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GSK responds quickly to query Avandia link to osteoporosis
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04 December 2007
GlaxoSmithKline has once again jumped to the defence of Avandia, this time in response to a study which claims that treatment with the controversial diabetes drug can lead to an increase in the risk of bone fractures and osteoporosis.
A paper published online in Nature Medicine (see article excerpt below), involved a study conducted in mice by a team of researchers at the Salk Institute for Biological Studies in La Jolla, California and its senior author Ronald Evans, claimed that long-term Avandia (rosiglitazone) usage in the treatment of type 2 diabetes "may cause osteoporosis due to both increased bone resorption and decreased bone formation".
However, Prof Evans added that "because Avandia is effective in controlling glucose and restoring the body's sensitivity to insulin, we do not recommend that people stop their treatment. You must balance the benefits against the complications."
Nothing new in findings
GSK moved swiftly in response, saying that the study "adds further context on the possible involvement of thiazolidinediones, such as Avandia and Takeda's Actos (pioglitazone), in osteoclast activity". However, its findings would appear to present nothing new.
The UK-based drugs major said that the TZD class of medicines "has already been widely studied in relation to bone fracture and fractures have been previously reported in female patients taking this class of drugs". Furthermore, there is a precaution for fracture on the labels of both Avandia and Actos and "this information has been widely communicated for both products through dear healthcare professional letters in Europe and the USA", GSK noted.
The company also noted that it evaluated the rates of bone fractures in a post hoc analysis of data from its massive ADOPT trial which showed that more women with bone fractures at peripheral sites (foot, hand and upper arm) were observed in the Avandia group versus those in the metformin or glyburide groups. However, "these peripheral fractures are not typical of those associated with osteoporosis", it added.
Furthermore, prior to this aforementioned analysis, there were no findings in the rosiglitazone clinical trial database to suggest that people with type 2 diabetes taking the drug were at increased risk for fractures. In addition, GSK claimed that the evidence to date from ADOPT shows no increase in spine or hip fractures, which are typically associated with osteoporosis, in patients treated with Avandia.
The company concluded by saying that it has "a comprehensive pre-clinical and clinical programme to better understand the underlying mechanism of the fractures" and the European Medicines Agency has agreed that the project is adequate.
This latest development comes just after the US Food and Drug Administration said that Avandia needs to carry a black box warning that will carry additional information stating that it may potentially increase the risk for heart attacks. The treatment has suffered a torrid time at the hands of its critics ever since a meta-analysis in May that was published in the New England Journal of Medicine raised concerns about cardiovascular risk and sales of the drug have suffered ever since.
By Kevin Grogan
Takeda's Actos linked to increased risk of fracture for women
12 March 2007
The US Food and Drug Administration and Japan's Takeda Pharmaceutical Co have informed doctors of an increased risk of fractures in women associated with the firm's diabetes drug Actos.
The warning comes after an analysis of the drugmaker's clinical trial database for Actos (pioglitazone) which showed more reports of fractures in female patients taking the treatment than those taking a comparator (either placebo or active).
The analysis included more than 8,100 patients taking Actos, and over 7,400 patients taking a placebo or alternative drug, the FDA said, noting that the majority of fractures were in the forearm, hand and wrist, foot, ankle, fibula and tibia. The patients were followed for up to three-and-a-half years and Takeda noted that the observed excess risk of fractures for women in this data set on pioglitazone is 0.8 fractures per 100 patient years of use.
In a letter to physicians, Dr Robert Spanheimer of Takeda wrote that the reason for the finding was unknown, but noted that "none of the pioglitazone studies in the database addressed, or were designed to study, the effect on bone, but fractures were collected as adverse events." Because of the limitations of the data available, he said multiple risk factors for fractures cannot be excluded as confounding variables but "further evaluation of these findings is ongoing," adding that there was no increased risk of fracture identified in men.
Takeda and the FDA said the risk of fracture should be considered before initiating pioglitazone therapy in women with type 2 diabetes and the company is working with the agency to sort out the correct wording to the label regarding the aforementioned risk.
The news comes less than a month after GlaxoSmithKline alerted doctors that long-term use of its diabetes drug Avandia (rosiglitazone), another thiazolidinedione like Actos, has been linked to an increased incidence of fractures in women. GSK informed the FDA of its findings after a safety review of the data from a large-scale clinical study of over 4,000 patients with type 2 diabetes.
Actos sales in the USA for the nine months ended December 31, 2006, reached 209 billion yen (about $1.77 billion), while Avandia brought in 324 million into GSK's coffers in the fourth quarter but the drugs are now facing competition from a newer class of diabetes products, the dipeptidyl peptidase-4 inhibitors, headed by Merck & Co's Januvia (sitagliptin).
Diabetes drug tied to bone loss
Randolph E. Schmid, The Associated Press
WASHINGTON - GlaxoSmithKline, which has a U.S. headquarters in Research Triangle Park, has received more potentially bad news about its popular diabetes drug, Avandia.
New research raises the possibility that long-term treatment with rosiglitazone, as Avandia is also called, could lead to osteoporosis.
While bones seem solid, they are constantly being broken down and rebuilt by the body. Researchers found that in mice, the drug increased the activity of cells that degrade bones, according to a study in this week's online issue of Nature Medicine.
An earlier study found a higher risk of fractures among women who take the drug. But this report is the first to attempt to explain the link between the drug and fractures.
Sales of the pill, GSK's second-best seller, were already expected to be down by more than $1 billion this year because the drug was linked months ago to a higher risk of heart attack.
GSK agreed to add a warning about heart risks to packages of Avandia, and to highlight it with a black box.
The recent finding "has led to a better understanding of the challenges associated with long-term treatment of patients with Type 2 diabetes," said Ronald M. Evans of the Salk Institute for Biological Studies in La Jolla, Calif., lead author of the report.
The News & Observer's attempts to reach GSK officials Sunday were unsuccessful.
Nearly 21 million people in the United States have diabetes. Avandia is widely used in people with Type 2, or adult onset, diabetes, the most common form of the disease.
Evans said the researchers were looking at different aspects of the diabetic mice and did not realize it would be possible to change the bone-removing activity.
The assumption had been that more brittle bones in diabetics were the result of a reduced bone-building activity, not increased bone removal.
"Considering the widespread use of these drugs and the known action in people, it is surprising that such a key observation had been missed," Evans said, adding that caution should be used with long-term treatment of patients with higher risks of fractures.
Using Avandia in combination with osteoporosis drugs could be beneficial, Evans said.
The research was funded by the Howard Hughes Medical Institute and the National Institutes of Health.
Seven GSK medicines have run into regulatory difficulties or delays this year.
Company spokeswoman Mary Anne Rhynes said last week that the regulatory setbacks are "a reflection of a more conservative FDA" and that the entire pharmaceutical industry is struggling with heightened regulatory scrutiny.
The FDA has increasingly zeroed in on drug safety since Merck pulled the painkiller Vioxx three years ago when a study suggested that Vioxx doubled the risk of heart attack.
(Staff writer Sabine Vollmer contributed to this report.)
Letter
Nature Medicine
Published online: 2 December 2007 | doi:10.1038/nm1672
PPAR- regulates osteoclastogenesis in mice
Yihong Wan1, Ling-Wa Chong1 & Ronald M Evans1
Abstract
Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Regulation of osteoclast function is central to the understanding of bone diseases such as osteoporosis, rheumatoid arthritis and osteopetrosis1. Although peroxisome proliferator-activated receptor-y (PPAR-y) has been shown to inhibit osteoblast differentiation2, 3, its role, if any, in osteoclasts is unknown. This is a clinically crucial question because PPAR-y agonists, "such as thiazolidinediones-" a class of insulin-sensitizing drugs, have been reported to cause a higher rate of fractures in human patients4, 5. Here we have uncovered a pro-osteoclastogenic effect of PPAR-y by using a Tie2Cre/flox mouse model in which PPAR-y is deleted in osteoclasts but not in osteoblasts. These mice develop osteopetrosis characterized by increased bone mass, reduced medullary cavity space and extramedullary hematopoiesis in the spleen. These defects are the result of impaired osteoclast differentiation and compromised receptor activator of nuclear factor-kB ligand signaling and can be rescued by bone marrow transplantation. Moreover, ligand activation of PPAR- by rosiglitazone exacerbates osteoclast differentiation in a receptor-dependent manner. Our examination of the underlying mechanisms suggested that PPAR-y functions as a direct regulator of c-fos expression, an essential mediator of osteoclastogenesis6. Therefore, PPAR-y and its ligands have a previously unrecognized role in promoting osteoclast differentiation and bone resorption.
Bone is a dynamic tissue that undergoes constant remodeling, balancing bone formation by osteoblasts and bone resorption by osteoclasts. Osteoclasts are multinucleated cells of hematopoietic lineage; in contrast, osteoblasts are of mesenchymal origin. Defects in osteoclastic bone resorption cause osteopetrosis, a disease associated with an increased skeletal mass and abnormally dense bone. In severely affected individuals, the medullary cavity is filled with endochondral new bone, leaving little space for hematopoietic cells. This results in extramedullary hematopoiesis, which can be reversed by hematopoietic stem cell transplantation1.
The nuclear receptor PPAR-y is an activator of adipogenesis7, 8, 9 and a repressor of osteoblastogenesis2, 3. However, the specific role of PPAR-y in osteoclast function has not been fully explored. PPAR-y agonists, such as thiazolidinediones (TZDs), have been shown to cause bone loss in both mice and rats10, 11, 12, 13, in part owing to increased bone resorption. In light of the increased rate of fractures reported in diabetic individuals treated with TZDs in clinical studies including the recent A Diabetes Outcome Progression Trial (ADOPT)4, 5, the potential role of PPAR-y in osteoclast function and bone resorption remains a clinically important issue.
To achieve a genetic separation of the effect of PPAR-y deletion on the hematopoietic lineages from the one it has on the mesenchymal lineages, we used Tie2Cre mice14 and homozygous PPAR-y flox (Ppargflox/flox, or gf/f) mice15 to specifically delete the PPAR-y gene in hematopoietic and endothelial cells via excision of the loxP-flanked (floxed) Pparg alleles16, 17. Tie2 is expressed in hematopoietic cells as early as 9.5 d postcoitum18. PCR analysis of genomic DNA showed that the floxed allele was efficiently deleted by Tie2Cre in all hematopoietic tissues, including the bone marrow, spleen, thymus and lymph node (Fig. 1a and Supplementary Fig. 1d online), but not in mesenchymal lineages, such as white adipose tissue. Consistent with these results, in the Tie2Cre/ROSA26-GFP Cre expression reporter mice, the flox deletion was virtually complete in each hematopoietic tissue, as evidenced by the shift of the entire FACS peak to GFP+ (Supplementary Fig. 1a). In addition, western blot analysis confirmed the reduction in PPAR-y protein abundance in both the bone marrow and the spleen in gf/f-Tie2Cre mutants (Supplementary Fig. 1b). Furthermore, quantitative RT-PCR analysis of individual cell types demonstrated that PPAR-y RNA expression was absent in osteoclasts and monocyte precursors, but remained normal in osteoblasts and adipocytes in the gf/f-Tie2Cre mutants (Supplementary Fig. 1c). Together, the genomic DNA, RNA, protein and FACS analyses show that PPAR-y expression was efficiently eliminated in the hematopoietic lineages but was unaltered in the mesenchymal lineages in gf/f-Tie2Cre mice.
An examination of the gf/f-Tie2Cre mice revealed splenomegaly (Fig. 1b) and pale bones (Fig. 1c). The spleen-to-body weight ratio was increased by 1.4- to 2-fold in adult mice, as well as in 18- and 5-d-old pups (Fig. 1d). There was a pronounced accumulation of megakarocytes in the spleen, indicative of extramedullary hematopoiesis (Fig. 1b). Quantification of the nucleated cells from bone marrow and spleen indicated that the cellularity was decreased by 35% in bone marrow but increased by 63% in spleen (Fig. 1e). At the clonogenic progenitor cell level, both erythroid and granulocytic/monocytic progenitors were less abundant in bone marrow and more abundant in spleen (Fig. 1e). At the hematopoietic stem cell (HSC) level, the percentage of the Lin-cKit+Sca1+ stem cell population was reduced by 58% in bone marrow but elevated by 275% in spleen (Fig. 1e). When the changes in total cell numbers were taken into account, the absolute numbers of HSCs were decreased by 73% in bone marrow but increased by 511% in spleen. In addition, the expression of several transcription factors crucial for HSC differentiation and/or self-renewal19 was also shifted from bone marrow to spleen (Fig. 1f). In total, these results show the presence of extramedullary hematopoiesis in the spleen of gf/f-Tie2Cre mice at both cellular and transcriptional levels. Notably, however, there were no significant differences in white blood cell (WBC), red blood cell and platelet counts (Fig. 1g) or in WBC differential count (Fig. 1h), suggesting that hematopoiesis was normal other than having shifted from bone to spleen.
Adult hematopoiesis mainly occurs in the bone marrow and is regulated by the osseous environment1, 20. Thus, we next examined bone volume and mineral density using microcomputed tomography (uCT) in vivo imaging (Fig. 2a). The bone volume fraction (BVF, or bone volume/tissue volume) was 40-70% higher in both the proximal and the distal regions of femur and tibia in the mutants (Fig. 2a), indicating a markedly increased bone volume with a reduced medullary cavity space. A similar increase in bone mineral density (BMD; 30-70%) was also observed (Fig. 2a). Consistently with this, static bone histomorphometry showed increased trabecular volume, thickness and number, with a decreased trabecular separation (Fig. 2b). Whole body X-ray analysis showed increased radio density in the skull, vertebrae and long bones (Fig. 2c). Furthermore, histological analyses confirmed the increased bone thickness and reduced marrow space (Fig. 2c). Moreover, in the wild-type (WT) controls, the bone had a uniform layered structure with parallel collagen fibers and smooth endosteal surface, typical of a normal lamellar bone. In contrast, in the mutants, the bone structure appeared irregular and the endosteal surface was uneven, reminiscent of woven bone (Fig. 2c). These results show that Tie2Cre-mediated PPAR-y deletion leads to increased bone mass, reduced medullary cavity space and altered bone remodeling, which is manifested as pale bones (Fig. 1c).
Because PPAR-y was deleted in the hematopoietic but not the mesenchymal lineages, this phenotype is a probable result of osteoclastic rather than osteoblastic defects. Indeed, tartrate-resistant acid phosphatase (TRAP) staining of femur sections revealed that the numbers of mature osteoclasts were lower in the mutants both in the trabecular bone and along the cortical bone shaft, whereas the numbers of alkaline phosphatase+ osteoblasts were unaffected (Fig. 2d-f). Consistently with this, dynamic histomorphometry by calcein double labeling showed that the bone formation rate and mineral apposition rate were unchanged (Fig. 2g). Furthermore, urine deoxypyridinoline levels were significantly lower in the mutants (Fig. 2h), suggesting decreased bone resorption; in contrast, serum alkaline phosphatase and osteocalcin levels (Fig. 2h) were normal, suggesting unaffected bone formation. The decrease in urine deoxypyridinoline at this time point assessed was 24%, but the cumulative effect of a continued decrease in bone resorption on bone volume and remodeling over time may be more profound. These results indicate that the osteopetrosis observed in the mutants was mainly due to decreased osteoclast number and bone resorption.
We next assessed the effect of PPAR-y deletion on receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation from spleen progenitor cells in an in vitro culture system21. After 6 d, many mature osteoclasts, represented by multinucleated TRAP+ cells, developed in the WT cultures (Fig. 3a). In contrast, multinucleated or TRAP+ cells were rarely observed in the mutant cultures (Fig. 3a). Because the numbers of stem and progenitor cells were actually higher in the mutant spleen (Fig. 1), the decreased osteoclast number was due not to a lack of progenitor cells but rather to intrinsic defects in the RANKL-mediated osteoclast differentiation. In addition, treatment with a PPAR- agonist, BRL 49653 (BRL, also known as rosiglitazone), stimulated osteoclast differentiation in WT cells, but not in mutant cells (Fig. 3a), indicating that the effect was PPAR-y-dependent. These results show a pro-osteoclastogenic role for PPAR-y and its ligand.
Mature osteoclasts are characterized by the expression of several genes that are crucial for extracellular matrix degradation and bone resorption22. Their gene products include TRAP, calcitonin receptor, carbonic anhydrase-2, cathepsin K and matrix metalloproteinase-9. In the RANKL-treated, spleen-derived macrophages, the expression of these genes was reduced by 35-80% in the mutants (Fig. 3b) and was induced by BRL only in the WT cells. These results confirm that PPAR-y and its ligand stimulate osteoclast differentiation at the transcriptional level. Similar and often more noticeable results were observed in bone marrow-osteoclast differentiation cultures (Supplementary Fig. 2 online).
Binding of RANKL to its receptor RANK triggers intricate and distinct signaling cascades that control lineage commitment and osteoclast activation23, 24. c-fos is an important mediator of osteoclastogenesis, and mice lacking c-fos develop osteopetrosis as a result of a block in osteoclast differentiation6. RANKL induces c-fos expression, which is required for the induction of nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent-1 (NFATc1) and fos-like antigen-1 (refs. 24,25). The expression of all three transcription factors was substantially reduced in mutant cells and was induced by BRL only in WT cells (Fig. 3b). In contrast, mature macrophage-related inflammatory genes such as monocyte chemoattractant protein-1 and tumor necrosis factor-a had a reciprocal pattern, with increased expression in the mutant cells and suppression by BRL in WT cells (Fig. 3b), which is consistent with the previously reported anti-inflammatory role of PPAR-y (ref. 26). Moreover, the expression of several genes that are common to macrophages and osteoclasts, including transcription factor PU.1, receptors MCSFR and RANK, and adaptor protein TRAF6, was unaffected by PPAR- deletion (Supplementary Fig. 3 online). These results suggest that the stimulatory effect of PPAR-y and its ligand is specific to osteoclast genes. The pro-osteoclastogenic role of PPAR-y was further illustrated by the observation that two PPAR-y antagonists, GW9662 (ref. 27) and T0070907 (ref. 28), inhibited osteoclast gene expression in a receptor-dependent manner by 40% and 70%, respectively (Fig. 3c).
Examination of gene expression during a time course after RANKL treatment showed that the induction of c-fos (encoded by Fos) and its target genes was highly impaired in the mutants (Fig. 3d). In contrast, RANKL-induced phosphorylation of c-Jun and degradation of IkBa, two other downstream signaling events, were intact (Fig. 3e). This suggests that PPAR-y deficiency selectively blocks the c-fos arm of the RANKL signaling pathways. Indeed, in a transient transfection assay in RAW264.7 cells, transcription from a 1.1-kilobase mouse c-fos promoter was upregulated with increasing amounts of PPAR-y and its heterodimer partner RXR-a, and it was further induced by BRL only in the presence of these receptors (Fig. 3f). Using both luciferase reporter assays of truncated c-fos promoters (Fig. 3f) and electrophoretic mobility shift assays (Supplementary Fig. 4 online), we identified two conserved PPAR- response elements (PPREs) in the c-fos promoter (Fig. 3f). The ligand-dependent binding of PPAR-y to these two PPREs in vivo was shown by chromatin immunoprecipitation (ChIP) assays with bone marrow-differentiated monocytes and macrophages (Fig. 3g). Notably, PPAR-y regulation of c-fos occurred both before (time 0) and after RANKL stimulation (Fig. 3d), suggesting that PPAR-y promotes both osteoclast lineage commitment and osteoclast maturation by maintaining the levels of c-fos in monocyte precursors and osteoclasts. In contrast with these results, osteoblast differentiation from bone marrow cells was unaffected, as evidenced by the unaltered expression of the regulators of osteoblastogenesis20 (Supplementary Fig. 5a online). Moreover, BRL inhibited osteoblast differentiation in cells from both WT and mutant mice, confirming that PPAR-y was not deleted in osteoblasts (Supplementary Fig. 5b). Together, these results show that PPAR- and its ligand promote osteoclast differentiation by directly regulating c-fos expression (Fig. 3h).
We next evaluated whether ectopic expression of c-fos can rescue the osteoclast differentiation blockade in the PPAR-y-deleted cells by using retrovirus-mediated gene transfer. The number of osteoclasts differentiated from mutant cells infected with c-fos virus was increased by fivefold compared to cells infected with control virus (Fig. 4a). Consistently with these results, quantitative RT-PCR analyses showed that c-fos overexpression effectively rescued the RANKL-mediated induction of osteoclast genes in the mutant cells without altering the expression of RANK (Fig. 4b). Moreover, BRL had no effect on the osteoclast gene expression rescued by the c-fos virus in the mutant cells (Fig. 4b). These results further demonstrate that PPAR- deficiency selectively blocks the c-fos arm of the RANKL signaling pathways.
We next tested whether the extramedullary hematopoiesis phenotype could be reversed by bone marrow transplantation. We used ubiquitin-GFP (Ub-GFP) transgenic bone marrow to monitor the reconstitution efficiency. First, we transplanted WT or mutant bone marrow into irradiated WT recipient mice (Fig. 4c). The mutant bone marrow recipients developed extramedullary hematopoiesis, as evidenced by increases in both spleen-to-body weight ratio and spleen cell number and a decrease in bone marrow cell number compared to the WT bone marrow recipients (Fig. 4c). The reverse transplantation, in which WT bone marrow was transplanted into mutant or WT recipients, rescued the extramedullary hematopoiesis, as evidenced by the comparable spleen weight and cell numbers between the two groups (Fig. 4d). FACS analyses showed that 94-96% of the blood cells were GFP+ and thus donor derived (Fig. 4c,d). Phenotype reversal by bone marrow transplantation further suggests that the osteopetrotic defect originates from the hematopoietic, rather than the mesenchymal, lineage.
Finally, to specifically address the predicted effects of gain of PPAR-y function on osteoclasts and bone resorption in vivo, we treated WT or gf/f-Tie2Cre mice with BRL at 10 mg/kg/d for 6 weeks (Supplementary Fig. 6 online). BRL treatment led to a significant increase in both deoxypyridinoline bone resorption marker levels (Supplementary Fig. 6a) and osteoclast numbers (Supplementary Fig. 6d) in WT but not in mutant mice. These results suggest that PPAR-y activation in vivo promotes osteoclast-mediated bone resorption in a receptor-dependent manner. Consistently with previous studies2, 10, 29, BRL also significantly decreased the abundance of the osteocalcin bone formation marker (Supplementary Fig. 6b), as well as osteoblast numbers (Supplementary Fig. 6e), in both WT and mutant mice. Consequently, BRL-mediated bone loss was partially alleviated in the mutants (15%) compared to WT controls (35%) (Supplementary Fig. 6c). This evidence suggests that, in addition to the established notion that TZDs can inhibit osteoblast differentiation and bone formation, stimulation of osteoclast differentiation and bone resorption may be a previously unrecognized pathway that further contributes to TZD-mediated bone loss (Supplementary Discussion and Supplementary Fig. 7 online).
In summary, this study reveals an unexpected role for PPAR- and its ligand in promoting osteoclast differentiation and bone resorption. Loss of function by targeted PPAR- deletion impairs osteoclast differentiation and bone resorption, resulting in osteopetrosis and extramedullary hematopoiesis. In contrast, gain of function by ligand activation of PPAR- accelerates osteoclast differentiation and bone resorption in a receptor-dependent manner. These findings have potential clinical implications, as they suggest that long-term rosiglitazone usage in the treatment of type 2 diabetes and insulin resistance may cause osteoporosis, owing to a combination of decreased bone formation and increased bone resorption. They also suggest that selective PPAR-y modulators may provide a new strategy for the treatment of bone diseases associated with increased osteoclast activity, such as osteoporosis and rheumatoid arthritis.
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