Purpose: Background: Dermal microdialysis (dMD) can directly monitor the rate and extent to which a topically administered drug becomes available in the dermis, at or near the site of action in the skin. In dMD, microdialysis probes traverse the dermis just beneath the skin surface, spanning the area of a test site. Using multiple test sites on the same subject, and replicate probes at each test site, it is feasible to compare the cutaneous pharmacokinetics (PK) of a drug from different products in parallel on the same subject. However, there are technical considerations related to the dMD experimental methods that must be characterized and optimized to ensure that the in vivo dMD study is sensitive and reproducible.
Objectives: The goals of this study were to assess: (i) the minimum distance required between test sites to prevent cross-talk between probes (due to potential lateral diffusion of drug in the dermis from a different test site); (ii) the sensitivity of the dMD method to detect differences in the local concentration of the drug, which is modulated by applying different doses of the topical product (3, 10, and 30 mg/cm2); (iii) the stability of dMD probe sampling over the 48-hour study duration.
Methods: Twenty-two microdialysis probes were inserted into the dorsum of an anesthetized Yucatan mini-pig using a scheme depicted in Figure 1. Two (duplicate) probes were inserted under each of the (6) test sites at 1 cm distance from each other. Metronidazole (MTZ) cream, 0.75% (Fougera) or a MTZ gel, 0.75% (Tolmar) was applied at different test sites, each at doses of 3, 10, or 30 mg/cm2 (Figure 1). Eight other dMD probes, designated to assess lateral diffusion (LD), were placed 1, 2, 3, or 4 cm away from the edge of the demarcated dose application area for each test site (Figure 1). An additional two dMD probes were used to evaluate the potential redistribution of MTZ in the skin due to systemic absorption and recirculation. All probes were perfused with 0.5µl/mL of lactated Ringer’s solution containing deuterated MTZ (MTZ-D3; 1µg/mL). Trans epidermal water loss and probe depth were measured in triplicate for each test site and probe. Samples were collected every 1 hour for 48 hours and were analyzed for MTZ and MTZ-D3 using a validated LC-MS/MS method (LLOQ: 0.4ng/mL; ULOQ: 200ng/mL). An area under the concentration vs. time curve (AUC) plot was used to compare the bioavailability of MTZ between each dose of the cream and gel. This study was replicated in two mini-pigs, and the results were combined for analysis. One AUC outlier value was identified for one probe using the Dean and Dixon Q-test.
Results: MTZ concentrations were slightly above the LLOQ in 47 of the 768 samples collected by the LD probes, and no samples contained MTZ above the LLOQ at the redistribution sites. The probe depth was relatively consistent across all probes (0.226 ± 0.077 cm; n=132) except for one probe (site A, cream, 3mg/cm2) whose depth was 0.069 ± 0.015cm (n=3). This probe was located much closer to the surface of the skin, and the AUC value was identified as an outlier relative to the other probes with the same treatment. The AUC value from this probe was excluded from the calculations to evaluate dose sensitivity. The AUC values correlated positively with the dose amount (R2>0.99) for both the gel and cream products. (Figure 2) The bioavailability of MTZ and the AUC values were substantially higher for the cream compared to the gel. Analysis of MTZ-D3 extraction efficiency in the first 24 hours compared to the second 24 hours did not show any significant difference, suggesting that probe recovery was stable during 48 hours of sampling. The maximum concentration (Cmax) of MTZ, time to maximum concentration (Tmax), and half-life of MTZ were difficult to estimate because the complete PK profile was not adequately characterized during the 48-hour study duration.
Conclusion: The negligible lateral diffusion and undetectable systemic redistribution observed suggest that the MTZ concentrations measured by the dMD probes can be specifically associated with the local bioavailability from the topical dose of the cream or gel. The results also indicate that the dMD method was sensitive to differences in the in vivo topical bioavailability of MTZ from each product, and selective to differences in bioavailability between cream and gel products of the same strength. A clinical dMD study using some of the study design parameters developed here, with a 10mg/cm2 dose of the MTZ cream or gel, may be suitable for further exploratory studies. However, other dose regimens and study durations need to be explored to better characterize the complete PK profile and compare the rate and extent to which topically administered MTZ becomes available in the dermis from different products.
Sharareh Senemar– Ph.D student, Long Island University
Tannaz Ramezanli– Division of Therapeutic Performance, Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA, Silver Spring, Maryland
Priyanka Ghosh– Pharmacologist, US Food and Drug Administration
Sam G Raney– Lead for Topical & Transdermal Drug Products, US Food and Drug Administration, Silver Spring, Maryland
Grazia Stagni– Associate professor, Long Island University, Brooklyn