Juan José Torrado

María Blanco

Magí Farré

Pere Roset

Alfredo García-Arieta

Laboratorios Gelos

Rationale and conditions for the requirement of chiral bioanalytical methods in bioequivalence studies

Keywords: Ibuprofen, Bioequivalence, Enantiomers, Chirality, Regulatory requirements

Purpose The aim of the present work was to assess the need for chiral bioanalytical methods in bioequivalence studies. Methods The samples from a bioequivalence study of two ibuprofen 2% oral suspensions that had shown bioequivalence for AUC and C max , but not for t max (medians of 2.0 and 0.75 h) with a non-chiral method were assayed with a chiral method to investigate whether there was an actual difference in the rate of absorption within the limits of C max and AUC bioequivalence.

Results

The non-chiral method and the sum of concentrations of both enantiomers obtained with the chiral method gave a similar outcome (90% CI C max non-chiral: 82.77-96.09, sum of enantiomers: 82.19-98.23; 90% CI AUC t non-chiral: 107.23-115.49, sum of enantiomers: 105.73-121.35). However, the chiral method showed differences in AUC and C max that resulted in non-bioequivalence for the individual enantiomers (90% CI C max S-ibuprofen: 76.05-91.36, R-ibuprofen: 87.84-113.05; 90% CI AUC t Sibuprofen: 96. 67-105.86, R-ibuprofen: 118.86-142.24). The differences in the pharmacokinetics of each enantiomer, and thus in the enantiomer concentration ratio, were dependent on the rate of absorption. Conclusions Due to the fact that in bioequivalence studies the rate of absorption of the new product is unknown, chiral bioanalytical methods should be employed for chiral drugs, such as ibuprofen, whose enantiomers exhibit different pharmacodynamic characteristics and whose enantiomer concentration ratio might be modified by the rate of absorption, irrespective of whether the eutomer is the minor enantiomer or the similarity of the pharmacokinetics of the enantiomers at a given rate of absorption.

Introduction

The need for chiral bioanalytical methods in bioequivalence studies is a controversial issue that has not been agreed upon by different regulatory agencies [START_REF]Note for guidance on the investigation of bioavailability and bioequivalence. 26-7-2001[END_REF][START_REF]Guidance for industry. Bioavailability and bioequivalence. Studies for orally adminsitered drug productsgeneral considerations[END_REF]. The FDA Guidance for Industry [START_REF]Guidance for industry. Bioavailability and bioequivalence. Studies for orally adminsitered drug productsgeneral considerations[END_REF] recommends the measurement of the racemate using a non-chiral assay for bioequivalence studies. Measurement of individual enantiomers in bioequivalence studies is only recommended when all of the following conditions are met: [START_REF]Note for guidance on the investigation of bioavailability and bioequivalence. 26-7-2001[END_REF] the enantiomers exhibit different pharmacodynamic characteristics, (2) the enantiomers exhibit different pharmacokinetic characteristics, (3) primary efficacy/safety resides with the minor enantiomer, and (4) non-linear absorption is present (as expressed by a change in the enantiomer concentration ratio with change in the input rate of the drug) for at least one of the enantiomers. In such cases, bioequivalence criteria should be applied to the enantiomers separately.

It is obvious that if enantiomers do not exhibit different pharmacodynamic characteristics, chiral bioanalytical methods are unnecessary. However, the other three conditions are debatable or difficult to understand.

It has been shown previously that even when the active (eutomer) and inactive enantiomer (distomer) exposure ratio is near unity, the non-chiral bioanalytical method may be biasing the results [3]. Obviously, if the eutomer were the minor enantiomer, the bias would be greater, but it does not mean that in other cases the bias is nonexistent.

The concept of non-linear absorption (expressed as a change in the enantiomer concentration ratio with a change in the input rate of the drug) has been confused frequently with non-linear pharmacokinetics (e.g. as required in the CPMP Notes for Guidance [START_REF]Note for guidance on the investigation of bioavailability and bioequivalence. 26-7-2001[END_REF][START_REF]Note for guidance on the investigation of chiral active substances[END_REF]). Boni et al. [5] illustrated with etodolac that this non-linear absorption may occur in drugs with linear kinetics both for the racemic and the individual enantiomers.

In addition, it must be taken into account that for those drugs where a change in the rate of absorption produces a change in the enantiomer concentration ratio, it could be understood that these enantiomers exhibit different pharmacokinetic characteristics, at least at some rates of absorption.

The need for chiral bioanalytical methods in bioequivalence studies has been discussed from a theoretical point of view [START_REF] Mehvar | Bioequivalence of chiral drugs. Stereospecific versus non-stereospecific methods[END_REF], or experimentally, changing the input rate artificially [5]. However, actual examples would be convenient to confirm if chiral bioanalytical methods are necessary in bioequivalence studies.

Ibuprofen is a chiral non-steroidal anti-inflammatory drug with the S-enantiomer possessing most of the beneficial anti-inflammatory activity [START_REF] Geisslinger | Pharmacological differences between R(-)-and S(+)-ibuprofen[END_REF]. Although enantiomer exposure ratio is near unity, the drug exhibits a complex and marked stereoselectivity in its pharmacokinetics [START_REF] Jamali | Pharmacokinetics of ibuprofen enantiomers in humans following oral administration of tablets with different absorption rates[END_REF]. In addition, according to the Biopharmaceutics Classification System (BCS) [START_REF] Amidon | A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability[END_REF][START_REF] Lobenberg | Modern bioavailability, bioequivalence and biopharmaceutics classification system. New scientific approaches to international regulatory standards[END_REF], ibuprofen is a highly soluble at neutral pH class II drug with complete oral absorption, but whose rate of absorption is highly dependent on formulation and manufacturing factors [START_REF] Lindenberg | Classification of orally administered drugs on the World Health Organization model list of essential medicines according to the biopharmaceutics classification system[END_REF][START_REF] Potthast | Biowaiver monographs for immediate release solid oral dosage forms: ibuprofen[END_REF] or even on the interaction with antacids [START_REF] Parojcic | Rationale for ibuprofen coadministration with antacids: potential interaction mechanisms affecting drug absorption[END_REF]. In other words, due to its low solubility at acidic pH, different formulations may show different in vivo dissolution rates, which lead to different rates of absorption [START_REF] Fornasini | Preliminary pharmacokinetic study of ibuprofen enantiomers after administration of a new oral formulation (ibuprofen arginine) to healthy male volunteers[END_REF]. For these reasons it was chosen as a model drug in a previous [3] and the present work.

The aim of the present work was to challenge the validity of the present regulatory requirements on chiral bioanalytical methods in bioequivalence studies. For this purpose, the samples from a bioequivalence study of two ibuprofen 2% oral suspensions that had shown bioequivalence for AUC and C max , but not for t max , with a non-chiral method, were assayed with a chiral bioanalytical method to investigate an actual difference in rate of absorption within the limits of C max and AUC bioequivalence.

Materials and methods

Study design

A randomized, open-label, single dose, balanced, two-way crossover study was conducted with the approval of the Ethical Committee (CEIC-IMAS) and the Spanish Agency for Medicines and Health Care Products. Written informed consent was obtained from subjects after explaining the nature and purpose of the study. The 18 volunteers (nine males and nine females) were non-smokers; were determined to be in good health by physical examination, complete blood count, urinalysis, and serum test on hepatic and renal function; and refrained from taking any drug including OTC products for at least 1 week prior to or during the study. After fasting overnight for 8 h, a dose of 600 mg of ibuprofen 2% suspension was orally administered with 220 mL of water, either as a new test product (Laboratorios Gelos, Spain; batch: R-002, content: 102%), or as the reference product (Dalsy, Laboratorios Knoll, Spain; batch: R-50, content: 103%). Blood was drawn before dosing and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12 and 24 h after dosing through an in-dwelling catheter placed in an antecubital vein in the forearm. The blood samples were centrifuged, and the plasma was collected and stored at -20°C until assayed. Seven days after this first administration, the alternative formulation was given to complete the sequence.

Assay procedure

Ibuprofen plasma concentrations were determined by the sponsor following a validated non-chiral method already described [START_REF] Farré | Study on the bioavailability in extent and rate of ibuprofen tablets[END_REF]. Subsequently, samples were assayed for ibuprofen enantiomers using a chiral AGP-α1 column of 100×4 mm ID (Chrom Tech, Sweden) according to the method previously published [3]. Ibuprofen enantiomers were extracted from 0.6 mL of plasma (to which 0.15 mL of 3 N HCl was added) with 5 mL of cyclohexane. After 15 min of vortexing, the samples were centrifuged at 1,200 g for 5 min, and 3 mL of the organic phase was separated. The organic extracts were evaporated to dryness in a Savant Speedvac concentrator (Savant Instrument, USA) at 45°C under vacuum. The residue was then reconstituted with 1.5 mL of mobile phase and an aliquot of 100 μL was injected into the HPLC.

The assay was performed with a modular Jasco (Japan) liquid chromatograph equipped with a DG-2080-53 3-line degasser, a LG 2080-02 ternary gradient unit, a PU-1580 pump, an AS2050 Plus autosampler (100 µl sampling loop) and a UV-1575 UV-visible detector. Integration of the peaks was performed with the program Borwin 1.5 for PC (JMBS Developments). Separation and determination of the proportions of Rand S-ibuprofen enantiomers were achieved with a chiral AGP column and UV detection at 220 nm. The mobile phase was 990 mL phosphate buffer with orthophosphoric acid (pH 6.9)/10 mL acetonitrile/204 μL N, N-dimethyloctylamine at a flow rate of 1.5 mL/min.

Under these conditions Rand S-ibuprofen enantiomers had retention times of 2.2 and 3.5 min respectively. Resolution determined by the USP 29 method was of 2.1. Figure 1 shows the chromatograms of standards of racemic ibuprofen (Fig. 1a), pure S-ibuprofen (Fig. 1b) and a representative test sample (Fig. 1c). Linearity of the method was studied in the range between 0.25 and 30 μg/mL of Rand S-ibuprofen (0.25, 0.5, 1, 5, 10, 15, 20 and 30 µg/mL) using as reference compounds racemic ibuprofen and pure S-ibuprofen supplied by Shasun (India). The results were analysed by linear regression, and correlation coefficients obtained were always at least 0.98 for both enantiomers. The lower quantification limit was 0.25 μg/mL for both enantiomers with relative standard deviation of 7.4 and 7.0% for the Rand S-ibuprofen enantiomers respectively. Accuracy (tested at 1, 15 and 30 μg/mL) ranged from 96.2 to 102.4%. Inter-day precision during in-study validation (tested at 0.75, 7.5 and 22.5 μg/mL) was not higher than 4.5 and 5.6% for the Rand S-enantiomers respectively. Ibuprofen concentration data reported as racemic were obtained as the sum of the two enantiomers.

Pharmacokinetics

The estimation of the pharmacokinetic parameters was performed from the plasma concentration-time data by non-compartmental analysis using WinNonlin Professional, version 2.0 (Pharsight, USA). C max and t max were obtained directly from the plasma concentration-time data. The AUC to the last time with measurable concentration (C t ) of the analyte (AUC 0-t ) was estimated by using the linear trapezoidal rule. The apparent elimination rate constant (k e ) was calculated by least squares log-linear regression from data of the last sample time points of each plasma concentration-time curve. The AUC extrapolated to infinity AUC 0À1 ð Þwas determined by adding the extrapolated area (C t /K e ) to the AUC 0-t .

Statistical analysis

The two-way analysis of variance (ANOVA) for crossover design was used to assess the effect of formulations, periods, sequences and subjects within sequence on logarithmically transformed data of AUC 0-t , AUC 0À1 and C max [START_REF] Chow | Design and analysis of bioavailability and bioequivalence studies, 2nd edn[END_REF]. The parametric analyses of the data were performed with WinNonlin Professional. The comparison of enantiomers within each formulation was carried out with a similar methodology, considering S-enantiomer as test and R-enantiomer as reference.

Results

Figures 2 and 3 show the plasma concentration-time profile of total ibuprofen (sum of Sand R-enantiomers) and that of the Sand R-enantiomers of both formulations respectively. The bioequivalence analyses based on the original non-chiral bioanalytical method, the sum of the enantiomers and the individual Sand R-enantiomers are summarised in Table 1.

The test product showed bioequivalence to the reference product for C max and AUC when considering either the non-chiral concentrations or the sum of enantiomers. However, the C max of the eutomer (S-ibuprofen) and the AUC of the distomer (R-ibuprofen) were not bioequivalent.

The mean relative bioavailability of the sum of enantiomers of the test product was 13% higher for AUC and 10% lower for C max compared to the reference one, and these differences were statistically significant since their 90% confidence intervals did not include 100%. The mean relative bioavailability of the eutomer of the test product was 1% higher for AUC and 17% lower for C max than the reference one, and the difference was significant for C max . Conversely, the mean relative bioavailability of the distomer of the test product was 30% higher for AUC and 0.4% lower for C max , and the difference in AUC was significant. Obviously, the point estimates of AUC and C max of the racemate are located in between those of the enantiomers.

The ratio of S-enantiomer to R-enantiomer in each product for the main pharmacokinetic parameters, presented in Table 2, shows that S-ibuprofen is the overall prevalent enantiomer in plasma and those ratios were higher with the reference product. Moreover, the S/R enantiomer ratio changed over time and showed a different profile between products, with higher eutomer/distomer ratio in the formulation with the faster rate of absorption (Fig. 4).

Discussion and conclusions

The test product showed a slower rate of absorption, with more than 30 min delay in t max , despite a similar C max , and its marketing authorisation was rejected since, for ibuprofen, onset of action is considered clinically important. However, this criterion is somewhat subjective, and it might have been approved since AUC and C max were bioequivalent, and alternative metrics such as partial AUC [17] are not used due to their high variability. Furthermore, if this drug had been one for which onset of action was not clinically relevant, the test product would have been approved based solely on AUC and C max . Figure 3 and Table 1, however, show that the difference in C max of the eutomer is larger and its 90% confidence interval lies outside the bioequivalence limits, which supports the rejection of the application. Importantly, this illustrates that even when C max is equivalent there is room for large differences in rate of absorption because C max is not a sensitive metric for detecting differences in rate of absorption [START_REF] Ring | Sensitivity of empirical metrics of rate of absorption in bioequivalence studies[END_REF][START_REF] Rostami-Hodjegan | Sensitivity of indirect metrics for assessing "rate" in bioequivalence studiesmoving the "goalposts" or changing the "game[END_REF]. These differences in rate of absorption may produce very different enantiomer ratios in products containing drugs with non-linear absorption as defined by the FDA [START_REF]Guidance for industry. Bioavailability and bioequivalence. Studies for orally adminsitered drug productsgeneral considerations[END_REF].

Interestingly, the AUCs of the eutomer (S-ibuprofen) were more similar between products, but the AUCs of the distomer (R-ibuprofen) were more different when compared with the original non-chiral analysis or the sum of enantiomers. This could be considered as clinically irrelevant but shows a different biopharmaceutical quality.

The logical observation that the point estimates of the sum of enantiomers is between the point estimates of the enantiomers demonstrates again [3] that the results of chiral assay may be biased even when the eutomer is not the minor one. The difference in eutomer exposure is detected with less accuracy by non-chiral assay. This 6.5% shift in the C max point estimate changed the conclusion of the study despite the fact that the eutomer is the enantiomer with a higher exposure (20% in case of the generic and 50% in case of the reference since it is formulation dependent). The failure to conclude equivalence does not seem to be due to a higher variability since the range of the 90% CI of C max is similar. In summary, data generated using non-stereospecific assays may not be Table 2 illustrates the concept of an enantiomer exposure ratio that is formulation dependent or absorption rate dependent. The S/R AUC ratio showed a 34% difference (154 vs. 120%) and the S/R C max ratio a 20% difference (121 vs. 101%) between products. In our previous study [3] differences were smaller, 10% in AUC (123 vs. 114%) and 6% in C max (112 vs. 106%).

This study reminds that it is important to distinguish between non-linear kinetics (dose-or time-dependency) and non-linear absorption, as defined by the FDA [START_REF]Guidance for industry. Bioavailability and bioequivalence. Studies for orally adminsitered drug productsgeneral considerations[END_REF], which may occur even in cases of linear kinetics, since ibuprofen shows non-linear kinetics due to saturation of protein binding only at doses greater than 600 mg. However, the inversion of R-ibuprofen to S-ibuprofen [START_REF] Brocks | The pharmacokinetics of ibuprofen in humans and animals[END_REF] is responsible of this non-linear absorption. This is consistent with the observations by Boni et al. [5], who showed that non-linear absorption may occur in a drug with linear pharmacokinetics in both the racemate and the individual enantiomers such as etodolac.

It could be considered that ibuprofen enantiomers show similar pharmacokinetic characteristics based on selected literature data. For example, in our previous work the S/R ratio was shown to be bioequivalent in that reference formulation (Neobrufen 600 mg tablet) [3]. However, in the present study, the reference product (Dalsy 100 mg/5 mL oral suspension) showed a point estimate of 121% for C max and 154% for AUC, suggesting that the kinetics was not similar between the two enantiomers. Therefore, the similarity of pharmacokinetics between enantiomers should not be considered a compelling justification to use non-chiral bioanalytical methods, unless this similarity is thoroughly proven at a large range of different absorption rates.

Based on the results of this investigation it can be concluded that only two of the four FDA conditions have to be fulfilled to require a chiral bioanalytical method in bioequivalence studies. Measurement of individual enantiomers in bioequivalence studies should be recommended when (1) the enantiomers exhibit different pharmacodynamic characteristics, and (2) non-linear absorption is present, as expressed by a change in the enantiomer concentration ratio with change in the input rate of the drug, for at least one of the enantiomers.

Fig. 1

 1 Fig. 1 HPLC chromatograms of standard racemic ibuprofen (a), S-ibuprofen (b) and a representative test sample (c)

Fig. 3 Fig. 2

 32 Fig. 3 Mean plasma concentration-time curve (n=18) of ibuprofen enantiomers after oral administration of 600 mg of ibuprofen in the reference and test formulations. Vertical bars show standard error of the mean

Fig. 4

 4 Fig.4Mean S/R enantiomer plasma concentration ratio-time curve (n=18) after oral administration of 600 mg of ibuprofen in the reference and test formulations (calculated as mean of the individual ratios per time-point). Vertical bars show standard error of the mean

Table 1

 1 Results of the bioequivalence analyses of test and reference formulations obtained by non-chiral bio-analytical method and chiral bioanalytical method (S-and R-ibuprofen) Test/reference ratios for AUC and C max based on log-transformed data are expressed as percentages a Non-bioequivalent results Table 2 AUC and C max data comparison regarding the enantiomer and drug formulation

		Non-chiral	Sum of enantiomers	S-enantiomer	R-enantiomer
	AUC 0-t , point estimate (%)	111.29	113.27	101.16	130.03
	AUC 0-t , 90% CI (%)	107.23-115.49	105.73-121.35	96.67-105.86	118.86-142.24 a
	AUC 0À1 , point estimate (%)	111.39	113.32	101.57	129.06
	AUC 0À1 , 90% CI (%)	107.29-115.65	105.98-121.17	97.18-106.16	118.19-140.92 a
	C max , point estimate (%)	89.18	89.85	83.36	99.65
	C max , 90% CI (%)	82.77-96.09	82.19-98.23	76.05-91.36 a	87.84-113.05
	t max of the test (h)	2.0	2.5	2.5	2.25
	t max of the reference (h)	0.75	0.875	1	0.75

Acknowledgements This work was partially supported by a grant of the Complutense University and Santander to the research group 910939.

Conflict of interest None


Sabrina Crépin
email: sabrina.crepin@chu-limoges.fr

Marie-Laure Laroche

Bernard Sarry

Louis Merle

M.-L Laroche

Osteonecrosis of the jaw induced by clodronate, an alkylbiphosphonate: case report and literature review

Keywords: Biphosphonate, Osteonecrosis of the jaw, Clodronate, Etidronate, Alkylbiphosphonate, Aminobiphosphonate

Purpose To report on a case of osteonecrosis of the jaw (ONJ) in a patient treated with clodronate, an alkylbiphosphonate, and to draw attention to the risk of ONJ following treatment with all biphosphonates, whether they are alkyl-or amino-biphosphonates.

Case report Beginning at age 58 years, a female patient took clodronate for almost 13 years for a metastatic bone cancer. She also underwent chemotherapy and radiotherapy. Three months after the end of biphosphonate therapy, she suffered from toothache, and tooth 27 (left maxillary second molar) was extracted. A maxillary focus of osteitis with an oral sinus communication was discovered, and a maxillofacial denture prosthesis was grafted in September 2006. Some days later, the patient consulted her dentist for an ulceration of the oral cavity floor in front of tooth 33 (left mandibular canine) extending to the left inferior side of the lip. In October 2006, teeth 33 and 34 (left mandibular first premolar) were extracted. No secondary infection occurred. A complete healing was only observed 3 months

after the last extraction. ONJ due to alkylbiphosphonate treatment was diagnosed as bone reconstruction and mucous cicatrisation were delayed. Methods A systematic review was carried out to identify all cases of alkylbiphosphonate-induced ONJ by searching the Medline and Cochrane databases using 'osteonecrosis of the jaw', 'jaw diseases', 'osteonecrosis', 'diphosphonate', 'biphosphonate' (amino-, alkyl-or the international nonproprietary name) as the main search items. The search was limited to English-and French-language articles published between 1966 and February 2010. Results Our search identified 27 cases of alkylbiphosphonateinduced ONJ in the literature. Among these cases, only ten patients were on alkylbiphosphonate monotherapy; in the other cases, aminobiphosphonates had also been used. The clinical presentation of the alkylbiphosphonate-induced ONJ was similar to that most often encountered with aminobiphosphonate treatment. The duration of exposure before onset was higher with alkylbiphosphonates than with aminobiphosphonates, and dental procedures before ONJ were frequent. Conclusion Osteonecrosis of the jaw has been widely reported with various aminobiphosphonates, but data on the role of alkylbiphosphonates are scarce. As these latter drugs are less potent, a high cumulative dose through long-term exposure would appear to be necessary and would favour ONJ. Although the degree of risk for ONJ occurrence in patients on alkylbiphosphonates remains uncertain, it would be wise to reconsider carefully the indications for using these agents and to apply preventive measures as is currently done for aminobiphosphonates.

Introduction

Biphosphonates are synthetic analogues of pyrophosphate, an endogenous regulator of bone mineralization. In this heterogeneous family, two types of biphosphonates share a common phosphorus-carbon-phosphorus backbone: [START_REF] Roelofs | Molecular mechanisms of action of bisphosphonates: current status[END_REF] alkylbiphosphonates, also called first-generation biphosphonates (clodronate, etidronate and tiludronate), which are devoid of amine function on the lateral chain; (2) aminobiphosphonates, which are considered to be second-(alendronate, pamidronate) or third-generation biphosphonates (zoledronate, risedronate, ibandronate) (Table 1). The amine function is responsible for the increased potency of the biphosphonates: the inhibition of bone resorption is greater with aminobiphosphonate than with alkylbiphosphonates [START_REF] Roelofs | Molecular mechanisms of action of bisphosphonates: current status[END_REF][START_REF] Rogers | Cellular and molecular mechanisms of action of bisphosphonates[END_REF]. Biphosphonates are used to treat numerous disorders that affect bone, such as osteoporosis, bone metastases, hypercalcemia of malignancy and multiple myeloma (Table 1). Since aminobiphosphonates have much stronger activities than alkylbiphosphonates, the former are more widely used.

Unfortunately, biphosphonates induce a serious adverse effect-osteonecrosis of the jaw (ONJ). Osteonecrosis is a necrotic lesion of bone tissue characterized by a slow progression and by failure to heal spontaneously. It manifests itself by pain, soft-tissue swelling, gingival bleeding, exposed bone and non-healing extraction socket, and it induces serious sequelae. These lesions can develop into an osteomyelitis with purulent material often containing Actinomyces. Radiographs may show regions of mottled bone [START_REF]Expert panel recommendations for the prevention, diagnosis, and treatment of osteonecrosis of the jaws[END_REF][START_REF] Khosla | Bisphosphonateassociated osteonecrosis of the jaw: report of a task force of the American Society for Bone and Mineral Research[END_REF]. A differential diagnosis of biphosphonate related-ONJ can be considered if all of the following three characteristics are present: (1) patient is currently undergoing or has previously received treatment with a biphosphonate; (2) patient has an exposed bone in the maxillofacial region that has persisted for >8 weeks; (3) patient has no history of radiotherapy to the jaw [START_REF] Colella | American Association of Oral and Maxillofacial Surgeons position paper: Bisphosphonaterelated osteonecrosis of the jaws-2009 update: the need to refine the BRONJ definition[END_REF].

The first ONJ cases due to aminobiphosphonate treatment were reported in 2003-2004 by Marx et al. [START_REF] Marx | Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic[END_REF] and Ruggiero et al. [START_REF] Ruggiero | Osteonecrosis of the jaws associated with the use of bisphosphonates: a review of 63 cases[END_REF]. Since then, more than 3,000 cases have been published [START_REF] Wimalawansa | Insight into bisphosphonate-associated osteomyelitis of the jaw: pathophysiology, mechanisms and clinical management[END_REF], almost all of which have been linked to aminobiphosphonate treatments. Risk factors such as administration route, indication, duration of treatment and administrated cumulative dose of biphosphonates have already been described. Several other factors also seem to favour ONJ occurrence, including dental risk factors (tooth extraction, periodontal disease, other surgical or invasive dental procedure), which were present in nearly 70% of reported cases, cancer and anti-cancer therapy (local radiotherapy, chemotherapy etc), corticosteroids and various comorbidities (diabetes, anaemia, among others) [START_REF] Wang | Osteonecrosis of the jaws associated with cancer chemotherapy[END_REF][START_REF] Fehm | Bisphosphonateinduced osteonecrosis of the jaw (ONJ): Incidence and risk factors in patients with breast cancer and gynecological malignancies[END_REF][START_REF] King | Osteonecrosis of the jaw in patients receiving intravenous or oral bisphosphonates[END_REF][START_REF] Mavrokokki | Nature and frequency of bisphosphonate-associated osteonecrosis of the jaws in Australia[END_REF]. Only a few cases have been reported with alkylbiphosphonates.

We report here on a case of ONJ in a patient who received an alkylbiphosphonate, clodronate, for treatment of bone metastasis from breast cancer. After a systematic review to identify all published cases of alkylbiphosphonate-induced ONJ, we discuss the risk factors of biphosphonate-induced ONJ.

Case report

In March 1987, the patient, then a 58-year-old woman, was operated on for cancer of the right breast and subsequently underwent locoregional irradiation. Six years later, she had bone metastasis (sternum and L4) and was treated with chemotherapy (5-fluorouracile, carboplatin, vinorelbine, mitoxantrone), hormone therapy (aminogluthetimide, leu- In May 2006, the patient consulted a dentist for the occurrence of pain near tooth 27 (left maxillary second molar). One month later, this infected tooth was extracted, and a maxillary focus of osteitis with an oral sinus communication was discovered. A maxillofacial denture prosthesis was grafted in September 2006. Some days later, the patient again consulted her dentist, this time for an ulceration of the oral cavity floor in front of tooth 33 (left mandibular canine) extending to the left inferior side of the lip. In October 2006, teeth 33 and 34 (left mandibular first premolar) were extracted. No secondary infection occurred. A complete healing was only observed 3 months after the last extraction. Based on the delayed bone reconstruction and mucous cicatrisation, we diagnosed ONJ.

Analysis using the Naranjo probability scale revealed that an association between clodronate and ONJ was probable [START_REF] Naranjo | A method for estimating the probability of adverse drug reactions[END_REF].

Case reports of alkylbiphosphonate-induced ONJ

A systematic review of the literature was performed by searching the Medline and Cochrane databases using Mesh and non-Mesh terms. The main search terms were 'osteonecrosis of the jaw', 'jaw diseases', 'osteonecrosis', 'diphosphonate' and 'biphosphonate' (amino-, alkyl-or international nonproprietary name). The search was limited to English-and French-language articles published between 1966 and February 2010. Titles and available abstracts were scanned for relevance in order to identify paper requiring further consideration. The literature review was completed by a search done by hand accessing the references cited in all relevant publications. Among the 1,211 references identified, nine articles were selected in which cases of ONJ were identified as being induced by alkylbiphosphonate treatment.

The results of the search are shown in Table 2. Twentyseven cases were identified: 25 with clodronate and two with etidronate [START_REF] Fehm | Bisphosphonateinduced osteonecrosis of the jaw (ONJ): Incidence and risk factors in patients with breast cancer and gynecological malignancies[END_REF][START_REF] Lannoy | Avascular osteonecrosis of the jaws induced by diphosphonates[END_REF][START_REF] Montazeri | Oral sodium clodronate induced osteonecrosis of the jaw in a patient with myeloma[END_REF][START_REF] Phal | Imaging findings of bisphosphonate-associated osteonecrosis of the jaws[END_REF][START_REF] Senel | Severe osteomyelitis of the mandible associated with the use of nonnitrogen-containing bisphosphonate (disodium clodronate): report of a case[END_REF][START_REF] Starck | Failure of osseointegrated dental implants after diphosphonate therapy for osteoporosis: a case report[END_REF][START_REF] Trenque | Osteonecrosis of the jaw with biphosphonates: french pharmacovigilance data (Abstr 138)[END_REF][START_REF] Favia | Osteonecrosis of the jaw correlated to bisphosphonate therapy in non-oncologic patients: clinicopathological features of 24 patients[END_REF][START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: an analysis of 31 cases with confocal laser scanning microscopy[END_REF]. Among these 27 cases, clodronate was used alone in eight cases and etidronate alone in two cases. Indications of biphosphonates were varied: six patients had multiple myeloma, 12 had solid cancers, five had osteoporosis, one had solid cancer associated with osteoporosis and two patients required orthopedic surgery (one indication was unknown). Eighteen (67%) of the patients had required dental procedures. The mean duration of exposure before onset of ONJ was 35 months when clodronate or etidronate was used alone.

Discussion

Reported cases of ONJ have been less frequently associated with alkylbiphosphonate treatments (27 cases to date plus the one discussed here) than with aminobiphosphonate treatments (>3,000). Our patient showed characteristics similar to those reported in earlier cases of alkylbiphosphonate-induced ONJ, namely, an indication of malignant disease and long period of alkylbiphosphonate treatment [in our case, almost 13 years (151 months) with a cumulative amount of close to 7 kg]. Other risk factors of ONJ are chemotherapy and dental procedure.

The main risk factors for developing ONJ linked to biphosphonate use are administration route, indication and duration of exposure.

Biphosphonates given intravenously are usually considered to be more potent than those administered by the oral route and, therefore, more of a risk factor for inducing ONJ, as evidenced by the higher estimated incidence of ONJ (0-10%) in patients receiving intravenous biphosphonates than in patients on oral therapy (<1%) [START_REF] Edwards | Pharmacovigilance and reporting oversight in US FDA fast-track process: bisphosphonates and osteonecrosis of the jaw[END_REF]. The risk depends on the specific biphosphonate given, the dose, the duration of treatment and the dental history of the patient. However, the incidence rate also depends on the reporting source. Manufacturer-sponsored epidemiological studies have reported incidence estimates of ONJ of 0.1-1.8% [START_REF] Hoff | Frequency and risk factors associated with osteonecrosis of the jaw in cancer patients treated with intravenous bisphosphonates[END_REF][START_REF]Osteonecrosis of the jaw[END_REF], while independent epidemiological studies from clinicians and the International Myeloma Foundation report somewhat higher incidence estimates of between 5 and 10%. The fairly low incidence of ONJ with oral biphosphonates could also be related to a poor bioavailability and to poor treatment compliance. As ONJ is now a well-known adverse effect of intravenous biphosphonates, one can be falsely reassuring about the safety of oral biphosphonates. According to Yarom et al., of all the ONJ patients in case series reported in the literature, the relative proportion of orally administrated biphosphonate-induced ONJ ranges from 2.5 to 27.3% [START_REF] Yarom | Osteonecrosis of the jaw induced by orally administered bisphosphonates: incidence, clinical features, predisposing factors and treatment outcome[END_REF]. In most cases, alendronate, an aminobiphosphonate, was the oral biphosphonate mentioned because it is the most widely used oral biphosphonate. These authors reported on a personal series of 11 female patients (8.9% of their ONJ patients on biphosphonates) who developed ONJ after monotherapy with oral biphosphonate. The extent of the relative risk with oral biphosphonate treatment is still being debated. In a casecontrol study nested within a cardiovascular cohort, Etminan et al. determined the adjusted relative risk for the occurrence of ONJ among oral biphosphonate users to be 2.9 [95% confidence interval (CI) 1.7-5.1] [START_REF] Etminan | Use of oral bisphosphonates and the risk of aseptic osteonecrosis: a nested case-control study[END_REF]. In contrast, in another study, Jeffcoast found no increased risk of ONJ with oral biphosphonates in 335 patients treated with alendronate for 2 years [START_REF] Jeffcoat | Safety of oral bisphosphonates: controlled studies on alveolar bone[END_REF]. Khan or other metabolic diseases was not causally linked to the development of ONJ [START_REF] Khan | Canadian consensus practice guidelines for bisphosphonate associated osteonecrosis of the jaw[END_REF] The administration route seen previously as a risk is in fact linked to the indication. ONJ is more often reported in patients with cancer, with the most frequently reported cancers being multiple myeloma, breast, prostate and lung cancers and all cases with bone metastasis. However, the type of malignant disease does not seem to be associated with an increased ONJ incidence [START_REF] King | Osteonecrosis of the jaw in patients receiving intravenous or oral bisphosphonates[END_REF][START_REF] Marx | Bisphosphonate-induced exposed bone (osteonecrosis/osteopetrosis) of the jaws: risk factors, recognition, prevention, and treatment[END_REF][START_REF] Bamias | Osteonecrosis of the jaw in cancer after treatment with bisphosphonates: incidence and risk factors[END_REF][START_REF] Vahtsevanos | Longitudinal cohort study of risk factors in cancer patients of bisphosphonate-related osteonecrosis of the jaw[END_REF]. In a recent meta-analysis performed on biphosphonates used in breast cancer treatment, ONJ occurred in 13 of the 5,312 patients receiving biphosphonates (0.24%) versus one of the 5,382 patients (0.018%) who were on placebo. In all cases, zoledronate, an aminobiphosphonate, was used. Treatment with zoledronate was significantly associated to the occurrence of ONJ [odds ratio (OR) 3.23, 95% CI 1.7-8] compared with no use [START_REF] Mauri | Osteonecrosis of the jaw and use of bisphosphonates in adjuvant breast cancer treatment: a meta-analysis[END_REF]. ONJ has also been reported in patients with non-malignant conditions, such as osteoporosis [START_REF] Favia | Osteonecrosis of the jaw correlated to bisphosphonate therapy in non-oncologic patients: clinicopathological features of 24 patients[END_REF][START_REF] Woo | Narrative [corrected] review: bisphosphonates and osteonecrosis of the jaws[END_REF]. In a retrospective survey, the frequency of ONJ in osteoporotic patients was 0.01-0.04% (mainly on weekly oral alendronate), and if a tooth extraction was carried out, the frequency increased up to 0.09-0.34% [START_REF] Mavrokokki | Nature and frequency of bisphosphonate-associated osteonecrosis of the jaws in Australia[END_REF]. In a review on ONJ in patients with osteoporosis treated with biphosphonates, the authors identified 26 cases in 11 publications. The biphosphonates prescribed were alendronate (23 patients), risedronate, pamidronate and alendronate and zoledronate (1 patient each); all of these are aminobiphosphonates [START_REF] Pazianas | A review of the literature on osteonecrosis of the jaw in patients with osteoporosis treated with oral bisphosphonates: prevalence, risk factors, and clinical characteristics[END_REF]. The most recent case series reported on 24 non-oncologic patients with ONJ who were treated with biphosphonates: 15 patients with alendronate, two with risedronate, two with ibandronate, three with clodronate, one with clodronate+alendronate and one with clodronate+risedronate. Five of these cases involved the use of alkylbiphosphonates. The indication was osteoporosis in 20 of the 24 patients [START_REF] Favia | Osteonecrosis of the jaw correlated to bisphosphonate therapy in non-oncologic patients: clinicopathological features of 24 patients[END_REF].

Drug exposure is also an important risk factor. In one study, the median duration of exposure to bisphosphonates was 39.3 months for patients with ONJ compared with 19 months for patients with no ONJ (p=0.001) [START_REF] Bamias | Osteonecrosis of the jaw in cancer after treatment with bisphosphonates: incidence and risk factors[END_REF]. When biphosphonates are administered by the intravenous route to treat skeletal complications of malignancy, the annual doses of pamidronate or zoledronate (two aminobiphosphonates), are four-to tenfold higher than those used to treat osteoporosis [START_REF] Watts | Osteonecrosis of the jaw[END_REF]. The mean time from first drug exposure to occurrence of ONJ is variable according to the drug used: 9.4-18 months for zoledronate, 14.3-24 months for pamidronate and 12.1 months for pamidronate+zoledronate [START_REF] Marx | Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic[END_REF][START_REF] Ruggiero | Osteonecrosis of the jaws associated with the use of bisphosphonates: a review of 63 cases[END_REF][START_REF] Mavrokokki | Nature and frequency of bisphosphonate-associated osteonecrosis of the jaws in Australia[END_REF][START_REF] Durie | Osteonecrosis of the jaw and bisphosphonates[END_REF][START_REF] Palaska | Bisphosphonates and time to osteonecrosis development[END_REF]. The estimated cumulative hazard rate of developing ONJ has been reported to be 1% at 1 year and 21% at 3 years or 15% at 4 years [START_REF] Bamias | Osteonecrosis of the jaw in cancer after treatment with bisphosphonates: incidence and risk factors[END_REF][START_REF] Dimopoulos | Osteonecrosis of the jaw in patients with multiple myeloma treated with bisphosphonates: evidence of increased risk after treatment with zoledronic acid[END_REF] with zoledronate and 7% at 3 years with pamidronate [START_REF] Bamias | Osteonecrosis of the jaw in cancer after treatment with bisphosphonates: incidence and risk factors[END_REF]. In cases of oral use, patients in some studies received biphosphonate for a mean duration of 24-40 months before the occurrence of ONJ [12, Etidronate ( 34], while in other case series, the mean duration was 4.6 and 5.6 years, respectively [START_REF] Palaska | Bisphosphonates and time to osteonecrosis development[END_REF][START_REF] Marx | Oral bisphosphonateinduced osteonecrosis: risk factors, prediction of risk using serum CTX testing, prevention, and treatment[END_REF]. Based on these results, it would appear that long-term treatment and high doses of biphosphonate are probable risk factors of ONJ onset.

To date, only a relatively few cases of alkylbiphosphonateinduced ONJ have been reported (etidronate, tiludronate and clodronate), including 25 cases of ONJ with clodronate and two cases with etidronate [START_REF] Fehm | Bisphosphonateinduced osteonecrosis of the jaw (ONJ): Incidence and risk factors in patients with breast cancer and gynecological malignancies[END_REF][START_REF] Lannoy | Avascular osteonecrosis of the jaws induced by diphosphonates[END_REF][START_REF] Montazeri | Oral sodium clodronate induced osteonecrosis of the jaw in a patient with myeloma[END_REF][START_REF] Phal | Imaging findings of bisphosphonate-associated osteonecrosis of the jaws[END_REF][START_REF] Senel | Severe osteomyelitis of the mandible associated with the use of nonnitrogen-containing bisphosphonate (disodium clodronate): report of a case[END_REF][START_REF] Starck | Failure of osseointegrated dental implants after diphosphonate therapy for osteoporosis: a case report[END_REF][START_REF] Trenque | Osteonecrosis of the jaw with biphosphonates: french pharmacovigilance data (Abstr 138)[END_REF][START_REF] Favia | Osteonecrosis of the jaw correlated to bisphosphonate therapy in non-oncologic patients: clinicopathological features of 24 patients[END_REF][START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: an analysis of 31 cases with confocal laser scanning microscopy[END_REF]; no case has been found with tiludronate. The clinical presentation of alkylbiphosphonate-induced ONJ is very similar to that of aminobiphosphonate-induced ONJ. In the reported cases, the patients underwent dental procedures at the same frequency as those receiving aminobiphosphonates (67 vs. 70%) [START_REF] King | Osteonecrosis of the jaw in patients receiving intravenous or oral bisphosphonates[END_REF][START_REF] Mavrokokki | Nature and frequency of bisphosphonate-associated osteonecrosis of the jaws in Australia[END_REF]. The duration of exposure before the onset of ONJ seemed to be longer with alkylbiphosphonates than with aminobiphosphonates: 35 months when clodronate or etidronate were used alone versus 9-18 months for zoledronate and 14-24 months for pamidronate, both used alone [START_REF] Marx | Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic[END_REF][START_REF] Ruggiero | Osteonecrosis of the jaws associated with the use of bisphosphonates: a review of 63 cases[END_REF][START_REF] Mavrokokki | Nature and frequency of bisphosphonate-associated osteonecrosis of the jaws in Australia[END_REF][START_REF] Montazeri | Oral sodium clodronate induced osteonecrosis of the jaw in a patient with myeloma[END_REF][START_REF] Starck | Failure of osseointegrated dental implants after diphosphonate therapy for osteoporosis: a case report[END_REF][START_REF] Trenque | Osteonecrosis of the jaw with biphosphonates: french pharmacovigilance data (Abstr 138)[END_REF][START_REF] Favia | Osteonecrosis of the jaw correlated to bisphosphonate therapy in non-oncologic patients: clinicopathological features of 24 patients[END_REF][START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: an analysis of 31 cases with confocal laser scanning microscopy[END_REF][START_REF] Durie | Osteonecrosis of the jaw and bisphosphonates[END_REF].

Of the biphosphonates, the ones most likely to induce ONJ are aminobiphosphonates [START_REF] Woo | Narrative [corrected] review: bisphosphonates and osteonecrosis of the jaws[END_REF][START_REF] Diel | Pathophysiology, risk factors and management of bisphosphonate-associated osteonecrosis of the jaw: Is there a diverse relationship of amino-and non-aminobisphosphonates?[END_REF][START_REF] Van Den Wyngaert | Bisphosphonates and osteonecrosis of the jaw: cause and effect or a post hoc fallacy?[END_REF], possibly because they are more potent than alkylbiphosphonates. Indeed, pamidronate, alendronate and zoledronate are respectively 10-, 100-and 1,000-fold more potent than clodronate. The exact mechanism involved in the occurrence of ONJ with biphosphonate is unknown. Different pharmacological mechanisms of biphosphonate activity have been reported, and mechanisms also seem to differ according to the biphosphonate generation. Osteoclasts metabolize alkylbiphosphonates into cytotoxic adenosine triphosphate analogues that accumulate in osteoclasts and induce apoptosis. Aminobiphosphonates inhibit the mevalonate pathway implicated in cytoskeleton organization [START_REF] Diel | Pathophysiology, risk factors and management of bisphosphonate-associated osteonecrosis of the jaw: Is there a diverse relationship of amino-and non-aminobisphosphonates?[END_REF]. The cortical bone of the mandibule has a much higher turnover than appendicular sites. Therefore, biphosphonates will be selectively concentrated into the jaw, thereby changing the balance of bone resorption and bone formation and resulting in reduced bone turnover. The biphosphonate concentration in the bone of the jaw may be sufficient to be directly toxic to the oral epithelium [START_REF] Reid | Is bisphosphonateassociated osteonecrosis of the jaw caused by soft tissue toxicity?[END_REF]. Biphosphonates inhibit proliferation, differentiation and migration of osteoclast precursors. Aminobiphosphonates also inhibit various proteins involved in cancer growth and metastasis and have an antiangiogenic effect [START_REF] Gutta | Bisphosphonates and osteonecrosis of the jaws: science and rationale[END_REF].

Bone necrosis is considered to be time and dose dependant because of the long half-life of biphosphonates in bones [START_REF] Woo | Narrative [corrected] review: bisphosphonates and osteonecrosis of the jaws[END_REF], which can amount to 10 years. Although the activity of clodronate is weaker than that of aminobiphosphonates, the osteoclast-inhibiting action of such a huge cumulated dose (7 kg in our case) was probably marked and protracted. We there conclude that it is essential to obtain a complete history for every patient treated with biphosphonates in whom ONJ is suspected.

Finally, it should be noted that as the action mechanisms of alkylbiphosphonates and aminobiphosphonates are slightly different and as aminobiphosphonates are more potent, these latter drugs tend to be prescribed more often.

Conclusion

The occurrence of ONJ is now a well-known adverse event associated with relatively long biphosphonate treatment. The intravenous route, duration of exposure, cumulative dose and aminobiphosphonates are recognized risk factors. Biphosphonates-seldom alkylbiphosphonates-are more likely to be involved in ONJ. The risk profile of patients treated with alkylbiphosphonates was similar to that of patients receiving aminobiphosphonate treatments (patients with malignant disease and history of recent dental procedures). As alkylbiphosphonates are less potent, longterm exposure seems to be necessary for a high cumulative dose and the development of ONJ. Two hypotheses could be advanced to explain the small numbers of case-reports with alkylbiphosphonates:

alkylbiphosphonates are prescribed less often than aminobiphosphonates; as the duration of treatment before ONJ occurrence is longer with alkylbiphosphonates, the relationship between ONJ and alkylbiphosphonate intake is more difficult to assess; however, in most of the cases reported with alkylbiphosphonates, aminobiphosphonates were also used.

Finally, there is probably a trend towards a moderate over-risk with alkylbiphosphonates, but data are still insufficient to quantitate this relationship with ONJ as we found only 28 cases of alkylbiphosphonate-induced ONJ (27 in the literature plus our case). Among these cases, only 40% of the patients were administered alkylbiphosphonates alone.

A Mayo Clinic consensus-statement for the use of bisphosphonates in multiple myeloma has recently been published. The statement recommends that the duration of biphosphonate therapy should no longer be indefinite and that pamidronate is the biphosphonate of choice [START_REF] Lacy | Mayo clinic consensus statement for the use of bisphosphonates in multiple myeloma[END_REF] among the aminobiphosphonates. The International Myeloma Working Group recommends clodronate [START_REF] Durie | Use of bisphosphonates in multiple myeloma: IMWG response to Mayo Clinic consensus statement[END_REF].

Physicians should be alerted to the risk of ONJ occurrence in patients on alkylbiphosphonates (clodronate, etidronate, tiludronate). As there is no effective therapy for biphosphonate-associated ONJ, specific measures should to be taken, such as dental care before the initiation of therapy, communication between professionals prescribing biphosphonates and dental professionals and careful choice of the indications for use.

  ; M, male; U, unknown b Time of onset after the beginning of the biphosphonates therapy c Months Eur J Clin Pharmacol (2010) 66:547-554

Table 1

 1 Description of available biphosphonates L5 for 1 year). She was also given clodronate by the oral route from August 1993 onwards. Biphosphonate treatment was stopped after the discovery of renal failure in March 2006. The cumulative dose of clodronate amounted to 6.8 kg within a 151-month period (almost 13 years).

	Type of biphosphonate	Molecule	Indications	Relative potency	Route
				relative to
				etidronate
	Alkylbiphosphonate (first-generation	Etidronate	Paget disease, malignancy-related hypercalcaemia	1	oral
	biphosphonate)	Clodronate	Malignancy-related hypercalcemia	10	oral/IV
		Tiludronate	Paget disease	10	oral
	Aminobiphosphonate
	Second-generation biphosphonate	Pamidronate Malignancy-related hypercalcaemia , bone metastasis,	100	IV
			multiple myeloma
		Alendronate	Osteoporosis	1,000	oral
	Third-generation biphosphonate	Ibandronate	Bone metastasis, malignancy-related hypercalcaemia,	5,000	oral/IV
			osteoporosis
		Risedronate	Osteoporosis	5,000	oral
		Zoledronate	Osteoporosis, Paget disease	10,000	IV
	IV, Intravenous

  et al. also reported that low-dose biphosphonate use in patients with osteoporosis

Table 2

 2 Review of cases of osteonecrosis of the jaw induced by alkylbiphosphonate treatment

	Associated drugs	or risk factors	(number of	patients)	Unknown							Chemotherapy							Chemotherapy						Chemotherapy (6)	Radiotherapy (3)	Corticosteroids (1)	Unknown (4)				Radiotherapy		Chemotherapy with	corticosteroids	Unknown
	Outcome				Unknown							Healing after	alveoplasty and	antibiotherapy					Healing after	alveoplasty and	antibiotherapy				Six healing	Five not healing	Three unknown					Unknown		Unknown	Unknown
	Time of onset b	[mean;	min-max]		Unknown							40 months							5 years						Zoledronate:	[23; 2-52] c	Clodronate:	[29; 7-73] c	Pamidronate:	[48; 29-67] c		Zoledronate:	11 months,	pamidronate:	44 months,	clodronate:	33 months	Unknown	6 months
	Duration of	treatment	[mean;	min-max]	Unknown							4 years							5 years						Zoledronate:	[23; 2-52] c	Clodronate	[17; 4-45] c	Pamidronate:	[39; 29-48] c		Zoledronate: 11	months,	pamidronate:	11 months,	clodronate: 2	days	Pamidronate:	27 cycles,	Zoledronate:	19 cycles,	Clodronate:	unknown	6 months
	Age (years) Underlying Presentation	[mean; condition (number (number of patients)	min-max] of patients)		64 Multiple myeloma Chronic nasal infection/	sinusitis. Computed	tomography scan:	maxillary sclerosis,	sequestrum and mucosal	thickening in the right	maxillary sinus	41 Multiple myeloma Unsatisfactorily healing	socket after dental	extraction. Biopsy: necrotic	bone fragment + acute and	chronic inflammatory	changes in soft tissue +	Actinomyces	72 Multiple myeloma Exposed alveolar bone after	tooth extraction. Biopsy :	necrotic bone + dense	infiltration of plasma cells	+ lymphocytes/leukocytes	+ Actinomyces	[63; 41-81] Solid cancer breast In ten cases, after tooth	cancer (11) extraction	Multiple myeloma (2)	Unknown (1)				60 Multiple myeloma Mandibular localization	Spontaneous onset	U Breast cancer Tooth extraction for	periodontitis. Maxilla	U Osteoporosis Failure of restored dental	implants
	Authors Sex (number	of patients) a			[16] M							[15] M							[17] F						[19] F(11)	M (3)						[14] U		[10] U	[18] U
	Molecule	(number of patients)			Clodronate +	pamidronate (1)						Clodronate (1)							Clodronate (1)						Clodronate +	zoledronate (9)	Clodronate +	pamidronate (2)	Clodronate +	pamidronate +	zoledronate (3)	Clodronate +	pamidronate +	zoledronate (1)	Clodronate +	pamidronate +	zoledronate (1)	Etidronate (1)

Eur J Clin Pharmacol (2010) 66:547-554

Conflict of interest Authors have no conflict of interest and no funding to declare.