BackgroundThe 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.ObjectiveTo 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.MethodsIn 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 plasma concentration) where the 90 % confidence interval of the geometric mean ratio between the T and R falls within 0.80–1.25.ResultsThe 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.ConclusionsdOFM 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).
The blood-brain barrier (BBB) limits substance transport to the brain and is therefore the major hurdle to overcome when developing neuroactive drugs. Herein, we report on cerebral open flow microperfusion (cOFM) as a new membrane-free technique for measuring substance transport across the intact BBB. The cOFM technique is based on a probe that is inserted into the brain, rupturing the BBB. The BBB is re-established within 15 days, which then allows sampling of interstitial brain fluid under physiological conditions. The aims of the present proof-of-concept study were to: (i) determine the time between cOFM probe insertion and BBB re-establishment; and (ii) demonstrate the ability of cOFM to sample the interstitial cerebral fluid with an intact BBB. The cOFM probe was inserted into the frontal lobe of Sprague-Dawley rats, resulting in BBB rupture. Re-establishment of the BBB was determined using Evans blue (EB) dye, which is an established marker for BBB intactness because it does not cross the intact BBB. Evaluating EB levels in the brain tissue indicated that the BBB was healed 11 days after probe insertion. To demonstrate transport across the healed BBB, we used sodium fluorescein (Naf), a sensitive, low molecular weight marker that can cross the intact BBB and can be used to monitor changes in BBB permeability. Significantly increased Naf levels were found in the interstitial fluid when hyperosmolar mannitol (known to open the BBB) was introduced via cOFM, which indicated partial opening of the BBB surrounding the cOFM probe. In conclusion, we show herein that cOFM allows monitoring of BBB permeability, which should be useful for measuring pharmacokinetics across the BBB and pharmacodynamics in the brain.
Blood-brain barrier (BBB) impairment in systemic inflammation leads to neuroinflammation. Several factors including cytokines, chemokines and signal transduction molecules are implicated in BBB dysfunction in response to systemic inflammation. Here, we have adopted a novel in vivo technique; namely, cerebral open flow microperfusion (cOFM), to perform time-dependent cytokine analysis (TNF-alpha, IL-6 and IL-10) in the frontal cortex of the rat brain in response to a single peripheral administration of lipopolysaccharide (LPS). In parallel, we monitored BBB function using sodium fluorescein as low molecular weight reporter in the cOFM sample. In response to the systemic LPS administration, we observed a rapid increase of TNF-alpha in the serum and brain, which coincides with the BBB disruption. Brain IL-6 and IL-10 synthesis was delayed by approximately 1 h. Our data demonstrate that cOFM can be used to monitor changes in brain cytokine levels and BBB disruption in a rat sepsis model.
Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging.
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