Eva Benfeldt

AAPS-FDA workshop white paper: Microdialysis principles, application, and regulatory perspectives

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (μD) is the only tool available that explicitly provides data on the extracellular space. Although μD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of μD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of μD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on μD as a tool in drug research and development.

Experimental itch in sodium lauryl sulphate-inflamed and normal skin in humans: a randomized, double-blind, placebo-controlled study of histamine and other inducers of itch.

Summary Background Investigations of pruritogenic substances in humans have involved intradermal injections in normal skin; itching of inflamed skin has been little studied.

Microdialysis sampling for investigations of bioavailability and bioequivalence of topically administered drugs: current state and future perspectives.

Microdialysis (MD) in the skin is a unique technique for in vivo sampling of topically as well as systemically administered drugs at the site of action, e.g. sampling the unbound tissue concentrations in the dermis and subcutaneous tissue. MD as a research method has undergone significant development, improvement and validation during the last decade and has proved to be a versatile, safe and valuable tool for pharmacokinetic and pharmacodynamic studies. This review gives an overview of the current state and future perspectives of dermal MD sampling. Methodological issues such as choice of instrumentation, calibration and experimental procedures are discussed along with the analytical considerations necessary for successful sampling. Clinical MD studies in the skin are reviewed with emphasis on pharmacokinetic studies of topically applied drugs with or without impairment of skin barrier function by skin disease or barrier perturbation. A comparison between MD and other tissue sampling techniques reveals the advantages and limitations of the method. Subsequently, an in-depth discussion of the application of MD for the evaluation of bioavailability and bioequivalence of topical formulations is concluded by the current regulatory point of view. The future perspective includes further expansion and validation of the use of MD in the experimental and clinical setting as well as in the optimization of the method for regulatory purposes, i.e. the commercialization of bioequivalent, generic drug products.

Feasibility of measuring lipophilic or protein-bound drugs in the dermis by in vivo microdialysis after topical or systemic drug administration.

Our aim was to assess the microdialysis technique for determining in vivo drug levels of a lipophilic and a protein-bound model drug in the dermis. Forearm skin of healthy volunteers received topical 2% fusidic acid or 0.1% betamethasone-17-valerate formulations twice daily as occluded treatment on irritative dermatitis. Microdialysis sampling in the dermis after 48 h was without measurable drug. Hairless rats received maximized treatment with occluded applications of 10% fusidic acid or 4% betamethasone-17-valerate in ethanol for 72 h followed by microdialysis. Mean levels of betamethasone-17-valerate were 11-45 ng/ml; fusidic acid was not measurable. Systemic administration in clinical doses to rats was without measurable drug levels; increasing doses to 312 mg/kg of fusidic acid and 158 mg/kg of betamethasone-17-valerate yielded betamethasone-17-valerate levels of 25-44 ng/ml and fusidic acid levels of 10-90 ng/ml. This study demonstrates the challenges arising when using microdialysis for measuring in vivo-drug levels. For the drugs chosen it was necessary to administer very high systemic doses or apply a high topical drug concentration to obtain measurable drug levels in the dialysates. Drug levels were in the nanomolar range and demonstrated reproducible and dynamic monitoring of in vivo drug levels in the skin. Using microdialysis for sampling highly protein-bound or lipophilic drugs in the skin requires very sensitive analytical methods, and the sensitivity of the analysis is likely to be the limiting factor.

Effect of barrier perturbation on cutaneous penetration of salicylic acid in hairless rats : In vivo pharmacokinetics using microdialysis and non-invasive quantification of barrier function

Abstract The penetration of topically applied drugs is altered in diseased or barrier-damaged skin. We used microdialysis in the dermis to measure salicylic acid (SA) penetration in hairless rats following application to normal (unmodified) skin ( n = 11) or skin with perturbed barrier function from (1) tape-stripping ( n = 5), (2) sodium lauryl sulphate (SLS) 2% for 24 h ( n = 3) or (3) delipidization by acetone ( n = 4). Prior to the experiment, transepidermal water loss (TEWL) and erythema were measured. Two microdialysis probes were inserted into the dermis on the side of the trunk and 5% SA in ethanol was applied in a chamber overlying the probes. Microdialysis sampling was continued for 4 h, followed by measurements of probe depth by ultrasound scanning. SA was detectable in all samples and rapidly increasing up to 130 min. Microdialysates collected between 80 and 200 min showed mean SA concentrations of 3 μg/ml in unmodified and acetone-treated skin, whereas mean SA concentrations were 280 μg/ml in SLS-pretreated skin and 530 μg/ml in tape-stripped skin ( P < 0.001). The penetration of SA correlated with barrier perturbation measured by TEWL ( P < 0.001) and erythema ( P < 0.001). A correlation between dermal probe depth and SA concentration was found in unmodified skin ( P = 0.04). Microdialysis sampling in anatomical regions remote from the dosed site excluded the possibility that SA levels measured were due to systemic absorption. Microdialysis sampling of cutaneous penetration was highly reproducible. Impaired barrier function, caused by irritant dermatitis or tape stripping, resulted in an 80- to 170-fold increase in the drug level in the dermis. This dramatic increase in drug penetration could be relevant to humans, in particular to topical treatment of skin diseases and to occupational toxicology.

Scratch Induction in the Rat by Intradermal Serotonin: a Model for Pruritus

Eight substances (histamine, compound 48/80, kallikrein, trypsin, papain, substance P, serotonin and platelet activating factor) were injected intradermally (volume 50 microl) into the rostral back (neck) of rats in order to establish an animal model for peripherally elicited pruritus. While serotonin induced excessive scratching at the site of injection, the other substances were weak or inactive. The dose-response relationship of serotonin was sigmoid, EC50=2.1 mg/ml (95% confidence interval: 1.0 to 4.3 mg/ml). Injections of serotonin 1 mg/ml into the caudal back elicited no scratching at all, i.e. neither at the site of injection nor elsewhere, so the experiment indicated no systemic effect of serotonin 1 mg/ml intradermally. Scratching was probably elicited histamine-independently, since histamine itself did not elicit scratching. The intra- and inter-observer variations were 3-4%. We conclude that serotonin is a reproducible local pruritogen eliciting scratching in the rat. The model may be useful in research and development of topical antipruritics of the nonhistaminic type as well as for various other purposes in pruritus research.

Application of dermal microdialysis for the evaluation of bioequivalence of a ketoprofen topical gel

The purpose was to investigate dermal microdialysis (DMD) for the assessment of the bioavailability of a ketoprofen topical gel formulation and to evaluate this technique as a tool for the determination of bioequivalence. Four microdialysis probes were inserted into the dermis on the volar aspect of the forearms of 18 human subjects and the probes were perfused with normal saline for 60 min. A ketoprofen (2.5%, m/m) gel formulation (50mg) was applied to the skin directly overlying the probes and samples were collected at 30 min intervals for 5h. With the probes still in place in the dermis each site was scanned by ultrasound to determine the implantation depth of these probes. Ketoprofen concentration in dialysates was determined by LC-MS/MS. The area under the curve obtained from the concentration-time profiles from pairs of application sites in each subject was evaluated in order to assess bioequivalence. Ninety percent confidence intervals were calculated using the two one-sided test procedure and limits of 80-125% based on log-transformed data were used as acceptance criteria to declare bioequivalence. The intra-subject variability was 10% between probes whereas inter-subject variability was 68% (n=18). Bioequivalence was confirmed with a power greater than 90%.

Distribution to the skin of penciclovir after oral famciclovir administration in healthy volunteers: comparison of the suction blister technique and cutaneous microdialysis.

Penciclovir is a drug active against herpes simplex viruses located in the epidermis basal layer. The aim of this study was to compare the suction blister technique and microdialysis as methods to measure the penciclovir concentration in the skin after a single dose (250 mg) of its prodrug, famciclovir. Suction blister fluid, microdialysates and plasma were sampled from 11 healthy volunteers for 5 h after famciclovir administration. Both the suction blister technique and microdialysis showed that penciclovir reaches the skin in concentrations sufficient to inhibit herpes virus replication. The maximum concentration in both suction blister fluid and in microdialysate was observed later than in plasma. The microdialysis concentration was decreased by cooling of the skin surface and by adrenaline-mediated vasoconstriction. The microdialysis recovery of penciclovir was studied with respect to the flow-rate of perfusion medium through the microdialysis probe. Microdialysis and the suction blister technique can be used to study the time-concentration profile of penciclovir in the skin and microdialysis allows a continuous sampling of the drug for a prolonged time after administration.

Microdialysis vs. suction blister technique for in vivo sampling of pharmacokinetics in the human dermis.

Our aim was to simultaneously investigate 2 techniques for in vivo sampling of peripheral compartment pharmacokinetics after systemic administration of acetylsalicylic acid. Ten volunteers were given 2 g acetylsalicylic acid orally. Blood samples and dialysates from 4 microdialysis probes inserted in the dermis of the forearm were collected for 5 h and suction blisters were raised 1-3 h after dosing. In microdialysates, both acetylsalicylic acid and the metabolite salicylic acid were measurable in the absence of hydrolysing enzymes. The mean Cmax (maximum concentration) of total, unbound salicylic acid was 9.5 microg/ml in microdialysates, 13.2 microg/ml in suction blister fluid and 56.5 microg/ml in plasma. Mean Tmax (time to Cmax) for salicylic acid was 188 and 161 min in plasma and microdialysates, respectively. The dermis-to-plasma Cmax ratio was 0.16+/-0.04 (mean +/- SD) by microdialysis sampling and 0.25+/-0.09 by the suction blister fluid method. Close correlations (p<0.01) were found between Cmax of salicylic acid in microdialysates and plasma, and between Cmax of salicylic acid in suction blister fluid and plasma. The 2 techniques were in excellent accordance with even closer correlation between maximum concentrations obtained by microdialysis and suction blister fluid sampling (p<0.001). However, comparing the tolerability of the sampling procedure, ease of analysis, and detail in chronology, microdialysis is superior for sampling in vivo pharmacokinetics in the dermis.

Dermal PK/PD of a lipophilic topical drug in psoriatic patients by continuous intradermal membrane-free sampling.

BACKGROUND Methodologies for continuous sampling of lipophilic drugs and high-molecular solutes in the dermis are currently lacking. We investigated the feasibility of sampling a lipophilic topical drug and the locally released biomarker in the dermis of non-lesional and lesional skin of psoriatic patients over 25h by means of membrane-free dermal open-flow microperfusion probes (dOFM) and novel wearable multi-channel pumps. METHODS Nine psoriatic patients received a topical p-38 inhibitor (BCT194, 0.5% cream) on a lesional and a non-lesional application site once daily for 8 days. Multiple dOFM sampling was performed for 25 h from each site on day 1 and day 8. Patients were mobile as dOFM probes were operated by a novel light-weight push-pull pump. Ultrasound was used to verify intradermal probe placement, cap-LC-MS/MS for BCT194 and ELISA for TNFα analysis. RESULTS dOFM was well tolerated and demonstrated significant drug concentrations in lesional as well as non-lesional skin after 8 days, but did not show significant differences between tissues. On day 8, TNFα release following probe insertion was significantly reduced compared to day 1. CONCLUSIONS Novel membrane-free probes and wearable multi-channel pumps allowed prolonged intradermal PK/PD profiling of a lipophilic topical drug in psoriatic patients. This initial study shows that dOFM overcomes limitations of microdialysis sampling methodology, and it demonstrates the potential for PK/PD studies of topical products and formulations in a clinical setting.