Date of Award


Document Type


Degree Name

Doctor of Philosophy in Pharmaceutical Sciences


Pharmaceutical Sciences

First Advisor

Grazia Stagni

Committee Chair and Members

Grazia Stagni, Chair

Kenza Benzeroual

Rutesh Dave

Amit Joshi


Calcium channel blocker/diltiazem, Dermal microdialysis, In vitro permeation profile/franz diffusion cell, Limonene, Permeation enhancer, Topical/transdermal drug product


The addition of chemical permeation enhancers (CPE) to a topical dermatological formulation is the prevalent approach to improve permeability of active pharmaceutical ingredients (API) across the stratum corneum. Terpenes have been used as CPEs for a long time since they are safe and non-irritating. Limonene is one such terpene majorly found in essential oils extracted from citrus fruits and many varieties of cannabis. Studies performed using Limonene as PE show that Limonene was able to increase the flux of the drug by many folds. Limonene acts as an agonist to transient receptor potential ankyrin 1 (TRPA1) receptor – the activity of which is highly Ca2+ dependent. Since, calcium is found in the entire epidermis, the purpose of this study was to evaluate whether the above-mentioned properties of Limonene are related to its penetration enhancing effects. The aim of this study was to investigate whether the permeation enhancers R-Limonene (R-L) & S-Limonene (S-L) might act by decreasing the extracellular Ca2+ concentration that it is responsible for the tight cell-cell adhesion in the stratum corneum. This was followed by testing the suitability of microdialysis technique for DPH and DTZ through recovery and retrodialysis (loss) experiments. Finally, in vivo experiments were performed on a rabbit model. A randomized, crossover, single-dose, four treatment study design was chosen so that each rabbit would receive the test and reference formulation. The four gel combinations – control (5% DPH); test (5% DPH + 1%R-L or 1% S-L); control + DTZ (5% DPH + DTZ); test + DTZ (5% DPH + 1% R-L or 5% DTZ) were applied to the rabbit’s dorsal surface on the same day and dermal concentration profiles of DPH were assessed using microdialysis. The pharmacokinetic parameters such as Cmax and AUCinf were estimated using Phoenix WinNonlin Non Compartmental Analysis and studied for comparison between different formulations. The gels containing R-L or S-L showed slightly higher dermal exposure of DPH compared to the control gel. Whereas the dermal exposure from the gel containing DTZ was not different from the control gel. The results support the hypothesis that Limonene inhibition of Ca+2 activity in the skin might contribute to its penetration enhancing effect, although more in-vivo experiments are necessary to further prove this hypothesis. The first step was to find the optimal concentration of limonene that enhances the penetration of a model drug, diphenhydramine (DPH) from topical dermatological gels. Seven gels containing 5% DPH were prepared with 0 (control) and three increasing concentrations of R-L or S-L (1.00, 1.75, and 2.50 %) using simple dispersion method. An in-vitro release study across regenerated cellulose membrane using Franz cell apparatus was conducted to evaluate any possible physical interaction by assessing release parameters like flux and cumulative amount permeated. Then, an ex vivo study using porcine ear skin was performed to select the optimal percentage of R-L and S-L. The 1.00% R-L and 1.00% S-L increased permeability of DPH by 82.37% and 111.51%, respectively compared to control formulation, whereas higher percentages were not significantly different from control gel. Then, the calcium channel blocker diltiazem (DTZ) was added to the selected formulations to evaluate whether it counteracts the penetration enhancing effects of R-L or S-L. These formulations were tested again in-vitro on regenerated cellulose membrane to evaluate physical interaction before proceeding to the in vivo studies. All gels were tested for consistency, transparency, and uniformity of content. Samples were analyzed with a bioanalytical method specifically developed and validated for DPH/DTZ combination.