Date of Award

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Pharmaceutical Sciences

First Advisor

Grazia Stagni

Committee Chair and Members

Grazia Stagni, Chair

Avinash Kumar

Yousuf Mohammad

Lakshmi Raghavan

Keywords

Bioavailability, Bioequivalence, Cutaneous permeation, Ex vivo dermal microdialysis, Pharmacokinetics, Pig study

Abstract

Clinical response to most topical dermatological drug products (TDDP) depends on the availability of the drug in the dermis. Dermal Microdialysis (dMD) is a sampling technique that permits measuring the concentration of a drug over time, in vivo, directly into the target tissue, the dermis. The pharmacokinetic parameters obtained from such studies may help to optimize the development of TDDP and potentially can be applied to the assessment of TDDP bioequivalence. However, these studies require several hours or even days of continuous sampling that makes it often stressful and unpractical for human subjects as well as animals. The goal of this dissertation was to develop a reliable and consistent ex-vivo dMD method to complement and assist in vivo dMD experiments.

In the first part of the project, we have developed and tested the ex-vivo dermal microdialysis method on two different experimental skin models using freshly excised porcine skin. Porcine skin was selected due to the close resemblance to human skin, it is advantageous in terms of availability and expense. For the microdialysis study, in-house dermal microdialysis probes were conveniently manufactured with controlled specifications and the microdialysis recovery process was screened with an in vitro setup to match the intended use. The in vitro microdialysis method was optimized for probe specification, analyte suitability, perfusion flow rate, and perfusate composition. A maximized, rapid, and steady recovery was demonstrated within a wide range of concentrations. For the ex vivo dermal microdialysis study, the two different skin models developed were: M1-- Full-thickness skin (≈0.25 cm) without subcutaneous fat layer placed on a hydrated 0.5 cm cellulose backing support, and M2 -- Full-thickness skin with subcutaneous fat layer (total thickness = 1.0 cm) placed directly on an aluminum boat, avoiding any kind of hydration. Both setups were tested on TDDP cream and gel of metronidazole (MTZ) for which both in vivo and IVPT data are available for comparison. The two different formulations, Metronidazole cream and gel, were compared side-by-side for the rate and extent of delivery to the dermis. The latter skin model was found suitable, manifesting data comparable to the available data from in vivo pig and IVPT (human cadaver) study. The selection of the best-fit-model was based on the comparative bioavailability response from the negative control, Metronidazole gel, resulting in a lower bioavailability profile (90% CI). Using this suitable ex vivo dMD model, site-specific results of the drug can be conveniently monitored in the dermis leading to dose-dependent rate and extent in concentration-time exposure.

The M2 model was further tested for the effect of temperature of the skin on the bioavailability profile of the drug. As reported in several pieces of literature, an increase in dermal exposure is expected with the rise in skin temperature. Superficial addition of heat to the skin was not feasible as it may change the thermodynamics of the formulation leading to alteration in permeation kinetics. Thus, the physiological temperature of ex vivo pig skin explant was achieved by providing continuous heat from the ventral side using a closed water-bath system. The process of supplementation of temperature did not impact the bioavailability profile, rather unfavorable damages to the skin microstructure due to thermal degradation was observed. Further studies with the proposed model ex vivo dMD model were conducted at ambient lab temperature.

Yet another aspect of the proposed model was to test its capability to determine bioequivalence (BE). The potential of using this model for BE testing was validated by comparing the BA of MetroCream with its USFDA-approved generic Metronidazole 0.75% cream. The overall BE estimation resulted in an ln-AUC of 91.65 (80.93, 104.88) and an ln-Cmax value of 87.56 (74.87,102.39). The fact that reference and test formulations can be tested simultaneously at multiple sites on a skin sample harvested from a single animal subject reduces the burden of vii-inter-subject variability. The experimental population size required to establish bioequivalence for topically applied drugs can be reduced.

Yet another aspect of the study was to design a mathematical model, based on the ex vivo dMD findings, to extend its predictability to in vivo outcomes. A first-of-its-kind unit impulse response method was applied in dermis tissue of skin explant to measure the absorption independent elimination parameters. The estimated parameters were employed to calculate the cumulative absorption of the drug from different topical formulations. The absorption profile of the developed model was time-scaled and absolute-scaled with a permeation scaling factor to map with available literature data on in vivo pig. The levy point-to-point regression coefficient was employed to predict the in vivo PK profile thereafter. With the current intervention, we propose a mathematical possibility to predict in vivo outcomes for a given topical dose. The studies presented were limited internal predictions only, and external validation with a different set of data is yet to be performed and to be undertaken at another time.

Overall, the studies presented in this work provides a foundation stone for an elaborate field of work that can be undertaken to minimize the use of animal in pharmacokinetic evaluations for topical and transdermal products. Ex vivo dermal microdialysis warrants testing on a plethora of drug molecules of different polarities to decide on the future of the technique. Regardless, the technique holds unmet potential and needs to be nurtured over time.

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