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Labrasol ALF and Transcutol HP (70%) ... of GSNO amount in formulation decreases the toxicity of this formulation; for GSNO (0.029M) ± 50% of cell viability.
Ana Henriques Mota1,2#, Marie Socha1, Philippe Maincent1 1 EA3452

CITHEFOR « Cibles Thérapeutiques, Formulation et Expertise préclinique du médicament », Faculté de Pharmacie, Université de Lorraine, 5, rue Albert Lebrun, BP 80403, 54001 Nancy, France 2 Escola de Ciências e Tecnologias da Saúde, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande, 376, 1749 - 024 Lisboa, Portugal #e-mail:

[email protected]

Introduction The principal reasons of death in western world are the cardiovascular diseases with 31% [1]. Among current treatment, the NO exhibits a vascular function at smooth muscle (vasodilatation) and a relaxation proliferation of cells (inhibition of activation and aggregation platelets; inhibition of adhesion of leucocytes at endothelium) [2]. The donors of Nitric Oxide (RSNO) are physiologic components and endogenous forms such as GSNO [3]. The RSNOs present cardiovascular homeostasis function, have a vasodilator action (local and systemic) and also an antiplatelet effect [3]. However, RSNO are very sensitive compounds, which have very short half-life after administration. Thus, it is very important to create news formulations in this therapeutic field. Among the innovative dosage forms, SMEDDS are microemulsions systems, which are known to increase solubility, bioavailability, and to potentiate action of drugs [4-5].

The purpose of the current study was to prepare and characterize SMEDDS of GSNO (in terms of size, stability, HLB, kinetic, cytotoxicity and incorporation efficiency), with a view to protecting GSNO and to increasing its bioavailability after oral administration.

Methods SMEDDS with GSNO were prepared through the self-emulsifying systems (Fig 1). The method used to make the incorporation of the drug was double emulsification (Fig. 3). • Labrasol ALF and Transcutol HP (70%) • Capryol (10%) • Solution of KH2PO4 (20%) • Labrasol ALF and Transcutol HP (70%) • Capryol (10%) • Solution of GSNO 0,01 M or 0,029 M (20%) Fig. 1 – Formulation of Blank SMEDDS and SMEDDS with GSNO.

Fig. 2 – Chemical structure of GSNO.

Fig. 3 – SMEDDS W/O/W.

SMEDDS were characterized as mentioned in figure 4. NO assay (in vitro release study by Griess-Saville and DAN-DAN Mercury methods)

Blank SMEDDS and SMEDDS with GSNO

Cytotoxicity study (MTT test in Caco-2 cells)

Fig. 4 – Studies of SMEDDS formulations.

Results

Size (d.nm)

Mean particle size was 210 ± 63 nm (n = 8) and 200 ± 21 nm (n = 4) for SMEDDS without and with drug, respectively. These formulations demonstrated a satisfactory stability during 9 days at room temperature when protected from light (graph. 1). The pseudoternary phase diagram of blank SMEDDS (fig. 5), the value of HLB was 3.9 ± 0.1 (n = 9) and 3.8 ± 0.1 (n = 5) for SMEDDS without and with drug, respectively. The results of release kinetic were not conclusive. 2700 2400 2100 1800 1500 1200 900 600 300 0 1 Blank Formulation

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Formulation GSNO 0,01M

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Formulation GSNO 0,029M

8 9 Time (days)

Graph. 1 – Size stability study over time (mean 210 ± 63 nm (n = 8) and 200 ± 21 nm (n = 4) for SMEDDS without and with drug, respectively).

Fig. 5 – Pseudoternary phase diagram of blank SMEDDS (blue: microemulsion area).

Graph. 2 – Cytotoxicity study.

In cytotoxicity study (graph. 2) it was observed that the increase of GSNO amount in formulation decreases the toxicity of this formulation; for GSNO (0.029M) ± 50% of cell viability and GSNO (0.01 M) ± 2%. The incorporation efficiency was between 3-8%.

Conclusion and Perspectives Based on the preliminary results obtained so far, SMEDDS look like a promising system for the oral administration of GSNO. However, it will be necessary to optimize release kinetic and enhance the incorporation efficiency. In addition it has the advantage that it should be easily scaled up at the industrial level.

Acknowledgements The authors are grateful to UPR 3349 CNRS, LRGP (Mr. R Schneider), for production of GSNO. The authors would like to thank to ANRF by financial support and Gattefosse for providing the excipients. References [1] WHO. The Impact of Chronic Disease in France. World Health Organization. http://www.who.int/chp/chronic_disease_report/france.pdf?ua=1. [2] Dahboul F. et al., PLoS ONE, 7 (9):1-8, 2012. [3] Parent, M. et al, European Journal of Pharmaceutics and Biopharmaceutics, 85:640-649, 2013. [4] Shakeel F., et al., Journal of Molecular Liquids, 182:57-63, 2013. [5] Liu Y., et al., International Journal of Pharmaceutics, 365:136-142, 2009.