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In this study, we discuss the origin of the slightly increased response of the charged aerosol detector when low-concentration polar drugs formulated with sodium chloride are analyzed by hydrophilic interaction liquid chromatography coupled to the charged aerosol detector. In the case of tromethamine mixed with saline solutions, we investigated several levels including the mobile phase, sample matrix, and detection. We show that the analysis of the rich-salted sample results in both interactions with the mobile phase modifiers and the stationary phase during the run time. With 150 mM NaCl as a compounding solution, a slight increase in the tromethamine peak area was observed (5.5%). Our study suggests that chloride ions in excess sequentially interact firstly with the counterions from the organic modifiers and secondly with the analyte via the stationary phase and the contribution of hydrophilic interaction liquid chromatography retention mechanisms. Because of these effects, the hydrophilic interaction liquid chromatography-charged aerosol detector analysis of drugs in saline solutions requires particular attention, and a correction factor for quantitative purposes that accounts for formulation ions remains appropriate. This article is protected by copyright. All rights reserved.

FOLFOX is the most common chemotherapy combination prescribed in colorectal cancer. It is composed of calcium levofolinate, 5-fluorouracil and oxaliplatin which demonstrated synergistic outcome. Nowadays, the lack of all-in-one formulation is due to the chemical composition of the pharmaceutical products and the highly pH-dependent stability of each drug. Herein, we aimed to investigate the stability of a ternary mixture of 5-fluorouracil, oxaliplatin and calcium levofolinate, knowing that coadministering these drugs would improve their efficacy. The effect of three pHs (5.0, 6.0 and 7.5) and two drug concentrations (8/3/6 and 1/1/1 mg/ml for 5-fluorouracil, oxaliplatin and calcium levofolinate, respectively) were examined. A high-performance liquid chromatography method was developed to separate and quantify the three drugs in one run. At higher concentrations, the ternary mixture was unstable regardless of pH. By reducing concentration, drug stability and compatibility in the mixture was improved at pH 5.0 for up to 3 days at +5°C ± 3 °C. In addition, binary mixtures provided stable properties at defined pHs. 5-fluorouracil/oxaliplatin mixture was stable at pH 5.0 over 48 hours while 5-fluorouracil/calcium levofolinate mixture was stable at pHs 6.0 and 7.5 up to 7 days.

Spironolactone is a potassium-sparing diuretic used as treatment for several diseases such as bronchopulmonary dysplasia, precocious puberty, hypertension or primary aldosteronism in child population. Because of the lack of paediatric formulations in the market, two types of solid oral formulation containing 2.5 and 10 mg of spironolactone were developed. During stability studies, excessive degradation was observed. As presented in this work, the degradation of spironolactone was linked to a drug excipient interaction between spironolactone and magnesium stearate via alkaline hydrolysis degradation pathway. In order to help the re-formulation with another lubricant, a complete drug excipient compatibility study was performed using thermal analysis techniques associated with chromatographic quantification of the drug after an isothermal stress testing. Spironolactone presents physicochemical incompatibilities with sodium benzoate, sodium stearyl fumarate, PEG 8000, PEG 20,000 and stearic acid. But no degradation was observed with PEG 20,000 and stearic acid indicating chemical compatibility. In total, talc, stearic acid and PEG 20,000 could be used for re-formulation of spironolactone paediatric drug.

International audience; The additive technology or 2D and 3D printing are increasingly used in various industrial fields, from aeronautics to mechanics but also in the fields of health such as dentistry or for bone reconstructions. These techniques have been studied for about fifteen years by the academic community in the pharmaceutical field (medical device and drug), and recently they have started to be applied to produce drugs in industry and in hospitals. Indeed, the Food and Drug Administration approved in August 2015 the marketing of the first drug printed by additive technique, then in 2018 the first clinical trial using 3D printed drugs was carried out in Great Britain by a hospital pharmacy. 2D-3D printing is presented as one of the tools of a more personalized medicine, the techniques of additive printing allowing the production of tabs containing several drugs in one tab (polypills) and the development of custom modified-releases drugs. This approach could allow better acceptance of the finished product and secure manufacturing. The objective of this work is to highlight relevant printing technologies for implementation in hospital pharmacies, and to see how these technologies could lead to a change in pharmaceutical practices, to improve patient care.

Oral lichen planus (OLP) is an ongoing and chronic inflammatory disease affecting the mucous membrane of the oral cavity. Currently, the treatment of choice consists in the direct application into the buccal cavity of semisolid formulations containing a corticosteroid molecule to decrease inflammatory signs and symptoms. However, this administration route has shown various disadvantages limiting its clinical use and efficacy. Indeed, the frequency of application and the incorrect use of the preparation may lead to a poor efficacy and limit the treatment compliance. Furthermore, the saliva clearance and the mechanical stress present in the buccal cavity also involve a decrease in the mucosal exposure to the drug. In this context, the design of a new pharmaceutical formulation, containing a steroidal anti-inflammatory, mucoadhesive, sprayable and exhibiting a sustained and controlled release seems to be suitable to overcome the main limitations of the existing pharmaceutical dosage forms. The present work reports the formulation, optimization and evaluation of the mucoadhesive and release properties of a poloxamer 407 thermosensitive hydrogel containing a poorly water-soluble corticosteroid, dexamethasone acetate (DMA), threaded into hydroxypropyl-beta-cyclodextrin (HP-&beta, CD) molecules. Firstly, physicochemical properties were assessed to ensure suitable complexation of DMA into HP-&beta, CD cavities. Then, rheological properties, in the presence and absence of various mucoadhesive agents, were determined and optimized. The hydration ratio (0.218&ndash, 0.191), the poloxamer 407 (15&ndash, 17 wt%) percentage and liquid-cyclodextrin state were optimized as a function of the gelation transition temperature, viscoelastic behavior and dynamic flow viscosity. Deformation and resistance properties were evaluated in the presence of various mucoadhesive compounds, being the sodium alginate and xanthan gum the most suitable to improve adhesion and mucoadhesion properties. Xanthan gum was shown as the best agent prolonging the hydrogel retention time up to 45 min. Furthermore, xanthan gum has been found as a relevant polymer matrix controlling drug release by diffusion and swelling processes in order to achieve therapeutic concentration for prolonged periods of time.

International audience; One of the main problems of colorectal cancer is not the treatment of the primary tumor but the metastatic stage. Means of metastatic spread is the invasion of the peritoneal cavity which leads to peritoneal metastasis (PM). PM cannot be easily cured, and the current treatments is rather heavy, combining cytoreductive surgery with intravenous and intraperitoneal chemotherapy. This therapeutic procedure is associated with significant morbidity, altered patient quality of life and poor prognosis. We postulated that development of a prophylactic treatment could be of high interest in this context. In this study, we formulated an anti-adhesive thermogel which contains chemotherapeutics to play a role of a barrier against tumor cells implantation, avoiding their adhesion and treating the remaining tumor cells with chemotherapy intraperitoneally in a mice model of PM. The bioavailability of the thermogel was tested intraperitoneally in mice. No sign of toxicity was observed in terms of change in body weight, anatomopathology and blood biomarkers. In vitro experiments proved that the thermogel induced limited adhesion of the tumor cells. Loading of oxaliplatin (Ox) and 5-Fluorouracil (5-FU) into the thermogel were able to significantly decreased peritoneal carcinomatosis index (PCI) (-58%) and ascites (-70%) in a murine model of peritoneal metastases. These pre-clinical results confirmed that smart thermogel associated with standard chemotherapy 5-FU and Ox could be a good candidate to decrease the risk of tumor cell implantation during cytoreductive surgery and prevent future metastatic process.

International audience; Peritoneum represents a frequent site of metastasis especially for digestive and ovarian primary cancers. The conventional approach to treat peritoneal metastasis consists in systemic chemotherapy, but the median survival associated is only a few months. Recent therapeutic developments result in an aggressive strategy associating cytoreductive surgery (CRS) and hyperthermic intra peritoneal chemotherapy (HIPEC). However, a recent study failed to show an improvement in the overall survival and relapse free survival of this combo in comparison to CRS alone. Confronted to a lack of guidelines, several drug delivery systems (DDS) had been developed and tested in animal models to offer an effective easy-to-use solution for surgeons to prevent peritoneal metastasis. In this work, we reviewed most of the strategies used to treat peritoneal metastasis (PM) from digestive or ovarian origin and concentrated on 3 different DDS strategies: particulates DDS, non particulates DDS (including implants, films and gels) and combination of both (in particular hydrogels loaded with particles).

Tromethamine (TMM), often encountered in a final drug product, exhibits interesting chemical properties as a counter ion, buffer, or active ingredient. European and US pharmacopeias propose titration against hydrogen chloride for TMM assays. However, this method can be a hindrance when using drugs containing low concentrations of TMM in complex buffered formulations. Due to the lack of chromophores and the high hydrophilicity of TMM, we performed a simple and reliable hydrophilic interaction chromatography coupled with a charged aerosol detector (HILIC-CAD) separation approach as an alternative for TMM analysis. An amide stationary phase and a mobile phase consisting of a binary mixture of acetonitrile and 10 mM ammonium formate, pH 3 (80/20, V/V) were used. As the CAD response deeply depends on parameters such as stationary phases and pH buffer, we investigated their impact and explored the optimal signal conditions. Including TMM analogs such as tris(hydroxymethyl) nitromethane and 2-amino-2-ethyl-1,3-propanediol allowed us to select these parameters appropriately. The effects of the evaporation temperature, flow rate, and power function value (PFV) on the CAD signal response were also studied and optimized. The method was validated according to the ICH Q2 R1 guidelines. A linear response (mean R2 > 0.997) covering the range for low TMM concentrations (170–520 μg/mL) was achieved. Satisfactory intra-day and inter-day precisions were obtained with RSDs lower than 1.9% and 2.8%, respectively. The trueness ranged from 99.6% to 101.2%, and the LOD was found to be 1.1 μg/mL. The HILIC-CAD method has been applied to a sterile TMM solution for injection.

Presenting many advantages, solid oral dosage forms (SODFs) are widely manufactured and frequently prescribed in older populations regardless of the specific characteristics of patients. Commonly, patients with dysphagia (swallowing disorders) experience difficulties taking SODFs, which may lead to non-adherence or misuse. SODF characteristics (e.g., size, shape, thickness) are likely to influence swallowability. Herein, we used the acceptability reference framework (the ClinSearch acceptability score test (CAST))&mdash, a 3D-map juxtaposing two acceptability profiles&mdash, to investigate the impact of tablet size on acceptability. We collected 938 observer reports on the tablet intake by patients &ge, 65 years in hospitals or care homes. As we might expect, tablets could be classified as accepted in older patients without dysphagia (n = 790), while not in those with swallowing disorders (n = 146). However, reducing the tablet size had a significant impact on acceptability in this subpopulation: tablets &lt, 6.5 mm appeared to be accepted by patients with swallowing disorders. Among the 309 distinct tablets assessed in this study, ranging in size from 4.7 to 21.5 mm, 83% are &ge, 6.5 mm and consequently may be poorly accepted by institutionalized older people and older inpatients suffering from dysphagia. This underlines the need to develop and prescribe medicines with the best adapted characteristics to reach an optimal acceptability in targeted users.

International audience; Spurred by high risk for local tumor recurrence and non-specific toxicity of systemic chemotherapy, clinicians have recently granted a growing interest to locoregional therapeutic strategies. In this perspective, we recently developed a multipurpose thermosensitive hydrogel based on reversible thermogelling properties of poloxamers P407 and P188, a bioadhesive excipient and antineoplastic effect of 5-fluorouracil (5-FU) for the local treatment of colorectal cancer (CRC) in ectopic CT26 murine models. Antitumor efficacy was assessed in mice following intratumoral (IT) injection mimicking neoadjuvant therapy and subcutaneous (SC) application after tumor excision simulating adjuvant therapy. Rheological characterization disclosed that P407/P188/alginate 20/2/1% w/v thermosensitive hydrogel is an injectable free-flowing solution at ambient temperature that undergoes a SOL-GEL transition at 26.0 degrees C +/- 0.6 degrees C and thereby forms in situ a non-flowing gel at physiological temperature. The generated gel presented an elastic behavior and responded according to a shear-thinning fluid upon shear rate. Although delayed by the addition of alginate 1% w/v, 5-FU is released mainly by diffusion mechanism. The local delivery of 5-FU from P407/P188/alginate/5-FU 20/2/1/0.5% w/v hydrogel in the preclinical tumor models led to a significant tumor growth delay. These results demonstrated that poloxamer-based thermo-sensitive hydrogels provide a simple and efficient means for local chemotherapeutics delivery.

Background In institutional care, oral liquid pharmaceutical products are widely prescribed for older patients, especially for those with swallowing disorders. As medicines acceptability is a key factor for compliance in the older population, this study investigated the acceptability of oral liquid pharmaceutical products in this targeted population. Methods An observational, multicenter, prospective study was conducted in eight geriatric hospitals and eight nursing homes in France. Observers reported several behaviours/events describing the many aspects of acceptability for various pharmaceutical products’ uses in patients aged 65 and older. Acceptability scores of oral liquid pharmaceutical products were obtained using an acceptability reference framework (CAST - ClinSearch Acceptability Score Test®): a 3D-map summarizing the different users’ behaviors, with two clusters defining the positively and negatively accepted profiles materialized by the green and red zones, respectively. Results Among 1288 patients included in the core study and supporting the acceptability reference framework, 340 assessments were related to the administration of an oral liquid pharmaceutical product. The mean age of these patients was 87 (Range [66-104y]; SD = 6.7), 68% were women and 16% had swallowing disorders. Globally, the oral liquid pharmaceutical products were classified as “positively accepted,” the barycenter of the 340 assessments, along with the entire confidence ellipses surrounding it, were positioned on the green zone of the map. Sub-populations presenting a different acceptability profile have also been identified. For patients with swallowing disorders, the oral liquid pharmaceutical products were classified as “negatively accepted,” the barycenter of the 53 assessments along with 87% of its confidence ellipses were associated with this profile. A gender difference was observed for unflavored oral liquids. In women, they were classified “negatively accepted,” the barycenter of the 68 assessments with 75% of its confidence ellipses were located in the red zone, while they were classified “positively accepted” in men. Conclusion This study showed that oral liquid pharmaceutical products are a suboptimal alternative to solid oral dosage forms in patients with swallowing disorders. To ensure an optimal acceptability, prescribers should also consider the presence of a taste-masker in these oral liquids. As highlighted herein, palatability remains crucial in older populations, especially for women.

In situ gelling delivery systems for the ocular administration of drugs has been a major focus of research over the past two decades, improving the treatment of diseases of the anterior segment of the eye by simple, safe, and reproducible drug administration. This drug delivery strategy results in high ocular bioavailability by avoiding rapid precorneal clearance resulting from nasolacrimal drainage and eye blinking. However, the development of such unconventional forms requires many parameters to be mastered, such as gelation time, viscoelastic behavior, mucoadhesion, and sustained release. In this review, we describe and assess the in vitro and in vivo methods available for in situ gelling ophthalmic delivery systems, highlighting the advantages of existing methods and, in some cases, the need for more relevant assays.

Intratumoral injection of biocompatible gels is increasingly used for the sustained delivery of drugs and vaccines to enhance the anti-cancer immune response. Granulocyte-macrophage colony stimulating factor (GM-CSF) has become an attractive adjuvant thanks to its ability to boost the antitumor immune response by inducing proliferation, maturation and migration of the dendritic-cells (DCs) and the differentiation of lymphocytes. Killed Mycobacteria, such as Heat-killed Mycobacterium tuberculosis (HKMT) have been used in several studies as TLR-2 agonist to increase maturation of DCs. In this study, we designed a mucoadhesive thermosensitive formulation for the local delivery of GM-CSF and HKMT in order to enhance DCs activation and improve the local antitumor immune response. This formulation was selected based on its elastic and mucoadhesive properties obtained thanks to rheological studies. More importantly, intratumoral residence time of the labelled gel and protein were evidenced by means of MRI and non invasive in vivo optical imaging. Then, the efficacy of the combination of immunomodulators loaded thermogel was demonstated in vitro and in vivo. The selected thermogel exhibits rheological properties which confer a good elasticity and increased residence time of the immunostimulatory agents in the tumor, thus increasing the recruitment of DCs and T cytotoxic CD8+ lymphocytes.

Palatability is a recognized driver of medicine acceptability in pediatrics but deemed less relevant in older populations due to sensory decline. Preliminary findings from an observational study implicated palatability problems with one Alzheimer&rsquo, s medicine. Among 1517 observer reports combining multiple measures on medicines uses in patients aged over 64, we focused on two original formulations of memantine (Ebixa&reg, tablets (n = 25) and oral solution (n = 60)). Evaluations were scored with an acceptability reference framework (CAST), the rodent Brief Access Taste Aversion (BATA) model tested aversiveness. Focusing on women treated with Ebixa&reg, (n = 54), the oral formulation sub-group was classified as &ldquo, negatively accepted&rdquo, while the coated tablet was associated with the &ldquo, positively accepted&rdquo, cluster. In men, both formulations belonged to the &ldquo, profile. Using BATA, the original oral solution was categorized as highly aversive/untolerated while solutions of excipients only were well tolerated. Furthermore, the number of licks was significantly lower in female than in male rats. These results revealed that medicine palatability remains important for acceptability in older populations. Moreover, converging results from humans and animal models highlighted that palatability profiles can significantly vary between the sexes. These drivers should be closely considered during drug development to enhance acceptability in this population.

International audience; Mydriasis is required prior to many eye examinations and ophthalmic surgeries. Nowadays, phenylephrine hydrochloride (PHE) and tropicamide (TPC) are extensively used to induce mydriasis. Several pharmaceutic dosage forms of these two active ingredients have been described. However, no optimal therapeutic strategy has reached the market. The present work focuses on the formulation and evaluation of a mucoadhesive ion-activated in situ gelling delivery system based on gellan gum and hydroxyethylcellulose (HEC) for the delivery of phenylephrine and tropicamide. First, in vitro drug release was studied to assess appropriate sustained drug delivery on the ocular surface region. Drug release mechanisms were explored and explained using mathematical modeling. Then, in situ gelling delivery systems were visualized using scanning electron microscopy illustrating the drug release phenomena involved. Afterward, cytotoxicity of the developed formulations was studied and compared with those of commercially available eye drops. Human epithelial corneal cells were used. Finally, mydriasis intensity and kinetic was investigated in vivo. Mydriasis pharmacodynamics was studied by non-invasive optical imaging on vigilant rabbits, allowing eye blinking and nasolacrimal drainage to occur physiologically. In situ gelling delivery systems mydriasis profiles exhibited a significant increase of intensity and duration compared with those of conventional eye drops. Efficient mydriasis was achieved following the administration of a single drop of in situ gel reducing the required amount of administered active ingredients by four- to eight-fold compared with classic eye drop regimen.

International audience; Introduction Medicine acceptability is a multi-faceted concept driven by both product and user characteristics. Although a key factor for treatment effectiveness, especially in vulnerable populations, knowledge of those medicine features that best promote individual user acceptability remains fragmented. Focusing on paracetamol, this study has explored the appropriateness of pharmaceutical products in different dosage forms to achieve adequate patient acceptability from infants to centenarians. Methods This observational, multicentre, prospective study was carried out in 10 hospitals, 8 nursing homes and over 150 community dispensaries. Observers reported several behaviours/events evaluating acceptability for 1016 different pharmaceutical product uses in paediatrics (= 65y.). Using mapping and clustering, a multivariate approach offered an intelligible reference framework for each population, providing comprehensive scores: positively or negatively accepted. Results Among all the evaluations supporting the acceptability reference frameworks, there were 502 reports on paracetamol products intake. Herein we focused on the 5 products with. 30 evaluations. Although oral suspension and powder for oral solution were positively-accepted in the paediatric group, the powder had a higher rate of negative patient reaction (p= 90y., capsule formulations appeared to be the best accepted product in patients without swallowing alterations, and thus could be a suitable alternative to the powder in this population. Conclusions By better integrating patient characteristics when choosing dosage forms, clinicians and caregivers may improve treatment acceptability and adherence. Moreover, hospitals and healthcare institutions could optimise purchasing to best suit their local population, disseminating information to help staff align specific dosage forms to targeted patients.

International audience; PurposeMedicine acceptability, which is of the utmost importance for vulnerable patients’ adherence, is driven by both user and product characteristics. Herein, a novel multivariate approach integrating the many aspects of acceptability is used to discriminate positively and negatively accepted medicines in the older population.MethodsAn observational study was carried out in eight hospitals and eight nursing homes to collect a large set of real-life data on medicines uses in older patients (≥65 years). Mapping and clustering explored these multiple observational measures and summarised the main information into an intelligible reference framework. Resampling statistics were used to validate the model’s reliability.ResultsA three-dimensional map and two clusters defining acceptability profiles, as positive or negative, emerged from the 1079 evaluations. Factors of interest (medicines, user features…) were positioned on the map at the barycentre of their evaluations and assigned to an acceptability profile. Focusing on patients’ ability to swallow, we have highlighted the tool’s efficacy in demonstrating the impact of user features on medicine acceptability.ConclusionsThis multivariate approach provides a relevant judgement criterion for this multi-dimensional concept. Facilitating the choice of the most appropriate dosage form to achieve optimal acceptability in a targeted population, this tool is of real potential to improve clinical decisions.

International audience; Calcium gluconate glucoheptonate (GGCa) is known to interact with glass containers, leading to the leaching of aluminum from the glass into the solution at toxic level. Therefore, plastic containers seem to be a preferable packaging alternative. Nevertheless, plastics contain potentially toxic additives which could be released into the solution. In order to study content container interaction between GGCa and two plastic containers (polypropylene PP and polyethylene PE containers), an HPLC-PDA method was developed to separate, detect and quantify eleven additives commonly found in plastic materials, with good limit of detection and quantification. This method was then applied to evaluate the compatibility between GGCa and the two plastic containers. After 3 months of storage at 25 degrees C, none of the eleven additives were detected in GGCa solutions. The safety concern threshold (SCT) and of the analytical evaluation threshold (AET) were evaluated to discriminate the need to identify and qualify unknown peaks. (C) 2018 Published by Elsevier B.V.

Buccal administration route is a promising way for a large number of drugs exhibiting a low oral bioavailability. The present work describes the formulation and evaluation of a mucoadhesive and thermosensitive in situ gelling delivery system based on poloxamer 407, poloxamer 188 and xanthan gum for buccal drug delivery. First, the mucoadhesion properties were evaluated using a tensile test. The effect of xanthan gum on the mucoadhesion force was demonstrated. Then, to assess the buccal residence time which reflects the mucoadhesion properties, the validation of a fluorescence probe for in vivo optical imaging experiment was conducted. Methyl-Cyanine 5 derivative (Me-Cy5) was used to label the hydrogels, dissolution tests and permeation studies through buccal epithelium cells showed that Me-Cy5 release from hydrogels was mainly due to an erosion mechanism and presented a limited penetration across epithelium cells. These results suggest that, Me-Cy5 is a suitable marker for thermosensitive in situ gelling delivery systems as the probe mostly stays entrapped in the hydrogel and do not cross the epithelial barrier. Buccal residence performance of the hydrogel was evaluated for the first time by non-invasive optical imaging after administration to mice. This technique is an interesting alternative compared to visual observations and sacrifice involved experiments, which could also be exploited to various administration routes.

International audience; Thermosensitive hydrogels developed for buccal delivery of salbutamol were prepared using poloxamer analogs ( Kolliphor (R) P407/P188), xanthan gum (Satiaxane (R) UCX930) and NaCl. P188 increased gelation temperature (Tsol-gel) by 2.5-5 degrees C, micellization temperature (3s. To obtain a suitable Tsol-gel at 28-34 degrees C, P407 and P188 concentrations were set to 18-19% and 1%. NaCl reduced Tsol-gel (>2 degrees C) out of the optimal range. Six formulations containing 0.05-0.1% Satiaxane (R) fulfilled the temperature criteria. Concerning the gel strength, 1% P188 had no significant effect, NaCl increased it at 20 degrees C, and Satiaxane (R) enhanced it at 20 degrees C and 37 degrees C. The release study using membrane-less (to mimic oral cavity) and membrane (to mimic buccal mucosa side) methods allowed a complete investigation showing that erosion and diffusion both contributed to the drug release but differed according to the formulation. In the membraneless method, simple P407 formulations had weak ability to retain salbutamol (T-80 = 35 min). P188 accelerated drug release. NaCl accelerated release in the membraneless method by 5-11 min but slightly reduced it in the membrane method. The hydrogels containing Satiaxane (R) exhibited the slowest release. In the membrane method, combination of P407/P188/Satiaxane (R) provided a sustained diffusion with a burst effect (T-25 = 9.6 min, T-80 = 97.8 min), which provides potential clinical interests. (c) 2014 Published by Elsevier B. V.

A rapid, sensitive and specific liquid chromatography coupled to tandem mass spectrometry method was developed for the simultaneous quantification pig plasma of ketamine and its two principal metabolites, norketamine and dehydronorketamine. Three extraction procoles were assessed including acetonitrile precipitation, Oase™ microplate extraction, and liquid-liquid extraction. Oase™ microplate extraction induced no significant matrix effect, important signal/noise ratio and good recoveries, ranging from 82 to 87% for the considered compounds. Using this extraction procedure, the assay was linear in the dynamic range 10-3000ng/mL (R2>0.99) regardless of the analytes. Intra- and inter-day accuracies were less than 12% for all compounds and intra- and inter-day precisions expressed as RSD were within

Since epidemiologic data have highlighted the positive effects of metformin to reduce cancer incidence and mortality, many in vitro and in vivo studies as well as a large number of clinical trials have been conducted in order to study its potential. The many anticancer actions of metformin lead to a cytostatic effect. Two distinct but not exclusive mechanisms can be implicated in these actions. First, by decreasing insulinemia and glycaemia, metformin can block the PI3K/MAPK signalling pathway implicated in cell growth. Second, metformin can directly act on cancer cells by targeting various pathways including tumour metabolism, inflammation, angiogenesis or cancer stem cells, mainly through the activation of the AMPK pathway. Nonetheless, although metformin alone displays chemopreventive properties, it does not seem to be sufficient to treat cancer, raising the need to be combined with other drugs (e.g. chemotherapy or glycolysis inhibitors) in order to synergistically reveal its cytotoxic action. However, in particular conditions such as specific mutations (e.g. LKB, p53 or OCT1) or low glucose availability, metformin alone does have cytotoxic effects. Thus, it is essential to consider the associated biomarkers in order to determine the potential of metformin in different types of cancers.

e15562Background: After radiofrequency ablation (RFA), cancer cells death releasing tumor antigens and expression of danger signals activate a specific T-cell response however inadequate to avoid r...

102 Background: Radiofrequency ablation (RFA) efficiency of liver tumors is compromised by high rates of relapse. Death of cancer cells by hyperthermia induced tumor antigen releasing, expression of danger signals that activate a specific T-cell response. This effect is ineffective to avoid recurrence. We propose to combine RFA with priming of a strong immune antitumor response as curative treatment of an aggressive colorectal cancer (CRC) in immunocompetent mouse. Methods: RFA was used to treat a CT26- luc tumor as primary lesion. In two distinct clinical situations, macroscopic or microscopic distant tumors were established as secondary lesions. The immune response was modulated by the injection, in the treated area, of a thermo-reversible hydrogel loaded by GM-CSF and BCG, targeting recruitment and maturation of dendritic cells. In mice with far large lesions, this strategy was combined with PD1checkpoint inhibition. The efficiency was assessed on survival, evolution of distant lesions, characterization of tumoral lymphocyte infiltration TNF-α and IFN-y expression in peripheral T lymphocytes. Results: The in situ immunogel injection after RFA resulted in prolonged survival of mice. Regression of distant lesions was related to the induction of a strong systemic antitumor immune response and a great improvement of tumor infiltration by CD3+ T lymphocytes. In adjuvant situation, the use of immunogel induced a complete cure of microscopic secondary lesions without another treatment. Immune escape of large secondary lesions was reversed by association of the RFA-immunogel vaccination with a systemic immune checkpoint inhibition, separately ineffective. Conclusions: Validation of this strategy, combining RFA of macroscopic lesions and activation of a strong immune response controlling the residual disease, could result in the design of a clinical assay including this approach within a standard treatment of colorectal liver metastases. The synergy between in situ immunomodulation as priming process and checkpoint blockade, ineffective alone in metastatic microsatellite stable CRC or after single RFA, allows reconsidering the use of checkpoint inhibitors in CRC.

Introduction La gestion des excursions de temperature d’un medicament thermosensible peut poser de reelles difficultes. Dans le cas d’une exposition a temperature ambiante de la methacholine par exemple, celle-ci s’hydrolyse rapidement en beta-methylcholine et acide acetique. Le retour a une conservation a +5 °C ± 3 °C, reduit, mais ne stoppe pas cette degradation. Une etude a ete conduite afin de determiner les temps maximum d’exposition a temperature ambiante de ce produit, et donc le delai maximal tolerable en cas d’excursion de temperature au cours de la chaine du froid. Materiels et methode Trois lots de cette preparation hospitaliere ont ete exposes initialement a +25 °C pendant 0, 7, 14, 21 et 28 jours puis conserve a +5 °C ± 3 °C jusqu’a peremption (18 mois). La degradation du produit est evaluee au regard de la teneur en methacholine, de la teneur en son produit de degradation (beta-methylcholine) et du pH. Les normes definies par les guidelines ICH et l’evaluation toxicologique de l’impurete sont les suivantes : a T0 le produit doit presenter des teneurs en beta-methylcholine inferieure a 1 % et une teneur en methacholine entre 95–105 % pour etre libere. A peremption, la preparation hospitaliere doit presenter une teneur en methacholine entre 95–105 % et une teneur en beta-methylcholine inferieure a 5 %. Resultats et discussion Pour les 3 lots, le suivi du pH montre une acidification croissante de la solution des T0 en fonction du temps d’exposition a +25 °C (perte de 0,2 a 0,3 unite de pH en 28 jours a +25 °C), indiquant ainsi une degradation de la solution en lien avec la formation d’acide acetique. La teneur en beta-methylcholine est a la limite de la non-conformite a peremption (4,96 % pour un lot) apres une exposition de 14 jours a +25 °C. La teneur en principe actif est conforme a T0 meme lors d’une exposition de 28 jours a +25 °C, mais devient non conforme a peremption des lors d’une exposition initiale de 7 jours a 25 °C pour un lot (94,5 % a T18 mois). Conclusion Au total, a partir de 7 jours consecutifs d’une exposition a +25 °C, la methacholine presente une non-conformite de teneur a peremption. Au regard de ces donnees, et en cas d’absence de rupture anterieure de la chaine du froid, une excursion unique de moins de 72 h a une temperature inferieure ou egale a +25 °C est jugee tolerable pour ce medicament qui devra alors etre utilise immediatement.

International audience; A salbutamol sulfate (SS)-Poloxamer bioadhesive hydrogel specially developed for buccal administration was investigated by studying interactions with TR146 human buccal epithelium cells (i.e. cellular toxicity (i) and trans-epithelial SS diffusion (ii)). The assessment of cell viability (MTT, Alamar Blue), membrane integrity (Neutral Red), and apoptosis assay (Hoechst 33342), were performed and associated to Digital Holographic Microscopy analysis. After the treatment of 2 h, SS solution induced drastic cellular alterations that were prevented by hydrogels in relation with the concentrations of poloxamer and xanthan gum. The formulation containing P407 19%/ P188 1%/Satiaxane 0.1% showed the best tolerance after single and multiple administrations and significantly reduced the trans-epithelial permeability from 5.00 +/- 0.29 (x10(3)) (SS solution) to 1.83 +/- 0.22 cm/h. Digital Holographic Microscopy images in good agreement with the viability data confirmed the great interest of this direct technique. In conclusion, the proposed hydrogels represent a safe and efficient buccal drug delivery platform. (C) 2015 Elsevier B.V. All rights reserved.

The purpose of this work was to study the in vitro equilibria and the adsorption kinetics of an ionizable drug, indomethacin, onto commercially available cationic polymeric microspheres: DEAE Trisacryl LS and QA Trisacryl LS. Isotherms were fitted to theoretical equations allowing accurate predictions of drug loading at different salt concentrations. Isotherm measurements were quickly obtained by simple column breakthrough experiments. The nature of the ion exchange group of the microspheres was observed to be preponderant for adsorption, as the tertiary amine derivative exhibited 53% more capacity than its quaternary amine counterpart. The maximum equilibrium uptake capacity in a 5 mM Tris–HCl buffer at pH 7.4 is 303 mmol/ml of particle volume, for DEAE microspheres. Transport properties of indomethacin into the tertiary amine microspheres were obtained in agitated contactor. Microbeads loading was completed in a 1–6 min range and was found to be controlled by pore diffusion mechanism. Equilibrium uptake data was fitted to the Langmuir and the mass action law models. Adsorption kinetics were fitted to a pore diffusion model. Good correlation was obtained between the theoretical models and the experimental data. The methodology outlined in this work provided a simple approach of estimating adsorption behavior of drugs onto ion-exchange macroporous microspheres. Although significant indomethacin loading was obtained onto the DEAE microspheres, the rapid rate of diffusion is not compatible with sustained release properties sought for this type of microspheres.

e14540 Background: Results of radiofrequency ablation (RFA), increasingly used to treat liver tumors, are compromised by local and systemic relapse. Hyperthermia related cancer cells death, release of tumor antigens and expression of danger signals activate a tumor-specific T-cells response. This effect remains ineffective to avoid recurrence. Therefore we propose to combine RFA with an activation of a solid immune antitumor response as curative treatment of a colorectal (CRC) metastatic disease in immunocompetent mouse. Methods: RFA was used to treat a CT26- luc tumor. In two distinct clinical situations, distant macroscopic or microscopic tumors were established as metastases before or at the time of RFA. Immune response was modulated by an injection in situof a thermo-reversible hydrogel loaded by GM-CSF and BCG, targeting dendritic cells. In the group of mice with large far lesions this strategy was combined with immune checkpoint inhibition. The efficiency was assessed on survival, evolution of distant lesions, characterization of lymphocyte infiltration in tumors and systemic immunity through specific TNF- α and IFN-y expression in spleen and draining lymph nodes. Results: The in situ immunogel injection after RFA resulted in a prolonged survival of mice. Regression of distant lesions was related to a strong systemic antitumor immune response and a great improvement of tumor infiltration by specific cytotoxic lymphocytes. In adjuvant situation, the use of immunogel induced a complete cure of microscopic secondary lesions without any treatment. Immune escape of large secondary lesions was reversed by association of RFA-immunogel vaccination with a systemic check point blockade, separately ineffective. Conclusions: Validation of this strategy, combining RFA of liver metastases and activation of a strong immune response controlling the residual disease, could result in a clinical assay including this approach within the standard treatment of CRC. Furthermorethe powerful synergy between RFA-in situ immunomodulation as a starter treatment and checkpoint blockade ineffective alone in CRC or after single RFA, allows reconsidering the use of immune checkpoint inhibitors in metastatic microsatellite stable CRC.

Objectif La ketamine (K) est un anesthesique utilise en medecine veterinaire et humaine. Chez l’homme, c’est un agent pharmacologique de reference indique pour induire des anesthesies courtes, mais egalement pour son action antalgique a faible dose, particulierement recherchee pour le traitement des douleurs aigues ou provoquees par les soins. Dans cette indication, en raison de la demi-vie courte de K et de l’effet de premier passage hepatique important, une forme buccale innovante a liberation modifiee a ete developpee. Une etude a ete realisee chez le porc pour valider le concept par comparaison des pharmacocinetiques de cette nouvelle forme a une solution de K administrees par voie sublinguale. Dans ce but, nous avons developpe le dosage plasmatique de la ketamine et de ses deux metabolites, la norketamine (NK), et la dehydronorketamine (DHNK), par chromatographie liquide couplee a la spectrometrie de masse en tandem (CL–SM/SM). Nous rapportons ici la validation analytique de cette methode et son application au dosage plasmatique chez l’animal. Materiel et methodes Chez des porcs dont l’anesthesie est induite par le propofol puis entretenue a l’isoflurane, la K est administree a la dose de (2–4–6) mg/kg pour la forme a liberation modifiee et a la dose de 4 mg/kg pour la solution. Des prelevements plasmatiques sur heparinate de lithium sont pratiques de facon sequentielle entre les temps 0 et 360 min apres l’administration. Les echantillons de sang sont centrifuges et le plasma est conserve a −80 °C jusqu’au moment du dosage. En utilisant la norketamine-d4 comme etalon interne et une prise d’essai de 100 μL, trois modes de preparation de echantillons ont ete testes : extraction liquide–liquide (chloroforme en milieu ammoniacal) ; simple deproteinisation par l’acetonitrile ; extraction sur microplaques (OASE®, Waters) pour eliminer les proteines et les phospholipides. Apres evaporations, les residus secs sont repris par 200 μL d’un melange eau–methanol–acide formique (97,4–2,5–0,1 % v/v) et 20 μL sont injectes dans le systeme CL–SM/SM. Les differents composes ont ete separes sur colonne Atlantis ® T3 100 A, 3 μm, 2,1 mm × 100 mm (Waters) en utilisant un gradient de methanol (debit : 300 μL/min). La detection a ete realisee a l’aide d’un triple quadripole Quantum Ultra® (ThermoFisher). Resultats Dans nos conditions, la DHNK, la NK et la K sont eluees en respectivement 1,97, 3,68 et 3,77 min. Les rendements d’extraction sont compris entre 75 et 85 % sauf pour la DHNK (65 %) lors de l’extraction par le chloroforme en milieu alcalin. La methode est lineaire pour les 3 molecules entre 25 et 2000 μg/L, avec des CV Conclusion Fiable, robuste et facile a mettre en œuvre, notre methode CL–SM/SM permet la quantification de la K et de ses metabolites en medecine veterinaire et humaine dans un contexte tant pharmacologique que toxicologique.

The treatment of asthma concerns a large population and generally necessitates an administration by the pulmonary route. This route has some drawbacks which could be overcome by the buccal route, like considered in this study expecting an improvement of the compliance and the bioavailability. The aim of this work was to develop and characterize a thermogelling mucoadhesive formulation containing salbutamol sulfate. The main properties of the formulation were: (i) thermogelation around 30°C to facilitate the administration by spray; (ii) strong mechanical properties and high mucoadhesive strength to maintain the formulation in situ; (iii) drug release kinetics adapted to the treatment. The excipients were selected with regard to the final formulation safety. The experimental work emphasized the great interest of certain techniques in the characterization of this kind of formulation, for example the drug release study using flow through apparatus, the cytotoxicity study using digital holographic microscopy and also the mucoadhesion study by in vivo imaging. The use of mathematical models provided a relevant understanding of drug release mechanisms. The conclusion of this study is favorable to a further development of the project for a future marketing.; Le traitement de l’asthme concerne une large population et fait appel très majoritairement à une administration par voie pulmonaire. Cette voie présente certains inconvénients qui pourraient être palliés par le recours à la voie buccale comme envisagé dans cette étude, avec une amélioration attendue de la compliance et de la biodisponibilité. Aussi ce travail a eu pour objectif de développer et caractériser une formulation mucoadhésive thermogélifiante à base de salbutamol. Au regard des contraintes liées à la voie buccale et à la prise en charge du traitement de l’asthme aigu, les principales propriétés pharmacotechniques attendues pour optimiser la biodisponibilité de cette formulation ont été : (i) une gélification optimale autour de 30°C après pulvérisation pour faciliter notamment l’administration ; (ii) des propriétés mécaniques et mucoadhésives importantes pour assurer un maintien de la forme au site d’absorption ; (iii) une cinétique de libération compatible avec le traitement de la phase aigüe de la pathologie. Le choix des excipients a été défini afin d’avoir la plus grande innocuité de la formulation finale. Les travaux expérimentaux ont mis en évidence l’intérêt de certaines techniques innovantes pour la caractérisation de ce type de formulation comme notamment l’étude de libération à l’aide de l’appareil de dissolution à flux continu, la détermination de la cytotoxicité par microscopie holographique digitale et l’évaluation de la mucoadhésion in vitro et in vivo par imagerie. Les résultats ont aussi permis de proposer des modèles mathématiques pour l’interprétation des mécanismes de libération du salbutamol et quantifier l’importance des phénomènes de diffusion et d’érosion. Les conclusions de cette étude sont favorables à la poursuite du développement en vue d’une commercialisation.