Jean-Luc Faillie
email: jl-faillie@chu-montpellier.fr

Pierre Petit

Jean-Louis Montastruc

Dominique Hillaire-Buys

J-L Faillie

J-L Montastruc

Dominique Hillaire

Jean-Luc Faillie

Jean-Louis Montastruc

Jean-Luc Faillie

Scientific evidence and controversies about pioglitazone and bladder cancer: which lessons can be drawn?

Pioglitazone, a peroxisome proliferator-activated receptors (PPAR) agonist has been authorized for the management of type 2 diabetes in 1999 in the US and in 2000 in Europe. Since, the risk of bladder cancer associated with pioglitazone use has been a serious concern. Following a warning from the French medicines agency, pioglitazone was suspended in France and Germany in June 2011. Elsewhere, restrictions of prescription were implemented, though, for both the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA), the risk-benefit ratio remains favorable. Since the development of pioglitazone, its risk assessment has suffered from several inaccuracies such as the alleged specificity to male rat, the untrustworthy selective agonism for PPAR  and the mistaken risk evaluation in the large PROactive trial (PROspective pioglitAzone Clinical Trial In macroVascular Events): one case showing a benign tumor was counted as a cancer case in the placebo group. It took until 2011 for epidemiological evidences to be sufficiently numerous and conclusive to initiate application of safety measures. Today, the increased risk of bladder cancer associated with pioglitazone seems to be real but the absolute risk is relatively low. However, in a context of weak efficacy in an extensive population of patients exposed, risk-benefit balance is now difficult to assess and restricted prescription do not ensure safety. For future risk management, the authors propose several suggestions that involve an increasing role of health authorities and academic organizations.

Background

Since the 1990s, the peroxisome proliferator-activated receptors (PPARs) are thought to play an important role in metabolic diseases such as obesity, insulin resistance, and coronary artery disease. Three subtypes of PPAR nuclear receptors have been described: PPARα, PPARδ/β, and PPARγ. PPARα is found in liver, muscle, kidney, and heart. Its role is to increase free fatty acid oxidation, to regulate lipoprotein concentrations and to provide antiinflammatory effects. The fibrates belong to the class of PPARα agonists. PPARδ/β is expressed in many tissues but markedly in brain, adipose tissue, and skin. PPARγ has high expression in fat, low expression in liver and muscle. Thiazolidinediones (TZD), also called "glitazones", are said to be synthetic ligands of PPARγ. By activating a number of genes in tissues, PPARγ agonists increase glucose and lipid uptake, increase glucose oxidation, decrease free fatty acid concentration, and decrease insulin resistance [1] . Three glitazones have been marketed for the management of type 2 diabetes mellitus: troglitazone, rosiglitazone and pioglitazone. The first to be marketed, troglitazone was withdrawn in 2000 for hepatotoxicity. [2] Rosiglitazone, authorized in 2000, was also withdrawn from European market in 2010 following evidence of an association with an increased cardiovascular risk in diabetic patients. [START_REF]European Medicines Agency recommends suspension of Avandia, Avandamet and Avaglim[END_REF] Pioglitazone has been authorized for type 2 diabetes in 1999 in the US [START_REF]Drug Products: ACTOS (pioglitazone hydrochloride)[END_REF] and in 2000 in Europe [START_REF]European public assessment reports[END_REF] . In 2011, exposure to pioglitazone was estimated at more than 20 million patient-years in the world. [6] In developed countries, bladder cancer is the 8 th more frequent cancer (4.1% of all cancers) with an age-standardized incidence rate of 9.1 per 100,000 persons per year. [START_REF][END_REF] Risk factors for bladder cancer include white ethnicity, male gender, age, personal or family history of bladder cancer, cigarette smoking, bladder birth defects, occupational exposure to aromatic amines and polycyclic aromatic hydrocarbons, drugs (e.g., cyclophosphamide), urinary schistosomiasis or pelvic radiation therapy (effects of low fluid consumption, common urinary tract infections or lithiasis have also been suggested but are still controversial). In 2011, regulatory decisions have been taken with respect to the potential risk of bladder cancer associated with pioglitazone use. Following a warning from the French medicines agency, pioglitazone was suspended in France and Germany in June 2011. [START_REF]Use of Medications Containing Pioglitazone (Actos®, Competact®) Suspended[END_REF][START_REF]Tandemact®): Increased incidence of bladder cancer[END_REF] Product information and conditions of prescription have been updated in the US and in Europe in order to take into account this potential association, though, for both the European Medicines Agency (EMA) and the Food and Drug Administration (FDA), the risk-benefit ratio remains favorable. Recently published data make it possible to draw reliable conclusions regarding the existence of bladder cancer risk associated with pioglitazone use. In order to draw lessons from this controversy, we review here the available scientific evidence, from preclinical studies to post-marketing pharmacoepidemiological studies and we discuss the regulatory management of this risk.

Biologic plausibility 2.1. Bladder cancer in rats and the crystalluria hypothesis

First label information for pioglitazone stated that preclinical studies reported occurrence of bladder cancer in male rats treated with pioglitazone at dose equivalent to recommended human oral dose. [START_REF] Actos Label Information | [END_REF] In 2005, a review of a two-year rodent carcinogenicity study for 11 PPAR agonists, including pioglitazone, determined that these compounds were multispecies, multi-sex and multi-site carcinogens. [START_REF] El-Hage | Rodent carcinogenicity findings associated with PPAR agonist and their regulatory recommendations[END_REF][START_REF] Aoki | Current Status of Carcinogenicity Assessment of Peroxisome Proliferator-Activated Receptor Agonists by the US FDA and a Mode-of-Action Approach to the Carcinogenic Potential[END_REF] Tumor types observed included transitional cell carcinomas of urothelium in rats of all strains. Regarding pioglitazone, the rationale involved the acidic urinary environment of rats which predisposed them to the formation of urinary microcrystals causing chronic irritation of bladder. This so called "crystalluria hypothesis" explained the occurrence of tumors secondary to mucosal irritation caused by urinary microcrystals in the ventral part of the bladder. Such a phenomenon was considered specific to rat and unlikely transferable to humans. [START_REF] Cohen | Effects of PPARgamma and combined agonists on the urinary tract of rats and other species[END_REF][START_REF] Dominick | Urothelial carcinogenesis in the urinary bladder of male rats treated with muraglitazar, a PPAR alpha/gamma agonist: Evidence for urolithiasis as the inciting event in the mode of action[END_REF][START_REF] Suzuki | Effects of pioglitazone, a peroxisome proliferatoractivated receptor gamma agonist, on the urine and urothelium of the rat[END_REF][START_REF] Tseng | Peroxisome proliferator-activated receptor agonists and bladder cancer: lessons from animal studies[END_REF] To our knowledge, the presence of urothelial calculi in patients participating to subsequent clinical trials was whether not extensively explored and/or not reported. Later, conflicting evidence emerged. In 2008, Long et al. concluded that urolithiasis was not an inciting event for urinary bladder of rats treated with naveglitazar, an in vitro -dominant PPAR / agonist. [START_REF] Long | Urothelial carcinogenesis in the urinary bladder of rats treated with naveglitazar, a gamma-dominant PPAR alpha/gamma agonist: lack of evidence for urolithiasis as an inciting event[END_REF] In 2011, Sato et al. showed that, while acid-forming diet reduced both amount of calculi and number of bladder cancer in male rats, diet did not reduce the incidence of hyperplasia in animals treated by pioglitazone during up to two years. [START_REF] Sato | Suppressive effects of acid-forming diet against the tumorigenic potential of pioglitazone hydrochloride in the urinary bladder of male rats[END_REF]6] Contradicting the "crystalluria hypothesis", hyperplastic response in urinary bladder of rat was not necessarily associated with microcrystal formation. These data suggested that occurrence of bladder cancer may not be specific to urinary environment of rats and thus, that a risk for humans cannot be brushed aside.

In vitro antitumor effects?

Numerous in vitro studies have supported the hypothesis of an antitumor activity of PPARγ agonists such as growth inhibition, induction of apoptosis, and modulatory effect on the proliferation/differentiation balance in urothelial cells. [START_REF] Suzuki | Effects of pioglitazone, a peroxisome proliferatoractivated receptor gamma agonist, on the urine and urothelium of the rat[END_REF][START_REF] Chaffer | PPARgamma-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma[END_REF][START_REF] Nakashiro | Role of peroxisome proliferator-activated receptor gamma and its ligands in non-neoplastic and neoplastic human urothelial cells[END_REF][START_REF] Guan | Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in human transitional bladder cancer and its role in inducing cell death[END_REF][START_REF] Yoshimura | Expression of peroxisome proliferator-activated receptors (PPARs) in human urinary bladder carcinoma and growth inhibition by its agonists[END_REF][START_REF] Kawakami | PPARgamma ligands suppress proliferation of human urothelial basal cells in vitro[END_REF] These positive results were inconsistent with observations made in animals (and further in humans) and may consequently illustrate a complex mechanism which is difficult to elucidate. [START_REF] Tseng | Peroxisome proliferator-activated receptor agonists and bladder cancer: lessons from animal studies[END_REF] For instance, it was shown that the potential antiproliferative effect of pioglitazone was lower than that of an endogenous ligand of PPAR (15d-PGJ2) or troglitazone (a specific PPAR  agonist). [START_REF] Nakashiro | Role of peroxisome proliferator-activated receptor gamma and its ligands in non-neoplastic and neoplastic human urothelial cells[END_REF] Furthermore, the positive expression of a differentiation factor for urothelial carcinoma (A-FABP) was inversely correlated to pioglitazone concentration while that of rosiglitazone and 15d-PGJ2 remained stable. [START_REF] Boiteux | A-FABP, a candidate progression marker of human transitional cell carcinoma of the bladder, is differentially regulated by PPAR in urothelial cancer cells[END_REF] In these in vitro studies, the suspected antiproliferative (proapoptotic) effect was rather described to be only effective in low grade of differentiation, at high concentration [START_REF] Nakashiro | Role of peroxisome proliferator-activated receptor gamma and its ligands in non-neoplastic and neoplastic human urothelial cells[END_REF][START_REF] Kassouf | Inhibition of bladder tumor growth by 1,1-bis(3'indolyl)-1-(p-substitutedphenyl)methanes: a new class of peroxisome proliferator-activated receptor gamma agonists[END_REF][START_REF] Chopra | Structurally diverse peroxisome proliferator-activated receptor agonists induce apoptosis in human uro-epithelial cells by a receptor-independent mechanism involving store-operated calcium channels[END_REF] and through a mechanism independent of the PPAR agonism. [START_REF] Chaffer | PPARgamma-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma[END_REF][START_REF] Wei | PPARgamma-independent antitumor effects of thiazolidinediones[END_REF]

Promoter and/or inductor effect?

Occurrence of bladder cancer in animals which did not show a priori preclinical lesions nor exposure to additional specific risk factors, suggests a mechanism of tumoral induction by the studied drug. This is a strong argument for a causative role of pioglitazone. Another argument for inductor effect is that pioglitazone may induce chromosomal and oxidative DNA damage by generating reactive oxygen species which can be inhibited by vitamin B12. [START_REF] Alzoubi | Evaluation of vitamin B12 effects on DNA damage induced by pioglitazone[END_REF] However, a promoter effect can also be evoked for PPAR agonists when interaction with other known risk factors leads to a potentiation of tumorigenic effects. Several studies suggest that diabetes can be considered as an independent risk factor for bladder cancer. [29- 31] Thus, occurrence of bladder cancer within relatively short period of exposure to pioglitazone in diabetic patients (see clinical trials and epidemiological studies sections) could be explained by a potentiation with the diabetic condition itself. Other risk factors can also be involved; Lubet et al. showed that bladder cancer incidence in rats exposed to hydroxybutyl(butyl)nitrosamine (a urinary bladder specific carcinogen) was significantly doubled when rosiglitazone was added at a dose equivalent to a standard human dose. [START_REF] Lubet | Rosiglitazone, a PPAR gamma agonist: potent promoter of hydroxybutyl(butyl)nitrosamine-induced urinary bladder cancers[END_REF] Another study exhibited that mice exposed to cigarette smoke during 6 months associated with high dose of pioglitazone showed enhanced urothelial tumorogenicity as compared with mice only exposed to cigarette smoke. [START_REF] Maestra | DNA damage in exfoliated cells and histopathological alterations in the urinary tract of mice exposed to cigarette smoke and treated with chemopreventive agents[END_REF] Potentiation was also observed with pioglitazone in a murine model of non-small cell lung cancer in mice: cancer progression and metastasis were promoted. [START_REF] Li | Activation of PPARgamma in myeloid cells promotes lung cancer progression and metastasis[END_REF] These examples are in favor of a promoter effect, PPAR agonists accelerating a tumorigenic process already initiated but still asymptomatic. Thus, in clinical trials and epidemiological studies, it is important to detect and consider all the bladder cancer cases which have occurred, even with short duration of exposure. Moreover, another main pharmacological mechanism remains unexplored: the potential role of pioglitazone metabolites. Active metabolites (such as M-III and M-IV) show equal or greater pharmacological action than pioglitazone (up to three fold) with a longer plasma elimination half-life (three to four fold). [START_REF]Actos: Product Information as approved by the CHMP on 20 october 2011[END_REF] Pioglitazone is principally metabolized by cytochrome P450 2C8 and by other isoforms in a lesser degree. Enzymatic inductors such as rifampicin, increase the level of these metabolites and may cause their accumulation in urine. [START_REF]Actos: Product Information as approved by the CHMP on 20 october 2011[END_REF][START_REF] Jaakkola | Effect of rifampicin on the pharmacokinetics of pioglitazone[END_REF] We can though question the role of tobacco which is a well-known enzymatic inductor. Unfortunately, during development, the search for potential deleterious effects of pioglitazone metabolites was poorly investigated.

Pioglitazone: a glitazar more than a glitazone

Pioglitazone has always been described as a selective PPARγ agonist. In order to improve both glucose and lipid metabolic parameters, dual / PPAR agonists were developed; these compounds were named "glitazars". However, glitazars, such as muraglitazar, have shown to dose-dependently increase the incidence of bladder tumors in male rats. [START_REF] Dominick | Urothelial carcinogenesis in the urinary bladder of male rats treated with muraglitazar, a PPAR alpha/gamma agonist: Evidence for urolithiasis as the inciting event in the mode of action[END_REF] Between 2004 and 2006, all the dual / PPAR agonists have been discontinued due to safety concerns, including occurrence of bladder tumors in rodents. [START_REF] Conlon | Goodbye glitazars?[END_REF] After careful consideration of pharmacodynamic data published after marketing, pioglitazone cannot be considered as a selective PPARγ agonist but in fact exhibits a pharmacological profile comparable to that of glitazars: a dual / PPAR activity at concentrations corresponding to human use. [START_REF] Hillaire-Buys | Stay vigilant: a glitazone (pioglitazone) can hide a glitazar![END_REF] Therefore, it may not be surprising that pioglitazone and some glitazars share the same urothelial toxicity.

Clinical trials

The uncertain effectiveness of pioglitazone

During development, reduction in hemoglobin A1c (HbA1c) levels was demonstrated in patients exposed to pioglitazone, but its long-term cardiovascular effectiveness was unknown. The PROspective PioglitAzone Clinical Trial In MacroVascular Events (PROactive) has been conducted to assess cardiovascular benefit of pioglitazone compared to placebo in 5,238 diabetic patients with extensive macrovascular disease. [START_REF] Dormandy | Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial[END_REF] Results published in 2005 indicated that, during a mean follow up of 34.5 months, a significant 0.5% decrease in HbA1c was shown in favor of pioglitazone. However, the authors did not observe a statistically significant reduction for the primary composite endpoint including all-cause mortality, non fatal myocardial infarction, stroke, acute coronary syndrome, endovascular or surgical intervention in coronary or leg arteries, and amputation above the ankle: HR=0.90 (95%CI: 0.80-1.02; p=0.095). They reported a significant reduction in secondary composite endpoint of death, myocardial infarction or stroke (HR=0.84; 95%CI: 0.72-0.98, p=0.027), result on which they based their conclusions. It is worth noting that this secondary endpoint was not clearly predefined in the publication of study protocol. [START_REF] Charbonnel | The prospective pioglitazone clinical trial in macrovascular events (PROactive): can pioglitazone reduce cardiovascular events in diabetes? Study design and baseline characteristics of 5238 patients[END_REF] To defend themselves from post-hoc analysis, authors explained later that this endpoint had been drawn during study but before data analysis. [41] Despite statement of authors, several points limit the evidences brought by this study: i) conclusions were drawn from results on secondary outcomes, ii) trial was placebo-controlled and not tested versus a reference drug, iii) included patients showed prior high cardiovascular risk (this is not a priori representative of pioglitazone-treated patients in real life) and iv) significant increased risk of heart failure was observed in the pioglitazone group. [START_REF] Dormandy | Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial[END_REF] Later, a meta-analysis of randomized controlled trials focusing on cardiovascular events associated with pioglitazone was conducted: 99 trials were selected. [START_REF] Lincoff | Pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus: a meta-analysis of randomized trials[END_REF] Among them, 10 were >12 months and 2 were >24 months in duration. In total, 16,390 patients were included. Twenty eligible trials (3,014 patients) not sponsored by the manufacturer were not included in the analysis. Composite endpoint including death, myocardial infarction or stroke was significantly decreased by 18% in the group of patients receiving pioglitazone. Serious heart failures were also significantly higher in the pioglitazone group (+41%), and a broader definition of cardiovascular risk combining death, myocardial infarction, stroke and serious heart failure did not show any cardiovascular benefit for pioglitazone: HR=0.96 (95%CI: 0.85-1.09), p=0.54. In 2008, another meta-analysis included 24 randomized trials, enrolling 11,268 patients in the pioglitazone and 9,912 patients in the comparator group (PROactive excluded). Unfortunately, only summary data without time to event information have been taken into account and many of the studies did not have specific cardiovascular endpoints. All-cause mortality was lower in the pioglitazone group (OR=0.30; 95%CI: 0.14-0.63) but not non-fatal coronary events. Increase in non-fatal heart failures was not statistically significant (OR=1.38; 95%CI: 0.90-2.12). The authors concluded that pioglitazone does not appear to be harmful in terms of cardiovascular events and all-cause deaths. [START_REF] Mannucci | Pioglitazone and cardiovascular risk. A comprehensive meta-analysis of randomized clinical trials[END_REF] All these data taken as a whole suggest that the clinical efficacy of pioglitazone in reducing cardiovascular events has not been clearly demonstrated since used methodologies can be criticized.

Assessment of bladder cancer risk in clinical trials

Premarketing clinical trials were often too short, too low on sample size and not specifically designed to measure occurrence of bladder cancer. In the first report of the PROactive trial, 14/2605 (0.5%) cases of bladder neoplasm were reported in the pioglitazone group versus 6/2633 (0.2%) in the placebo group, this difference did not reach statistical significance (p=0.069). [START_REF] Dormandy | Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial[END_REF] An overview of the PROactive data published in 2009, revealed that one case in the placebo group exhibited in fact a benign histology. Unfortunately, no new estimation of risk was reported at that time. [START_REF] Dormandy | Safety and tolerability of pioglitazone in high-risk patients with type 2 diabetes: an overview of data from PROactive[END_REF] Taking into account only five cancers in the placebo group, the overall incidence was statistically lesser than in the pioglitazone group: 0.19% (n=5) versus 0.54% (n=14) respectively (p=0.040). [START_REF] Hillaire-Buys | Pioglitazone and bladder cancer[END_REF] Recalculated crude risk ratio for bladder cancer was 2.83 (95% CI 1.02-7.85). Authors of the PROactive study explained that, after a blinded review of bladder cancer cases, which eliminated the cases reported within one year of randomization or who showed known risk factors for bladder cancer, only two cases in the pioglitazone group and one in the placebo group were left. The Data and Safety Monitoring Committee concluded that these numbers were too small to consider bladder cancer as a safety issue. [START_REF] Dormandy | Safety and tolerability of pioglitazone in high-risk patients with type 2 diabetes: an overview of data from PROactive[END_REF] An extended study was planned to monitor PROactive patients over a longer period (up to 10 years). The results have not been published to date but interim reports were submitted to EMA by the manufacturer: a pooled analysis of data from the double-blinded period and the 6-year observational period showed that, overall, bladder cancer was reported in 25 subjects (0.9%) who received pioglitazone and 20 subjects (0.8%) never exposed to pioglitazone (HR=0.98, 95%CI: 0.6-1.8). [6] In two other 72 weeks double-blinded randomized multicenter trials conducted between 2003 and 2006, incidence of bladder neoplasms was not reported. [START_REF] Nissen | Comparison of pioglitazone vs glimepiride on progression of coronary atherosclerosis in patients with type 2 diabetes: the PERISCOPE randomized controlled trial[END_REF][START_REF] Mazzone | Effect of pioglitazone compared with glimepiride on carotid intima-media thickness in type 2 diabetes: a randomized trial[END_REF] 4. Observational studies 4.1.

Analysis of spontaneous reports

In a review of cases based on spontaneous reports performed within the manufacturer's global database, 68 cases of bladder cancer associated with pioglitazone were found for the period 31 July 1999 to 15 March 2011. [6] After elimination of cases with confounding factors, there were 13 ). [48] ROR measures the disproportionality of reporting in pharmacovigilance databases; although it cannot fully be interpreted as a Risk Ratio, these findings supported a significant risk of bladder cancer associated with pioglitazone. However, analysis of adverse event reporting databases is always susceptible to reporting bias due to important under-reporting. On the contrary, over-reporting can be observed due to media or notoriety bias. In order to take this phenomenon into account, the authors performed a yearby-year analysis and found a significant relationship as soon as 2004 (ROR=4.77, 95%CI: 1.30-15.88), i.e. before the first PROactive publication. [48] 4.2.

Studies using large medical databases

Inherent limits of observational studies

Observational studies which evaluate risk of bladder cancer associated with pioglitazone use are susceptible to numerous biases which can potentially limit the interpretation of their results. Firstly, as it can be criticized for randomized controlled trials, duration of follow-up in available observational studies could be often too short to detect occurrence of cancer, specifically inducted cancer. For study of promoted cancer, shorter lengths of follow-up can be sufficient. Secondly, even if some studies use very large medical databases, analysis of rare events (which is the case here) sometimes cannot involve enough cases to provide sufficient statistical power. Thirdly, it seems that patients with type 2 diabetes show an increased risk of bladder cancer [START_REF] Larsson | Diabetes mellitus and risk of bladder cancer: a metaanalysis[END_REF][START_REF] Mackenzie | Diabetes and risk of bladder cancer: evidence from a case-control study in New England[END_REF][START_REF] Giovannucci | Diabetes and cancer: a consensus report[END_REF] and that this risk increase with diabetes duration. [START_REF] Mackenzie | Diabetes and risk of bladder cancer: evidence from a case-control study in New England[END_REF] Consequently, observational studies have to deal with the risk of errors related to the potential difference in severity of diabetes among patients exposed to pioglitazone and other diabetic patients. As pioglitazone is often prescribed as a second line therapy, indication bias represents a major issue, which can partially be taken into account in adjusting the results for duration of diabetes or duration of therapy. For the same reason, it is important that these studies include elderly patients. Fourthly, changes in pharmacotherapy over time can be frequent in patients with type 2 diabetes; these changes cannot always be considered or may require complex analyses that limit interpretation. In addition, information bias is often difficult to rule out: in numerous observational studies, prescription of glucose-lowering agents is used as a proxy for definition of diabetes. Drug use is also defined by prescriptions written by practitioners or dispensed in pharmacy. Thus, it is difficult to know whether these prescriptions correspond to actual exposure. Bladder cancer occurrence mentioned in health insurance databases, hospital discharge diagnosis or primary care medical records are also subjected to potentially substantial information bias as cancer might not be completely validated. Finally, information on significant risk factors for bladder cancer such as cigarette smoking, race/ethnicity, history of urinary tract diseases, are sometimes missing and some others such as occupational or environmental exposure to carcinogenic chemicals are rarely available. As a result, potential confounders sometimes cannot be adjusted resulting in biased estimation of the associated risk.

Systematic reviews and meta-analyses

In study of rare events, meta-analyses are a useful way to increase statistical power by pooling risk estimations from different studies. However, one should keep in mind that study biases are reflected in meta-analysis results and that a better level of evidence is reached when it implicates randomized controlled trials rather than observational studies. In December 2011, revaluation of pioglitazone by EMA revealed the results of an unpublished meta-analysis conducted by the manufacturer from its clinical trial database. Thirty six trials were included involving 22,000 patients (PROactive study was analyzed separately). Study duration was less than one year for twenty four studies, between 1 and 2 years for 6 studies and more than 2 years for 6 studies. Including all cases of bladder cancer, even those that happened within one year of treatment, there were 19 cases in the pioglitazone group (0.15%) versus 7 in the comparator group (0.07%), resulting in a HR of 2.64 (95%CI: 1.11-6.31, p=0.029). This risk was not significant when cases occurring within the first year of treatment were excluded. [6] In June and July 2012, two systematic reviews of published studies regarding the association between pioglitazone therapy and bladder cancer were published. At the time of their implementation, publications from the UK GPRD [START_REF] Azoulay | The use of pioglitazone and the risk of bladder cancer in people with type 2 diabetes: nested case-control study[END_REF]50] or THIN [51] were not available. The first meta-analysis by Zhu et al. included a clinical trial and observational studies together. [START_REF] Zhu | Increased risk of bladder cancer with pioglitazone therapy in patients with diabetes: A meta-analysis[END_REF] Five studies were selected: the PROactive randomized trial [START_REF] Dormandy | Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial[END_REF] , the KPNC study by Lewis et al. [53] , the French study by Neumann et al [54] and the two Taiwanese studies [55,56] including 2,350,908 diabetic patients in total. Overall pooled RR was 1.17 (95%CI: 1.03-1.32). RR for cumulative treatment duration >24 months and cumulative dose >28,000 mg were 1.38 (95%CI: 1.12-1.70) and 1.58 (95%CI: 1.12-2.06) respectively. It is worth noting that the numbers of cases from the PROactive study taken into account in this review were still incorrect (14 versus 6). The second meta-analysis conducted by Colmers et al. studied the risk of bladder associated with both pioglitazone and rosiglitazone. [START_REF] Colmers | Use of thiazolidinediones and the risk of bladder cancer among people with type 2 diabetes: a meta-analysis[END_REF] Three cohort studies were selected for the pioglitazone meta-analysis: Lewis et al. [53] , Neumann et al. [54] and Tseng et al. [56] . The risk of bladder cancer was significantly increased with the use of pioglitazone (pooled RR=1.22, 95%CI: 1.07-1.39). In contrast, the specific meta-analysis for rosiglitazone did not show any significant association. The principal investigator of this study, cited in a subsequent comment, stated that a re-run of the metaanalysis for bladder cancer risk with pioglitazone use, including the GPRD nested case control study by Azoulay et al. [START_REF] Azoulay | The use of pioglitazone and the risk of bladder cancer in people with type 2 diabetes: nested case-control study[END_REF] , found a pooled risk ratio increased from 1.22 to 1.26. [START_REF] Kermode-Scott | Meta-analysis confirms raised risk of bladder cancer from pioglitazone[END_REF] In January 2013, two other meta-analyses found similar overall risk for pioglitazone: RR was 1.20 (95% CI: 1.07-1.34) for Bosetti et al. who included six observationnal studies [59] and HR was 1.23 (95% CI: 1.09-1.39) for Ferwana et al. who included six observational or experimental studies. [START_REF] Ferwana | Pioglitazone and risk of bladder cancer: a meta-analysis of controlled studies[END_REF]

Regulatory management of the bladder cancer risk

At the time of the first authorization of pioglitazone in the US, a key point for the risk management was the information and recommendations included in the drug label regarding the occurrence of bladder cancer in male rats in preclinical studies. In 1999, the label information stated that: "Benign and/or malignant transitional cell neoplasms were observed in male rats at 4 mg/kg/day and above (approximately equal to the maximum recommended human oral dose based on mg/m²). The relationship of these findings in male rats to humans is unclear". [START_REF] Actos Label Information | [END_REF] It is worth noting that this last sentence has been anecdotally removed during a label revision in June 2003. In 2000, for European authorization, data from animal carcinogenesis were also added to the Summary of Product Characteristics in the "Preclinical Safety" section. In both cases no recommendation has been specified to prescribers, especially for the selection and monitoring of patients potentially at risk. At this time, regulators asked to implement a post-marketing surveillance study to monitor treated patients [6] and the KPNC cohort was planned to start in 2003 for a duration of ten years. Since then, and during the following decade, minor changes appeared in the product information but no regulatory decision or recommendations were clearly stated.

In July 2004, the following sentence was added to FDA label precautions section: "Urinary tract tumors have been reported in rodents taking experimental drugs with dual PPAR / activity; however, Actos is a selective agonist for PPAR ." [61] This last statement was inappropriate, as pharmacological profile of pioglitazone appears to be comparable to that of the dual PPAR / agonists. [START_REF] Hillaire-Buys | Stay vigilant: a glitazone (pioglitazone) can hide a glitazar![END_REF] This part was removed from the drug label in August 2006.

In September 2009, under requirement of the FDA, a Risk Evaluation and Mitigation Strategy (REMS) was implemented for pioglitazone products in the US. A medication guide was dispensed with each prescription noting that "bladder cancer occurred in a few more people who were taking pioglitazone than in people who were taking other diabetes medicines. There were too few cases to know if the bladder cancer was related to pioglitazone." [START_REF]Actos Medication Guide[END_REF] In September 2010, after the publication of the third interim analysis of the KPNC cohort study (from 2003 to 2008), the FDA issued an alert on its website mentioning "potential increased risk of bladder cancer after two years exposure. At this time FDA has not concluded that Actos increases the risk of bladder cancer. Its review is ongoing." [START_REF]Drug Safety Communication: Ongoing Safety Review of Actos (pioglitazone) and Potential Increased Risk of Bladder Cancer After Two Years Exposure[END_REF] In April 2011, the European Commission requested EMA's Committee for Medicinal Products for Human Use (CHMP) to assess the impact of available information on the risk-benefit balance for the pioglitazone products. In June 2011, following the results of the retrospective cohort from the French health insurance database, the French medicines agency (Afssaps) decided the suspension of medications containing pioglitazone in France. [START_REF]Use of Medications Containing Pioglitazone (Actos®, Competact®) Suspended[END_REF] Soon after, the Federal Institute for Drugs and Medical Devices of Germany (BfArM) confirmed similar action. [START_REF]Tandemact®): Increased incidence of bladder cancer[END_REF] In July 2011, CHMP concluded to the existence of a small increased risk of bladder cancer associated with the use of pioglitazone but "could not identify any of the currently approved indications, where the benefits would specifically no longer outweigh the risks." [6] CHMP recommended restrictions of use to be introduced to the product information in order to ensure that the risk-benefit of pioglitazone remains positive:

-Adequacy of response to treatment (e.g. reduction in HbA1c) should be reviewed 3-6 months after initiation of therapy and pioglitazone should be discontinued failing adequate response.

-Advise to start treatment with the lowest available dose.

-Contraindication for patients with current bladder cancer or a history of bladder cancer or macroscopic haematuria. -Macroscopic haematuria should be investigated before starting pioglitazone therapy.

-Patients should promptly report macroscopic haematuria or other symptoms such as dysuria or urinary urgency. -Combination use with insulin should be considered with caution in elderly patients.

-Risk of bladder cancer was added to the table of adverse reactions, under the header "Neoplasms benign, malignant and unspecified (including cysts and polyps)" with the frequency "uncommon" for all indications. [6] In addition, risk minimization measures such as educational materials, prescriber guide, a Dear Healthcare Professional Communication letter and an update of the risk management plan were implemented. CHMP also concluded that "systematic bladder cancer screening for monitoring purposes was considered as unrealistic." Finally, further analyses including a pan-European epidemiological study were asked by the CHMP. [6] These conclusions were made public in July 2011 and, on its website, the EMA "recommends new contraindications and warnings for pioglitazone to reduce small increased risk of bladder cancer" [START_REF]new contra-indications and warnings for pioglitazone to reduce small increased risk of bladder cancer[END_REF] whereas "positive benefit-risk balance is confirmed as second and third line treatment." [START_REF]Positive benefit-risk balance confirmed as second and third line treatment[END_REF] In July 2011, a warning was added to the pioglitazone US label information: "Preclinical and clinical trial data, and results from an observational study suggest an increased risk of bladder cancer in pioglitazone users. The observational data further suggest that the risk increases with duration of use. Do not use in patients with active bladder cancer. Use caution when using in patients with a prior history of bladder cancer." [START_REF] Actos Label Information | [END_REF] In December 2011, Actos Summary of Product Characteristics for EU was updated with the CHMP recommendations. [START_REF]Actos: Product Information as approved by the CHMP on 20 october 2011[END_REF] Figure 1 presents a summary timeline of scientific evidence and regulatory management.

Comments and perspectives

The burden of type 2 diabetes mellitus is massive and still growing, diabetic patients are expected to be 30 million by 2030. [START_REF] Wild | Global prevalence of diabetes: estimates for the year 2000 and projections for 2030[END_REF] In 2011, sales for Actos represented $3.4 billion in the United States. [68] The patent for Actos has expiried in 2012 and numerous generic products are entering the market. Whatever the fate of pioglitazone in the future, we can point several comments about different aspects of this matter.

Should pioglitazone have been licensed in the first place?

In terms of benefit for the patients, we can legitimately question whether authorizing pioglitazone was worth after all. In 1999, we knew from the premarketing data that pioglitazone showed a mild reduction in HbA1c and, except frequent fluid retention, a relatively acceptable tolerance. Pioglitazone was authorized making the assumption that results from a surrogate endpoint such as HbA1c, compared to placebo, will transform into benefits in terms of cardiovascular morbidity and mortality. This assumption to be wrong. No evidence has proven that tight HbA1c control really the risk of death from a cardiovascular event. [START_REF] Montori | Glycemic control in type 2 diabetes: time for an evidencebased about-face?[END_REF] More than ten years after its initial marketing, no well-designed long-term study has yet strictly confirmed the real benefit of pioglitazone in reducing cardiovascular diseases incidence. It is another example of the well known (but unfortunately often forgotten) difference in drug evaluation between clinically significant (morbi-mortality, quality of life) and surrogate (here biological, HbA1c) endpoints.

A modest but critical risk of bladder cancer

It would not be not ethical and neither practical to implement a randomized control trial to definitively answer to the question regarding bladder cancer risk. Although some studies such as Azoulay's achieve the best standards, it is obvious that no observational study would equal the level of proof of a randomized control trial. However, evidence can be approached by the combination of different data from different fields and different designs, so the increased risk of bladder cancer associated with pioglitazone seems real to us. Observational studies analyzed in this article indicate that the basal risk of bladder cancer in diabetic patients is estimated at 50 to 100/100,000 person-years, which corresponds to less than 1 case per 1000 patients for one year. Risk estimates for pioglitazone do not seem, at first glance, to represent a significant impact in terms of public health. Moreover, the majority of cases associated with pioglitazone use showed low-grade lesions. [6] However, this risk must not be neglected. Firstly because the dramatic increase in prevalence of diabetes over the last 25 years and its projection to the future suggest that a small risk of serious adverse event associated with a popular blockbuster will consequently affect an extensive population of patients. Given the worldwide exposure to pioglitazone estimated to over 20 million patient-years in 2011 [6] , risk ratio ranging from 1.2 to 2.6 (which are observed for overall effect in meta-analyses) could hypothetically stand for approximately 2,000 to 18,000 additional bladder cancer cases. Secondly because, notwithstanding an a priori low lethality, bladder cancer still represents a consequent deterioration of the quality of life for affected patients. Finally, because this risk seems mainly avoidable without a great loss of opportunity for the patients and the society. Indeed, real benefit of pioglitazone in reducing cardiovascular diseases or total mortality is not proved, its real cost-effectiveness is not comparatively assessed and alternative glucose-lowering drugs are available.

Lost opportunities for risk management

It seems clear that before authorization, premarketing data brought to mind a doubt about the risk of bladder cancer associated with pioglitazone. Even if bladder cancers which occurred in male rats treated with pioglitazone were claimed at this time to be specific to rats, a strict precautionary approach would have led to delay the authorization until deep evaluation of this risk. A less strict approach could have been, at least, to advise prescribers to closely monitor their patients with this potential risk in mind. None of these approaches were chosen.

In 2005, by excluding the benign tumor in the placebo group, a correct analysis of the PROactive data would have revealed a significant association between pioglitazone use and bladder cancer in a randomized trial and would have strengthened the doubt. [START_REF] Hillaire-Buys | Pioglitazone and bladder cancer[END_REF] Another inaccuracy lies in the definition of pioglitazone as a selective agonist for PPAR: pioglitazone now appears to have similar pharmacological profile as the dual PPAR / agonists, which were discontinued before 2006 due to safety concerns, including bladder tumors. [START_REF] Conlon | Goodbye glitazars?[END_REF][START_REF] Hillaire-Buys | Stay vigilant: a glitazone (pioglitazone) can hide a glitazar![END_REF] Whatever the cause of these inaccuracies may be, the opportunity was lost to give in time to the regulators the right information to properly assess the safety profile of pioglitazone. It took until 2011 for pharmacoepidemiological evidences to be sufficiently numerous and conclusive to initiate the application of safety measures such as prescription restriction, close monitoring and review of adequacy of response to treatment. The first lesson that we can draw from this sequence of events is that, if the opportunity to implement regulatory actions to prevent potential side effects before drug authorization is missed, it takes many years to make changes once the drug is marketed. Secondly, it is crucial to be particularly vigilant with the safety information provided before and after-marketing. In a context of weak efficacy, the doubt always has to benefit to the patient.

The difficulty of screening and monitoring treated patients

In the example of pioglitazone, a great difficulty lies in identifying the patients who are more at risk to develop a bladder cancer. Cystoscopy associated with biopsy is the examination of choice to detect bladder cancer but it is totally unfeasible as a systematic screening for bladder lesion in order to select or monitor pioglitazone-treated patients. Recommendations to not accumulate known and detectable risk factors to the prescription of pioglitazone and to clinically monitor treated patients have been implemented but it only represents an approach by default. In other words, patient history and clinical urinary symptoms including macroscopic haematuria, which are the criteria now recommended for selection and monitoring of patient, may be insufficiently reliable to avoid exposure of patients at risk. Pioglitazone can be useful in the population of good responder and well tolerant patients but it appears that practitioners cannot really count neither on HbA1c to assess it cardiovascular effectiveness nor on restricted prescription to ensure its safety. This problematic situation is now left in the hands of the prescribers who may consequently encounter complex patient management with medico-legal responsibility.

Conclusions

The pioglitazone example is giving us a good opportunity to think about how the drug regulatory system is working. With the new therapeutic agents which are in development, in the field of diabetes as in the other therapeutic fields, this complex situation will happen again and risk management will have to be more proactive. As a basis for reflection, some solutions can be proposed:

-Do not neglect basic pharmacological studies (pharmacokinetic and pharmacodynamic) and strictly analyze the slightest doubt. -Do not authorize drugs without well-designed long-term studies comparing clinical outcomes to reference medications and not only to placebo.

-Always ensure that the results of any study are disclosed; widely spread available information to the medical community and the public. -Conduct rapidly post-marketing studies (clinical trials and observational studies) with the highest level of proof to assess the safety, the effectiveness and the cost effectiveness of newly marketed drugs. -Ensure that these studies are independent from pharmaceutical firms.

-If a risk of serious adverse event is suspected, strict and reliable recommendations for prescription and monitoring must be immediately implemented. -Suspend the drug whose risk-benefit balance is judged negative and in cases of doubt decision have to be taken only in the patient's interest. In our opinion, any improvement implies an increasing role of health authorities and academic organizations. No solution would satisfy everyone involved (health authorities, pharmaceutical industries, clinical community and patients) but we must think about the patient first and give ourselves the means to comply with the principal precept of medical ethics: "primum non nocere".

Figure 1 .

 1 Figure 1. Timeline for main scientific information and regulatory actions about pioglitazone and bladder cancers.

  cases left. Fourteen additional cases (including one patient who died as a result of cerebral metastasis) were found in the EudraVigilance database from 16 March 2011 to 24 May 2011, which leads to 82 cases in total. In June 2011, in an independent case/non-case analysis from the US FDA Adverse Event Reporting System (AERS) database, Piccinni et al. found 31 cases associated with pioglitazone between the years 2004 and 2009; Reporting Odds Ratio (ROR) was 4.30 (95%CI: 2.82-6.52

Table 1

 1 presents the design and characteristics of the principal observational studies focusing on bladder cancer risk associated with pioglitazone use. Results and comments are shown in table 2.

Table 1 .

 1 Characteristics of observational studies investigating the risk of bladder cancer associated with pioglitazone use

	Tables
	Authors	Year, Reference	Data source	Funding	Study design	Type of comparison	Time period	Study population	Bladder cancer definition	Exposure assessment	Adjusted confounding factors
	Piccinni et al.	2011, [48]	US FDA AERS database	Independent	Case/non case study	Not applicable	Jan 2004-Dec 2009	37,841 reports concerning pioglitazone	Relevant Preferred Terms of MedDRA® classification	Drug names medicines referred to pioglitazone containing	Age, sex, other glucose-lowering drugs
	Oliveria et al.	2008, [70]	US pharmacy claims database	Unknown	Retrospective cohort	Use of TZD vs. other diabetes medications	Jan 2000 -Dec 2004	191,223 diabetic patients, ≥18 years old, no bladder cancer during the 12 months prior to follow-up	Hospital diagnoses and treatment received	Ever use, median duration of follow-up: 3.9y	Age, sex, schistosomiasis, and pelvic radiation
								193,099 diabetic patients, ≥40			Age, race, sex, other glucose-
	Lewis et al.	2011, [53] 2012, [71]	Californian healthcare system KPNC, US	Takeda Pharma-ceuticals	Prospective cohort	Use of pioglitazone vs. other diabetes medications or dietary therapy	Jan 1997-Apr 2008 Jan 1997-Dec 2010	years old, no prior bladder cancer or within 6 months of entry date Pioglitazone: Comparators: 30,173 162,926	Inpatient and outpatient medical diagnoses	New users (8% were exposed within 4 months of entry into the cohort), median of exposure: 2y	lowering drugs, socioeconomic status, smoking, occupational exposures, urinary diseases or symptoms, HbA1C levels, duration of diabetes in the
												cohort, comorbidities
								1,491,060 diabetic patients (at
								least one prescription for a	Hospital
	Neumann et al.	2011, [54]	French health insurance SNIIRAM	French medicines agency	Retrospective cohort	Use of pioglitazone vs. other diabetes medications	Dec 2006 -Dec 2009	glucose-lowering drug in 2006), 40 to 79 years, no prior bladder cancer or within 6 months of entry date	discharge diagnosis (ICD-10) and specific therapeutic	Prevalent and new users, median of exposure: 1.5y	Age, sex, other glucose-lowering drugs, proxy for disease duration
								Pioglitazone:	Comparators:	procedure
								155,535	1,335,525
	Li et al.	2012, [50]	Primary care medical record database GPRD, UK	Unfunded	Retrospective cohort (matched with propensity score)	Use of pioglitazone vs. other diabetes medications	Jan 2001 -Dec 2010	207,714 diabetic patients, ≥40 years old, no prior bladder cancer or within 90 days of entry date Pioglitazone: Comparators: 23,548 184,166	Primary care medical record	New user? median of follow-up: 3.5y	Age, sex, other glucose-lowering drugs , duration of diabetes, smoking status and body mass index
	Tseng et al.	2012, [56]	Taiwanese National Health Insurance	Taiwanese National Health Research Institutes	Retrospective cohort	Use of pioglitazone vs. other diabetes medications	Jan 2006 -Dec 2009	54,928 diabetic patients Pioglitazone: Comparators: 2,545 52,383	Reimbursement records (ICD-9)	Prevalent and previous users	Age, other glucose-lowering drugs, diabetes duration, comorbidities

Table 2 .

 2 Risk estimations for bladder cancer associated with pioglitazone use in observational studies Not available ROR cannot be interpreted as RR. Probable reporting bias. Limited notoriety bias: significant relation in 2004 before the publication of the PROactive study (year by year analysis).Pioglitazone use was not analyzed separately. Smoking status, occupational exposure, history of urinary pathology, duration of diabetes, and prior use of glucose-lowering agents were not taken into account as confounders. Potential information bias due to study of automated claims data. Probable lack of statistical power. Few prevalent users at entry date: limited confounding by previous exposure. Restricted to a localized region in the United States (unknown representativeness). Substantial loss of follow-up rate. Poor adjustment for duration of diabetes. Nested case control study: collected data for smoking suggested no confounding effect. Changes in method for measurement of exposure duration not fully detailed nor explained. Significant increased risks observed in the subgroup of non-smokers for high exposure.

	Authors	Year, Reference	No. of bladder cancers and incidence rate	Assessment of dose and duration	Risk Ratio estimates (95% CI)	RR for men (95% CI)	Comments
	Piccinni et al. ROR: 4.30 (2.82-6.52) Oliveria 2011, [48] 31 reports of bladder cancer concerning pioglitazone Not available TZD: 1.05 (0.71-1.54) 2008, [70] Overall: n=178 Not available TZD combined with other oral glucose-et al. lowering drugs:	Not available
						1.92 (0.89-4.13)
					Ever use vs. never use	1.2 (0.9-1.5)	1.1 (0.9-1.5)
	Lewis et al.	2011, [53]	Pioglitazone: 90 81.5/100,000 PY	Comparators: 791 68.8/100,000 PY	Duration of therapy > 24 months 1.4 (1.03-2.0)	1.6 (1.2-2.3)
					Cumulative dose > 28,000 mg	1.4 (0.96-2.1)	1.8 (1.2-2.6)
					Ever use vs. never use	1.07 (0.87-1.30)	1.02 (0.82-1.27)
	Lewis et al.	2012, [71] Pioglitazone: 137 Comparators: 952	Duration of therapy > 48 months 1.38 (0.97-1.96)	1.41 (0.96-2.06)
					Cumulative dose > 35,000 mg	1.25 (0.91-1.74)	1.43 (1.03-1.99)
	Neumann et al	2011, [54]	Pioglitazone: 175 49.4/100,000 PY	Comparators: 1,841 42.8/100,000 PY	Ever use vs. never Duration of therapy > 24 months 1.36 (1.04-1.79 1.22 (1.05-1.43) Cumulative dose > 28,000 mg 1.75 (1.22-2.50)	1.28 (1.09-1.51) 1.44 (1.09-1.91) 1.88 (1.30-2.71)	Large sample size and good population representativeness. High prevalent users rate (>60%), 38,925 (25.0%) filled a prescription for pioglitazone in January 2006 and 59,296 (38.1%) during the first 6 months of 2006. Patients >79 years old not included. Poor adjustment for duration of diabetes, no adjustment for smocking.
	Li et al.	2012, [50]	Pioglitazone: 66 80.2/100,000 PY	Comparators: 803 81.8/100,000 PY	Ever use vs. never Follow up time >24 months	1.16 (0.83-1.62) 1.20 (0.74-1.93)	Not available	No analysis of cumulative exposure (analysis of follow up time only). Probable lack of statistical power.
								All bladder cancer in ever users occurred within a duration of
	Tseng et al.	2012, [56]	Pioglitazone: 10 105/100,000 PY	Comparators: 155 79/100,000 PY	Ever use vs. never	1.31 (0.66-2.58)	Not available	therapy <24 months. Asian population. Probable lack of statistical power. No adjustment for biochemical data, obesity, smoking, lifestyle, diet, occupational exposure, and genetic
								parameters
	Mamtani et al.	2012, [51]	Pioglitazone: 41 123.8/100,000 PY	Rosiglitazone: 86 106.6/100,000 PY	Ever use vs. never >5 years of use Ever use vs. never	1.14 (0.79-1.66) 1.59 (0.74-3.43) TZD: 0.93 (0.68-1.29)	Not available	Comparison of pioglitazone users vs. SU users was not performed. Authors concluded to a TZD class effect.
			TZD: 60	SU: 137			Not available
					>5 years of use	TZD: 3.25 (1.08-9.71)

Acknowledgments

No sources of funding were used to assist in the preparation of this manuscript.