Development of novel bone forming agents from natural and synthetic sources

Abstract

Osteoporosis is a disease of bones that leads to an increased risk of fracture. In osteoporosis the bone mineral density (BMD) is reduced, bone micro architecture is deteriorating, and the amount and variety of proteins in bone is altered. Osteoporosis is defined by the World Health Organization (WHO) as a bone mineral density that is 2.5 standard deviations or more below the mean peak bone mass as measured by DXA. The term "established osteoporosis" includes the presence of a fragility fracture. The disease may be classified as primary type 1, primary type 2, or secondary [1].The form of osteoporosis most common in women after menopause is referred to as primary type 1 or postmenopausal osteoporosis. Primary type 2 osteoporosis or senile osteoporosis occurs after age 75 and is seen in both females and males at a ratio of 2:1. Osteoporosis is an increasing public health problem for the ageing society. For India's growing aging population, osteoporosis will emerge as one of the major drains of public fund unless indigenous and cost-effective therapies are developed [2]. For women at menopause, a widely used timely estrogen hormone replacement therapy (HRT) has been discontinued due to cardio-vascular side effects [3]. All currently available, approved therapies for osteoporosis are anti-resorptive [4]. Anti-resorptive agents reduce fracture risk that is not more than 50% of the baseline risk. However, despite their great value, the anti-resorptive agents are not generally associated with increases in bone mass to any significant extent. Increase in bone mass requires discovery of agents that will enhance osteoblast functions – popularly ascribed as bone anabolic/osteogenic therapy by which bone formation is directly stimulated. Currently available anabolic therapies for treatment of bone loss include PTH, NaF and strontium ranelate [5-7]. PTH enhances the recruitment of pre-osteoblasts from marrow stromal cells, induces the maturation of lining osteoblasts and also reduces osteoblast apoptosis [8]. Recombinant human PTH (Teriparatide) has been approved in USA as monotherapy for the treatment of postmenopausal women with osteoporosis and men with low bone density and osteoporosis [9]. Intermittent PTH has demonstrated increase in cancellous bone mass at several sites, but has no effect on cortical bone [10]. The other most prevalent anabolic agent, NaF stimulates the osteoblasts to lay down osteoid and leads to increase in bone mass [11]. However, the use of fluoride has been associated with marked increases in vertebral BMD but it also increases the risk of non-vertebral fractures [12]. Very recently, strontium ranelate (Protelos) has been introduced as dual action agent that enhances osteoblast function as well as inhibits osteoclast function [13]. Since, strontium acts by activating calcium-sensing receptor (a cell surface G protein-coupled receptor) that acutely regulates PTH secretion, strontium could pose the risk of adversely modulating systemic calcium homeostasis [14]. In addition to these three available bone anabolic therapies, several other candidates are currently under investigation for their potential bone anabolic action. These are insulin-like growth factor (IGF-1), bone morphogenetic protein (BMP-2), and statins (HMG coenzyme A reductase inhibitors) [15-17]. Sclerostin deficiency in humans, together with the data from SOST knockout mice, suggests that sclerostin inhibition might be a viable approach for the development of novel anabolic agents [18, 19]. Of note, the marked increase in bone mass found in these genetic cases of life-long sclerostin deficiency includes the early stage of life that is normally characterized by rapid skeletal growth and bone mass accrual. As such, it is not known what the extent and magnitude of sclerostin's role is in the control of bone formation and bone mass during the clinically important later stages of life, when bone mass accrual has ceased and when the incidence of bone-related disorders, such as postmenopausal osteoporosis (PMO), is highest [20, 21] Odanacatib is a powerful, reversible nonpeptidic biaryl inhibitor of cathepsin K that inactivates the proteolytic activity of cathepsin k. It is synthesized by replacing the P2-P3 amide bond of an aminoacetronintrile dipeptide 1 with a phenyl ring. This results in a powerful, selective inhibitor with the capacity to inhibit cathepsin K in osteoclasts [22, 23]. Tissue selective estrogen complexes (TSEC), the pairings of a selective estrogen receptor modulator (SERM) with estrogen, have been investigated in recent years arising from the need for effective menopausal therapies with tolerability profiles better than those of currently available treatment options [24, 25]. Combining the right SERM and estrogen could result in a blend of activities providing a favourable clinical profile for menopausal women, optimally having the benefits of each of its individual components with improved tolerability [26, 27]. Natural foods, such as soy, contain phytoestrogens (plant-produced substances that act like estrogen in the body). Phytoestrogens, found in many edible plants are diverse groups of biologically active compounds with structural similarity to estradiol [28, 29]. The major estrogenic isoflavones including daidzein, genistein, and biochanin A, have been shown to have important role in reducing symptoms associated with estrogen deficiency disorders [30-33]. These compounds may be protective against osteoporosis due to their ability to exert osteogenic and anti-resorptive actions on bone, particularly on bone turnover and growth [34, 35]. High dietary intakes of these isoflavones have been reported to increase BMD in lumbar spine of Japanese [36], Chinese [37] and American [38] postmenopausal women. In general, individuals who consume diets high in phytoestrogens – particularly phytoestrogens that belong to the subgroup of isoflavones - seem to maintain bone density [39]. Therefore, isoflavones are of interest as potential osteoporosis prevention. Much more longterm controlled research is needed to prove their bone protective effect. The present study was designed to: 1. To evaluate the natural and synthetic products for osteogenic activity using rat calvarial osteoblast culture. These cultures will be used to assay the in vitro bone formation and mineralization in response to test agents. Pertinently, age related bone loss occurs because of decreased osteoblastic activity. 2. To evaluate the in vivo efficacy of osteogenic agent in growing Sprague-Dawley rats. For this test agent(s) will be administered to immature (21 day old), Sprague-Dawley rats to study their anti-osteoporosis activity using biochemical markers of bone formation viz. serum alkaline phosphatase and osteocalcin, and bone histomorphometry. 3. Analysis of gene expression involved in osteoblastogenesis and those expressed on osteoblasts and affecting osteoclastogenesis will be carried out in presence or absence of test agent(s) by Real-time PCR. Besides, signaling studies to understand the molecular mode of action of test agent(s) will also be carried out. 4. Bone turnover rate in in vivo condition is coupled by the activity of both the cell types, osteoblasts and osteoclasts. An in vitro osteoblast-osteoclast co-culture model will thus be established which would serve to study the cross talk between these two cell types in presence of test agent(s). 5. To evaluate the estrogen agonist and antagonist activity using three day immature rat bioassay. Pertinently, HRT/ERT or estrogenic molecules are known to increase risk of uterine hypertrophy, cancerous breasts etc. after longterm administration (Korach et al., 1991). Agent(s) showing no or negligible estrogenic activity in these test systems would be identified for detailed evaluation.