 and d 13 indicate day −6, day −5 and day 13 relative to the starting day of dosing             
 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
               
               
                 Urinary analysis - Females 
               
               
                   
               
             
            
               
                 Control 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62551 
                 W62552 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 −8 
                 −7 
                 13 
                 −8 
                 −7 
                 13 
               
               
                   
               
               
                 VOLUME 
                 ml 
                 21 
                 21 
                 43 
                 18 
                 53 
                 53 
               
               
                 CREAT 
                 μmol/l 
                 16420 
                 16420 
                 9560 
                 14300 
                 6700 
                 5380 
               
               
                 NTx 
                 nM BCE 
                 — 
                 9248 
                 7824 
                 — 
                 5053 
                 4695 
               
               
                 CTx 
                 μg/l 
                 — 
                 19280 
                 17916 
                 — 
                 12014 
                 10557 
               
               
                 D-PYR 
                 nmol/l 
                 — 
                 2500 
                 2748 
                 — 
                 1397 
                 2159 
               
               
                 LDH 
                 IU/L 
                 10.0 
                   
                 15.0 
                 9.0 
                   
                 25.0 
               
               
                 NAG 
                 IU/l 
                 19.2 
                   
                 4.2 
                 10.3 
                   
                 3.5 
               
               
                 Na+ 
                 mmol/l 
                 110 
                   
                 44 
                 140 
                   
                 64 
               
               
                 K+ 
                 mmol/l 
                 82 
                   
                 122 
                 124 
                   
                 87 
               
               
                 Cl− 
                 mmol/l 
                 24 
                   
                 73 
                 72 
                   
                 56 
               
               
                 Ca2+ 
                 mmol/l 
                 2.90 
                   
                 16.10 
                 11.90 
                   
                 19.50 
               
               
                 I.PHOS 
                 mmol/l 
                 88.2 
                   
                 7.7 
                 20.3 
                   
                 3.5 
               
               
                 Mg2+ 
                 mmol/l 
                 2.35 
                   
                 7.20 
                 9.00 
                   
                 5.45 
               
               
                 Na/Crea 
                 mM/mM 
                 6.70 
                   
                 4.60 
                 9.80 
                   
                 11.90 
               
               
                 K/Crea 
                 mM/mM 
                 5.00 
                   
                 12.80 
                 8.70 
                   
                 16.20 
               
               
                 Cl/Crea 
                 mM/mM 
                 1.50 
                   
                 7.60 
                 5.10 
                   
                 10.50 
               
               
                 Ca/Crea 
                 mM/mM 
                 0.18 
                   
                 1.68 
                 0.83 
                   
                 3.63 
               
               
                 Pho/Crea 
                 mM/mM 
                 5.37 
                   
                 0.81 
                 1.42 
                   
                 0.64 
               
               
                 Mg/Crea 
                 mM/mM 
                 0.10 
                   
                 0.80 
                 0.60 
                   
                 1.00 
               
               
                 LDH/crea 
                 IU/mM 
                 0.61 
                   
                 1.57 
                 0.63 
                   
                 4.65 
               
               
                 NAG/crea 
                 IU/mM 
                 1.17 
                   
                 0.44 
                 0.72 
                   
                 0.65 
               
               
                 NTx/Crea 
                 nME/mM 
                   
                 563 
                 818 
                   
                 754 
                 873 
               
               
                 CTx/Crea 
                 μg/μm. 
                   
                 1174 
                 1874 
                   
                 1793 
                 1962 
               
               
                 Pyr/Crea 
                 nM/mM 
                   
                 152 
                 288 
                   
                 209 
                 401 
               
               
                   
               
            
           
           
               
            
               
                 Salmon Calcitonin 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62553 
                 W62554 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 −8 
                 −7 
                 13 
                 −8 
                 −7 
                 13 
               
               
                   
               
               
                 VOLUME 
                 ml 
                 11 
                 58 
                 67 
                 32 
                 14 
                 49 
               
               
                 CREAT 
                 μmol/l 
                 10780 
                 6920 
                 4800 
                 11260 
                 13380 
                 4200 
               
               
                 NTx 
                 nM BCE 
                 — 
                 4624 
                 3465 
                 — 
                 7393 
                 2812 
               
               
                 CTx 
                 μg/l 
                 — 
                 6983 
                 5392 
                 — 
                 13411 
                 5631 
               
               
                 D-PYR 
                 nmol/l 
                 — 
                 2762 
                 1644 
                 — 
                 2016 
                 1110 
               
               
                 LDH 
                 IU/L 
                 14.0 
                   
                 6.0 
                 6.0 
                   
                 36.0 
               
               
                 NAG 
                 IU/l 
                 10.2 
                   
                 2.8 
                 1.2 
                   
                 2.7 
               
               
                 Na+ 
                 mmol/l 
                 98 
                   
                 40 
                 156 
                   
                 32 
               
               
                 K+ 
                 mmol/l 
                 104 
                   
                 53 
                 172 
                   
                 57 
               
               
                 Cl− 
                 mmol/l 
                 31 
                   
                 63 
                 156 
                   
                 65 
               
               
                 Ca2+ 
                 mmol/l 
                 3.00 
                   
                 17.55 
                 3.50 
                   
                 12.70 
               
               
                 I.PHOS 
                 mmol/l 
                 25.4 
                   
                 5.1 
                 10.8 
                   
                 5.8 
               
               
                 Mg2+ 
                 mmol/l 
                 3.35 
                   
                 5.40 
                 3.80 
                   
                 4.85 
               
               
                 Na/Crea 
                 mM/mM 
                 9.10 
                   
                 8.30 
                 13.90 
                   
                 7.60 
               
               
                 K/Crea 
                 mM/mM 
                 9.60 
                   
                 11.10 
                 15.20 
                   
                 13.50 
               
               
                 Cl/Crea 
                 mM/mM 
                 2.90 
                   
                 13.20 
                 13.80 
                   
                 15.40 
               
               
                 Ca/Crea 
                 mM/mM 
                 0.28 
                   
                 3.66 
                 0.31 
                   
                 3.02 
               
               
                 Pho/Crea 
                 mM/mM 
                 2.35 
                   
                 1.05 
                 0.96 
                   
                 1.38 
               
               
                 Mg/Crea 
                 mM/mM 
                 0.30 
                   
                 1.10 
                 0.30 
                   
                 1.20 
               
               
                 LDH/crea 
                 IU/mM 
                 1.30 
                   
                 1.25 
                 0.53 
                   
                 8.57 
               
               
                 NAG/crea 
                 IU/mM 
                 0.95 
                   
                 0.58 
                 0.11 
                   
                 0.64 
               
               
                 NTx/Crea 
                 nME/mM 
                   
                 668 
                 722 
                   
                 553 
                 670 
               
               
                 CTx/Crea 
                 μg/μm. 
                   
                 1009 
                 1123 
                   
                 1002 
                 1341 
               
               
                 Pyr/Crea 
                 nM/mM 
                   
                 399 
                 343 
                   
                 151 
                 264 
               
               
                   
               
            
           
           
               
            
               
                 PTS893 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62555 
                 W62556 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 −8 
                 −7 
                 13 
                 −8 
                 −7 
                 13 
               
               
                   
               
               
                 VOLUME 
                 ml 
                 14 
                 15 
                 52 
                 39 
                 69 
                 42 
               
               
                 CREAT 
                 μmol/l 
                 19160 
                 18240 
                 5620 
                 14060 
                 7600 
                 8060 
               
               
                 NTx 
                 nM BCE 
                 — 
                 10499 
                 2514 
                 — 
                 4818 
                 5679 
               
               
                 CTx 
                 μg/l 
                 — 
                 21919 
                 3813 
                 — 
                 8877 
                 11236 
               
               
                 D-PYR 
                 nmol/l 
                 — 
                 2963 
                 1356 
                 — 
                 1377 
                 2036 
               
               
                 LDH 
                 IU/L 
                 11.0 
                   
                 10.0 
                 18.0 
                   
                 9.0 
               
               
                 NAG 
                 IU/l 
                 0.5 
                   
                 1.2 
                 5.9 
                   
                 5.1 
               
               
                 Na+ 
                 mmol/l 
                 145 
                   
                 71 
                 118 
                   
                 146 
               
               
                 K+ 
                 mmol/l 
                 302 
                   
                 150 
                 164 
                   
                 70 
               
               
                 Cl− 
                 mmol/l 
                 119 
                   
                 101 
                 53 
                   
                 133 
               
               
                 Ca2+ 
                 mmol/l 
                 11.50 
                   
                 20.05 
                 6.60 
                   
                 12.35 
               
               
                 I.PHOS 
                 mmol/l 
                 0.2 
                   
                 0.1 
                 7.6 
                   
                 2.9 
               
               
                 Mg2+ 
                 mmol/l 
                 7.35 
                   
                 6.90 
                 4.00 
                   
                 5.90 
               
               
                 Na/Crea 
                 mM/mM 
                 7.60 
                   
                 12.60 
                 8.40 
                   
                 18.10 
               
               
                 K/Crea 
                 mM/mM 
                 15.80 
                   
                 26.80 
                 11.70 
                   
                 8.60 
               
               
                 Cl/Crea 
                 mM/mM 
                 6.20 
                   
                 18.00 
                 3.70 
                   
                 16.50 
               
               
                 Ca/Crea 
                 mM/mM 
                 0.60 
                   
                 3.57 
                 0.47 
                   
                 1.53 
               
               
                 Pho/Crea 
                 mM/mM 
                 0.01 
                   
                 0.02 
                 0.54 
                   
                 0.36 
               
               
                 Mg/Crea 
                 mM/mM 
                 0.40 
                   
                 1.20 
                 0.30 
                   
                 0.70 
               
               
                 LDH/crea 
                 IU/mM 
                 0.57 
                   
                 1.78 
                 1.28 
                   
                 1.12 
               
               
                 NAG/crea 
                 IU/mM 
                 0.03 
                   
                 0.21 
                 0.42 
                   
                 0.63 
               
               
                 NTx/Crea 
                 nME/mM 
                   
                 576 
                 447 
                   
                 634 
                 705 
               
               
                 CTx/Crea 
                 μg/μm. 
                   
                 1202 
                 679 
                   
                 1168 
                 1394 
               
               
                 Pyr/Crea 
                 nM/mM 
                   
                 163 
                 241 
                   
                 181 
                 253 
               
               
                   
               
               
                   d −8, d −7 and d 13 indicate day −8, day −7 and day 13 relative to the starting day of dosing    
               
            
           
         
       
     
      The salmon calcitonin group presented with moderate decreases in serum somatomedin (S.MLD, see TABLES 9 and 10).  
               TABLE 9                       Hormones - Males                  Control                         Animal no.   W62501   W62502                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               ACTH   pg/ml   91   63   87   117   136   150       CORTISOL   nmol/l   2183   1415   1328   1378   1020   1348       ALDOST   pg/ml   316   433   484   501   644   622       INSULIN   mU/l   26.0   33.0   37.0   12.0   30.0   9.0       GLUCAG   pg/ml   791   486   704   577   353   585       C-PEPTI   ng/ml   n/a   5.20   5.50   n/a   3.60   1.60       GASTRIN   pg/ml   n/a   105   93   n/a   147   148       T3   nmol/l   1.34   2.61   2.94   2.19   2.73   2.50       T4   nmol/l   56   61   44   57   68   48       TSH   mUI/l   0.17   0.18   0.42   0.00   0.05   0.04       IPH   pg/ml   103   75   108   174   173   155       CT   pg/ml   5.9   4.6   4.8   16.4   15.0   13.1       VD25-H   nmol/l   49   47   54   76   71   58       VD1-25dh   pmol/l   n/a   —   —   n/a   —   —       OSTEO   ng/ml   n/a   26   34   n/a   41   40       CTx   nmol/l   10   15   20   17   19   20       ICTP   ng/ml   18   13   19   26   16   15       PICP   ng/ml   n/a   311   395   n/a   610   495       G.H.   ng/ml   13.8   7.0   16.2   15.2   3.6   17.2       S.STA   pg/ml   n/a   —   —   n/a   —   —       S.MED   ng/ml   n/a   888   1185   n/a   793   689       PROLACT   ng/ml   0.0   3.3   3.6   21.6   22.5   22.5       TESTO   nmol/l   10.5   8.4   n.s.   7.9   4.7   n.s.       ESTR   nmol/l   n/a   n/a   n/a   n/a   n/a   n/a       PROG   pmol/l   n/a   n/a   n/a   n/a   n/a   n/a                         Salmon Calcitonin                         Animal no.   W62503   W62504                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               ACTH   pg/ml   98   87   87   115   78   73       CORTISOL   nmol/l   2316   979   1611   1578   1523   1709       ALDOST   pg/ml   983   1058   819   465   987   977       INSULIN   mU/l   13.0   14.0   17.0   4.0   10.0   22.0       GLUCAG   pg/ml   905   247   428   869   218   503       C-PEPTI   ng/ml   n/a   1.70   1.80   n/a   1.20   2.30       GASTRIN   pg/ml   n/a   83   88   n/a   128   136       T3   nmol/l   1.06   2.35   2.51   1.48   1.65   1.90       T4   nmol/l   53   64   47   62   79   65       TSH   mUI/l   0.99   1.12   1.03   0.14   0.41   0.40       IPH   pg/ml   213   75   78   99   62   71       CT   pg/ml   6.7   4.0   2.4   5.1   2.5   4.9       VD25-H   nmol/l   63   50   49   62   44   45       VD1-25dh   pmol/l   n/a   —   —   n/a   —   —       OSTEO   ng/ml   n/a   33   41   n/a   27   30       CTx   nmol/l   12   26   38   18   22   24       ICTP   ng/ml   21   15   15   22   21   20       PICP   ng/ml   n/a   284   363   n/a   361   439       G.H.   ng/ml   11.5   1.7   16.2   14.6   13.6   15.7       S.STA   pg/ml   n/a   —   —   n/a   —   —       S.MED   ng/ml   n/a   268   332   n/a   307   384       PROLACT   ng/ml   8.1   8.6   4.6   0.0   0.0   6.6       TESTO   nmol/l   8.5   3.6   n.s.   9.5   7.3   n.s.       ESTR   nmol/l   n/a   n/a   n/a   n/a   n/a   n/a       PROG   pmol/l   n/a   n/a   n/a   n/a   n/a   n/a                         PTS893                         Animal no.   W62505   W62506                                             Test   Units   d −6   d 7   d 13   d −6   d 7   d 13               ACTH   pg/ml   96   101   83   115   88   91       CORTISOL   nmol/l   1662   1156   1299   1506   1432   1212       ALDOST   pg/ml   265   380   592   141   471   651       INSULIN   mU/l   16.0   22.0   14.0   12.0   38.0   10.0       GLUCAG   pg/ml   858   656   786   694   497   739       C-PEPTI   ng/ml   n/a   2.90   2.10   n/a   4.40   2.40       GASTRIN   pg/ml   n/a   84   78   n/a   98   94       T3   nmol/l   2.48   3.47   3.55   1.38   2.76   2.43       T4   nmol/l   84   90   68   59   80   56       TSH   mUI/l   0.22   0.40   0.15   0.00   0.07   0.03       IPH   pg/ml   123   96   78   71   62   55       CT   pg/ml   6.1   4.0   4.6   10.4   7.8   7.6       VD25-H   nmol/l   77   62   50   88   62   50       VD1-25dh   pmol/l   n/a   —   —   n/a   —   —       OSTEO   ng/ml   n/a   43   55   n/a   32   42       CTx   nmol/l   19   20   31   12   12   16       ICTP   ng/ml   28   23   22   18   16   18       PICP   ng/ml   n/a   420   500   n/a   774   706       G.H.   ng/ml   13.4   15.8   12.1   8.5   11.6   14.0       S.STA   pg/ml   n/a   —   —   n/a   —   —       S.MED   ng/ml   n/a   749   914   n/a   828   867       PROLACT   ng/ml   7.1   15.7   7.5   7.5   5.5   2.2       TESTO   nmol/l   11.8   10.5   n.s.   5.3   3.7   n.s.       ESTR   nmol/l   n/a   n/a   n/a   n/a   n/a   n/a       PROG   pmol/l   n/a   n/a   n/a   n/a   n/a   n/a                 d −6, d 7 and d 13 indicate day −6, day 7 and day 13 relative to the starting day of dosing             
 
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
               
               
                 Hormones - Females 
               
               
                   
               
             
            
               
                 Control 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62551 
                 W62552 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 ACTH 
                 pg/ml 
                 146 
                 276 
                 121 
                 58 
                 60 
                 101 
               
               
                 CORTISOL 
                 nmol/l 
                 1983 
                 1546 
                 827 
                 1894 
                 837 
                 818 
               
               
                 ALDOST 
                 pg/ml 
                 244 
                 953 
                 312 
                 149 
                 90 
                 199 
               
               
                 INSULIN 
                 mU/l 
                 8.0 
                 12.0 
                 7.0 
                 2.0 
                 29.0 
                 21.0 
               
               
                 GLUCAG 
                 pg/ml 
                 729 
                 779 
                 583 
                 818 
                 507 
                 514 
               
               
                 C-PEPTI 
                 ng/ml 
                 n/a 
                 2.40 
                 1.40 
                 n/a 
                 3.30 
                 2.30 
               
               
                 GASTRIN 
                 pg/ml 
                 n/a 
                 84 
                 102 
                 n/a 
                 90 
                 92 
               
               
                 T3 
                 nmol/l 
                 2.22 
                 2.95 
                 3.40 
                 2.04 
                 3.09 
                 3.23 
               
               
                 T4 
                 nmol/l 
                 78 
                 67 
                 59 
                 51 
                 50 
                 49 
               
               
                 TSH 
                 mUI/l 
                 0.14 
                 0.27 
                 0.49 
                 0.15 
                 0.54 
                 0.50 
               
               
                 IPH 
                 pg/ml 
                 155 
                 149 
                 129 
                 145 
                 129 
                 112 
               
               
                 CT 
                 pg/ml 
                 4.70 
                 3.90 
                 4.10 
                 11.50 
                 11.60 
                 11.20 
               
               
                 VD25-H 
                 nmol/l 
                 64 
                 59 
                 51 
                 80 
                 78 
                 70 
               
               
                 VD1-25dh 
                 pmol/l 
                 n/a 
                 — 
                 — 
                 n/a 
                 — 
                 — 
               
               
                 OSTEO 
                 ng/ml 
                 n/a 
                 37 
                 39 
                 n/a 
                 34 
                 39 
               
               
                 CTx 
                 nmol/l 
                 11 
                 26 
                 28 
                 12 
                 16 
                 20 
               
               
                 ICTP 
                 ng/ml 
                 21 
                 23 
                 22 
                 19 
                 16 
                 15 
               
               
                 PICP 
                 ng/ml 
                 n/a 
                 864 
                 503 
                 n/a 
                 339 
                 298 
               
               
                 G.H. 
                 ng/ml 
                 8.5 
                 13.4 
                 1.7 
                 7.0 
                 12.0 
                 4.5 
               
               
                 S.STA 
                 pg/ml 
                 n/a 
                 — 
                 — 
                 n/a 
                 — 
                 — 
               
               
                 S.MED 
                 ng/ml 
                 n/a 
                 696 
                 839 
                 n/a 
                 1173 
                 1527 
               
               
                 PROLACT 
                 ng/ml 
                 4.30 
                 8.30 
                 5.90 
                 2.90 
                 0.00 
                 0.00 
               
               
                 TESTO 
                 nmol/l 
               
               
                 ESTR 
                 nmol/l 
                 58 
                 64 
                 61 
                 48 
                 45 
                 60 
               
               
                 PROG 
                 pmol/l 
                 3.40 
                 3.50 
                 1.70 
                 2.70 
                 1.10 
                 1.40 
               
               
                   
               
            
           
           
               
            
               
                 Salmon Calcitonin 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62553 
                 W62554 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 ACTH 
                 pg/ml 
                 72 
                 129 
                 97 
                 157 
                 233 
                 141 
               
               
                 CORTISOL 
                 nmol/l 
                 1536 
                 1220 
                 1202 
                 1222 
                 1705 
                 1128 
               
               
                 ALDOST 
                 pg/ml 
                 185 
                 948 
                 523 
                 155 
                 1073 
                 457 
               
               
                 INSULIN 
                 mU/l 
                 12.0 
                 8.0 
                 9.0 
                 20.0 
                 18.0 
                 24.0 
               
               
                 GLUCAG 
                 pg/ml 
                 585 
                 295 
                 258 
                 619 
                 594 
                 303 
               
               
                 C-PEPTI 
                 ng/ml 
                 n/a 
                 1.60 
                 1.00 
                 n/a 
                 1.50 
                 2.20 
               
               
                 GASTRIN 
                 pg/ml 
                 n/a 
                 83 
                 84 
                 n/a 
                 91 
                 84 
               
               
                 T3 
                 nmol/l 
                 1.17 
                 1.68 
                 1.51 
                 1.43 
                 1.51 
                 2.00 
               
               
                 T4 
                 nmol/l 
                 58 
                 76 
                 60 
                 61 
                 87 
                 60 
               
               
                 TSH 
                 mUI/l 
                 0.81 
                 1.31 
                 1.16 
                 0.08 
                 0.34 
                 0.41 
               
               
                 IPH 
                 pg/ml 
                 59 
                 47 
                 58 
                 145 
                 82 
                 53 
               
               
                 CT 
                 pg/ml 
                 3.10 
                 6.40 
                 4.90 
                 7.00 
                 3.60 
                 2.30 
               
               
                 VD25-H 
                 nmol/l 
                 61 
                 43 
                 40 
                 72 
                 56 
                 60 
               
               
                 VD1-25dh 
                 pmol/l 
                 n/a 
                 — 
                 — 
                 n/a 
                 — 
                 — 
               
               
                 OSTEO 
                 ng/ml 
                 n/a 
                 21 
                 25 
                 n/a 
                 35 
                 35 
               
               
                 CTx 
                 nmol/l 
                 12 
                 21 
                 25 
                 17 
                 34 
                 28 
               
               
                 ICTP 
                 ng/ml 
                 28 
                 28 
                 24 
                 29 
                 30 
                 24 
               
               
                 PICP 
                 ng/ml 
                 n/a 
                 115 
                 142 
                 n/a 
                 240 
                 287 
               
               
                 G.H. 
                 ng/ml 
                 6.3 
                 15.2 
                 8.6 
                 5.1 
                 17.9 
                 13.1 
               
               
                 S.STA 
                 pg/ml 
                 n/a 
                 — 
                 — 
                 n/a 
                 — 
                 — 
               
               
                 S.MED 
                 ng/ml 
                 n/a 
                 374 
                 297 
                 n/a 
                 204 
                 488 
               
               
                 PROLACT 
                 ng/ml 
                 0.00 
                 2.30 
                 4.30 
                 19.30 
                 20.20 
                 24.40 
               
               
                 TESTO 
                 nmol/l 
               
               
                 ESTR 
                 nmol/l 
                 47 
                 63 
                 59 
                 141 
                 82 
                 170 
               
               
                 PROG 
                 pmol/l 
                 1.80 
                 1.90 
                 1.50 
                 2.60 
                 4.00 
                 1.60 
               
               
                   
               
            
           
           
               
            
               
                 PTS893 
               
            
           
           
               
               
               
            
               
                 Animal no. 
                 W62555 
                 W62556 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Test 
                 Units 
                 d −8 
                 d 7 
                 d 13 
                 d −8 
                 d 7 
                 d 13 
               
               
                   
               
               
                 ACTH 
                 pg/ml 
                 109 
                 104 
                 110 
                 95 
                 132 
                 126 
               
               
                 CORTISOL 
                 nmol/l 
                 1482 
                 1331 
                 917 
                 1532 
                 1253 
                 1375 
               
               
                 ALDOST 
                 pg/ml 
                 314 
                 217 
                 330 
                 210 
                 228 
                 226 
               
               
                 INSULIN 
                 mU/l 
                 1.0 
                 22.0 
                 19.0 
                 15.0 
                 30.0 
                 22.0 
               
               
                 GLUCAG 
                 pg/ml 
                 711 
                 591 
                 657 
                 696 
                 437 
                 380 
               
               
                 C-PEPTI 
                 ng/ml 
                 n/a 
                 3.00 
                 2.40 
                 n/a 
                 3.80 
                 3.50 
               
               
                 GASTRIN 
                 pg/ml 
                 n/a 
                 83 
                 82 
                 n/a 
                 96 
                 91 
               
               
                 T3 
                 nmol/l 
                 2.08 
                 2.74 
                 2.63 
                 1.98 
                 2.69 
                 2.05 
               
               
                 T4 
                 nmol/l 
                 72 
                 56 
                 55 
                 59 
                 61 
                 45 
               
               
                 TSH 
                 mUI/l 
                 0.34 
                 0.14 
                 0.25 
                 0.88 
                 0.89 
                 0.69 
               
               
                 IPH 
                 pg/ml 
                 95 
                 45 
                 64 
                 111 
                 67 
                 58 
               
               
                 CT 
                 pg/ml 
                 2.50 
                 1.90 
                 2.70 
                 1.80 
                 2.90 
                 2.80 
               
               
                 VD25-H 
                 nmol/l 
                 72 
                 53 
                 47 
                 55 
                 44 
                 43 
               
               
                 VD1-25dh 
                 pmol/l 
                 n/a 
                 — 
                 — 
                 n/a 
                 — 
                 — 
               
               
                 OSTEO 
                 ng/ml 
                 n/a 
                 38 
                 43 
                 n/a 
                 32 
                 36 
               
               
                 CTx 
                 nmol/l 
                 13 
                 11 
                 15 
                 17 
                 14 
                 14 
               
               
                 ICTP 
                 ng/ml 
                 22 
                 16 
                 16 
                 20 
                 15 
                 15 
               
               
                 PICP 
                 ng/ml 
                 n/a 
                 612 
                 436 
                 n/a 
                 478 
                 393 
               
               
                 G.H. 
                 ng/ml 
                 3.5 
                 1.5 
                 0.0 
                 1.1 
                 8.2 
                 11.8 
               
               
                 S.STA 
                 pg/ml 
                 n/a 
                 — 
                 — 
                 n/a 
                 — 
                 — 
               
               
                 S.MED 
                 ng/ml 
                 n/a 
                 533 
                 502 
                 n/a 
                 432 
                 589 
               
               
                 PROLACT 
                 ng/ml 
                 0.00 
                 0.20 
                 3.20 
                 9.90 
                 5.70 
                 3.60 
               
               
                 TESTO 
                 nmol/l 
               
               
                 ESTR 
                 nmol/l 
                 67 
                 68 
                 60 
                 59 
                 66 
                 57 
               
               
                 PROG 
                 pmol/l 
                 2.80 
                 1.70 
                 1.50 
                 2.40 
                 2.20 
                 2.40 
               
               
                   
               
               
                   d −8, d 7 and d 13 indicate day −8, day 7 and day 13 relative to the starting day of dosing    
               
            
           
         
       
     
      Tissue sampling. Animals were killed by deep anaesthesia induced by intravenous injection of Pentothal®, followed by exsanguinations. All relevant tissues were sampled for histopathology and gene expression profiling. The following tissue samples were processed for analysis: liver, kidney, pituitary, muscle, bone, duodenum, spleen and trachea. Samples for histopathology were fixed in phosphate-buffered 10% formalin. Bone demineralization was performed with 10% formic acid. Tissue samples were embedded in Paraplast® and sectioned at 4 microns, for staining with haematoxylin and eosin. Samples for gene expression profiling were quickly frozen in liquid nitrogen immediately after excision, stored on dry ice and subsequently in a deep-freezer at approximately −80° C. until further use. All selected tissues for gene expression profiling were examined histopathologically.  
      Histopathology. Histopathological examination of the tissues selected for gene profiling analysis exhibited a normal spectrum of incidental lesions which were in terms of severity and distribution of lesions not different to the controls in all groups of treatment.  
      A slightly higher incidence of inflammatory and regenerative changes in the kidneys of females administered salmon calcitonin was observed. These changes were not considered to be relevant, since no records of kidney toxicity exist after 40 years of calcitonin therapeutic use.  
      Bone sections were stained for osteonectin, osteopontin and osteocalcin and were evaluated histopathologically. Histomorphometry of the bone tissue was performed regarding parameters for bone resorption and synthesis (osteoid formation).  
      The osteonectin, osteopontin, and osteocalcin staining of the tibia showed no difference between the groups one (control) and two (salmon calcitonin). Osteonectin exhibited a major enlargement and deterioration of the epiphysial growth plate of animal no 2553 due to a severe non-treatment related pathological status (severe, subacute epiphysiolysis).  
      Histomorphometry of bone tissue was performed to determine parameters related to bone resorption and bone synthesis (osteoid formation).  
      The results (see, TABLES 11 and 12) showed that salmon calcitonin increased trabecular volume and thickness in about a 17% in tibia, but not in vertebra. PTS893 reduced the cortical thickness (18%) and increased the cortical porosity (54%) in tibia (T), but not in vertebra (V). In contrast, PTS893 induced an increase in osteoid volumne (37% T, 213% V) and surface (49% T, 37% V), as well as an increase in the osteoblast surface (40% T, 24% V), in both tibia and vertebra, respectively.  
               TABLE 11                          Histomorphometry Tibia (Average Males and Females)                                                                 Tb Th   Tb N   Tb Sp       Ct Th                           BT/TV %   μm   mm −1     μm   Ct Por %   μm   OS/BS %   OV/BV %   ES/BS %   Obs/BS %                                                                     Control   20.70   106.32   1.95   407.20   2.53   1583.13   40.00   8.76   5.73   17.53           17.72   97.99   1.81   454.90   2.59   976.66   33.37   8.51   4.70   12.77           28.74   109.18   2.63   270.69   1.21   1036.24   29.45   5.79   10.19   11.70           20.15   103.59   1.94   410.62   1.19   1031.89   29.19   5.29   5.71   15.80       mean   21.83   104.27   2.08   385.85   1.88   1156.98   33.00   7.09   6.58   14.45       SD   4.79   4.77   0.37   79.79   0.78   285.39   5.04   1.80   2.45   2.69       sCT   32.28   140.64   2.30   295.01   2.10   895.98   42.71   11.72   5.02   18.32           25.00   122.19   2.05   366.51   1.98   1022.55   31.58   5.86   2.31   6.37           29.96   129.05   2.32   301.75   1.61   939.32   35.21   5.03   6.89   18.58           16.08   115.65   1.39   603.45   2.40   1178.70   30.37   4.01   5.61   19.36       mean   25.83   126.88   2.01   391.68   2.02   1009.14   34.97   6.65   4.95   15.66       SD   7.17   10.68   0.43   144.81   0.33   124.65   5.56   3.46   1.93   6.21       PTS893   19.69   129.22   1.52   526.99   2.76   1022.62   54.84   11.24   4.62   16.16           16.65   93.20   1.79   466.69   2.94   893.43   43.57   9.61   4.76   21.25           25.74   120.52   2.13   347.63   2.94   950.33   43.63   8.14   4.21   18.46           24.78   126.07   1.97   382.61   2.95   939.53   54.97   9.95   2.85   25.25       mean   21.72   117.25   1.85   430.98   2.90   951.48   49.25   9.74   4.11   20.28       SD   4.30   16.43   0.26   81.20   0.09   53.46   6.53   1.28   0.87   3.91                 sCT: salmon Calcitonin;            SD: Standard deviation            BV/TV trabecular bone volume;            Tb. Th. Trabecular thickness;            Tb. N. Trabecular number;            Tb. Sp. Trabecular Separation;            Ct. Por. Cortical porosity;            Ct, Th. Cortical thickness;            OS/BS osteoid surface;            OV/BV osteoid volume;            ES/BS eroded surface;            Obs/BS osteoblast surface.             
 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                   
               
               
                 Histomorphometry Vertebra (Average Males and Females) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Tb Th 
                 Tb N 
                 Tb Sp 
                   
                 Ct Th 
                   
                   
                   
                   
               
               
                   
                 BT/TV % 
                 μm 
                 mm −1   
                 μm 
                 Ct Por % 
                 μm 
                 OS/BS % 
                 OV/BV % 
                 ES/BS % 
                 Obs/BS % 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Control 
                 21.67 
                 179.80 
                 1.21 
                 649.79 
                 0.88 
                 887.91 
                 23.61 
                 1.22 
                 8.94 
                 16.14 
               
               
                   
                 15.85 
                 144.89 
                 1.09 
                 769.35 
                 0.26 
                 639.93 
                 20.77 
                 2.02 
                 8.81 
                 5.96 
               
               
                   
                 19.54 
                 122.91 
                 1.59 
                 506.23 
                 0.87 
                 416.48 
                 17.91 
                 1.58 
                 5.85 
                 4.07 
               
               
                   
                 21.95 
                 131.30 
                 1.67 
                 466.91 
                 0.85 
                 604.45 
                 11.58 
                 0.97 
                 1.82 
                 4.79 
               
               
                 mean 
                 19.75 
                 144.72 
                 1.39 
                 598.07 
                 0.71 
                 637.20 
                 18.47 
                 1.45 
                 6.36 
                 7.74 
               
               
                 SD 
                 2.82 
                 25.07 
                 0.28 
                 138.62 
                 0.30 
                 193.78 
                 5.15 
                 0.45 
                 3.34 
                 5.65 
               
               
                 sCT 
                 17.32 
                 113.29 
                 1.53 
                 540.84 
                 1.70 
                 705.10 
                 3.95 
                 0.46 
                 11.60 
                 3.21 
               
               
                   
                 19.33 
                 144.31 
                 1.34 
                 602.15 
                 1.18 
                 810.09 
                 5.82 
                 0.86 
                 2.55 
                 3.97 
               
               
                   
                 20.11 
                 118.49 
                 1.70 
                 470.71 
                 1.18 
                 576.42 
                 11.48 
                 1.43 
                 4.93 
                 6.81 
               
               
                   
                 19.46 
                 123.71 
                 1.57 
                 511.96 
                 0.12 
                 907.16 
                 4.91 
                 0.32 
                 3.47 
                 1.23 
               
               
                 mean 
                 19.06 
                 124.95 
                 1.53 
                 531.42 
                 1.05 
                 749.69 
                 6.54 
                 0.77 
                 5.64 
                 3.80 
               
               
                 SD 
                 1.21 
                 13.59 
                 0.15 
                 55.24 
                 0.66 
                 141.96 
                 3.38 
                 0.50 
                 4.09 
                 2.31 
               
               
                 PTS893 
                 15.15 
                 105.46 
                 1.44 
                 590.67 
                 1.49 
                 707.43 
                 18.84 
                 3.24 
                 9.31 
                 10.36 
               
               
                   
                 20.23 
                 118.79 
                 1.70 
                 468.39 
                 1.45 
                 629.35 
                 41.28 
                 8.42 
                 2.30 
                 9.07 
               
               
                   
                 23.56 
                 134.66 
                 1.75 
                 436.79 
                 0.41 
                 740.87 
                 23.65 
                 3.49 
                 2.55 
                 10.47 
               
               
                   
                 24.86 
                 134.82 
                 1.84 
                 407.56 
                 0.92 
                 624.35 
                 17.66 
                 2.66 
                 3.96 
                 8.33 
               
               
                 mean 
                 20.95 
                 123.43 
                 1.68 
                 475.85 
                 1.07 
                 675.50 
                 25.36 
                 4.45 
                 4.53 
                 9.56 
               
               
                 SD 
                 4.33 
                 14.15 
                 0.17 
                 80.47 
                 0.51 
                 57.85 
                 10.93 
                 2.67 
                 3.27 
                 1.04 
               
               
                   
               
               
                   sCT: salmon Calcitonin;    
               
               
                   SD: Standard deviation    
               
               
                   BV/TV trabecular bone volume;    
               
               
                   Tb. Th. Trabecular thickness;    
               
               
                   Tb. N. Trabecular number;    
               
               
                   Tb. Sp. Trabecular Separation;    
               
               
                   Ct. Por. Cortical porosity;    
               
               
                   Ct, Th. Cortical thickness;    
               
               
                   OS/BS osteoid surface;    
               
               
                   OV/BV osteoid volume;    
               
               
                   ES/BS eroded surface;    
               
               
                   Obs/BS osteoblast surface.    
               
            
           
         
       
     
      Histomorphometry showed inconsistent results between tibial and vertebral bone, except for an increase in osteoid synthesis induced by PTS893. This effect is well documented for parathyroid hormone, when administered in a discontinuous way.  
      RNA extraction andpurificationz. A set of tissues was selected for gene expression profiling. These set included samples from kidney, bone, muscle, duodenum, pituitary and liver. In particular, diaphyseal bone from femur and tibia were processed for gene expression profiling. Briefly, total RNA was obtained by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol®, Invitrogen Life Technologies, Carlsbad, Calif. USA) from each frozen tissue section and the total RNA was then purified on an affinity resin (RNeasy®, Qiagen) according to the manufacturer&#39;s instructions. Total RNA was quantified by the absorbance at λ=260 nm (A260 nm), and the purity was estimated by the ratio A260 nm/A280 nm. Integrity of the RNA molecules was confirmed by non-denaturing agarose gel electrophoresis. RNA was stored at approximately −80° C. until analysis. One part of each individual RNA sample was kept for the analysis of critical genes by means of Real-time PCR.  
      Hybridization assay. Transcript profiling by means of GeneChip® expression probe arrays was done in the laboratories of the Genomics Factory EU, as recommended by the manufacturer of the GeneChip® system ( GeneChip Expression Analysis Technical Manual , Affymetrix Inc., Santa Clara, Calif. USA). HG-U95Av2 GeneChip® expression probe arrays (Affymetrix, Santa Clara Calif. USA) were used. Double stranded cDNA was synthesized with a starting amount of approximately 5 μg full-length total RNA using the Superscript Choice System (Invitrogen Life Technologies) in the presence of a T7-(dT) 24 DNA oligonucleotide primer. Following synthesis, the cDNA was purified by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation. The purified cDNA was then transcribed in vitro using the BioArray® High Yield RNA Transcript Labelling Kit (ENZO) in the presence of biotinylated ribonucleotides form biotin labelled cRNA. The labelled cRNA was then purified on an affinity resin (Rneasy®, Qiagen), quantified and fragmented. An amount of approximately 10 μg labelled cRNA was hybridized for approximately 16 hours at 45° C. to an expression probe array. The array was then washed and stained twice with streptavidin-phycoerythrin (Molecular Probes) using the GeneChip Fluidics Workstation 400 (Affymetrix). The array was then scanned twice using a confocal laser scanner (GeneArray® Scanner, Agilent) resulting in one scanned image. This resulting “.data-file” was processed using the Micro Array Analysis Suite version 4 (MAS4) program (Affymetrix) into a “.cel-file”. The “.cel file” was captured and loaded into the Affymetrix GeneChip Laboratory Information Management System (LIMS). The LIMS database is connected to a UNIX Sun Solaris server through a network filing system that allows for the average intensities for all probes cells (CEL file) to be downloaded into an Oracle database. Raw data was converted to expression levels using a “target intensity” of 150. The numerical values displayed are weighted averages of the signal intensities of the probe-pairs comprised in a probe-set for a given transcript sequence (AvgDiff value). The data were checked for quality and loaded into the GeneSpring® software versions 4.2.4 and 5 (Silicon Genetics, Calif. USA) for analysis.  
      Data analysis. Data analysis was performed with the Silicon Genetics software package GeneSpring version 4.2.1 and 5. Average difference values below 20 were set to 20. Various filtering and clustering tools in these programs were used to explore the data sets and identify transcript level changes that inform on altered cellular and tissue functions and that can be used to establish working hypotheses on the modes of action of the compound.  
      The threshold range for considering as up or down regulation was determined within the context of the biological interpretation of the EXAMPLE.  
      The information content of these data sets is a conjunction of numerical changes and biological information. The decision to consider a specific gene relevant was based on a conjunction of numerical changes identified by comparative and statistical algorithms and the relationship to other modulated genes that point to a common biological theme. The weight of that relationship was assessed by the analyst through a review of the relevant scientific literature.  
      Increase and decrease reported here refer to transcript abundance, unless specifically stated.  
      Gene expression profiling. Multi-organ comparative gene profiling analysis was performed in the group administered salmon calcitonin at 50 μg/animnal/day. The organs chosen for analysis were liver, kidney, pituitary, skeletal muscle, bone, duodenum, spleen and trachea.  
               TABLE 13                          Multi-Organ Gene Expression Profiling of Salmon Calcitonin                                             GeneChip ®                                   expression probe       set identifier   Coding Gene   bone   kidney   liver   muscle   pituitary   trachea                                                     36611_at   acid phosphatase 1 isoform a           −1.33       −1.33           32714_s_at   activin A receptor type II-like 1   −1.62           −1.83       39314_at   activin A type IIB receptor precursor           −1.12   1.41   −4.15       35915_at   activin beta-C chain.   −1.21           −2.41   −1.67       36621_at   alpha-2-HS-glycoprotein   1.33   1.53               1.12       34588_i_at   amelogenin           −1.61       37747_at   annexin V       −1.30   1.87       −2.58       40376_at   arylsulfatase E precursor       −1.59       39326_at   ATPase H(+)-vacuolar   −1.57   −2.80           −1.62       38814_at   ATFase H(+)-vacuolar subunit   1.22       33741_at   ATPase, H+ transport, lysosomal   1.23               −1.50       33033_at   ATPase, H+ transporting, lysosomal   −1.29       −3.19       −1.43   1.23       38814_at   ATPase, H+ transporting, lysosomal               1.30   −1.28   1.14       38126_at   biglycan                   1.75   −1.61       39407_at   bone morphogenetic protein 1           −1.20       −1.55       31399_at   bone morphogenetic protein 10   1.44   1.45   −1.31   −1.77       1113_at   bone morphogenetic protein 2A   −1.12   2.63               1.29       1831_at   bone morphogenetic protein 5   −1.43   1.39   1.40       1733_at   bone morphogenetic protein 6       −1.37   −1.17   −1.64   −1.27   −1.1           precursor       34500_at   calcium binding protein 1 (calbrain)       2.31           1.21       31670_s_at   calcium/calmodulin-dependent protein       1.17       1.57   −1.28   1.60           kinase (CaM kinase) II gamma       1751_g_at   calreticulin   −4.03   −1.60       1.67       32067_at   cAMP responsive element modulator   1.39       −1.24       −1.50           (CREM)       39241_at   carbonic anhydrase I   −2.68       1.18       −1.69       40095_at   carbonic anhydrase II   −1.69       40163_r_at   cartilage oligomeric matrix protein   2.36   5.61           precursor       128_at   cathepsin k   1.18           1.35   −2.33       129_g_at   cathepsin k   1.20   −1.54       1.17   −1.28       38466_at   cathepsin k   1.27           1.40       −1.19       40718_at   cathepsin w   −1.31       −1.54       2.05       32833_at   CDC-like kinase 1   1.63       646_s_at   CDC-like kinase 2 isoform hclk2/139   1.19           1.86       38112_g_at   chondroitin sulphate proteoglycan 2       −2.16       1.51       −1.68           (versican)       32642_at   chondroitin sulphate proteoglycan 3                   −1.49           (neurocan)       31493_s_at   chorionic somatomammotropin                   −1.59           hormone 1       40714_at   chymotrypsin C (caldecrin)                   1.39   3.22       35474_s_at   Collagen type 1 and PDGFB fusion               −7.30       −3.35           transcript       598_at   collagen type II alpha-1   −1.38       1.69   −1.27   2.77   −3.02       32488_at   collagen type III alpha 1   −1.41   −1.59   −1.53   −3.20   −1.89   −1.35       38952_s_at   collagen type IV alpha-2   1.23               −1.73       35379_at   collagen type IX alpha1   −2.22   −3.28       38722_at   collagen type VI alpha-1       −3.38   −1.13   −1.42       34802_at   collagen type VI alpha-2 (AA 570-998)   −1.37       −1.10   −1.39       −1.28       37892_at   collagen type XI alpha-1   1.24               −2.46   −1.51       1026_s_at   collagen type XI alpha2   −1.20   −1.32           1.15   −2.20       1027_at   collagen type XI alpha2   1.11       −1.25           1.37       32305_at   collagen, type I, alpha 2       −1.45               −1.54       39333_at   collagen, type IV, alpha 1                       −1.49       39925_at   collagen, type IX, alpha 2                   −2.38   −1.36       38420_at   collagen, type V, alpha 2       −1.29   −1.18   −1.11       −1.10       41351_at   collagen, type VI, alpha 1       −2.29       −1.27   −1.50       41350_at   collagen, type VI, alpha 1 precursor                       −3.55       35168_f_at   collagen, type XVI, alpha 1                       −1.59       35169_at   collagen, type XVI, alpha 1                       −1.18       39632_at   collagenase 3 (matrix metalloproteinase   1.20           13)       36638_at   connective tissue growth factor                   −2.11       40697_at   cyclin A2   −1.60       34736_at   cyclin B1   −2.83       36650_at   cyclin D2   1.21       35249_at   cyclin E2   −2.95       1206_at   cyclin-dependent kinase 5   1.56                   −1.54       799_at   cyclin-dependent kinase 5, regulatory   1.32           subunit 1 (p35)       41546_at   cyclin-dependent kinase 6   1.15   1.52           1.34       2031_s_at   cyclin-dependent kinase inhibitor 1A   1.95           (p21, Cip1)       35816_at   cystatin B (stefin B)   1.57       806_at   cytokine-inducible kinase   1.20               1.35       40049_at   death-associated protein kinase 1                   −1.47   −1.29       33903_at   death-associated protein kinase 3   −1.22       34029_at   dentin matrix acidic phosphoprotein 1   1.65           (DMP1)       40186_at   dual specificity phosphatase 9                       1.59       37996_s_at   dystrophia myotonica-protein kinase       1.25               −1.50       342_at   ectonucleotide Pyrophosphatase/   1.45           Phosphodiesterase 1;       343_s_at   ectonucleotide pyrophosphatase/   1.11           −1.42           phosphodiesterase 1;       33602_at   endothelial differentiation, G protein       1.15           2.24   −1.66           coupled receptor 6 precursor       1442_at   oestrogen receptor   1.47   1.23           1.60       33670_at   oestrogen receptor   1.30       1487_at   oestrogen receptor-related protein   1.11           −1.52       1.24       38882_r_at   oestrogen-responsive B box protein   1.22       −1.51           (EBBP)       39945_at   fibroblast activation protein   −1.27               −1.48   −1.32       996_at   fibroblast growth factor 1 (acidic)       1.17       −1.41       41586_at   fibroblast growth factor 18       2.06       1730_s_at   fibroblast growth factor 4       1.55               1.46       424_s_at   fibroblast growth factor receptor.   −1.17   −1.59       40131_at   follistatin-like 1   −1.31       40132_g_at   follistatin-like 1                   −1.22   1.15       33510_s_at   glutamate receptor, metabotropic 1   1.26           −1.31       33269_at   GPI1 N-acetylglucosaminyl transferase   1.24           component Gpi1       1401_g_at   granulocyte-macrophage   −3.07   2.24           colony-stimulating factor (CSF1)       1911_s_at   growth arrest and       1.84       −3.84       1.24           DNA-damage-inducible, alpha       37615_at   growth factor receptor-bound protein   1.21               −1.61           10       32845_at   heparan sulphate proteoglycan 2           1.27           −1.11           (perlecan)       32778_at   inositol 1,4,5-triphosphate receptor,       1.75           −2.57   1.20           type 1       32779_s_at   inositol 1,4,5-triphosphate receptor,           1.21   2.02           type 1       756_at   inositol 1,4,5-triphosphate receptor,                       1.24           type 2       34209_at   inositol 1,4,5-trisphosphate 3-kinase       2.29       1.42   −1.36   1.75           isoenzyme       33506_at   inositol polyphosphate 4-phosphatase   1.12   1.66   2.09       1.27           type I-beta       172_at   inositol polyphosphate-5-phosphatase,           −1.22   −1.15       32697_at   inositol(myo)-1(or   −1.36   −2.70           1.61           4)-monophosphatase 1       36496_at   inositol(myo)-1(or                       1.13           4)-monophosphatase 2       2079_s_at   insulin-like growth factor (IGF-II)               −1.32   1.15   −1.31       36782_s_at   insulin-like growth factor 2                       −1.69           (somatomedin A)       1232_s_at   insulin-like growth factor binding   −1.31           −1.53           protein       40422_at   insulin-like growth factor binding                   −2.97   −1.16           protein 2       1586_at   insulin-like growth factor binding   1.45       −1.16   1.70           protein 3       37319_at   insulin-like growth factor binding   2.17           1.58       −1.52           protein 3       41420_at   insulin-like growth factor binding       1.15       −2.66           protein 5       1741_s_at   insulin-like growth factor binding   −2.49               −2.17   −1.22           protein-2       1464_at   insulin-like growth factor II precursor   1.18   1.10           −1.26       1591_s_at   insulin-like growth factor II precursor   1.41                   −2.80       33082_at   integrin alpha 10 subunit   1.33           −2.32   −1.18       1100_at   interleukin-1 receptor-associated kinase               1.39   −1.48       2005_s_at   Janus kinase 3               −1.51       1.57       40060_r_at   LIM protein (similar to rat protein   1.44               −1.68   −1.31           kinase C-binding enigma)       36811_at   lysyl oxidase-like protein   −1.44   1.14       1.30   −1.19       1433_g_at   MAD, mothers against decapentaplegic   1.14   −1.13       −1.61   −1.65   −1.69           homolog 3       34655_at   MAGUKs (membrane-associated   1.23           guanylate kinase homologues       35652_g_at   MAP kinase kinase kinase (MTK1)   1.14       33246_at   MAPK13: mitogen-activated protein           −1.24   −1.13   −1.91   1.65           kinase 13       41280_r_at   MAPK8IP1: mitogen-activated protein   −1.31   1.92           1.58           kinase 8 interacting protein 1       2004_at   MEK kinase               1.13   −1.62   1.16       1509_at   metalloproteinase   −1.42       −1.11       −1.23   −1.18       976_s_at   mitogen-activated protein kinase 1   −1.61       34006_s_at   mitogen-activated protein kinase 8   1.32       1844_s_at   mitogen-activated protein kinase kinase 1                   −1.60   1.15       35694_at   mitogen-activated protein kinase kinase               1.26           kinase kinase 4       1469_at   mitogen-activated protein       1.13   −1.30           1.16           kinase-activated protein kinase 2       1637_at   mitogen-activated protein               1.11       1.34           kinase-activated protein kinase 3       37565_at   MMD: monocyte to macrophage   1.28   −2.48               −1.28           differentiation-associated       38307_at   neurochondrin,           2.80       −1.39       39144_at   nuclear factor of activated T-cells,       2.72       1.42       −1.70           cytoplasmic, calcineurin-dependent 1       41202_s_at   OS-4 protein (OS-4)   1.24               −1.72       1451_s_at   OSF-2os osteoblast specific factor-2   −1.65               −2.06   1.56           (periostin)       467_at   osteoclast stimulating factor (OSF)   −1.23   −1.50   −1.58       −4.12       33814_at   PAK4   1.16           −1.33   1.11       38757_at   PDGF associated protein.   −1.89               −1.15   1.20       146_at   phosphatidylinositol 4-kinase, catalytic,               1.19       1.23           beta polypeptide       34496_at   phosphatidylinositol glycan, class L       2.34   1.34   1.51       34169_s_at   phosphatidylinositol polyphosphate                   −1.33   1.49           5-phosphotase, isoform b       37412_at   phosphatidylinositol-4-phosphate   −1.87       −1.31           5-kinase isoform C (−1)       37253_at   phosphatidylinositol-4-phosphate       1.17       −1.13       1.11           5-kinase, type I, beta       35741_at   phosphatidylinositol-4-phosphate               −1.18       −1.18           5-kinase, type II, beta       751_at   phosphatidylinositol-glycan-class C   1.14   1.19           −1.22           (PIG-C)       666_at   phosphodiesterase 4A, cAMP-specific   1.33       −1.32       −1.18       38526_at   phosphodiesterase 4D, cAMP-specific   1.30   1.15               3.51           (dunce (Drosophila)-homolog           phosphodiesterase E3)       38921_at   phosphodiesterase IB,   1.52               1.42   1.12           calmodulin-dependent       31699_at   phosphoinositide-3-kinase   1.56       −1.56       36287_at   phosphoinositide-3-kinase, catalytic,   1.31           gamma polypeptide       35665_at   phosphoinositide-3-kinase, class 3                   −1.11   1.21       364_s_at   phospholipase C b3   1.22       901_g_at   phospholipase C, beta 4   −1.20       1.41       −1.55       1293_s_at   phospholipase D   −1.26       38023_at   phosphotidylinositol transfer protein       2.25       1.33   1.55   1.71       38269_at   PKD2 Protein kinase D2           1.34       32306_g_at   preprocollagen type I alpha-2   1.19       −1.38       −1.75   −1.31       35473_at   preprocollagen type I alpha1.   −2.72   −1.37       −3.94       −2.70       32307_s_at   procollagen   1.13       −1.26   −2.44   −1.56   −1.82       37605_at   procollagen alpha 1 type II               −1.84       −1.61       36184_at   procollagen-lysine 5-dioxygenase       2.52       −2.15       −1.30       37037_at   procollagen-proline, 2-oxoglutarate           1.87   1.46   −1.67   1.29           4-dioxygenase (proline 4-hydroxylase),           alpha polypeptide I       37633_s_at   progestagen-associated endometrial       2.00           protein (placental protein 14,           pregnancy-associated endometrial           alpha-2-globulin, alpha uterine protein)       36109_at   prolidase (imidodipeptidase) PEPD:   −2.55               −2.05       1884_s_at   proliferating cell nuclear antigen   −1.85       36666_at   prolyl 4-hydroxylase beta   1.95           1.37   2.08       718_at   protease, serine, 11 (IGF binding)   −1.30               −1.81   −1.30       719_g_at   protease, serine, 11 (IGF binding)   −1.43               −1.97   −1.27       385_at   proteasome (prosome, macropain)   1.36       −1.29           subunit, beta type, 10       37431_at   protein inhibitor of activated STAT X                   −1.23   1.28       39183_at   protein kinase 1 PCTAIRE   −1.17       39711_at   protein kinase C substrate 80K-H                       1.31       1437_at   protein kinase C, alpha           −2.06           1.82       36359_at   protein kinase, cAMP-dependent,   1.39       1.14   −1.49   1.30   1.13           catalytic, gamma       1091_at   protein kinase, cAMP-dependent,   1.65   −1.80       2.06           regulatory, type I, beta       116_at   protein kinase, cAMP-dependent,   1.28           −1.18           regulatory, type II, alpha       33633_at   purinergic receptor P2Y, G-protein   1.90   −1.82           coupled, 11       32737_at   RAC2 Ras-related C3 botulinum toxin   1.16                   1.22           substrate 2 (rho family, small GTP           binding protein Rac2)       1007_s_at   receptor tyrosine kinase DDR   1.21       1048_at   retinoid X receptor-gamma   1.47               1.47       41404_at   ribosomal protein S6 kinase   −1.67   −1.40       −1.83       −1.40       865_at   ribosomal protein S6 kinase, 90 kD,       −1.42               1.27           polypeptide 3       32290_at   SCAMP1: secretory carrier membrane   2.50               −1.27   −1.39           protein 1 (vesicular transport)       34342_s_at   secreted phosphoprotein 1       1.15               −3.01           (osteopontin, bone sialoprotein I, early           T-lymphocyte activation 1)       39166_s_at   serine (or cysteine) proteinase       −2.82   1.56   2.04       −1.29           inhibitor, clade H (heat shock protein           47), member 2       36217_at   serine/threonine kinase 38   1.54                   −1.59       1223_at   serine/threonine protein kinase           2.42       32447_at   SF-1; Steroidogenic factor-1   8.76       1.59       1.27   −2.01       33338_at   signal transducer and activator of           −1.14   1.15   −2.11   −1.93           transcription 1       1244_at   signal transducer and activator of                       1.57           transcription 2, 113 kD       40458_at   signal transducer and activator of                   1.14   1.39           transcription 5A       506_s_at   signal transducer and activator of               1.32   2.60           transcription 5A       41222_at   signal transducer and activator of   1.44           1.14   −1.46           transcription 6 (STAT6)       1950_s_at   Smad 3               −2.44       −1.16       38889_at   Smad anchor for receptor activation,       1.28           −1.14   −1.51           isoform 1       1013_at   Smad5                   −2.62   1.22       1955_s_at   SMAD6 (inhibits BMP/Smad1   1.19               −1.37           (MADH1)       37718_at   SNF-1 related kinase   1.49       −1.13   1.18       35883_at   Spi-B transcription factor (SPI1/PU.1       3.76           −2.96   1.15           related)       472_at   Stat5b (stat5b)   −1.42   −1.28           −1.83   −2.50       38669_at   Ste20-related serine/threonine kinase   1.24               −1.78       38374_at   TEIG; TGFB inducible early growth   1.18               −1.79           response       224_at   TGFB inducible early growth response;   1.26               −2.69           TIEG       36940_at   TGFB1-induced anti-apoptotic factor 1   1.22           1.28   −1.38       32217_at   TGF-beta induced apoptosis protein 12   1.40   1.55       1.12       41445_at   TGF-beta precursor   1.14   1.11       1890_at   TGF-beta superfamily protein   1.74   1.85   1.12       1.38       40631_at   Tob   −1.14           1.28   −2.09       32219_at   tousled-like kinase 1       −1.16       1897_at   transforming growth factor, beta       1.18               1.12           receptor III (betaglycan, 300 kD)       1735_g_at   transforming growth factor-beta 3       −1.15       −4.45   −1.39   −2.23       1767_s_at   transforming growth factor-beta 3   −1.71   1.41       −1.71           (TGF-beta 3)       40581_at   TRIO: triple functional domain   1.65   1.62       1.34       −1.42           (PTPRF interacting)       32272_at   tubulin alpha   −1.20           1.18       330_s_at   tubulin alpha 1   −1.80       1.23   −1.20   −1.19       40567_at   tubulin alpha 3   −1.39       −1.18           −1.10       685_f_at   tubulin alpha isotype H2-alpha   −4.36       1.32       2.13       151_s_at   tubulin beta   −1.40   −1.14   1.16   1.22   1.16       33678_i_at   tubulin beta 2   −1.15           1.75       33679_f_at   tubulin beta 2   −1.31           1.45       709_at   tubulin beta 3   −1.18               −1.35   1.20       471_f_at   tubulin beta 4   −1.38           1.50       39399_at   tubulin beta, cofactor D   −1.85               −4.69       32098_at   type VI collagen alpha 2 chain                       −3.79           precursor       1651_at   ubiquitin carrier protein E2-C   −3.74       1953_at   vascular endothelial growth factor   1.40       36101_s_at   vascular endothelial growth factor   1.45       37268_at   vascular endothelial growth factor B               −1.58       36140_at   Y box binding protein-1   2.30   1.86       2.36   −2.72                  
 
      In addition, the effect of PTS893 was assessed in bone.  
               TABLE 14                          Gene-Profiling Analysis of Salmon Calcitonin and PTS893 in Bone                             GeneChip ®       Fold Increase           Expression Probe       Salmon   Fold Increase       Set Identifier   Coding Gene   Calcitonin   PTS893                                     38909_at   25-hydroxyvitamin D3 1-alpha-hydroxylase       −1.14       32714_s_at   activin A receptor type II-like 1   −1.62       35915_at   activin beta-C chain.   −1.21       39279_at   activin type II receptor       1.24       39383_at   adenylate cyclase 6, isoform a       −1.22       38965_at   aggrecan 1       2.03       39206_s_at   aggrecan 1       1.41       36621_at   alpha-2-HS-glycoprotein   1.33       34589_f_at   Amelogenin   1.10   −3.10       39326_at   ATPase H(+) vacuolar   −1.57   −1.19       38814_at   ATPase H(+) vacuolar   1.22       33741_at   ATPase, H+ transport, lysosomal   1.23       33033_at   ATPase, H+ transporting, lysosomal   −1.29   −1.17       40328_at   bHLH transcription factor       2.57       39407_at   bone morphogenetic protein 1       1.16       31399_at   bone morphogenetic protein 10   1.44   1.20       1113_at   bone morphogenetic protein 2A   −1.12   −1.13       40367_at   bone morphogenetic protein 2A       −1.18       1114_at   bone morphogenetic protein 2B or BMP4       −1.70       1831_at   bone morphogenetic protein 5   −1.43   −1.60       1733_at   bone morphogenetic protein 6 precursor       1.27       40333_at   bone morphogenetic protein-4 (hBMP-4)       −1.42       34847_s_at   calcium/calmodulin-dependent protein kinase (CaM kinase) II       1.13           beta       33935_at   calcyclin binding protein       1.41       1751_g_at   Calreticulin   −4.03       32067_at   cAMP responsive element modulator (CREM)   1.39   2.75       39241_at   carbonic anhydrase I   −2.68       40095_at   carbonic anhydrase II   −1.69       40163_r_at   cartilage oligomeric matrix protein precursor   2.36       128_at   cathepsin k   1.18       129_g_at   cathepsin k   1.20       38466_at   cathepsin k   1.27       40718_at   cathepsin w   −1.31       32833_at   CDC-like kinase 1   1.63       646_s_at   CDC-like kinase 2 isoform hclk2/139   1.19       34763_at   chondroitin sulphate proteoglycan 6       −1.18       598_at   collagen type II alpha-1   −1.38   −1.19       32488_at   collagen type III alpha 1   −1.41       38952_s_at   collagen type IV alpha-2   1.23   1.44       35379_at   collagen type IX alpha 1   −2.22       34802_at   collagen type VI alpha-2 (AA 570-998)   −1.37       38566_at   collagen type X alpha-1       1.67       37892_at   collagen type XI alpha-1   1.24   1.18       1026_s_at   collagen type XI alpha2   −1.20       1027_at   collagen type XI alpha2   1.11       39632_at   collagenase 3 (matrix metalloproteinase 13)   1.20       36638_at   connective tissue growth factor.       −1.32       1943_at   cyclin A       −74       40697_at   cyclin A2   −1.60   −1.39       34736_at   cyclin B1   −2.83       39251_at   cyclin C       −2.03       1983_at   cyclin D2       −1.28       36650_at   cyclin D2   1.21       35249_at   cyclin E2   −2.95       1649_at   cyclin G1 interacting protein       1.31       1913_at   cyclin G2       −1.29       160024_at   cyclin-dependent kinase (CDC2-like) 10 PISSLRE       1.53       1942_s_at   cyclin-dependent kinase 4       −1.22       1206_at   cyclin-dependent kinase 5   1.56       40549_at   cyclin-dependent kinase 5       −1.40       799_at   cyclin-dependent kinase 5, regulatory subunit 1 (p35)   1.32       41546_at   cyclin-dependent kinase 6   1.15       2031_s_at   cyclin-dependent kinase inhibitor 1A (p21, Cip1)   1.95       1787_at   cyclin-dependent kinase inhibitor 1C       1.18       38673_s_at   cyclin-dependent kinase inhibitor 1C       1.13       39545_at   cyclin-dependent kinase inhibitor 1C       1.24       1797_at   cyclin-dependent kinase inhibitor 2D (p19, inhibits CDK4)       −1.21       35816_at   cystatin B (stefln B)   1.57       806_at   cytokine-inducible kinase   1.20       40049_at   death-associated protein kinase 1       −1.30       33903_at   death-associated protein kinase 3   −1.22   −7.73       34029_at   dentin matrix acidic phosphoprotein 1 (DMP1)   1.65       38059_g_at   dermatopontin       1.72       343_s_at   ectonucleotide pyrophosphatase/phosphodiesterase 1   1.11       342_at   ectonucleotide Pyrophosphatase/Phosphodiesterase 1   1.45       1442_at   oestrogen receptor   1.47       33670_at   oestrogen receptor   1.30       1487_at   oestrogen receptor-related protein   1.11       38882_r_at   oestrogen-responsive B box protein (EBBP)   1.22       38902_r_at   oestrogen-responsive B box protein (EBBP)       1.23       39945_at   fibroblast activation protein   −1.27       424_s_at   fibroblast growth factor receptor.   −1.17       466_at   general transcription factor II,       1.34       1102_s_at   glucocorticoid receptor alpha       1.43       33510_s_at   glutamate receptor, metabotropic 1   1.26   1.23       33269_at   GPI1 N-acetylglucosaminyl transferase component Gpil   1.24   1.21       41476_at   G-protein alpha subunit 11       1.24       1401_g_at   granulocyte-macrophage colony-stimulating factor (CSF1)   −3.07   −2.57       1911_s_at   growth arrest and DNA-damage-inducible protein (gadd45)       2.87       888_s_at   growth differentiation factor 1       −1.43       37615_at   growth factor receptor-bound protein 10   1.21       33929_at   heparan sulphate proteoglycan (glypican).       2.00       39757_at   heparan sulphate proteoglycan core protein       1.10       755_at   inositol 1,4,5-trisphosphate receptor type 1       1.27       33506_at   inositol polyphosphate 4-phosphatase type I-beta   1.12   −1.24       33290_at   inositol polyphosphate 5-phosphatase (5ptase)       −1.20       32697_at   inositol(myo)-1(or 4)-monophosphatase 1   −1.36       1975_s_at   insulin-like growth factor 1       −1.41       1501_at   insulin-like growth factor 1 (somatomedin C)       −1.12       1232_s_at   insulin-like growth factor binding protein   −1.31       40422_at   insulin-like growth factor binding protein 2       −1.27       1586_at   insulin-like growth factor binding protein 3   1.45       37319_at   insulin-like growth factor binding protein 3   2.17       1737_s_at   insulin-like growth factor binding protein 4       1.13       41420_at   insulin-like growth factor binding protein 5       1.18       1396_at   insulin-like growth factor binding protein 5       1.62       1678_g_at   insulin-like growth factor binding protein 5       1.44       38650_at   insulin-like growth factor binding protein 5       1.53       1741_s_at   insulin-like growth factor binding protein-2   −2.49   −2.11       1464_at   insulin-like growth factor II precursor   1.18       1591_s_at   insulin-like growth factorn II precursor   1.41   1.31       39781_at   insulin-like growth factor-binding protein 4       1.16       33082_at   integrin alpha 10 subunit   1.33       35131_at   integrin-binding sialoprotein (bone sialoprotein, bone       1.15           sialoprotein II)       40060_r_at   LIM protein (similar to rat protein kinase C-binding enigma)   1.44   1.32       36184_at   lysyl hydroxylase (PLOD) procollagen-lysine, 2-oxoglutarate 5       −1.40           dioxygenase       34795_at   lysyl hydroxylase isoform 2 (PLOD2)       1.49       36811_at   lysyl oxidase-like protein   −1.44       1433_g_at   MAD, mothers against decapentaplegic homolog 3   1.14   1.73       34655_at   MAGUKs (membrane-associated guanylate kinase homologues   1.23       36179_at   MAP kinase activated protein kinase 2       1.18       35652_g_at   MAP kinase kinase kinase (MTK1)   1.14       41279_f_at   MAPK8IP1 Mitogen-activated protein kinase 8 interacting       1.25           protein 1       41280_r_at   MAPK8IP1: mitogen-activated protein kinase 8 interacting   −1.31   −1.31           protein 1       1509_at   Metalloproteinase   −1.42       976_s_at   mitogen-activated protein kinase 1   −1.61   1.12       34006_s_at   mitogen-activated protein kinase 8   1.32       1439_s_at   mitogen-activated protein kinase-activated protein kinase 2       1.78       37565_at   MMD: monocyte to macrophage differentiation-associated   1.28   1.30       38369_at   myeloid differentiation primary response gene (88)       −1.10       1052_s_at   NF-IL6-beta protein       1.30       36472_at   N-myc and STAT interacter-       −1.35       38354_at   nuclear factor NF-IL6 (AA 1-345)       1.92       33106_at   nuclear orphan receptor LXR-alpha nuclear receptor subfamily       3.29           1, group H, member 3       33381_at   nuclear receptor co-activator       1.11       279_at   nuclear receptor subfamily 4, group A, member 1       2.30       280_g_at   nuclear receptor subfamily 4, group A, member 1       3.08       37623_at   nuclear receptor subfamily 4, group A, member 2 Member of the       27.72           steroid/thyroid hormone receptor family       547_s_at   nuclear receptor subfamily 4, group A, member 2 Member of the       26.77           steroid/thyroid hormone receptor family       190_at   nuclear receptor subfamily 4, group A, member 3 Member of       5.45           steroid/thyroid receptor family of nuclear hormone receptors       41202_s_at   OS-4 protein (OS-4)   1.24       1451_s_at   OSF-2os osteoblast specific factor-2 (periostin)   −1.65       38822_at   O-sialoglycoprotein endopeptidase       2.43       467_at   osteoclast stimulating factor (OSF   −1.23       35107_at   osteoprotegerin ligand       3.33       33814_at   PAK4 protein   1.16       38757_at   PDGF associated protein.   −1.89       40253_at   phosphatidylinositol 4-kinase (NPIK-C).   1.77       37412_at   phosphatidylinositol-4-phosphate 5-kinase isoform C (−1)   −1.87       751_at   phosphatidylinositol-glycan-class C (PIG-C)   1.14   −1.25       666_at   phosphodiesterase 4A, cAMP-specific   1.33   1.30       38526_at   phosphodiesterase 4D, cAMP-specific   1.30   3.53       38921_at   phosphodiesterase IB, calmodulin-dependent   1.52       38944_at   phosphodiesterase IB, calmodulin-dependent       1.17       32029_at   phosphoinositide dependent protein kinase-1 (3)       1.16       31699_at   phosphoinositide-3-kinase   1.56   1.16       1085_s_at   phospholipase C       −1.14       364_s_at   phospholipase C b3   1.22       901_g_at   phospholipase C, beta 4   −1.20       1293_s_at   phospholipase D   −1.26       32306_g_at   preprocollagen type I alpha-2   1.19       35473_at   preprocollagen type I alpha 1.   −2.72       38951_at   PRKCQ Protein kinase C, theta       1.43       32307_s_at   procollagen   1.13       34494_at   procollagen I-N proteinase.       1.92       37605_at   procollagen type II alpha 1       1.91       36109_at   prolidase (imidodipeptidase) PEPD   −2.55       1884_s_at   proliferating cell nuclear antigen   −1.85       34390_at   prolyl 4-hydroxylase alpha (II) subunit       1.19       37037_at   prolyl 4-hydroxylase alpha subunit       1.20       36666_at   prolyl 4-hydroxylase beta   1.95       36533_at   prostacyclin synthase       1.20       718_at   protease, serine, 11 (IGF binding)   −1.30       719_g_at   protease, serine, 11 (IGF binding)   −1.43       385_at   proteasome (prosome, macropain) subunit, beta type, 10   1.36       39183_at   protein kinase 1 PCTAIRE   −1.17       37698_at   protein kinase A (PRKA) anchor protein 1       1.29       39711_at   protein kinase C substrate 80K-H       1.13       39161_at   protein kinase Njmu-R1       1.21       35348_at   protein kinase, AMP-activated, beta 1 non-catalytic subunit       2.10       36359_at   protein kinase, cAMP-dependent, catalytic, gamma   1.39       546_at   protein kinase, cAMP-dependent, catalytic, inhibitor alpha       1.14       227_g_at   protein kinase, cAMP-dependent, regulatory, type I, alpha       1.18       41768_at   protein kinase, cAMP-dependent, regulatory, type I, alpha       1.15       1091_at   protein kinase, cAMP-dependent, regulatory, type I, beta   1.65       116_at   protein kinase, cAMP-dependent, regulatory, type II, alpha   1.28       33633_at   purinergic receptor P2Y, G-protein coupled, 11   1.90       32737_at   RAC2 Ras-related C3 botulinum toxin substrate 2 (rho family,   1.16           small GTP binding protein Rac2)       40299_at   RE2 G-protein coupled receptor       1.24       35668_at   receptor (calcitonin) activity modifying protein 1 RAMP 1       1.34       40696_at   receptor (TNFRSF)-interacting serine-threonine kinase 1       1.12       1007_s_at   receptor tyrosine kinase DDR   1.21       37701_at   regulator of G-protein signalling 2, 24 kD       2.06       1048_at   retinoid X receptor-gamma   1.47   1.34       36217_at   serine/threonine kinase 38   1.54       41544_at   serum-inducible kinase       1.16       32447_at   SF-1; Steroidogenic factor-1   8.76       36487_at   short stature homeobox 2,       −1.46       41222_at   signal transducer and activator of transcription 6 (STAT6)   1.44       1955_s_at   SMAD6 (inhibits BMP/Smad1 (MADH1) signalling)   1.19       37718_at   SNF-1 related kinase   1.49   1.19       35883_at   Spi-B       −2.80       1244_at   Stat2       −1.12       506_s_at   Stat5A       1.16       38994_at   STAT-induced STAT inhibitor-2       1.25       38669_at   Ste20-related serine/threonine kinase   1.24   1.65       37152_at   steroid hormone receptor superfamily       1.19       35844_at   syndecan 4       1.37       38374_at   TEIG; TGFB inducible early growth response   1.18       38427_at   TEIG; TGFB inducible early growth response       1.38       32080_at   tetracycline transporter-like protein       1.41       224_at   TGFB inducible early growth response; TIEG   1.26       36940_at   TGFB1-induced anti-apoptotic factor 1   1.22   1.60       32217_at   TGF-beta induced apotosis protein 12   1.40       41445_at   TGF-beta precursor   1.14       1890_at   TGF-beta superfamily protein   1.74       40631_at   Tob   −1.14   1.59       39358_at   transcriptional co-repressor nuclear receptor co-repressor 2       1.42       1385_at   transforming growth factor induced protein       1.36       1830_s_at   transforming growth factor-beta       1.17       1767_s_at   transforming growth factor-beta 3 (TGF-beta 3)   −1.71   −1.63       40581_at   TRIO: triple functional domain (PTPRF interacting)   1.65   1.56       32272_at   tubulin alpha   −1.20       685_f_at   tubulin alpha isotype H2-alpha   −4.36   −1.79       330_s_at   tubulin alpha, 1,   −1.80   −1.15       151_s_at   tubulin beta   −1.40       39399_at   tubulin beta cofactor D   −1.85       471_f_at   tubulin beta, 4   −1.38       40567_at   tubulin, alpha 3   −1.39       709_at   tubulin, beta 3   −1.18       33678_i_at   tubulin, beta, 2   −1.15       33679_f_at   tubulin, beta, 2   −1.31       1651_at   ubiquitin carrier protein E2-C   −3.74   −1.22       32548_at   unactive progesterone receptor       −1.33       1953_at   vascular endothelial growth factor   1.40   1.20       36101_s_at   vascular endothelial growth factor   1.45   1.44       36140_at   Y box binding protein-1   2.30   5.49                 − numbers = fold down-regulated            + numbers = fold up-regulated             
 
      Real-time PCR. Based on the DNA microarray data a set of transcripts was chosen for quantitative analysis by real time-PCR (RT-PCR).  
      Briefly, the method exploits the SyBr Green dye which intercalates into double stranded DNA. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the SyBr Green dye. Reactions are characterised by the point in time during cycling when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the starting copy number of nucleic acid target, the sooner a significant increase in fluorescence is observed.  
      From each RNA sample, cDNA was made using an Applied Biosystem kit (Applied Biosystems # N808-0234) following the recommendation of the manufacturer. The PCR mixture was prepared using the SyBr Green Universal PCR Master Mix (Applied Biosystems # 4309155) as follows: 5 μl cDNA template, 400 nM of each primer, 0.2 mM deoxynucleotide triphosphates, 1 mM MgCl2 and 0.5 U Taq DNA polymerase, 5 μl SyBr Green PCR buffer and RNase free water up to a final volume of 50 μl. The PCR was performed using the ABI Prism 7700 Sequence Detection System, after a step at 95° C. for 10 min, the step-cycle program was performed for a total of 40 cycles as follows: 95° C. for 30 s, 60° C. for 1 min. A negative control was included: PCR reaction mixture with water in place of the cDNA sample.  
      The initial template concentration was determined based on the threshold cycle. The threshold cycle is the PCR cycle at which fluorescence is first detected above background and has been shown to be inversely proportional to the number of target copies present in the sample. Quantification was performed by calculating the unknown target concentration relative to an absolute standard and by normalizing to a validated endogenous control such as a housekeeping gene (β-actin). Results are presented as percentage of control, once the ratio between the numbers of molecule for the gene of interest divided by the number of molecule for beta-actin has been calculated.  
      Based on the DNA microarray data the following set of transcripts was chosen for quantitative analysis by RT-PCR: adhesion receptor CD44, angiopoietin, bone morphogenetic protein 5, carbonic anhydrase II, cartilage oligomeric matrix protein, cathepsin K, osteopontin, pre-pro-alpha-2 type I collagen, Spi-B and Y-box binding protein.  
               TABLE 15                          Real Time PCR Results                             GeneChip ®       Treatment Effect   Treatment Effect       Expression Probe       Salmon Calcitonin   PTS893       Set Identifier   Coding Gene   (% respect to control)   (% respect to control)               1372_at   adhesion receptor   No change   No change           CD44       1929_at   angiopoietin-1   No change   No change       1831_at   bone morphogenetic   +16   +18           protein 5       40095_at   carbonic anhydrase II   −60   No change       40161_at   cartilage oligomeric   +34.23   No change           matrix protein       128_at   cathepsin K   +67.2   No change       2092_s_at   osteopontin   No change   No change       32306_g_at   pre-pro-alpha-2 type I   +38   +62           collagen       35883_at   Spi-B   −44   −18       36140_at   Y-box binding   +14   +26           protein (bone)       36140_at   Y-box binding   +15   n.a.           protein (kidney)       36140_at   Y-box binding   −26   n.a.           protein (muscle)                 n.a.: not applicable             
 
      RT-PCR confirmed in most of the cases the changes observed in the gene profiling analysis, as it was the case for bone morphogenetic protein 5, carbonic anhydrase II, cathepsin K, cartilage oligomeric matrix protein, pre-pro-alpha-2 type I collagen, Spi-B and Y-Box binding protein. No changes were however detected in the level of expression of adhesion receptor CD44, angiopoietin-1 and osteopontin.  
      Analysis. Calcitonin is known to exert an effect on the differentiation, survival and resorptive activity of osteoclasts, resulting in a decreased osteoclastic activity. Pondel M,  Intl. J. Exp. Pathol . 81(6): 405-22 (2000). These effects could be reconstructed by multi-organ gene profiling (TABLE 16).  
               TABLE 16                          Effects on Osteoclasts                                     Salmon           Function   Coding genes   Calcitonin   PTS893               Osteoclast   PU.1 (SPI1)   B, K, P, T   B       determination.   Granulocyte to macrophage   B, K   B       survival and   colony-stimulating factor (CSF1)       differentiation   Monocyte to macrophage   B, K, T   B           differentiation associate (MMD)           Osteoclast stimulating factor 1   B, K, L, P           (Autocrine stimulation of           osteoclast resorptive activity)       Bone resorption   H+ ATP-ases   ALL   B       by osteoclast   Carbonic anhydrase I, II.   B, L, P           Cathepsin K   ALL           ODF/OPGL: osteoprotegerin       B           ligand       Osteoclast   Tubulins   ALL       motility   PAK4 protein   B, M, P                 Multi-organ gene expression profiling in salmon calcitonin treated animals. Organs where changes in expression were seen are displayed.            B = bone;            K = kidney;            M = muscle;            P = pituitary;            L = liver;            T = trachea.             
 
      Salmon calcitonin seems to exert a paracrine regulation of the osteoclast resorptive activity, through the regulation of cystatin expression in the osteoblast, as shown in TABLE 17.  
               TABLE 17                          Gene Expression Profiling: Osteoclast Function                                 GeneChip ®                       expression       probe set       Average   Average   Fold       identifier   Coding Gene   controls   sCT   change                                         40729_s_at   ATPase, H+ transporting,   204   327   1.6           lysosomal (vacuolar proton           pump) subunit G isoform 2       37367_at   ATPase, H+ transporting,   272   328   1.2           lysosomal 31 kDa, V1           subunit E isoform 1       40568_at   ATPase, H+ transporting,   938   1132   1.21           lysosomal 56/58 kDa, V1           subunit B, isoform 2       39241_at   carbonic anhydrase I   1266   441   −2.87       128_at   cathepsin K (pycnodysostosis)   5690   7821   1.37       129_g_at   cathepsin K (pycnodysostosis)   5036   6757   1.34       38466_at   cathepsin K (pycnodysostosis)   5494   7267   1.32       36611_at   acid phosphatase 1, soluble   254   331   1.3                  
 
      PU.1 is involved in the initial stages of osteoclastogenesis. Tondravi M M et al.,  Nature  386(6620): 81-4 (1997). CSF-1 is imperative for macrophage maturation; it binds to its receptor c-fms on early osteoclast precursors, providing signals required for their survival and proliferation. Teitelbaum S L,  Science  289(5484):1504-1508 (2000).  
      Interestingly, PTS893 also regulates the genes implicated in osteoclast differentiation and survival, SPI1, CSF-1 and MMD. This osteoclast regulation has not been previously described.  
      Salmon calcitonin was shown to regulate the expression of the gene coding for osteoclast stimulating factor (OSF), which is an intracellular protein produced by osteoclasts that indirectly induces osteoclast formation and bone resorption. Reddy S et al.,  J. Cell Physiol . 177 (4): 636-45 (1998). This would imply an autocrine effect of salmon calcitonin in the regulation of the osteoclast function, which is described here for the first time.  
      In addition, salmon calcitonin seems to exert a paracrine regulation of the osteoclast resorptive activity, through the regulation of cystatin expression in the osteoblast. Carbonic anhydrase I, II, H + -ATPases and cathepsin K are the main effectors for dissolving bone mineral and matrix degradation. Blair H C et al.,  Biochem . (2002). Regulation of tubulins and PAK4 genes can be related to the effect of calcitonin on osteoclast motility PAK 4. Zaidi M et al.,  Bone  30(5): 655-63 (2002); Jaffer Z M &amp; Chemoff J,  Intl. J. Biochem. Cell Biol . 34(7): 713-7 (2002).  
      These results show modulating effects of calcitonin on genes affecting the direct, autocrine, paracrine and endocrine regulation of the osteoblast function (TABLE 18). These data support the hypothesis that attributes a bone anabolic effect to calcitonin.  
               TABLE 18                          Effects on Osteoblasts                                     Salmon           Function   Coding Gene   Calcitonin   PTS893               Antagonists of   Cystatins   B           cathepsins:   Alpha-2-HS-Glycoprotein   B, K, T       antiresorptive activity   Bone Morphogenetic Proteins   ALL   B       Autocrine/paracrine   Fibroblast Growth Factors   B, K, M, P, T   B       regulation of   IL6/LIF       B       osteoblast function   Insulin-like Growth Factors   ALL   B           TGFs   B, K, M, P   B           Tob   B, M, P   B           Vascular Endothelial Growth Factor   B, M   X       Endocrine regulation   Activin   B, L, M, P   B       of osteoblast function   Oestrogen receptor   ALL           Retinoic receptor X   B, P   B           Steroidogenic factor   B, L, P, T           nuclear receptors (steroid/thyroid       B           family)       Transcription factor   Y-box binding protein   B, K, M, P   B       that regulates collagen       type 1 synthesis                 Multi-organ gene expression profiling in salmon calcitonin treated animals. Organs where changes in expression were seen are displayed.            B = bone;            K = kidney;            M = muscle;            P = pituitary;            L = liver;            T = trachea.             
 
      The results of this EXAMPLE show modulating effects of calcitonin on genes affecting the direct, autocrine, paracrine and endocrine regulation of the osteoblast function. These data support the hypothesis that attributes a bone anabolic effect to calcitonin.  
      Three families of growth factors, the transforming growth factor betas (TGF-βs), insulin-like growth factors (IGFs), and bone morphogenetic proteins (BMPs), are considered to be principal local regulators of osteogenesis. Bone morphogenetic proteins are thought to have their major effects on early precursor bone cell replication and osteoblast commitment. In contrast, TGB-βs are thought to be the most potent inducers of committed bone cell replication and osteoblast matrix production, while IGFs appear to integrate and extend the effect of both factors. McCarthy T L et al.,  Crit Rev. Oral Biol. Med . 11(4): 409-22 (2000). These results support the fact that both salnon calcitonin and PTS893 are able to regulate these local and systemic factors implicated in bone metabolism.  
      The fact that salmon calcitonin regulates α2-HS glycoprotein (AHSG), which blocks TGF-β-dependent signalling in osteoblastic cells, also supports this role. Mice lacking AHSG display growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis. Szweras M et al.,  J. Biol. Chem ., 277(22): 19991-19997 (2002).  
      Salmon calcitonin and PTS893 were also shown to modulate the expression of the genes coding for vascular endothelial growth factor (VEGF). VEGF is known for playing a key role in normal and pathological angiogenesis. The critical role of angiogenesis for successful osteogenesis during the endochondral ossification is well documented. VEGF indirectly induces proliferation and differentiation of osteoblasts by stimulating endothelial cells to produce osteoanabolic growth factors. Wang D S et al.,  Endocrinology  138(7): 2953-62 (1997). In addition, VEGF stimulates chemotactic migration of primary human osteoblasts, suggesting a functional role in bone formation and remodelling. Mayr-Wohlfahrt U et al.,  Bone  30 (3): 472-7 (2002).  
      The effects of parathyroid hormone on osteoblast for mediating both bone resorption and formation have been widely described. Swarthout J T et al.,  Gene  282(1-2):1-17 (2002). It was here possible to confirm the effect of PTS893 on cytokines like interleukin 6 (IL-6), which mediates the paracrine activation of osteoclast differentiation and activity. Greenfield E M et al.,  Life Sci . 65:1087-102 (1999). PTS893 also produced a strong up-regulation on nuclear receptors (steroid/thyroid family).  
               TABLE 19                          Gene Expression Profiling: Growth Factors and Hormones                                 GeneChip ®                       expression       probe set       Average       identifier   Coding Gene   controls   Average sCT   Fold change                                         39407_at   bone morphogenetic protein 1   448   607   1.36       1122_f_at   chorionic gonadotropin, beta polypeptide   263   380   1.44       39945_at   fibroblast activation protein, alpha   636   436   −1.46       1970_s_at   fibroblast growth factor receptor 2   184   108   −1.69           (bacteria-expressed kinase, keratinocyte           growth factor receptor, craniofacial           dysostosis 1, Crouzon syndrome, Pfeiffer           syndrome, Jackson-Weiss syndrome)       32254_at   follistatin-like 3 (secreted glycoprotein)   1514   2209   1.46       38737_at   insulin-like growth factor 1 (somatomedin   66   37   −1.79           C)       36782_s_at   insulin-like growth factor 2 (somatomedin   212   323   1.52           A)       1591_s_at   insulin-like growth factor 2 (somatomedin   293   402   1.37           A)       40422_at   insulin-like growth factor binding protein   181   105   −1.73           2, 36 kDa       37319_at   insulin-like growth factor binding protein 3   495   1561   3.15       1586_at   insulin-like growth factor binding protein 3   428   722   1.69       37319_at   insulin-like growth factor binding protein 3   604   879   1.46       1586_at   insulin-like growth factor binding protein 3   355   445   1.25       1451_s_at   osteoblast specific factor 2 (fasciclin I-   538   292   −1.84           like) periostin       532_at   parathyroid hormone receptor 1   1337   1849   1.38       234_s_at   pleiotrophin (osteoblast specific factor 1)   710   507   −1.4       34820_at   pleiotrophin (heparin binding growth   422   329   −1.28           factor 8, neurite growth-promoting factor           1)       1897_at   transforming growth factor beta 1 induced   176   296   1.68           transcript 1       1385_at   transforming growth factor, beta-induced,   187   292   1.57           68 kDa       39588_at   tumour necrosis factor (ligand)   176   127   −1.39           superfamily, member 12       31410_at   tumour necrosis factor (ligand)   197   128   −1.54           superfamily, member 4       38631_at   tumour necrosis factor receptor   134   240   1.79           superfamily, member 13B       35150_at   tumour necrosis factor receptor   443   298   −1.48           superfamily, member 5       595_at   tumour necrosis factor, alpha-induced   118   191   1.62           protein 3       1953_at   vascular endothelial growth factor   351   557   1.59       36100_at   vascular endothelial growth factor   282   407   1.45       1953_at   vascular endothelial growth factor   521   629   1.21       37268_at   vascular endothelial growth factor B   379   504   1.33       39091_at   vitamin A responsive; cytoskeleton related   421   299   −1.41                  
 
      Both calcitonin and parathyroid hormone receptors belong to the G-protein receptor superfamily. After receptor stimulation, signal transduction is mediated by adenylate cyclase/cAMP/protein kinase, Phospholipase C, Phospholipase D, and MAPK (as a late effecter) pathways in the case of calcitonin, and by adenylate cyclase and phospholipase C in the case of parathyroid hormone. Gene profiling analysis allowed the reconstruction of these pathways, showing genes that were modulated by the treatment and that are localised at different levels of the signal transduction pathway.  
               TABLE 20                          Effects on Signal Transduction and Cell Cycle                                     Salmon           Function   Coding Gene   Calcitonin   PTS893               Signal   Adenylate cyclase       B       transduction.   Calcyclin binding protein       B           Calreticulin   B, K, M           CREM   B, L, P   B           CDC Kinase   B, M           MAPK   ALL   B           Protein kinases   ALL           Phosphatidylinositol pathway   ALL   B           Phosphodiesterase (IB, 4A, 4B)   ALL   B           Phospholipase (C, D)   ALL   B           PCNA   B           SMAD pathway   ALL   B           STAT pathway   ALL   B       Cell cycle   Cyclins (A, A2, B1, C, D2,   B   B           E2, G1, G2)           Cyclin-dependent kinases 5, 6, 10   B, K, P, T   B           Cyclin-dependent kinases   B   B           inhibitor 1A, 1C, 2D)                 Multi-organ gene expression profiling in salmon calcitonin treated animals. Organs where changes in expression were seen are displayed.            B = bone;            K = kidney;            M = muscle;            P = pituitary;            L = liver;            T = trachea.             
 
      Salmon calcitonin seems also to exert a direct influence on cell cycle, since changes in cyclins and cyclin-related proteins could be also observed, as shown in TABLE 21.  
               TABLE 21                          Gene Expression Profiling: Signal Transduction                                 GeneChip ®                       expression       probe set       Average   Average   Fold       identifier   Coding Gene   controls   sCT   change                                         769_s_at   annexin A2   8393   6969   −1.2       32066_g_at   cAMP responsive element   168   231   1.38           modulator       40777_at   catenin (cadherin-associated   3688   4689   1.27           protein), beta 1, 88 kDa       40697_at   cyclin A2   212   128   −1.65       40697_at   cyclin A2   272   175   −1.56       1943_at   cyclin A2   121   83   −1.45       2020_at   cyclin D1 (PRAD1:   238   135   −1.76           parathyroid adenomatosis 1)       36650_at   cyclin D2   204   312   1.53       40225_at   cyclin G associated kinase   827   1011   1.22       31700_at   G protein-coupled receptor 35   844   591   −1.43       41074_at   G protein-coupled receptor 49   242   146   −1.66       33082_at   integrin, alpha 10   171   243   1.42       33082_at   integrin, alpha 10   228   289   1.26       33411_g_at   integrin, alpha 6   65   35   −1.86       33410_at   integrin, alpha 6   201   90   −2.22       38297_at   phosphatidylinositol transfer   753   1006   1.34           protein, membrane-associated       31904_at   phosphodiesterase 2A, cGMP-   555   740   1.33           stimulated       38269_at   protein kinase D2   747   1067   1.43       36008_at   protein tyrosine phosphatase   518   376   −1.38           type IVA, member 3       35361_at   PTEN induced putative   95   255   2.69           kinase 1       35178_at   WNT inhibitory factor 1   1746   2127   1.22                  
 
      Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signalling in osteoblasts. Smad pathway as well as Tob as one of their regulators were also identified as genes modulated by the sCT and PTS893 treatment, in agreement with the hypothesised effect of both compounds on BMP regulation of bone remodelling. Within this context, both compounds seem to exert a direct influence on cell cycle, since changes in cyclins and cyclin-related proteins could be also observed.  
      Both compounds regulate also synthesis and degradation of extracellular matrix components (TABLE 22).  
               TABLE 22                          Effects on Extracellular Matrix                                     Salmon           Function   Coding Gene   Calcitonin   PTS893               Cell attachment.   Integrins   B, M, P   B       Signal transduction.       Collagen digestion   Collagenase   B           Matrix metalloproteinases I, II   B, L, P, T       Collagen synthesis   Procollagen endopeptidase/proteinase       B           Lysyl hydroxylase       B       Extracellular matrix   Aggrecan       B       component   Cartilage Oligomeric Matrix Protein   B, K,           Precursor           Collagen type I, type II, type III, type IV,   ALL   B           type V, type VI, type IX, type X, type XI,           type XIII, type XIV, type XV, and/or type XVI)           Chondroitin sulphate proteoglycan   K, M, T   B           Dermatopontin       B           Heparan sulphate proteoglycan   L, T   B           Syndecan       B                 Multi-organ gene expression profiling in salmon calcitonin-treated animals. Organs where changes in expression were seen are displayed.            B = bone;            K = kidney;            M = muscle;            P = pituitary;            L = liver;            T = trachea.             
 
      Salmon calcitonin regulates also the synthesis and degradation of extracellular matrix components, as shown in TABLE 23.  
               TABLE 23                          Gene Expression Profiling: Extracellular Matrix                                 GeneChip ®                       expression       probe set       Average   Average   Fold       identifier   Coding Gene   controls   sCT   change                                         36253_at   bone gamma-carboxyglutamate (gla)   26305   33265   1.26           protein (osteocalcin)       32094_at   carbohydrate (chondroitin 6)   253   130   −1.95           sulfotransferase 3       32094_at   carbohydrate (chondroitin 6)   292   241   −1.21           sulfotransferase 3       41447_at   carbohydrate (chondroitin) synthase 1   192   107   −1.79       34042_at   chondroadherin   7965   10266   1.29       32306_g_at   collagen, type I, alpha 2   7740   9337   1.21       32488_at   collagen, type III, alpha 1 (Ehlers-Danlos   2399   1294   −1.85           syndrome type IV, autosomal dominant)       34802_at   collagen, type VI, alpha 2   2374   1500   −1.58       35816_at   cystatin B (stefin B)   983   1897   1.93       34029_at   dentin matrix acidic phosphoprotein   216   587   2.72       38059_g_at   dermatopontin   695   962   1.38       38057_at   dermatopontin   1090   1381   1.27       33929_at   glypican 1   235   163   −1.44       39350_at   glypican 3   64   50   −1.29       37176_at   hyaluronoglucosaminidase 1   109   266   2.43       1546_at   hyaluronoglucosaminidase 1   49   79   1.59       36683_at   matrix Gla protein   65   117   1.8       609_f_at   metallothionein 1B (functional)   2693   3485   1.29       870_f_at   metallothionein 3 (growth inhibitory factor   1744   2296   1.32           (neurotrophic))       38307_at   neurochondrin   452   696   1.54       34342_s_at   secreted phosphoprotein 1 (osteopontin,   16370   21279   1.3           bone sialoprotein I)       38127_at   syndecan 1   534   346   −1.54       1693_s_at   tissue inhibitor of metalloproteinase 1   4549   5522   1.21           (erythroid potentiating activity,           collagenase inhibitor)       2092_s_at   secreted phosphoprotein 1 (osteopontin,   7748   9576   1.24           bone sialoprotein I, early T-lymphocyte           activation 1)       38308_g_at   neurochondrin   679   490   −1.39                  
 
      Of particular interest is the regulation of the Y-Box binding protein (YB-1), which appears to be modulated by both treatments and in four out of six organs analysed in the salmon calcitonin group. YB-1 is a protein that interacts with a TGF-β response element in the distal region of the collagen alpha 1(I) gene. YB-1 protein activates the collagen promoter and translocates into the nucleus during TGF-β addition to fibroblasts, suggesting a role for this protein in TGF-β signalling. Sun W et al.,  Matrix Biol . 20(8): 527-41 (2001).  
      In addition, salmon calcitonin and PTS893 regulated some aspects of the mineralization of the bone extracellular matrix, since changes in amelogenin, dentin and ectonucleotide pyrophosphatases were observed.  
               TABLE 20                          Effects on Mineralization and Visualisation                                     Salmon           Function   Coding Gene   Calcitonin   PTS893               Cement component   Amelogenin   B, L   B       Mineral matrix protein   Dentin   B   B       Enzyme for synthesis of   Ectonucleotide   B, M       inorganic Pi   pyrophosphatases       Growth factor   VEGF   B, M   B       vascularisation                 Multi-organ gene expression profiling in salmon calcitonin treated animals. Organs where changes in expression were seen are displayed.            B = bone;            K = kidney;            M = muscle;            P = pituitary;            L = liver;            T = trachea.             
 
      All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. In addition, all GenBank accession numbers, Unigene Cluster numbers and protein accession numbers cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each such number was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.  
      The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatus within the scope of the invention, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications and variations are intended to fall within the scope of the appended claims. The present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.