Preparation And In Vitro Characteristics Of Insulin Buccal Adhesive Tablets

AimInsulin is the most important drug for treating diabetes. However, to present, insulin is administrated mainly by injection, which would put great physical and mental burden on patients, for diabetic patients often need insulin administration for long term or even life time. In addition, the cost of injected forms of insulin is high, putting heavy economic burden on patients. Therefore, development of non-injected forms of insulin administration is one of the most interesting areas, including pulmonary, buccal, oral delivery, nasal, rectal and transdermal delivery et al. However, effective progress has not been made until now. Buccal drug delivery has been paid more and more attention for its many advantages. Buccal drug delivery can avoid degradation of drugs in gastrointestinal tract and first pass effect in liver. Buccal mucosa has large absorption surface, low protease activities and abundant vessels. The tolerance of buccal mucosa is well which could reduce relevant adverse effects. In addition, buccal drug delivery could be better accepted by patients for it is a convenient administration system. The key point of developing buccal drug delivery of insulin is how to improve the bioavailability of insulin, which is difficult to permeate and transport across the buccal mucosa and absorbed to the circulation because of its large molecular weight, approximately 6000. The aim of this study is to develop safe and effective insulin buccal adhesive tablets, by screening safe and high performance permeation enhancer and refining preparation technology of insulin buccal adhesive tablets, which would promote the permeation and transport of insulin across buccal mucosa. Methods1 . Establishment of human buccal epithelium model for studying permeation and transport of insulin using TR146 cell line. TR146 cells were cultured in Millicell culture plate inserts to establish three dimension human buccal epithelium model. The growth condition of cell layer was evaluated by measuring the trans-epithelium electric resistance (TEER) of cell layers at different times. Detection method of insulin concentration was established using (high performance liquid chromatogram) HPLC, which were used to study the permeation and transport of insulin across TR146 multilayer to estimate the integrity of TR146 cell layers.2. Screening of permeation enhancer in vitro using TR146 buccal epithelium cell model. Different concentrations of permeation enhancers NAC, SDC, SNP, GSH, Gln, CS, Arg, Azone and SPC were studied to evaluate their effects on promoting the permeation and transport of insulin across TR146 multilayer. The apparent permeability coefficient (Papp) of insulin and the enhancement ratio (ER) of each enhancer were calculated by measuring the transported amount of insulin across the TR146 multilayer model using HPLC. The effects of each enhancer of different concentrations on TR146 cell activity were studied with MTT experiment by calculating the cell relative enzyme activity. Effects on permeation and transport of insulin and toxic effect on the TR146 cell of enhancers were combined to screen safe and effective permeation enhancers.3. Preparation and in vitro characteristics of insulin buccal adhesive tablets. Twelve formulations F1-F12, weighting 80 mg/per tablet, containing 2 mg insulin were prepared, using CS, AP, CP, HPMC, CMC-Na and SA as adhesive materials. The adhesive layer were prepared by lyopyilization. Then one side of the adhesive layer were added with an impermeable backing layer consisting of 10% ethylcellulose solution, and dried open-air at room temperature. Evaluation was performed on each formulation by investigating their appearance characteristics, determining the weight, in vitro adhesive strength, swelling percent and in vitro releasing law of insulin buccal adhesive tablets. Results1. Establishment of TR146 buccal epithelium cell model.①The TEER of TR146 cell layer increased in a straight line at 2-6 days, increased slowly at 6-24 days, and arrived at peak value of plateau phase at 24-30 days, when the TEER is 129.4±9.7 ??cm2 (n = 50).②Control group showed the largest value of permeation rate and ultimate accumulated permeation amount of insulin, whose accumulated permeation amount of insulin at 30min and 240min were 1591.95±104.29μg and 2449.91±129.52μg respectively. Permeation rate and ultimate accumulated permeation amount of insulin at groups of 4, 8, 12, 16, 20, 24 d decreased with the time. 28 d and 32 d group showed the most slow permeation rate, whose ultimate accumulated permeation amount are 248.56±93.11μg and 177.29±59.52μg respectively, implying the TR146 multilayer was stable at this time and can be used as a reliable model to study permeation and transport of insulin.2. In vitro screening of permeation enhancers①Single enhancers 5% NAC, SDC, GSH, CS, Arg, Azone, SPC, SNP, 1% SNP, and mixed enhancers 5% CS/1% Azone, 5% SPC/1% CS, 5% SPC/1% Azone could promote the permeation and transport of insulin across TR146 multilayer buccal epithelium model significantly (p < 0.05 compared with control group).②MTT studies showed that, relative cell enzyme activities of 1% SDC, Arg, 5% Gln, Azone, SDC, SNP, Arg and 5% CS/5% Azone groups were significantly decreased compared with that of control group (p < 0.05), while that of other groups were not decreased significantly, whose toxic to cells were trivial and can be used as safe enhancers.3. Preparation and in vitro characteristics of insulin buccal adhesive tablets①Insulin buccal adhesive tablets prepared by lyopyilization has spongelike porosity structures, which can promote the release of insulin. The impermeable layer ensured directional release of insulin and avoiding drug lose.②F5, F7 and F12 were poorly formulated and further investigations were not performed on them. For other well formed formulations, the in vitro adhesive strength were F6 (137.97±7.33 g/cm2)>F8>F11>F1>F9>F4>F10>F3>F2 (66.1±6.65 g/cm2); the swelling percent were F6>F11>F8>F1>F4>F3>F10>F9>F2. The formulations with the largest in vitro release were F2, F9 and F10.Conclusion1. The human buccal epithelium model established by TR146 cells was stable and reliable with well reproducibility, which can be used to study permeation and transport of drug across buccal musosa.2. Single enhancers 5% NAC, GSH, CS, SPC, 1% SNP and mixed enhancers 5% CS/1% Azone, 5% SPC/1% CS, 5% SPC/1% Azone 5% NAC can greatly enhance the permeation and transport of insulin across TR146 multilayer, with little toxicity effects on the activity of TR146 cells, which would be safe and effective permeation enhancers for insulin transport.3. Formulations of insulin buccal adhesive tablets F2 using AP as primary adhesive materials and F9, F10 using CP as primary adhesive materials have suitable bioadhasive, well swelling rate and in vitro drug release characteristics, whihc can be used for further studies.