Hassane Sadou Yayé

Lamia Hassani

Philippe-Henri Secrétan

Martine Babiard

Haroun Aouati

Agnès Bellanger

Patrick Tilleul

Najet Yagoubi

Bernard Do

Ivo B Rietveld

Physical and chemical stability of a generic etoposide formulation as an alternative to etoposide phosphate

Keywords:

A method to quantify the decomposition products from etoposide was developed and validated. Degradation kinetics were studied and degradations products structures were elucidated using LC-HR-MS n and computational methods. Physico-chemical stability of etoposide in ready-to-use chloride or glucose solutions was determined The article is a direct consequence of the need to use alternative pharmaceuticals due to a global shortage of etoposide phosphate

Abstract

The generic Mylan ® etoposide (ETP) has been investigated as an alternative for Etopophos ® , in part due to a global shortage of the latter. The generic alternative is different both in its formulation and in its very limited stability (6 h at 25°C against 4 days for Etopophos ® ) once reconstituted in ready-to-use chloride or glucose solutions. Its intrinsic stability has been

Introduction

Stock-outs of medicinal products of major therapeutic interest have increased lately.

According to the ANSM (French National Agency for Medicines and Health Products) up to 530 stock-out alerts were reported in 2017, a 30% increase compared to 2016 [START_REF]Progress reports in 2017[END_REF]. The problem is in particular marked in a hospital context, where drug products are frequently subject to additional reconstitution and/or reformulation steps; hence the need to reassess the stability of modified formulations in many cases obtained from new manufacturers. In fact, the time span of drug expiry or "effective stability" is often well beyond that mentioned in the Summary of Manufacturers' Product Characteristics [START_REF] Bardo-Brouard | Stability of ipilimumab in its original vial after opening allows its use for at least 4 weeks and facilitates pooling of residues[END_REF][START_REF] Young | Evaluation of the physicochemical and functional stability of diluted REMSIMA ® upon extended storage-A study compliant with NHS (UK) guidance[END_REF]. It is in this context that at the end of 2016, due to a worldwide shortage of the prodrug Etopophos® (ETPO), we sought to replace it by a generic supplied by Mylan® (ETP) formulated with the drug etoposide itself.

Etoposide, chemically known as 4'-demethylepipodophyllotoxin 9-(4,6-O-R-Ethylidene-β-D-Glucopyranoside) or (5S,5aR,8aR,9R)-5-[[(2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano [3,2-d] [START_REF]Progress reports in 2017[END_REF][START_REF] Young | Evaluation of the physicochemical and functional stability of diluted REMSIMA ® upon extended storage-A study compliant with NHS (UK) guidance[END_REF]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5H- [START_REF] Bardo-Brouard | Stability of ipilimumab in its original vial after opening allows its use for at least 4 weeks and facilitates pooling of residues[END_REF]benzofuro [6,5-f] [START_REF]Progress reports in 2017[END_REF][START_REF] Young | Evaluation of the physicochemical and functional stability of diluted REMSIMA ® upon extended storage-A study compliant with NHS (UK) guidance[END_REF]benzodioxol-8-one, is a semi-synthetic derivative of podophyllotoxin that shows antitumor activity and is used to treat a broad spectrum of malignancies, among which lung cancer and leukemia [START_REF] Clark | The clinical pharmacology of etoposide and teniposide[END_REF][START_REF] Najar | Pharmaceutical and pharmacological approaches for bioavailability enhancement of etoposide[END_REF].

There are some notable dissimilarities between the previous and the new formulations. First, Etopophos is a lyophilized powder containing etoposide phosphate, which is a water-soluble (solubility of 150 mM) etoposide prodrug. The compound is rapidly and completely converted to ETP after intravenous injection with similar pharmacological properties and bioavailability compared to ETP. However, etoposide is poorly soluble in water [START_REF] O'neil | The Merck Index -An Encyclopedia of Chemicals, Drugs, and Biologicals[END_REF] and the formulation of Mylan® contains organic solvents and uses polysorbate 80 to increase its solubility [START_REF] Feng | Preparation and in vitro evaluation of etoposideloaded PLGA microspheres for pulmonary drug delivery[END_REF].

Moreover, the excipients differ from that of etopophos, which contains trisodium citrate and dextran 40, while ETP Mylan ® contains macrogol 300, anhydrous citric acid, polysorbate 80, benzyl alcohol and ethanol.

According to the Summary of manufacturers Product Characteristics (SmPC) the stability of ETP Mylan ® (6 hours at 25°C) is much less than that of etopophos (24 hours at 25°C and 7 days at 4°C) [START_REF]Summary of manufacturers Product Characteristics (SmPC) Etopophos ® Bristol Meyers Squib[END_REF][START_REF]Summary of manufacturers Product Characteristics (SmPC) Etoposide Mylan-France ® 20 mg mL -1[END_REF]. These stipulated stability ranges are a consequence of the design space and the control strategies [START_REF]HHS, International Conference on Harmonisation; Guidance on Q8 Pharmaceutical Development; availability. Notice[END_REF] of the manufacturers.

Nonetheless, pharmaceutical formulations identical to the one of ETP Mylan ® are accompanied with different and sometimes even contradictory shelf-life recommendations after reconstitution for patient use. For instance, Sandoz ® identifies the concentration as the key factor impacting the stability of the admixture listing 7 days until expiry for a concentration of 0.2 mg mL -1 and 12 hours for a concentration of 0.4 mg mL -1 irrespective of the storage temperature either around ambient temperature (between 15°C to 30°C) or in a refrigerator (between 2°C to 8°C) [START_REF]Summary of manufacturers Product Characteristics (SmPC) Etoposide Injection USP Sandoz-Canada, 20 mg mL-1[END_REF]. Accord healthcare limited ® links the stability to the nature of the reconstituted solution at ambient temperature (between 20°C to 25°C) with 96 hours in a sodium chloride solution (0.9 % w/v) and 48 hours in a glucose solution (5% w/v)) [START_REF]Summary of manufacturers Product Characteristics (SmPC) Etoposide 20 mg/ml Accord healthcare limited[END_REF]. Mylan Pharmaceuticals ® ULC-Canada recommends a 24-hour time window for the use of the admixture at ambient temperature (between 15°C to 30°C) irrespective of the concentration between 0.2 and 0.4 mg mL -1 [START_REF]Summary of manufacturers Product Characteristics (SmPC) Etoposide Mylan Pharmaceuticals ® ULC-Canada. Etoposide injection[END_REF].

The Parisian Hospitals, depending on the French SmPC for Etoposide Mylan ® were confronted with the reduced shelf life of the replacing product, which resulted in organizational complications for the hospital production units along with a worsening in patient care, due to the expiration of formulations before administration, lack of available preparations, and the necessity to administer large volumes.

Thus, in agreement with Mylan ® Laboratories, this study was carried out to extend the knowledge space [START_REF]HHS, International Conference on Harmonisation; guidance on Q9 Quality Risk Management; availability. Notice[END_REF] involving the stability behavior of reconstituted ETP improving the identification and the ranges of critical quality parameters defined in the SmPC in terms of effectiveness and safety. Impacts were explored of the concentration ("clinically common" = 0.68 mM (0.4 mg mL -1 ) and "clinically high" = 1 mM (0.6 mg mL -1 )), of the nature of the environment (NaCl 0.9% or Glucose 5%), of the interaction between content and container, and of the storage and conditioning temperatures on the drug product behavior. Degradation kinetics have been evaluated and the generated degradation products have been characterized by LC-HR-MS n , followed by an analysis of the chemical and physical processes governing the drug product instability. In addition, density functional theory (DFT) has been used to calculate the HOMO -LUMO energy gap, a molecular electrostatic potential map and the Mulliken charges in a further analysis of the fragility of the drug. In silico QSAR toxicity studies have been performed to compare the mutagenicity and developmental toxicity of the degradation products with the theoretical data of ETP.

Materials and methods

Materials and reagents

ETP vials of 34 mM (20 mg mL -1 ) were kindly provided by Mylan ® Laboratory (Saint-Priest, France). The ETP standard reference (European Pharmacopoeia, Sigma Aldrich, St Quentin-Fallavier, France) was used to validate the analytical method. Bags filled with 100 mL of sodium chloride (0.9% NaCl) or with glucose 5% (G5%) were obtained from Freeflex ® (Fresenius Kabi France SA, Sevres, France) and Easyflex ® (Macopharma, Mouvaux, France), respectively. Chemoprotect ® Microspike and Take Set ® (Codan®, Bischwiller, France), each equipped with a Luer-Lock connection system were stuck respectively into the drug vial and into the bags. Syringes purchased from Becton Dickinson® (Rungis, France) were connected to the Microspike for the withdrawal of the drug from the vials, and then injected into the bags. The Take Set ® was maintained in each bag and served for the sampling of the drug mixture by means of a syringe. Purified water (Fresenius, Sevres, France), formic acid and acetonitrile (VWR, Fontenay-sous-Bois, France) and high grade methanol (Merck, Guyancourt, France) were used for analytical purposes.

Preparation, storage, and stability study

NHS Pharmaceutical Quality Assurance Committee requirements [START_REF] Santillo | Standard Protocol for Deriving and Assessment of Stability, Part 1: Aseptic Preparations (Small Molecules), 4 edition NHS Pharmaceutical Quality Assurance Committee October[END_REF] were used as the basis for the study design (Table 1). Etoposide solutions in bags (n=128) were produced by diluting ETP Mylan ® in NaCl 0.9% or G5% to obtain final concentrations of 0.68 and 1 mM, respectively. Easyflex® and Freeflex® bags, made of polypropylene were used for the preparation. The preparations were carried out under a controlled atmosphere (ISO 5), partly under a laminar airflow containment hood and under aseptic isolators. The bags were stored in a refrigerator at 5 ± 0.1°C or at 25 ± 0.2°C in a drying oven (Thermofisher, Waltham, Massachusetts, USA). The stability of each preparation was tested in triplicate on day 0, 1, 3, 7, 14, 21 and 28. Various parameters were monitored: clearness by visual examination, turbidity by turbidimetry, pH, and characterization of degradation products by LC-HR-MS n . Twelve bags stored at ambient temperature were monitored on a semi-annual basis for 1 year. Table 1. Study design for robust stability study according to NHS Pharmaceutical Quality Assurance Committee [START_REF] Santillo | Standard Protocol for Deriving and Assessment of Stability, Part 1: Aseptic Preparations (Small Molecules), 4 edition NHS Pharmaceutical Quality Assurance Committee October[END_REF].

Instrumentation and analytical procedures

The concentration of etoposide in the different solutions has been monitored as a function of time with an LC system (Dionex UltiMate 3000 HPLC system, Les Ulis, France) equipped with a quaternary pump, a vacuum degasser, an autosampler and a UV detector.

The controlling software was Chromeleon® software version 6.80 SR11 (Dionex, Les Ulis, France). The selected analytical column was a Phenomenex C18 (250 nm x 4.6 nm; 5 µm), maintained at 25°C. The flow-rate, the injection volume, and the detection wavelength were set at 1 mL min -1 , 100 µL and 220 nm, respectively. In order to prevent any co-elution and include late eluting analytes, the LC parameters were optimized in terms of mobile phase composition, buffer concentration, pH, and temperature as described by Bakshi and Singh enabling the development of stability-indicating assay method (SIAM) capable of discriminating ETP and its degradation products or excipients without interferences with appropriate resolution values (Rs >1.5) and sufficiently sensitive to detect and quantify degradation products [START_REF] Bakshi | Development of validated stability-indicating assay methodscritical review[END_REF]. As a result, the optimum composition of the mobile phase consisted of ultrapure water adjusted to pH 3.8 by formic acid (solvent A) and of acetonitrile (solvent B) in gradient mode (0-12 min: 70% A; 12-15 min: 70/50% A; 15-20 min: 50/70% A). [START_REF] Bakshi | Development of validated stability-indicating assay methodscritical review[END_REF][START_REF] Hubert | Quantative analytical procedures: Harmonization of the approaches: Part II -Statistics[END_REF]. No special protection against ambient light was employed, as ETP is not considered sensitive to light.

The identification and structural elucidation of the degradation products have been carried out by LC-HR-MS with the formerly described LC system coupled to an electrospray-LTQ-Orbitrap Velos Pro system consisting of a double linear trap followed by an orbital trap (Thermo Fisher Scientific, CA, USA). Analyses were carried out in positive ion mode using the following conditions: source voltage 3.4 kV, source and capillary temperatures 300°C and 350°C, respectively, sheath gas and auxiliary gas nitrogen flows 40 and 20 arbitrary units, respectively S-Lens at 60%, 35% CEL (collision energy level) for high resolution fragmentation studies, mass range 100-800 m/z, a standard solution was used daily for external mass calibration and MS data processing was carried out using Xcalibur ® software (version 2.2 SP 1.48).

The pH was determined by means of an S40 SevenMulti pH meter (Mettler-Toledo, Greifensee, Switzerland) and limpidity and turbidity of the prepared ETP solutions were assessed at 620 nm using a spectrophotometer DU 640B (Beckman Coulter, Villepinte, France) on storage days 0, 1, 3, 7, 14, 21 and 28.

Lastly, interaction between content and container was investigated by exploring the topography of the bag's surfaces (Easyflex ® and Freeflex ® ) in the presence of ETP on storage days 0 and 7, using atomic force microscopy Innova AFM (Bruker, Palaiseau, France). The cantilever resonance frequency was 320 kHz using a silicon probe NCHV (Bruker) with a stiffness of 42 N/m. The scan rate was between 0.5 and 1 Hz, using tapping mode. For each bag, images of three different areas were recorded. The reference images were compared to those of the bags that had been in contact with ETP.

Validation protocol

ETP standard and drug product

Standard ETP was used as well as the commercial form of ETP provided by Mylan ® . Specificity was established on the basis of separations and peak purity analysed using the photodiode array detector. Linearity and accuracy were determined by defining five concentrations of ETP Mylan ® (102, 136, 170, 204, 238 µM) on three consecutive days.

Validation of linearity was defined by comparing slopes and intercepts at the origin between both products. For reproducibility and repeatability, 6 solutions of 170 µM were injected on three consecutive days. Limits of detection (LOD) and quantitation (LOQ) were determined by graphical method (signal-to-noise ratio) [START_REF]HHS, International Conference on Harmonisation; guidance on Q1A stability testing of new drug substances and products; availability. Notice[END_REF]. A solution of ETP 1 mM was prepared and subjected to -20°C and to +40°C to optimize the HPLC method.

For the quantification of the etoposide and its degradation products, the solutions were diluted 5 times with water to ensure that they all fell within the range of linearity of the method.

Degradation products (DP): Collection and quantification

Degradation products were observed when the product was exposed to 40°C. This meant that etoposide was temperature sensitive and degraded by thermolysis and/or thermocatalysis. As the degradation products were not commercially available, in order to obtain larger quantities of DPs for LC-MS characterization purposes, we have further forced degradation by exposing at 60°C while verifying that only the intensities of the DPs change but that the impurity profiles remain comparable. Subsequently, the DPs were separated using the HPLC and collected after the detector. The sample corresponding to each DP was kept open over a week at 25 °C to concentrate the fraction by solvent evaporation. The concentrations of degradation products were expressed in relative contents, i.e. by normalizing of areas using Chromeleon® software. The chosen method should provide a content estimate very close to the calibration quantification approach since the UV spectra of D1 and D2 have maxima comparable to that of etoposide, suggesting that the different response factors are equivalent under the chosen detection conditions. Therefore, the linearity and the accuracy for the quantification of the degradation products by HPLC were evaluated at the concentration range of 2.1 -19.2 µM in comparison with ETP.

Degradation kinetics

ETP degradation kinetics were evaluated at 40°C using LC-UV. ETP samples at 0.68 mM were prepared in triplicate and placed in an oven at 40°C (Thermofisher, USA) then removed and analyzed by HPLC at 0, 1, 2, 3, 7, 14, 21, and 28 days. A rate constant (k) and half-life (t1/2) were obtained from the experimental data.

Simultaneously, the collected degradation products were subjected to the same stress conditions as those of the ETP degradation conditions (pH at 3.8, in saline and glucose solution) to complete the mass spectrometry elucidation of the degradation pathways.

Stability study

For a better understanding of the phenomena around the physical and/or the chemical instability, two sets of stress testing experiments were conducted on ETP reconstituted solutions. First, ETP solutions (n=3) were frozen at -20°C for 2 hours then defrosted at ambient temperature. Second, ETP solutions (n=3) were subjected to 4 cycles of thermal variation, successively for one hour at 5°C and for one hour at 40°C. Possible interactions between the container (Easyflex ® and Freeflex ® ) and the content giving rise to modifications of the container's surface were investigated by AFM.

Computational details

In order to obtain stable molecular structures, the geometrical parameters of ETP were optimized using the hybrid density functional B3LYP method with the 6-31G(d,p) basis set.

The calculations were performed with the Gaussian 09 program. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) analysis provided the most likely charge transfer within the molecule from the ground state (HOMO) to the first excited state (LUMO). The HOMO-LUMO energy gap (∆E) reflecting the chemical activity of the molecule was also determined. Molecular electrostatic potential (MEP) analysis has been used to highlight the reactive sites of ETP [START_REF] Suhasini | Vibrational and electronic investigations, thermodynamic parameters, HOMO and LUMO analysis on Lornoxicam by density functional theory[END_REF]. In addition, Mulliken population analysis was performed to determine the differences in electronegativity of atoms within the molecule.

In silico toxicological assessment

After structural elucidation and intrinsic stability assessment, toxicity of degradation products was evaluated by in silico strategy. Two prediction softwares were used. The first one is Toxtree, aimed to predict the toxicity thresholds based on Cramer classification [START_REF] Kroes | European branch of the International Life Sciences Institute, Structure-based thresholds of toxicological concern (TTC): guidance for application to substances present at low levels in the diet[END_REF]. The other one, T.E.S.T (Toxicity Estimation Software Tool, version 4.1) was used for the evaluation of potential developmental toxicity and of mutagenic properties as per Ames test.

It appeals to a consensual-based approach making an average of the data produced from several in silico strategies [START_REF] Benigni | Mechanisms of Chemical Carcinogenicity and Mutagenicity: A Review with Implications for Predictive Toxicology[END_REF][START_REF] Hansen | Benchmark Data Set for in Silico Prediction of Ames Mutagenicity[END_REF].

Results and discussion

HPLC method optimization and validation

The chromatographic peaks of the API and the excipients are well-resolved demonstrating that the method can be used to accurately analyze the drug and its DPs without interferences. Two degradation products generated under stress conditions at 40°C and labeled DP1 and DP2, possess relative retention times of 0.37 and 0.92 compared to the peak of ETP (Figure 1). The HPLC response was linear for both the standard and the commercial form of ETP with respect to the concentration range 102 -238 µM, yielding correlation coefficients R of 0.9807 and 0.9911 respectively. The method was found to be precise with an RSD of 1.9%. LOD and LOQ were found to be 0.69 µM and 2.10 µM (Table 2).

As the standards of degradation products were not available, in view of the identical maximum wavelength absorption exhibited by ETP and its DPs (Figure S1 in the Supplementary materials), the method validation of the degradation products was performed with DP1 assuming a similar molar extinction coefficient to that of the standard solution of ETP. The method was found suitable for the quantification of the degradation products (Table 2). Table 2; Validation parameters of the HPLC method for ETP standard, ETP Mylan ® and the degradation products (DP1).

Physical-chemical stability of the drug product

Degradation kinetics

At ambient temperature (25 °C), DP1 appeared rapidly after just 2 days of storage; however, it remained below the identification threshold for up to 21 days of storage, which is within the recommendations of the International Council for Harmonization on the content and qualification of impurities in new drug products (< 0.2% for a Maximum Daily Dose comprised between 10 mg -2 g) [START_REF]Food and Drug Administration, HHS, International Conference on Harmonisation; revised guidance on Q3B(R) Impurities in New Drug Products; Availability. Notice[END_REF]. A second degradation product (DP2) was observed after 7 and 50 days of exposure at 40°C and 25°C, respectively. Neither of the degradation products was detected in the samples stored at 5°C. The formation of the degradation products increased proportionally with increasing temperature (Figure 1). The decomposition related to the formation of DP1 was fitted with a zeroth order kinetic model leading to a half-life (t1/2) of 119 days and a kinetic constant ( ) of 0.0028 mM day -1 for ETP in the prepared solutions at 25 °C.

Long term stability of reconstituted ETP

ETP Mylan ® solutions, produced under aseptic conditions in polypropylene bags (Freeflex ® , n = 64, and Easyflex ® , n = 64), did not show any significant degradation or precipitation over a period of 21 days at 25°C irrespective of the tested concentrations, container material, or solutions (based on a 5 % threshold). However, at 5°C, two Easyflex ® bags with ETP of 0.68 and 1 mM diluted with G5% showed a precipitation at day 15 and 21, respectively, demonstrated by an increase in absorbance at 620 nm. The concentration decreased dramatically down to 80% (day 15) and 65% (day 21) due to precipitation. These bags have been left out of further analysis. For all other bags irrespective of the temperature, clearness results were strongly correlated with turbidimetry measurements demonstrating an absence of precipitation; the concentration variation was found to be <5% over the test period of 28 days (Figure 2 and Table S1 in the Supplementary materials). The pH value remained stable over time (The standard deviation < ± 2.5%) irrespective of temperature, duration of storage, appearance of degradation products or precipitation (Figure 2).

Exploration of the surface topography (AFM)

As illustrated in Figure S2 (Supplementary materials), on the whole, the surface morphologies of the 2 types of bags (Freelex® and Easyflex®) were homogenous and reproducible. The roughness values of the surface of the reference bag compared to that in contact with ETP were 20.3 ± 0.7 nm / 17.1± 1.5 nm and 17.8 ± 0.8 / 19.2 ± 0.4 for Freeflex and Easyflex bags respectively at 20 x 20 μm 2 scales. At this stage, no particular modification as a result of the interaction between ETP and the polymers of the bags was observed. It can therefore be concluded that etoposide does not degrade the polypropylene bags. Moreover, etoposide does not appear to be adsorbed to the bags, indicating a complete absence of interactions between the API and its container.

Physical stability

Exposed to a freezing process at -20°C, the bags showed precipitation after defrosting at 20 °C (ambient temperature) rendering the solutions not fit for medical use. Those subjected to a successive thermal shock (refrigerator (5°C) and heat (40 °C)) remained clear. As ETP is poorly soluble in water, excipients (ethanol, Macrogol 300 and polysorbate 80) were employed in the formulation to increase water-solubility. Despite the favorable miscibility and solubility of the excipients with water, during the injection of ETP in the bags of sodium chloride or glucose solution, a temporary whitish veil is observed. Rapid stirring quickly disperses the Mylan ® formulation and prevents any precipitation, as demonstrated by a dozen of ETP bags stored for over 1 year at ambient temperature that remain clear.

The cold increases the supersaturation of ETP, which has a very low water-solubility of about 50 µM at 25 °C [START_REF] Gennaro | Remington: The Science and Practice of Pharmacy, 19th Ed[END_REF]. This is clearly confirmed by the precipitation of ETP in the bags that were subjected to -20°C. The precipitate has been analyzed by X-ray diffraction and was found to be amorphous in nature (Figure S3 in the Supplementary materials). Precipitation was also observed in two solutions stored at 5°C, which indicates that the Mylan ® formulation is, from a thermodynamic point of view, not stable at 5°C. Nonetheless, only 2 solutions of 64 in total showed precipitation, implying that the formulation provides an active kinetic barrier to precipitation. It is not certain whether the Mylan ® formulation is thermodynamically stable at 25°C, however no precipitation has been observed in solutions that were properly mixed after introduction of the ETP with its excipients in the bags with NaCl or glucose solutions.

Characterization of degradation products by LC-HR-MS n

The fragmentation pattern of ETP is a key factor to get access to the structure of its degradation products. The origin of each fragment was established through MS n studies and confirmed by high-resolution mass spectrometry analysis. The mass error was calculated to obtain the most probable elemental composition (Table 3). For the sake of simplicity, the nominal m/z has been considered. The ETP mass spectrum represented in Figure 3a shows that the molecular ion of ETP [M+H]+ has an m/z of 589 and exhibits eight daughter ions at m/z 435, 401, 383, 363, 299, 247, 229 and 185. Most of the fragments have already been described in the literature [START_REF] Chen | Highly sensitive liquid chromatography-electrospray mass spectrometry (LC-MS) method for the determination of etoposide levels in human serum and plasma[END_REF][START_REF] Kosjek | Biodegradability of the anticancer drug etoposide and identification of the transformation products[END_REF]. The fragmentation pattern shows two main parallel fragmentation pathways (Figure 3b). DP2 (C21H21O8 + ) the late onset degradation product has already been described in the literature as 4'-dimethylepipodophyllotoxin [START_REF] Gupta | Synthesis and structure-activity relationships among glycosidic derivatives of 4'-demethylepipodophyllotoxin and epipodophyllotoxin, showing VM26-and VP16-213-like activities[END_REF].

Table 3. Structures, accurate masses with errors, elemental compositions and MS n and relevant product ions of Etoposide and its degradation products.

Postulated degradation pathways of ETP in the drug product

After reconstitution in saline or glucose solution, ETP was shown susceptible to temperature.

The appearance and increase of the two DPs are correlated with the increase in temperature Furthermore, the separate samples of DP1 and DP2 collected by HPLC as described in the experimental section, showed no interconversion between each other while subjected to pH 3.8 at 40°C, suggesting independent degradation pathways. It is proposed that an initial protonation of the ether results mainly in DP1 (major product) and to a lesser extent in DP2 (minor product). In the literature, ETP was described to undergo a ring-opening reaction of the lactone ring in acidic media (pH<4) and epimerization (pH>6) [START_REF] Gómez-Bombarelli | Mechanisms of Lactone Hydrolysis in 622 Acidic Conditions[END_REF]. Such mechanism was not observed in the present study.

Toxicological assessment

The potential toxicity of the identified impurities was evaluated and compared with the ones of the precursor using quantitative structure-activity relationship (QSAR) tools. As described in Table S2 (Supplementary materials), the analysis did not identify any structural alerts related to mutagenicity for ETP or its degradation products based on its most up-to-date database. However, T.E.S.T has associated ETP, DP1 and DP2 to developmental toxicity risks. Based on the Cramer rules, Toxtree indicated that ETP and its degradation products might belong to class III, which includes unknown and unsafe products. Overall, the apparition of degradation products does not seem to lead to additional toxicity.

Conclusion

The present study provides information about the chemical and physical stability of reconstituted etoposide in aqueous solutions containing either NaCl or glucose (0.68 or 1 mM). Whereas in solutions kept at 25°C a slow increase in degradation products DP1 and DP2 could be observed, no decomposition was apparent in solutions kept at 5°C for at least 28 days. Chemically, the stability depends on hydrolysis followed by the departure of the glycoside moiety as the main degradation pathway of diluted ETP. No effect of the presence of sodium chloride or glucose has been observed.

In relation to the physical stability, solutions kept at 25°C did not show any precipitation, whereas at 5°C two solutions contained amorphous precipitates after 14 and 21 days. From this perspective, storage under cold conditions must be avoided as recommended by Sandoz® (SmPC Etoposide Sandoz®, 2015). Although the saturation concentration for the samples at 25°C is not known, the excipients appear to protect the active ingredient ETP against precipitation quite well, even at 5°C.

It is clear that the chemical and physical stability behaviors require different storage conditions and limit therefore the total storage time of the reconstituted product. Considering the identification / qualification threshold of the impurities [START_REF] Kosjek | Biodegradability of the anticancer drug etoposide and identification of the transformation products[END_REF] and the physical properties, ETP prepared under good manufacturing practices (i.e. in particular well controlled mixing when introducing the Mylan ® formulation in the respective NaCl and glucose solutions) remained unchanged over a period of 21 days at 25 °C irrespective of concentration, nature of the solvents (NaCl or glucose), or type of container. The occurrence of the degradation products above the defined acceptance limits does not appear to be associated with additional toxicity. In the light of these results, by applying a safety margin, the stability period after reconstitution may be set at a period of less than 21 days. In relation to the shelf life of the previously used reconstituted etopophos, i.e. 4 days at 25 °C, the stability data of the present replacement product after reconstitution provide hospitals with an opportunity to improve its production organization, while guaranteeing patient safety. These conclusions should be applicable too to other ETP-based proprietary pharmaceuticals with the same qualitative and quantitative compositions.

Since this study has been carried out, more than 20,000 reconstituted solutions have been produced by the Parisian Hospitals and no precipitation or excess degradation products have been observed within the 21 day limit. As a result, the study contributes to diminishing the cost of discarding ready-to-use preparations exceeding the limited official expiry date avoiding the environmental consequences. In addition, it provides the hospitals with a more flexible planning for the reserve of reconstituted solutions leading to less error-prone patient care. Lastly, the study also demonstrates the need of harmonization of the SmPCs on an international level, in particular for formulations that are the same in constituent and concentration even if produced by different manufacturers. Table 1. Study design for robust stability study according to NHS Pharmaceutical Quality Assurance Committee [START_REF] Santillo | Standard Protocol for Deriving and Assessment of Stability, Part 1: Aseptic Preparations (Small Molecules), 4 edition NHS Pharmaceutical Quality Assurance Committee October[END_REF].

Figure 1 .

 1 Figure 1. Chromatograms of ETP and degradation products (DPs) for several time intervals and storage temperatures.

Figure 2 .

 2 Figure 2. Long term stability of reconstituted etoposide Mylan ® (ETP): a) pH variation; b) Concentration variation of (ETP) for 0.68 and 1 mM in sodium chloride (Easyflex® bags); c) Concentration variation of ETP for 0.68 and 1 mM in G5% (Easyflex® bags)

  First, loss of dimethoxyphenyl has been identified, characterized by the transitions of m/z 589 m/z 435, m/z 401 m/z 247 and m/z 383 m/z 229. Then, rearrangement and a neutral loss of the glucopyranosyl moiety by O-dealkylation has been observed, illustrated by the transitions of m/z 589 m/z 401, m/z 589 m/z 383 and m/z 435 m/z 229. The fragment of m/z 383, the most intense peak, is generated both by a rearrangement leading to the departure of a 206 Da moiety (589 383) or by water loss through the ion m/z 401. The two fragmentation paths lead to a common fragment, with an intensive peak at m/z 229 (4,6,13-trioxatetracyclohexadeca-1(9),2,7,15-tetraen-12-one) through the loss of two moieties on either side of the four-ring core, namely the glycoside and the dimethoxyphenyl groups. Furthermore, the four-ring core breaks up through different mechanisms, such as the departure of formic acid (H2CO2) from the strained lactone ring demonstrated by the transitions m/z 383 m/z 337 and 229 m/z 185, or by rearrangement and neutral losses resulting from the departure of propadiene C5H8O3 (m/z 383 m/z 267) and carbon dioxide (m/z 383 m/z 299). The fragments of m/z 383 and 401 were identified to have the same structures and fragmentation patterns as DP1 and DP2 respectively. The accurate mass of the protonated ion of 383.1112 matched the molecular formula of DP1 (C21H19O7 + ). The structure was supported by the fragmentation pattern illustrated in figure 3b. Hence, compared to ETP, in DP1 the transition corresponding to the neutral loss of the glucopyranosyl derivative (C8H12O5 or C8H14O6) was absent implying that this moiety was impacted by degradation. Hence, DP1 is postulated to be 10-(4-hydroxy-3,5-dimethoxyphenyl)-4,6,13-trioxatetracyclohexadeca-1(9),2,7,15-tetraen-12-one.

Figure 3 .

 3 Figure 3. ETP MS spectrum (a) and fragmentation pattern (b).

  and were evidenced by high resolution mass spectrometry. The degradation occurred mainly through a mechanism of acid-catalyzed hydrolysis resulting in the departure of the glycoside chain. Due to its C-O bond, etoposide undergoes O-dealkylation by hydroxylation of α-carbon to form an unstable hemiacetal intermediate, which spontaneously cleaves to D2 and a carbonyl compound, D2' (Figure S4 in the Supplementary materials). Dehydration of D2 results in the formation of D1. The chemical degradation mechanism is supported by the theoretical molecular orbital analysis. Analysis of the frontier molecular orbitals: As illustrated in Figure 4.B, the HOMO is essentially localized on the phenyl ring (C23, C33, C36, C39) and on the oxygen atoms of the 4-hydroxy-3,5-dimethoxyphenyl moiety. The glycoside moiety does clearly not contribute to the HOMO. The LUMO is mainly located on the 1,3-benzodioxole moiety; the lactone ring (O2, O5 and C21) and the ether oxygen O1 contribute too (Figure 4.B). The calculated values of the ETP HOMO and LUMO energies are -0.2026 eV and -0.0154 eV respectively, leading to a HOMO-LUMO gap of ∆E (ETP) = 0.1872 eV. The small value indicates that onlylittle energy is required for excitation. ETP is therefore expected to be easily polarizable with a relatively high chemical reactivity[START_REF] Rauk | Orbital Interaction Theory of Organic Chemistry[END_REF][START_REF] Powell | A first-principles density-functional calculation of the electronic and vibrational structure of the key melanin monomers[END_REF].ETP electrostatic potential and Mulliken charges. The calculated electrostatic potential has been provided in Figure4.C. The potential increases from strongly negative values in red < orange < yellow < green < blue, with blue reflecting the highest positive values. The color code of the map was set between -7.480 au (deepest red) and 7.480 au (deepest blue). As illustrated in Figure4.C, the most negative electrostatic potential regions (red) were located around oxygen atoms O13, O5, O6 and O8. In contrast, the areas colored in blue, zones with the highest positive potential are comparable to the position of the LUMO. The Mulliken charges distribution obtained from the optimized ETP (Figure4.A) demonstrated that the C21 (lactone), C38 (1,3-dioxole), and C21 have the biggest positive atomic charges of 0.637e, 0.567e and 0.522e respectively. This is due to the large negative charges of the O2 (-0.490e) and O5 (-0.516e) of the lactone, the O9 (-0.553e) and O10 (-0.552e) of the 1,3-dioxole and the O1 (-0.546e) of the ether.It can be concluded that the reactive regions are localized around the polarized sites. The formation of DP1 and DP2 are the result of reactions involving the ether O1 directly connected to the electropositive carbon atom C22 of the glycoside. The effect of temperature has clearly been demonstrated in the ETP degradation process.

Figure 4 .

 4 Figure 4. A) Optimized structure of ETP; B) The frontier molecular orbitals and related energies; C) Molecular electrostatic potential (MEP) surface map for ETP

Figure 4 :

 4 Figure 4: A) Optimized structure of ETP; B) The frontier molecular orbitals and related energies; C) Molecular electrostatic potential (MEP) surface map for ETP

Acknowledgements

We sincerely acknowledge Mylan Laboratories and APHP for supporting the project. The authors declare no conflict of interest.


Jean-Philippe Collet

PhD Michel Zeitouni

MD Niki Procopi

MD; Jean-Sébastien Hulot

MD, PhD; Johanne Silvain

PhD Mathieu Kerneis

MD Daniel Thomas

MD Benoit Lattuca

MD Olivier Barthelemy

MD Yoan Lavie-Badie

MD; Jean-Baptiste B Esteve

MD Laurent Payot

MD Delphine Brugier

PhD Izolina Lopes

BSc Abdourahmane Diallo

PhD Eric Vicaut

PhD ; G Montalescot

MD Gilles Montalescot
email: gilles.montalescot@aphp.fr

Long-term Evolution of Premature Coronary Artery Disease Brief title: Evolution of premature coronary artery disease

Keywords: Premature coronary artery disease, young, myocardial infarction, long-term outcomes, recurrent event Abbreviations AFIJI: Appraisal of risk Factors in young Ischemic patients Justifying aggressive Intervention CABG: Coronary Artery Bypass Graft CAD: Coronary Artery Disease HIV: Human Immunodeficiency Virus MACE: Major Adverse Cardiovascular Events NSTEMI: Non ST-Segment Elevation Myocardial Infarction PCI: Percutaneous Coronary Intervention 4 STEMI: ST-Segment Elevation Myocardial Infarction AdjHR: adjusted hazard ratio

Background: The long-term evolution of premature coronary artery disease (CAD) is unknown. Objectives: To describe the evolution of coronary atherosclerosis in young patients and identify the risk factors of poor outcomes. Methods: Participants aged 45 years or less with acute or stable obstructive coronary artery disease were prospectively enrolled and followed-up. The primary endpoint was all-cause death, myocardial infarction, refractory angina requiring coronary revascularization and ischemic stroke.

Results: 880 patients with premature CAD were included. They were aged 40.1 years (±5.7), mainly males, smokers, with a family history of CAD or hypercholesterolemia. . At baseline presentation, 91.2 % underwent coronary revascularization, predominantly for acute MI (78.8%). Over a follow-up to 20 years, one third (n=264) presented a total of 399 ischemic events, of whom 36% had at least a second recurrent event. Myocardial infarction was the most frequent first recurrent event (n=131/264) mostly related to new coronary lesions (17.3 % vs 7.8%, p=0.01, HR=1.45, 95% CI [1.09-1.93] for new versus initial culprit lesion). All-cause death (n=55, 6.3%) occurred at 8.4 years (median time). Ethnic origin (sub-Saharan African vs Caucasian, adjHR: 1.95, 95% CI [1.13-3.35], p=0.02), inflammatory disease (adjHR: 1.58 95%CI [1.05-2.36], p=0.03)) and persistent smoking (adjHR: 2.32, 95% [1.63-3.28], p<0.01) were the strongest correlates of a first recurrent event. When considering all recurrent events, the same factors and Asian ethnicity predicted poor outcome, but persistent smoking had the greatest impact on prognosis. Conclusions: Premature CAD is an aggressive disease despite the currently recommended prevention measures with high rates of recurrent events and mortality. Ethnicity and concomitant inflammatory disease are associated with a poor prognosis along with insufficient control of risk factors.

CONDENSED ABSTRACT:

This prospective cohort study reports the contemporary natural history of obstructive CAD over 20 years in 880 patients aged ≤45 years. Ischemic recurrences were frequent (4.68 per 100 patient-years) with a fast progression towards multivessel disease. Ethnicity, chronic inflammation and behavior-related risk factors were the major contributors of poor prognosis. When considering all recurrent events, multivessel disease also predicted poor outcome, but persistent smoking had the greatest impact on prognosis. Premature CAD carries a high risk burden compared with atherothrombosis in middle-aged patients. Specific prevention should be implemented in these young patients to prevent the rapid progression of the disease.

Introduction

Ischemic heart disease accounts for the majority of premature deaths in the world [START_REF] Yusuf | Cardiovascular Risk and Events in 17 Low-, Middle-, and High-Income Countries[END_REF].

Coronary artery disease (CAD) risk factors explain more than 90% of the attributable risk of CAD, of which nearly half can be reduced by the adoption of a heart-healthy lifestyle [START_REF] Khera | Genetic Risk, Adherence to a Healthy Lifestyle, and Coronary Disease[END_REF][START_REF] Yusuf | Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study[END_REF].

However, the incidence of CAD increases in many regions of the world and starts at an earlier age, despite major advances in the prevention and treatment of atherosclerosis [START_REF] Arora | Twenty Year Trends and Sex Differences in Young Adults Hospitalized With Acute Myocardial Infarction[END_REF]. The long-term evolution of these young CAD patients in the contemporary era of secondary prevention remains, however, poorly known.

Premature CAD, defined as the occurrence of symptomatic obstructive coronary atherothrombotic lesions before the age of 45 years, has been described in a few registries of limited size, and restricted to the description of the patients baseline characteristics in a retrospective approach [START_REF] Lawesson | Gender perspective on risk factors, coronary lesions and long-term outcome in young patients with ST-elevation myocardial infarction[END_REF][START_REF] Avezum | Impact of age on management and outcome of acute coronary syndrome: observations from the Global Registry of Acute Coronary Events (GRACE)[END_REF][START_REF] Puymirat | Association of changes in clinical characteristics and management with improvement in survival among patients with ST-elevation myocardial infarction[END_REF][START_REF] Singh | Study of young patients with myocardial infarction: Design and rationale of the YOUNG-MI Registry[END_REF][START_REF] Mohammad | Prevalence and risk factors of premature coronary artery disease in patients undergoing coronary angiography in Kurdistan, Iraq[END_REF][START_REF] Sadeghi | Premature coronary heart disease and traditional risk factors-can we do better?[END_REF]. Neither the long-term outcome nor the precise contribution and evolution of the risk factors have been precisely reported. Such data are critical to know better the burden of CAD and its prognosis under optimal treatment, when the disease starts two decades in advance.

The Appraisal of risk Factors in young Ischemic patients Justifying aggressive

Intervention (AFIJI) multicenter prospective cohort was launched in 1996 to better characterize the patient profile, treatment and follow-up of premature CAD in the contemporary era of percutaneous myocardial revascularization, arterial coronary bypass graft (CABG) and potent secondary prevention pharmacotherapy. The goal was also to provide these young patients with optimal cardiovascular prevention measures and follow their evolution after a first unexpected serious cardiac event. We tested the hypothesis whether secondary events were more likely related to new coronary lesion or to the initial culprit lesion.

Methods

Study design and eligibility

Between April 1, 1996, and June 2017, 880 patients aged <45 years who survived the first manifestation of CAD were enrolled in the prospective ongoing AFIJI multicenter cohort study. This program was designed to identify risk factors of premature coronary artery disease and to provide a continuous prospective long-term follow-up [START_REF] Collet | Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis[END_REF][START_REF] Collet | Cytochrome P450 2C19 polymorphism in young patients treated with clopidogrel after myocardial infarction: a cohort study[END_REF][START_REF] Hulot | CYP2C19 but not PON1 genetic variants influence clopidogrel pharmacokinetics, pharmacodynamics, and clinical efficacy in post-myocardial infarction patients[END_REF]. Premature CAD was defined as the occurrence of an acute myocardial infarction or a symptomatic myocardial ischemia with an obstructive coronary artery disease (stenosis ≥ 70 %) before the age of 45 years. Myocardial infarction due to non-obstructive coronary artery disease was an exclusion criterion when ischemia or necrosis was not confirmed by cardiac MRI, as well as myocarditis, Tako-Tsubo myocardiopathy and coronary spasm. This prospective study was approved by the local Ethics committee, sponsored by the Assistance Publique-Hôpitaux de Paris, supported and driven by the ACTION Study Group. All patients provided a written informed consent prior to enrolment.

Data collection and follow-up

Baseline characteristics including risk factors, medical history, ethnicity and treatments were reported prospectively as previously described [START_REF] Collet | Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis[END_REF][START_REF] Collet | Cytochrome P450 2C19 polymorphism in young patients treated with clopidogrel after myocardial infarction: a cohort study[END_REF][START_REF] Hulot | CYP2C19 but not PON1 genetic variants influence clopidogrel pharmacokinetics, pharmacodynamics, and clinical efficacy in post-myocardial infarction patients[END_REF]. Familial history of CAD was defined as any coronary event that occurred in first-degree relatives before 60 years of age in men and 65 years in women.

Participants were followed-up by general cardiologists with also regular visits to participating tertiary centers (at least once every two years). All information was collected and updated in all patients, presenting or not an event. General follow-up surveys were carried out regularly (2003/2007/2009/2012/2017) to collect additional clinical information including risk factors, symptoms, treatments, regular stress tests and echocardiograms as well as the socioprofessional status. Incentives to participate in educational programs were also launched. In addition to classical risk factors, the presence of chronic inflammatory or immunosuppressive disease such as HIV infection, viral hepatitis or any other chronic inflammatory disease including cancer was recorded. Baseline and repeated coronary angiograms were reviewed by independent investigators at the ACTION study group angiography core laboratory. New ischemic events were classified as being related to the initial treated coronary lesion or to a new lesion on another coronary segment or artery.

Study objectives

The primary study objective was to determine the rate of a first recurrent major adverse cardiovascular event (MACE) at maximal follow-up and the related independent risk factors of MACE. The second objective was to determine whether recurrent events were related to the initial culprit lesion or to the occurrence of new lesions. The third objective was to determine the rate of repeated recurrences and identify their related independent correlates.

Endpoint Definitions

The primary endpoint was a composite of all cause death, myocardial infarction, refractory angina leading to coronary revascularization and ischemic stroke. Ischemic stroke was defined as an acute episode of focal or global neurological dysfunction as result of cerebral infarction. Myocardial infarction was defined as type 1 according to the Third Universal Definition of 2012 [START_REF] Thygesen | Third universal definition of myocardial infarction[END_REF]. Secondary endpoints were the individual components of the primary endpoint related to a new lesion site, the occurrence of heart failure (NYHA>2) and major bleedings according to the TIMI definition. Events were adjudicated by physicians not involved in the recruitment and follow-up of patients in the AFIJI program.

Statistical Analysis

Participants were classified according to the occurrence of a first MACE during the follow-up in the study. Continuous variables are presented as mean and standard deviation (SD) or median, as appropriate and compared using Student t-test in case of Gaussian distribution or Mann-Whitney U test in case of non-Gaussian distribution. Categorical variables are presented as counts and percentages and compared using Chi square test or Fisher's exact test in case of low number of events. Cumulative incidence rates were calculated and expressed as the number of new cardiovascular events divided by the number of patient-years of follow-up (number of events per 100 patient-years).

The primary analyses were based on the occurrence of a first MACE. To describe the frequency of cardiovascular events according to time, we used Kaplan-Meier curves for cumulative event-free survival for the prespecified primary endpoint. Follow-up of patients was censored at the 30 th of June 2017 irrespective of whether a first recurrent event corresponding to the primary endpoint had occurred. For the first set of analyses, the time to first cardiovascular event was also compared according to whether the event was related to the initial culprit lesion or to a new lesion using log-rank tests.

The secondary analyses involved the time evolution of diabetes and smoking status. Time to recurrences and time to risk factor evaluations were stratified and matched between patients with and without recurrences. For each variable, we evaluated the cumulative exposures from baseline to the first, second or third recurrent event for patients with MACEs, and to the last known status within the corresponding period of time for patients without recurrence. A generalized estimating equation with a trend test was used to assess the differences in the cumulative exposures of each risk factor between patients with or without recurrence.

The third set of analyses assessed the independent factors of recurrences. Because of the time-dependence between risk factors like LDL-C, smoking or diabetes and events, a stratified Cox procedure including these time-dependent covariates was performed to identify the independent variables associated with a first occurrence of MACE. This cox model was stratified according to the time delay from the disease onset to follow-up and recurrent MACE as previously described [START_REF] Ahmed | Modeling survival in colon cancer: a methodological review[END_REF]. Variables with a univariate p-value<0.2 as well as age, initial presentation (myocardial infarction or stable coronary artery disease), ethnicity, cardiovascular risk factors (familial history of CAD, active smoking, hypertension and dyslipidemia), LDL-C level, inflammatory disease and revascularization status were included in the model. Eventually, a pooled analysis using repeated measurements with time-dependent covariates was performed to identify the risk factors associated with multiple recurrences. The two-sided significance level was fixed at 5%. All the analyses were performed using the SAS software (9.4, SAS Institute, Cary NC, USA).

Results

Baseline characteristics

Patients were predominantly young males, active smokers, with a frequent family history of CAD and hypercholesterolemia while diabetes was less common. Mean LDL-cholesterol at baseline was 1.69±1.3 g/dL (Table 1). One out of ten patients had a chronic inflammatory or immunosuppressive disease including HIV (4.0%), viral hepatitis (1.3%), systemic auto-immune disease including polyarthritis or systemic lupus with antiphospholipid syndrome (1.3%) and cancer (3.5%) (Table 1). Acute myocardial infarction due to single vessel disease with subsequent percutaneous coronary revascularization was the most frequent clinical pattern. Few patients presented with coronary dissection (n=10) or coronary thrombosis after heavy physical exertion (n=11) without underlying stenosis. Secondary prevention treatments were used as recommended per guidelines and did not differ according to the occurrence of MACE (Table 1).

Fewer patients were exposed to ticagrelor in the MACE group versus the event-free group (5.6% versus 10.1%, p=0.006) and very few patients were on oral anticoagulation (Table 1).

Time to first event analyses

Detailed cardiovascular outcomes and vital status were obtained for all patients, except for five patients who were lost to follow-up (0.57%). The description of lipid-lowering therapies along follow-up and dual antiplatelet therapy durations are displayed in online table 1 and2. Over the 20-year follow-up, 30% of patients (n=264) presented a total of 399 MACEs corresponding to 4.68 (4.23-5.18) events per 100 patient-years (Table 2). Myocardial infarction was the most frequent first recurrent event (2.6 per 100 patient-years) while all-cause death (1.60 per 100 patient-years) and stroke (0.70 per 100 patient-years) were less frequent. The first nonfatal recurrent ischemic event was related to a new lesion site in 152 patients and to the initial culprit lesion in 69 patients (17.3% vs 7.8%, p=0.01, HR=1.45, 95% CI [1.09; 1.93] for new versus initial lesion, respectively) (Central Illustration). Among the 522 patients with single coronary vessel disease at baseline, 112 evolved towards symptomatic multivessel disease. Death as the first recurrent event was three times less frequent than myocardial infarction and occurred at a much later stage (8.4 vs. 3.7 years). Stroke occurred in less than 1% of patients (Table 2).

Patients with a first MACE were more likely to have uncontrolled risk factors with more frequent active smoking, diabetes, and with a higher LDL-cholesterol level prior to the first recurrent event; they were also more likely to be of Sub-Saharan Africa or of Asian origin (Table 1). Patients with multivessel disease were also more likely to suffer from an ischemic recurrence, as well as patients initially treated by CABG.

Twenty-one patients (2.4%) developed severe heart failure, five patients underwent heart transplantation and thirteen had a major bleeding. More than one-third of the patients of the AFIJI registry (295/880) had an event during follow-up as defined according to secondary endpoints.

Time to second or third recurrence

Of the 255 patients with a first non-fatal recurrence, 81 patients (36.0 %) had at least a second recurrent MACE (table 2).. The continuous monitoring of risk factors showed more frequent new diabetes and a greater exposure to active smoking before each recurrent MACE (Figure 1 A andB). The exposure to active smoking decreased but remained high in patients with multiple recurrences (from 50.9% to 35.0%) compared with patients free or ischemic recurrences (41.8% to 1.5%). Interestingly, one out of three patients with multiple recurrences eventually developed diabetes as compared to one out of ten among event-free patients.

Independent risk factors of poor outcome

The multivariate stratified Cox regression model based on repeated measurements with time-dependent covariates demonstrated that persistent smoking was the greatest correlate of a first recurrent event (adjHR: 2.32, 95% [1.63-3.28], p<0.01). Ethnic origin, (sub-Saharan African vs Caucasian, adjHR: 1.95, 95% CI [1.13-3.35], p=0.02), diabetes, (adjHR: 1.75, 95%CI [1.20 -2.55], p<0.01) as well as chronic inflammatory disease (adjHR: 1.58 95%CI [1.05-2.36], p=0.03 and multivessel disease at baseline were strong independent risk factors of a first recurrent MACE. When considering all recurrent events, the same factors plus hypertension and Asian ethnicity predicted poor outcome, but persistent smoking had by far the strongest impact on prognosis (adjHR: 2.70, 95% CI [2.05-3.55], p<.001) (Figure 2 A and B, Online Table 3 and4).

Discussion

The AFIJI cohort provides a prospective description of the contemporary long-term evolution of coronary atherothrombosis occurring in a cohort of young patients. Recurrent events were frequent, occurred early in the course of the disease and were mainly due to new coronary lesions. Insufficient control of modifiable risk factors, concomitant chronic inflammatory disease and Asian/sub-Saharan ethnicities were the major factors associated with a poor prognosis. Our findings shed light on where our efforts should focus to blunt this unfavorable evolution in one out of three patients who presented a first event before the age of 45 years.

All patients of the AFIJI registry were all-comers screened during hospital stay or at the outpatient clinic of the investigating centers. The large majority of patients had angiographically established CAD with and most of them presented with ST-elevation myocardial infarction. The few who did not undergo coronary revascularization presented with a myocardial infarction due to a coronary dissection (mostly in women) or to acute coronary thrombosis without significant underlying coronary stenosis. The high rate of recurrent myocardial infarction in the AFIJI registry demonstrates that premature CAD is an evolving disease. This high rate of ischemic events persisted through the different therapeutic eras, in spite of the advent of drug eluting stents, potent P2Y12 inhibitors and second-generation statins (Online Figure 1). Half of recurrences occurred within the first 4 years and 75% within the first 10 years of follow-up. Most of the secondary prevention trials testing the long-term benefits of secondary prevention interventions reported lower event rates than AFIJI, certainly because of a less aggressive disease in older patients, a shorter follow-up of these studies and consideration of the first recurrent event only(16-19) (online table 5). Remarkably, the first recurrent MACE on optimal secondary prevention occurred before the age of 60 in 98% of our patients, an age corresponding to the median age of CAD revelation in the general population.

Multivessel disease at baseline was logically an independent predictor of first recurrence and multiple recurrences. This was also true for patients who underwent CABG surgery with recurrent events in more than half of them. The progression of atherothrombosis from single to multivessel disease paralleled the number of recurrences. The PROSPECT study reported that major recurrent events were equally distributed between culprit and non-culprit lesions and we report here that new coronary lesions were involved in 2 out of 3 patients suggesting a fast progression of the disease in these young patients [START_REF] Stone | A prospective natural-history study of coronary atherosclerosis[END_REF]. This aggressive progression towards new lesion sites demonstrates the need for non-invasive multi-modal strategies able to capture the evolution and instability of subclinical atherosclerosis in young patients [START_REF] Ahmadi | Do plaques rapidly progress prior to myocardial infarction? The interplay between plaque vulnerability and progression[END_REF]. Among the promising technical advances, computed tomography angiography have enabled to describe the remodeling and necrotic aspects of plaques and relate them to the risk of coronary events [START_REF] Motoyama | Computed tomographic angiography characteristics of atherosclerotic plaques subsequently resulting in acute coronary syndrome[END_REF].

Poor control of cardiovascular risk factors is obviously of paramount prognostic importance. One out of two patients was still an active smokers at the time of the first recurrence, a rate that is consistent with recent European surveys [START_REF] Kotseva | EUROASPIRE IV: A European Society of Cardiology survey on the lifestyle, risk factor and therapeutic management of coronary patients from 24 European countries[END_REF]. A 7-fold decrease of active smoking was obtained with education programs but still 11% of the patients were active smokers at the end of our follow-up. Our data indirectly confirm that smoking cessation is the most efficient secondary prevention measure, especially before the age of 40 years [START_REF] Critchley | Mortality risk reduction associated with smoking cessation in patients with coronary heart disease: a systematic review[END_REF]. The other modifiable risk factors -diabetes and hypertension -were also independently associated with multiple recurrent MACEs, emphasizing the need for an aggressive secondary prevention in this population. Larger infarct size, poor recovery from myocardial injury, comorbidities and persistent inflammation are the known consequences of persistent active smoking triggering coronary recurrences, heart failure and cardiovascular death [START_REF] Haig | Current Smoking and Prognosis After Acute ST-Segment Elevation Myocardial Infarction: New Pathophysiological Insights[END_REF][START_REF] Sharma | Meta-Analysis Comparing Outcomes of Smokers Versus Nonsmokers With Acute Coronary Syndrome Underwent Percutaneous Coronary Intervention[END_REF]. The average level of LDL cholesterol in these young patients was noticeably higher than the average usual MI population [START_REF] Guerin | Association of Serum Cholesterol Efflux Capacity With Mortality in Patients With ST-Segment Elevation Myocardial Infarction[END_REF]. Although improving over time, the proportion of patients with on-treatment LDL cholesterol plateau levels above 0.7 g/L was high, suggesting unrecognized heterozygous familial hypercholesterolemia but also insufficient treatment intensity (online Table 1). [START_REF] Singh | Familial Hypercholesterolemia Among Young Adults With Myocardial Infarction[END_REF][START_REF] Singh | Cardiovascular Risk and Statin Eligibility of Young Adults After an MI: Partners YOUNG-MI Registry[END_REF].

The increasing proportion of new onset of diabetes over time in patients with recurrent adverse event is another intriguing finding which indicates partial failure of our behavioral programs with respect to physical activity and diet.

The 10% of patients with chronic inflammatory disease had accelerated atherothrombosis with more frequent recurrent events. Chronic inflammation together with the cardiometabolic effects of corticosteroids or antiretroviral drugs may have participated to CAD progression. High platelet reactivity on P2Y12 inhibitors and potential drug-drug interactions are also additional explanatory factors [START_REF] Hauguel-Moreau | Platelet reactivity in human immunodeficiency virus infected patients on dual antiplatelet therapy for an acute coronary syndrome: the EVERE2ST-HIV study[END_REF]. Ethnicity is a non-modifiable risk factor involving both genetics and habits -that impacted long-term outcomes of young patients [START_REF] Meadows | Ethnic differences in cardiovascular risks and mortality in atherothrombotic disease: insights from the Reduction of Atherothrombosis for Continued Health (REACH) registry[END_REF]. Young sub-Saharan African patients carried the higher burden of cardiovascular disease. Previous similar findings have associated this higher risk with a more frequent hypertension, diet habits and unequal access to prevention and healthcare [START_REF] Howard | Association of Clinical and Social Factors With Excess Hypertension Risk in Black Compared With White US Adults[END_REF]. Asian ethnicity is particular to the AFIJI registry recruitment with patients originating from south-east Asia where the prevalence of diabetes and multivessel disease is high. Several Indian and South-east Asian registries have described an early onset of coronary artery disease, with a median age of 55 years old compared to 65 years old in western countries [START_REF] Sharma | Premature Coronary Artery Disease in Indians and its Associated Risk Factors[END_REF].

Ethnicity, chronic inflammatory disease, familial history of premature CAD and early menopause have been listed as risk-enhancers in the American guidelines on blood cholesterol management [START_REF] Grundy | AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines[END_REF]. The present results display additional evidence and variables to support tailored secondary prevention strategies for these young patients. Non-invasive imaging methods to better predict the potential evolution of the non-culprit coronary plaques in these specific subgroups needs further investigation. In particular, CT scan data combined with deep phenotyping including LDL-cholesterol on statin therapy may further help refinement of the intensity of lipid-lowering therapies with dedicated machine learning algorithms [START_REF] Grundy | AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines[END_REF][START_REF] Tota-Maharaj | Coronary artery calcium for the prediction of mortality in young adults <45 years old and elderly adults >75 years old[END_REF][START_REF] Kanaya | Comparing Coronary Artery Calcium among U.S. South Asians with Four Racial/Ethnic Groups: The MASALA and MESA studies[END_REF].

Whether we can improve the long-term prognosis of these patients is a pending issue.

Better control of the risk factors and treatment adherence are known challenges for secondary prevention. It is likely that a more aggressive secondary prevention using PCSK9 inhibitors and new antidiabetic drugs, which reduce both MACE and mortality, would be of incremental value in this high-risk population with frequent dyslipidemia and new onset of diabetes [START_REF] Sabatine | Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease[END_REF][START_REF] Zinman | Cardiovascular Outcomes, and Mortality in Type 2 Diabetes[END_REF][START_REF] Mann | Liraglutide and Renal Outcomes in Type 2 Diabetes[END_REF].

Lifestyle changes is often difficult to obtain but the potential benefit of the Mediterranean diet and physical activity are well demonstrated [START_REF] Khera | Genetic Risk, Adherence to a Healthy Lifestyle, and Coronary Disease[END_REF][START_REF] Estruch | Primary Prevention of Cardiovascular Disease with a Mediterranean Diet Supplemented with Extra-Virgin Olive Oil or Nuts[END_REF]. The residual risk related to chronic inflammation is another target in this young population. Interleukin-1β inhibition with canakinumab was effective in reducing cardiovascular events in a different high-risk atherosclerosis population, particularly those with elevated markers of inflammation [START_REF] Ridker | Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease[END_REF]. Antiinflammatory therapy may have cardiovascular efficacy in AFIJI-type patients considering the independent risk prediction of inflammatory disease we observed. Whether tailored therapy according to inflammatory biomarkers may improve outcome of these young patients needs to be addressed.

Limitations

There are several limitations inherent to this long-term prospective registry. First, advances in secondary pharmacological prevention treatment and in myocardial revascularization technology may have created a time-confounding bias not entirely addressed by the use of time-dependent variables and the time-stratified cox model. Similarly, statistical modelling could only partially balance the discrepancies in the measurement of time-dependent variables between patients with and without recurrent events. Second, asymptomatic patients did not undergo systematic coronary imaging investigations and we may have underestimated progression of the disease in some of these patients. Third, this AFIJI cohort outlines the role of ethnicity which may be confounded by other socio-economic factors. Finally, changes in physical activity or diet were not captured in this survey as well as psychological traits, all being additional prognostic factors of coronary disease.

Conclusions

Premature coronary artery disease is a chronic and aggressive disease, with a high rate of recurrences and a rapid evolution towards multivessel disease. Ethnicity and chronic inflammation, along with traditional modifiable risk factors including pursuit of smoking and new onset of diabetes appear to be important factors of poor outcomes. Intensification of the current secondary prevention measures is desirable to prevent the evolution of the disease towards multivessel disease, repeated coronary events and heart failure.

CLINICAL PERSPECTIVES

Competency in Medical Knowledge: Ethnicity, chronic inflammation and behavior-related risk factors are the most important contributors to the poor prognosis of young patients with premature coronary artery disease.

Translational Outlook: More research is needed to design and validate specific programs that reduce the incidence of ischemic events in patients with premature coronary artery disease through modification of behavioral and biological risk factors.

Figure 1 :

 1 Figure 1: Cumulative exposure to time-dependent major risk factors according to the

Figure 2 :

 2 Figure 2: Hazard Ratio plot of multivariate stratified Cox Model using repeated

Table 1 :

 1 Baseline patient's characteristics according to occurrence of a first MACE.Footnote: CAD stands for coronary artery disease, LDL-Cholesterol for low-density lipoprotein cholesterol, MINOCA for myocardial infarction with non-obstructive coronary artery disease, LVEF for left ventricle ejection fraction, CABG for coronary artery bypass grafting, PCI for percutaneous coronary intervention, DES for drug-eluting stent, BMS for bare-metal stent, ARB for angiotensin II receptor blockers, ACE-inhibitors for Angiotensin converting enzyme inhibitors. *corresponds to the values observed before the first recurrence.

	Baseline characteristics	Total Population n= 880	Recurrent events n = 264	n=616 No recurrent events	P value
	Age -year (mean)	40.1 ± 5.7	39.6±5.7	40.2±5.7	0.1
	age -year (median)	41.4 (36.5-44.2)	41.5 (36.9-44.3) 41.1 (36.0-44.0)
	Age < 35 %	160 (18.2%)	58 (22.0%)	102 (16.6%)
	Female gender	117 (13.3%)	33 (12.5%)	84 (13.6%)	0.1
	Body mass index (kg/m 2 )	26.1 ± 4.3	26.1 ± 4.7	26.1 ± 4.2
	Ethnic group
	White European	638 (72.5%)	189 (71.6%)	449 (72.9%)	0.3
	North Africa & Middle East	166 (18.9%)	45 (17.0%)	121 (19.6%)	0.5
	Sub-Saharan Africa	46 (5.2%)	16 (6.1%)	30 (4.9%)	0.2
	Asian continent	30 (3.4%)	14 (5.3%)	16 (2.6%)	0.02
	Admission event
	Myocardial infarction	693 (78.8%)	195 (73.9%)	498 (80.8%)	0.8
	Anterior	311 (35.3%)	87 (32.6%)	224 (36.3%)
	Inferior	274 (31.1%)	72 (27.7%)	202 (32.8%)
	Lateral	63 (7.2%)	19 (7.2%)	44 (7.1%)
	Non-specific electric signs	45 (5.1%)	17 (6.4%)	28 (4.5%)
	Stable angina	187 (21.3%)	69 (26.1%)	118 (19.1%)	0.5
	Risk factors*
	Familial history of CAD	359 (40.8%)	121 (45.8%)	238 (38.6%)	0.09
	Active cigarette smoking	680 (77.3%)	210 (79.6%)*	470 (76.3%)*	0.1
	Dyslipidaemia	443 (50.3%)	155 (58.7%)	288 (46.8%)	0.01
	LDL-Cholesterol (g/L)	1.69±1.30	1.99±1.46*	1.56±1.20*	0.004
	Arterial hypertension	178 (20.3%)	75 (28.4%)	103 (16.7%)	0.002
	Diabetes	94 (10.7%)	41 (15.5%)*	53 (8.6%)*	<0.001
	Creatinine Clearance	130.3±44.9	126.7 ± 58.8	131.9±37.4	0.3

Table 2 :

 2 Major adverse cardiovascular events. Footnote: * (%) is the percentage of patients with a second recurrent MACE out of patients who had a first non-fatal recurrent MACE ; ** (%) is the percentage of patients with 3 or more recurrent MACE out of patients with a second non-fatal second recurrent MACE. STEMI stands for ST-segment elevation Myocardial Infarction, NSTEMI for Non-ST-segment elevation myocardial infarction, PCI for Percutaneous Coronary Intervention, CABG: coronary artery bypass grafting.

		1 st recurrence	Time in years	2nd recurrence	Recurrences ≥ 3	Total Events
		n=264/880	median, (25 th and	n=81/225*	n=54/70**	Counts
		(30.0%)	75 th percentile)	(36.0%)	(77.1%)
	All-cause Death	39 (4.4)	8.4, (3.2-15.9)	11 (4.9)	5 (7.1)	55
	Myocardial Infarction	131 (14.9)	3.7, (0.8 -7.8)	40 (17.8)	38 (54.3)	209
	STEMI	47 (5.3)	4.1, (0.8-7.9)	10 (4.4)	12 (17.1)	69
	NSTEMI	84 (9.5)	3.5, (0.7-7.7)	30 (13.3)	26 (37.1)	140
	Refractory Ischemia requiring revascularization	88 (10.0)	5.1, (1.4-11)	28 (12.4)	11 (15.7)	127
	PCI	66 (7.5)	6.3, (1.7-10.7)	18 (8.0)	2 (2.9)	85
	CABG	22 (2.5)	2.8, (0.9-12.3)	10 (4.4)	9 (12.9)	41
	Ischemic Stroke	6 (0.7)	5.4, (1.8-8.4)	2 (0.9)	0 (0)	8
						399 total events
	Primary endpoint	264 (30.0)	4.2, (1.3-9.8)			4.68 (4.23-5.18)
					events per 100 patient-years

The study was led by the ACTION Study Group at the Institute of Cardiology of Pitié-Salpêtrière Hospital. (www.action-coeur.org) Disclosures JP Collet has received research grants from AstraZeneca, Bayer, Bristol-Myers Squibb, Daiichi-Sankyo, Eli-Lilly, Fédération Française de Cardiologie, Lead-Up, Medtronic, MSD, Sanofi-Aventis, WebMD. M. Zeitouni has received research grants from Institut Servier and Federation Française de Cardiologie. Jean-Sebastien Hulot has received research grants (to the institution) from Fédération Française de Cardiologie, Institut Servier, Sanofi; consulting fees from Novartis, Servier; J. Silvain has received research grants from Amed, Amgen, Algorythm, Astra-Zeneca, Bayer, Daiichi-Sankyo, Eli Lilly, Fondation de France, Gilead Science, Iroko Cardio, Sanofi-Aventis and Saint-Jude Medical. Mathieu Kerneis has received research grants from Féfération Francaise de Cardiologie and Institut Servier. Daniel Thomas has received sponsorship to attend scientific meetings and speaker or consultancy honoraria from Pfizer, Novartis and Pierre Fabre Santé. B. Lattuca has received research grants from Biotronik, Daiichi-Sankyo and Fédération Française de Cardiologie; consultant fees from Daiichi-Sankyo and Eli Lilly; and lecture fees from AstraZeneca and Medtronic. Lilly; consultancy from Pfizer, Sanofi, LFB, Abbottand grants from Boehringer and Sanofi. G Montalescot has received research grants from Abbott, Amgen, Actelion, AstraZeneca, Bayer,