Detailed pharmacokinetic characterization of advanced topical acyclovir formulations with IVPT and in vivo Open Flow Microperfusion.

Successful treatment of herpes simplex viruses is currently limited by a lack of effective topical drugs. Commonly used topical acyclovir products only reduce the duration of lesions by a few days. Optimizing topical formulations to achieve an enhanced acyclovir solubility and penetration could increase the efficacy of topically applied acyclovir, but new formulations need to show reliable acyclovir delivery into at least the epidermis/dermis and need to provide sustained acyclovir release for extended time periods. The aim of this study was to compare pharmacokinetic data from in vitro permeation testing (IVPT) and preclinical dermal open flow microperfusion (dOFM) experiments regarding the penetration behavior of different acyclovir formulations relative to the reference product Zovirax® 5% cream. Four test formulations that delivered the best penetration data in IVPT were further tested using continuous dOFM in vivo dermal sampling. The use of dOFM identified one of the four tested formulations to perform significantly better than the other three tested formulations and the reference product. In vivo dOFM data showed differences in the dermal acyclovir concentration that had not been detected by using IVPT. Improved acyclovir delivery to the dermis was likely achieved by the new formulation that uses a much lower drug load compared to the reference product. This optimized formulation was able to achieve a dermal concentration similar to oral application and can thus provide the opportunity of more efficacious topical HSV-1 treatment with less side effects than oral systemic treatment.

Comparison of cerebral Open Flow Microperfusion and Microdialysis when sampling small lipophilic and small hydrophilic substances.

BACKGROUND: Assessment of drug concentration in the brain interstitial fluid (ISF) is crucial for development of brain active drugs, which are mainly small, lipophilic substances able to cross the blood-brain barrier (BBB). We aimed to compare the applicability of cerebral Open Flow Microperfusion (cOFM) and Microdialysis (MD) to sample the lipophilic substance amitriptyline (AMI), its metabolites Hydroxyamitriptyline (HYA), Nortriptyline (NOR), Amitriptyline-N-Oxide (ANO), deuterated water (D2O) and the hydrophilic substance sodium fluorescein (Naf) in brain ISF. NEW METHOD: cOFM has been refined to yield increased spatial resolution and performance. COMPARISON OF COFM AND MD AND RESULTS: Performance of cOFM and MD was assessed by in vivo AUC ratios of probe samples (AUCCOFM/AUCMD) and the in vivo relative recovery of D2O (RRvv,D2O). Adsorption of AMI and Naf to MD and cOFM was assessed by the in vitro relative recovery (RRvt) prior to the in vivo experiments. The in vivo AUC ratio of AMI and RRvv,D2O was about two times higher for cOFM than for MD (AUCOFM/AUCMD = 2.0, RRvv,D2O(cOFM)/RRvv,D2O(MD) = 2.1). cOFM detected all investigated AMI metabolites except NOR. MD did not detect HYA, NOR, ANO and Naf. In vitro adsorption of AMI and Naf to the MD membrane was strong (RRvt,AMI = 4.4%, RRvt,Naf = 1.5%) but unspecific adsorption to cOFM was negligibly small (RRvt,AMI = 98% and RRvt,Naf = 98%). CONCLUSIONS: cOFM showed better performance when sampling AMI and its metabolites, Naf and D2O, and had an about two times higher RRvv,D2O than MD. MD did not detect HYA, NOR, ANO and Naf, most likely due to membrane adsorption.

Quantification of acyclovir in dermal interstitial fluid and human serum by ultra-high-performance liquid-high-resolution tandem mass spectrometry for topical bioequivalence evaluation.

Time-concentration curves for the topical anti-viral drug acyclovir can provide valuable information for drug development. Open flow microperfusion is used for continuous sampling of dermal interstitial fluid but it requires validated methods for subsequent sample analysis. Therefore, we developed a sensitive, selective and high-throughput ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry method to determine acyclovir in human dermal interstitial fluid and serum. We validated the method over a concentration range of 0.1-25 ng/mL for a sample volume of just 20 µL and employed cation-exchange solid-phase extraction in a fully automated sample treatment procedure. Short- and long-term sample stability data and the analysis of 5000 samples from a clinical trial demonstrate the successful application of our method.

Open Flow Microperfusion as a Dermal Pharmacokinetic Approach to Evaluate Topical Bioequivalence.

BACKGROUND: The availability of generic topical dermatological drug products is constrained by the limited methods established to assess topical bioequivalence (BE). A novel cutaneous pharmacokinetic approach, dermal open-flow microperfusion (dOFM), can continuously assess the rate and extent to which a topical drug becomes available in the dermis, to compare in vivo dermal bioavailability (BA) and support BE evaluations for topical products. OBJECTIVE: To evaluate whether dOFM is an accurate, sensitive, and reproducible in vivo method to characterize the intradermal BA of acyclovir from 5 % acyclovir creams, comparing a reference (R) product either to itself or to a different test (T) product. METHODS: In a single-center clinical study, R or T products were applied to six randomized treatment sites on the skin of 20 healthy human subjects. Two dOFM probes were inserted in each treatment site to monitor the intradermal acyclovir concentration for 36 h. Comparative BA (of R vs. R and T vs. R) was evaluated based on conventional BE criteria for pharmacokinetic endpoints (area under the curve and maximum dermal concentration) where the 90 % confidence interval of the geometric mean ratio between the T and R falls within 0.80-1.25. RESULTS: The positive control products (R vs. R) were accurately and reproducibly confirmed to be bioequivalent, while the negative control products (T vs. R) were sensitively discriminated not to be bioequivalent. CONCLUSIONS: dOFM accurately, sensitively, and reproducibly characterized the dermal BA in a manner that can support BE evaluations for topical acyclovir 5 % creams in a study with n = 40 (20 subjects in this study).

Matrix removal in state of the art sample preparation methods for serum by charged aerosol detection and metabolomics-based LC-MS.

Investigations into sample preparation procedures usually focus on analyte recovery with no information provided about the fate of other components of the sample (matrix). For many analyses, however, and particularly those using liquid chromatography-mass spectrometry (LC-MS), quantitative measurements are greatly influenced by sample matrix. Using the example of the drug amitriptyline and three of its metabolites in serum, we performed a comprehensive investigation of nine commonly used sample clean-up procedures in terms of their suitability for preparing serum samples. We were monitoring the undesired matrix compounds using a combination of charged aerosol detection (CAD), LC-CAD, and a metabolomics-based LC-MS/MS approach. In this way, we compared analyte recovery of protein precipitation-, liquid-liquid-, solid-phase- and hybrid solid-phase extraction methods. Although all methods provided acceptable recoveries, the highest recovery was obtained by protein precipitation with acetonitrile/formic acid (amitriptyline 113%, nortriptyline 92%, 10-hydroxyamitriptyline 89%, and amitriptyline N-oxide 96%). The quantification of matrix removal by LC-CAD showed that the solid phase extraction method (SPE) provided the lowest remaining matrix load (48-123 µg mL(-1)), which is a 10-40 fold better matrix clean-up than the precipitation- or hybrid solid phase extraction methods. The metabolomics profiles of eleven compound classes, comprising 70 matrix compounds showed the trends of compound class removal for each sample preparation strategy. The collective data set of analyte recovery, matrix removal and matrix compound profile was used to assess the effectiveness of each sample preparation method. The best performance in matrix clean-up and practical handling of small sample volumes was showed by the SPE techniques, particularly HLB SPE. CAD proved to be an effective tool for revealing the considerable differences between the sample preparation methods. This detector can be used to follow matrix compound elution during chromatographic separations, and the facile monitoring of matrix signal can assist in avoiding unfavourable matrix effects on analyte quantification.