Local vasoregulative interventions impact drug concentrations in the skin after topical laser‐assisted delivery

Abstract

Introduction

The ability of ablative fractional lasers (AFL) to enhance topical drug uptake is well established. After AFL delivery, however, drug clearance by local vasculature is poorly understood. Modifications in vascular clearance may enhance AFL‐assisted drug concentrations and prolong drug dwell time in the skin. Aiming to assess the role and modifiability of vascular clearance after AFL‐assisted delivery, this study examined the impact of vasoregulative interventions on AFL‐assisted 5‐fluorouracil (5‐FU) concentrations in in vivo skin.

Methods

5‐FU uptake was assessed in intact and AFL‐exposed skin in a live pig model. After fractional CO₂ laser exposure (15 mJ/microbeam, 5% density), vasoregulative intervention using topical brimonidine cream, epinephrine solution, or pulsed dye laser (PDL) was performed in designated treatment areas, followed by a single 5% 5‐FU cream application. At 0, 1, 4, 48, and 72 h, 5‐FU concentrations were measured in 500 and 1500 μm skin layers by mass spectrometry (n = 6). A supplemental assessment of blood flow following AFL ± vasoregulation was performed using optical coherence tomography (OCT) in a human volunteer.

Results

Compared to intact skin, AFL facilitated a prompt peak in 5‐FU delivery that remained elevated up to 4 hours (1500 μm: 1.5 vs. 31.8 ng/ml [1 hour, p = 0.002]; 5.3 vs. 14.5 ng/ml [4 hours, p = 0.039]). However, AFL's impact was transient, with 5‐FU concentrations comparable to intact skin at later time points. Overall, vasoregulative intervention with brimonidine or PDL led to significantly higher peak 5‐FU concentrations, prolonging the drug's dwell time in the skin versus AFL delivery alone. As such, brimonidine and PDL led to twofold higher 5‐FU concentrations than AFL alone in both skin layers by 1 hour (e.g., 500 μm: 107 ng/ml [brimonidine]; 96.9 ng/ml [PDL], 46.6 ng/ml [AFL alone], p ≤ 0.024), and remained significantly elevated at 4 hours (p ≤ 0.024). A similar pattern was observed for epinephrine, although trends remained nonsignificant (p ≥ 0.09). Prolonged 5‐FU delivery was provided by PDL, resulting in sustained drug deposition compared to AFL alone at both 48 and 72 hours in the superficial skin layer (p ≤ 0.024). Supporting drug delivery findings, OCT revealed that increases in local blood flow after AFL were mitigated in test areas also exposed to PDL, brimonidine, or epinephrine, with PDL providing the greatest, sustained reduction in flow over 48 hours.

Conclusion

Vasoregulative intervention in conjunction with AFL‐assisted delivery enhances and prolongs 5‐FU deposition in in vivo skin.

INTRODUCTION

The use of laser‐assisted drug delivery (LADD) to enhance uptake of topical agents has in recent years evolved from experimental practice to a commonly applied tool in dermatological settings. Particularly ablative fractional lasers (AFL), with their ability to generate customizable microchannels through the stratum corneum to access underlying skin layers, have emerged as an effective modality for LADD. ¹ , ² Compared to conventional topical application, AFL‐assisted drug delivery can facilitate greater and deeper cutaneous drug deposition, more uniform biodistribution, and accelerated uptake. ¹ In the clinic, these benefits have translated into improved therapeutic efficacy, shortened treatment times, and reduced reliance on patient adherence for a range of indications, including actinic keratosis, keratinocyte carcinoma (KC), scars, keloids, and photodamage. ³ , ⁴ , ⁵ , ⁶

In the past decade, topical drug uptake after AFL‐assisted delivery has been widely investigated, shedding light on the impact of specific laser settings, laser channel morphology, and individual drugs' physicochemical properties. ¹ , ³ In contrast, drug clearance post‐LADD remains poorly understood. For a topical treatment to be effective, not only adequate but sustained drug concentrations at the target site are often needed. This aspect is particularly important in the context of pharmacological treatment of KC, where insufficient drug concentrations or exposure durations can result in treatment failure. ⁷ , ⁸ In intact skin, the dermal blood supply's significant contribution to the clearance of topically applied agents is well known  ⁹ , ¹⁰ In AFL‐treated skin, on the other hand, an understanding of the interplay between AFL and local microcirculation as well as its impact on cutaneous drug clearance remains lacking.

Emerging evidence suggests that the dwell time of individual agents in the skin after AFL‐assisted delivery is limited by vascular clearance. Mass spectrometry drug imaging studies reveal the notable disappearance of 5‐fluorouracil (5‐FU) from human basal cell carcinomas (BCC) shortly after AFL‐assisted application. ¹¹ Similarly, quantitative studies of chemotherapeutics show that prompt AFL‐assisted peaks in in vivo drug deposition are followed by rapid declines in cutaneous concentrations in the hours following application. ¹² , ¹³ , ¹⁴ , ¹⁵ A problem of accelerated drug clearance may in part explain why Hsu et al. ¹⁵ reported a suboptimal 67% clearance rate in 12 superficial BCCs following a single AFL‐assisted application of 5‐FU under occlusion. ¹⁵

In drug delivery literature, several vasoregulative approaches have been used in experimental and clinical settings to sustain therapeutic concentrations by slowing vascular drug clearance. Such approaches include vasoconstriction by pharmacological action, as well as by physical stimuli such as temperature. ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰  Aiming to assess the role and modifiability of vascular clearance after AFL‐assisted delivery, this study examines the impact of three different vasoregulative interventions on AFL‐assisted 5‐FU concentrations in in vivo skin.

MATERIALS AND METHODS

Animal

Inclusion of one female Yorkshire swine (80 kg) was approved by the Massachusetts General Hospital's Institutional Animal Care and Use Committee. General anesthesia was performed on study Days 0, 2 (48 hours) and 3 (72 hours) using intramuscular telazol/xylazine (4.4 and 2.2 mg/kg) and isoflurane 2.0% inhalation (2.0 L/min). Blankets maintained core temperature during procedures. At the study's conclusion on Day 3, euthanasia was performed with intravenous pentobarbital 100 mg/kg under general anesthesia.

Study design

The study compared AFL‐assisted 5‐FU delivery with or without vasoregulative intervention. Three different vasoregulative approaches were evaluated: pharmacological vasoconstriction by topical epinephrine (EPI) solution (α₁‐adrenergic receptor agonist; 183.2 Da) or brimonidine (BRI) gel (α₂‐adrenergic receptor agonist; 442.2 Da), and photothermal vascular obliteration by pulsed dye laser (PDL). At study initiation, an approximately 40 × 35 cm skin area was cleaned, trimmed, and 2 × 2 cm test sites were demarcated on the pig's paraspinal region. Test sites were designated to receive one of six interventions: (1) AFL + 5‐FU cream; (2) AFL + EPI + 5‐FU; (3) AFL + BRI + 5‐FU; (4) AFL + PDL + 5‐FU; (5) 5‐FU or (6) no treatment (negative control). Cutaneous concentrations of 5‐FU were subsequently measured 1, 4, 48, and 72 h after application.

AFL and channel dimensions

AFL irradiation was performed using an Ultrapulse CO₂ laser with a DeepFx handpiece (10,600 nm; Lumenis). Laser‐induced channels were generated using a single pulse at 15 mJ/microbeam (mJ/mb), 5% density (196 microscopic ablation zone [MAZ]/cm²), 0.12 mm spot size, and pulse duration of ~250 µs without overlap. Laser‐channel dimensions were measured in representative histological images of vertical skin sections originating from four 6‐mm punch biopsies immediately after irradiation. Distinct laser channel dimensions (n = 10) were measured using NPD.view2 software (Hamamatsu Photonics) as previously described (Figure 1). ²¹

Figure 1.

Laser channel morphology and study methods. (A) Laser channel measurements (n = 10) were performed using histological hematoxylin and eosin sections originating from four skin biopsies taken immediately after ablative fractional CO₂ laser irradiation. Microthermal treatment zones, defined as the vertical column of heat‐altered tissue extending beyond the ablated portion of the laser channel had a mean depth of 351 μm (range: 320–369 μm). Mean depth of MAZ, corresponding to the ablated (open) channel, was 267 μm (range: 202–294 μm). Mean MAZ width was 123 μm (range: 88–142 μm). The coagulation zone, defined as the width of the broadest apparent layer of tissue coagulation identified at the lower two‐thirds of each MAZ, measured 65 μm (range: 58–76 μm). (B) Digital photo of customized wells made from DuoDerm Hydroactive™ dressings (ConvaTec, Flintshire, UK). Wells secured to the paraspinal region of the pig were used to ensure the containment of topical cream and liquid agents. Pictured are wells for two interventions shown before 5‐FU application: AFL + 5‐FU with apparent erythema (left) and AFL + EPI + 5‐FU with EPI under occlusion (right). Comparatively less erythema due to EPI‐mediated vasoconstriction can be noted in the test areas. AFL, ablative fractional laser; EPI,  epinephrine; 5‐FU, 5‐fluorouracil; MAZ, microscopic ablation zones.

Vasoregulative interventions

Immediately after AFL, customized wells made from DuoDerm Hydroactive™ dressings (ConvaTec) were secured to all test areas to ensure the containment of topical agents (Figure 1). Subsequently, 0.5 ml EPI solution (Xylocaine® lidocaine HCL + EPI; 10 mg/ml + 10 µg/ml; APP Fresnius Kapi) or 0.2 ml BRI gel (Mirvaso® 0.33%; Galderma) was deposited in designated sites. After 15 minutes, both agents were gently removed with a cotton swab and 0.2 ml 5% 5‐FU cream (Efudix®; Meda Pharmaceuticals) was applied to all designated areas. Wells were promptly occluded with Tegaderm® (3M Denmark), leaving 5‐FU cream on the skin until evaluation at 1, 4, 48, or 72 hours.

In test areas designated for vasoregulation with PDL, irradiation was performed 5 minutes after AFL using a 595 nm laser (V‐beam; Candela Corp.). To achieve sufficient capillary damage as indicated by purpura, the following laser settings were applied: 6 J/cm² pulse energy, 1.5 millisecond pulse duration, and 10 mm spot size. A total of five pulses covered each test area.

Drug pharmacokinetics and biodistribution

For analysis of intracutaneous pharmacokinetics and biodistribution, a total of 144 punch biopsies (6 mm) were taken from test areas 1, 4, 48, and 72 hours after 5‐FU application (six samples per intervention per time point). Samples were oriented, flash‐frozen in TissueTek® OCT Compound (Sakura Finetek USA, Inc.), and stored at −80°C. Frozen, 30‐μm‐thick horizontal sections were collected at skin depths of 500 μm (+10%) and 1500 μm (+10%) from each biopsy using a cryostat. The resulting depth‐specific skin sections were stored at −80°C until analysis.

5‐FU detection

Before mass spectrometry analysis, 5‐FU was passively extracted from individual skin sections in 0.5 ml phosphate‐buffered saline overnight at 4°C. Three hundred and seventy‐five microliters of extract was transferred to sampling vials for drug quantification on an Agilent 6430 Triple Quad System (Agilent Technologies) in negative polarity MRM mode (129 → 42 transition). The applied 2.1 × 100 mm HILIC column (Agilent Technologies) with a 2.7 μm particle size was kept at 25°C. Mobile phase A consisted of 100% acetonitrile, while mobile phase B was 10 mM ammonium formate. The gradient of mobile phase A was 90% to 50% over 5 min. The mobile phase flow rate was 0.4 ml/min, resulting in a 5‐FU retention time of 0.77 min. A set of 5‐FU calibration standards ranging from 1 to 600 ng/ml were prepared by serial dilution of stock solution and concurrently analyzed. The limit of quantification was 5 ng/ml.

Optical coherence tomography (OCT) imaging

The vascular impact of AFL and vasoregulative interventions (i.e., EPI, BRI, and PDL) was visualized using noninvasive OCT. On the inner forearm of a human volunteer, four interventions, AFL, AFL + EPI, AFL + BRI, and AFL + PDL (single pulse), as well as untreated skin, were evaluated 1, 4, and 48 hours after AFL using a prototype OCT system as previously described. ²² , ²³ The system comprised a wavelength‐swept laser source constructed using a polygon‐mirror scanner enabling simultaneous acquisition of structural intensity and angiography measurements. The laser source operated at a 50 kHz A‐line rate with its spectrum centered at 1310 nm and spanning a bandwidth of nearly 130 nm. The output laser beam was directed through a single‐mode fiber into a two‐dimensional galvanometer scanning mirror system and an objective lens to focus on the skin. The imaging system enabled a full field of view of 1.1 × 1.1 cm with a lateral resolution of 15 μm and an axial resolution of 6 μm. A contact adhesive was used to minimize the effects of motion of the subject on the acquired data.

Angiography and vasculature index

Binarized angiography images were constructed from OCT output data using a complex differential variance technique. ²² In the chosen colormap, darker red areas in the full‐field, en face vascular images indicated a higher amount of blood flow, while pink areas indicated a lower amount of blood flow. A detailed explanation of the algorithm used and the extraction of vascular contrast images from OCT data is described previously. ²² Regions of interest (ROIs) were manually selected from the images, by avoiding nontreated regions in the test area, regions with motion artifacts, or regions overlapping with the contact adhesive's crosshairs. Within each image's ROI (highlighted in green in Figure 4B), a mean of angiography data was computed, referred to as the vasculature index.

Figure 4.

Impact of vasoregulative intervention on blood flow in in vivo human skin. (A) Digital photos were taken 30 minutes after AFL exposure ± subsequent vasoregulation with topical EPI, BRI, or a single pulse of PDL. AFL alone induced discernable erythema in irradiated skin. In contrast, blanching developed in test areas exposed to AFL + EPI and AFL + BRI, while AFL + PDL resulted in clinical purpura as a marker for vessel hemorrhage. (B) OCT revealed notable differences in blood flow and resulting (C) vasculature index after AFL ± vasoregulation. While vasculature indexes in untreated skin remained low through the study, notable increases in blood flow were shown throughout the 48‐hour assessment period in test areas exposed to AFL alone, peaking at the earliest time point 1 hour after irradiation. This effect was mitigated by all three vasoregulative approaches, although with differing lengths of duration. Thus, suppression of AFL‐mediated increases in blood flow had worn off by 4 hours for AFL + EPI and by 48 hours for AFL + BRI. In comparison, vasculature indexes in areas exposed to AFL + PDL remained low and similar to that of untreated skin throughout the study. AFL, ablative fractional laser; BRI, brimonidine; EPI,  epinephrine; OCT, optical coherence tomography; PDL, pulsed dye laser.

Statistics

Shapiro–Wilk tests for normality indicated significant deviations from the normal distribution in 5‐FU concentration data. Nonparametric Kruskal–Wallis and Mann–Whitney U tests with Bonferroni correction for multiple comparisons were therefore used to compare interventions. An unpaired test design was used, as matching over time was not maintained. p Values were exact, two‐sided, and statistically significant when <0.05. Statistical analyses and graphical presentations were performed using SPSS version 24 (IBM Corporation) and GraphPad Prism version 7.00 (GraphPad Software, Inc.), respectively.

RESULTS

Cutaneous 5‐FU uptake

Compared to intact skin, AFL facilitated a prompt peak in 5‐FU delivery with significantly higher early concentrations in superficial and deep skin layers (1 hour: 500 μm: 12.5 vs. 46.5 ng/ml [p = 0.002]; 1500 μm: 1.5 vs. 31.8 ng/ml [p = 0.002]) (Figure 2). The impact of AFL was most pronounced in the deep skin layer, remaining elevated at 4 hours versus intact skin (1500 μm: 5.3 vs. 14.5 ng/ml [p = 0.039]). By 48 and 72 hours, however, 5‐FU detection in AFL‐exposed skin fell to levels comparable to unexposed skin.

Figure 2.

Impact of vasoregulative intervention on in vivo intracutaneous 5‐FU uptake. Measured at 500 and 1500 μm skin depth, AFL‐assisted drug delivery led to significant enhancement in early 5‐FU uptake (θ), but was comparable to intact skin between 48 and 72 hours. Vasoregulation by BRI and PDL, but not EPI, significantly increased intracutaneous 5‐FU concentrations versus AFL alone measured at 1 and 4 hours at both measured skin depths (*). At the earliest measured time point, BRI and PDL further provided significantly higher concentrations than EPI in the deep, 1500 μm skin layer (Ω). After 48 hours, only the effect of PDL remained significant versus AFL alone, limited to the 500 μm skin compartment (Ψ). BRI, brimonidine; EPI,  epinephrine; 5‐FU, 5‐fluorouracil; PDL, pulsed dye laser.

In the early phase of delivery, all vasoregulative approaches enhanced peak 5‐FU concentrations compared to AFL alone (Figures 2 and 3). Thus, by the first hour, BRI and PDL pretreatment led to a doubling of 5‐FU uptake in both skin compartments (i.e., AFL vs. AFL + BRI: 31.8 vs. 72.7 ng/ml [1500 μm; p = 0.012]; AFL vs. AFL + PDL: 31.8 vs. 62.6 ng/ml [1500 μm; p = 0.012]). While a similar, although less pronounced tendency was observed for EPI, trends remained nonsignificant after posthoc statistical analysis.

Figure 3.

Digital photography of skin reactions over time in vivo porcine skin with and without vasoregulative intervention Compared to untreated skin, AFL induced clearly discernable erythema, shown 1 and 4 hours after irradiation. Pharmacological interventions with EPI or BRI diminished the erythematous reaction produced by AFL, depicted at corresponding timepoints. At 48 and 72 hours, AFL, EPI, and BRI test areas were indistinguishable. In contrast, exposure to pulsed dye laser resulted in obvious purpura that was sustained over the entire study period. AFL, ablative fractional laser; BRI, brimonidine; EPI,  epinephrine.

Vasoregulative intervention maintained 5‐FU concentrations in the 500 μm skin compartment using EPI and PDL at 4 hours (AFL vs. AFL + EPI: 36.1 vs. 66.2 ng/ml [p = 0.5]; AFL vs. AFL + PDL: 36.1 vs. 98.5 ng/ml [p = 0.024]) (Figure 2). A similar pattern of sustained deposition was not observed in the deeper 1500 μm skin layer, as indicated by declining 5‐FU concentrations despite vasoregulative intervention between 1 and 4 hours. Despite diminishing 5‐FU levels, however, concentrations still remained higher than AFL delivery without vasoregulative intervention (4 hours: AFL vs. AFL + EPI, 14.5 vs. 26.3 ng/ml [p = 0.492]; vs. AFL + BRI, 42.3 ng/ml [p = 0.012]; vs. AFL + PDL, 38.4 ng/ml [p = 0.024]).

In the late stages of assessment at 48 and 72 hours, pharmacological vasoregulative interventions, EPI and BRI, did not alter 5‐FU deposition versus AFL alone (Figure 2). In contrast, PDL was associated with significantly extended 5‐FU delivery still limited to the superficial skin layer (AFL vs. AFL + PDL; 48 hours: 11 vs. 43.8 ng/ml [p = 0.024]; 72 hours: 7 vs. 22 ng/ml [p = 0.012]).

Direct comparisons of the three vasoregulative approaches revealed that BRI and PDL provided significantly higher 5‐FU uptake compared to EPI in the deep skin layer at 1 hour (EPI vs. BRI: 50.8 vs. 72.7 ng/m [p = 0.036]; EPI vs. PDL: 50.8 vs. 62.6 ng/ml [p = 0.012]) (Figure 2). At all other time points, differences remained nonsignificant. Similarly, no statistically significant difference between PDL and BRI was identified after posthoc correction for multiple comparisons at any time point or skin depth.

Vascular imaging

Angiography images of human skin with and without vasoregulation are shown in Figure 4. Measured from 1 to 48 hours, AFL produced appreciable increases in blood flow, resulting in elevated vasculature indexes compared to untreated adjacent skin (untreated vs. AFL; 1 hour: 0.14 vs. 0.52; 4 hours: 0.09 vs. 0.44; 48 hours: 0.17 vs. 0.38). AFL's augmenting impact on blood flow was thus most pronounced at early time points and remained detectable 48 hours after laser exposure. Vasoregulation with EPI, BRI, or PDL initially reduced AFL‐mediated increases in vasculature index (EPI vs. BRI vs. PDL: 0.24 vs. 0.21 vs. 0.13 [1 hour]); an effect that appeared to taper by 4 hours for EPI and by 48 hours for BRI. On the other hand, in skin areas exposed to AFL + PDL, vasculature indexes appeared consistently lower than in skin exposed to AFL alone, indicating sustained reductions in blood flow throughout the experiment.

DISCUSSION

The impact of vasoregulative intervention in topical LADD has not previously been examined. This study's findings indicate that early vascular drug clearance is a modifiable parameter in AFL‐assisted delivery in live skin. Overall, preceding vasoregulative intervention with BRI and PDL led to significantly enhanced 5‐FU concentrations compared to AFL + 5‐FU alone measured at 500 and 1500 μm skin depths at 1–4 hours. At later stages 48–72 hours after 5‐FU application, only the enhancing effects of PDL appeared to be sustained, with augmented 5‐FU concentrations in the 500 μm skin layer. Shown by vascular imaging of in vivo human skin, AFL led to local increases in blood flow that remained elevated versus untreated skin throughout 48 hours. These effects were partially reduced by EPI, BRI, and PDL with varying degrees of duration. A reduction in local blood flow and consequent lower vascular drug clearance might explain the enhancing impact of BRI and PDL on AFL‐assisted 5‐FU concentrations in in vivo skin.

Cutaneous drug clearance is primarily controlled by two parameters: (1) vasculature (i.e., total area/density, wall thickness, and blood flow) and (2) specific drug properties (i.e., diffusivity, partitioning coefficient, concentration/depletion, bound fraction, metabolism). ⁹ , ²⁴ It has been postulated that local blood circulation removes at least 50% and possibly close to 90% of agents applied to intact skin. ²⁴ Illustrated by vascular imaging and digital photography in this study, AFL prompts near‐immediate increases in local blood flow, generating an enhanced washout‐effect versus intact skin. Unlike the slow and gradual rise in superficial drug uptake over intact skin, the rapid peak and trough in cutaneous drug concentrations shown previously for some agents after AFL delivery may reflect this phenomenon. ¹² , ¹³ , ¹⁴

After posthoc analysis, PDL and BRI but not EPI facilitated statistically significant enhancements in 5‐FU uptake after AFL delivery. This finding may be explained by the duration of each intervention's effects. While BRI is a selective α₂‐adrenergic receptor agonist reported to reach maximum vasoconstrictive effect on intact skin between 3 and 6 hours, ²⁵ EPI's vasoconstrictive effect on α₁‐adrenergic receptors occurs within minutes and lasts between 2 and 10 minutes. ²⁶ Although EPI led to reduced vasculature indexes assessed by imaging 1 hour after application, the peak impact of EPI likely occurred earlier. In addition, the study employed an EPI product commonly available in dermatological clinics that also contained lidocaine, an agent with vasodilating effects and a longer half‐life than EPI. ²⁷ Lidocaine may thus to some extent have diminished EPI's impact on drug washout. In contrast, BRI's mitigating effect on blood flow remained discernable at 4 hours, corresponding to the herein demonstrated enhanced 5‐FU concentrations at 500 and 1500 μm skin depth. Expectedly, the most enduring impact on vasculature was produced by PDL, which leads to photothermal obliteration of vessels in irradiated skin areas. Sustained PDL‐mediated reductions in blood flow are the likely cause of significantly enhanced 5‐FU detection at the superficial, 500 μm skin compartment at 48 and 72 hours. Similar effects were not maintained in the deep skin layer by PDL, potentially due to insufficient laser penetration at this depth.

In the clinical setting, PDL's sustained effect on cutaneous 5‐FU concentrations is particularly interesting in clinical contexts of BCC treatment and scar therapy. For both indications, clinical studies demonstrate the efficacy of PDL‐based therapy via (1) selective targeting of lesion vessels and (2) nonselective thermal injury/remodeling produced by high pulse energies and multiple passes. ²⁸ , ²⁹ , ³⁰ Our study introduces a third potential rationale for a PDL‐based approach: reduced drug clearance when combined with AFL‐assisted topical treatments such as 5‐FU.

More generally, LADD with combined vasoregulative intervention may offer a new means to slow systemic uptake and retain drugs in the skin after delivery. This is desirable in instances where drug exposure duration is a limiting factor in topical treatment efficacy. As such, topical agents whose mechanism of action hinges on prolonged exposure (i.e., 5‐FU acts through enzymatic inhibition and is S phase‐specific) may benefit more from vasoregulative intervention versus drugs that do not require extended dwell times (i.e., cisplatin is phase nonspecific and causes direct, dose‐dependent DNA damage). ⁷ , ⁸ , ¹⁴ , ³¹ Furthermore, agents whose action is oxygen‐dependent, such as photodynamic therapy, may be negatively impacted by reducing blood flow. Finally, it should be noted that increasing peak drug concentration and dwell time might also lead to exaggerated local side effects ³² or impaired healing due to PDL‐mediated vascular damage. While the study did not examine these aspects, the appropriateness of vasoregulative intervention should thus be evaluated based on the individual patient, disease indication, and drug mechanism of action/properties.

At present, most existing LADD literature is derived from the in vitro setting. ¹ Our results call into question the transferability of in vitro data to the real‐life setting, since in vivo laser–tissue interactions and the herein demonstrated contribution of vasculature to drug clearance are poorly modeled in vitro. Our findings furthermore carry clear clinical implications since many topical treatment regimens require sustained drug concentrations for adequate efficacy. Previously, Nguyen et al. ³³ treated 12 primary superficial BCCs with a fractional CO₂ laser (identical to the one used in this study) at similar settings (10 mJ/mb, 5% density, and 0.12 mm spot size), followed by a single 5‐FU application under occlusion for 7 days. Authors reported BCC clearance rates of 67% at least 9 months later. ¹⁵ Given the rapid rate of drug removal following AFL‐assisted 5‐FU application shown here and in our previous BCC study, ¹¹ , ³⁴ it is possible that the brevity of 5‐FU exposure in treated skin contributed to lower therapeutic efficacy.

The study assessed drug uptake in in vivo pigskin. Rosenberg et al. ³⁵ recently showed that porcine skin represents an appropriate model for AFL‐assisted 5‐FU delivery experiments with a good correlation with human skin. Crucially, porcine vasculature resembles that of human skin based on blood vessel size and number as well as vascular organization in a superficial and deep plexus. ³⁶ , ³⁷ Nonetheless, variation between patients and the herein described standardized experiment should be expected. While the depth of the superficial, papillary plexus is reported to be between 100 and 300 µm in pigs, the papillary dermis in humans is located at a depth of 100–150 µm. ³⁸ , ³⁹ , ⁴⁰  Human skin furthermore shows greater inter‐ and intra‐individual variability in drug uptake, particularly in the setting of dermatological diseases that impact skin barrier. ¹

Strengths of this study include the in vivo design, assessment of multiple skin depths, and sensitive mass spectrometry‐based drug detection. ¹ Authors chose to examine a brief 15‐minute application of BRI and EPI based on two considerations: (1) preceding observations revealed this application time produced obvious clinical blanching and (2) this application time would also be feasible for clinical implementation in combination with LADD procedures. It is possible, however, that optimal effects of EPI and BRI would be achieved with longer applications. Other study limitations include the low sample size, limited number of time points, lacking assessment of impact on healing and safety, and inability to perform vascular imaging on the study animal due to breathing artifacts. All in all, future in vivo studies are needed to further characterize drug clearance and optimize vasoregulative approaches after topical AFL‐assisted drug delivery.

CONCLUSION

Vasoregulative intervention in conjunction with AFL‐assisted delivery enhances and prolongs 5‐FU deposition in in vivo skin. Reducing vascular clearance by vasoregulative intervention may be used to maintain therapeutic drug concentrations in treated skin after AFL‐assisted delivery.

Wenande E, Gundavarapu SC, Tam J, Bhayana B, Thomas CN, Farinelli WA, et al. Local vasoregulative interventions impact drug concentrations in the skin after topical laser‐assisted delivery. Lasers Surg Med. 2022;54:1288–1297. 10.1002/lsm.23558