Denosumab is a fully human monoclonal antibody against RANK ligand (RANKL) marketed under the name of Prolia® (Pralia® in Japan) for osteoporosis and Xgeva® for the prevention of skeletal complications in oncology. The discovery of the essential role of RANK ligand and of RANK signaling in osteoclast differentiation, activity, and survival led the way to developing denosumab. Denosumab acts by binding to and inhibiting RANKL from binding to RANK, leading to the loss of osteoclasts from bone surfaces.

RANK ligand is one of the two cytokines that are essential and sufficient to induce osteoclast differentiation. The other is the macrophage colony-stimulating factor (M-CSF). M-CSF binds to c-Fms, a single transmembrane domain receptor of the tyrosine kinase family, and RANKL binds to RANK, a single transmembrane receptor of the tumor necrosis factor (TNF) receptor family, which forms trimers upon ligand binding. The role of M-CSF is to first activate the proliferation and survival of cells of the monocyte–macrophage lineage and the expression of RANK, allowing the action of RANKL, which, together with M-CSF, constitutes an absolute requirement for commitment to and progression of early precursors along the osteoclast lineage. The differentiation step from osteoclast precursors to multinucleated osteoclasts is then induced by RANKL, again through the M-CSF-dependent expression of RANK at the cell surface of these early precursors. M-CSF and RANKL are both secreted by bone marrow stromal cells and osteoblasts, whereas RANKL is also secreted by T cells and to a lesser extent by B cells. The other component of the RANKL/RANK system is osteoprotegerin (OPG), which is a shed extracellular portion of the RANK receptor . Most of the cells producing RANKL also produce this decoy RANK receptor, OPG, which acts as an antagonist to RANK signaling and osteoclastogenesis by scavenging RANKL in the extracellular environment. OPG is also a regulated molecule, and it is the ratio between RANKL and OPG which determines the level of activation of RANK and therefore the extent to which osteoclast production and function is activated. All these molecules are thought to act in a paracrine manner and to regulate bone resorption locally. The overall endocrine regulation of skeletal homeostasis involves interactions between systemic hormones and the RANKL/RANK/OPG system. The expression and secretion of both RANKL and OPG are regulated by several calcium-regulating hormones, including estrogens, parathyroid hormone (PTH), and vitamin D3.

The first attempts to develop therapeutics based on these pathways utilized a chimeric OPG-Fc fusion protein to antagonize RANKL. However, the formation of neutralizing antibodies against OPG after administration of the fusion protein and its potential cross-reactivity with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) led to the more attractive strategy of inhibiting RANKL directly using denosumab.

The key pharmacological differences between denosumab and the bisphosphonates reside in the distribution of the drugs within bone and their effects on precursors and mature osteoclasts [15]. This may explain differences in the degree and rapidity of reduction of bone resorption, their potential differential effects on trabecular and cortical bone, and the reversibility of their actions.

In the FREEDOM pivotal phase 3 clinical trial, denosumab was shown to significantly reduce vertebral, non-vertebral, and hip fractures by 68 %, 20 %, and 40 %, respectively, compared with placebo. In the FREEDOM study, 7808 women with postmenopausal osteoporosis were randomized to receive denosumab 60 mg subcutaneously once every 6 months or placebo [16]. FREEDOM is the acronym for the Fracture Reduction Evaluation of Denosumab in Osteoporosis every 6 Months study.

These and further studies involving over 12,000 patients have confirmed the efficacy and overall safety of denosumab [17–20], out to 8 years of treatment in the ongoing extension studies. Denosumab treatment increased BMD at the total hip, lumbar spine, and/or femoral neck and reduced markers of bone turnover to a significantly greater extent than oral bisphosphonates in women who had not received bisphosphonates at all or in the recent past or in those who had switched from alendronate to denosumab treatment.

There are several respects in which the differences between bisphosphonates and denosumab have practical consequences. It appears that the effects of denosumab to increase BMD continue on prolonged treatment, whereas they may plateau earlier on bisphosphonates.

Denosumab may also have a greater impact on protecting cortical bone than bisphosphonates, which is likely to contribute to its efficacy in reducing fractures.

However, the effects of denosumab rapidly reverse when treatment stops which means that ensuring compliance is even more important if potential benefits are to be sustained. Another difference is the potential use of denosumab in patients with impaired renal function, in whom use of bisphosphonates is contraindicated.

The recognition that the decline in estrogen production at the menopause was associated with bone loss dates back to the time of Albright more than half a century ago. The use of estrogens as HRT (ERT or hormone replacement therapy) in postmenopausal women was therefore a logical approach toward alleviating the symptoms and consequences of the menopause. After the link between menopause and osteoporosis was first identified, estrogen treatment became the standard means for preventing bone loss, even though there was no fracture data. This changed dramatically when results from the Women’s Health Initiative (WHI) study were published, which showed an increase in heart attacks and breast cancer [21]. Even though the risks were small, this prompted a large drop off in estrogen use. In later analyses, the WHI study showed that estrogen reduced fractures and actually prevented heart attacks in the 50–60-year age group. Estrogen alone appeared to be safer to use than estrogen plus the progestin medroxyprogesterone acetate and actually reduced breast cancer. The overall benefits and risks of estrogens are now better appreciated and are being continuously reappraised [22, 23]. In one analysis of hysterectomized women, it was estimated that by avoiding estrogen, there had been ∼50,000 extra deaths from 2002 to 2011 [24]. Currently, it is widely accepted that estrogens are effective in relieving menopausal symptoms and in preventing perimenopausal bone loss, as well as having other potential benefits, but their use as first-line therapy in osteoporosis is no longer appropriate.

The way in which estrogens act on bone and indeed on other tissues is complex. Estrogens may have direct effects on bone cells, but many of their effects may be indirect, mediated, for example, by reducing the actions of the RANK-ligand system on osteoclast differentiation. Estrogens exert their cellular effects mainly by binding to estrogen receptors (α & β), which are members of the large family of nuclear hormone receptors that regulate the transcription of specific genes. The different cofactors present in various tissues result in differential effects of individual ER ligands in key target tissues [25].

Estrogens can also exert rapid cellular responses by non-genomic mechanisms by binding to another type of receptor, called the G protein-coupled estrogen receptor 1 (GPER), formerly referred to as G protein-coupled receptor 30 (GPR30). GPER is an integral membrane protein with high affinity for estradiol and is a member of the rhodopsin-like family of G protein-coupled receptors.

Selective estrogen receptor modulators (SERMs) are a diverse group of naturally occurring or synthetic nonsteroidal compounds that exhibit tissue-specific estrogen receptor (ER) agonist or antagonist activity, i.e., they can act as estrogens on some tissues but antiestrogens on others. The biochemical basis for differential actions on various target tissues is most likely to be due to the different conformation changes induced in the receptor after binding to individual ligands. These conformational changes in the ER in turn result in different patterns of binding to various coactivators and corepressor molecules , thereby leading to distinctive patterns of gene transcription and protein expression in the various target tissues.

The pharmacological profile of individual SERMs is therefore determined by the effects observed on the key target tissues that are important for human health. An ideal SERM would therefore have positive effects on the cardiovascular system and bone , without stimulating breast or endometrial tissue and raising the risk of cancer. Attempts to meet this ideal profile have been partially successful with currently approved SERMs such as raloxifene and bazedoxifene. However, it has so far proved impossible to wean out the adverse effects of provoking hot flushes or venous thrombosis from any of the individual SERMs in current clinical use. There are several comprehensive reviews of estrogens and SERMs available [26, 27].

Many different molecules with estrogen agonist or antagonist activity have been synthesized and evaluated experimentally. Several have been examined for their clinical potential. Some have failed to progress on account of unacceptable side effects or lack of desired efficacy. These include droloxifene, idoxifene, and arzoxifene . Overall, there have been more failures than successes in clinical development. There are currently only two SERMs approved for use of osteoporosis, namely, raloxifene and bazedoxifene (Fig. 2.3).

The earliest SERMs developed for clinical use were agents such as clomiphene , still used for induction of ovulation, and tamoxifen used as an antiestrogen in the management of breast cancer. Currently, other FDA-approved SERMs include toremifene, also used for prevention and treatment of breast cancer, and ospemifene approved for treatment of dyspareunia from menopausal vaginal atrophy. Raloxifene is approved for the prevention and treatment of osteoporosis and prevention of invasive breast cancer. Bazedoxifene is also approved in many countries either as a single agent but interestingly also as a combined preparation with estrogen. This tissue-selective estrogen complex (TSEC) involves a pairing of conjugated equine estrogens with bazedoxifene and is approved by the FDA. This pairing is designed to reduce the risk of endometrial hyperplasia that can occur with the estrogenic component of the TSEC without the need for a progestogen in women with a uterus. The combination also allows for the estrogen to control hot flushes and to prevent bone loss without stimulating the breast or the endometrium.

Although bisphosphonates remain first-line therapy for most patients, SERMs such as raloxifene and bazedoxifene provide a safe and effective alternative to bisphosphonates for women who are unable to tolerate bisphosphonates and for younger women with an increased risk of fracture who may remain on therapy for many years [28, 29].

Strontium ranelate emerged in the 1990s as a potential treatment for osteoporosis. It was initially portrayed as an uncoupling agent, which stimulated bone formation while decreasing bone resorption. This was an attractive profile for a new drug for osteoporosis. The proposed dual mechanism of action even led to the introduction of the term “DABA” (dual-acting bone agent).

Two large pivotal phase 3 trials (Spinal Osteoporosis Therapeutic Intervention (SOTI) and Treatment of Peripheral Osteoporosis (TROPOS) ) were conducted within a total of 6740 Caucasian women with postmenopausal osteoporosis. In these trials, strontium ranelate showed efficacy in reducing fractures [34].

Strontium is the active component, while ranelate is an anion that enabled patent protection. Despite its apparent efficacy in reducing fractures, the mechanism by which this is achieved remains unclear [35]. The effects on bone resorption and formation are small, but there are substantial changes in BMD measurements largely due to substitution of strontium for calcium in bone mineral. It is known that strontium apatites have different solubility and physical characteristics than the calcium-containing hydroxyapatites and related minerals normally present in bone. Strontium salts have been used in the dental field as toothpaste additives for their protective effects on mineral dissolution. The simple classification of strontium as a dual-acting agent or an antiresorptive drug does not properly reflect these other properties on the physicochemical behavior of bone mineral, which may be an important part of its pharmacological actions.

Strontium ranelate has been quite widely used in osteoporosis treatment in several countries particularly in the elderly. Safety concerns about sensitivity reactions, venous thrombosis, and adverse cardiovascular effects are now limiting its use.

Calcitonin is a peptide hormone secreted by the C-cells of the thyroid gland, which lowers blood calcium by inhibiting bone resorption. It acts directly on osteoclasts to produce a temporary cessation of their resorptive activity. Calcitonins were first used therapeutically in the 1970s for Paget’s disease of bone, in which they were moderately active. The potencies of calcitonins vary according to the species of origin, and porcine, human, and salmon calcitonins have all been used to varying extents in clinical practice. Salmon calcitonin emerged as the preferred of these, predominantly and curiously because it is more potent than human. It was eventually developed as a nasal inhalation rather than being given by injection. Studies with salmon calcitonin in osteoporosis only showed weak anti-fracture effects, and its use has been superseded by more effective treatments. Recent studies with oral calcitonin have also proved disappointing.

Calcitonin is still sometimes used for pain relief after vertebral fracture. The nasal forms of calcitonin have been recently withdrawn by the CHMP because of concerns about cancer risk.

Cathepsin K (Cat K) is one of the primary enzymes involved in degrading type I collagen, the major component of the organic bone matrix . Cathepsin K is a member of the papain family of cysteine proteases and is highly expressed by activated osteoclasts but also in other cell types, giving rise to potential off-target effects (the skin, lungs, etc.). Cathepsins are lysosomal proteases that belong to the papain-like cysteine protease family. There are 11 different types (B, C, F, H, K, L, O, S, V, X, and W). There is incomplete homology of these enzymes among animal species, making animal experiments sometimes misleading. Making selective inhibitors is difficult but key to success. There has been intense activity in the pharmaceutical industry to create inhibitors as illustrated by the many patents that have been issued [36] (Fig. 2.4).

Loss-of-function mutations in the cathepsin K gene lead to pycnodysostosis , a disorder characterized by osteosclerosis, bone fragility, and decreased bone turnover. This discovery led to a flurry of research activity especially in the mid-1990s. Cathepsin K therefore became an attractive therapeutic target in osteoporosis and also potentially in osteoarthritis and bone cancers. The concept is scientifically interesting and illustrates the process of modern drug design and development, based on identifying a target (cathepsin K) and then using medicinal chemistry to design and synthesize selective inhibitors [37]. Key components of a clinically viable inhibitor are oral bioavailability, high selectivity over related cathepsins, and a covalent, reversible warhead to bind to the active-site cysteine of the enzyme.

There have been many hurdles encountered during the discovery and development of cathepsin K inhibitors as drugs. These include species differences in amino acid sequences in the critical site of the target enzyme, species differences in bone metabolism, and also discrepancies between pharmacokinetic and pharmacodynamic (PK/PD) profiles due to unique tissue distribution of the inhibitor affecting both efficacy and side effects, originating from idiosyncratic intracellular or tissue distribution of some classes of compounds [38]. The antiresorptive properties of several cathepsin K inhibitors have been studied in phase I and phase II clinical trials. Phase III studies have recently been completed for odanacatib, the only cathepsin K inhibitor for which fracture reduction has been shown. Overall and despite the involvement of “big Pharma,” there have been more failures than successes in this field so far, with several “failures” in the development of Cat K inhibitors (e.g., balicatib, relacatib; see below).