Raphaelle Fanciullino

Optimization of trans-Resveratrol bioavailability for human therapy

We have developed an innovative soluble galenic form to overcome the low absorption of trans-Resveratrol (t-Res) as a dry powder. We present here data on pharmacokinetics, bioavailability, and toxicity of t-Res in human volunteers treated with this soluble form, plus additional data on biological effects in rodents. Fifteen healthy volunteers of both sexes received 40 mg of t-Res in two forms, the soluble formulation (caplets) and the original powder (capsules), in a crossover design. Blood samples were collected at 15 min, 30 min, and every hour for 5 h. Plasma concentrations of t-Res and its metabolites were analyzed by liquid chromatography and mass spectrometry. The single dose (40 mg) of the soluble t-Res was well absorbed and elicited biologically efficient blood levels (0.1-6 μM) for several hours, despite metabolization into glucuronide and sulfate conjugates coupled to renal elimination. In contrast, t-Res administered as a dry powder barely elicited efficient blood levels for a short duration. The new formulation led to 8.8-fold higher t-Res levels in plasma versus the powder. t-Res metabolism was not modified and neither intolerance nor toxicity were observed during the study and the following week. The soluble formulation elicited a robust anti-inflammatory effect in various tissues of mice fed a high-fat diet, while dry powder t-Res was almost inactive. Our data suggest that significant improvements in t-Res bioavailability and efficiency can be obtained by this soluble galenic form, also allowing lower doses. The use of t-Res in human therapy is thus greatly facilitated and the toxicity risk is reduced.

Early severe toxicities after capecitabine intake: possible implication of a cytidine deaminase extensive metabolizer profile

We report here the case of a 19-year-old female patient who suffered from extremely severe toxicities (G4 mucitis, fever, diarrhea, alteration of general state) while undergoing low-dose capecitabine treatment for her metastatic corticosurrenaloma. The severe toxicities stopped as soon as treatment was suspended. Interestingly, this patient was not deficient in DPD, a pharmacogenetic syndrome usually associated with increased risk of developing severe/lethal toxicities in patients undergoing fluoropyrimidine therapy, and she had been treated previously with 5-FU with a good tolerance. We then hypothesized that cytidine deaminase (CDA) extensive phenotype could be responsible for the severe toxicities observed with capecitabine. CDA is affected by genetic polymorphism, with subsequent acquisition of either deficient or extensive metabolizer profile. Phenotypic investigations confirmed that CDA activity in this patient was +180% higher than the ones usually recorded in the general population. This strongly suggests that the extensive activation of triple-prodrug capecitabine could have occurred in this patient, resulting in overexposure to 5-FU and its cytotoxic metabolites eventually. This case report suggest for the first time that severe toxicities with a capecitabine-containing protocol could be, at least in part, linked with an extensive-CDA syndrome. The case reported here suggests therefore that besides DPD, screening for CDA activity could be of interest to ensure a better safety in the handling of oral capecitabine at the bedside.

In Vitro and In Vivo Evaluation of Lipofufol, a New Triple Stealth Liposomal Formulation of Modulated 5-Fu: Impact on Efficacy and Toxicity

PurposeDrug resistance and severe toxicities are limitations when handling 5-FU. We have developed a triple liposomal formulation of 5-FU combined to 2′-deoxyinosine and folinic acid to improve its efficacy-toxicity balance.MethodsStealth liposomes were obtained using the thin-film method. Antiproliferative activity was tested on human colorectal and breast cancer models using sensitive (HT29) and resistant (SW620, LS174t, MDA231) cell lines. In vivo, pharmacokinetics, biodistribution and safety studies were performed in rodents. Finally, efficacy was evaluated using two tumor-bearing mice models (LS174 and MDA231) with response and survival as main endpoints.ResultsLipoFufol is a 120-nm pegylated liposome, displaying 20–30% encapsulation rates. In vitro, antiproliferative activities were higher than 5-FU, and matched that of FolFox combination in colorectal models, but not in breast. Drug monitoring showed an optimized pharmacokinetics profile with reduced clearance and prolonged half-life. Liposome accumulation in tumors was shown by fluorescence-based biodistribution studies. Beside, milder neutropenia was observed when giving LipoFufol to animals with transient partial DPD-deficiency, as compared with standard 5-FU. In LS174t-bearing mice, higher response and 55% longer survival were achieved with Lipofufol, as compared with 5-FU.ConclusionThe issues of drug-resistance and drug-related toxicity can be both addressed using a stealth liposomal formulation of modulated 5-FU.

Nucleoside analogs: ready to enter the era of precision medicine?

ABSTRACT Introduction: The term ‘precision medicine’ has garnered significant attention in the oncological setting in relation to attempts to optimize anticancer treatment. Precision medicine is mostly associated with oral targeted therapies and biotherapies, however, to date classic cytotoxics still remain the backbone of most regimens for treating solid tumors or in hematology, both in children and in adults. Among the existing cytotoxic therapies, nucleosides are widely used for treating a variety of cancerous diseases, alone or as part of combination therapies. Areas covered: Several markers at the tumor or the germinal levels have been identified as being associated with clinical outcome (e.g. CDA, DPD, EONFS1, hENT1, TYMS, MTHFR), however little effort has been made to implement bioguided therapy with nucleoside analogs. Still, growing clinical evidence has demonstrated how the efficacy-toxicity balance of these drugs could be improved by developing bioguided strategies at the bedside. This review covers the current knowledge regarding putative markers to be used with nucleoside analogs, what is known on their pharmacokinetic/pharmacodynamic relationships, and provides clues for implementing precision medicine with those old, yet pivotal drugs. Expert opinion: Through a variety of strategies ranging from pharmacogenetics, tumor genomics and pharmacokinetically-driven adaptive dosing procedures, nucleoside analogs could enter the era of precision medicine in oncology.

Biodistribution, Tumor Uptake and Efficacy of 5-FU-Loaded Liposomes: Why Size Matters

ABSTRACTPurposeWe have investigated the impact of particle size on the biodistribution, tumor uptake and antiproliferative efficacy of 5-FU-loaded liposomes.MethodsThree different batches of pegylated liposomes varying in size (i.e., 70, 120 and 250xa0nm respectively) were tested. The active compounds encapsulated were an equimolar mix of 5-FU, 2′-deoxyinosine and folinic acid. Liposomes were subsequently tested on the human breast cancer model MDA231 cells, a model previously found to be resistant to 5-FU. In vitro, antiproliferative efficacy and microscopy studies of liposomes uptake were carried out. In vivo, comparative biodistribution and efficacy studies were performed in tumor-bearing mice.ResultsDifference in size did not change in vitro antiproliferative activity. Fluorescence-Microscopy studies showed that liposomes were mainly uptaken by tumor cells through a direct internalization process, regardless of their size. Biodistribution profiles in tumor-bearing mice revealed higher accumulation of small liposomes in tumors throughout time as compared with normal and large liposomes (pu2009<u20090.05). Additionally, we observed that the bigger were the tumors, the more vascularised they were and the greater was the difference in accumulation between small and large liposomes. Consequently, in vivo efficacy studies showed at study conclusion that a 68% reduction in tumor size was achieved with small liposomes (pu2009<u20090.05), whereas larger liposomes failed to reduce significantly tumor growth. Similarly, at study conclusion a trend towards higher survival-rate in animals treated with smaller liposomes was observed.ConclusionThis study suggests that particle size is critical to achieve higher selectivity and efficacy in experimental oncology, including in resistant tumors.

Abstract 2208: Cytidine deaminase deficiency and severe toxicities upon aza-cytidine intake: Clinical observations and experimental evidences.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnAzacytidine is a nucleosidic analog used for treating a variety of haematological malignancies. Azacytidine pharmacokinetics is dependent upon a liver detoxification step driven by cytidine deaminase (CDA). CDA is affected by several genetic polymorphisms leading to a wide inter-individual variability in resulting enzymatic activities. In particurlar, dysregulated CDA has been associated with life-threatening toxicities with several nucleosidic analogs such as after cytarabine, capecitabine and gemcitabine intake. However, little is known about the impact of CDA deficiency on the clinical outcome of patients treated with azacytidine. In our institute, a lethal toxicity after pancytopenia has been observed with a 82-years old female patient upon aza-cytidine intake. CDA deficiency was evidenced after retrospective phenotyping (functional activity was found to be reduced by 65% as compared with standard CDA values: 1.2 VS. 3.5 U/mg), thus strongly suggesting that impaired ability to detoxify azacytidine could be the culprit indeed for this case. To further establish the exact role CDA-deficiency could have played in this toxic-death, a non-clinical study was undertaken in rodents. To mimic CDA deficiency, 8 mice were pre-treated with 100 mg/kg of tetrahydrouridine I.P. before being administered with 24 mg/kg of azacytidine. Another group of 8 mice received 24 mg/kg azacytidine only, and were therefore considered as control-CDA. Four mice were used as untreated controls when evaluating tolerance to the treatment. Neutrophils were monitored by flow-cytometry before and after treatment over 10 days as a surrogate marker for global azacytidine-related toxicities. CDA was measured using the same test than the one used at bedside and showed that the two groups differed markedly in their CDA phenotypes (ie, 7.7 VS. 0.98 U/mg) and that deep CDA deficiency (Poor Metabolizer: PM status) was achieved indeed in THU-treated animals. Pharmacokinetics study was carried out on a satellite group (T10, T20, T40 and T60 min) by revered-phase HPLC-UV analysis and showed a marked overexposure in azacytidine plasma levels in PM animals displaying CDA-deficiency. In full line with this observation, longer and deeper neutropenia was observed in this PM subset, as compared with the mice with normal CDA activity treated with the same dosage of azacytidine. Overall, our experimental data strongly support the hypothesis that CDA deficiency is a condition associated with increased risk to undergo life-threatening toxicities after azacytidine treatment. Detecting PM patients should help to secure the use of azacytidine at bedside.nnCitation Format: Raphaelle Fanciullino, Cindy Serdjebi, Severine Mollard, Regis Costello, LHoucine Ouafik, Joseph Ciccolini, Cedric Mercier. Cytidine deaminase deficiency and severe toxicities upon aza-cytidine intake: Clinical observations and experimental evidences. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2208. doi:10.1158/1538-7445.AM2013-2208

CDA as a predictive marker for life-threatening toxicities in patients with AML treated with cytarabine

Cytarabine (Ara-C) is the backbone of acute myeloid leukemia (AML) chemotherapy. Little is known about possible risk factors predictive for the frequent (ie, up to 16%) life-threatening or lethal toxicities caused by Ara-C. Ara-C is detoxified in the liver by a single enzyme, cytidine deaminase (CDA), coded by a gene known to be highly polymorphic. In this proof-of-concept study, we particularly investigated the role of the CDA poor metabolizer (PM) phenotype in Ara-C toxicities. CDA phenotyping (measurement of CDA residual activity in serum) and genotyping (search for the CDA*2 allelic variant) were performed in 58 adult patients with AML treated with the standard 7+3 (Ara-C + anthracyclines) protocol. Statistically significantly lower CDA activity was observed in patients experiencing severe/lethal toxicities as compared with patients who did not (1.5 ± 0.7 U/mg vs 3.95 ± 3.1 U/mg; Student t test P < .001). Subsequent receiver operating characteristic analysis identified a threshold in CDA activity (ie, 2 U/mg) associated with PM syndrome and increased risk of developing severe toxicities. Five percent of patients experienced lethal toxicities, all displaying CDA PM status (1.3 ± 0.5 U/mg). In terms of efficacy, a trend toward higher response rates and longer progression-free survival and overall survival were observed in patients with low CDA activity. Taken together, the results of this study strongly suggest that CDA is a predictive marker of life-threatening toxicities in patients with AML receiving induction therapy with standard Ara-C.

Abstract 4506: Computational-driven metronomics: application to gemcitabine in neuroblastoma-bearing mice

Metronomics (i.e., repeated administration of small doses of drugs over a long period of time) is an attractive strategy to reduce treatment-related toxicities while possibly offering unexpected novel mechanisms of action such as anti-angiogenesis or immuno-stimulatory properties. Defining an optimal metronomic schedule remains uneasy because of the variety of possible combinations between dosing, frequency and duration. Our group has developed innovative modeling tools to optimize metronomic schedules. Here, we have used this computational approach to test whether metronomics gemcitabine would make better than standard gemcitabine in mice bearing human neuroblastoma. In silico simulation suggested that shifting from standard 100 mg/kg/w over 4 consecutive weeks to the metronomic 1 mg/kg/d schedule for 28 consecutive days would achieve higher antitumoral efficacy while being well tolerated. To test this hypothesis, 100 000 human GI-ME-N neuroblastoma cells stably transfected with luciferase were subcutaneously grafted in 40 nude mice. Mice were next split in 3 groups: control, 100 mg/kg/w gemcitabine (STD-GEM) and 1 mg/kg/d gemcitabine (MetroGem-1). An additional satellite group was treated with 0.5 mg/kg/d (MetroGem-0.5). Metro-Gem was administrated using an osmotic pump implanted subcutaneously. Efficacy (i.e., comparison in tumor growth) was the main endpoint. Pharmacokinetics, tumor inflammation, and vascular density were the secondary endpoints. Gemcitabine assay showed that whereas Cmax up to 80 μg/ml were reached in mice treated with STD-GEM, steady-state concentrations of 0.1 μg/ml only were observed in animals undergoing MetroGem-1. Measuring cathepsin expression by fluorescence imaging suggested reduced inflammation in mice treated with the metronomic schedule. Similarly, fluorescence imaging showed smaller vascular density in mice treated with MetroGem-1 or -0.5 as compared with control or STD-GEM animals. To further check a possible anti-angiogenic effect, tumor perfusion rate was measuring next using luciferine, and fully confirmed that mice treated with Metro-Gem exhibited reduced blood flow as compared with other groups. Finally, bioluminescence monitoring of tumor growth showed that whereas STD-GEM was totally ineffective, both MetroGem-1 and MetroGem-0.5 achieved 60% reduction in tumor mass at the end of the treatment (p Citation Format: Joseph Ciccolini, Eddy Pasquier, Aurelie Lombard, Sarah Giacometti, Christian Faivre, Raphaelle Fanciullino, Cindy Serdjebi, Dominique Barbolosi, Nicolas Andre. Computational-driven metronomics: application to gemcitabine in neuroblastoma-bearing mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4506. doi:10.1158/1538-7445.AM2015-4506

Seek and destroy: improving PK/PD profiles of anticancer agents with nanoparticles

ABSTRACT Introduction: The Pharmacokinetics/pharmacodynamics (PK/PD) relationships with cytotoxics are usually based on a steepening concentration–effect relationship; the greater the drug amount, the greater the effect. The Maximum Tolerated Dose paradigm, finding the balance between efficacy, while keeping toxicities at their manageable level, has been the rule of thumb for the last 50-years. Developing nanodrugs is an appealing strategy to help broaden this therapeutic window. The fact that efficacy and toxicity with cytotoxics are intricately linked is primarily due to the complete lack of specificity toward the tumor tissue during their distribution phase. Because nanoparticles are expected to better target tumor tissue while sparing healthy cells, accumulating large amounts of cytotoxics in tumors could be achieved in a safer way. Areas covered: This review aims at presenting how nanodrugs present unique features leading to reconsidering PK/PD relationships of anticancer agents. Expert commentary: The constant interplay between carrier PK, interactions with cancer cells, payload release, payload PK, target expression and target engagement, makes picturing the exact PK/PD relationships of nanodrugs particularly challenging. However, those improved PK/PD relationships now make the once contradictory higher efficacy and lower toxicities requirement an achievable goal in cancer patients.

Turning cold tumors into hot tumors: harnessing the potential of tumor immunity using nanoparticles

ABSTRACT Introduction: Immune checkpoint inhibitors have considerably changed the landscape of oncology. However apart from world-acclaimed success stories limited to melanoma and lung cancer, many solid tumors failed to respond to immune checkpoint inhibitors due to limited immunogenicity, unfavorable tumor micro-environments (TME), lack of infiltrating T lymphocytes or increases in Tregs. Areas covered: Combinatorial strategies are foreseen as the future of immunotherapy and using cytotoxics or modulating agents is expected to boost the efficacy of immune checkpoint inhibitors. In this respect, nanoparticles displaying unique pharmacokinetic features such as tumor targeting properties, optimal payload delivery and long-lasting interferences with TME, are promising candidates for such combinations. This review covers the basis, expectancies, limits and pitfalls of future combination between nanoparticles and immune check point inhibitors. Expert opinion: Nanoparticles allow optimal delivery of variety of payloads in tumors while sparing healthy tissue, thus triggering immunogenic cell death. Depleting tumor stroma could further help immune cells and monoclonal antibodies to better circulate in the TME, plus immune-modulating properties of the charged cytotoxics. Finally, nanoparticles themselves present immunogenicity and antigenicity likely to boost immune response at the tumor level.