Adipose tissue is the largest endocrine organ in the human body; it serves considerable role in the energy homeostasis and several metabolic processes. It also has a multifaceted associa‐ tion with the bone. It is a general phenomenon that with increasing age the fat tissue in the bone marrow increased while the bone mass decreased. It is because both the osteoblast and adipocyte are developed from the same cell lineage the mesenchymal stromal cells (MSCs). The segregation tendency to preadipocytes or preosteoblasts is competitive and being inhibited by each other. Nevertheless, published literature advocates that through adipokines, adipose tissue have intricate effects on bone metabolism [63]. The family of adipokines, including leptin, adiponectin, chemerin, resistin, omentin, visfatin and vaspin, take part in several physiological, biochemical and pathological processes, including, the glucose and lipid oxidation, energy expenditure, immunity, reproduction, inflammation, and others. Because of growing evidences regarding interaction of adipokines and bone metabolism, the researchers are paying special attention to their relationship.

Leptin is a diverse 16 kDa peptide hormone, which belong to the family of helical cytokine with lengthy chain (figure 3) [64]. Leptin is an important member of adipokine family that helps in regulation of food intake, energy homeostasis, reproduction, metabolism, immune function, bone physiology, tissue remodeling, neuroendocrine function and others [65].

Research has shown that leptin plays significant role in the regulation of body weight and BMD. Experimental evidence in mice showed that they appeared obese and have increased bone density when they were congenitally absent in leptin (ob/ob), indicating that leptin has a role in loss of bone density as well as fatty tissue [66].

**Figure 3.** Leptin structure [64]

A meta-analysis provided a proof on homocysteine and fracture risk, displaying that HHcy augments the fracture risk [56]. A very important evidence of effect of HHcy on fracture risk was provided by another meta-analysis including more than ten thousand subjects, demon‐

In spite of substantial evidence that indicating the direct damaging role of HHcy on bone metabolism, the role of serum tHcy in bone loss is still unclear. HHcy can be reduced by vit B12, and folate therapy, so by designing randomized controlled trials (RCTs) of B-vitamins vs. placebo, it is quite likely to establish whether tHcy is a contributory risk factor for bone fracture or not [62].There are few RCTs available in literature but because of various reasons these are

Based on evidences, provided in the literature, it seems that HHcy may have negative role in bone metabolism generally and in the process of osteoporosis particularly. However, more interventional studies are required to confirm the unfavorable role of HHcy, particularly in subjects who are at the risk of developing low bone mass. It is suggested that there is a need to develop several multicenter, well-designed trials on determining the effects of Hcy-lowering treatment on the prevention and/or management of osteoporosis. These longitudinal trials would determine the accurate nature of Hcy and bone interaction and that will establish the outcome of different bisphosphonate therapies on Hcy levels in treating osteoporosis.

Adipose tissue is the largest endocrine organ in the human body; it serves considerable role in the energy homeostasis and several metabolic processes. It also has a multifaceted associa‐ tion with the bone. It is a general phenomenon that with increasing age the fat tissue in the bone marrow increased while the bone mass decreased. It is because both the osteoblast and adipocyte are developed from the same cell lineage the mesenchymal stromal cells (MSCs). The segregation tendency to preadipocytes or preosteoblasts is competitive and being inhibited by each other. Nevertheless, published literature advocates that through adipokines, adipose tissue have intricate effects on bone metabolism [63]. The family of adipokines, including leptin, adiponectin, chemerin, resistin, omentin, visfatin and vaspin, take part in several physiological, biochemical and pathological processes, including, the glucose and lipid oxidation, energy expenditure, immunity, reproduction, inflammation, and others. Because of growing evidences regarding interaction of adipokines and bone metabolism, the researchers

Leptin is a diverse 16 kDa peptide hormone, which belong to the family of helical cytokine with lengthy chain (figure 3) [64]. Leptin is an important member of adipokine family that helps in regulation of food intake, energy homeostasis, reproduction, metabolism, immune function, bone physiology, tissue remodeling, neuroendocrine function and others [65].

strated 4% augmented risk of fracture by per μmol/L raise in Hcy level [57].

**3.1. Expectations**

44 Advances in Osteoporosis

not up to the mark.

**4. Leptin**

are paying special attention to their relationship.

In a study, leptin replacement demonstrated the improvement of abnormalities in ob/ob mice in the form of decreasing food intake, energy loss, and temperature of body, infertility and immune function [67]. Since then, the knowledge which has been obtained from various studies indicating that leptin is not only important in control of body weight but also play imperative role in angiogenesis, hematopoiesis, blood pressure, immune function, lymphoid organ homeostasis, T lymphocyte systems, fertility, and bone formation [68].

Leptin is produced primarily from adipocytes according to adipose tissues quantity in the body. Brown fat tissue, stomach, placenta, ovary, bone marrow, liver, pituitary and mammary epithelial cells also secrete leptin [69]. Researchers observed that leptin is encoded by ob gene (LEP) (ob - obese and LEP - Leptin), according to the name that was first proposed, 'leptos', derived from the Greek word meaning thin, as ob protein is considered to be one of the molecules that regulates energy balance in mice [70].

#### **4.1. Leptin receptor**

Leptin's receptors were first isolated by cloning from mouse choroid plexus, these receptors belong to the cytokine family. There are six leptin receptors, which are divided into secretory (ob-Re), long (ob-Rb), and short forms (four short form receptors) [71-72]. The molecular receptor structure of the leptin and helical cytokine (class I) is similar. These homodimer receptors are able to activate the Janus kinase (JAK), and JAK is capable of starting as an activator of STAT – signal transducer and activator of transcription (Figure 4) [73]. Leptin signals through the activator system of JAK transcription, are associated with the form obRb (isoform long form) that would alter the expression of hypothalamic neuropeptides [74-75]. The ob-Rb is found in high levels in the hypothalamic nuclei [71] and its activity helps in mediating signal transduction by leptin in the hypothalamus, while the other leptin receptor activity (short form) is not strong enough in the functioning of leptin [72].

**Figure 4.** Leptin receptor [73]

#### **4.2. Leptin and bone interaction: conflicts and evidences**

Bone mass has been said to be regulated by leptin. The interaction of leptin with bone is multifaceted, related with location of bone. It also depends on the fact that whether leptin has a direct action on osteoblasts through receptors or it acts indirectly through the hypothalamus, it has the ability to both stimulate and inhibit bone formation [76-79]. Reduction in serum leptin levels are linked with decreased intake of food that might results in reduction in formation of bone and growth especially in children and adolescence [80-81]. In mice, lack of leptin also causes loss of bone and increased adiposity in bone marrow [82]. Leptin's enhanced levels and bone mass are associated with each other in obesity [83]. Leptin instead of supporting adipocyte phenotype of bone marrow stromal cells (BMSCs) sup‐ ports osteoblast demonstrated by in vitro studies [78, 84-85]. Similarly, in vitro studies have demonstrated that in ovariectomized rats there was reduction in bone loss and suspen‐ sion of tail after leptin treatment [86-87].

A study in Caucasian woman demonstrated that serum leptin had an inverse relationship with BMD, another study observed a positive connection [88-89]. A large-scale study established that serum leptin has no association with BMD [90]. Similarly, in another study no relation established between leptin and BMD [91]. Furthermore, it was suggested that leptin might be a forecaster of BMD in females with low BMD [92]. Recently, in Pakistan, we investigated correlation of serum leptin with BMD in postmenopausal osteoporotic females but no rela‐ tionship was found [31].

In vitro data shows that serum leptin supports the segregation and development of the osteoblast lineage cells and controls development of osteoclast [92-93]. Some clinical data have shown an inverse relationship between bone resorption markers and serum leptin concentra‐ tions in postmenopausal women [89, 94] while other found a negative relationship [95-96].

Recently, Scotece et al., (2014) described that leptin level could be a valuable risk marker for osteoporosis [97]. A study by Suh et al., (2013) found that changes in serum osteocalcin were linked with leptin levels. It seems that there is connection between adipose tissue and bone [98]. On the other hand, Mohiti-Ardekani et al., (2014) reported that there is no correlation of circulating leptin concentrations with BMD and bone biochemical markers including osteo‐ calcin [99].

#### **4.3. Indirect role of leptin in bone metabolism**

receptors are able to activate the Janus kinase (JAK), and JAK is capable of starting as an activator of STAT – signal transducer and activator of transcription (Figure 4) [73]. Leptin signals through the activator system of JAK transcription, are associated with the form obRb (isoform long form) that would alter the expression of hypothalamic neuropeptides [74-75]. The ob-Rb is found in high levels in the hypothalamic nuclei [71] and its activity helps in mediating signal transduction by leptin in the hypothalamus, while the other leptin receptor

activity (short form) is not strong enough in the functioning of leptin [72].

**Figure 4.** Leptin receptor [73]

46 Advances in Osteoporosis

**4.2. Leptin and bone interaction: conflicts and evidences**

Bone mass has been said to be regulated by leptin. The interaction of leptin with bone is multifaceted, related with location of bone. It also depends on the fact that whether leptin has a direct action on osteoblasts through receptors or it acts indirectly through the hypothalamus, it has the ability to both stimulate and inhibit bone formation [76-79]. Reduction in serum leptin levels are linked with decreased intake of food that might results Leptin has both direct and indirect effects on bone. Indirect mechanisms have been revealed by experimental studies in mutant rats and mice that cannot synthesize leptin or do not have receptors for it. Leptin is carried across the blood brain barrier (BBB) by special receptors, which are located on the endothelial cells, the obRa receptors. In the brain leptin binds with obRb receptors which are located in hypothalamus and activates them. Binding of leptin with its receptors stimulate the expression of a hypothalamic osteoblast inhibitory factor (HOBIF) that lowers the ability of osteoblast to make bone matrix [76-77, 100-101]. And because of this mechanism, obese ob (Lep)–/– mice, due to lack of leptin have an unusually elevated bone mass.

Leptin stimulates bone formation via beta-1-adrenergic receptors and stimulation of the somatotropin-IGF-1 system [102]. Leptin inhibits activity of osteo-catabolic neuropeptide Y (NPY) and stimulates activity of osteo-anabolic systems, such as cocain and amphetamineregulating transcript, via hypothalamic relays [103]. Moreover, leptin suppresses bone formation through beta-2-adrenergic receptors located in bone and inhibits serotonin produc‐ tion in the brain that result in loss of trabecular bone formation [104].

#### **4.4. Direct role of leptin in bone metabolism**

This is achieved by directly inhibiting the BMSCs to generate osteoclast while stimulating them to differentiate into osteoblasts [84, 93]. The BMSCs can be discriminated mainly into adipo‐ cytes or osteoblast cell family. These adipocytes in bone marrow provide leptin, which is able to inhibit adipogenesis discrimination of BMSC. It can excite discrimination of osteoblasts [84] while another study have pointed out that extremely elevated level of leptin causes apoptosis of BMSCs. A study reported that human osteoblasts, can begin producing and secreting leptin in the delayed matrix-mineralizing stage or shifting to osteocytes [85]. Proliferation of cultured human osteoblasts is also stimulated with leptin and it causes human BMSCs to express collagen-I, osteocalcin & alkaline phosphatase, and matrix mineralization [84,105]. These in vitro studies demonstrated that effects of leptin are dual in the microenvironment of bone and it also depends on presence of the leptin levels locally.

In the last decade, researchers proposed leptin to be a strong inhibitor of synthesis of bone because they could not find that osteoblast have long isoforms of the leptin receptors (ob-R) [76, 100]. In hypothalamus, and in many peripheral tissues expression of a large number of long isoform of ob-R have been confirmed [69].

Leptin receptors are also expressed by BMSCs, osteoblasts, osteoclasts and chondrocytes [84]. The signaling pathway in osteoblast, by which leptin acts, is osteoprotegerin (OPG)/RANKL (Receptor Activator for Nuclear factor κB Ligand). There was alteration found in the OPG/ RANKL expression profile after treatment with leptin [93]. Burguera et al., (2001) have also established that bone loss in ovariectomized rats is reduced by elevating osteoprotegerin mRNA in osteoblasts [87].

For verifying the central effect, infusion of leptin was given into the brain and it was found that leptin also inhibits the bone formation through central nervous system. One important finding was that the lower doses inhibited the bone formation as compared to the doses which was essential for the loss of body weight [100-101]. It was observed in the experimental ob/ob mice that this central effect is due to activation of sympathetic part of autonomic nervous system. This activation of sympathetic system by leptin is very important in controlling energy homeostasis and numerous physiological functions [100].

In mice, food restriction decreased longitudinal growth and bone mass, but administration of leptin restored skeletal growth, in spite of low energy intake and raised serum osteocalcin, that is a bone formation marker [106].

In patients of hypothalamic amenorrhea, who have relative leptin deficiency, recombinant leptin treatment improves markers of bone formation [107]. It seems that leptin affects bone centrally through the sympathetic nervous system, which is a main downstream mediator [108]. Leptin deficiency seems to reduce adrenergic tone [109] and to influence the discharge of noradrenaline from sympathetic nerve fibers [110]. Noradrenaline binds to α -adrenergic receptors on osteoblasts and restrains formation of bone [108-109,111].

For observing direct and indirect or central and peripheral effects in vitro/in vivo, many researchers used supraphysiological doses of leptin in their studies [78,84,85,87], so it's difficult to draw accurate conclusion from these studies.

The mechanism of leptin function is highly intricate, because of the several receptor isoforms, localized expression and activation of leptin. Still, the role of leptin is not completely under‐ stood and the heterogeneity in its functions and its involvement in several body systems make its role challenging, particularly in the field of metabolism and endocrinology, and generally in medicine. Further studies are essentially needed for better understanding the role of leptin in several metabolic and non-metabolic functions that may also elucidate its role in health and disease and particularly in the pathophysiology of osteoporosis.

**GSK** glycogen synthase kinase, **VSMC** vascular smooth muscle cells **BBB** Blood Brain Barrier, **HOBIF** Hypothalamic osteoblast inhibitory factor, **OPG** osteoprotegrin, **RANKL** receptor activator of nuclear factor-κB ligand, **OPN** Osteo‐ pontin

**Table 2.** Effects of leptin on different Bone components

#### **4.5. Expectations**

**4.4. Direct role of leptin in bone metabolism**

48 Advances in Osteoporosis

it also depends on presence of the leptin levels locally.

homeostasis and numerous physiological functions [100].

to draw accurate conclusion from these studies.

receptors on osteoblasts and restrains formation of bone [108-109,111].

long isoform of ob-R have been confirmed [69].

mRNA in osteoblasts [87].

is a bone formation marker [106].

This is achieved by directly inhibiting the BMSCs to generate osteoclast while stimulating them to differentiate into osteoblasts [84, 93]. The BMSCs can be discriminated mainly into adipo‐ cytes or osteoblast cell family. These adipocytes in bone marrow provide leptin, which is able to inhibit adipogenesis discrimination of BMSC. It can excite discrimination of osteoblasts [84] while another study have pointed out that extremely elevated level of leptin causes apoptosis of BMSCs. A study reported that human osteoblasts, can begin producing and secreting leptin in the delayed matrix-mineralizing stage or shifting to osteocytes [85]. Proliferation of cultured human osteoblasts is also stimulated with leptin and it causes human BMSCs to express collagen-I, osteocalcin & alkaline phosphatase, and matrix mineralization [84,105]. These in vitro studies demonstrated that effects of leptin are dual in the microenvironment of bone and

In the last decade, researchers proposed leptin to be a strong inhibitor of synthesis of bone because they could not find that osteoblast have long isoforms of the leptin receptors (ob-R) [76, 100]. In hypothalamus, and in many peripheral tissues expression of a large number of

Leptin receptors are also expressed by BMSCs, osteoblasts, osteoclasts and chondrocytes [84]. The signaling pathway in osteoblast, by which leptin acts, is osteoprotegerin (OPG)/RANKL (Receptor Activator for Nuclear factor κB Ligand). There was alteration found in the OPG/ RANKL expression profile after treatment with leptin [93]. Burguera et al., (2001) have also established that bone loss in ovariectomized rats is reduced by elevating osteoprotegerin

For verifying the central effect, infusion of leptin was given into the brain and it was found that leptin also inhibits the bone formation through central nervous system. One important finding was that the lower doses inhibited the bone formation as compared to the doses which was essential for the loss of body weight [100-101]. It was observed in the experimental ob/ob mice that this central effect is due to activation of sympathetic part of autonomic nervous system. This activation of sympathetic system by leptin is very important in controlling energy

In mice, food restriction decreased longitudinal growth and bone mass, but administration of leptin restored skeletal growth, in spite of low energy intake and raised serum osteocalcin, that

In patients of hypothalamic amenorrhea, who have relative leptin deficiency, recombinant leptin treatment improves markers of bone formation [107]. It seems that leptin affects bone centrally through the sympathetic nervous system, which is a main downstream mediator [108]. Leptin deficiency seems to reduce adrenergic tone [109] and to influence the discharge of noradrenaline from sympathetic nerve fibers [110]. Noradrenaline binds to α -adrenergic

For observing direct and indirect or central and peripheral effects in vitro/in vivo, many researchers used supraphysiological doses of leptin in their studies [78,84,85,87], so it's difficult

Leptin replacement therapy in animals and humans has shown promising results, but, whether in postmenopausal osteoporotic patients, its use as a drug or drug target may give some therapeutic benefits; it's a basic question that is yet to be explored.

Therefore, in future, it could be expected that administration of some therapeutic agents that target the leptin secretion would contribute in decreasing the severity of the disease in postmenopausal women. Future studies may elucidate such agents and their role in alleviating progression and spread of osteoporosis. Serum leptin effects on bone metabolism are incon‐ sistent and intricate, so there is still need to investigate the precise effects of leptin on bone by designing some clinical trials and longitudinal studies. Such studies would improve our understanding regarding the mechanisms underlying leptin role in bone metabolism and how it could be targeted particularly to treat osteoporosis or other bone disorders. Still, several venues need to be explored to explicate its undiscovered functions in human body especially, its role in terms of agonist and/or antagonist for the bone forming cells.

#### **5. Conclusion**

Evidences, shown by the results of several studies have clearly exhibited the role and involve‐ ment of leptin and Hcy in pathophysiology of osteoporosis at various levels but still their exact role, generally in bone metabolism and particularly in osteoporosis is not clear. We still need well designed experimental and clinical studies to validate the specific involvement of these two biochemical agents in bone pathophysiology or as a diagnostic & therapeutic markers, and also their precise involvement in osteoporosis and their synergistic, antagonists and/or agonists' influences on bone forming or destroying cells and other bone proteins.

It seems in future, leptin and Hcy could be the emerging therapies and new targets for treatment of osteoporosis. However, it would be too early to validate the role of Hcy and leptin in osteoporosis before addressing some important queries: like identifying their actual mechanism of action, site/s of their targets in the body for influencing bone metabolism, the effects of presently available anti-resorptive treatment on Hcy and leptin levels. Although, various studies have revealed mechanism of Hcy and leptin action, still further clarification regarding bone specific receptors, peripheral or central or both actions and their interaction with osteoporotic risk factors need to be investigated.

Numerous basic and clinical studies are available which are helping us to understand their role in health and diseases but still these are far from completion. Our future research should be focused on these important questions that whether bringing change in their level and/or activity may help us in preventing and treating osteoporosis, and interaction of Hcy and leptin with several other hormones and peptides and consequences of their increased/decreased levels at various tissues and organs level. Such questions require to be addressed for estab‐ lishing their precise role in bone metabolism. Therefore, further studies are needed to reveal their character as novel therapeutic agents and/or targets.

#### **6. Few key points**

#### **Author details**

**5. Conclusion**

50 Advances in Osteoporosis

**6. Few key points**

Evidences, shown by the results of several studies have clearly exhibited the role and involve‐ ment of leptin and Hcy in pathophysiology of osteoporosis at various levels but still their exact role, generally in bone metabolism and particularly in osteoporosis is not clear. We still need well designed experimental and clinical studies to validate the specific involvement of these two biochemical agents in bone pathophysiology or as a diagnostic & therapeutic markers, and also their precise involvement in osteoporosis and their synergistic, antagonists and/or

It seems in future, leptin and Hcy could be the emerging therapies and new targets for treatment of osteoporosis. However, it would be too early to validate the role of Hcy and leptin in osteoporosis before addressing some important queries: like identifying their actual mechanism of action, site/s of their targets in the body for influencing bone metabolism, the effects of presently available anti-resorptive treatment on Hcy and leptin levels. Although, various studies have revealed mechanism of Hcy and leptin action, still further clarification regarding bone specific receptors, peripheral or central or both actions and their interaction

Numerous basic and clinical studies are available which are helping us to understand their role in health and diseases but still these are far from completion. Our future research should be focused on these important questions that whether bringing change in their level and/or activity may help us in preventing and treating osteoporosis, and interaction of Hcy and leptin with several other hormones and peptides and consequences of their increased/decreased levels at various tissues and organs level. Such questions require to be addressed for estab‐ lishing their precise role in bone metabolism. Therefore, further studies are needed to reveal

agonists' influences on bone forming or destroying cells and other bone proteins.

with osteoporotic risk factors need to be investigated.

their character as novel therapeutic agents and/or targets.

**•** Each μmol/L raise in Hcy level augments 4% fracture risk.

control development of osteoclast.

somatotropin-IGF-1 system

into osteoblasts.

**•** Homocysteine (Hcy) and leptin are new risk factor for primary osteoporosis.

**•** Hcy level could be modulated by nutritional conditions, and physical activity.

reducing bone blood supply and directly interacting with bone matrix.

**•** Hcy interacts with bone by enhancing osteoclastic and reducing osteoblastic activity,

**•** Serum leptin supports the segregation and development of the osteoblast lineage cells and

**•** Leptin stimulates bone formation via beta-1-adrenergic receptors and stimulation of the

**•** Leptin directly inhibits the BMSCs to generate osteoclast and stimulate them to differentiate

Mukhtiar Baig1 , Saba Tariq2 and Sundus Tariq3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Clinical Biochemistry, Faculty of Medicine, Rabigh, King Abdulaziz University, Jeddah, KSA

2 Department of Pharmacology, University Medical & Dental College, The University of Faisalabad, Faisalabad, Pakistan

3 Department of Physiology, University Medical & Dental College, The University of Faisalabad, Faisalabad, Pakistan

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### **Biological Effects of Skeletal Renin-Angiotensin System in Osteoporosis**

Yan Zhang and Liang Wang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59299

#### **1. Introduction**

#### **1.1. Classical renin-angiotensin system**

The renin–angiotensin system (RAS) is an endocrine system that governs body fluid, electro‐ lyte balance and blood pressure. Within the classical RAS, angiotensinogen (AGT) secreted by liver is enzymatically cleaved to angiotensin (ANG) I by kidney-derived renin. ANG I is, hereafter, cleaved by angiotensin-converting enzyme (ACE) to generate the effector hormone ANG II, which exerts various biological actions through its receptors, ANG II type 1 receptor (AT1) and ANG II type 2 receptor (AT2). The initial reaction between the enzyme renin and the substrate AGT is the rate-limiting step of the RAS [1].

As the RAS is a hormonal cascade that is thought to act as a master controller of blood pressure and fluid balance within the body [2], the systematic RAS has been an important target of antihypertensive medications. There are several groups of drugs in this category that affect different parts of the RAS axis, including ACE inhibitors (ACEI) and angiotensin receptor blockers (ARB), both of which are widely used for anti-hypertension treatment [3]. Addition‐ ally, Aliskiren, the first orally active direct renin inhibitor approved for clinical use, is a small molecule competitive inhibitor that specifically inhibits the enzymatic activity of renin [4, 5], consequently it could effectively suppress the rate-limiting step within RAS cascade to reduce the production of ANG II. The recent evidences have shown the effective blood pressure control of Aliskiren, generally well tolerated as monotherapy or in combination with other antihypertensive drugs [3, 6].

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Tissue renin–angiotensin system**

From an evolutionary point of view, it is cost-effective to have a common system to potentiate and effect the actions of the regulating hormones. Interestingly, the RAS is also found in primitive animals without a closed circulatory system, which indicates that the system is far more than a mediator of vasoconstriction [7]. It is now evident that the components of RAS, in addition to the classical pathway, are produced and acting locally in multiple tissues, a concept known as tissue RAS [7]. The local effects of tissue RAS are diverse and depend on the specific tissues involved.

The functional tissue RAS is postulated to participate in various physiological and pathological processes such as insulin secretion [8], glomerular sclerosis [9], renal inflamma‐ tion [10], atherosclerosis [11], cardiac hypertrophy [12], brain ischemia [13] and follicular development and endometrial cancer in female reproductive tract [14]. A growing body of studies has demonstrated that the diabetic complications, such as cardiovascular disease [15], nephropathy [16] and retinopathy [17], are caused by the high activity of tissue RAS and the increased production of ANG II in local tissues, and the clinical practice has revealed that these pathological alterations associated with diabetes were significantly improved in response to the treatment with RAS inhibitors [15-17]. Additionally, hyperglycemia, obesity, hypertension, and cortisol, well-known risk factors of metabolic disease, are all stimula‐ tors on tissue RAS, whereas glucagon-like peptide-1, vitamin D, and aerobic exercise could, to some extent, prevent metabolic disease through inhibiting tissue RAS [7]. Thus, the factors and drugs suppressing tissue RAS activity have potential in improving RAS-involved tissue injuries.

Recent in vivo studies showed that the components of RAS, such as renin, ACE, and ANG II receptors, were expressed in the local milieu of bone [1, 18-20], and in vitro study identified the expression of ANG II receptors in primary osteoblasts derived from newborn mouse calvaria [1], indicating the components of RAS are expressed locally in bone microenviron‐ ment. Our further animal studies demonstrated that the local RAS in bone was involved in age-related osteoporosis of aging mice [21], and bone deteriorations of mice with either obstructive nephropathy [22] or type 1 diabetes [23]. Other groups elucidated the involvement of skeletal RAS in the process of fracture healing in a mouse femur fracture model [24], and the steroid-induced osteonecrosis in rabbits [20] as well as the development of postmenopausal osteoporosis in ovariectomized (OVX) animal models [25, 26] and glucocorticoid-induced osteoporosis [19]. Therefore, it concludes that the local RAS exists in bone tissue and plays an important role in local bone metabolism.

#### **3. Action of angiotensin II on bone**

ANG II has been postulated to be able to act upon the cells involved in bone metabolism through receptors located in osteoblasts and osteoclasts or regulate blood flow in bone marrow capillaries. At the end of last century, the studies showed that ANG II stimulat‐ ed DNA and collagen synthesis and decreased alkaline phosphatase (ALP) activity in bone cell populations derived from calvariae of fetal rat [27] and newborn rat [28]. Similar effects of ANG II were observed in osteoblastic ROS17/2.8 cells [29] and human adult bone cells obtained by collagenase digestion from trabecular bone [27]. The clonal cell analysis, autoradiographic studies, and receptor subtype analysis suggested that ANG II might be intimately involved in the proliferation of the osteoblast-rich populations of cells through the AT1 receptor [27, 28], which also plays one of the essential roles in bone metabolism as a mechanoreceptor of osteoblasts [30].

**2. Tissue renin–angiotensin system**

the specific tissues involved.

62 Advances in Osteoporosis

important role in local bone metabolism.

**3. Action of angiotensin II on bone**

injuries.

From an evolutionary point of view, it is cost-effective to have a common system to potentiate and effect the actions of the regulating hormones. Interestingly, the RAS is also found in primitive animals without a closed circulatory system, which indicates that the system is far more than a mediator of vasoconstriction [7]. It is now evident that the components of RAS, in addition to the classical pathway, are produced and acting locally in multiple tissues, a concept known as tissue RAS [7]. The local effects of tissue RAS are diverse and depend on

The functional tissue RAS is postulated to participate in various physiological and pathological processes such as insulin secretion [8], glomerular sclerosis [9], renal inflamma‐ tion [10], atherosclerosis [11], cardiac hypertrophy [12], brain ischemia [13] and follicular development and endometrial cancer in female reproductive tract [14]. A growing body of studies has demonstrated that the diabetic complications, such as cardiovascular disease [15], nephropathy [16] and retinopathy [17], are caused by the high activity of tissue RAS and the increased production of ANG II in local tissues, and the clinical practice has revealed that these pathological alterations associated with diabetes were significantly improved in response to the treatment with RAS inhibitors [15-17]. Additionally, hyperglycemia, obesity, hypertension, and cortisol, well-known risk factors of metabolic disease, are all stimula‐ tors on tissue RAS, whereas glucagon-like peptide-1, vitamin D, and aerobic exercise could, to some extent, prevent metabolic disease through inhibiting tissue RAS [7]. Thus, the factors and drugs suppressing tissue RAS activity have potential in improving RAS-involved tissue

Recent in vivo studies showed that the components of RAS, such as renin, ACE, and ANG II receptors, were expressed in the local milieu of bone [1, 18-20], and in vitro study identified the expression of ANG II receptors in primary osteoblasts derived from newborn mouse calvaria [1], indicating the components of RAS are expressed locally in bone microenviron‐ ment. Our further animal studies demonstrated that the local RAS in bone was involved in age-related osteoporosis of aging mice [21], and bone deteriorations of mice with either obstructive nephropathy [22] or type 1 diabetes [23]. Other groups elucidated the involvement of skeletal RAS in the process of fracture healing in a mouse femur fracture model [24], and the steroid-induced osteonecrosis in rabbits [20] as well as the development of postmenopausal osteoporosis in ovariectomized (OVX) animal models [25, 26] and glucocorticoid-induced osteoporosis [19]. Therefore, it concludes that the local RAS exists in bone tissue and plays an

ANG II has been postulated to be able to act upon the cells involved in bone metabolism through receptors located in osteoblasts and osteoclasts or regulate blood flow in bone marrow capillaries. At the end of last century, the studies showed that ANG II stimulat‐ When investigating the direct effects of ANG II on matured osteoblasts, the results revealed that ANG II inhibited the expression of mRNA for osteocalcin, which is a protein that is specifically expressed during maturation of osteoblastic cells, decreased the activity of ALP, the number and the total area of mineralized nodules as well as reduced the accumulation of calcium in cells and the matrix layer [31]. Besides, the ANG II-involved impairment of bone formation may be attributed that it altered the expression of Cbfa1 by activating the cAMP signaling pathway and subsequently reduced osteoblast number and osteoblastic function [32]. SOST, which encodes sclerostin, is a secretory product of osteocytes that counters Wnt signaling, thereby negatively regulates bone formation [33]. The AT1-involved inhibition on bone formation was highly correlated with its regulation on downstream factor SOST, as the decreased SOST expression in osteocytes was observed in AT1-deficient mice [34]. Further‐ more, the treatment with ANG II strikingly increased the expressions of matrix metallopro‐ teinase (MMP)-3 and-13 through MAPK signaling pathways via the AT1 in osteoblastic ROS17/2.8 cells, suggesting that ANG II stimulated the degradation process that occurs during extracellular matrix (ECM) turnover in osteoid by increasing the production of MMP-3 and-13 in osteoblasts [29]. Additionally, ANG II induced mitochondrial dysfunction and promoted apoptosis via JNK signaling pathway in primary mouse calvaria osteoblast [35]. Taken together, the target genes including Cbfa1, SOST, MMP-3 and MMP-13, and the signaling pathways like MAPK and JNK are involved in the mediation of ANG II on osteoblastic function and bone formation.

Osteoblast modulates osteoclast differentiation by producing both positive and negative regulators, most notably receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), respectively [36]. Of note, it has been recently found that ANG II could induce the differentiation of bone marrow mononuclear cells to multinuclear cells and the number of multinuclear cells in osteoclasts as well as increase tartrate-resistant acid phosphatase (TRAP) positive multinuclear osteoclasts due to its stimulation on the expression of osteoclastogenesissupporting cytokine, RANKL in osteoblasts, leading to the activation of osteoclasts [1, 25], whereas these effects were completely blocked by either ANG II type 1 receptor blockade (olmesartan) or mitogen-activated protein kinase kinase inhibitors (U0126) [25]. Importantly, ANG II itself had no capacity to induce osteoclast differentiation and did not potentiate osteoclast formation triggered by RANKL, while it stimulated the formation of osteoclasts in the co-culture system of primary osteoblasts and bone marrow macrophages in a dosedependent manner [1, 32]. Taken together, these results suggested that ANG II stimulates osteoclastogenesis by acting on osteoblastic cells (i.e., 'the soil cells'), but not through a direct action on hematopoietic 'seed cells' [1].

It was found that the TRAP activity and the TRAP-positive stained area were significantly increased in the tibia of OVX rats with systemic administration of ANG II at a subpresser dose (200 ng/kg/min), and the treatment with ANG II significantly induced the ovariectomyinduced increase in urinary level of deoxypyridinoline [25]. The ratio of ALP to TRAP was significantly decreased in the tibia of OVX rats upon to ANG II treatment. These results suggested that ANG II accelerated the turnover of bone metabolism, which is similar to the typical pattern in elderly postmenopausal women who are at high risk for osteoporosis [25]. Of importance, the bone density as assessed by double energy X ray absorptiometry (DEXA) was significantly decreased in the tibia of OVX rats by ANG II. These results suggested that ANG II directly accelerated estrogen deficiency-induced osteoporosis.

#### **4. Interaction of AT1 and AT2 on bone**

Previous studies have focused on the ANG II–AT1 interactions since these are the best described and considered the most important. However, the system is complex and several other components probably play significant roles as well [7]. Several publications raise the possibility that AT1 and AT2 carry out negative cross-talk within fibroblasts and vascular endothelial cells with respect to each other's signaling pathways and responses [37]. This may be of particular importance when the AT1 are pharmacologically blocked.

*Asaba* et al. determined the relative contribution of the two receptors for transducing the osteoclastogenesis-supporting function of ANG II in osteoblasts by knocking down the expression of each of the receptors with siRNA in primary osteoblasts in culture [1]. In AT1-knockdown osteoblasts, the stimulatory effect of ANG II on osteoclast formation was somewhat enhanced. In AT2-knockdown osteoblasts, in contrast, the osteoclastogenic potential was markedly attenuated [1], which was consistent with that AT2 deficiency increased bone mass of distal metaphyseal regions of femoral in mice as well as the treatment with AT2 blocker PD123319 suppressed ANG II-induced increase in the num‐ ber of osteoclasts in organ cultures of bone [18]. *Asaba* et al.'s study suggested that the action of ANG II on osteoblasts in terms of stimulating osteoclastogenesis was mainly mediated through the AT2 and AT1 might exert an inhibitory effect on AT2. These findings in osteoblasts are consistent with the notion that the functions of AT1 and AT2 are in many cases counter-regulatory to each other [7]. However, they are contrary to the conclusion that AT2 is the protective arm of RAS and counterbalances pathological processes and enable recovery from disease [38]. Thus, further studies are needed to dissect the signal‐ ing pathways downstream of each receptor in osteoblasts.

#### **5. ACE inhibitors and osteoporosis**

osteoclastogenesis by acting on osteoblastic cells (i.e., 'the soil cells'), but not through a direct

It was found that the TRAP activity and the TRAP-positive stained area were significantly increased in the tibia of OVX rats with systemic administration of ANG II at a subpresser dose (200 ng/kg/min), and the treatment with ANG II significantly induced the ovariectomyinduced increase in urinary level of deoxypyridinoline [25]. The ratio of ALP to TRAP was significantly decreased in the tibia of OVX rats upon to ANG II treatment. These results suggested that ANG II accelerated the turnover of bone metabolism, which is similar to the typical pattern in elderly postmenopausal women who are at high risk for osteoporosis [25]. Of importance, the bone density as assessed by double energy X ray absorptiometry (DEXA) was significantly decreased in the tibia of OVX rats by ANG II. These results suggested that

Previous studies have focused on the ANG II–AT1 interactions since these are the best described and considered the most important. However, the system is complex and several other components probably play significant roles as well [7]. Several publications raise the possibility that AT1 and AT2 carry out negative cross-talk within fibroblasts and vascular endothelial cells with respect to each other's signaling pathways and responses [37]. This may

*Asaba* et al. determined the relative contribution of the two receptors for transducing the osteoclastogenesis-supporting function of ANG II in osteoblasts by knocking down the expression of each of the receptors with siRNA in primary osteoblasts in culture [1]. In AT1-knockdown osteoblasts, the stimulatory effect of ANG II on osteoclast formation was somewhat enhanced. In AT2-knockdown osteoblasts, in contrast, the osteoclastogenic potential was markedly attenuated [1], which was consistent with that AT2 deficiency increased bone mass of distal metaphyseal regions of femoral in mice as well as the treatment with AT2 blocker PD123319 suppressed ANG II-induced increase in the num‐ ber of osteoclasts in organ cultures of bone [18]. *Asaba* et al.'s study suggested that the action of ANG II on osteoblasts in terms of stimulating osteoclastogenesis was mainly mediated through the AT2 and AT1 might exert an inhibitory effect on AT2. These findings in osteoblasts are consistent with the notion that the functions of AT1 and AT2 are in many cases counter-regulatory to each other [7]. However, they are contrary to the conclusion that AT2 is the protective arm of RAS and counterbalances pathological processes and enable recovery from disease [38]. Thus, further studies are needed to dissect the signal‐

ANG II directly accelerated estrogen deficiency-induced osteoporosis.

be of particular importance when the AT1 are pharmacologically blocked.

ing pathways downstream of each receptor in osteoblasts.

**4. Interaction of AT1 and AT2 on bone**

action on hematopoietic 'seed cells' [1].

64 Advances in Osteoporosis

Osteoporosis, hypertension, diabetes are major chronic diseases in older subjects and the latter two are well known to be high risk factors for osteoporosis. As ACE inhibitors are usually prescribed for hypertension, cardiac failure, and diabetic nephropathy [23, 39], it is important to know the prospective effects of ACEI on bones of these patients taking ACEI treatment.

Previously, most of clinical studies demonstrated that patients treated with ACEI showed an increased bone mineral density (BMD) and a reduced fracture risk [40-45]. The menopausal and hypertensive women who followed treatment with ACEI fosinopril did not present the physiological loss of bone mass that affected to menopausal women without treatment [43]. A large case–control analysis carried out in the UK, suggested a possibly decreased fracture risk associated with longer-term use of ACE inhibitors [40], and in an open prospective study including 134 patients with low to moderate hypertension and stable BMD, the plasma calcium and 25-hydroxyvitamin D levels were both increased in patients treated with the ACEI quinapril [41]. It also significantly increased BMD of lumbar spine in female subjects with ACE DD genotype, which could induce a higher level of ANG II [41]. The research group from Hong Kong performed two large scale cohort studies which investigated the risk factors for osteo‐ porotic fractures in Hong Kong-dwelling elderly Chinese, and their data concluded that male ACEI users had higher BMD at the total hip, female neck and lumbar spine than non-users. Likewise, female ACEI users also had higher BMD than non-users, although only significant at the femoral neck [42].

While, in the contrary to the above mentioned beneficial effects of ACEI on bone health, the recent emerging evidences indicated that ACEI use did not change the rate and risk of fracture [46], and even led to greater bone loss [39, 47, 48]. The same research group from Hong Kong recently also stated that female continuous users of ACEI had increased bone loss both in total hip and femur neck [47]. A large sample size study in American men also supported this theory by showing that ACEI use was associated with increased bone loss [39], moreover, another prospective study-a cohort study of atomic bomb survivors in Japan, demonstrated that ACEI use was associated with increased bone loss of femoral neck in older Japanese [48].

Similarly in animal studies, the use of ACEI enalapril (10-20 mg/kg, i.g.) did not show positive effects on bone function of OVX mice [49] or OVX spontaneously hypertensive rats (SHR) [50], and the administration of enalapril (0.4 mg/kg, i.p.) in a dose recommended for the treatment of hypertension did not cause significant changes in bone density, the ash and mineral content or morphometric parameters of the femur in female Wistar rats [51]. Another ACEI moexipril, when given alone at oral dose of 10 mg/kg, had no effect on the cancellous bone site in either OVX or sham-operated rats and did not hamper the osteoprotective effects of 17beta-estradiol [52]. Even though the treatment of Tsukuba hypertensive mouse with enalapril improved osteoporosis [1] as well as the OVX rats in response to the treatment with ACEI captopril (1 or 5 mg/kg) showed the increased trabecular area of lumbar vertebrae (L4) and the improved biomechanical properties by increasing L5 break stress and elastic modulus [26], our recent published article elucidated that the treatment with captopril (10 mg/kg, i.g.) significantly elevated serum level of TRAP 5b, and had a trend to decrease BMD of trabecular bone and damage micro-architecture of proximal tibial head and distal femoral end in type 1 diabetic mice [23].

Based on the facts that ANG II locally in bone tissue has detrimental effects to bone function and ACE is the major enzyme producing ANG II, it is surprising that ACEI could not improve even accelerate bone loss in both humans and animals. Since the modest changes in ACE level affect the levels of its substrates much more than its products, indicating that relatively small changes in the levels of ACE affect kinin level more than ANG II level [53], a possible reason comes from the regulation of ACE on kinin-kallikrein system within which bradykinin can stimulate bone resorption and reduce BMD [47]. Another possible explanation we should consider is that although short-term ACEI therapy was associated with decreased ANG II level, there were some evidences that long-term ACE inhibition resulted in a return of ANG II towards baseline level, so-called 'ACE escape' [47]. The complete mechanisms have not yet been fully demonstrated. More research needs to be carried out to clarify the influence of the ACEI treatment on bone health as this might be of clinical relevance when antihypertensive therapy is initiated, particularly in hypertensive women who typically suffer from a concom‐ itant rapid onset of osteoporosis after menopause [52].

#### **6. Angiotensin receptor blockers and osteoporosis**

The clinical profiles of users of ACEI and ARB were very similar. In the USA, ARB was usually prescribed when ACEI was not tolerated, thus explaining the smaller number of ARB users [39] and limited human studies of ARB and BMD or fracture risk in the literatures [46]. The completed studies on the affections of ARB on bone function in human and animals have shown contradictory results.

The recent population-based, retrospective cohort study with propensity score-matching using administrative databases in Ontario, Canada to examine the risk of osteoporosis-related fractures in hypertensive elderly patients treated with ARBs versus ACEIs, showed that there was no significant difference between the effects of ARBs and ACE inhibitors on hip and other osteoporotic fractures [54]. A large cohort study on Medicare beneficiaries with a diagnosis of hypertension initiating single-drug therapy for anti-hypertension treatment suggested the increasingly protective effect of ARB on relative fracture risk over time [46]. While, the study with large sample size of community-dwelling older adults from six different geographic regions demonstrated that the use of ARBs did not have any significant overall effect on bone loss in older men [39].

The contradictory results about the actions of ARBs on bone metabolism were also shown among animal studies. The treatment with telmisartan, olmesartan, and losartan, could reduce bone loss of OVX mice [49], attenuate the ovariectomy-induced decrease in BMD [25], and increase bone strength, mass and trabecular connections of OVX rats femur [55, 56], respectively. Moreover, telmisartan partially protected from thiazolidinedione-in‐ duced bone loss by actively blocking thiazolidinedione-induced anti-osteoblastic activity via maintaining PPARγ serine 112 phosphorylation [57], and promoted fracture healing in

a mice model [58]. However, some studies reported ARBs did not cause significant changes of bone properties in normal female rats [51], type 2 diabetic mice [57], OVX rats [59] or orchiectomized rats [60]. Importantly, it was noted that in some animal models ARBs may lead to more bone injuries [1, 61]. The treatment of transgenic Tsukuba hypertensive mouse with losartan resulted in exacerbation of the low bone mass phenotype [1]. The study in our group demonstrated a trend of losartan to promote the loss of bone mass and the deteriorations of trabecular bone micro-architecture in type 1 diabetic mice due to the compensatory stimulation of bone RAS activation as shown by the up-regulation of renin and ANG II expression in bone tissue [61].

#### **7. Perspective**

damage micro-architecture of proximal tibial head and distal femoral end in type 1 diabetic

Based on the facts that ANG II locally in bone tissue has detrimental effects to bone function and ACE is the major enzyme producing ANG II, it is surprising that ACEI could not improve even accelerate bone loss in both humans and animals. Since the modest changes in ACE level affect the levels of its substrates much more than its products, indicating that relatively small changes in the levels of ACE affect kinin level more than ANG II level [53], a possible reason comes from the regulation of ACE on kinin-kallikrein system within which bradykinin can stimulate bone resorption and reduce BMD [47]. Another possible explanation we should consider is that although short-term ACEI therapy was associated with decreased ANG II level, there were some evidences that long-term ACE inhibition resulted in a return of ANG II towards baseline level, so-called 'ACE escape' [47]. The complete mechanisms have not yet been fully demonstrated. More research needs to be carried out to clarify the influence of the ACEI treatment on bone health as this might be of clinical relevance when antihypertensive therapy is initiated, particularly in hypertensive women who typically suffer from a concom‐

The clinical profiles of users of ACEI and ARB were very similar. In the USA, ARB was usually prescribed when ACEI was not tolerated, thus explaining the smaller number of ARB users [39] and limited human studies of ARB and BMD or fracture risk in the literatures [46]. The completed studies on the affections of ARB on bone function in human and animals have

The recent population-based, retrospective cohort study with propensity score-matching using administrative databases in Ontario, Canada to examine the risk of osteoporosis-related fractures in hypertensive elderly patients treated with ARBs versus ACEIs, showed that there was no significant difference between the effects of ARBs and ACE inhibitors on hip and other osteoporotic fractures [54]. A large cohort study on Medicare beneficiaries with a diagnosis of hypertension initiating single-drug therapy for anti-hypertension treatment suggested the increasingly protective effect of ARB on relative fracture risk over time [46]. While, the study with large sample size of community-dwelling older adults from six different geographic regions demonstrated that the use of ARBs did not have any significant overall effect on bone

The contradictory results about the actions of ARBs on bone metabolism were also shown among animal studies. The treatment with telmisartan, olmesartan, and losartan, could reduce bone loss of OVX mice [49], attenuate the ovariectomy-induced decrease in BMD [25], and increase bone strength, mass and trabecular connections of OVX rats femur [55, 56], respectively. Moreover, telmisartan partially protected from thiazolidinedione-in‐ duced bone loss by actively blocking thiazolidinedione-induced anti-osteoblastic activity via maintaining PPARγ serine 112 phosphorylation [57], and promoted fracture healing in

itant rapid onset of osteoporosis after menopause [52].

shown contradictory results.

loss in older men [39].

**6. Angiotensin receptor blockers and osteoporosis**

mice [23].

66 Advances in Osteoporosis

It has been argued that neither ACEIs nor ARBs completely block the RAS cascade due to the disruption of the feedback inhibition of renin production [62]. The increase in renin activity stimulates the conversion of ANG I and ultimately ANG II, which largely limits the efficacy of RAS inhibition [63]. The increased renin can also act through the prorenin/renin receptor, which may cause tissue damages independent of ANG II [64]. Thus, as compared to single treatment with RAS inhibitors, whether combining renin inhibitor, like Aliskiren, with ARB or ACEI could generate better therapeutic effects on tissue injuries, such as osteoporosis, should be further clarified.

As discussed in this chapter, RAS locally plays a key role in the modulation of bone metabo‐ lism. However, over the past 10 years, several studies have presented evidences for the existence of a new arm of the RAS, namely the ACE2/ANG-(1–7)/Mas axis [65]. The identifi‐ cation of the ACE homolog, ACE2 as a key ANG-(1–7)-forming enzyme, unravels the existence of a distinct enzymatic pathway for the production of ANG-(1-7), which has a broad range of effects in different organs and tissues that goes beyond its initially described cardiovascular and renal actions [66]. This heptapeptide exerts its actions through binding to a G proteincoupled receptor Mas, distinct from AT1 and AT2 [67]. It is now accepted that the ACE2/ANG- (1–7)/Mas axis is able to counteract most of the deleterious actions of the ACE/ANG II/AT1 axis, especially in pathological conditions [68] such as cardiac dysfunction, increased blood pressure, decreased baroreflex function, endothelial dysfunction, reduced reproductive function, increased thrombogenesis [66]. Thus, how the cross-talk and the interaction between the dual axis systems of RAS contribute to the maintainance of bone metabolism needs to be further investigated and elucidated for better understanding the molecular mechanism of bone metabolic diseases.

#### **Acknowledgements**

This work was sponsored by National Natural Science Foundation of China (No. 81202894).

#### **Author details**

Yan Zhang1\* and Liang Wang2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Center for Systems Biomedical Sciences, University of Shanghai for Science and Technology, Shanghai, China

2 Department of Orthopaedics, The 309th hospital of Chinese People's Liberation Army, Beijing, China

#### **References**

[9] Zhang Z, Zhang Y, Ning G, Deb DK, Kong J, Li YC. Combination therapy with AT1 receptor blocker and vitamin D analog markedly ameliorates diabetic nephropathy. Proc Natl Acad Sci USA 2008;105(41) 15896-15901.

**Author details**

68 Advances in Osteoporosis

Shanghai, China

Beijing, China

**References**

Yan Zhang1\* and Liang Wang2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Center for Systems Biomedical Sciences, University of Shanghai for Science and Technology,

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### **Pharmacological Treatment of Osteoporosis**

Jorge Malouf, Berta Magallares and Roberto Güerri

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59469

#### **1. Introduction**

Osteoporosis can be divided into two principle strands, clinical osteoporosis and densitometric osteoporosis. Clinical osteoporosis involves the identification of a fragility fracture and does not need densitometry for treatment to begin. Densitometric osteoporosis is identified via an assessment of bone mineral density. Approaches to treatment depend on the global fracture risk and the outcomes of densitometric tests.

The initial stage of the pharmacological treatment of osteoporosis is to identify the pathology of the primary condition or to determine whether the loss of bone density is a secondary symptom of a separate condition. Where secondary osteoporosis is identified, priority is given to the treatment of the primary condition. The option of pharmacological therapy must only be contemplated if the risk of fracture is too elevated, given that the intention behind phar‐ macological treatment in osteoporosis is to reduce the fracture risk. Based on World Health Organization figures, less than half of patients presenting a fragility fracture have been diagnosed with densitometric osteoporosis [1]. Once a course of medication has begun, longterm management must address improvements to lifestyle and take aspects of, security, cost, and compliance into account. As such, it is absolutely necessary to assess and make determi‐ nations on the basis of cost, assessment of cost-efficiency, and the adaptability of patients to drug security.

#### **2. Antiresorptives**

#### **2.1. Calcitonin**

Calcitonin binds to osteoclasts and hinders bone resorption. The use of salmon calcitonin has previously been widespread as a result of its extreme potency in humans, a result of its greater affinity for the human calcitonin receptor.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

Calcitonin is now no longer a treatment for osteoporosis, having been supplanted by other treatments. Following a European risk-benefit analysis, the scientific committee of the Europe‐ anMedicinesAgency(CHMP)advisedthattreatmentsusingcalcitoninshouldonlybedeployed in short-term scenarios.. Treatments using injectable calcitonin should be confined to the shortterm in Paget's disease, the prevention of acute bone loss as a result of sudden immobiliza‐ tion and hypercalcemia resulting from cancer. In addition to this, calcitonin has been proven to be effective in treating pain resulting from fractures of the vertebral column. [2-4]

#### **2.2. Hormonal Replacement Therapy (HRT)**

HRT is a form of treatment which deploys varying doses of estrogen, sometimes on its own, sometimes in combination with progestagens. The calculated risk of fracture, based on principal cohort trials of postmenopausal women treated with HRT over the long term, indicate an appreciable lowering of the likelihood of both vertebral fracture (RR=0.6; CI 95%: 0.36 to 0.99) and wrist fracture (RR=0.39; CI 95%: 0.24 to 0.64), but a non-significant lowering of the likelihood of hip fracture (RR=0.64; CI 95%: 0.32 to 1.04). The WHI trial (Women's Health Initiative), a randomised clinical trial (RCT) that assessed postmenopausal women randomly assigned to combined HRT (combined equine estrogen 0.625mg daily plus medroxyproges‐ terone 2.5mg daily) or a placebo, recorded, following 5.2 years of treatment, a decrease in hip fracture risk of 34% (hazard ratio [HR]=0.66; CI 95%: 0.45 to 0.98), in clinical vertebral fractures of 34% (HR=0.66; CI 95%: 0.44 to 0.98) and in any fracture of 24% (HR=0.76; CI 95%: 0.69 to 0.85) [5,6]. In the same investigation, the cohort taking estrogen on its own demonstrated comparable outcomes, however the treatment was put on hold as a result of an adverse riskbenefit ratio. In two meta-analyses of RCT's, a decrease of 27% (RR=0.73; CI 95%: 0.56 to 0.94) in non-vertebral fractures and a trend towards a reduction of vertebral fractures (RR=0.66; CI 95%: 0.41 to 1.07) was recorded [7]. Nonetheless, neither the HERS (The Heart and Estrogen + Progestin Replacement Study) RCT nor the subsequent group, the HERS II study (Hulley et al., 1998), were able to register a decrease of the risk of hip fractures or of other locations (RR=1.04; CI 95%: 0.87 to 1.25) in patients with a history of cardiovascular disease [8].

The British National Institute of Health and Clinical Excellence published a meta-analysis of RCTs on HRT efficacy (with estrogen alone or combined) compared with placebo/nontreatment in postmenopausal women or those with surgical menopause [9]. The outcomes were organised according to the location of the fracture and the RCT used as the basis for the calculation of the relative risk was also identified. The outcomes are outlined in table 1.

**Table 1.** Relative fracture risk in NICE meta-analysis

#### *2.2.1. Security*

Calcitonin is now no longer a treatment for osteoporosis, having been supplanted by other treatments. Following a European risk-benefit analysis, the scientific committee of the Europe‐ anMedicinesAgency(CHMP)advisedthattreatmentsusingcalcitoninshouldonlybedeployed in short-term scenarios.. Treatments using injectable calcitonin should be confined to the shortterm in Paget's disease, the prevention of acute bone loss as a result of sudden immobiliza‐ tion and hypercalcemia resulting from cancer. In addition to this, calcitonin has been proven to

HRT is a form of treatment which deploys varying doses of estrogen, sometimes on its own, sometimes in combination with progestagens. The calculated risk of fracture, based on principal cohort trials of postmenopausal women treated with HRT over the long term, indicate an appreciable lowering of the likelihood of both vertebral fracture (RR=0.6; CI 95%: 0.36 to 0.99) and wrist fracture (RR=0.39; CI 95%: 0.24 to 0.64), but a non-significant lowering of the likelihood of hip fracture (RR=0.64; CI 95%: 0.32 to 1.04). The WHI trial (Women's Health Initiative), a randomised clinical trial (RCT) that assessed postmenopausal women randomly assigned to combined HRT (combined equine estrogen 0.625mg daily plus medroxyproges‐ terone 2.5mg daily) or a placebo, recorded, following 5.2 years of treatment, a decrease in hip fracture risk of 34% (hazard ratio [HR]=0.66; CI 95%: 0.45 to 0.98), in clinical vertebral fractures of 34% (HR=0.66; CI 95%: 0.44 to 0.98) and in any fracture of 24% (HR=0.76; CI 95%: 0.69 to 0.85) [5,6]. In the same investigation, the cohort taking estrogen on its own demonstrated comparable outcomes, however the treatment was put on hold as a result of an adverse riskbenefit ratio. In two meta-analyses of RCT's, a decrease of 27% (RR=0.73; CI 95%: 0.56 to 0.94) in non-vertebral fractures and a trend towards a reduction of vertebral fractures (RR=0.66; CI 95%: 0.41 to 1.07) was recorded [7]. Nonetheless, neither the HERS (The Heart and Estrogen + Progestin Replacement Study) RCT nor the subsequent group, the HERS II study (Hulley et al., 1998), were able to register a decrease of the risk of hip fractures or of other locations

(RR=1.04; CI 95%: 0.87 to 1.25) in patients with a history of cardiovascular disease [8].

The British National Institute of Health and Clinical Excellence published a meta-analysis of RCTs on HRT efficacy (with estrogen alone or combined) compared with placebo/nontreatment in postmenopausal women or those with surgical menopause [9]. The outcomes were organised according to the location of the fracture and the RCT used as the basis for the calculation of the relative risk was also identified. The outcomes are outlined in table 1.

**Fracture Location Nr of RCTs n RESULTS References** Vertebral fracture 4 RCTs 11,842 RR=0.55; CI 95%: 0.46 to 0.66 [10-13] Non-vertebral fracture 3 RCTs 11,774 RR=0.73; CI 95%: 0.65 to 0.81 [10, 11,14] Hip fracture 2 RCTs 11,745 RR=0.63; CI 95%: 0.42 to 0.93 [11,14] Any type of fracture 3 RCTs 11,556 RR=0.70; CI 95%: 0.63 to 0.78 [14-16]

be effective in treating pain resulting from fractures of the vertebral column. [2-4]

**2.2. Hormonal Replacement Therapy (HRT)**

76 Advances in Osteoporosis

**Table 1.** Relative fracture risk in NICE meta-analysis

#### *<IP_ADDRESS>. Vascular illness*

A thorough and methodical review of five RCTs looking at HRT with estrogen and two looking at combined HRT estrogen plus progesterone, failed to display compelling variance in the occurrence of acute coronary events (including acute myocardial infarction) between the cohort subject to intervention and the control cohort [7]. A combination of the outcomes of three studies contrasting estrogenic therapy to a placebo [11, 17] reported an odds ratio (OR) of 1.34 (IC 95%: 1.07 to 1.68) for cerebral vascular events. The combined outcomes of the studies that contrasted estrogen plus progesterone combined treatment with a placebo [5, 18], indicated an elevated risk of ictus (OR=1.28; CI 95%: 1.05 to 1.57) in the cohort subject to intervention. Out of four thorough and methodical reviews of observational trials looking at women treated with HRT [19-22], three of these indicated a significant decrease in the global mortality risk for acute coronary events. A recently published, thorough and methodical review, that compensated for selection bias of inclusion and analysis, did not reveal any link between the THS and the incidence, and mortality of acute coronary events [22].

The WHI primary prevention trial indicated a distinct elevation of the risk of acute coronary events (41%), starting the second year of treatment (29 instances in the treatment cohort, compared with 21 instances for 10,000 women per year in the general population) [5]. This elevated risk was greater in non-mortal coronary incidents (RR=1.50; CI 95%: 1.08 to 2.08) than in the mortal coronary incidents (RR=1.20; CI 95%: 0.58 to 2.50). The RCTs of HRT with estrogens alone, in both primary and secondary prevention, failed to indicate any positive impact on cerebrovascular illness [23]. In addition, the WHI study cohort with estrogen indicated an elevated risk of cerebrovascular incidents.

#### *<IP_ADDRESS>. Venous thrombotic events*

In a thorough and methodical review, McLean et al. indicated that estrogen patients treated with estrogen demonstrate an elevated risk of major venous thromboembolic incidents (OR=1.36; CI 95%: 1.01 to 1.86) compared to the placebo cohort [7]. A further thorough and methodical review assessing the impact of HRT (estrogen with or without progestagens) encompassed 12 studies (3 RCTs, 8 case-control studies and 1 cohort study) and indicated an elevated risk of thromboembolism (RR=2.14; CI 95%: 1.64 to 2.81). This risk was elevated in the first two years of the therapy and it varied according to the dose [24].

#### *<IP_ADDRESS>. Breast cancer*

A thorough and methodical review of 4 RCTs proved that patients treated with estrogens alone have a lower risk of breast cancer (OR=0.79; CI 95%: 0.66 to 0.93) than those treated with the placebo [7]. On the other hand, patients treated with estrogen and progestin have a higher risk of breast cancer (OR=1.28; CI 95%: 1.03 to 1.60) than those treated with the placebo [5, 18, 24].

Nonetheless the combined HRT cohort of the WHI study presented an elevated risk of invasive breast cancer [5]. This elevated risk occurred following the fourth year of treatment (RR=1.26; CI 95 %: 1.0 to 1.59), with a propensity to rise in line with the treatment's longevity (38 instances compared with 30 for 10,000 women per year).

#### *<IP_ADDRESS>. Endometrial cancer*

The treatment of estrogen alone elevates the risk of subsequent endometrial hyperplasia and cancer [25,26]. A meta-analysis including 29 observational studies reported a demonstrable elevation of the risk of endometrial cancer, with or without combined estrogens (RR=2.3; CI 95%: 2.1 to 2.5) [20]. This risk is directly related to the treatment's longevity and continues to be raised for a maximum of 5 years or more following the termination of treatment.

#### *<IP_ADDRESS>. Ovarian cancer*

Recently published thorough and methodical reviews of observational trials indicate an elevated incidence of ovarian cancer amongst women undergoing treatment, particularly longterm therapies (more than 10 years) [28,28]. Two cohort trials of postmenopausal women who underwent treatment for a period of more than 10 years corroborate this elevated risk of ovarian cancer (RR=2.2; CI 95%: 1.53 to 3.17), as well as an elevated mortality risk (RR=1.59; CI 95%: 1.13 to 2.25) [29, 30].

We can conclude that HRT is an effective therapy both for postmenopausal osteoporosis and for the management of fracture risk. Nevertheless, even taking this conclusion into account, the use of combined HRT is not recommeneded for periods greater than 5 years, given the possible risk factors linked with treatments using a daily dose equivalent to 50 pg of estradiol. When HRT treatment is indicated, it should be prescribed at a low dosage (equivalent to estrogen transdermal patches of 25 mcg), only using higher doses if it is absolutely essential to do so. Estrogens and progestagens are only advised for the treatment of women with intact uteri. The level of the progestagen dose should be determined on the basis of the estrogen dose. In instances where a hysterectomy was carried out as a result of endometrial cancer, HRT should not involve combined estrogen and progestagens. Continuous combined HRT treat‐ ment should only commence following one whole year of menopause.

#### **2.3. Selective Estrogen Receptor Modulators (SERMs)**

Selective estrogen receptor modulators are medications with a selective impact on the estrogen receptor. They can function as estrogen receptor (ER) agonists in some tissues while in other tissues functioning as estrogen receptor antagonists. As a result of their selective estrogenagonist behaviour within a variety of tissues, SERMs may be indicated as an alternative option for the prevention or treatment of conditions like osteoporosis, which are the result of a deficiency of estrogen, where avoiding the negatives effects of estrogens is a priority.

#### *2.3.1. Differences between SERMs*

At present there are two forms of SERM, which are distinguished by their chemical structure: triphenylethylene derivatives, for example tamoxifen and toremifene, and benzothiophene derivatives, for example raloxifene and bazedoxifene. Tamoxifen and toremifene on the one hand are indicated for use in the treatment of breast cancer. Raloxifene on the other hand is used for the prevention and treatment of osteoporosis and in addition the prevention of breast cancer. All SERMs have been linked with an elevated occurrence of pulmonary thromboemb‐ olism and with the start of hot flushes, however they also impact in a positive manner on the lipid profile.

The SERMs vary distinctively with regards to tissue specificity. Bazedoxifene appears to have a lower impact on the uterus than estradiol jointly with raloxifene in animal experiments as a result of reduced estrogen receptor alpha agonistic effects.

#### *2.3.2. Raloxifene*

CI 95 %: 1.0 to 1.59), with a propensity to rise in line with the treatment's longevity (38 instances

The treatment of estrogen alone elevates the risk of subsequent endometrial hyperplasia and cancer [25,26]. A meta-analysis including 29 observational studies reported a demonstrable elevation of the risk of endometrial cancer, with or without combined estrogens (RR=2.3; CI 95%: 2.1 to 2.5) [20]. This risk is directly related to the treatment's longevity and continues to

Recently published thorough and methodical reviews of observational trials indicate an elevated incidence of ovarian cancer amongst women undergoing treatment, particularly longterm therapies (more than 10 years) [28,28]. Two cohort trials of postmenopausal women who underwent treatment for a period of more than 10 years corroborate this elevated risk of ovarian cancer (RR=2.2; CI 95%: 1.53 to 3.17), as well as an elevated mortality risk (RR=1.59; CI

We can conclude that HRT is an effective therapy both for postmenopausal osteoporosis and for the management of fracture risk. Nevertheless, even taking this conclusion into account, the use of combined HRT is not recommeneded for periods greater than 5 years, given the possible risk factors linked with treatments using a daily dose equivalent to 50 pg of estradiol. When HRT treatment is indicated, it should be prescribed at a low dosage (equivalent to estrogen transdermal patches of 25 mcg), only using higher doses if it is absolutely essential to do so. Estrogens and progestagens are only advised for the treatment of women with intact uteri. The level of the progestagen dose should be determined on the basis of the estrogen dose. In instances where a hysterectomy was carried out as a result of endometrial cancer, HRT should not involve combined estrogen and progestagens. Continuous combined HRT treat‐

Selective estrogen receptor modulators are medications with a selective impact on the estrogen receptor. They can function as estrogen receptor (ER) agonists in some tissues while in other tissues functioning as estrogen receptor antagonists. As a result of their selective estrogenagonist behaviour within a variety of tissues, SERMs may be indicated as an alternative option for the prevention or treatment of conditions like osteoporosis, which are the result of a

At present there are two forms of SERM, which are distinguished by their chemical structure: triphenylethylene derivatives, for example tamoxifen and toremifene, and benzothiophene derivatives, for example raloxifene and bazedoxifene. Tamoxifen and toremifene on the one

deficiency of estrogen, where avoiding the negatives effects of estrogens is a priority.

ment should only commence following one whole year of menopause.

**2.3. Selective Estrogen Receptor Modulators (SERMs)**

be raised for a maximum of 5 years or more following the termination of treatment.

compared with 30 for 10,000 women per year).

*<IP_ADDRESS>. Endometrial cancer*

78 Advances in Osteoporosis

*<IP_ADDRESS>. Ovarian cancer*

95%: 1.13 to 2.25) [29, 30].

*2.3.1. Differences between SERMs*

Raloxifene acts as estrogen agonist in bone and other systems but not in reproductive tissue. Many trials have proved the effectiveness of raloxifene for preserving bone in the early postmenopausal phase. In a meta-analysis of seven studies (four treatment and three preven‐ tion studies) which looked at the impact of raloxifene versus a placebo on bone mineral density, raloxifene augmented bone mineral density within the lumbar spine following a two year period of treatment [31]. A trial of 601 women, five years following the menopause, who were given a daily dose of 30, 60 or 150mg of raloxifene over two years, indicated an augmentation of their bone mineral density in spine and hip, whereas those subjected to the placebo presented reduced bone mineral density in the same locations [32]. In contrast with the results from the placebo, the average alteration in BMD with 60mg of raloxifene was 2.4% in the spine and 2.4% at the total hip (p<0.001 versus placebo). Postmenopausal women presenting low bone mass and osteoporosis were monitored over eight years in the study entitled 'Multiple Outcomes of Raloxifene Evaluation' (MORE, n=7,705) and its sister trial entitled 'Continuing Outcomes Relevant to Evista' (CORE, n=4,011) [33]. In relation to fractures, whilst raloxifene treatment led to a decreased risk of vertebral fracture, it failed to demonstrate a reduced risk of non-vertebral fractures. Nevertheless, in a meta-analysis of RCTs contrasting the effects of raloxifene with those of a placebo, raloxifene typically led to a decreased risk of vertebral fractures in postmenopausal women (OR=0.6; CI 95%: 0.5-0.7).

The results of the MORE trial indicated that, following a raloxifene treatment period of four years, at 60mg per day, the cumulative relative risk of one or more vertebral fractures was 0.64 (IC 95%: 0.53 - 0.76), compared with treatment using a placebo.

Verus placebo, treatment with 60mg of raloxifene was also linked to a decrease of 65% to 78% in occurrences of invasive breast cancer and invasive breast cancer with positive estrogen receptor (both p <0.05).

#### *<IP_ADDRESS>. Adverse Effects*

The MORE and CORE studies reported a link between raloxifene and an elevated incidence (1.7 times) of thromboembolism (TE), versus treatment using a placebo (95% CI: 0.93-3.14; risk difference total of 0.9/1,000 women-years) [34]. In a meta-analysis of nine trials, raloxifene treatment was linked with an elevated incidence of deep venous thrombosis and pulmonary embolism (OR=1.5; CI 95%: 1.1-2.1 and OR=1.9; 95% CI: 1.0-3.5, respectively) [35]. The RUTH trial ('Raloxifene Use for The Heart'), which studied 10,101 postmenopausal women with an average age of 68 and presenting with coronary heart disease, indicated a link between raloxifene and an elevated incidence of fatal stroke (HR=1.49; 95% CI: 1.00-2.24, a rise in the absolute risk of 0.7/1,000 women-years) as well as an elevated risk of thromboembolism (HR=1.44; 95% CI: 1.06-1.95, a rise in the absolute risk of 1.2/1,000 women-years) in comparison with the placebo results. No elevated risk of myocardial infarction or other coronary events was indicated in the RUTH trial. Nevertheless, in line with the observations regarding thromboembolism and pulmonary embolism, the outcomes of a recent review of a sub-cohort of the trial indicated that age had an impact on the occurrence of coronary events. For women of 60 years or under, the rate of occurrence of coronary events was distinctly reduced with raloxifene (50 cases), compared with the placebo group (84 cases; HR=0.59; 95% CI: 0.41 to 0.83, p=0.003). Raloxifene was also linked with an elevated occurrence of hot flushes, especially amongst women with recent menopause onset [36].

We can conclude that raloxifene provides an alternative option within osteoporosis therapies for specific patients. The drug's profile relating to heart disease and breast cancer is sound but its links to an elevated risk of venous thrombosis should be taken into account in its use as a treatment.

#### *<IP_ADDRESS>. Bazedoxifene*

Bazedoxifene is a third-generation SERM. Some key differences have been demonstrated between the generations regarding their impact on the uterus and on breast tissue in particular [37]. The drug was developed with raloxifene as a template and by replacing the benzothio‐ phene core with an indole ring [38].

In a phase II trial of healthy postmenopausal women, oral doses of bazedoxifene 2.5, 5, 10, 20, 30, or 40mg per day were as a rule well-tolerated and did not aggravate the endometrium. In addition, bazedoxifene 30 and 40 mg resulted in a notably reduced increase in the thickness of the endometrium and distinctly lowered the occurrence of uterine bleeding versus results from the placebo. In a two-year phase III trial of postmenopausal women at risk of osteoporosis, bazedoxifene 10, 20, and 40mg were proven to prevent bone loss and decrease bone turnover and were linked with a positive endometrial, ovarian, and breast security profile [39, 40].

A phase III, multi-centre, double-blind, randomised, controlled trial was formulated with the sole purpose of assessing the effectiveness of bazedoxifene in fracture prevention. The trial looked at 7,492 healthy postmenopausal women presenting with osteoporosis both with or without prevalent vertebral fractures. The women were randomly assigned to 20 or 40mg per day of bazedoxifene, 60mg of raloxifene, or to a placebo plus 1200mg of calcium and 400IU of vitamin D. The primary outcome was the occurrence of new vertebral fractures following a three-year treatment period. Secondary indicators included clinical vertebral fractures, worsening of vertebral fractures, non-vertebral fractures, breast cancer incidence, and varia‐ tions in height. Both bazedoxifene 20 and 40mg reduced the occurrence of vertebral fractures to a similar extent as raloxifene versus the placebo. The occurrence at 36 months of new vertebral fractures was 2.3%, 2.5%, 2.3%, and 4.1% in the bazedoxifene 20mg, bazedoxifene 40mg, raloxifene 60mg, and placebo cohorts, respectively, with a distinct lowering of the relative incidence for new vertebral fractures of 42%, 37%, and 42%, respectively, versus placebo. There was no overall impact on non-vertebral fractures, with incidence rates of 5.7% and 5.6% for the bazedoxifene 20 and 40mg cohorts, respectively, versus 5.9% for the raloxifene cohort and 6.3% for the placebo cohort. Nevertheless, in a later review of women with elevated fracture risk (poor femoral neck T-score and multiple vertebral fractures, n=1,772), bazedoxi‐ fene 20mg reduced the incidence of non-vertebral fracture by 50% and 44% reduction relative to the placebo (HR=0.50; 95% CI: 0.28–0.90; p=0.02) and raloxifene 60mg (HR=0.56; 95% CI: 0.31–1.01; p=0.05), respectively [41].

#### *<IP_ADDRESS>. Safety*

trial ('Raloxifene Use for The Heart'), which studied 10,101 postmenopausal women with an average age of 68 and presenting with coronary heart disease, indicated a link between raloxifene and an elevated incidence of fatal stroke (HR=1.49; 95% CI: 1.00-2.24, a rise in the absolute risk of 0.7/1,000 women-years) as well as an elevated risk of thromboembolism (HR=1.44; 95% CI: 1.06-1.95, a rise in the absolute risk of 1.2/1,000 women-years) in comparison with the placebo results. No elevated risk of myocardial infarction or other coronary events was indicated in the RUTH trial. Nevertheless, in line with the observations regarding thromboembolism and pulmonary embolism, the outcomes of a recent review of a sub-cohort of the trial indicated that age had an impact on the occurrence of coronary events. For women of 60 years or under, the rate of occurrence of coronary events was distinctly reduced with raloxifene (50 cases), compared with the placebo group (84 cases; HR=0.59; 95% CI: 0.41 to 0.83, p=0.003). Raloxifene was also linked with an elevated occurrence of hot flushes, especially

We can conclude that raloxifene provides an alternative option within osteoporosis therapies for specific patients. The drug's profile relating to heart disease and breast cancer is sound but its links to an elevated risk of venous thrombosis should be taken into account in its use as a

Bazedoxifene is a third-generation SERM. Some key differences have been demonstrated between the generations regarding their impact on the uterus and on breast tissue in particular [37]. The drug was developed with raloxifene as a template and by replacing the benzothio‐

In a phase II trial of healthy postmenopausal women, oral doses of bazedoxifene 2.5, 5, 10, 20, 30, or 40mg per day were as a rule well-tolerated and did not aggravate the endometrium. In addition, bazedoxifene 30 and 40 mg resulted in a notably reduced increase in the thickness of the endometrium and distinctly lowered the occurrence of uterine bleeding versus results from the placebo. In a two-year phase III trial of postmenopausal women at risk of osteoporosis, bazedoxifene 10, 20, and 40mg were proven to prevent bone loss and decrease bone turnover and were linked with a positive endometrial, ovarian, and breast security profile [39, 40]. A phase III, multi-centre, double-blind, randomised, controlled trial was formulated with the sole purpose of assessing the effectiveness of bazedoxifene in fracture prevention. The trial looked at 7,492 healthy postmenopausal women presenting with osteoporosis both with or without prevalent vertebral fractures. The women were randomly assigned to 20 or 40mg per day of bazedoxifene, 60mg of raloxifene, or to a placebo plus 1200mg of calcium and 400IU of vitamin D. The primary outcome was the occurrence of new vertebral fractures following a three-year treatment period. Secondary indicators included clinical vertebral fractures, worsening of vertebral fractures, non-vertebral fractures, breast cancer incidence, and varia‐ tions in height. Both bazedoxifene 20 and 40mg reduced the occurrence of vertebral fractures to a similar extent as raloxifene versus the placebo. The occurrence at 36 months of new vertebral fractures was 2.3%, 2.5%, 2.3%, and 4.1% in the bazedoxifene 20mg, bazedoxifene 40mg, raloxifene 60mg, and placebo cohorts, respectively, with a distinct lowering of the

amongst women with recent menopause onset [36].

treatment.

80 Advances in Osteoporosis

*<IP_ADDRESS>. Bazedoxifene*

phene core with an indole ring [38].

Miller et al. demonstrated that deep venous thromboembolism was uncommon with baze‐ doxifene (0% to 0.6% with varying dosage levels after two years) and similar to the placebo (0.3%). The rate of occurrence and the intensity of hot flushes were comparable with raloxifene, but slightly elevated versus placebo [40]. In the trial by Silverman et al., leg cramps (10.9% to 11.7% with varying dosage after three years) and deep venous thromboembolism (0.4% to 0.5% with varying dosage after three years) were decidedly more prevalent with bazedoxifene compared with the placebo (8.2% for leg cramps and 0.2% for deep venous thromboembolism), while fibrocystic breast disease was markedly less frequent. No distinction in risk levels between bazedoxifene and placebo was noted for myocardial infarction, strokes (ischemic or hemorraghic), or retinal vein thrombosis [40-43].

We can conclude that bazedoxifene appears to have improved selectivity in contrast with other SERMs. The impact of bazedoxifene on the skeleton is not dissimilar to raloxifene, and bazedoxifene may be employed in the same way as raloxifene. The usefulness of bazedoxifene possibly lies in its risk profile being distinct to that of raloxifene, particularly with regards to uterine safety, and bazedoxifene may therefore present another option for the prevention and treatment of osteoporosis.

#### *2.3.3. Lasofoxifene*

Lasofoxifene is a powerful third-generation SERM. It has a distinct structure compared to firstand second-generation SERMs (raloxifene, tamoxifen and clomiphene or idoxifene). Lasofox‐ ifene displays powerful estrogenic and anti-estrogenic activity in vitro and in vivo, targeting any areas with estrogens receptors, including bone, uterus, breast, blood vessels, and liver. Lasofoxifene has been analysed in postmenopausal women with regards to the prevention and treatment of osteoporosis. Security and tolerance levels of lasofoxifene is similar to that of raloxifene, however nonadherence rates as a result of adverse events are greater with lasofox‐ ifene. Despite these indications, results demonstrate that lasofoxifene treatment may lead to greater endometrial thickness versus the placebo, despite there being no evidence of an elevated incidence of endometrial hyperplasia or cancer.

The PEARL study, a three-year pivotal fracture study, showed that lasofoxifene elevated lumbar spine and femoral neck BMD by approximately 3%. Furthermore, vertebral fractures saw a decrease of 42%, and non-vertebral fractures of 27%, with a decrease in markers of bone turnover. Nevertheless, lasofoxifene did not reduce the risk of hip fractures [43].

#### **2.4. Bisphosphonates**

#### *2.4.1. Analysis and mode of action*

Bisphosphonates are a member of a class of antiresorptive agents whose antifracture action is well-documented through randomised controlled studies. There have been no studies to compare different bisphosphonates, a fact which has prevented the identification of a definite order of effectiveness for treatment.

Bisphosphonates lower fracture risk as a result of its inhibitory action of osteoclasts, which enables the osteoblasts to synthesize bone in the resorption spaces and some bone lacunae. This produces an augmentation in bone mass. However, the bisphosphonates also increase bone quality, by conserving the bone architecture, as demonstrated in studies which have analysed the biopsies of treated patients and control subjects.

Bisphosphonates comprise pyrophosphate analogs in which the central oxygen has been replaced by a carbon atom and two side chains (R1 and R2). Two phosphate chains are vital to enable the drug to bind to bone and to have an antiresorptive effect.

#### *2.4.2. Etidronate*

Etidronate was the original bisphosphonate used in osteoporosis therapy. It is no longer used in current practice. Its greatest asset is most likely its cost. It augments bone mass in the spine and femur and lowers the risk of vertebral fractures, however it has not demonstrated a reduction in the incidence of femoral fractures [44,45].

#### *2.4.3. Clodronate*

Clodronate has been deployed in postmenopausal osteoporosis therapy in oral and intrave‐ nous treatments. The trials indicate that it reduces the risk of bone loss in the vertebral spine in comparison to control subjects, and it presents similar results to estrogens after two years. In a six-year long study, it was also demonstrated to lower the incidence rate of vertebral fractures. McCloskey et al. carried out a three-year, double-blind, controlled study to observe the impact of oral clodronate (800mg per day) on fracture rates. In this study, clodronate was linked with a distinct improvement in the mean lumbar spine and hip BMD. Furthermore, it significantly lowered the risk of vertebral fracture (relative risk, 0,54; 95% CI, 0,37-0,80; p<0,0001). Despite these outcomes, subsequent to the introduction of powerful nitrogen bisphosphonates, the first-generation bisphosphonates have been reduced to a therapy of last resort [46].

#### *2.4.4. Alendronate (alendronic acid)*

Alendronate is one of the most commonly deployed bisphosphonates. It augments vertebral bone mass approximately 6-8% and 3-6% at the hip in postmenopausal osteoporotic women after a three-year treatment. It demonstrates a reduction in vertebral and non-vertebral fractures of around 50% in this time period. In male osteoporosis, it has demonstrated improvements in bone mass of 5% after two years of treatment.

Alendronate is given orally, in doses of 70mg/week, fasting with 200 ml of water. The patient is prohibited from consuming solids or liquids for 30 minutes after treatment and must remain standing for this time.

The decisive study of alendronate, the FIT (Fracture Intervention Trial), demonstrated that the incidence of clinical fracture was reduced for the alendronate cohort compared to the control cohort (139 (13.6%) versus 183 (18.2%); relative hazard=0.72 (0.58-0.90)). The corresponding risk of hip and wrist fracture for the alendronate cohort when compared to the placebo cohort were 0.49 (0.23-0.99) and 0.52 (0.31-0.87) [47]. Ensrud et al. provided an assessment of a subset of FIT subjects who were patients with an elevated risk of fracture. The outcomes of this analysis demonstrate a decisive 47% lowering of the risk of new vertebral fractures in the alendronate cohort when set against the control cohort. A number of other papers have been generated from the FIT study, addressing multiple symptomatic fractures, bone mineral density, biochemical markers of formation and resorption, fracture prevention in osteopenic women, impact of alendronate continuation versus discontinuation, and the impact on women who lost bone over the course of treatment [48-51].

We can conclude that alendronate is a well-tolerated, secure and efficacious treatment method for postmenopausal osteoporosis, male osteoporosis, and glucocorticoid induced osteoporosis (GIOP).

#### *2.4.5. Risedronate*

saw a decrease of 42%, and non-vertebral fractures of 27%, with a decrease in markers of bone

Bisphosphonates are a member of a class of antiresorptive agents whose antifracture action is well-documented through randomised controlled studies. There have been no studies to compare different bisphosphonates, a fact which has prevented the identification of a definite

Bisphosphonates lower fracture risk as a result of its inhibitory action of osteoclasts, which enables the osteoblasts to synthesize bone in the resorption spaces and some bone lacunae. This produces an augmentation in bone mass. However, the bisphosphonates also increase bone quality, by conserving the bone architecture, as demonstrated in studies which have

Bisphosphonates comprise pyrophosphate analogs in which the central oxygen has been replaced by a carbon atom and two side chains (R1 and R2). Two phosphate chains are vital

Etidronate was the original bisphosphonate used in osteoporosis therapy. It is no longer used in current practice. Its greatest asset is most likely its cost. It augments bone mass in the spine and femur and lowers the risk of vertebral fractures, however it has not demonstrated a

Clodronate has been deployed in postmenopausal osteoporosis therapy in oral and intrave‐ nous treatments. The trials indicate that it reduces the risk of bone loss in the vertebral spine in comparison to control subjects, and it presents similar results to estrogens after two years. In a six-year long study, it was also demonstrated to lower the incidence rate of vertebral fractures. McCloskey et al. carried out a three-year, double-blind, controlled study to observe the impact of oral clodronate (800mg per day) on fracture rates. In this study, clodronate was linked with a distinct improvement in the mean lumbar spine and hip BMD. Furthermore, it significantly lowered the risk of vertebral fracture (relative risk, 0,54; 95% CI, 0,37-0,80; p<0,0001). Despite these outcomes, subsequent to the introduction of powerful nitrogen bisphosphonates, the first-generation bisphosphonates have been reduced to a therapy of last

Alendronate is one of the most commonly deployed bisphosphonates. It augments vertebral bone mass approximately 6-8% and 3-6% at the hip in postmenopausal osteoporotic women

turnover. Nevertheless, lasofoxifene did not reduce the risk of hip fractures [43].

**2.4. Bisphosphonates**

82 Advances in Osteoporosis

*2.4.2. Etidronate*

*2.4.3. Clodronate*

resort [46].

*2.4.4. Alendronate (alendronic acid)*

*2.4.1. Analysis and mode of action*

order of effectiveness for treatment.

analysed the biopsies of treated patients and control subjects.

reduction in the incidence of femoral fractures [44,45].

to enable the drug to bind to bone and to have an antiresorptive effect.

This treatment has been proven to improve bone mass in spine and hip and to considerably lower the incidence of fracture in postmenopausal women. Treatment of postmenopausal women with osteoporosis with risedronate over a three-year period has produced a reduction in the risk of vertebral fractures in roughly 50% and non-vertebral fractures in 39% of subjects. At the hip, the fracture reduction rate is between 40 and 60%. After a five-year period, the outcomes are comparable. The treatment has demonstrated its anti-fracture efficacy after a sixmonth course. In other trials it has been proven that this reduction in risk was still present following a seven-year period of treatment, and was accompanied by a positive security profile. One of the principal studies of risedronate [52] looked at 5,445 women aged 70 to 79 years with osteoporosis (T-score at the femoral neck greater than -4 SD below the mean or lower than -3 plus a non-skeletal risk factor for hip fracture, such as poor gait or a tendency to fall) and 3,886 women aged at least 80 years with a minimum of one non-skeletal risk factor for hip fracture or poor BMD at the femoral neck (T-score below -4 or below -3 plus a hip-axis length of 11.1cm or greater). The subjects were given a treatment at random of either oral risedronate (2.5 or 5.0mg per day) or a placebo, over a three-year period. The outcomes indicated that the risk of hip fracture among subjects given risedronate was 2.8%, versus 3.9% among those given the placebo (relative risk, 0.7; 95% CI, 0.6 to 0.9; p=0.02). In the cohort of women with osteoporosis (70 to 79 years old), the risk of hip fracture among subjects given risedronate was 1.9%, versus 3.2% among subjects given the placebo (relative risk, 0.6; 95% CI, 0.4 to 0.9; p=0.009). In the cohort of subjects chosen principally for non-skeletal risk factors (those at least 80 years old), the risk of hip fracture was 4.2% for subjects given risedronate and 5.1% for those given the placebo (p=0.35) [52].

To assess the impact on vertebral fracture risk, Reginster et al. carried out a randomised, double-blind, controlled trial to evaluate the effectiveness and security of risedronate for reducing the risk of vertebral fractures in postmenopausal women with established osteopo‐ rosis. The trial was carried out at 80 locations in Europe and Australia. In total, 1,226 postme‐ nopausal women with two or more prevalent vertebral fractures were given risedronate 2.5mg or 5mg per day or a placebo. Whilst the trial was carried out over three years, the 2.5mg cohort was ended by protocol amendment after two years. Risedronate 5mg lowered the incidence of new vertebral fractures by 49% over three years in comparison with the placebo (p<0.001). A distinct decrease of 61% was witnessed over the initial year alone (p=0.001). The decreased incidence of fracture was comparable in both cohorts after two years. The incidences of nonvertebral fracture saw a decrease of 33% in relation to the placebo figures over three years (p=0.06). Risedronate produced a distinct elevation in BMD at the spine and hip within a sixmonth period. We can conclude that risedronate 5mg was an efficacious and well-tolerated treatment for severe postmenopausal osteoporosis, decreasing the risk of vertebral fractures and increasing bone density in women with chronic osteoporosis [53].

#### *2.4.6. Ibandronate*

In trials lasting three years, ibandronate has been proven to decrease the risk of vertebral fractures (52%) and improve vertebral BMD (6.5%) whilst not having a substantial negative impact on bone histology. It has also shown to be very efficacious in reducing bone loss in GIOP (glucocorticoid induced osteoporosis). In women with severe osteoporosis T scores (<-3), it decreases the risk of non-vertebral fractures up to 69% [54].

Randomised clinical studies such as MOPS (Monthly Oral Pilot Study) or MOBILE (Monthly Oral Ibandronate in Ladies) have indicated that the ibandronate monthly dosage is just as efficacious and safe as the daily dosage. Amongst the general population of the pivotal trial (BONE, Oral Ibandronate Osteoporosis Vertebral Fracture Trial in North America and Europe), the likelihood of adverse incidents of the gastrointestinal tract in both the daily and the intermittent treatment cohorts was similar to the control cohort. Dyspepsia was the only adverse incident with a marginally greater rate in subjects undergoing therapy with ibandronate [55].

#### *2.4.7. Zoledronate (zoledronic acid)*

Zoledronic acid is a third-generation bisphosphonate. It is roughly as powerful as alendronate, risedronate and ibandronate, however the application of this drug intravenously prevents any negative impact and in fact augments the bioavailability, whilst also improving compliance to 100%.

The HORIZON trial (Health Outcomes and Reduced Incidence with Zoledonic Acid Once Yearly Pivotal Fracture Trial) was a global, multi-centre, double-blind, controlled study of postmenopausal women with osteoporosis, whose goal was to demonstrate the increase efficacy of intravenous zoledronic acid 5mg compared with a control. Subjects presented with densitometric osteoporosis or densitometric osteopenia with a minimum of 2 mild to moderate vertebral fractures [56]. Over 7,700 women were involved in the trial and were monitored over a three-year period; particular scrutiny was made of new fractures, bone remodeling bio‐ chemical markers and densitometric developments. On completion of the trial, subjects that had been treated with zoledronic acid presented a decrease in the vertebral fracture risk of 70%. The decrease was comparable for the first two years of the trial, varying from 60% to 71%. In addition, subjects given zoledronate presented a decrease of 41% in hip fracture incidence and 25% in non-vertebral fracture incidence. The outcomes of bone density and biochemical bone remodeling markers were also markedly improved for the cohort given zoledronic acid. Furthermore, bone mineral density was elevated to over 6% in the lumbar spine and total hip, and to over 5% in the femoral neck. The biochemical markers of bone remodeling, after the initial transfusion of zoledronic acid, decreased significantly as anticipated, and stayed stable throughout the remainder of the trial [57].

Many subjects experienced adverse effects over the course of the trial, with a greater occurrence of these in the zoledronate cohort. This variance was explained by post-infusion syndrome, which commonly manifested itself 24-48 hours after the zoledronic acid infusion and dissi‐ pated three days after infusion. The syndrome presented with mild fever, myalgias, flu-like symptoms, headache and/or arthralgias and was dissipated with analgesic, non-steroid antiinflammatory drugs or acetaminophen. A few subjects presented with passing renal function deterioration 9 to 11 days post-infusion, however these instances were of no clinical transcen‐ dence [57].

Perhaps the most significant conclusion in relation to zoledronate therapy is the 28% decrease in mortality of any cause, which was demonstrated in a cohort of over 2,000 subjects with femur fracture [56].

We can conclude that zoledronate therapy is extremely efficacious in the reduction of vertebral, non-vertebral and hip fractures. It reduces mortality, independent of the cause, following a femur fracture.Moreover, it is a low-risk therapy that avoids the gastrointestinal adverse events and high nonadherence rates that are commonly encountered with other bisphospho‐ nates, but it should be dispensed and regulated with great caution when treating individuals with severe renal function impairment.

#### *2.4.8. Safety of bisphosphonates*

0.4 to 0.9; p=0.009). In the cohort of subjects chosen principally for non-skeletal risk factors (those at least 80 years old), the risk of hip fracture was 4.2% for subjects given risedronate and

To assess the impact on vertebral fracture risk, Reginster et al. carried out a randomised, double-blind, controlled trial to evaluate the effectiveness and security of risedronate for reducing the risk of vertebral fractures in postmenopausal women with established osteopo‐ rosis. The trial was carried out at 80 locations in Europe and Australia. In total, 1,226 postme‐ nopausal women with two or more prevalent vertebral fractures were given risedronate 2.5mg or 5mg per day or a placebo. Whilst the trial was carried out over three years, the 2.5mg cohort was ended by protocol amendment after two years. Risedronate 5mg lowered the incidence of new vertebral fractures by 49% over three years in comparison with the placebo (p<0.001). A distinct decrease of 61% was witnessed over the initial year alone (p=0.001). The decreased incidence of fracture was comparable in both cohorts after two years. The incidences of nonvertebral fracture saw a decrease of 33% in relation to the placebo figures over three years (p=0.06). Risedronate produced a distinct elevation in BMD at the spine and hip within a sixmonth period. We can conclude that risedronate 5mg was an efficacious and well-tolerated treatment for severe postmenopausal osteoporosis, decreasing the risk of vertebral fractures

In trials lasting three years, ibandronate has been proven to decrease the risk of vertebral fractures (52%) and improve vertebral BMD (6.5%) whilst not having a substantial negative impact on bone histology. It has also shown to be very efficacious in reducing bone loss in GIOP (glucocorticoid induced osteoporosis). In women with severe osteoporosis T scores (<-3),

Randomised clinical studies such as MOPS (Monthly Oral Pilot Study) or MOBILE (Monthly Oral Ibandronate in Ladies) have indicated that the ibandronate monthly dosage is just as efficacious and safe as the daily dosage. Amongst the general population of the pivotal trial (BONE, Oral Ibandronate Osteoporosis Vertebral Fracture Trial in North America and Europe), the likelihood of adverse incidents of the gastrointestinal tract in both the daily and the intermittent treatment cohorts was similar to the control cohort. Dyspepsia was the only adverse incident with a marginally greater rate in subjects undergoing therapy with

Zoledronic acid is a third-generation bisphosphonate. It is roughly as powerful as alendronate, risedronate and ibandronate, however the application of this drug intravenously prevents any negative impact and in fact augments the bioavailability, whilst also improving compliance to

The HORIZON trial (Health Outcomes and Reduced Incidence with Zoledonic Acid Once Yearly Pivotal Fracture Trial) was a global, multi-centre, double-blind, controlled study of

and increasing bone density in women with chronic osteoporosis [53].

it decreases the risk of non-vertebral fractures up to 69% [54].

5.1% for those given the placebo (p=0.35) [52].

*2.4.6. Ibandronate*

84 Advances in Osteoporosis

ibandronate [55].

100%.

*2.4.7. Zoledronate (zoledronic acid)*