Twenty healthy human subjects were enrolled (Caucasian, 7 women, 13 men, age 28 ± 5 years).
Topical Study Drugs
Acyclovir cream 5 % (Zovirax®; Valeant, Bridgewater, NJ, USA) was used as the R product. Acyclovir cream 5 % (Aciclovir 1A Pharma—Creme; 1A Pharma GmbH, Vienna, Austria), which has a different formulation composition and from which the topical BA of acyclovir was observed to be different relative to the R product (unpublished results from exploratory, pilot, in vivo dOFM studies), was used as the T product.
dOFM probes were inserted intradermally (two probes at each of the three treatment sites on each limb) and dermal interstitial fluid samples were continuously collected (1 µL/min) from a pre-dose baseline time period (−1 h to 0 h) to 36 h post-dose. The dOFM devices (sterile probes, wearable pumps, accessories; CE certified for human use) were developed by HEALTH—Joanneum Research GmbH (Graz, Austria). The devices and their clinical use have been described in detail previously [23, 24]. In this study, the newest version of a CE-certified dOFM probe (DEA15003) was used. Like its precursors, this new dOFM probe is a highly flexible linear probe with a demarcated 15-mm fully permeable section, and it also has imprinted position markers to facilitate the precise positioning of the permeable section of the probe below the treatment area. The probe’s outer diameter is 0.5 mm and it is inserted over a length of 30 mm using a standard 0.9-mm (outer diameter) hollow insertion needle.
After enrollment and qualification of study subjects based upon the protocol inclusion and exclusion criteria, a set of three treatment sites (referred to as the ‘test triad’) was demarcated on each thigh as depicted in Fig. 2 (providing a total of six treatment sites per subject, each 5.5 cm2). Twelve dOFM probes were inserted intradermally (two replicate probes per treatment site) and dermal interstitial fluid was continuously sampled at 1 µL/min using sterile perfusate that included 1 % albumin and 600 mg/dL glucose. The skin at a treatment site was cooled using a sterile ice bag prior to probe insertion as well as during the initial hour following probe insertion.
At baseline, the transepidermal water loss was measured on the skin of each leg (Aquaflux AF200; Biox Ltd, London, UK) and the baseline serum and dOFM samples were collected.
At t = 0, 15 mg cream/cm2 of each of the R and T products were applied to the respective treatment sites on the skin using a tared spatula. The spreading procedure was of standardized duration (1 min) for all treatment sites and care was taken to consistently dose the cream as a homogenous layer. The R product was applied at two sites on each thigh (R
1 central, R
2 non-central) and T non-central (Fig. 2a) in a randomized order, using treatment randomization sequences of either ‘R
1–T’ or ‘T–R
2’ according to a randomization scheme that had been predefined in the study protocol to rule out the impact of anatomical location or procedural factors. Treatment sites were protected by a transparent, non-occlusive, dome-shaped, perforated plastic shield (Fig. 2b) and samples of the continuous perfusion from the dOFM probes in the dermis were collected at 4-h intervals, up to 36 h post-dose. Glucose in dOFM samples was measured at the bedside (Super GL; Dr. Müller Gerätebau GmbH, Freital, Germany) and the relative glucose loss from the perfusate (relative to 600 mg/dL in %) was calculated to monitor the exchange rate (‘relative recovery’) across the probe.
Study subjects rested in bed most of the time and slept in a supine position during the night. The sampling procedure during the night was identical to that used during the day. After termination of the study at 36 h, the intradermal position of each probe was assessed by a longitudinal ultrasound scan (GE LOGIQ e R6 device with linear 22 MHz probe; GE Healthcare, Vienna, Austria).
To reduce perturbations to the probes and to minimize variability in the kinetic data, any torsional strains or skin stretching from anatomical flexion at the test triads across 36 h were minimized through the use of self-adhesive stabilization rings (HEALTH-Joanneum Research GmbH). As additional controls to reduce experimental variability and optimize the precision and reproducibility of the study, room temperature and relative humidity were tightly controlled throughout the study (22 ± 1 °C, 40–60 % relative humidity).
Acyclovir determination: Samples (20 µL of perfusate plus D4-deuterated acyclovir internal standard) were processed by solid-phase extraction (Oasis MCX µElution plate; Waters, Milford, MA, USA). Samples were washed with formic acid/water (5 %/95 %, v/v) followed by methanol; eluted with NH4OH/methanol (5 %/95 %, v/v); evaporated to dryness; and finally reconstituted in 20 µL methanol/water (5 %/95 %, v/v). High-performance liquid chromatography (HPLC) analysis of acyclovir in dOFM samples was performed with an Ultimate 3000 HPLC (Thermo Fisher Scientific, Waltham, MA, USA); ACQUITY-UPLC-HSS-T3 column (50 × 1.0 mm; Waters) using an isocratic method and a methanol/water (95 %/5 %, v/v) mobile phase with a flow rate of 300 µL/min and an injection volume of 3 µL. Acyclovir detection and quantification was performed by mass spectrometry (MS) using a Q-Exactive (Thermo) MS/MS in positive heated-electrospray mode (m/z 226.0935–152.05635 for acyclovir and 230.1196–152.05635 for the acyclovir-D4 internal standard).
Pharmacokinetic Data Evaluation
All clinical data were collected with an electronic case report form (eCRF) within OpenClinica Enterprise Edition. The eCRF system was hosted by Joanneum Research GmbH and is 21 CFR Part 11 compliant. All data management activities were performed and documented according to international standards and the data management plan.
The statistical analysis plan predefined data analysis, including criteria for the identification and treatment of possible outliers from intradermal 36-h concentration profiles. Possible outliers were identified by a statistical approach comparing each value with the moving median. Values that were <50 or >200 % of the moving median were reanalyzed and the reanalyzed values were used. Values that were still regarded as outliers according to this criterion were imputed by the arithmetic mean value of the preceding and subsequent time point samples, or by using the carry-last-value-forward approach in situations where there was no subsequent timepoint, to obtain complete profiles for PK endpoint calculation. The dermal PK endpoint AUC0–36h was calculated as sum of the dOFM sample concentrations over 36 h of continuous sampling. The dermal PK endpoint C
max was identified as the maximum concentration during the same duration.
An exploratory pilot study in six subjects where an even higher topical acyclovir dose was applied over 36 h showed no detectable acyclovir in the central test sites when the central test sites were left untreated, and it also did not show any detectable acyclovir in serum. Therefore, there was no evidence of any potential for cross-talk between test sites and all 40 test triads were considered independent for BE analysis in this proof-of-principle study.
BE criteria were used to evaluate the dermal PK endpoints for the positive control for BE (R
2 vs. R
1) and the negative control for BE (T vs. R
1) based on the typical acceptance criteria for BE. The dermal PK endpoints AUC0–36h and C
max were log transformed prior to analysis. The results were evaluated to determine whether the 90 % confidence interval of the mean ratios of T/R fell within the conventional BE limits of 0.80–1.25.