Lydia Codetta Pan

Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2−/− mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2−/− mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.

Pyrazolinone-piperidine dipeptide growth hormone secretagogues (GHSs) : Discovery of capromorelin

Novel pyrazolinone-piperidine dipeptide derivatives were synthesized and evaluated as growth hormone secretagogues (GHSs). Two analogues, capromorelin (5, CP-424391-18, hGHS-R1a K(i)=7 nM, rat pituicyte EC(50)=3 nM) and the des-methyl analogue 5c (hGHS-R1a K(i)=17 nM, rat pituicyte EC(50)=3 nM), increased plasma GH levels in an anesthesized rat model, with ED(50) values less than 0.05 mg/kg iv. Capromorelin showed enhanced intestinal absorption in rodent models and exhibited superior pharmacokinetic properties, including high bioavailabilities in two animal species [F(rat)=65%, F(dog)=44%]. This short-duration GHS was orally active in canine models and was selected as a development candidate for the treatment of musculoskeletal frailty in elderly adults.

Discovery and biological characterization of capromorelin analogues with extended half-lives

New tert-butyl, picolyl and fluorinated analogues of capromorelin (3), a short-acting growth hormone secretagogue (GHS), were prepared as part of a program to identify long-acting GHSs that increase 24-h plasma IGF-1 levels. Compounds 4c and 4d (ACD LogD values >or=2.9) displayed extended plasma elimination half-lives in dogs, primarily due to high volumes of distribution, but showed weak GH secretagogue activities in rats (ED(50)s>10 mg/kg). A less lipophilic derivative 4 (ACD LogD=1.6) exhibited a shorter canine half-life, but stimulated GH secretion in two animal species. Repeat oral dosing of 4 in dogs for 29 days (6 mg/kg) resulted in a significant down-regulation of the post dose GH response and a 60 and 40% increase in IGF-1 levels relative to pre-dose levels at the 8- and 24-h post dose time points. Compound 4 (CP-464709-18) has been selected as a development candidate for the treatment of frailty.

Common mechanism for the estrogen agonist and antagonist activities of droloxifene

The incidence of postmenopausal osteoporosis is increasing as the population ages. Even though estrogen replacement therapy has proven beneficial in reducing the number of skeletal fractures, the known risks and associated side‐effects of estrogen replacement therapy make compliance poor. Recent research has focused on the development of tissue specific estrogen agonist/anatagonists such as droloxifene which can prevent estrogen deficiency‐induced bone loss without causing uterine hypertrophy. Furthermore, droloxifene acts as a full estrogen antagonist on breast tissue and is being evaluated for treatment of advanced breast cancer. In this report we propose a common mechanism of action for droloxifene that underlies its estrogen agonist and antagonist effects in different tissues. Droloxifene and estrogen, which have identical effects on bone in vivo, both induced p53 expression and apoptosis in cells of in vitro rat bone marrow cultures resulting in a decrease in the number of bone‐resorbing osteoclasts. Droloxifene is growth inhibitory in MCF‐7 human breast cancer cells and therefore acts as an antagonist, whereas estrogen is mitogenic to these cells and acts as an agonist. Droloxifene, but not estrogen, induced p53 expression and apoptosis in MCF‐7 cells. These results indicate that the induction of apoptosis by droloxifene may be the common mechanism for both its estrogen agonist effects in bone and its antagonist effects in breast tissue. J. Cell. Biochem. 65:159–171.

Efficacy of a growth hormone-releasing peptide mimetic in cardiac ischemia/reperfusion injury

The cardioprotective efficacy of the pyrazolinone-piperidine dipeptide growth hormone secretagogue (GHS) CP-424,391 was studied in an in vivo rabbit model of ischemia and reperfusion. CP-424,391 was administered at 25 mg/kg p.o. x 7 days. Ischemia was induced by left coronary artery occlusion for 30 min, after which the heart was reperfused for 2 h. At the end of reperfusion, animals were euthanized and the infarct size was determined. The area at risk of infarct was not different between the control (45.8+/-3.7%, n=6) and CP-424,391-treated groups (36.9+/-4.3%, n=11). The infarct size of the control animals was 49.5+/-7.1% and was significantly (P<0.05) lower in the CP-424,391-treated group (infarct size=17.3+/-3.0). There was a trend, albeit not significant, for the left ventricular function to recover to a greater extent in CP-424,391-treated rabbits. Thus, the treatment of rabbits for 7 days with CP-424,391 was cardioprotective against ischemia/reperfusion injury.

Molecular cloning and characterization of the canine prostaglandin E receptor EP2 subtype

Prostaglandin E2 (PGE2) binds to four G-protein coupled cell surface receptors (EP1-EP4) and has been implicated as a local mediator of bone anabolism via a cyclic AMP mediated pathway following activation of the EP2 and/or EP4 receptor subtype. A canine kidney cDNA library was screened using a human EP2 probe, and a clone with an open reading frame of 1083 bp, potentially encoding a protein of 361 amino acids, was characterized. This open reading frame has 89% identity to the human EP2 cDNA at the nucleotide level and 87% identity at the predicted protein level. Scatchard analysis of a CHO cell line stably transfected with canine EP2 yielded a dissociation constant of 22 nM for PGE2. Competition binding studies, using 3H-PGE2 as ligand, demonstrated specific displacement by PGE2, Prostaglandin E1, Prostaglandin A3, and butaprost (an EP2 selective ligand), but not by ligands with selectivity for the related DP, FP, IP, or TP receptors. Specific ligand binding also resulted in increased levels of cAMP in EP2 transfected cells with no evidence of short-term, ligand-induced desensitization. Northern blot analysis revealed two transcripts of 3300 and 2400 bp in canine lung, and reverse-transcription polymerase chain reaction showed expression in all tissues examined. Southern blot analysis suggests the presence of a single-copy gene for EP2 in the dog.

Isolation and Culture of Rodent Osteoprogenitor Cells

Osteoblasts are the cells responsible for formation of new bone throughout life. Rats are one of the most widely studied mammalian species in skeletal biology and serve as useful models for many aspects of human skeletal physiology. The availability of genetically modified mice as research tools has greatly enabled our understanding of how specific genes contribute to the process of skeletogenesis. In order to explore the impact of biochemical, genetic, or pharmacological manipulation on bone formation, various osteogenic cell culture systems have been developed. Two of the most widely accepted rodent osteogenic culture models, using osteoprogenitor cells isolated from calvaria or bone marrow, are described in this chapter.