Preparation Of Nobiletin In Situ Gel And Evaluation Of Its Brain Targeting Following Intranasal Administration

OBJECTIVESThe purpose of this paper was to develop a thermosensitive in situ gel including nobiletin (NOB) for intranasal administration. We evaluated drug release behavior of NOB nasal in situ gel in vitro to speculate the drug release mechanism in vivo. Finally, through the comparision among three NOB formulations:NOB nasal in situ gel, NOB solution following intranasal administration and NOB solution following intravenous injection administration, to evaluate the pharmacokinetics of NOB nasal in situ gel and to testify the probability of brain targeting of NOB by intranasal administration.METHODS1. Preformulation StudyWe determinated dissolubility of NOB in defferent solvents and surfactants, and distribution coefficient in oil-water by n-octyl alcohol/water system. The effects of different formulation additives on the thermodynamic properties of thermosensitive in situ gel were studied.2. Preparation of NOB nasal in situ gelUsing sol-gel transition temperature (Tsol-gel) as the index, the basic blank formulation of nasal in situ gel was expored by orthogonal design, and determinated optimization formulation by central composite design. We could get NOB nasal in situ gel by adding NOB in the blank in situ gel. 3. Investigate the release behavior of NOB nasal in situ gel in vitroWe used the Franz diffusion cell method, membrane free method and dialysis cell method to evaluate the release behavior of NOB nasal in situ gel in vitro.4. Established the assay method of NOB in biological samplesNOB in plasma or brain tissue fluid was extracted with ethyle acetate and N-hexane after precipitating by acetonitrile. Then the organic phase was condensed. Nimodipine as the internal standard, the reconstitution was chromatographed on the Kromasil C18 column (4.6 mm×150 mm,5μm) at 40℃with mobile phase methanol-water (65:35, v/v), the flow rate was 0.8 mL·min-1 and detected at 335 nm.5. Pharmacokinetic study of NOB nasal in situ gelSprague-Dawley rats were administrated with NOB solution by nasal delivery, or NOB in situ gel by nasal delivery or NOB solution by intravenous injection separately, collecting blood samples and brain tissue samples at different time points. We determined drug concentration in blood plasma and brain tissue by HPLC, in order to study the pharmacokinetics and the brain targeting of NOB nasal in situ gel.RESULTS1. NOB dissolubility was very tiny in water (0.04 mg·L-1), and the greater in ethanol was 14.01 mg·L-1. The distribution coefficient in oil-water was logPwater=2.97. The Tsol-gel of nasal in situ gel significantly showed concentration-dependence of Poloxamer 407; and it also showed that NOB and the additives [Poloxamer 188, PEG 200, ethanol,1,2-propylene glycol, Tween-80, HP-β-CD and NaCl] had the effect on Tsol-gel of nasal thermosensitive in situ gel, which would provide the basis for the formulation of NOB nasal in situ gel.2. With the orthogonal design and central composite design, the optimization formulation of NOB nasal thermosensitive in situ gel was 0.3%NOB+12%EtOH+12%PEG200+18%P407+1.4%P188+0.9% NaCl+55.4%H2O. Its Tsol-gel was 33-34℃.3. The release behavior of NOB nasal in situ gel was mainly by erosion, also in accompany with diffusion feature. The drug of NOB nasal in situ gel was completely released in 120 min.4. The linear range of NOB concentration was 7.8-500 ng·mL-1, and the limit of quantification was 7.8 ng·mL-1. The extraction recoveries were above 80% and the methodology recoveries were 97-104%. The intra-assay and inter-assay RSDs were less than 13.2%.5. Compared with the solution by nasal administration, the relative bioavailability of in situ gel by nasal administration was similary and about 45%, and the brain tissue local bioavailability was lower. The former was 53.2% and the latter was 44.0%. About the drug targeting index (DTI), the in situ gel by nasal administration was similar to the solution following intravenous injection, the DTI of the solution following nasal administration was higher and about 1.2, which indicated that the solution following nasal administration had the better characteristic of brain targeting.CONCLUSIONS1. This study investigated the preparation methods of NOB thermosentive in situ gel, which may provide the basis for the development of NOB nasal preparations.2. This project has certainly proved the favourable feature of brain targeting of NOB by nasal delivery.