Purpose: Since parenteral insulin can only be administered clinically to treat diabetes mellitus, many non-parenteral administrations of insulin as alternative methods have been studied in the past. Although many publications are available in the literature, a mechanistic understanding of sublingual permeation which will facilitate the optimization of insulin administration has not been explored extensively. Due to hydrophilic property of insulin, sublingual insulin is reported to permeate through membrane by passive diffusion pathway. However, because of the large molecular weight of insulin, it tends to have low permeability. To achieve satisfactory membrane permeability, the use of permeation enhancer becomes a prerequisite for any insulin formulation for sublingual administration. Therefore, it is imperative to screen the permeation enhancers and/or their combination and then optimize their concentration. HPβCD (2-hydroxypropyl-β-cyclodextrin) has been well explored as a permeation enhancer with insulin for sublingual as well as other routes such as nasal and oral administration. Furthermore, poloxamer 188 has been investigated recently as a permeation enhancer for nasal administration. Hence, the current investigation includes exploring the combination effect of these two permeation enhancers using Box-Behnken design (BBD) to optimize the permeation of insulin solution administered sublingually.
Methods: As per Table 1, the independent (X1: concentration of insulin, X2: concentration of HPβCD, and X3: concentration of poloxamer 188) and dependent (Y1: insulin permeated at 60 min and Y2: insulin permeation flux) variables were used to generate BBD using FusionPro software (S-Matrix Corporation, version 7.3.20). As a result, 15 formulations were generated as displayed in Table 2 and subjected to in vitro permeation evaluation. A setup of side-by-side permeation apparatus was used for the in vitro permeation evaluation using cellulose acetate membrane as artificial membrane. The artificial membrane was used to minimize use of animals for the experiment and to screen the suitable formulation for further evaluations during early stage of formulation development. Each formulation was placed in donor compartment (pH 6.8), while phosphate buffer solution (pH 7.4) was placed in receptor compartment. Samples were withdrawn at predetermined time intervals from the receptor compartment. All samples were analyzed for insulin content by Nanodrop spectrophotometer at 214 nm. Upon analyzing the relationship among the independent and dependent variables by FusionPro software, an optimized formulation was obtained and tested using MatTek Corp. buccal tissue model for permeation of insulin. Furthermore, insulin stability studies of the formulation were performed in artificial saliva and phosphate buffer solution (pH 6.8) for 2 h, respectively.
Results: As per the Pareto ranking obtained from FusionPro, it was seen that the highest effect on Y1 was the interaction of X1 and X2, while on Y2 was interaction of X2 and X3. The regression equations generated for each response variable were as follows:
Y1 = + 4.963200 - 0.256300 X1- 0.512600 X2+ 0.051260 (X1 × X2)
Y2 = + 68.221170 + 1.337953 X1 - 5.805021 X2 - 159.730280 X3 + 76.838187 (X3)² - 0.267591 (X1 × X2) + 14.423321 (X2 × X3)
The positive sign of variable in regression equations indicates that the response value increases with the increase in the variable and vice versa. The optimization goal was set at maximum to get the highest response for both Y1 and Y2 variables. At all settings the desirability value was found to be 0.8 wherein the desirability target was 1. The highest insulin permeated at 60 min and insulin permeation flux from the optimized formulation were found to be 14.483 IU and 22.630 IU/cm2/min, respectively, and was similar to the formulation F4. In addition, the insulin content analyzed form insulin stability studies of the optimized formulation was found to be 96.8 % and 97.2 % in artificial saliva and phosphate buffer solution, respectively and indicating insulin was stable at 37° C both in artificial saliva and phosphate buffer solution.
Conclusion: Using design of experiments approach, such as BBB, it was seen that the selected formulation shows promise for development of insulin solution dosage form for sublingual administration.
Senshang Lin– St johns university, New York