Linda Brewer

David Williams

A Moore

Current and Future Treatment Options in Osteoporosis

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Introduction

Osteoporosis is a metabolic bone disease that results in bone fragility and increased susceptibility to fracture. As populations age globally, the incidence of osteoporosis-related fractures will rise substantially over the coming decades. Even if age-adjusted incidence rates remain stable, the estimated number of hip fractures worldwide will rise from 1.7 million in 1990 to 6.3 million in 2050. [1] Approximately 1 in 2 women and 1 in 4 men aged over 50 years will have an osteoporosis-related fracture in their lifetime. The increase in medical visits, hospitalisations, need for community supports and nursing home admissions will pose a major economic burden on healthcare systems. Currently the combined annual cost of all osteoporotic fractures has been estimated at $20 billion for the US and $30 billion for the EU. [2] In order to offer patients greater protection against osteoporosis-related fractures an increasing number of newer therapies have been developed. Knowledge of underlying mechanisms of osteoporosis has advanced in recent years allowing novel agents to be introduced (Figure 1). Bone is continually remodelled to replace microdamage and to adapt bone density to patterns of usage. Agerelated bone loss results in 0.5% decline in bone mass per year. This occurs because of an imbalance between the activity of osteoclasts (bone-resorbing cells) and osteoblasts (bone-forming cells) and multiple factors are now known to regulate this balance. In this article we briefly summarize the evidence of established treatments before outlining a number of newer agents and prescribing considerations for this common condition.

Determining Fracture Risk

Prevention of osteoporosis is preferable to treatment as microarchitectural changes associated with bone loss are largely irreversible. Although BMD is the best available non-invasive assessment of bone strength, other factors contribute to bone quality including the mineralisation and rate of bone turnover. [START_REF] Heaney | Remodelling and skeletal fragility[END_REF] Markers of bone formation, turnover and resorption have been used in some trials to assess drug efficacy. Studies also show their highly significant correlation with subsequent rate of bone loss and future fracture risk [START_REF] Bruyere | Loss of hip bone mineral density over time in association with spine and hip fracture incidence in osteoporotic postmenopausal women[END_REF] and subgroups with high turnover at baseline have been shown in some studies to gain most from antiresorptive therapy.

[ 5-7] Pharmacological treatments may stabilise or increase BMD but are unlikely to fully restore bone quality and strength. Dual energy x-ray absorptiometry (DXA) is the most widely used method for measuring BMD as it gives very precise measurements at clinically relevant sites. The greatest relative risk of fracture is in individuals with lower BMD but the absolute number of fractures in those with BMD T-scores in the osteopenic range is greater than those with T-scores in the osteoporotic range. Therefore assessment of risk factors that are independent of BMD is important for fracture prediction. [START_REF] Cummings | Risk factors for hip fracture in white women[END_REF] Advanced age and previous fracture are the most significant of these validated risk factors (Table II). In 2008 the World Health Organisation (WHO) introduced FRAX ® , the Fracture Risk Assessment Tool, which estimates the 10 year probability of hip fracture or major osteoporotic fractures combined. [START_REF] Kanis | FRAX and the assessment of fracture probability in men and women from the UK[END_REF] It has been validated in 11 independent cohorts and allows estimation of 10year fracture probability from an individual's set of risk factors alone or in combination with BMD.

Patients can be stratified according to fracture risk instead of using BMD alone and intervention can therefore be effectively targeted to those at highest risk of fracture (Figure 2). The FRAX ® tool enables the estimation of risk based on a wider range of risk factors, and treatment efficacy is dependent on the level of risk in the analyzed population. Furthermore thresholds for cost-effective pharmacological intervention have been calculated using fracture and economic data that are country-specific.

[10]

FRAX ® can also be used to assess the cost-effectiveness of individual drugs to treat osteoporosis. [11,12] Since the introduction of FRAX ® , two new fracture clinical risk scores (QFractureScores) have been developed and validated in the UK. [13] In addition to traditional variables already included in the FRAX Application of this approach of fracture risk assessment to individual patients allows clinicians to make more informed decisions when considering whether or not to commence pharmacological intervention.

Anti-resorptive agents

Calcium & Vitamin D

Calcium and vitamin D are necessary for normal skeletal homeostasis and are considered the first step in osteoporosis treatment. Inadequate intake of calcium and vitamin D leads to reduced calcium absorption, a compensatory rise in parathyroid hormone, higher bone remodelling rates and increased bone loss. Vitamin D deficiency is also linked to reduced muscle function and increased risk of falling and its replacement has been shown to reduce the rate of falling amongst older individuals. [14] The positive association between calcium and vitamin D levels and bone mineral density (BMD) has been well documented. [15][16][17] However their replacement, either alone or in combination, has been shown to lower fracture rates in some trials but not in others. Calcium supplementation alone has been shown

to reduce the incidence of osteoporotic fracture compared with placebo (HR 0.66) only in patients who are over 80% compliant.

[18]

The relationship between vitamin D replacement and fracture prevention is unclear when first examined and many studies using various doses of 25OHD and differently aged study populations have attempted to clarify this relationship. Despite some inconsistent trial results it appears that vitamin D supplementation studies that had positive results achieved higher vitamin D levels. From available evidence, it seems that individuals will benefit by having their 25OHD levels no lower than 70 nmol/l. Vitamin D protects against fracture by decreasing PTH and increasing bone mass and also by lowering risk of falling.

[19] A higher level may provide additional benefit, but this has not been established. Supplemental calcium, used in many of the trials, both positive and negative, may be essential for benefit but this remains uncertain. The recommended dose of calcium, usually in the form of calcium carbonate, is 1g daily and common side effects include dyspepsia and constipation.

However an increased risk of myocardial infarction (HR 1.31, 95% CI 1.02 to 1.67) in patients on calcium supplements, without co-prescribed vitamin D, was recently reported in a meta-analysis by Bolland and colleagues [20] which raises concerns about its widespread use. Due to limited available evidence of it's benefit when used alone, it is advised that calcium should not be prescribed to patients with osteoporosis unless they are also receiving vitamin D and/or an effective treatment for osteoporosis.

Bisphosphonates

Bisphosphonates are now widely used in the treatment of osteoporosis as they inhibit bone resorption with relatively few side effects. There are two classes of bisphosphonate: the nitrogen-containing and non nitrogen-containing bisphosphonates all of which have a structure similar to pyrophosphate and thereby inhibit the activation of enzymes that utilise pyrophosphate. [21] Alendronate, risedronate, ibandronate and zoledronic acid are all nitrogen-containing bisphosphontes and have demonstrated efficacy in fracture risk reduction. For these bisphosphonates, the direct intracellular target is the enzyme farnesyl diphosphate synthase in the HMG-CoA reductase biosynthetic pathway. Its inhibition suppresses a process, protein prenylation, resulting in disruption to vital signalling pathways necessary for osteoclastic bone resorption. Bisphophonate molecules preferentially bind to calcium and therefore accumulate to a high concentration in bones where they are ingested by osteoclasts.

Alendronate and risdeonate are most commonly given in weekly oral formulations and zoledronic acid (ZA) is administered annually as an intravenous infusion. Ibandronate comes in monthly oral or 3 monthly intravenous formulations. Alendronate has been studied in postmenopausal women with low BMD, [22,23] with or without prevalent vertebral fractures and subgroup analysis of those women with documented osteoporosis reported a reduction in hip fractures of over 50%. Treatment also demonstrated a reduction of 44-50% in vertebral fractures. Risedronate also has documented efficacy in improving BMD and fracture risk in postmenopausal women with osteoporosis and vertebral fractures. A reduction in the risk of vertebral and non vertebral fractures of 41% and 49% respectively has been reported [START_REF] Harris | Effects of risedronate treatment on vertebral and non-vertebral fractures in women with postmenopausal osteoporosis: a randomized controlled trial. Vertebral Efficacy With Risedronate Therapy (VERT) Study Group[END_REF] and in older women (70-79 years) with osteoporosis treatment reduced the rate of hip fracture by 30%. [START_REF] Mcclung | Effect of risedronate on the risk of hip fracture in elderly women[END_REF] Positive data on risk reduction of hip and vertebral fracture is also available for Ibandronate, showing similar results to other bisphosphonates. [26, 27] Annual ZA reduces vertebral and hip fracture by 70% and 41% respectively compared with placebo [START_REF] Black | Once-yearly zolendronic acid for treatment of postmenopausal osteoporosis[END_REF] and offers 35% relative reduction in recurrent fracture and all-cause mortality (HR 0.72) when given after 2 weeks post hip fracture. [29, 30] Although oral bisphosphonates are well tolerated if swallowed as indicated (Table 1), caution is advised with a background of gastrointestinal complaints. Recent reports have proposed a link between bisphosphonate-related oesophagitis and oesophageal cancer [START_REF] Wysowski | Reports of esophageal cancer with oral bisphosphonate use[END_REF] and this association has been investigated using data from the UK General Practice Research database and reported in two separate studies. At a follow-up period of four and a half years, Cardwell et al reported no increase in the risk of oesophageal or gastric cancer with bisphosphonate use. [START_REF] Cardwell | Exposure to Oral Bisphosphonates and risk of Esophageal cancer[END_REF] In contrast the case-control analysis by Green et al. [START_REF] Green | Oral bisphosphonates and risk of cancer of oesophagus, stomach, and colorectum: case-control analysis within a UK primary care cohort[END_REF] found an increase in the risk of oesophageal cancer with 10 or more prescriptions for oral bisphosphonates and with prescriptions over a five year period (an increase in incidence of 1 per 1000 to 2 per 1000 population aged 60-79 years with five years' use of oral bisphosphonates). This study had a longer follow-up period of 7.7 years. However, neither study validated diagnoses by medical records nor provided information on whether drugs were taken according to directions. Even if oral bisphosphonates do increase the risk of oesophageal cancer, the incidence in the population would be expected to remain relatively low. As evidence to date is inconclusive, cautious prescribing of bisphosphonates, patient education and active follow-up are advised.

Adverse skeletal events in the form of atypical fragility fractures and osteonecrosis of the jaw in patients on bisphosphonates have been described but remain relatively rare. It is plausible that atypical fragility fractures could follow from suppression of bone remodelling, but high-quality studies proving causality are lacking. A recent secondary analysis of 3 large bisphosphonate trials found no significant increase in the risk of atypical fracture associated with bisphosphonate use for up to 10 years. [START_REF] Black | Bisphosphonates and Fractures of the Subtrochanteric or Diaphyseal Femur[END_REF] However the study was not adequately powered for definitive conclusions. A further British cohort study using national healthcare data found alendronate-treated patients to be at higher risk of hip and subtrochanteric/diaphyseal fracture than matched control subjects. [35] Large cumulative doses of alendronate were not however associated with a greater absolute risk of subtrochanteric/diaphyseal fractures than small cumulative doses, suggesting that these fractures could be due to osteoporosis rather than to alendronate.

Osteonecrosis of the jaw (ONJ) has been described in patients receiving bisphosphonate therapy and to date several aetiological theories have been proposed including inhibition of bone remodelling, effect on soft tissues, and antiangiogenic effect of bisphosphonates. However almost all cases to date have been described in cancer patients on high dose intravenous formulations and are often associated with dental procedures. The European Medicines Agency in a recent review concluded that the risk of ONJ is higher in those patients receiving intravenous bisphosphonates for cancer than for patients receiving oral therapy.

[36]

Hormone replacement therapy

Numerous randomised placebo-controlled studies including the Womens health initiative (WHI) [START_REF] Rossouw | Effects of hormone therapy on bone mineral density: results from the postmenopausal estrogen/progestin interventions (PEPI) trial. The Writing Group for the PEPI[END_REF] and the postmenopausal oestrogen/progesterone interventions (PEPI) [38] have established that the postmenopausal decrease in BMD is attenuated by oestrogen, resulting in a lower risk of fracture.

However data from the WHI also suggested that oestrogen/progesterone therapy increases the incidence of breast cancer, coronary heart disease, stroke and venous thromboembolism. Unopposed oestrogen therapy has been shown to increase stroke and thromboembolism only. Therefore as overall health risks exceed benefits, hormone replacement is not considered first-line therapy in the management of postmenopausal osteoporosis.

Selective Estrogen Receptor Modulators

Raloxifene, lasofoxifene and bazedoxifene are currently the SERMs available for use in the treatment of osteoporosis and their effects on vertebral and nonvertebral fractures have been studied in numerous trials reporting beneficial results. The first SERM used to treat osteoporosis, raloxifene, demonstrated a reduced risk of vertebral fractures (HR 0.7) but not nonvertebral fracture in a large 3 year study of postmenopausal women. [START_REF] Ettinger | Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators[END_REF] However, subsequent post hoc analyses reported a decrease in new vertebral and nonvertebral fracture risk in the subgroup of women with severe vertebral fractures at baseline. [START_REF] Delmas | Severity of prevalent vertebral fractures and the risk of subsequent vertebral and nonvertebral fractures: results from the MORE trial[END_REF] Bazedoxifene significantly reduced the risk of new vertebral fracture in postmenopausal women with osteoporosis and decreased the risk of nonvertebral fracture in subjects at higher fracture risk [START_REF] Silverman | Efficacy of bazedoxifene in reducing new vertebral fracture risk in postmenopausal women with osteoporosis: results from a 3-year, randomized, placebo-, and active-controlled clinical trial[END_REF] and in the recent PEARL study of postmenopausal women with osteoporosis, lasofoxifene was associated with reduced risk of both nonvertebral and vertebral fractures. [START_REF] Cummings | PEARL Study Investigators. Lasofoxifene in postmenopausal women with osteoporosis[END_REF] Although SERMs have demonstrated benefits with regard to ER-positive breast cancer, coronary heart disease, and stroke, the main concern with treatment is the three-fold increased risk of venous thromboembolism. Ospemifene and arzoxifene are new SERM molecules currently under investigation. [43,44]

Anabolic therapy

Parathyroid Hormone

Recombinant Parathyroid Hormone (PTH) 1-34 and 1-84 are anabolic agents which work by stimulating preosteoblasts to mature into bone-forming osteoblasts. When given at low dose by daily subcutaneous injection they enhance bone remodelling. Bone formation begins within the first month of treatment and bone resorption begins after 6 months. [START_REF] Black | The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis[END_REF] Therefore during the first year of treatment bone remodelling is in a positive balance. In the Fracture Prevention Trial, postmenopausal women with previous vertebral fracture were randomised to PTH (20 or 40 mcg) or placebo. BMD in the 20mcg and 40mcg groups increased both at the lumbar spine (9% and 13% respectively) and at the femoral neck (3% and 6% respectively). However overall fracture risk reduction was proportionately greater and found to be very similar for both dosage groups. In the 20 mcg group, where side effects were less common, the respective relative risks of vertebral and non-vertebral fractures were 0.35 and 0.47. Subgroup analysis showed that the benefits were independent of age, baseline BMD and prevalent vertebral fractures.

The changes in BMD seen with PTH are early and of greater magnitude than those seen with other treatments and it therefore has a role in patients with severe established disease who are at particularly high risk for fragility fractures; T score ≤ -3.5 without fracture or T score ≤ -2.5 with fragility fracture. However the strongest evidence to date in support of the efficacy of PTH in reducing vertebral and non-vertebral fractures showed that its use is likely to be most effective in patients with pre-existing fragility fractures and not necessarily associated with very low BMD score. [46] The main concern with long term treatment is development of osteosarcoma as this was seen in rat models with high dose treatment. [START_REF] Vahle | Skeletal changes in rats given daily subcutaneous injections of recombinant human parathyroid hormone (1-34) for 2 years and relevance to human safety[END_REF] Fortunately in practice this has not been observed and very few long term side effects have been reported. Due to concern regarding long term safety and plateau in bone formation with treatment beyond 18 months, treatment is limited to low dose for a maximum of 2 years. PTH is not usually considered first-line therapy due to cost, route of administration and paucity of strong data on nonvertebral fracture reduction. It has a role however in patients with severe disease and when other therapies are not tolerated or have failed. Anti-resorptives should however be reintroduced following discontinuation of PTH to preserve the BMD gained on treatment, as demonstrated in an extension of the PaTH study.

[48]

Dual actionanti-resorptive and anabolic

Strontium ranelate

Strontium ranelate has a unique mode of action: it dissociates bone remodelling by reducing bone resorption while allowing continued bone formation. Its effect on vertebral fractures was assessed in SOTI, a trial in which postmenopausal women received daily strontium ranelate or placebo for three years.

[49]

A relative risk reduction in vertebral fractures was seen in favour of strontium; 49% after 1 year and 41% over 3 years (RR 0.59). Strontium ranelate increased bone mineral density at month 36 by 14.4% at the lumbar spine and 8.3% at the femoral neck (P<0.001 for both). Efficacy was subsequently assessed in prevention of non-vertebral fractures in TROPOS, a double-blind 5-year study of 5091 postmenopausal women. [START_REF] Seeman | Strontium ranelate reduces the risk of nonvertebral fractures in postmenopausal women with osteoporosis: Treatment of Peripheral Osteoporosis (TROPOS) study[END_REF] Relative risk for major fragility fractures was reduced in the strontium group by 19% compared with the placebo group. On extension of these studies to 8 years, BMD of lumbar spine, femoral neck and total hip continued to increase with some evidence for sustained anti-fracture efficacy and strontium was found to be safe and well tolerated. [51] Women ≥ 80 years are at higher relative risk of vertebral and non-vertebral fractures and subgroup analysis of these studies concluded an early and sustained reduction of both vertebral and nonvertebral fractures with strontium in these patients. [START_REF] Seeman | Strontium ranelate reduces the risk of vertebral and nonvertebral fractures in women eighty years of age and older[END_REF] Caution has been advised when interpreting BMD values when on strontium as a proportion of the change in value may be due to accumulation of strontium in the bone tissue however an increase in femoral neck BMD has been shown to be associated with a proportional reduction in vertebral fracture incidence. [START_REF] Bruyere | Relationship between bone mineral density changes and fracture risk reduction in patients treated with strontium ranelate[END_REF] Side effects include transient nausea, diarrhoea and headache. A slight excess of venous thromboembolism (VTE) in patients with a history of thromboembolism has been noted in some but not all trials. A recent large retrospective cohort study reported a greater association of VTE in osteoporotic compared with nonosteoporotic patients (RR 1.75; CI 1.09 to 1.84) but no increased incidence in those treated with strontium compared with alendronate.

[54]

Novel therapeutic Agents for the management of osteoporosis

Denosumab Denosumab (AMG 162) is a human monoclonal antibody with a high affinity and specificity for RANKL, a key mediator of osteoclastic bone resorption. By reducing RANKL binding to the osteoclast receptor RANK, there is a decrease in differentiation, activity, and survival of osteoclasts, lowering the rate of bone resorption. [START_REF] Lewiecki | Denosumab: an investigational drug for the management of postmenopausal osteoporosis[END_REF] In a dose-ranging study the efficacy and safety of subcutaneously administered denosumab were evaluated in 412 postmenopausal women (mean age 62 years) with low BMD. [START_REF] Mcclung | AMG 162 Bone Loss Study Group. Denosumab in postmenopausal women with low bone mineral density[END_REF] Patients were randomised to receive denosumab or alendronate. Denosumab resulted in an increase in BMD from baseline at the lumbar spine of up to 6.7 percent (vs 4.6 % with alendronate)

and at the total hip of up to 3.6 percent (vs 2.1 % with alendronate). This study was extended to 24 months and a sustained increase in BMD at total hip, lumbar spine and distal radius was recorded with no increase in adverse events. [START_REF] Lewiecki | Two year treatment with denosumab (AMG 162) in a randomized phase 2 study of postmenopausal women with low BMD[END_REF] The effects on bone turnover were found to be fully reversible on discontinuation of the drug but restored with subsequent treatment. [START_REF] Miller | Effect of denosumab on bone density and turnover in postmenopausal women with low bone mass after long-term continued, discontinued, and restarting of therapy: a randomised blinded phase 2 clinical trial[END_REF] A further larger study of 1 189 postmenopausal women randomised to denosumab 60mg 6 monthly or alendronate 70mg weekly showed significant increase in BMD at all measured skeletal sites in patients on denosumab at 12 months (3.5% vs 2.6%, p<0.0001). There was a significantly greater reduction of bone turnover markers and the safety profile was similar for both treatments.

[59]

The FREEDOM trial of 7868 women aged 60 to 90 years with osteoporosis (BMD T score between -2.5 and -4.0) at the lumbar spine or total hip showed very promising results. [START_REF] Cummings | Denosumab for prevention of fractures in postmenopausal women with osteoporosis[END_REF] Subjects were assigned to receive either 60 mg of denosumab or placebo subcutaneously every 6 months for 36 months. Denosumab reduced the risk of new radiographic vertebral fracture by 68% and reduced the risk of hip fracture by 40%. It also reduced the risk of nonvertebral fracture by 20%. Adverse events were similar to placebo indicating a favourable safety profile. In this large study there was no increase in the risk of cancer, infection, cardiovascular disease, delayed fracture healing or hypocalcemia and no adverse reactions to the injection of denosumab. Since the publication of this study, suspected cases of ONJ have been described in patients on denosumab [61, 62] but to date these case reports remain very limited. Significantly greater treatment adherence was observed for subcutaneous administration of denosumab every 6 months than for oral alendronate once weekly [START_REF] Kendler | Adherence, preference, and satisfaction of postmenopausal women taking denosumab or alendronate[END_REF] with a risk ratio for non-adherence at 12 months of 0.58 (p = 0.043) for denosumab compared with alendronate.

Patient preference and satisfaction were also significantly greater for denosumab. The FDA (Food and drug administration) and EMEA (European Medicines Agency) have approved denosumab for the treatment of postmenopausal osteoporosis and it is currently available for use in clinical practice.

Odanacatib

Cathepsin K is a lysosomal cysteine protease expressed in abundance by osteoclasts which degrades Type I collagen and is responsible for degradation of the organic bone matrix during bone remodelling.

Odanacatib (MK-822) is a potent and selective inhibitor of Cathepsin K [START_REF] Gauthier | The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K[END_REF] and has been shown in studies to reduce osteoclast-mediated bone resorption. In two double-blind, randomized, placebocontrolled studies, postmenopausal females received odanacatib 2.5mg once weekly for 3 weeks or once daily for 21 days. It was well tolerated and had a long half-life (t ½ 66-93 h) consistent with onceweekly dosing. A reduction in biochemical markers of bone resorption of up to 81% was seen. [START_REF] Stoch | Effect of the cathepsin K inhibitor odanacatib on bone resorption biomarkers in healthy postmenopausal women: two double-blind, randomized, placebo-controlled phase I studies[END_REF] A recent study of 399 women with BMD T-scores between -2.0 and -3.5 at the lumbar spine or femoral neck was performed whereby participants were randomly assigned to receive placebo or one of four doses of odanacatib (3, 10, 25 or 50mg weekly) over 24 months. After 24 months, lumbar spine and total hip BMD increased 5.5% and 3.2% respectively in patients on 50-mg odanacatib compared with placebo. [START_REF] Bone | Odanacatib, a Cathepsin-K Inhibitor for Osteoporosis: A Two-Year Study in Postmenopausal Women With Low Bone Density[END_REF] Unlike other less selective cathepsin inhibitors which caused skin reactions, the safety and tolerability of odanacatib are similar to placebo. Results of ongoing large scale trials to evaluate the effects of odanacatib on fracture risk in postmenopausal women are awaited.

Glucagon-like peptide 2 (GLP-2)

There is a circadian pattern to bone remodelling, as shown by biochemical assessments, whereby bone resorption is reduced during the daytime and increased at night, and this pattern has been linked

to food intake. GLP-2 is released in the small intestine almost immediately after food intake and its secretion is therefore reduced overnight with nocturnal fasting. The presence of GLP-2 receptors in osteoclasts suggests that GLP-2 may form part of an 'entero-osseous axis', coordinating nutrientdependent bone turnover. As bone resorption occurs at increased rate during this fasting period it was hypothesised that administration of GLP-2 at bedtime may reduce this rate of bone resorption overnight. A double-blind placebo-controlled trial of 60 postmenopausal women administered GLP-2 daily for 14 days showed a significant and sustained reduction in bone resorption (Ctx) with the added advantage of not decreasing bone formation (OC), unlike other antiresorptive agents

. [67] The investigators then extended the study period to four months. This phase II, placebo-controlled trial of 160 postmenopausal women treated with GLP-2 (3 doses) over 120 days similarly resulted in a reduction in nocturnal rise of C-telopeptide and no effect on osteocalcin and an increase in hip BMD

(1.1% with highest dose of 3.2mg, p=0.007). [START_REF] Henriksen | Four month treatment with GLP-2 significantly increases hip BMD: a randomized, placebo-controlled, dose-ranging study in postmenopausal women with low BMD[END_REF] The overall rates of adverse events in the treatment groups were similar and there were no signs of tachyphlaxis or antibody production against GLP-

Wnt signalling: Inhibitors of Sclerostin and Dickkopf 1(Dkk-1)

Wnt proteins are a large family of extracellular glycoproteins involved in many cellular processes. The Wnt/beta-catenin signaling pathway has emerged as a key regulator of bone homeostasis and recently neutralizing inhibitors have emerged as a possible therapeutic option for the management of osteoporosis. [START_REF] Hoeppner | Wnt signaling as a therapeutic target for bone diseases[END_REF] These include antibodies to Sclerostin and Dkk-1 and also inhibitors of secreted Frizzled-related protein 1 and glycogen synthase kinase 3beta. Sclerostin is secreted by osteocytes and genetic studies have shown that it is a key negative regulator of bone formation. Its inhibition was assessed using a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl-AbII).

[70]

When ovariectomized rats were administered Scl-Abll for 5 weeks robust anabolic effects were noted on trabecular, periosteal, endocortical, and intracortical surfaces. It resulted in complete reversal of bone loss at several skeletal sites but also further increased bone mass and bone strength to levels greater than those found in control rats. In the first human study a single subcutaneous dose of Scl-AbII (AMG 785) was administered to 72 healthy men and postmenopausal women. It resulted in an increase in P1NP and osteocalcin and a dose related decrease in the bone marker CTx at day 84 of treatment. An increase of 5.3% in BMD was seen at the lumbar spine and of 2.8% at the total hip. Neutralising antibodies developed in 2 patients but had no effect on the pharmacokinetics of this agent. [START_REF] Padhi | Single-dose, placebo-controlled, randomised study of AMG 785, a sclerostin monoclonal antibody[END_REF] Dkk-1 is a negative regulator of bone homeostasis and levels are significantly higher in osteoporotic women compared with controls. [START_REF] Anastasilakis | The effect of teriparatide on serum Dickkopf-1 levels in postmenopausal women with established osteoporosis[END_REF] Manipulation of its function and expression has therapeutic significance for the treatment of osteoporosis and progressive Dkk-1 reduction has been shown to increase trabecular and cortical bone mass in mice and primates. [START_REF] Macdonald | Bone mass is inversely proportional to Dkk1 levels in mice[END_REF][START_REF] Betts | The application of target information and preclinical pharmacokinetic/ pharmacodynamic modeling in predicting clinical doses of a Dickkopf-1 antibody for osteoporosis[END_REF] Human studies showing beneficial effects are currently underway.

Calcium Sensing Receptor, Activin Receptor Fusion Protein & Integrin aub3

Potential future targets for osteoporosis include calcium sensing receptor, activin receptor fusion protein and integrin aub3. Antagonists to calcium sensing receptor result in transient PTH surge with each oral dose similar to that seen with intermittently administered exogenous PTH. Activin, a cofactor for osteoclastogenesis, binds to activin receptor IIA and an antibody to it has shown a reduction Ctelopeptide in postmenopausal women. Antibodies to alpha(v)beta(3) integrin, expressed on osteoclasts, inhibit bone resorption in vitro and in vivo, suggesting an important role in regulating osteoclast function. Although interesting novel targets, future studies will determine long-term tolerability and safety. It is likely to be some years before these agents are introduced to clinical practice.

Prescribing Considerations

Osteoporosis is predominantly a disease of older adults so, when prescribing, other factors such as cognition, compliance, co-morbidities and route of administration must be taken into consideration.

Medication effectiveness needs to be balanced against tolerability in individual patients. All patients should be on calcium supplementation and should be vitamin D replete. For the well, compliant older person, with no contra-indications, oral weekly bisphosphonate therapy or oral daily strontium are considered first line. However hip fracture is the main concern amongst older patients with osteoporosis and bisphosphonates have well documented hip fracture risk reduction which is maintained over many years.

Repletion of Vitamin D is particularly important in the frailest older patients, such as nursing home residents or older patients with poor mobility and limited sunlight exposure. It may be given intramuscularly if compliance is uncertain. [START_REF] Nugent | The effect of intramuscular vitamin D (cholecalciferol) on serum 25OH vitamin D levels in older female acute hospital admissions[END_REF] It also suppresses parathyroid hormone, increases bone mineral density and reduces falls [START_REF] Harwood | Nottingham Neck of Femur (NONOF) Study. A randomised, controlled comparison of different calcium and vitamin D supplementation regimens in elderly women after hip fracture: The Nottingham Neck of Femur (NONOF) Study[END_REF] and hip fracture.

In patients who have difficulty swallowing medications or in those intolerant of oral bisphosphonates due to upper gastrointestinal disturbance, annual intravenous zolendronic acid or subcutaneous denosumab are useful options. It should also be considered when compliance with oral therapy remains an issue in community-dwelling patients.

More than half of all fractures in the community occur in patients with osteopenia. However most studies assessing antifracture efficacy of drugs include osteoporotic patients who are at higher absolute risk for fracture. In cases of osteopenia, treatment with strontium can be considered. Pooled data from SOTI and TROPOS reported significant antifracture efficacy of strontium ranelate in women with lumbar spine (LS) and femoral neck (FN) osteopenia. [START_REF] Seeman | Strontium ranelate reduces the risk of vertebral fractures in patients with osteopenia[END_REF] The risk of clinical vertebral fractures was reduced by 60% with alendronate in a subgroup of patients from the FIT 1 study with FN osteopenia and prevalent fractures . [START_REF] Quandt | Effect of alendronate on vertebral fracture risk in women with bone mineral density T scores of -1.6 to -2.5 at the femoral neck[END_REF] Further studies are needed to determine effective therapies to reduce hip fracture risk in patients with osteopenia.

PTH is recommended as alternative treatment to bisphosphonates or strontium in the setting of a contraindication, intolerance to or failure of these drugs. Despite PTH's known efficacy, due to its high cost and without very strong evidence to support superiority to other treatments in antifracture efficacy, its use is likely to remain relatively restricted.

Prescribing in patients with reduced GFR

Osteoporosis in older persons often co-exists with chronic kidney disease (CKD) and could be included in the broad spectrum of metabolic bone conditions that accompany a decline in renal function. As the population prevalence of CKD is increasing, the management of osteoporosis in these patients is a frequently encountered problem. In advanced stages of CKD it is important to discriminate between osteoporosis and other forms of renal osteodystrophy, which can also impair bone strength and lead to fracture. WHO criteria for diagnosing osteoporosis can be applied in CKD stages 1 to 3, once biochemical profiling is normal. In these cases current registered therapies for osteoporosis have proven safety and can be used. In the setting of fragility fractures in more advanced stages of kidney disease the diagnosis and management of osteoporosis is less clear.

Careful assessment of bone turnover using serum levels of parathyroid hormone and bone-specific alkaline phosphatase is needed. In some cases, double-tetracycline labelled bone biopsy may be indicated to outrule adynamic bone disease before considering anti-resorptive treatment. In fracture patients with osteoporosis and stages 4 and 5 CKD treatment is difficult as therapies remain unlicenced in this setting. However post hoc analyses of trials using risedronate, alendronate and raloxifene have reported tolerability of these drugs in stage 4 CKD. [START_REF] Miller | Safety and efficacy of risedronate in patients with age-related reduced renal function as estimated by the Cockcroft and Gault method: a pooled analysis of nine clinical trials[END_REF][START_REF] Jamal | Alendronate treatment in women with normal to severely impaired renal function: an analysis of the fracture intervention trial[END_REF][START_REF] Ishani | The effect of raloxifene treatment in postmenopausal women with CKD[END_REF] No efficacy data however supports their use in end stage kidney disease. If recurrent fragility fractures pose a great risk to these patients it may be reasonable, based on known pharmacokinetics, to reduce the dose of bisphosphonate by half after clear diagnosis is made and consent from the patient is sought. In patients with renal osteodystrophy on dialysis, ibandronate has shown significant increase in BMD and decrease in BTM. [START_REF] Bergner | Treatment of reduced bone density with ibandronate in dialysis patients[END_REF] Alendronate has also shown a bone preserving effect in patients on haemodialysis. [START_REF] Wetmore | Effects of short-term alendronate on bone mineral density in haemodialysis patients[END_REF] In all patients with advanced CKD supplementation with active vitamin D is essential to reduce the effects of secondary hyperparathyroidism. In summary no consensus exists to guide our treatment decisions in cases of osteoporosis in stages 4 and 5 CKD and further large-scale prospective studies are needed. As denosumab is a monoclonal antibody, it is not cleared by the kidneys, which makes it a safer option for people who have renal disease. In patients with varying degrees of renal function, including patients on dialysis, the degree of renal impairment had no effect on the pharmacokinetics of denosumab. Therefore no dose adjustments of this drug are required with different degrees of renal impairment.

Conclusion.

The incidence of osteoporosis-related fractures will rise substantially over the coming decades as the population ages. This increased incidence will have significant social and economic implications for

23

and is a well tolerated therapy which has shown good levels of patient satisfaction. This exciting new drug is likely to be a very useful alternative therapy in current and future clinical practice. Novel agents such as Odanacatib, a cathepsin K inhibitor and Glucagon-like peptide 2 are currently under investigation for the treatment of osteoporosis and may prove to be interesting future therapies for osteoporosis.

When treating older patients with osteoporosis many factors must be taken into consideration. These include the presence of co-morbidities such as cognitive impairment, gastro-oesophageal disease and chronic kidney disease which can limit the use of some treatments. Compliance difficulties may be an issue with some drug formulations and routes of administration and this should be strongly considered to ensure the most appropriate drug is prescribed to individual older patients with high fracture risk.

Drug Administration

Bisphosphonates

Oral bisphosphonates should be taken first thing in the morning with a full glass of water.

As less than 1% of the dose is absorbed, it is always taken on an empty stomach. For at least 30 minutes after administration eating and lying down should be avoided.

Strontium ranelate

Dissolved in water and given the slow absorption is taken at bedtime. Calcium containing foods may reduce bioavailability by 60 to 70% and should be avoided for at least 2 hours before taking strontium.

PTH

Administered as a daily subcutaneous injection into the thigh or abdominal wall.

®

  algorithm, further fracture risk factors are included such as history of falls, type 2 diabetes, cardiovascular disease, asthma, use of hormone replacement therapy (HRT) and use of tricyclic antidepressants. No BMD score is used in the calculation and patients as young as 30 can be assessed. It can be used to estimate fracture risk at one, two, five, and 10 years. Validation statistics, especially for the hip fracture algorithm, suggest that the QFractureScores are likely to be effective at identifying patients at high risk of fracture and suggested favourable performance compared with FRAX ® .

2 .

 2 The results indicate that GLP-2 produces a substantial decrease in bone resorption without suppression of bone formation thereby changing the bone remodelling balance in favour of bone formation, particularly at the hip. If this disassociation between bone resorption and formation can be sustained with no adverse effects, it may prove an interesting future therapy for osteoporosis. Studies are also currently underway looking at GLP-1 as a possible anabolic agent to improve deficiencies in bone formation associated with glucose intolerance.

  most countries, particularly in the developed world. In recent years our understanding of normal bone physiology and the mechanisms underlying development of osteoporosis has significantly advanced and this has led to the development of new therapies. Currently we have good anti-fracture data to support the use of many available anti-resorptive and anabolic drugs and supplementation of calcium and vitamin D. Newer agents, particularly denosumab, will increase the therapeutic options for clinicians in the coming years. Determination of future fracture risk, evaluation of co-morbidities, measurement of renal function and estimation of potential compliance difficulties are all important considerations for the prescribing physician to take into account when deciding upon which agents to use in individual patients.

Figure 1 .

 1 Figure 1. Signalling pathways involved in osteogenesis and osteoclastogenesis in bone remodelling. Reproduced with permission of BioMed Central Ltd. Originally published by Lane et al. in Arthritis Research and Therapy 2009,11:228.

Figure 2 .

 2 Figure 2. Management algorithm for the assessment of individuals at risk of fracture by the use of clinical risk factors and bone mineral density (BMD). Image used with permission of the World Health Organisation Collaborating Centre for Metabolic Bone Diseases, University of Sheffield. FRAX® is registered to Professor JA Kanis, University of Sheffield, UK. Also with kind permission from Springer Science & Business Media: Osteoporosis International, Development and use of FRAX® in Osteoporosis (2010) 21:(Suppl 2)S407-S413.

  Borgström F, Ström O, Coelho J et al. The cost-effectiveness of strontium ranelate in the UK for the management of osteoporosis. Osteoporos Int. 2010 Feb;21(2):339-49. Epub 2009 Jun 10 Bischoff-Ferrari HA.Jean Mayer. Therapy of osteoporosis with calcium and vitamin D. J Bone Miner Res. 2007 Dec;22 Suppl 2:V59-6. 20 Bolland MJ, Avenell A, Baron J et al. Effect of calcium supplements on risk of myocardial infarction and cardiovascular events: meta-analysis. BMJ 2010;341:c3691. 21 Dunford JE. Molecular Targets of the Nitrogen Containing Bisphosphonates: -The Molecular Pharmacology of Prenyl Synthase Inhibition. Curr Pharm Des. 2010 Aug 20. [Epub ahead of print] 22 Black DM, Cummings SR, Karpf DB et al. Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Fracture Intervention Trial Research Group. Lancet. 1996 Dec 7;348(9041):1535-41.

	13
	Hippisley-Cox J, Coupland C. Predicating risk of osteoporotic fracture in men and women in England
	and Wales: prospective derivation and validation of QFractureScores. BMJ. 2009;339:b4229.
	14
	Holick MF. Optimal vitamin D status for the prevention and treatment of osteoporosis. Drugs Aging.
	2007;24(12):1017-29.
	15
	Bischoff-Ferrari, HA, Dietrich, T, Orav, E et al. Positive association between 25-hydroxy vitamin D
	levels and bone mineral density: a population-based study of younger and older adults. Am J Med
	2004;116:634.
	16
	Jackson RD, LaCroix AZ, Gass M, et al. Calcium plus vitamin D supplementation and the risk of
	fractures. N Engl J Med 2006;354:669
	17
	Dawson-Hughes B, Harris SS, Krall EA et al.. Effect of calcium and vitamin D supplementation on
	bone density in men and women 65 years of age or older. N Engl J med 1997;337:670
	18
	Prince RL, Devine A, Dhaliwal SS et al. Effects of Calcium Supplementation on Clinical Fracture and
	Bone Structure. Results of a 5-Year, Double-blind, Placebo-Controlled Trial in Elderly Women. Arch
	Intern Med. 2006;166:869-875.
	19
	Dawson-Hughes B,

Table 1 .

 1 Instructions for administration of osteoporosis medications.

Table 2 .

 2 Risk factors for osteoporosis.

	Advanced age
	Previous fracture
	Low body weight (<58kg)
	Family history of hip fracture
	Long term Glucocorticoid therapy
	Cigarette smoking
	Excess alcohol

Informative Summary

The incidence of osteoporosis is rising which will result in an increased incidence of fragility fractures in older people. This will lead to a rise in medical visits, admissions to hospitals and a reduction in independent living. Overall this will pose large economic strain on countries and healthcare systems and prevention of such trends is very important. A variety of medical therapies have been developed in recent years to protect against a reduction in bone mineral density and strength in an effort to reduce fracture risk in high risk populations. Fracture risk is assessed using known risk factors, including age and previous fracture, along with dual energy x-ray absorptiometry measurements where available. Clinical risk scores have recently been developed which are validated algorithms used to predict fracture risk over many years.


Mirva Steinhoff

Markus Fischer

Angelika Paschke
email: angelika.paschke@uni-hamburg.de

Angelika Paschke-Kratzin

Comparison of extraction

Keywords: Extraction, Immunoassays Additives/Contaminants: Allergens, Process contaminants Food Types: Bakery products, Eggs, Milk Food allergy, hidden allergens, extraction of allergens, hen´s egg, cow's milk 32 33 34

published or not. The documents may come

Introduction

36

Cow's milk and hen´s egg as well as their products belong to eight food or food groups which 37 are responsible for about 90 % of allergic reactions [START_REF] Hefle | Food allergy and the food industry[END_REF]. The 38 consumption of small amounts can already cause life-threatening or lethal reactions (Vieluf et 39 al., 2002). Nevertheless, allergic persons can unintentionally consume allergenic material if 40 these are unexpectedly part of a food product e.g. because of food contamination / cross contact within the manufacturing process [START_REF] Hefle | Food allergy and the food industry[END_REF]. The threshold doses for 42 triggering symptoms after ingestion of the offending food are strongly dependent on the 43 patient's individual susceptibility and the allergic potential of the particular food. Clinical data

44

shows that exposure to even about 1 mg of the allergen can elicit adverse reactions up to Wiesbaden, Germany). In the case of matrix addition, 1 g matrix (wheat starch, fat powder, 118 dry cake mix) was also weight in the centrifuge tube and the dry material was mixed 5 sec 119 with a vortex mixer (VWR international, Darmstadt, Germany; mixing frequency 2,500 rpm)

129

For examination of extraction duration, protein isolation as described above was interrupted The isolated proteins separated by LDS -PAGE were transferred electrophoretically from the 172 gel onto the surface of a nitrocellulose membrane (2 µm, Whatman, Kent, UK) using semidry 173 blotting method. In brief, dried membranes were blocked with blocking solution (containing 174 0.5 % Tween 20 ® , 0.9 % sodium chloride and 0.6 % tris base) and incubated overnight with 175 sheep anti-casein (1 : 400), rabbit anti-whey (1 : 200000) and rabbit anti-ovalbumin

176

(1 : 200000) antibodies, respectively, at room temperature. Membranes were then incubated whole egg powder, powdered egg yolk, powdered egg white) and streptavidin -horseradish

198

For all the egg materials which were examined, the extraction agents I and J aqueous solution 199 gave the highest protein yields (Table 2). In contrast to the extractants mentioned above, the 200 protein contents of the extracts prepared using saline solutions (A, B, C, D), G or H to extract 201 egg proteins from egg material led to recovery rates lower than 30 %. Where egg proteins 202 were isolated from egg powder in dry cake mix, solutions I and J gave the highest protein 203 recovery rates. Extracting proteins from egg material in wheat starch and fat powder as food 204 matrix, the highest protein contents were measured in the extracts prepared using solutions I 205 and J. But also E achieved high recovery of protein from egg material in wheat starch (Table 206 2).

208

In this study, optimization of pH-value of extractants as well as extraction temperature was 209 carried out. Therefore, solution I (pH 7.4) and J (pH 8.5) were additionally prepared having a 210 pH of 9.4 and 11.4 (I) as well as 10.5 and 12.5 (J), respectively. Extraction procedures were 211 simultaneously performed at three different extraction temperatures (4 °C, RT = room 212 temperature and 40 °C). The estimated recovery rates are presented in Tables 3 and4.

213

Comparing the protein yields there is not much of a difference. The deviation of the obtained 214 recovery rates is of the order of the calculated standard deviation.

308

The evaluation of recovery rates of extracted milk and egg material using eleven different 309 solutions led to similar results. The solutions I and J achieved the highest protein yields.

310

In the case of milk samples, the extractants had only minor impact on the extraction 311 efficiency. On the other hand, the extraction efficiency of egg material was much more 312 sensitive to the solution used for the protein extraction. The major difference between milk 313 and egg is the fat content. Because of the higher fat content of egg material, extractants 314 containing a detergent yielded higher protein recovery rates as compared to those without.

315

Emulsifying properties of egg-lecithin alone are not sufficient to extract egg powder properly.

316

These results clearly indicate that the composition of the extractant is of particular 317 importance for the efficiency and quality of the extract.

  were additionally prepared with pH-values adjusted to 10.5 and 12.5 and 9.4 and 11.4, 110 respectively. Adjustment of pH was carried out with 0.1 M NaOH. procedure was carried out as follows. According to the protein content 114 measured via Kjeldahl method, 30 mg milk powder, 100 mg sweet whey, 100 mg acid whey, 115 25 mg whole egg powder, 15 mg dried egg white and 30 mg dried egg yolk, respectively, 116 were weighed into a 50 mL polypropylene centrifuge tube (Nunc GmbH & Co. KG, 117

  120 before extractant was added. Protein extraction was carried out by suspending sample 121 material with 20 mL of extraction solution. The homogenate was extracted over night on a 122 laboratory shaker (Bühler, Tübingen, Germany; shaking frequency 150 rpm) at room 123 temperature and centrifuged afterwards 30 min at 10,000 rpm (Sigma-Aldrich Chemie 124 GmbH, Munich, Germany). Solids were removed by filtration (Macherey-Nagel GmbH & Co. 125 KG, Düren, Germany; filter paper grade MN 616). Investigations with the aim to optimize 126 extraction temperature were additionally carried out at 4 °C on a laboratory shaker in a 127 standard refrigerator (Linde, München, Germany) and at 40 °C using a laboratory incubator 128 (IRC-1-U Clim-o-shake, Adolf Kühner AG, Birsfelden, Swiss; shaking frequency 150 rpm).

  180 peroxidase. Staining solution containing TMB and DONS (0.06 % 3, 3´, 5 , 5´ -181 Tetramethylbenzidine, 0.2 % dioctylsodiumsulphosuccinate, 25 % ethanol, 0.7 % citric acid, 182 1 % Na 2 HPO 4 in water) was used for staining. in the extracts were estimated by relating theoretical calculated 187 protein content measured via the Kjeldahl method to total protein yield of the extract 188 measured with Lowry or Bradford method (data not shown

Food

  J with a pH of 12.5. Conversely, any characteristic proteins detected after 251 electrophoretic separation of the extracts made from solution I with a pH of 11.4 or the 252 detection of the characteristic proteins was much weaker. 253 254 The band intensities obtained via silver staining of milk and egg proteins isolated at different 255 temperatures were almost identical. Extracts obtained at the end of varied extraction 256 durations using solution I (pH 7.4) and J (pH 8.5) were electrophoretically separated and 257 proteins were detected via silver staining. Regardless of extractant and extracted material, 258 band intensities of characteristic proteins did not differ in terms of extraction duration (extractants ensure comprehensive isolation of intact allergens 263 specific immunochemical staining on nitrocellulose membrane after LDS -PAGE and 264 Western Blotting was performed (Figures 3 and 4). The extracts derived with solution I, J and 265 E present most strongly stained bands of ovalbumine compared to solutions G and H in the 266 immunoblots. The results of unspecific staining procedure as well as the determination of the 267 protein concentration are reflected in the results of immunochemical staining. Rabbit anti-268 ovalbumin antibody used for detection of albumin also detects conalbumin (76 kDa) as 269 characteristic egg protein. Quite similar results were obtained by immunochemical detection 270 of milk proteins. Band intensities of milk proteins of purchased extracts looked rather similar 271 to the extracts described in this paper and Immunoblots showed only slight variations in 272 protein recovery rates. Anti-casein antibody used for detection of casein also detects bovine 273 serum albumin (BSA; 76 kDa) as characteristic milk protein. 274 275 The influence of wheat starch and dry cake mix on extraction efficiency and quality was 276 investigated by comparing the extract properties prepared with matrix addition and without as 277 described above. Addition of 8 M urea as extractant to wheat starch or dry cake mix 278 Page 10 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk a strong enhancement of extract viscosity. Those solutions were 279 unemployable for further analysis. Band intensities referring to silver and immunochemical 280 staining do not differ noteworthy comparing the extracts made with matrix addition or without 281 (Figures 3 and 4). Considering dilution factors, extracts of solution I, J as well as E present 282 most strongly stained bands of ovalbumine compared to solutions G and H in the 283 immunoblots. Purchased extracts showed similar band intensities. The immunoblots affirm 284 the slight variation in protein recovery rates obtained for extracts made of dairy products. 285 286 Comparing the band intensities of the characteristic proteins in the extracts prepared with 287 solution I (pH 7.4 and pH 9.4) and J (pH 8.5 and 10.5), respectively, no differences 288 appeared. Any characteristic proteins could be visualized in the extracts made of solution J 289 pH 12.5 whilst the detection of the characteristic proteins extracted via solution I" pH 11.4 290 was much weaker. 291 292 The band intensities obtained via immunochemical staining of milk and egg proteins isolated 293 at different temperatures were almost identical. Regardless of extractant and extracted 294 material, the band intensity of the characteristic proteins did not differ in terms of extraction 295 duration (data not shown). 296 297 Discussion 298The ultimate prerequisite for sensitive analytical allergen detection is an efficient and reliable 299 were calculated by comparing the total protein concentration of the 302 food sample with the protein yields of the extracts. The photometric detection, which was 303 much more suitable for the screening of the extracts than the Kjeldahl procedure, only 304 provides a relative but not an absolute quantification of the proteins. Nevertheless, the 305 relative quantification was sufficient to assess the efficiency of the different extractants.

Page

  extracts by LDS -PAGE followed by silver staining as well as immunoblotting 321 confirmed the results of protein quantification. Solution I as well as J led to the strongest 322 bands of characteristic milk and egg proteins, respectively. However, it has to be considered, 323 that silver staining generally possesses a lower sensitivity than photometrical protein 324 quantification. Slight variations concerning protein content in the extracts cannot be 325 discriminated after electrophoretical separation. 326 327 Detection of selected proteins by western blotting clearly demonstrated that both extraction 328 solutions had no negative influence onto the antigenicity of the extracted allergenic proteins. 329 Solution I and J are the most qualified extractants to isolate proteins of milk. In case of hen´s 330 eggs, they are the only suitable solutions for the efficient extraction of proteins. Hence, hen´s 331 egg is more sensitive to the extractant than milk. 332 333 Optimization of the extraction procedure: temperature and pH 334 1 hour at room temperature guarantees high protein yields without 393 any negative influence on the allergens antigenicity.

  reactions. New Trends in Allergy V, 5th,Davos, Switzerland, Sept. 15-17, 2000.

  Newsome WH, Abbott MA. 2000. Determination of egg proteins in food products 455 by enzyme immunoassay. J AOAC Int. 83: 139-143.

Fig. 2 :Fig. 3 : 19 Fig. 4 :

 23194 Fig. 2: LDS -PAGE and silver staining patterns of proteins extracted from egg powder using

Table 1 :

 1 Protein recovery rates (%) estimated using different extractants to isolate milk proteins out of milk material or out of milk material in matrix (WM = without matrix; WS = wheat starch; FP = fat powder; CM = cake mix)

	Extractant

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y

Table 2 :

 2 Protein recovery rates (%) estimated using different extractants to isolate egg proteins out of egg material or out of egg material in matrix

	(WM = without matrix; WS = wheat starch; FP = fat powder; CM = cake mix)
		1	2	3	4	5	6	7	8	9	10	11	12
		WM 27 ± 2 26 ± 2 20 ± 1 23 ± 2 108 ± 3	74 ± 5	26 ± 1 22 ± 1 119 ± 11 112 ± 3 64 ± 2 87
	Whole egg	WS 22 ± 1 22 ± 2 22 ± 2 21 ± 1 108 ± 10 FP 20 ± 1 22 ± 1 18 ± 2 41 ± 2 75 ± 1	--	21 ± 2 27 ± 2 100 ± 4 117 ± 4 70 ± 0 84 22 ± 2 30 ± 2 125 ± 0 134 ± 5 64 ± 6 97
		CM 29 ± 2 25 ± 1 21 ± 4 28 ± 3	78 ± 1	-	27 ± 3 30 ± 3 100 ± 2 124 ± 4 45 ± 2 112
		WM 37 ± 2 36 ± 1 38 ± 0 30 ± 2	86 ± 0	63 ± 4	79 ± 2 49 ± 2 129 ± 6 113 ± 5 16 ± 0 71
	Egg yolk	WS 38 ± 1 38 ± 2 39 ± 4 30 ± 2 FP 81 ± 6 70 ± 4 70 ± 11 75 ± 4	83 ± 4 77 ± 2	--	62 ± 1 45 ± 1 111 ± 9 108 ± 2 20 ± 1 73 86 ± 6 33 ± 2 151 ± 9 139 ± 4 94 ± 7 90
		CM 38 ± 2 38 ± 1 50 ± 3 45 ± 4	85 ± 5	-	107 ± 8 35 ± 3 113 ± 12 133 ± 6 51 ± 1 83
		WM 76 ± 2 76 ± 7 61 ± 11 53 ± 1 121 ± 3 116 ± 8 37 ± 2 20 ± 0 138 ± 16 123 ± 5 77 ± 2 86
	Egg white	WS 76 ± 3 79 ± 1 71 ± 1 69 ± 1 120 ± 3 FP 15 ± 1 14 ± 0 19 ± 0 16 ± 0 90 ± 1	--	39 ± 1 21 ± 2 135 ± 9 132 ± 2 88 ± 2 95 39 ± 2 32 ± 1 130 ± 10 145 ± 4 24 ± 2 111
		CM 16 ± 2 19 ± 2 22 ± 8 26 ± 4	78 ± 0	-	52 ± 6 44 ± 1 120 ± 4 141 ± 9 41 ± 2 102

Table 3 :

 3 Protein recovery rates (%) using solution I and J, respectively, at different pH-values to isolate milk proteins from milk material and from milk material in certain matrices at different temperatures. (WM = without matrix; WS = wheat starch; CM = cake mix)

	Recovery rates [%]

Table 4 :

 4 Protein recovery rates (%) using solution I and J, respectively, at different pH-values to isolate egg proteins from egg material and from egg material in certain matrices at different temperatures. (WM = without matrix; WS = wheat starch; CM = cake mix)

	Recovery rates [%]

Table 5 :

 5 Protein recovery rates obtained by varied extraction durations at three temperatures using solution I (pH 7.4) and J (pH 8.5) to isolate milk proteins from skim milk (WM = without matrix; CM = cake mix).

	Extractant Matrix Temp.	0	0.5	Extraction duration [h] 1 2	4	8
		4 °C		82 ± 0 90 ± 1 85 ± 2 85 ± 6 82 ± 2
	WM	RT	81 ± 1	81 ± 2 97 ± 1 89 ± 3 80 ± 7 85 ± 4
	J	40 °C		82 ± 3 89 ± 3 78 ± 1 82 ± 3 84 ± 3
	(pH 8.5)	4 °C		89 ± 8 98 ± 3 92 ± 7 99 ± 2 88 ± 5
	CM	RT	80 ± 1	80 ± 6 90 ± 9 98 ± 5 75 ± 7 86 ± 0
		40 °C		82 ± 4 88 ± 7 87 ± 1 96 ± 2 91 ± 7
		4 °C		88 ± 1 101 ± 9 84 ± 2 86 ± 1 82 ± 0
	WM	RT	96 ± 1	94 ± 2 102 ± 4 86 ± 0 114 ± 9 86 ± 1
	I	40 °C		84 ± 7 112 ± 8 83 ± 1 87 ± 1 88 ± 8
	(pH 7.4)	4 °C		90 ± 4 83 ± 1 80 ± 5 86 ± 2 83 ± 1
	CM	RT	78 ± 4	80 ± 0 90 ± 6 83 ± 4 80 ± 6 77 ± 7
		40 °C		83 ± 3 92 ± 5 76 ± 7 93 ± 9 78 ± 3

Table 6 :

 6 Protein recovery rates obtained by varied extraction durations at three temperatures using solution I and J to isolate egg proteins from whole egg powder (WM = without matrix; CM = cake mix).

	Extractant Matrix Temp.	0	0.5	Extraction duration [h] 1 2	4	8
		4 °C		106 ± 5 105 ± 1 104 ± 4 106 ± 3 104 ± 4
	WM	RT	107 ± 4	110 ± 3 108 ± 3 104 ± 2 104 ± 1 109 ± 2
	J	40 °C		110 ± 0 109 ± 2 109 ± 4 106 ± 3 107 ± 1
	(pH 8.5)	4 °C		116 ± 6 105 ± 3 112 ± 1 105 ± 4 113 ± 8
	CM	RT	115 ± 6	119 ± 12 117 ± 5 112 ± 4 111 ± 3 112 ± 6
		40 °C		116 ± 0 122 ± 3 120 ± 2 123 ± 0 111 ± 6
		4 °C		96 ± 2	99 ± 2	99 ± 5 104 ± 2 104 ± 3
	WM	RT	94 ± 6	96 ± 4	93 ± 3	98 ± 2	94 ± 3	96 ± 6
	I	40 °C		99 ± 3	103 ± 3 95 ± 2 101 ± 0 101 ± 3
	(pH 7.4)	4 °C		92 ± 1	93 ± 1	92 ± 1	89 ± 6	91 ± 0
	CM	RT	91 ± 2	90 ± 0	99 ± 1	93 ± 2	94 ± 4	93 ± 5
		40 °C		112 ± 3 112 ± 6 117 ± 3 117 ± 2 108 ± 0

anaphylactic shocks in individuals with IgE-mediated food allergies[START_REF] Besler | Stability of food allergens and allergenicity of 402 processed foods[END_REF]

Afterwards the gel was developed in 2.5 % sodium carbonate (2.5 % Na 2 CO 3 , 0.01%

consecutively with biotinylated rabbit anti-sheep serum (1 : 100000; milk powder) or

biotinylated goat anti-rabbit serum (1 : 7500; powdered sweet whey, powdered acid whey,

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

With the aim to investigate optimum extraction duration, extraction procedures were

proteins. Evaluation of recovery rates, results of unspecific and specific staining of the

industrieller Forschungsvereinigungen). Special thanks go to B. Jacobsen, K. Ramcke and S.

casein and its application to retail and milk-allergic complaint foods. J Food Prot. 67:

Moneret-Vautrin DA, Kanny G. 2004. Update on threshold doses of food allergens:

342: 214-220.

Schubert S. 2003. Charakterisierung und Isolierung von Allergenen der Tomate

(Lycopersicon lycopersicum (L.) Karst. ex Farw.)[dissertation]. [Institut für Biochemie

und Lebensmittelchemie]: Universität Hamburg.

Vieluf I, Besler M, Paschke A, Steinhart H, Vieluf D. 2002. Practical approach to adverse

Acknowledgments

396

This study was supported by the AiF-project AiF 14805 N (AiF: Arbeitsgemeinschaft

Schwarz for technical support.

399

Implications for patients and the food industry. Curr Opin Allergy Clin Immunol. 4:


Mark Dechesne
email: mdechesne@campusdenhaag.nl

Deradicalization: Not soft, but strategic

Keywords: Deradicalization, Psychology, Terrorism, Persuasion, Strategy

come   L'archive ouverte pluridisciplinaire

Deradicalization: Not soft, but strategic

Deradicalization is hot. In 2007, Time magazine [START_REF] Ripley | Future revolutions: Reverse radicalism[END_REF] reviewed what they considered the most revolutionary ideas of the 21st century. Reversing radicalism, deradicalization, figured prominently among them. The Time article marks quite a change from the initial response to the September 11 th attacks in 2001 that involved a widespread call for an all out military campaign targeted at all those threatening freedom. However, deradicalization should not be considered soft. It is strategic. Deradicalization seeks to prevent further escalation of violence. Moreover, and this makes disengagement and deradicalization different, deradicalization creates the conditions conducive to dialogue. Deradicalization is therefore an important strategic tool in the fight against terrorism. And not surprisingly, then, it is being embraced by many. In Europe, countries such as the Netherlands and the UK follow with great interest developments in Asia and North Africa where the first results of large-scale deradicalization attempts are being published [START_REF] Neuman | Prisons and Terrorism. Radicalisation and De-Radicalisation in 15 Countries. International Centre for the Study of Radicalisation and Political Violence[END_REF]. In this article, partly based on recent publications in the field of deradicalization, I will discuss the nature of deradicalization, evaluate existing deradicalization programs, and identify areas of attention to enhance understanding and efficiency of deradicalization.

Four observations

Let me start with some general observations. These observations form the basis for a general characterization of the phenomenon.

I have already mentioned the first observation: Deradicalization is not soft, but strategic. Terrorism can create a "jiujutsu" dynamic [START_REF] Mccauley | Jujitsu politics: terrorism and response to terrorism[END_REF]. That is, harsh, military reactions against terrorism may alienate potential sympathizers with a terrorist cause and expand the terrorists' pool of recruits. Effective counterterrorism policy should then strive to break the cycle of violent action and response. Deradicalization, whereby radicals abandon violence and are brought back into society, may constitute an effective tool to do so.

The second: Deradicalization is not new. While the increased number of publications on the topic suggests that deradicalization is a modern invention, it was already applied in the seventies [START_REF] Kruglanski | Inside the Terrorist Mind[END_REF]. The PLO dismantled its Black September and brought them to a more moderate path through methods that resemble those of deradicalization. Members of the Black September were given the prospect of marriage with a carefully selected bride and of a reward of $ 3,000 for marriage with an additional bonus of $ 5,000 for a first child. Individuals in the program also got an apartment with cooking facilities, refrigerator and television, in addition to a desk job at the PLO. The British government experimented with temporary prison leaves and vocational training as ways to bring back IRA convicts to civil society. The Italian government used special repentance laws to facilitate Red Brigade members to abandon the organization.

Third observation: Deradicalization may arise spontaneously. Deradicalization may not require any coordinated action to get it underway [START_REF]Leaving Terrorism Behind: Individual and Collective Disengagement[END_REF]. There can be several reasons for why members of terrorist organization may spontaneously deradicalize. The peer pressure in an organization can be experienced as unpleasant. A terrorist organization is composed of many violent people, and that violence can also be directed inwards. Members of the group may also develop self-doubts, or question whether the ideals of the organization will ever be achieved, or start doubting whether reality is living up to expectation. Tanja Nijmijer, a Dutch member of FARC, for example, frequently notes in her recovered diaries that her everyday life is far off from the idealistic commitment the FARC organization attempts to publically portray [START_REF] Gumber | Woman's diary of a jungle guerrilla's life. The Independent[END_REF].

Fourth observation: It is important to differentiate between physical and psychological forms of disengagement.

For a number of reasons, a member of a terrorist organization may no longer be able to engage in violence but may maintain a firm belief in the righteousness of the cause. Deradicalization pertains to the disappearance of radical thoughts, not just radical behavior. Arrests, forced change within the organization, exclusion from the group, or a change in interest, can all lead to physical withdrawal from violent engagement without psychological deradicalization. Also, a lack of resources, goals, or the presence of alternative goals, may leave even the most devoted member of the terrorist organization out of work. Behavior may be blocked, but there is no change in attitudes.

The nature of radicalization and the workings of deradicalization programs

The nature of deradicalization can thus be summarized as in Figure 1. The figure describes deradicalization using two dimensions. The first, vertical dimension pertains to psychological change (top) vs behavioral change (bottom). The second, horizontal dimension pertains the locus of change. On the left, endogenous influences pertain to those changes in behavior or thought that are self-initiated. On the right, exogenous influences pertain to changes in radical thoughts/behaviors that are brought about by external influences. From the current perspective, deradicalization, i.e. the systematic moderation of radical thoughts, should above all implicate 1) an attempt to create the circumstances under which individuals become more open to alternative viewpoints; and 2) an attempt to externally bring the radicalized individual to more moderate viewpoints. If there is no self-initiated openness to change beliefs, external attempts to moderate radical beliefs are unlikely to be successful. If there is no external regulation of self-initiated belief change, deradicalization may loose its strategic use. Going back to Figure 1, then, deradicalization programs should pertain to both the left and right upper quadrant of the figure .   With this scheme in mind, I have studied a number of publications reporting on deradicalization programs [START_REF] Horgan | Disengaging from Terrorism[END_REF]. These programs include the Exit Programs initiated by Tjore Bjørgo in Scandinavia and Germany that pertain to the extreme right. The programs also include Asian (in Saoudi Arabia, Yemen, Singapore, and Indonesia) deradicalization programs targeting Jihadis. Let me summarize my primary findings in the form of propositions. Proposition 1: "Deradicalization" programs for the extreme right primarily target the behavioral aspects of involvement in an extremist organization. It is questionable, then, whether these programs provide adequate insight into deradicalization.

The EXIT programs set up by Tjore Bjorgo in several Scandinavian countries and Germany for the extreme right primarily seek to facilitate the disengagement from extremist group [8]. It thus targets those who have already decided themselves to disengage. The programs promote opportunities for neo-Nazi's who wish to exit, by coaching them through the EXIT program. Furthermore, the programs use various means to influence the behavior of the radicals, including the involvement of parents to detect signals of radicalization and to stop further engagement in the extreme right, and the use of "serious conversations" to be undertaken by parents or police. These conversations do not involve a substantive discussion on radical right-wing ideology, but rather seek to emphasize the serious repercussions that involvement in extreme right-wing groups might have. The EXIT programs never seek to influence the individuals' worldview through external influence. As a result, these programs may be less suited to reflect on the possibilities and impossibilities of deradicalization.

Proposition 2: There is insufficient insight into what motivates people to deradicalize.

This is partly due to the setup of deradicalization programs. Virtually all programs that seek to deradicalize Islamic radicals are based on establishing a dialogue between the radical and an authority taking a moderate stance [9]. The rationale is that through this dialogue, the beliefs of the radical will gradually shift towards the moderate viewpoint. In Singapore and Yemen, the moderate position is taken by a clergyman. In Saudi Arabia, a team of clergymen, psychologists, consultants and external communication advisors, is involved. Indonesia uses prominent former radicals.

Most Asian deradicalization programs target prisoners. As a reward for participating in the program, prisoners are sometimes promised release. This, however, yields a peculiar situation. People can participate because of a very practical reward: freedom. As a result, it is hard to determine the sincerity of the motives for participating. But the effectiveness of these deradicalization programs cannot be assessed without taking into account what motivated the deradicalization process in the first place. Thus, there is insufficient insight into what really motivates people to deradicalize. Proposition 3: Insights from psychology are still insufficiently used to increase effectiveness of deradicalization.

The consideration of the EXIT programs and Islamic deradicalization efforts suggests that they fail to implicate both of the two crucial elements of deradicalization: promoting inner openness for a change of mind, and presenting alternative views from the outside. The discipline of psychology has much to say about how to optimize the conditions for both. It is remarkable, then, that in the radicalization literature very little attention is paid to psychological research, in particular research in the area of social cognition which specializes in attitude and belief change.

How can social psychological insights be use to better understand the deradicalization process? Perhaps a line of research that I conducted together with colleagues from the University of Arizona and the University of British Columbia can clarify [START_REF] Kosloff | Smearing the opposition: Implicit and explicit stigmatization of the 2008 U.S. Presidential candidates and the current U.S. President[END_REF]. Just before the US presidential elections in 2008 we investigated the circumstances under which people are willing to believe that Barack Obama is a Muslim. The research may not be directly related to the issue of terrorism, but it is related to the willingness to adopt false beliefs and the creation of the circumstances under which such beliefs are adopted or discarded.

In three studies, participants were asked about their political preferences. Moreover, half the subjects were asked to indicate their skin-color just prior to the experiment. Because all subjects were Caucasian, the question induced a sense of difference between the participant and Barack Obama for half of the participants. Republican participants were more likely to associate Barack Obama with Islam than Democratic participants. Also, the subtle induction of a sense of difference based on race lead participants to more strongly associate Barack Obama with Islam and the belief that he is a Muslim.

Hence, it appears that people are particularly sensitive to the information that Obama is a Muslim when (1) they already disagreed with him ideologically, [START_REF] Neuman | Prisons and Terrorism. Radicalisation and De-Radicalisation in 15 Countries. International Centre for the Study of Radicalisation and Political Violence[END_REF] when the psychological distance between the participant and Barack Obama was subtly enlarged by pointing to differences in skin color. Our research thus suggests that achieving a sense of similarity, a sense of "we-ness", may very well be an important condition under which deradicalization can work. First one creates the perception of similarity between the radical and the person seeking to deradicalize, and then and only then it becomes possible to influence the radical's belief. This is, of course, highly consistent with the idea proposed in this article that deradicalization should contain two critical elements: promoting inner openness for a change of mind, and presenting alternative views from the outside.

Conclusion

Deradicalization may be considered the jewel in the crown of modern counterterrorism techniques. It's a potentially very valuable strategic tool enabling to break a dangerous spiral of violence between terrorists and counterterrorists. At this point, however, it is difficult to determine whether deradicalization works, because it is not sufficiently clear how exactly deradicalization processes work. Research can make an important contribution to optimize efficiency of deradicalization for counterterrorism purposes.

The starting point for this research must be that deradicalization is not soft, but strategic. Implementing a strategic operation involves consideration of goals and assessment and evaluation of a situation, the identification of problem areas that need to be addressed, and a plan for how to address the problems areas. When deradicalization is considered within a frame of strategy, psychological questions will inevitably need to be answered: What does the "architecture" of radical ideas look like? What exactly do you want change, and where is it mentally represented? What methods are most effective to bring about belief change? Under what circumstances can deradicalization best take place? What "incentives" encourage deradicalization? Are there individual differences?

In a counterterrorism climate that increasingly becomes to see the value of deradicalization, answering these questions should be assigned high priority.

Figure 1 :

 1 Figure 1: Two-dimensional structure of deradicalization.