This method is quite invasive and causes some discomfort to the volunteers. Therefore, there are few, dermatopharmacologic studies on human volunteers to be found in the litera-ture. The procedure consists of placing a spe-cific bell-shaped device with several holes in the base, a Dermovac® cap (Medko Medical, Espoo, Finland), onto the skin surface, and subsequently applying a partial negative pres-sure of 200-300 mm Hg for 2-3 hours. The skin is raised through the 4 to 8-mm holes, thus disrupting the dermo-epidermal junction and forming a blister, which progressively fills with interstitial fluid and serum [60], given the proximity to the dermal capillaries.
For dermatopharmacokinetic studies, the drug is generally applied before the blister suction, so that it gradually fills the blister during its
growth. Furthermore, multiple blisters are necessary for each drug concentration time-points. The blister fluid carrying the absorbed drug is subsequently withdrawn by the means of an insulin syringe, and analyzed for drug content, thus allowing the local bioavailability to be determined from a concentration-time profile. Depending on the blister fluid compo-sition and the possible drug binding, however, artifacts of drug levels may appear, and ex-treme caution should be exercised when in-terpreting the results.
Treffel et al. [61,62] compared two formu-lations (an emulsion and an oil) containing bergamot oil (including citropten and bergap-ten, used as a cosmetic tanning product) on 10 cm2-areas of volar forearms of volunteers.
Suction blisters were raised on the treated sites and sampled after 100, 160, and 220 minutes. In order to optimize the analytical sensitivity (HPLC), seven blisters were neces-sary for each time-point. Results showed higher blister fluid concentrations for the emulsion than for the oil.
Another group [59] studied the uptake of a labeled topical acitrecin (a retinoid used for the treatment of psoriasis and various kerati-nization disorders) formulation (330 µg/ml) on the upper buttocks of volunteers, under occlusion (polypropylene chambers). After 24 hours, the chamber was removed, a 4-mm suction blister was raised, sampled 2 hours later, and finally analyzed by liquid scintilla-tion counting. Drug levels in the blister fluid
were extremely low, suggesting removal of acitrecin by the close dermal capillary net-work, since levels in the epidermis, asssessed by shave and punch biopsy, showed compara-ble levels to those observed after oral admini-stration.
Microdialysis
Despite its invasiveness, microdialysis is a promising in vivo sampling technique for lo-cal cutaneous bioavailability/bioequivalence assessment, since it allows continuous collec-tion of dermal interstitial fluid loaded with absorbed drug, thus permitting an optimal monitoring of drug levels. This method has been used for a number of years in neuro-pharmacological studies on animals, but its use in dermatopharmacological studies in human is relatively recent [63,64], since fea-sibility was first tested on animal skin [65,66].
The idea of dermal microdialysis is that topi-cally absorbed drug may be collected in the skin compartment where it is normally re-moved by the blood capillaries, so that local levels may be measured. The device consists of a 0.2 to 0.5-mm diameter probe, a short portion of which (10-15 mm) is made of a semi-permeable dialysis membrane with a molecular weight cutoff of 20 kDa, allowing exchange of fluids but excluding bulky en-dogenous macromolecules such as proteins.
The dialysate is therefore generally ready for analysis, with little or no sample preparation.
Two kinds of probes are usually used: a single-lumen and a dual-lumen. In the former type (Fig. 5A) the membrane is located in the middle of the probe lenght, and the perfusate flow is mono-directional: a point of entry and a point of exit are therefore necessary, which increases the invasiveness. In the latter type (Fig. 5B) the membrane is on the tip of the probe, and a concentric dual lumen allows the perfusion fluid to flow in a bi-directional way,
Figure 5: Schematic diagram of two currently used mi-crodialysis probes for dermatopharmacokinetic experi-ments. In both cases, a semi-permeable dialysis mem-brane collects the absorbed free drug. (A) Single-lumen probe: the membrane is located in the middle of the probe, and the perfusate flow is mono-directional. (B) Dual-lumen probe: the membrane is on the tip of the probe, and the perfusate flow is bi-directional.
formulation
thereby reducing the puncture to a single point. The probe enters the skin via a guide catheter, and is settled parallel to the skin sur-face at a depth of 0.5 to 1 mm. This operation may cause some discomfort, and is usually carried out under local anaesthesia. Effects of tissue trauma following insertion of the probe have been studied by Groth et al. on animals [67] and on humans [68]. The author ob-served a local inflammation resulting in oe-dema of the skin and subsequent increase in skin thickness, as assessed by ultrasonogra-phy. This effect may increase the diffusion area, and thereby result in increased drug re-covery. A minimum equilibrium period of two hours (i.e., the time required for the skin to return to baseline range) was therefore rec-ommended after probe insertion.
Once the probe is in place, an isotonic fluid is perfused through the probe at very low rates (typically, 0.5 to 10 µl/min), and the diffusing free (unbound) drug is collected continuously. The lower the flow, the higher will be the relative recovery of the drug (ratio of the drug concentration in the perfusate over the drug concentration in the dialysed tissue), since a higher equilibration time allows a higher uptake of the drug in the probe [69].
Constant and reproducible positioning of the probe are essential for correct interpreta-tion of the data, since lower posiinterpreta-tioning may result in collection of lower drug levels, due to competitive removal by the dermal capillar-ies. For bioequivalence studies, this issue is
particularly important, since lower levels col-lected from one formulation may lead to er-roneus interpretation of the drug bioavailabil-ity. However, since a microdialysis probe can be left in place for many days, it may be pos-sible to sequentially test two different formu-lations on the same skin site.
Deeper positioning of the probe may allow the assessment of the local bioavailability of anti-inflammatory drugs in the subcutaneous tissue or even in the muscle. Albeit more in-vasive, these depths have been recently at-tained, on human volunteers, by, respectively, Müller et al. [70] and Tegeder et al. [71]. The first group studied the local bioavailability of diclofenac gel in the anterior side of the thigh, by inserting the microdialysis probe at depths varying from 4 to 10 mm. After a single dose administration, effective tissue levels were attained in 40% of the subjects. The second group compared the uptake of oral and topical ibuprofen applied to the thigh, again, but the probe was inserted at 4-5 mm (subcutis) and 25-30 mm (muscle). The relevant finding was that the topical administration led to greater drug concentrations in the subcutaneous tissue than those obtained after oral administration, whereas in the muscle the concentrations were almost identical.