Comparative Study of Dermal Pharmacokinetics Between Topical Drugs Using Open Flow Microperfusion in a Pig Model.

PURPOSE: Accurate methods to determine dermal pharmacokinetics are important to increase the rate of clinical success in topical drug development. We investigated in an in vivo pig model whether the unbound drug concentration in the interstitial fluid as determined by dermal open flow microperfusion (dOFM) is a more reliable measure of dermal exposure compared to dermal biopsies for seven prescription or investigational drugs. In addition, we verified standard dOFM measurement using a recirculation approach and compared dosing frequencies (QD versus BID) and dose strengths (high versus low drug concentrations). METHODS: Domestic pigs were topically administered seven different drugs twice daily in two studies. On day 7, drug exposures in the dermis were assessed in two ways: (1) dOFM provided the total and unbound drug concentrations in dermal interstitial fluid, and (2) clean punch biopsies after heat separation provided the total concentrations in the upper and lower dermis. RESULTS: dOFM showed sufficient intra-study precision to distinguish interstitial fluid concentrations between different drugs, dose frequencies and dose strengths, and had good reproducibility between studies. Biopsy concentrations showed much higher and more variable values. Standard dOFM measurements were consistent with values obtained with the recirculation approach. CONCLUSIONS: dOFM pig model is a robust and reproducible method to directly determine topical drug concentration in dermal interstitial fluid. Dermal biopsies were a less reliable measure of dermal exposure due to possible contributions from drug bound to tissue and drug associated with skin appendages.

Topical Delivery Systems Effectively Transport Analgesics to Areas of Localized Pain via Direct Diffusion.

Topical delivery systems (TDSs) enable the direct transport of analgesics into areas of localized pain and thus minimize the side effects of administration routes that rely on systemic drug distribution. For musculoskeletal pain, clinicians frequently prescribe topical products containing lidocaine or diclofenac. This study assessed whether drug delivery from a TDS into muscle tissue occurs mainly via direct diffusion or systemic transport. An investigational TDS containing 108 mg lidocaine (SP-103, 5.4% lidocaine), a commercially available TDS containing 36 mg lidocaine (ZTlido®, 1.8% lidocaine), and a topical pain relief gel (Pennsaid®, 2% diclofenac) were tested. Using open flow microperfusion (OFM), interstitial fluid from the dermis, subcutaneous adipose tissue (SAT), and muscle was continuously sampled to assess drug penetration in all tissue layers. Ex vivo and in vivo experiments showed a higher diffusive transport of lidocaine compared to diclofenac. The data showed a clear contribution of diffusive transport to lidocaine concentration, with SP-103 5.4% resulting in a significantly higher lidocaine concentration in muscle tissue than commercially available ZTlido® (p = 0.008). These results indicate that SP-103 5.4% is highly effective in delivering lidocaine into muscle tissue in areas of localized pain for the treatment of musculoskeletal pain disorders (e.g., lower back pain).

Small volume rapid equilibrium dialysis (RED) measures effects of interstitial parameters on the protein-bound fraction of topical drugs.

The importance of plasma protein binding in the early stages of drug development is well recognized. Free and bound drug fractions in plasma are routinely determined with well-established methods. However, for physiological fluids with a small accessible volume and low protein concentrations, such as dermal interstitial fluid (dISF) validated methods are currently missing. Due to the low protein concentration and highly dynamic processes in the dermis, protein binding data obtained from plasma samples may underestimate in-vivo efficacy. This study aimed to validate a small volume rapid equilibrium dialysis (RED) for low protein samples, as a tool to examine drug-protein binding directly in the biological fluid at the site of action. The sample volume required for RED was successfully downscaled to 50 µl and plasma protein binding values of the four model drugs were consistent with previous studies with an average recovery of 88 ± 8% which makes all tested drugs suitable for small volume RED. Inter- and intra-batch variability showed sufficient reproducibility across RED plates. Small volume RED was successfully applied to assess the effects of interstitial parameters, including the evaluation of the major binding protein and the effects of binding protein concentration, drug concentration, and pH on the protein-bound drug fraction using 2% HSA and/or diluted human plasma as a surrogate for dISF.

Quantification of the Therapeutic Antibody Ocrelizumab in Mouse Brain Interstitial Fluid Using Cerebral Open Flow Microperfusion and Simultaneous Monitoring of the Blood-Brain Barrier Integrity.

The increasing relevance of improved therapeutic monoclonal antibodies (mAbs) to treat neurodegenerative diseases has strengthened the need to reliably measure their brain pharmacokinetic (PK) profiles. The aim of this study was, therefore, to absolutely quantify the therapeutic antibody ocrelizumab (OCR) as a model antibody in mouse brain interstitial fluid (ISF), and to record its PK profile by using cerebral open flow microperfusion (cOFM). Further, to monitor the blood-brain barrier (BBB) integrity using an endogenous antibody with a similar molecular size as OCR. The study was conducted on 13 male mice. Direct and absolute OCR quantification was performed with cOFM in combination with zero flow rate, and subsequent bioanalysis of the obtained cerebral ISF samples. For PK profile recording, cerebral ISF samples were collected bi-hourly, and brain tissue and plasma were collected once at the end of the sampling period. The BBB integrity was monitored during the entire PK profile recording by using endogenous mouse immunoglobulin G1. We directly and absolutely quantified OCR and recorded its brain PK profile over 96 h. The BBB remained intact during the PK profile recording. The resulting data provide the basis for reliable PK assessment of therapeutic antibodies in the brain thus favoring the further development of therapeutic monoclonal antibodies.

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.

Atraumatic access to human glioblastoma in a xenograft animal model by cerebral open flow microperfusion.

BACKGROUND: Orthotopic xenograft studies promote the development of targeted/personalized therapies to improve the still poor life expectancy of glioblastoma patients. NEW METHOD: We implemented an atraumatic access to glioblastoma with cerebral Open Flow Microperfusion (cOFM) by implantation of xenograft cells in rat brain with intact blood brain barrier (BBB) and subsequent development of a xenograft glioblastoma at the interface between the cOFM probe and surrounding brain tissue. Human glioma U87MG cells were implanted at a well-defined position into immunodeficient Rowett nude rat´s brain via cOFM (cOFM group) and syringe (control group). Characteristics of the mature tumors from both groups were assessed. RESULTS: For the first time xenograft cells were successfully introduced into rat brain with intact BBB using cOFM, and the tumor tissue developing around the cOFM probe was unaffected by the presence of the probe. Thereby an atraumatic access to the tumor was created. The success rate of glioblastoma development in the cOFM group was high (>70%). The mature cOFM-induced tumors (20-23 days after cell-implantation) resembled the syringe-induced ones and showed typical features of human glioblastoma. COMPARISON WITH EXISTING METHOD: Examining xenograft tumor microenvironment with currently available methods inevitably causes trauma that could affect the reliability of obtained data. CONCLUSION: This novel atraumatic access to human glioblastoma in rat brain provides the possibility to collect interstitial fluid from functional tumor tissue in vivo without trauma generation. Thereby, reliable data can be generated promoting drug research, biomarker identification, and enabling investigation of the BBB of an intact tumor.

In vivo monitoring of brain pharmacokinetics and pharmacodynamics with cerebral open flow microperfusion.

In vivo investigation of brain pharmacokinetics and pharmacodynamics (PK/PD) is an integral part of neurological drug development. However, drugs intended to act in the brain may reach it at very low concentrations due to the protective effect of the blood-brain barrier (BBB). Consequently, very sensitive measurement methods are required to investigate PK/PD of drugs in the brain. Also, these methods must be capable of continuously assessing cerebral drug concentrations with verifiable intact BBB, as disrupted BBB may lead to compound efflux from blood into brain and to biased results. To date, only a few techniques are available that can sensitively measure drug concentrations in the brain over time; one of which is cerebral open flow microperfusion (cOFM). cOFM's key features are that it enables measurement of cerebral compound concentrations with intact BBB, induces only minor tissue reactions, and that no scar formation occurs around the probe. The membrane-free cOFM probes collect diluted cerebral interstitial fluid (ISF) samples that are containing the whole molecule spectrum of the ISF. Further, combining cOFM with an in vivo calibration protocol (e.g. Zero Flow Rate) enables absolute quantification of compounds in cerebral ISF. In general, three critical aspects have to be considered when measuring cerebral drug concentrations and recording PK/PD profiles with cOFM: (a) the BBB integrity during sampling, (b) the status of the brain tissue next to the cOFM probe during sampling, and (c) the strategy to absolutely quantify drugs in cerebral ISF. This work aims to review recent applications of cOFM for PK/PD assessment with a special focus on these critical aspects.

Dermal open flow microperfusion for PK-based clinical bioequivalence studies of topical drug products.

Topically applied drug products have experienced an extraordinary price increase in the United States, mostly due to a lack of generic products. Generic drug development is hindered by high costs and risks associated with clinical endpoint studies required to show bioequivalence (BE) of prospective generic products relative to their reference products. There is a continued need for cost- and time-efficient alternatives to clinical endpoint studies to determine BE of topically applied dermal drug products. Cutaneous PK-based BE studies present such an alternative and dOFM (dermal open flow microperfusion) has already been successfully used in several verifications studies to show an accurate and sensitive assessment of the rate and extent at which drugs become available in the skin. dOFM technology is discussed as well as the dOFM setup of clinical pilot and main studies to achieve BE assessment with a minimum number of participants and an outlook is given on the use of dOFM technology for other drug products.

ImmUniverse Consortium: Multi-omics integrative approach in personalized medicine for immune-mediated inflammatory diseases.

Immune mediated inflammatory diseases (IMIDs) are a heterogeneous group of debilitating, multifactorial and unrelated conditions featured by a dysregulated immune response leading to destructive chronic inflammation. The immune dysregulation can affect various organ systems: gut (e.g., inflammatory bowel disease), joints (e.g., rheumatoid arthritis), skin (e.g., psoriasis, atopic dermatitis), resulting in significant morbidity, reduced quality of life, increased risk for comorbidities, and premature death. As there are no reliable disease progression and therapy response biomarkers currently available, it is very hard to predict how the disease will develop and which treatments will be effective in a given patient. In addition, a considerable proportion of patients do not respond sufficiently to the treatment. ImmUniverse is a large collaborative consortium of 27 partners funded by the Innovative Medicine Initiative (IMI), which is sponsored by the European Union (Horizon 2020) and in-kind contributions of participating pharmaceutical companies within the European Federation of Pharmaceutical Industries and Associations (EFPIA). ImmUniverse aims to advance our understanding of the molecular mechanisms underlying two immune-mediated diseases, ulcerative colitis (UC) and atopic dermatitis (AD), by pursuing an integrative multi-omics approach. As a consequence of the heterogeneity among IMIDs patients, a comprehensive, evidence-based identification of novel biomarkers is necessary to enable appropriate patient stratification that would account for the inter-individual differences in disease severity, drug efficacy, side effects or prognosis. This would guide clinicians in the management of patients and represent a major step towards personalized medicine. ImmUniverse will combine the existing and novel advanced technologies, including multi-omics, to characterize both the tissue microenvironment and blood. This comprehensive, systems biology-oriented approach will allow for identification and validation of tissue and circulating biomarker signatures as well as mechanistic principles, which will provide information about disease severity and future disease progression. This truly makes the ImmUniverse Consortium an unparalleled approach.

Advanced Online Monitoring of In Vitro Human 3D Full-Thickness Skin Equivalents.

Skin equivalents and skin explants are widely used for dermal penetration studies in the pharmacological development of drugs. Environmental parameters, such as the incubation and culture conditions affect cellular responses and thus the relevance of the experimental outcome. However, available systems such as the Franz diffusion chamber, only measure in the receiving culture medium, rather than assessing the actual conditions for cells in the tissue. We developed a sampling design that combines open flow microperfusion (OFM) sampling technology for continuous concentration measurements directly in the tissue with microfluidic biosensors for online monitoring of culture parameters. We tested our design with real-time measurements of oxygen, glucose, lactate, and pH in full-thickness skin equivalent and skin explants. Furthermore, we compared dermal penetration for acyclovir, lidocaine, and diclofenac in skin equivalents and skin explants. We observed differences in oxygen, glucose, and drug concentrations in skin equivalents compared to the respective culture medium and to skin explants.

The Impact of Prolonged Inflammation on Wound Healing.

The treatment of chronic wounds still challenges modern medicine because of these wounds' heterogenic pathophysiology. Processes such as inflammation, ischemia and bacterial infection play major roles in the progression of a chronic wound. In recent years, preclinical wound models have been used to understand the underlying processes of chronic wound formation. However, the wound models used to investigate chronic wounds often lack translatability from preclinical models to patients, and often do not take exaggerated inflammation into consideration. Therefore, we aimed to investigate prolonged inflammation in a porcine wound model by using resiquimod, a TLR7 and TLR8 agonist. Pigs received full thickness excisional wounds, where resiquimod was applied daily for 6 days, and untreated wounds served as controls. Dressing change, visual documentation and wound scoring were performed daily. Biopsies were collected for histological as well as gene expression analysis. Resiquimod application on full thickness wounds induced a visible inflammation of wounds, resulting in delayed wound healing compared to non-treated control wounds. Gene expression analysis revealed high levels of IL6, MMP1 and CD68 expression after resiquimod application, and histological analysis showed increased immune cell infiltration. By using resiquimod, we were able to show that prolonged inflammation delayed wound healing, which is often observed in chronic wounds in patients. The model we used shows the importance of inflammation in wound healing and gives an insight into the progression of chronic wounds.

Optimization of topical formulations using a combination of in vitro methods to quantify the transdermal passive diffusion of drugs.

This paper describes a new approach to the early-stage optimization of topical products and selection of lead formulation candidates. It demonstrates the application of open flow microperfusion in vitro in conjunction with the Franz diffusion cell to compare time-resolved, 24-hour profiles of diclofenac passive diffusion through all skin layers (including the skin barrier, dermis, and subcutis) resulting from nine topical formulations of different composition. The technique was successfully validated for in vitro sampling of diclofenac in interstitial fluid. A multi-compartmental model integrating the two datasets was analyzed and revealed that the passive diffusion of diclofenac through the dermis and subcutis does not correlate with its diffusion through the skin barrier and cannot be predicted using Franz diffusion cell data alone. The combined application of the two techniques provides a new, convenient tool for product development and selection enabling the comparison of topical formulation candidates and their impact on drug delivery through all skin layers. This approach can also generate the experimental data required to improve the robustness of mechanistic PBPK models, and when combined with clinical sampling via open flow microperfusion - for the development of better in vivo-in vitro correlative models.

miRNAs as Regulators of the Early Local Response to Burn Injuries.

In burn injuries, risk factors and limitations to treatment success are difficult to assess clinically. However, local cellular responses are characterized by specific gene-expression patterns. MicroRNAs (miRNAs) are single-stranded, non-coding RNAs that regulate mRNA expression on a posttranscriptional level. Secreted through exosome-like vesicles (ELV), miRNAs are intracellular signalers and epigenetic regulators. To date, their role in the regulation of the early burn response remains unclear. Here, we identified 43 miRNAs as potential regulators of the early burn response through the bioinformatics analysis of an existing dataset. We used an established human ex vivo skin model of a deep partial-thickness burn to characterize ELVs and miRNAs in dermal interstitial fluid (dISF). Moreover, we identified miR-497-5p as stably downregulated in tissue and dISF in the early phase after a burn injury. MiR-218-5p and miR-212-3p were downregulated in dISF, but not in tissue. Target genes of the miRNAs were mainly upregulated in tissue post-burn. The altered levels of miRNAs in dISF of thermally injured skin mark them as new biomarker candidates for burn injuries. To our knowledge, this is the first study to report miRNAs altered in the dISF in the early phase of deep partial-thickness burns.

Characterizing Cutaneous Drug Delivery Using Open-Flow Microperfusion and Mass Spectrometry Imaging.

Traditionally, cutaneous drug delivery is studied by skin accumulation or skin permeation, while alternative techniques may enable the interactions between the drug and the skin to be studied in more detail. Time-resolved skin profiling for pharmacokinetic monitoring of two Janus Kinase (JAK) inhibitors, tofacitinib and LEO 37319A, was performed using dermal open-flow microperfusion (dOFM) for sampling of perfusate in an ex vivo and in vivo setup in pig skin. Additionally, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed to investigate depth-resolved skin distributions at defined time points ex vivo in human skin. By dOFM, higher skin concentrations were observed for tofacitinib compared to LEO 37319A, which was supported by the lower molecular weight, higher solubility, lipophilicity, and degree of protein binding. Using MALDI-MSI, the two compounds were observed to show different skin distributions, which was interpreted to be caused by the difference in the ability of the two molecules to interact with the skin compartments. In conclusion, the techniques assessed time- and depth-resolved skin concentrations and were able to show differences in the pharmacokinetic profiles of two JAK inhibitors. Thus, evidence shows that the two techniques can be used as complementary methods to support decision making in drug development.

OFM-recirculation and OFM-suction: advanced in-vivo open flow microperfusion (OFM) methods for direct and absolute quantification of albumin in interstitial fluid.

OBJECTIVE: To implement OFM-recirculation and OFM-suction capable of direct and absolute in-vivo quantification of albumin in the ISF of pigs. APPROACH: OFM-recirculation and OFM-suction were used to collect ISF in-vivo in pigs and lymph was collected from the same pigs after OFM sampling. Blood was collected before and after OFM sampling, plasma was isolated and mean albumin plasma concentrations per pig were used to yield albumin ISF-to-plasma ratios. We characterized the quality of the collected undiluted ISF via (1) stable albumin ISF-to-plasma ratio in OFM-recirculation and in OFM-suction samples, (2) comparison of albumin ISF-to-plasma ratios from OFM-recirculation and OFM-suction and (3) comparison of normalized albumin concentrations in the ISF and lymph. MAIN RESULTS: Both advanced OFM methods were successfully implemented and albumin was quantified from the collected ISF samples. OFM-recirculation reached stable albumin ISF-to-plasma ratios after 20 recirculation cycles. Absolute ISF albumin concentrations were 11.2 mg ml-1 (OFM-recirculation) and 14.2 mg ml-1 (OFM-suction). Albumin ISF-to-plasma ratios were 0.39 ± 0.04 (OFM -recirculation) and 0.47 ± 0.1 (OFM-suction). SIGNIFICANCE: Knowledge of the ISF protein content is of major importance when assessing PK/PD effects, especially of highly protein bound drugs. Up to now, only blood albumin values have been available to determine the degree of protein binding in several tissues. OFM-recirculation and OFM-suction allow direct, absolute quantification of albumin in ISF for the first time and enable investigation of the degree of protein binding of a drug directly in its target tissue.

A novel human ex vivo skin model to study early local responses to burn injuries.

Burn injuries initiate numerous processes such as heat shock response, inflammation and tissue regeneration. Reliable burn models are needed to elucidate the exact sequence of local events to be able to better predict when local inflammation triggers systemic inflammatory processes. In contrast to other ex vivo skin culture approaches, we used fresh abdominal skin explants to introduce contact burn injuries. Histological and ultrastructural analyses confirmed a partial-thickness burn pathology. Gene expression patterns and cytokine production profiles of key mediators of the local inflammation, heat shock response, and tissue regeneration were analyzed for 24 h after burn injury. We found significantly increased expression of factors involved in tissue regeneration and inflammation soon after burn injury. To investigate purely inflammation-mediated reactions we injected lipopolysaccharide into the dermis. In comparison to burn injury, lipopolysaccharide injection initiated an inflammatory response while expression patterns of heat shock and tissue regeneration genes were unaffected for the duration of the experiment. This novel ex vivo human skin model is suitable to study the local, early responses to skin injuries such as burns while maintaining an intact overall tissue structure and it gives valuable insights into local mechanisms at the very beginning of the wound healing process after burn injuries.

Evaluating Dermal Pharmacokinetics and Pharmacodymanic Effect of Soft Topical PDE4 Inhibitors: Open Flow Microperfusion and Skin Biopsies.

PURPOSE: To investigate the difference in clinical efficacy in AD patients between two topical PDE4 inhibitors using dermal open flow microperfusion and cAMP as a pharmacodynamic read-out in fresh human skin explants. METHODS: Clinical formulations were applied to intact or barrier disrupted human skin explants and both skin biopsy samples and dermal interstitial fluid was sampled for measuring drug concentration. Furthermore, cAMP levels were determined in the skin biopsies as a measure of target engagement. RESULTS: Elevated cAMP levels were observed with LEO 29102 while no evidence of target engagement was obtained with LEO 39652. In barrier impaired skin the dISF concentration of LEO 29102 was 2100 nM while only 33 nM for LEO 39652. For both compounds the concentrations measured in skin punch biopsies were 7-33-fold higher than the dISF concentrations. CONCLUSIONS: Low unbound drug concentration in dISF in combination with minimal target engagement of LEO 39652 in barrier impaired human skin explants supports that lack of clinical efficacy of LEO 39652 in AD patients is likely due to insufficient drug availability at the target. We conclude that dOFM together with a pharmacodynamic target engagement biomarker are strong techniques for establishing skin PK/PD relations and that skin biopsies should be used with caution.

Variability of Skin Pharmacokinetic Data: Insights from a Topical Bioequivalence Study Using Dermal Open Flow Microperfusion.

PURPOSE: Dermal open flow microperfusion (dOFM) has previously demonstrated its utility to assess the bioequivalence (BE) of topical drug products in a clinical study. We aimed to characterize the sources of variability in the dermal pharmacokinetic data from that study. METHODS: Exploratory statistical analyses were performed with multivariate data from a clinical dOFM-study in 20 healthy adults evaluating the BE, or lack thereof, of Austrian test (T) and U.S. reference (R) acyclovir cream, 5% products. RESULTS: The overall variability of logAUC values (CV: 39% for R and 45% for T) was dominated by inter-subject variability (R: 82%, T: 91%) which correlated best with the subject's skin conductance. Intra-subject variability was 18% (R) and 9% (T) of the overall variability; skin treatment sites or methodological factors did not significantly contribute to that variability. CONCLUSIONS: Inter-subject variability was the major component of overall variability for acyclovir, and treatment site location did not significantly influence intra-subject variability. These results support a dOFM BE study design with T and R products assessed simultaneously on the same subject, where T and R treatment sites do not necessarily need to be next to each other. Localized variation in skin microstructure may be primarily responsible for intra-subject variability.

A novel human ex-vivo burn model and the local cooling effect of a bacterial nanocellulose-based wound dressing.

BACKGROUND: Burn wound progression is a significant problem as burns initially thought to be superficial can actually become full thickness over time. Cooling is an efficient method to reduce burn wound conversion. However, if the cooling agent is below room temperature, depending on the wound size the patient is at risk of hypothermia. Additionally, tissue perfusion is reduced leading to an aggravation of burn wound progression. We investigated if wound dressings based on non-pre-cooled bacterial nanocellulose (BNC) with a high water content cool a burn just by evaporation and reduce the intradermal damages in the skin. MATERIAL AND METHODS: In a human ex-vivo model, skin explants underwent contact burns using a 100 °C hot steel block. The burned areas were divided into two groups of which one was cooled with a BNC-based wound dressing. Intradermal temperature probes measured temperature in cooled and uncooled burn sites over 24 h. For histological assessments of the burned areas biopsies were taken at different time points. High mobility group box-1 (HMBG1) staining served as marker for cell vitality and necrosis in the different skin layers. RESULTS: Intradermal temperature measurement showed that application of the BNC-based wound dressing reduced temperature significantly in burned skin. This cooling effect resulted in a maximum temperature difference of 6.4 ± 1.9 °C and a significant mean reduction of the area under the curve in the first hour after burn of 62% (p < 0.0001). The histological results showed less necrosis and less dermal-epidermal separation in the cooled areas. The HMGB1 staining revealed more vital cells in the cooled group than in the uncooled group. CONCLUSION: Based on our results, BNC-based wound dressings cool a burn. Intradermal temperature as well as thermal damage of the tissue was reduced. The tested BNC-based wound dressing can be used without pre-cooling to cool a burn as well as to reduce the burn BNC-based wound progression through its evaporation cooling effect.