| Objective:The purpose of this study was to prepare and evaluate an in-situ gel of rizatriptan benzoate(RZTB)nasal spray for the treatment of migraine.Furthermore,in vivo and vitro evaluation were studied which provides basis for its further study.Methods:The content of RZTB was determined by HPLC method.The related properties of RZTB and the effects of acid,alkali,oxidants,high temperature and light on RZTB were investigated.Poloxamer 407(P407)and poloxamer 188(P188)as gel materials to prepare in-situ gel of RZTB nasal spray.Nasal perfusion experiment in rats and cell penetration test were performed to screening the absorption enhancer.The proportion of each component in the formulation was determined by gel temperature,viscosity,particle size distribution,spray angle and content of drug at each spray.The pharmacokinetic parameters and distribution of drug in brain were performed in rats.The effects of RZTB and absorption enhancers on the survival of Calu-3 cells were investigated by MTT assay.After continuous administration,the rat nasal mucosa was sectioned to investigate the toxicity of the drug on nasal mucosa.The quality of RZTB in-situ gel nasal spray was evaluated.Results:Wavelength of 280 nm was selected to determine RZTB.The precision and recovery both met the requirements of the methodology.The oil-water partition coefficient was-0.90.The solubility was similar under various pH conditions and the highest solubility was found in pH 4.0 buffer solution,which was 62.87 mg/mL.RZTB is relatively sTab.to light,but it would induce a small amount of impurities under high temperature and alkaline conditions.RZTB is sensitive to acids and oxidants which impurities were 3.98%and 6.15%,respectively.Nasal perfusion experiments in rats showed that 1%propylene glycol,1%Tween 80,1%HP-β-CD,0.5%CS and 0.5%CMCS can all promote the absorption of RZTB.CMCS had a relatively good absorption promoting effect.The results of cell penetrated experiments showed that the cumulative amount of RZTB had a concentration-dependent manner.There was no significant difference between RZTB solution with absorption promoter and without absorption enhancer(p>0.05).Therefore,the optimal formulation of RZTB in-situ gel nasal spray is:20%P407,2%P188,5%glucose,0.02%benzalkonium bromide,0.1%CMCS and 0.5%RZTB.The results of pharmacokinetic test showed that Tmax of nasal administration was 10 min,while the Tmax of oral administration was 60 min.In addition,about three folds increase in the Cmax value of nasal saline(224 ± 72 ng/mL)compared with oral saline(75± 16 ng/mL),and nasal gel has the most Cmax value(388 ± 63 ng/mL).The AUC0-6h of oral saline,nasal saline and nasal gel were 157 ± 33,281 ± 13,445 ± 49 ng/mL·h,respectively.Furthermore,relative bioavailability AUCN.S0-6h/AUCO.S 0-6h was 1.79 ± 0.15%and AUCN.G0-6h=G AUCO.S 0-6H was 2.84 ± 0.07%.In addition,the drug concentrations in the brain at 30 and 60 min after nasal gel solution administration were 130 ± 6 ng/mL and 117 ± 8 ng/mL,respectively,higher than that of the oral solution(58 ± 7 ng/mL,21 ± 5 ng/mL)and nasal solution administration(78 ± 5 ng/mL,50 ± 4 ng/mL).The AUC0-2h of oral solution administration,nasal solution administration and nasal gel solution administration were 68± 10 ng/mL·h,86 ± 5 ng/mL·h and 138 ±5 ng/mL·h,respectively.The results of in vitro cytotoxicity of RZTB and different absorption enhances indicated that concentration of RZTB among 6.25～800 μg/mL was safe in 48 hours.1%propylene glycol,1%HP-β-CD and 0.5%CMCS had no obvious cytotoxicity in 24 hours to the Calu-3 cells,whereas cell survival rate of 0.5%Tween 80 and 0.5%CS were below 80%.The results of nasal toxicity test concluded that in-situ gel of RZTB had no significant effect on morphology and cell differentiation of nasal mucosa in rats.The quality of RZTB in-situ gel nasal spray was conformed to the requirements of pharmacopoeia.Conclusion:This study successfully prepared a formulation of in-situ gel of RZTB nasal spray.Cell tests in vitro and pharmacokinetic trials in vivo were carried out.Furthermore,toxicity tests of cells and nasal mucosal were performed.It provides the basis for the research of nasal delivery for the triptans. |
