VCOM Carolinas Research Day 2023

Biomedical Studies


Carsten Steinmetz, OMS-II; Kyle Kleiman, OMS-II; Joseph Metz, DO; Lindsey Richardson, OMS-III; Jaime Foushee, PharmD; Patrick D. Ellis Fisher, PhD; David Eagerton, PhD; Luciana Schwab, PhD Edward Via College of Osteopathic Medicine-Carolinas Campus - Carolinas Campus; Spartanburg, SC

Abstract # BIOM-6



Discussion and Conclusions

Context: Dysplastic oral leukoplakia (OL) lesions, or white patches within the oral mucosa, are premalignant lesions that may progress to oral squamous cell carcinoma (OSCC). 1 Data suggest anywhere from 11-36% of OL will transform to OSCC if left untreated, depending on the length of follow up and degree of dysplasia. 2 Several clinical studies have utilized either vitamin A or vitamin D synthetic derivatives in topical formulations for the treatment of dysplastic OL with some success 3-11 but using a combination of the two drugs together has not been examined. In addition, to the best of our knowledge, cell culture studies using oral dysplastic cell lines treated with either vitamin A derivatives, vitamin D derivatives, or a combination of both are lacking. Vitamin A and vitamin D derivatives have distinct unique mechanisms, which when used together could exhibit synergistic effects. Objective: To determine the effect of a drug combination of a vitamin A and a vitamin D derivative on the viability of human dysplastic oral keratinocytes, compared to monotherapy with each agent. It is hypothesized that the combination of these synthetic derivatives would result in a synergistic cytotoxic effect, compared to the use of each drug individually. • A dysplastic oral keratinocyte (DOK) cell line was obtained and cultured according to cell provider instructions. • Cells were plated in 96 well plates, at 1000 cells per well on triplicate wells, and treated with the drugs or vehicle control for 24, 48, and 72 hours. • Cells were treated with: • Vehicle control (DMSO) • 25, 50, 100, 200, or 300 µM of single drug retinoic acid (aka retinoic acid) • 0.5, 1, 10, 50, or 100 µM of single drug calcipotriene • Five different combinations of the two vitamin derivatives together at a fixed ratio of 4:1, retinoic acid:calcipotriene as follows: 12.5+3.1, 25+6.3, 50+12.5, 100+25, and 200+50 µM. • Cell viability was measured utilizing an MTT assay. • The effect of the combination between calcipotriene and retinoic acid on cell viability and synergism was calculated for the 72-hour time point utilizing CompuSyn software. 12-13 Methods

• The combination of retinoic acid and calcipotriene had a more profound effect on cell viability than either drug alone after 48 or 72 hours. • Results from CompuSyn software indicate synergism and the potential for favorable dose reduction for 2 of the 5 studied combinations. • Favorable dose reduction may allow for lower concentrations of each medication to be applied topically to OL lesions, which could limit drug toxicity. • Limitations of the study include the in vitro cell culture design, with continuous application of drug treatments, which may not translate to the intermittent topical application of these agents in a living human model. • Additional studies are necessary to determine the IC50 for calcipotriene. This may allow for the calculation of a drug concentration that will give a more accurate dose-response curve. Other project ideas include the development of an interprofessional OL educational program to increase the early identification of OL lesions.

Figure 2. Cell viability with calcipotriene or retinoic acid monotherapy

Figure 3. Cell viability with calcipotriene and retinoic acid combination therapy


Figure 4. Oral leukoplakia classifications 14

Verrucous Non homogenous OL

Nodular Non Homogenous OL

Homogenous OL

Speckled Non homogenous OL

Table 1. Quantitative assessment of the inhibitory effect of calcipotriene and retinoic acid combination therapy using the combination index (CI) method


1. Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J. Oral Pathol Med . 2007;36:575-80. 2. Lee JJ, Hong WK, Hittelman WN, et al. Predicting Cancer Development in Oral Leukoplakia: Ten Years of Translational Research. Clin Cancer Res . 2000;6(5): 1702-10. Additional references:

Figure 1. Experimental design

• The half-maximal inhibitory concentration (IC50) was able to be determined for retinoic acid, but not for calcipotriene. • Results show that the addition of the two drugs together had a more profound inhibitory effect on cell viability: the IC50 for retinoic acid monotherapy was 90 µM, while IC50 for retinoic acid + 0.25x calcipotriene was 50 µM at 72 hours. • Furthermore, analysis of the experimental data in combination with simulation values from the CompuSyn software indicated a slight synergism for 2 of the 5 combinations tested here and a favorable dose reduction of 1.4-fold for retinoic acid.

Treat with retinoic acid, calcipotriene, or combination therapy

Culture dysplastic cell line

Measure cell viability using MTT assay


The authors would like to thank VCOM’s Research Department for providing financial support for this project.


2 0 2 3 R e s e a r c h R e c o g n i t i o n D a y

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