PBPK modeling of recombinant factor IX Fc fusion protein (rFIXFc) and rFIX to characterize the binding to type 4 collagen in the extravascular space.

Patients with severe and sometimes moderate hemophilia B are prophylactically treated with factor IX concentrates to prevent bleeding. For some time now, various extended terminal half-life (EHL) recombinant factor IX concentrates are available allowing less frequent administration during prophylaxis in comparison to standard half-life recombinant FIX (rFIX). Especially, recombinant FIX-Fc fusion protein (rFIXFc; Alprolix®) exhibits a rapid distribution phase, potentially due to binding to type IV collagen (Col4) in the extravascular space. Studies suggest that the presence of extravascular rFIXFc is protective against bleeding as without measurable FIX activity in plasma, and no extra bleeding seems to occur. The physiologically based pharmacokinetic (PBPK) model for rFIXFc which we describe in this study, is able to accurately predict the observed concentration-time profiles of rFIXFc in plasma and is able to quantify the binding of rFIXFc to Col4 in the extravascular space after an intravenous dose of 50 IU/kg rFIXFc in a male population. Our model predicts that the total AUC of rFIXFc bound to Col4 in the extravascular space is approximately 19 times higher compared to the AUC of rFIXFc in plasma. This suggests that rFIXFc present in the extravascular compartment may play an important role in achieving hemostasis after rFIXFc administration. Further studies on extravascular distribution of rFIXFc and the distribution profile of other EHL-FIX concentrates are needed to evaluate the predictions of our PBPK model and to investigate its clinical relevance.

Characteristics of FXa-storing platelets in hemophilia B mice and the influence of alcohol on the platelets.

Platelet-stored activated blood coagulation factor X (FXa) has great potential in the gene therapy of hemophilia B (HB). However, we still need to understand more about the properties of FXa-storing platelets and how dietary factors affect them. We created transgenic mice called 2bFXa-HB, which had stable expression and storage of FXa in their platelets, resulting in the alleviation of the bleeding disorder in these mice. Even after inducing anti-factor IX (FIX) inhibitors in 2bFXa-HB mice, the hemorrhage phenotype could still be rescued by the expression of FXa. The activation capacity of 2bFXa-HB platelets remained unchanged, and there were no signs of elevated thrombotic risk in these mice. In an acute alcohol exposure mouse model, a single administration of alcohol reduced both the number of platelets and their activation capacity, as well as impaired coagulation function. However, it did not increase the markers of thrombotic risk in either 2bFXa-HB or HB mice. These results suggest that FXa storage in platelets is safe and effective for treatment of HB, but alcohol could impair the therapeutic effect of FXa-containing platelets.

Phase 1-2 Trial of AAVS3 Gene Therapy in Patients with Hemophilia B.

BACKGROUND: FLT180a (verbrinacogene setparvovec) is a liver-directed adeno-associated virus (AAV) gene therapy that uses a synthetic capsid and a gain-of-function protein to normalize factor IX levels in patients with hemophilia B. METHODS: In this multicenter, open-label, phase 1-2 trial, we assessed the safety and efficacy of varying doses of FLT180a in patients with severe or moderately severe hemophilia B (factor IX level, ≤2% of normal value). All the patients received glucocorticoids with or without tacrolimus for immunosuppression to decrease the risk of vector-related immune responses. After 26 weeks, patients were enrolled in a long-term follow-up study. The primary end points were safety and efficacy, as assessed by factor IX levels at week 26. RESULTS: Ten patients received one of four FLT180a doses of vector genomes (vg) per kilogram of body weight: 3.84×1011 vg, 6.40×1011 vg, 8.32×1011 vg, or 1.28×1012 vg. After receiving the infusion, all the patients had dose-dependent increases in factor IX levels. At a median follow-up of 27.2 months (range, 19.1 to 42.4), sustained factor IX activity was observed in all the patients except one, who resumed factor IX prophylaxis. As of the data-cutoff date (September 20, 2021), five patients had normal factor IX levels (range, 51 to 78%), three patients had levels from 23 to 43%, and one had a level of 260%. Of the reported adverse events, approximately 10% were related to FLT180a and 24% to immunosuppression. Increases in liver aminotransferase levels were the most common FLT180a-related adverse events. Late increases in aminotransferase levels occurred in patients who had received prolonged tacrolimus beyond the glucocorticoid taper. A serious adverse event of arteriovenous fistula thrombosis occurred in the patient with high factor IX levels. CONCLUSIONS: Sustained factor IX levels in the normal range were observed with low doses of FLT180a but necessitated immunosuppression with glucocorticoids with or without tacrolimus. (Funded by Freeline Therapeutics; ClinicalTrials.gov numbers, NCT03369444 and NCT03641703; EudraCT numbers, 2017-000852-24 and 2017-005080-40.).

Dexamethasone Transiently Enhances Transgene Expression in the Liver When Administered at Late-Phase Post Long-Term Adeno-Associated Virus Transduction.

Glucocorticoids have anti-inflammatory and immunosuppressive functions and have commonly been used for preventing liver toxicity after the systemic application of a high dose of adeno-associated virus (AAV) vector for gene therapy. Clinical studies have reported that glucocorticoids have rescued factor IX (FIX) expression in patients with hemophilia B who showed a reduced FIX expression at 6 to 10 weeks post-AAV vector administration. In this study, we explored whether glucocorticoids could affect transgene expression in AAV targeted livers in animal models. When dexamethasone was applied before AAV9/FIX vector administration in the wild-type C57BL/6 mice, FIX expression was much higher than that of the control mice at any time point. More importantly, FIX expression transiently increased after dexamethasone was administered at week 6 or later post-AAV injection regardless of the various dexamethasone treatments applied. The transient enhancement in transgene expression was observed once there were one to several consecutive dexamethasone treatments completed. A similar result was also achieved in other wild-type BALB/c and hemophilia B mice that were treated with AAV9/FIX and dexamethasone. This mechanism study demonstrated that the administration of dexamethasone did not change either AAV genome copy number or transgene expression at the transcription level but transiently decreased interferon beta (IFN-ß) and tumor necrosis factor alpha (TNF-α) expression in the livers of mice at a later time after AAV injection. Next, we studied the effect of dexamethasone on late transgene expression in hemophilia B dogs. Dexamethasone was administered 1 year after AAV9/FIX injection. Inconsistent with the results in mice, no significant change of FIX expression was observed in hemophilia B dogs. In summary, the results from this study indicate that dexamethasone may have various effects on transgene expression in AAV-transduced livers in different species, which provides valuable information about the rational application of dexamethasone in future clinical studies.

Gene Therapy in Hemophilia: Recent Advances.

Hemophilia is a monogenic mutational disease affecting coagulation factor VIII or factor IX genes. The palliative treatment of choice is based on the use of safe and effective recombinant clotting factors. Advanced therapies will be curative, ensuring stable and durable concentrations of the defective circulating factor. Results have so far been encouraging in terms of levels and times of expression using mainly adeno-associated vectors. However, these therapies are associated with immunogenicity and hepatotoxicity. Optimizing the vector serotypes and the transgene (variants) will boost clotting efficacy, thus increasing the viability of these protocols. It is essential that both physicians and patients be informed about the potential benefits and risks of the new therapies, and a register of gene therapy patients be kept with information of the efficacy and long-term adverse events associated with the treatments administered. In the context of hemophilia, gene therapy may result in (particularly indirect) cost savings and in a more equitable allocation of treatments. In the case of hemophilia A, further research is needed into how to effectively package the large factor VIII gene into the vector; and in the case of hemophilia B, the priority should be to optimize both the vector serotype, reducing its immunogenicity and hepatotoxicity, and the transgene, boosting its clotting efficacy so as to minimize the amount of vector administered and decrease the incidence of adverse events without compromising the efficacy of the protein expressed.

Tailoring the effect of antithrombin-targeting therapy in haemophilia A using in silico thrombin generation.

Factor (F) VIII deficiency causes bleeding in haemophilia A patients because of the reduced formation of procoagulant enzyme thrombin, which is needed to make the blood clot. We measured the dynamics of coagulation in haemophilia A patients by measuring thrombin generation (TG). Additionally, we quantified the procoagulant process of prothrombin conversion and anticoagulant process of thrombin inhibitor complex formation. In haemophilia A, prothrombin conversion is severely reduced, causing TG to be low. Nevertheless, the thrombin inactivation capacity of these patients is comparable to that in healthy subjects, leading to a severe imbalance between procoagulant and anticoagulant processes and a subsequent increased bleeding risk. A novel therapy in haemophilia A is the targeting of anticoagulant pathway, e.g. thrombin inhibitor antithrombin (AT), to restore the haemostatic balance. We simulated the effect of AT reduction on TG in silico. Lowering AT levels restored TG dose-dependently and an AT reduction of 90-95% led to almost normal TG in most patients . However, the variation in response to AT reduction was large between patients, indicating that this approach should be tailored to each individual patients. Ideally, TG and thrombin dynamics simulation could in the future contribute to the management of patients undergoing AT targeting therapy.

Serpins, New Therapeutic Targets for Hemophilia.

Hemostasis is a tightly regulated process characterized by a finely tuned balance between procoagulant and anticoagulant systems. Among inherited hemostatic conditions, hemophilia is one of the most well-known bleeding disorders. Hemophilia A (HA) and B (HB) are due to deficiencies in coagulation factor VIII (FVIII) or FIX, respectively, leading to unwanted bleeding. Until recently, hemophilia treatment has consisted of prophylactic replacement therapy using plasma-derived or recombinant FVIII in cases of HA or FIX in cases of HB. Because FVIII and FIX deficiencies lead to an imbalance between procoagulant and anticoagulant systems, a recent upcoming strategy implies blocking of endogenous anticoagulant proteins to compensate for the procoagulant factor deficit, thus restoring hemostatic equilibrium. Important physiological proteins of the anticoagulant pathways belong to the serpin (serine protease inhibitor) family and, recently, different experimental and clinical studies have demonstrated that targeting natural serpins could decrease bleeding in hemophilia. Here, we aim to review the different, recent studies demonstrating that blocking serpins such as antithrombin, protein Z-dependent protease inhibitor, and protease nexin-1 or modifying a serpin like α1-antitrypsin could rebalance coagulation in hemophilia. Furthermore, we underline the potential therapeutic use of serpins for the treatment of hemophilia.

Factor VIII-driven changes in activated factor IX explored by hydrogen-deuterium exchange mass spectrometry.

The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system.

Is the Detection of Factor IX Inhibitors in Hemophilia B Orphan than Factor VIII Inhibitors in Hemophilia A? A Concise, Systematic Review.

OBJECTIVE: Development of inhibitors in hemophilia A and B comprise significant challenge for patients, hematologists, and health provider systems. It has recommended by the World Federation of Hemophilia (WFH) to check inhibitors every 3-4 months. The incidence of inhibitor in hemophilia B is lower than hemophilia A. Here, it tried to unravel whether the detection of inhibitors in hemophilia B neglected compared to hemophilia A or not? METHODS: A comprehensive review carried out using six international and local medical search engines on published contributions about inhibitors in hemophilia A and B in Iran. RESULTS: From 699 titles, 12 relevant papers were selected. The mean of factor VIII inhibitors in hemophilia A was 14.8%. The mean of factor IX inhibitors in hemophilia B was 6%. The minimum and maximum reported percentages of factor VIII inhibitors were 4% and 19.6%, while the minimum and maximum of reported percentages of factor IX inhibitors were 0% and 11.8%, respectively. The inhibitors in hemophilia A had reported in 6 papers. One paper had covered the inhibitors in hemophilia B. There were five papers on inhibitors in both hemophilia A and B. The comparison between the reported patients showed that 3020 patients with hemophilia A and 314 patients with hemophilia B had studied. CONCLUSION: Consistent with the lower frequency of hemophilia B and the lower development of inhibitors in hemophilia B compared to hemophilia A, it was concluded that hemophilia B had not neglected in Iran. It seems to be rational that each country, check rates of detection of inhibitors in hemophilia B to identify whether it has neglected or not.

Advances in gene therapy for hemophilia: basis, current status, and future perspectives.

Hemophilia is a congenital hemorrhagic disease caused by genetic abnormalities in coagulation factor VIII or factor IX. Current conventional therapy to prevent bleeding requires frequent intravenous injections of coagulation factor concentrates from early childhood. Accordingly, gene therapy for hemophilia remains an exciting future prospect for patients and their families, due to its potential to cure the disease through a one-time treatment. After a series of successes in basic research, recent clinical trials have demonstrated clear efficacy of gene therapy for hemophilia using adeno-associated virus (AAV) vectors. Although this is likely to alter the paradigm of hemophilia care in the near future, it will be important to overcome immune responses against AAV. Gene therapy for hemophilia cannot be given to patients with anti-AAV capsid-neutralizing antibodies, and cellular immunity with CD8+ T cells should be controlled for sustained expression. Furthermore, long-term therapeutic effects should be closely observed because of the failure of the AAV vector genome to replicate during cell division. This review focuses on the basis of gene therapy, current successes of clinical trials, and the future direction of hemophilia gene therapy.

Hemophilia A and B mice, but not VWF-/-mice, display bone defects in congenital development and remodeling after injury.

While joint damage is the primary co-morbidity of hemophilia, osteoporosis and osteopenia are also observed. Coagulation factor VIII deficient (FVIII-/-) mice develop an osteoporotic phenotype in the absence of induced hemarthrosis that is exacerbated two weeks after an induced joint injury. Here we have compared comprehensively the bone health of clotting factor VIII, factor IX, and Von Willebrand Factor knockout (FVIII-/-, FIX-/-, and VWF-/- respectively) mice both in the absence of joint hemorrhage and following induced joint injury. We found FVIII-/- and FIX-/- mice, but not VWF-/- mice, developmentally have an osteoporotic phenotype. Unilateral induced hemarthrosis causes further bone damage in both FVIII-/- and FIX-/- mice, but has little effect on VWF-/- bone health, indicating that the FVIII.VWF complex is not required for normal bone remodeling in vivo. To further investigate the bone healing following hemarthrosis in hemophilia we examined a two week time course using microCT, serum chemistry, and histological analysis. Elevated ratio of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL), increased osterix+ osteoblastic cells, and decreased smoothness of the cortical bone surface were evident within several days of injury, indicative of acute heterotopic mineralization along the cortical surface. This was closely followed by increased interleukin-6 (IL-6) levels, increased osteoclast numbers, and significant trabecular bone loss. Uncoupled and disorganized bone formation and resorption continued for the duration of the study resulting in significant deterioration of the joint. Further elucidation of the shared mechanisms underlying abnormal bone homeostasis in the absence of FVIII or FIX is needed to guide evidence-based approaches to the screening and treatment of the prevalent bone defects in hemophilia A and B.

Real-life experience in switching to new extended half-life products at European haemophilia centres.

The concept of replacement therapy in haemophilia is changing significantly thanks to the switch from standard products to extended half-life products. These novel drugs are showing beneficial effects overcoming current prophylaxis limitations by reducing the infusion frequency, maintaining a higher trough level to ensure a lower risk of bleeding, and making treatment significantly less distressing to patients by improving the quality of life. Real-life data on the efficacy of novel drugs and their impact on routine management of haemophilia A and B patients are still limited. This manuscript reports the results of a European survey conducted by the European Association for Haemophilia and Allied Disorders (EAHAD) at the beginning of 2018 on the clinical management of patients using extended half-life recombinant FVIII and FIX fusion products, since at the time of the survey none of the PEGylated products were available yet. We report data on the efficacy of these novel drugs by 33 European haemophilia centres that have already switched to extended half-life fusion products, showing a significant reduction in the number of infusions and a satisfactory trough levels in the clinical care of haemophilia patients, with a greater impact for haemophilia B.

Performance evaluation of Revohem™ FVIII chromogenic and Revohem™ FIX chromogenic in the CS-5100 autoanalyser.

INTRODUCTION: Chromogenic substrate assay (CSA) reagents Revohem™ FVIII and Revohem™ FIX are now available as in vitro diagnostic reagents for autoanalysers in Japan. In this study, we evaluated the performance of these reagents in the CS-5100 automated coagulation analyser. METHODS: We assessed within-run and between-day imprecision, on-board stability and frozen-storage stability of Revohem FVIII and FIX. Sensitivity to lupus anticoagulant (LA) was examined using LA-positive patient plasma. Correlations were analysed using plasma samples from normal individuals and patients with haemophilia A (HA) or B (HB) or von Willebrand disease (VWD). RESULTS: Imprecision was <2% for Revohem FVIII and <6.5% for Revohem FIX. On-board storage of Revohem FVIII resulted in a <10% decrease in FVIII levels from baseline at 24 hours, whereas Revohem FIX showed a >10% decrease at 8 hours. Revohem FVIII showed good stability while frozen for 22 days. Although Revohem FIX showed degradation due to freeze-thawing, a new calibration improved stability up to 22 days. Interference from LA was not observed with Revohem FVIII or FIX. The FVIII CSA-CSA correlation was excellent in normal (r = 0.9924), HA (r = 0.9945) and VWD (r = 0.9914). The FVIII CSA-OSA correlation was good in normal (r = 0.8468) and excellent in HA (r = 0.975) and VWD (r = 0.9936). The FIX CSA-OSA correlation was fair in normal (r = 0.4791) and excellent in HB (r = 0.9501). CONCLUSION: Revohem FVIII and FIX both showed excellent performance in the CS-5100 analyser. These reagents could be useful in routine laboratory testing for diagnosing and treating haemophilia.

Genetic-Based Approaches to Inherited Metabolic Liver Diseases.

In vertebrates, the liver is the central metabolic organ of the body, which carries out an estimated 500 functions that range from general detoxification to protein synthesis, bile production, metabolism of fats, carbohydrates, proteins, bilirubin, vitamin and mineral storage and it even has an immune function. Hepatocytes are considered the professional liver cells, which carry out all of these functions. With such a variety of tasks to perform, it is not surprising that more than 400 rare monogenic disorders of hepatic origin have been described. For many of these, liver transplantation remains the only curative strategy, however, this is limited by organ availability and requires lifelong immune suppression. The fact that liver transplantation is curative led to the assumption that the restoration of the expression of the defective gene would result in the resolution of the disease. Indeed, liver-directed gene therapy trials for hemophilia A and B have demonstrated the potential of gene therapy to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Thus, liver-directed gene therapy and gene editing strategies have emerged as promising alternatives to transplantation in inherited monogenic liver disorders. Herein, we review the advances and limitations of gene therapy for such disorders, covering therapeutic strategies based on gene addition and gene editing and the exciting clinical results obtained with the use of ribonucleic acid as therapeutic molecules.

Phenotypic characterization of haemophilia B - Understanding the underlying biology of coagulation factor IX.

Haemophilia B is a recessive, X-linked bleeding disorder due to inherited deficiency in vitamin K-dependent coagulation factor IX (FIX). FIX activity levels, as a basis for the definition of disease severity, do not clearly correlate with bleeding phenotype, likely due to the multiple steps regulating coagulation. Timely, with the availability of extended half-life products and successful steps in gene therapy, haemophilia B therapy is in an active developmental phase. Therefore, increased knowledge of the factors contributing to the variation of haemostatic and clinical outcome and response to therapy is welcomed. FIX acts at the crossroads of both the extrinsic and intrinsic pathways, and on the platelet procoagulant membrane at the site of vascular injury, and therefore, FIX biology is targeted for multiple effectors and regulators. The synthesis, cellular and molecular interactions, and elimination routes of FIX are not as well studied as for FVIII. The specific roles of magnesium in both platelet adhesion and FIX activation, and of vascular collagen at the haemostatic site of platelet adhesion and FIX residence are of particular interest. Biochemical and translational research on these issues should improve our understanding of the mechanisms involved, leading to the development of relevant assays that measure both haemostasis and treatment response. The latter is becoming increasingly important in the new era of haemophilia management and ultimately may lead to improved treatment strategies individually tailored to a patient's needs and cost-efficiency.

Prophylactic administration of glycoPEGylated factor IX provides protection and joint outcome superior to recombinant factor IX after induced joint bleeding.

BACKGROUND: Following induced joint hemorrhage, hemophilia B results in the abnormal persistence of iron deposition, inflammation, and neovascularity of the synovial tissue, as well as deterioration of the bone articular surface and strength. Previously, we demonstrated that a factor IX (FIX) replacement protein with extended circulating FIX activity, glycoPEGylated FIX nonacog beta pegol (N9-GP), could improve synovial and osteochondral parameters in F9 knockout mice when administered after joint injury. OBJECTIVE: We explored the use of N9-GP prior to unilateral joint hemorrhage and compared to unmodified recombinant FIX (rFIX). METHODS: Pharmacodynamics, histology, and microcomputed tomography were used to assess the effects of prophylactic administration of glycoPEGylated FIX. RESULTS: In comparison to rFIX, N9-GP significantly improved soft tissue histological parameters, as well as bone outcome at 2 weeks post injury, while performing equally in reduction of blood present in the joint space assessed 1 day after injury. CONCLUSIONS: These results indicate that, in comparison to rFIX, the prophylactic use of extended half-life FIX provides superior protection from bleeding-induced joint damage, manifested by improved correction of histologic parameters.

Regional variation and cost implications of prescribed extended half-life factor concentrates among U.S. Haemophilia Treatment Centres for patients with moderate and severe haemophilia.

BACKGROUND: Extended half-life (EHL) factor VIII (FVIII) and IX (FIX) products are intended to decrease the burden of prophylaxis for patients with haemophilia A or B. Whether these newer concentrates have led to meaningful clinical practice change remains vague. AIM: To characterize the longitudinal use of standard (SHL) and EHL factor concentrates at haemophilia treatment centres (HTCs), using the ATHNdataset, a US database of 138 ATHN-affiliated HTCs. METHODS: Factor concentrate use among moderate and severe haemophilia A and B patients without inhibitors was analysed at three time points over 18 months. RESULTS: Use of EHL concentrates rose from 10% of patients to 22% during this study. EHL FVIII prophylaxis is prescribed to the minority of patients, 28%; EHL FIX now predominates for prophylaxis, 52%. Rates of prescribed EHL products varied significantly by age group and HTC region. Median prescribed prophylaxis for SHL compared to EHL products was FVIII 6240 and 5200 and FIX 6968 and FIX 3900 IU/kg/y, respectively. On-demand EHL use has grown but has minimal contribution to overall usage (2%). CONCLUSION: Haemophilia treatment centre region and patient age impact the rate of adoption of EHL products; however, EHL prescribing continues to rise nationally, particularly for EHL FIX. Careful attention to annual cost of prophylaxis is imperative as the decrease in median EHL prophylaxis consumption is not offset by the higher unit cost of these products. It is unclear how further growth in use of EHLs will be impacted by emerging non-factor replacement and gene therapies.

Increased bone resorption in hemophilia.

In patients with hemophilia, osteoporosis is frequently observed for which the etiology remains unclear. The aim of this paper is to review the available experimental evidence indicating the presence of this disorder in patients with hemophilia, explore the potential mechanisms which may lead to reduced bone mineral density (BMD) and speculate on useful interventions to circumvent it. A narrative review of the English literature up to April 2018 was performed. The available evidence demonstrates an increased rate of bone resorption and an excess of osteoporosis among patients with hemophilia. FVIII and FIX may act through at least two pathways: promoting bone formation by a thrombin-mediated mitogenic effect on osteoblasts and by cytokine-mediated osteoclast activity. Another potential indirect mechanism mediated through the RANK-RANKL pathway has been suggested but remains controversial. The role of confounders such as lack of activity and immobility must be considered.