Cyanine derivative as a suitable marker for thermosensitive in situ gelling delivery systems: In vitro and in vivo validation of a sustained buccal drug delivery.

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.

Poloxamer bioadhesive hydrogel for buccal drug delivery: Cytotoxicity and trans-epithelial permeability evaluations using TR146 human buccal epithelial cell line.

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 2h, 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 (×10(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.

Influence of additives on a thermosensitive hydrogel for buccal delivery of salbutamol: relation between micellization, gelation, mechanic and release properties.

Thermosensitive hydrogels developed for buccal delivery of salbutamol were prepared using poloxamer analogs (Kolliphor(®) P407/P188), xanthan gum (Satiaxane(®) UCX930) and NaCl. P188 increased gelation temperature (Tsol-gel) by 2.5-5°C, micellization temperature (<1°C) and gelation time by >3s. To obtain a suitable Tsol-gel at 28-34°C, P407 and P188 concentrations were set to 18-19% and 1%. NaCl reduced Tsol-gel (>2°C) out of the optimal range. Six formulations containing 0.05-0.1% Satiaxane(®) fulfilled the temperature criteria. Concerning the gel strength, 1% P188 had no significant effect, NaCl increased it at 20°C, and Satiaxane(®) enhanced it at 20°C and 37°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 (T80=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(®) exhibited the slowest release. In the membrane method, combination of P407/P188/Satiaxane(®) provided a sustained diffusion with a burst effect (T25=9.6 min, T80=97.8 min), which provides potential clinical interests.

Altered HepG2 cell models using etomoxir versus tert-butylhydroperoxide.

Energetic failure which occurs in both ischemia/reperfusion and acute drug-induced hepatotoxicity is frequently associated with oxidative stress. This study displays the setting of a new cell culture model for hepatic energetic failure, i.e., HepG2 models modified by etomoxir [ETO] addition [0.1 mM to 1 mM] and compares the cell impact versus tert-butylhydroperoxide [TBOOH; 0.2 mM], an oxidative stress inducer. As it was observed with Minimum Essential Medium (MEM) without any interfering agent, decreasing temperature drastically lowered adenosine triphosphate (ATP) levels, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability test, and protein content, compared to 37 °C (p=0.02, p<0.001 and p<0.001, respectively), but to a larger extent in the presence of ETO or TBOOH. The alteration was generally highly dependent on the ETO concentration, time, and temperature. At 37 °C 24 h after (T24h), regarding ETO concentration, R² correlation ratio was 0.65 (p<0.001), 0.70 (p<0.001), and 0.89 (p<0.001) for ATP levels, protein content, and viability, respectively. The lowest ETO concentration producing a significant effect was 0.25 mM. Concerning time dependency (i.e., T24h versus after 5 h (T5h)), at 37 °C with ETO, ATP level continued to significantly decrease between T5h and T24h. In a similar way, at 37 °C, the MTT viability test decrease was accelerated only between T5h and T24h for ETO concentrations higher than 0.5 mM (p=0.016 and p=0.0001 for 0.75 and 1 mM, respectively). On the contrary, with TBOOH, comparing T24h versus T5h, cellular indicators were improved but generally remained lower than MEM without any interfering agent at T24h, suggesting that TBOOH action was time limited probably in relation with its oxidation in cell medium. This study confirms the interest of altered ETO cell model to screen agents (or formulation) prone to prevent or treat energetic depletion in relation with oxidative stress.

Freeze-drying of ATP entrapped in cationic, low lipid liposomes.

Concerning the instability of ATP liposomes formulated to easily diffuse through the liver (size approximately 100 nm), this work targets the key parameters that influence the freeze-drying of a preparation that combines cholesterol, DOTAP and phosphatidylcholine (either natural soybean or egg (SPC or EPC) or hydrogenated (HSPC)). After freeze-drying blank liposomes, size increased significantly when initial lipid concentration was lowered from 20 to 5mM (p=0.0018). With low lipid concentration preparation (5mM), SPC limited size increase (SI) more efficiently compared to EPC or HSPC. With SPC and EPC, sucrose showed better size results compared to trehalose (Lyoprotectant/Lipid ratio (w/w) avoiding any SI: approximately 5 and approximately 10 (for SPC), approximately 10 and approximately 15 (for EPC), for sucrose and trehalose, respectively), but the opposite was evidenced with HSPC liposomes where a Trehalose/Lipid ratio of 25 barely prevented SI. In addition, slow versus quick cooling rate led to limiting SI for HSPC liposomes (p=0.0035). With sucrose or trehalose at both Lyoprotectant/Lipid ratios ensuring size stabilisation (10:1 and 15:1, respectively), ATP leakage ranged between 38.8+/-7.9% and 58.2+/-1.4%. In conclusion, this study emphasizes that using strict size maintenance as the primary objective does not result in drug complete retention inside the liposome core.

Formulation and evaluation of ATP-containing liposomes including lactosylated ASGPr ligand.

An original ligand (Lac-10-Chol) designed to interact with asialoglycoprotein receptors to potentially target hepatocyte was synthesised by grafting a lactose head to a cholesteryl structure, which was then included in liposomes. Preliminary formulation tests led to the selection of conventional formulations based on soybean phosphatidylcholine/cholesterol/DOTAP (+/- DOPE) (+/- Lac-10-Chol) that present reproducible absolute entrapment value (1.45 +/- 0.10%), with a size of 109 +/- 7 nm and a slight positive charge (3.77 +/- 1.59 mV). Cell viability (via the MTT test), expressed as the percentage of nontreated cells in HepG2 cells, was very close to the control. Internalization tests evidenced an intracellular penetration of fluorescent liposomes, but no specific ligand effect was demonstrated (P > 0.05). Nevertheless, regarding the adenosine triphosphate (ATP) assay, a slight increase was obtained with liposome loaded with ATP incorporating Lac-10-chol after 24 hours (P < 0.05).

L-cysteine encapsulation in liposomes: effect of phospholipids nature on entrapment efficiency and stability.

Liposomal entrapment of L-cysteine (L-CySH) could be a solution to enhance its oxidative stability and its intracellular bioavailability for glutathione (GSH) synthesis. This study addresses the influence of different factors (i.e. pH value (6.3 vs 7.4), antioxidant agents (EDTA or tocopherol (TO and nature of phosphatidylcholine (PC) (Soybean PC (SPC) vs hydrogenated SPC (HSPC)) to formulate and optimize Large Unilamellar Vesicles (LUVs) of L-CySH composed of PC/Cholesterol/ Phosphatidylglycerol (6:3:1). pH decrease (p = 0.0002) and substitution of SPC by HSPC (p < 0.001) reduced L-CySH oxidation. EE% (entrapment efficiency) varied from 0.98% +/- 0.54 (SPC, pH 7.4) to 6.46% +/- 1.37 (HSPC, pH 6.3) and was improved by decreasing pH (p = 0.011) and using HSPC (p < 0.0001). An immediate release of L-CySH was observed with SPC. On the contrary, with HSPC at pH 6.3, 42.0% +/- 1.2 and 73.0% +/- 1.7 remained encapsulated after 24h at 25 degrees C and 4 degrees C, respectively. In conclusion, HSPC offering both stronger rigidity and lesser propensity for peroxidation led to optimize L-CySH liposomal stability.

A review of poloxamer 407 pharmaceutical and pharmacological characteristics.

Poloxamer 407 copolymer (ethylene oxide and propylene oxide blocks) shows thermoreversible properties, which is of the utmost interest in optimising drug formulation (fluid state at room temperature facilitating administration and gel state above sol-gel transition temperature at body temperature promoting prolonged release of pharmacological agents). Pharmaceutical evaluation consists in determining the rheological behaviour (flow curve or oscillatory studies), sol-gel transition temperature, in vitro drug release using either synthetic or physiological membrane and (bio)adhesion characteristics. Poloxamer 407 formulations led to enhanced solubilisation of poorly water-soluble drugs and prolonged release profile for many galenic applications (e.g., oral, rectal, topical, ophthalmic, nasal and injectable preparations) but did not clearly show any relevant advantages when used alone. Combination with other excipients like Poloxamer 188 or mucoadhesive polymers promotes Poloxamer 407 action by optimising sol-gel transition temperature or increasing bioadhesive properties. Inclusion of liposomes or micro(nano)particles in Poloxamer 407 formulations offers interesting prospects, as well. Besides these promising data, Poloxamer 407 has been held responsible for lipidic profile alteration and possible renal toxicity, which compromises its development for parenteral applications. In addition, new findings have demonstrated immuno-modulation and cytotoxicity-promoting properties of Poloxamer 407 revealing significant pharmacological interest and, hence, human trials are in progress to specify these potential applications.

A double-blind sham controlled study of right prefrontal repetitive transcranial magnetic stimulation (rTMS): therapeutic and cognitive effect in medication free unipolar depression during 4 weeks.

BACKGROUND: Transcranial magnetic stimulation (TMS) has become a therapeutic tool in psychiatric diseases. METHODOLOGY: The objective was to evaluate the efficacy of TMS in unipolar depression: the percentage of responders (>50% HDRS reduction) and remission (HDRS score < or =8, after four weeks of active TMS treatment in depressed patients free of any antidepressive agent versus placebo-TMS. RESULTS: 27 patients were randomized in two groups: rTMS (N=11) versus sham TMS (N=16). Statistical differences were detected between sham and TMS treated groups on remission (0/16 versus 4/11 p=0.032, 1/16 versus 6/11 0.028 and 1/16 versus 7/11 p=0.011 at day 14, day 21 and day 28, respectively) and on response (2/16 versus 5/11 at day 14 (NS), 2/16 versus 7/11 p=0.0115 at day 21 and 1/16 versus 7/11 (p=0.025) day 28, respectively, using the exact Fisher test). Significant differences were observed between day 1 versus day 8 (p<0.01), day 15, day 21 and day 28 (p<0.001) in TMS group and only versus day 21 (p<0.01) and day 28 (p<0.05) for the sham group. ANOVA comparison between TMS and sham groups was significant at day 14 and day 28 (p<0.05). LIMITATIONS: The few number of patients. CONCLUSION: Our study has shown an efficacy of right rTMS in free medication unipolar depression over a month. Nevertheless, number of patients included is limited and multicentric studies will be necessary to specify the antidepressive action of TMS.

Liver function tests during treatment with antipsychotic drugs: a case series of 23 patients.

Atypical antipsychotics represent a new class of medication for the treatment of schizophrenia and their use is associated with a reduction of neurological side effects. This article reports the result of the systematic clinical and biological supervision of hepatic enzymes on 23 schizophrenic inpatients treated by atypical antipsychotic during 2 weeks at Days 1 (D1), 7 (D7), and 14 (D14) in a naturalistic study during 6 months. The drug administrated was limited to four medications--risperidone, amisulpride, olanzapine, and clozapine--but other psychotropic agents were prescribed. Six cases of biological cytolytic hepatitis were observed. Due to the numerous risk factors and the frequency of "routine" conditions, careful supervision of the hepatic function is needed to prevent this kind of side effect.