Abstract
Radiation therapy is a mainstream strategy in the treatment of several cancer types that are surgically unresectable. Unfortunately, cancer patients often suffer from unintended consequences of radiotherapy, including the development of skin inflammation (dermatitis), which may progress to fibrosis. These morbid complications often require interruption of radiotherapy and threaten the relapse of underlying cancer. Current treatment options for radiation dermatitis are suboptimal and compel the need to develop safer, more effective therapies. In this study, we assessed the biophysical properties of topically-formulated esomeprazole (here referred to as dermaprazole) and performed proof-of-concept studies to evaluate its efficacy in vitro and in vivo. We found that dermaprazole induced nuclear translocation of erythroid 2-related factor 2 (Nrf2) and significantly upregulated heme oxygenase 1 (HO1) gene and protein expression in a 3D human skin model. Our animal study demonstrated that dermaprazole improved macroscopic appearance of the irradiated skin and accelerated healing of the wounds. Histopathology data corroborated the photographic evidence and confirmed that both prophylactically and therapeutically administered dermaprazole conferred potent anti-inflammatory and antifibrotic effects. Gene expression data showed that dermaprazole downregulated several pro-oxidant, pro-inflammatory and profibrotic genes. In conclusion, topical formulation of the FDA-approved drug esomeprazole is highly effective in attenuating dermal inflammation and fibrosis.
Editor’s note.
The online version of this article (DOI: 10.1667/RR15398.1) contains supplementary information that is available to all authorized users.
INTRODUCTION
Radiation therapy is part of the treatment regimen for many patients with solid tumors (1). Unfortunately, many cancer patients suffer from off-target effects of radiotherapy, including the development of widespread skin inflammation (radiation dermatitis) and progressive fibrosis that could lead to painful and disfiguring scarring of the face, chest, head and neck area. Acute radiation dermatitis is characterized by excessive skin inflammation that involves epidermal, dermal and vascular tissues and occurs in up to 95% of patients who receive radiotherapy (2). Up to 25% of the patients develop severe skin reactions that can lead to necrosis and scarring. Unfortunately, these complications could interrupt the treatment plan and threaten relapse of the underlying cancer (3–5).
Symptoms of radiation dermatitis vary in onset and duration. According to the National Cancer Institute Common Toxicity Criteria-Adverse Events (NCI-CTCAE) (6) and the Radiation Therapy Oncology Group (RTOG) (7, 8) toxicity scoring system, mild dermatitis (grade 1), characterized by mild redness (erythema), hyperpigmentation, epidermal thickening (hyperkeratosis) or dry desquamation, appears shortly after initiation of radiation treatment. Moderate dermatitis (grade 2) occurs within two weeks of the completion of therapy and manifests as painful and intense erythema, loss of hair from the root (epilation), epidermal necrosis, blisters and edema. In severe dermatitis (grade 3 and 4), moist desquamation occurs prominently and may lead to persistent inflammation, full-thickness skin necrosis and severely painful ulceration that is prone to infection. The acute and milder skin effects occur almost immediately after receiving doses of 2–40 Gy, whereas chronic effects occur several months-to-years after exposure to high-dose radiation (>45 Gy) (9). These structural and functional impairments are driven in part by exquisite sensitivity of hair follicle stem cells, basal keratinocytes and melanocytes to radiation. Radiation ionizes cellular water to promote the production of reactive oxygen species (ROS) and adducts that are involved in DNA damage (10). Furthermore, recruitment of circulating inflammatory cells to the local vasculature and increased levels of inflammatory molecules (11) exacerbates the injury and compromises skin integrity, leading to increased susceptibility to infection, delayed wound healing, fibrous thickening and irreversible scarring.
Recently, we discovered that a class of FDA-approved drug, proton pump inhibitors (PPIs), regulates the nitric oxide (NO) synthase (NOS) pathway mainly driven by the inducible (iNOS) isoform (12). This isoform is expressed in the skin by resident keratinocytes, dendritic, endothelial and epithelial cells in response to inflammatory stimuli, such as radiation or chemotherapy (13–15). The NO catalyzed by iNOS (from L-arginine) is short-lived and rapidly oxidizes to peroxynitrite (OONO–), a highly reactive molecule that is involved in the generation of nitrotyrosine, which is a more stable product involved in tissue inflammation. In addition, a number of cytokines that are released in response to chemoradiation therapy (16) are known to enhance NO production (17).
We found that regulation of iNOS pathway by PPIs is due to their direct interaction with dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that breaks down asymmetric dimethylarginine (ADMA), the endogenous and competitive NOS inhibitor (Fig. 1). Subsequently, we showed that esomeprazole is the most potent PPI that controls the profibrotic enzyme DDAH and several inflammatory cytokines. Some of the PPI regulated pro-inflammatory cytokines include nuclear factor kappa B (NFκB), tumor necrosis factor alpha (TNFα), interleukins (IL1β and IL6) and adhesion molecules (VCAM1 and ICAM1) (18). Many of these esomeprazole-regulated pro-inflammatory cytokines are reported to be pathologically upregulated in radiation dermatitis (11), in part, due to activation of these molecules by the ROS signaling that is activated in response to ionizing radiation.
FIG. 1.
Regulation of the NOS/DDAH pathway by PPIs. PPIs directly inhibit DDAH enzymatic activity resulting in accumulation of the endogenous substrate ADMA. ADMA is a competitive NOS inhibitor and limits the production of reactive oxygen and nitrogenous species resulting in reduced tissue inflammation and fibrosis. Physiologically, oxidation of L-arginine in the presence of NOS generates nitric oxide. iNOS = inducible nitric oxide synthase; DDAH = dimethylarginine dimethylaminohydrolase; ADMA = asymmetric dimethylarginine; PPIs = proton pump inhibitors.
In this regard, we found that esomeprazole significantly upregulates the antioxidant molecule heme oxygenase 1 (HO1) in fibroblasts, endothelial and epithelial cells (18). Several studies have reported that ionizing radiation disrupts the oxidant/antioxidant balance by depleting endogenous antioxidants such as glutathione peroxidases and heme oxygenases including HO1 (19). This phenomenon favors the production of tissue reactive species such as hydroxyl, peroxides and superoxide radicals. The oxidative stress and uncontrolled inflammation seen in radiation dermatitis are barriers to accelerated wound healing. By contrast, administration of antioxidants or anti-inflammatory molecules in preclinical models has been reported to inhibit radiation-induced oxidative stress and ameliorate radiation dermatitis. For example, scavenging ROS with the glutathione precursor N-acetylcysteine has been shown to be protective from radiation-induced dermatitis in a rat model (20). In addition, Reisman et al. (21) demonstrated that activation of the major antioxidant and transcription factor erythroid 2-related factor 2 (Nrf2) with a triterpenoid (RTA 408) upregulated several antioxidant enzymes such as glutathione, thioredoxins and superoxide dismutase, as well as downregulated pro-inflammatory molecules including interleukins and adhesion molecules to protect animals from radiation dermatitis. Biologically, activation of Nrf2 involves its translocation to the nuclei and subsequent stimulation of antioxidant enzymes such as HO1. Similar to the upregulation of Nrf2, pharmacological or genetic overexpression of HO1 has been shown to suppress radiation-induced dermatitis (22). Interestingly, PPIs are able to directly scavenge ROS in various cell types (23). In addition, we recently reported that esomeprazole downregulates the generation of extracellular matrix (ECM) components (fibronectin and collagen) by fibroblasts in response to transforming growth factor beta (TGFβ), a cytokine reported to be instantly upregulated by radiation to play a pathophysiological role in radiation-induced tissue fibrosis (24).
Collectively, the antioxidant, anti-inflammatory and antifibrotic properties of esomeprazole in cell culture and in in vivo studies encouraged us to reformulate the oral preparation into a topical cream (which we coined dermaprazole) and assess its biological efficacy in a 3D human skin model and in an animal model of radiation-induced dermatitis.
MATERIALS AND METHODS
Formulation of Esomeprazole
Esomeprazole was formulated into a topical product (i.e., dermaprazole) for evaluation of efficacy in regulating radiation-induced inflammation and fibrosis. Detailed description of the formulation and characterization process is provided in Supplementary Materials and Methods (https://dx.doi.org/10.1667/RR15398.1.S1).
Animal Study Approval
The animal study was reviewed and approved by Baylor College of Medicine’s Institutional Animal Care and Use Committee (IACUC no. AN-7127). All other reagents used in this study, including the human 3D skin model, were obtained from commercial sources.
In Vitro Study: 3D Human Skin Model
A three-dimensional (3D) human skin model (EpiDermFT, MatTek Corp., Ashland, MA) was used to validate the ability of formulated esomeprazole to engage biological targets. Total RNA was isolated from the 3D skin for gene expression studies. Detailed information about the 3D human skin model, as well as gene and protein expression studies, are provided in the Supplementary Materials and Methods (https://dx.doi.org/10.1667/RR15398.1.S1).
In Vivo Study: Mouse Model of Radiation-Induced Dermatitis
We conducted a 30-day study using 10-week-old male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) to assess the efficacy of dermaprazole in mitigating radiation-induced dermatitis. The experiment consisted of one group receiving no radiation (group 1) and five groups receiving radiation (groups 2–6) (Supplementary Table S2; https://dx.doi.org/10.1667/RR15398.1.S1) from an X-ray source. Dermatitis was induced in groups 2–6 by exposing the animals’ left flank to a 30 Gy dose. The efficacy of dermaprazole was evaluated in a prophylactic and therapeutic course. The in vivo study, including histopathology and immunohistochemistry, is described in detail in Supplementary Materials and Methods.
Western Blot Analysis
We performed Western blot analysis of erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO1) proteins in homogenates derived from irradiated EpidermFT tissues (Fig. 3). Nuclear and cytoplasmic proteins were fractionated and Nrf2 was probed in the nuclear fraction using rabbit anti-Nrf2 antibody (1:250) and the housekeeping gene histone H3 was probed using rabbit anti-histone H3 antibody (1:3,000) (both from Abcam®, Cambridge, MA). HO1 was probed in the cytoplasmic fraction using rabbit anti-HO1 (1:500; Enzo Life Sciences Inc., Farmingdale, NY) and GAPDH was detected using mouse anti-GAPDH antibody (1:5,000; Thermo Fisher Scientifice™ Inc., Waltham, MA). The secondary antibody used was anti-rabbit monoclonal (1:5,000; GE Healthcare Bio-Sciences, Pittsburgh, PA) or anti-mouse monoclonal (1:5,000).
FIG. 3.
Western blot analysis of erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO1) proteins in homogenates derived from irradiated EpidermFT tissues. Nuclear and cytoplasmic proteins were fractionated and Nrf2 was probed in the nuclear fraction using rabbit anti-Nrf2 antibody. The housekeeping gene histone H3 was probed using rabbit anti-histone H3 antibody. HO1 was probed in the cytoplasmic fraction using rabbit anti-HO1 and GAPDH was detected using mouse anti-GAPDH antibody. The secondary antibody used was anti-rabbit monoclonal or anti-mouse monoclonal. The data show that dermaprazole upregulated the protein expression of Nrf2 and HO1.
We performed Western blot analysis of heme oxygenase 1 (HO1) protein in homogenates derived from nonirradiated EpidermFT tissue (Fig. 4). Dermaprazole (1–2%) was applied on the tissue topically and the viable EpidermFT tissue was incubated at 378C/5% CO2 for 24 h. HO1 was probed using rabbit anti-HO1 (1:500; Enzo Life Sciences) and GAPDH was detected using mouse anti-GAPDH antibody (1:5,000; Thermo Fisher Scientific). The secondary antibody used was anti-rabbit monoclonal (1:5,000; GE Healthcare).
FIG. 4.
Western blot analysis of heme oxygenase 1 (HO1) protein in homogenates derived from nonirradiated EpidermFT tissue. Dermaprazole (1–2%) was applied on the tissue topically and the viable EpidermFT tissue was incubated at 37°C/5%CO2 for 24 h. HO1 was probed using rabbit anti-HO1 and GAPDH was detected using mouse anti-GAPDH antibody. The secondary antibody used was anti-rabbit monoclonal. The data show that dermaprazole upregulated HO1 expression in the absence of radiation.
Statistical Analysis
The number of animals per study group was calculated using power and sample size calculation (Vanderbilt University, Nashville, TN). Both parametric and nonparametric data were analyzed using one-way analysis of variance (ANOVA) (GraphPad Prism; La Jolla, CA) unless indicated otherwise. Multiple groups were compared using ANOVA followed by Bonferroni post hoc test and differences between two groups were compared using unpaired t test. All data are expressed as mean ± SEM unless indicated otherwise. Differences were considered statistically significant at P < 0.05.
RESULTS
Dermaprazole is a Homogenous Product with Consistency in Appearance, Color and Odor
Our study of the grittiness, color and odor of dermaprazole over time shows that the product has homogenous appearance with relatively stable odor and light brown color at low drug strength (0.01–2%), which increased in intensity towards dark brown over concentration, temperature and time (data not shown). Given the reproducible stability of dermaprazole at 1–2%, we evaluated the in vitro and in vivo efficacy of the product at these drug strengths.
Physicochemical Stability of Dermaprazole
Our liquid chromatography-mass spectrometry (LC-MS) study showed that the concentration of esomeprazole molecules recovered from the cream was proportional to the strength of dermaprazole formulation, with the lowest strength of dermaprazole (i.e., 0.01%) correlating to the lowest recovery of esomeprazole molecules (Supplementary Table S1; https://dx.doi.org/10.1667/RR15398.1.S1). As expected, the vehicle control showed no presence of esomeprazole molecules. In addition, the chromatogram data showed that the signature of the peak intensity and acquisition time for esomeprazole in the cream after 1 month of storage (day 32) correlated well with that of freshly prepared dermaprazole cream (day 0) and esomeprazole powder that was not formulated into a cream (Supplementary Fig. S1; https://dx.doi.org/10.1667/RR15398.1.S1). Meanwhile, the atomic force microscopy scanning data showed that dermaprazole blended well into the cream base that was used as a carrier and formed a relatively stiffer and less adhesive micelle-like product compared to the cream base alone (Supplementary Fig. S2).
Retention and Permeability of Dermaprazole
Our drug permeation study showed that the various strengths of dermaprazole maintained variable skin retention properties, as well as permeability through dermal membrane over a course of time. More specifically, measurement of drug concentration in the receptor compartments of the Franz diffusion cell demonstrated that the lowest dermaprazole strengths (0.01% to 1%) do not show appreciable increase in the concentration of drug in the receptor chamber over time relative to baseline, while the highest dermaprazole strengths (10–20%) showed significant increase in the amount of drug released over time (Supplementary Fig. S3; https://dx.doi.org/10.1667/RR15398.1.S1).
Induction of Nrf2-HO1 Pathway by Dermaprazole in 3D Human Skin Model
Topical application of dermaprazole for 24 h onto radiation-exposed 3D full-thickness human skin resulted in robust induction of the gene (Fig. 2) and protein (Fig. 3) expression of Nrf2 and HO1 in the dermal layer of the tissue. Our subcellular fractionation study also showed that Nrf2, which is physiologically compartmentalized in the cytoplasm of cells, was translocated into the nuclei upon treatment with dermaprazole (Fig. 3, top row). Although one of the possible mechanisms for the nuclear translocation of Nrf2 is inhibition of Keap1 by dermaprazole, no change in Keap1 expression was observed upon dermaprazole treatment (Supplementary Fig. S4; https://dx.doi.org/10.1667/RR15398.1.S1). Intriguingly, the expression of HO1 was significantly induced in nonirradiated human skin (Fig.4), suggesting that the major antioxidant enzyme HO1 can be upregulated by dermaprazole despite exposure to radiation and oxidative stress response.
FIG. 2.
Gene expression profiling of the transcription factor erythroid 2-related factor 2 (Nrf2) and the antioxidant enzyme heme oxygenase 1 (HO1) in irradiated EpidermFT tissue homogenates from a 3D human skin model. The EpidermFT was exposed to various strengths of dermaprazole, vehicle cream or the steroid hydrocortisone (1%) for 24 h. Fold change normalized to the vehicle control is shown. Data are from duplicate experiments. *P < 0.05 compared to the expression of Nrf2 in the vehicle and +P < 0.05 compared to the expression of HO1 in the vehicle group.
Efficacy of Dermaprazole in Suppressing Radiation-Induced Dermatitis
Our digital photography data from the mouse model of radiation-induced dermatitis revealed that treatment of animals with dermaprazole had substantial effect in improving the macroscopic appearance of the irradiated skin by day 16 and resulted in complete or nearly complete healing of the wound at the irradiation site in over 60% of the animals in both prophylactic dermaprazole groups by the end of the study at day 30 (Fig. 5). Intriguingly, therapeutic administration of dermaprazole after irradiation at the full dose also resulted in significant closure of the wounds and skin appearance comparable to that of animals treated prophylactically with dermaprazole (Fig. 5). Meanwhile, moisturizing of the irradiation site with vehicle cream also improved the appearance of the skin. However, treatment with 1% hydrocortisone had no effect on wound healing in 90% of the animals in this model and, as a result, the animals in the corticosteroid group had severe skin necrosis at both 16 days and 30 days postirradiation (Fig. 5). Assessment of the skin by a dermatologist revealed that treatment with dermaprazole, whether administered prophylactically or therapeutically, significantly reduced the degree of dermatitis by day 16 and normalized the appearance of the skin by day 30 (Supplementary Fig. S5; https://dx.doi.org/10.1667/RR15398.1.S1).
FIG. 5.
Topical application of the PPI esomeprazole improves skin appearance in a fractionated radiation-induced model of dermatitis. Mice were irradiated (2 × 15 Gy) on days 0 and 7. Topical esomeprazole (i.e., dermaprazole), vehicle (base) cream or the corticosteroid hydrocortisone was applied once daily on the indicated days (days 1–30 for the prophylactic group and days 10–30 for the therapeutic group). Representative images from the same animals are shown.
Dermaprazole Reduces Dermal Inflammation: Histological and Genetic Evidence
Consistent with the macroscopic improvement in skin appearance upon dermaprazole treatment, H&E staining confirmed that prophylactic or therapeutic treatment of the irradiated skin with dermaprazole reduced epidermal thickening at days 16 and 30 (Fig. 6A). In addition, the histological scores of ulceration, necrosis, parakeratosis/crust and overall inflammation were significantly reduced by dermaprazole treatment. The calculated mean score for each of these parameters showed that 1% dermaprazole administered in a prophylactic course reduced ulceration by 15%, necrosis by 18%, parakeratosis/crust by twofold, inflammation by twofold, and epidermal thickening by approximately sixfold at day 16 compared to the steroid control. Encouragingly, the trend continued throughout the treatment period with reductions at day 30 by twofold, fivefold, 88%, 86% and 86%, respectively (Supplementary Fig. S6; https://dx.doi.org/10.1667/RR15398.1.S1). Similarly, 2% prophylactic dermaprazole reduced these scores by 1.5-fold, 6.5-fold, 88%, 1.3-fold and 100%, respectively, compared to the steroid control at day 30, while the 2% therapeutic dermaprazole reduced these histological scores by 1.3-fold, 4-fold, 70%, 100% and 80%, respectively. In addition, immunohistochemical staining of paraffin-embedded skin tissues for the pan-leukocyte marker CD11b and the macrophage specific marker F4/80 showed that dermaprazole reduced recruitment of these cells into the injury site (Supplementary Fig. S7; https://dx.doi.org/10.1667/RR15398.1.S1). As expected, immunohistochemical staining for the iNOS-induced 3-nitrotyrosine showed intense staining in the vehicle and steroid groups but was diminished in the dermaprazole group (Supplementary Fig. S8A; https://dx.doi.org/10.1667/RR15398.1.S1). This observation is corroborated with the data measuring circulating levels of NO (Supplementary Fig. S8B; https://dx.doi.org/10.1667/RR15398.1.S1).
FIG. 6.
Histological data showing that dermaprazole improves skin histology in a radiation dermatitis model. Panel A: H&E stained irradiated skin tissue from animals treated with vehicle cream, dermaprazole or hydrocortisone once daily on the indicated days. Panel B: Masson’s trichrome stain was used to assess the degree of dermal fibrosis. Increased collagen deposition (blue stain) is observed in the vehicle and steroid groups. Treatment with dermaprazole inhibited collagen deposition. Representative images are shown at 20× magnification. The scale bar shown in the vehicle group at day 16 is 50 03BCm and applies to all the images.
In addition, gene expression study probing markers of inflammation confirmed that dermaprazole treatment significantly downregulated the mRNA expression of many of the classic pro-inflammatory cytokines, including TNFα, IL1β, NFkB, TLR4, IL6, ICAM1, VCAM1 and iNOS (Supplementary Fig. S9; https://dx.doi.org/10.1667/RR15398.1.S1).
Dermaprazole Reduces Dermal Fibrosis: Immunohistochemical and Genetic Evidence
Masson’s trichrome stain of collagen in skin tissue explanted from the radiation dermatitis model showed that treatment with dermaprazole notably reduced collagen accumulation on days 16 and 30 compared to vehicle or corticosteroid controls (Fig. 6B), indicating greater inhibition of fibrotic changes upon dermaprazole treatment. The trichrome stain scores indicated that there was a significantly lower degree of fibrotic changes in the dermaprazole treatment groups (Supplementary Fig. S10; https://dx.doi.org/10.1667/RR15398.1.S1).
The gene expression study for profibrotic markers also confirmed that dermaprazole significantly downregulated the expression of TGFβ, DDAH1, collagen 1, 3, 5 and fibronectin (Supplementary Fig. S11; https://dx.doi.org/10.1667/RR15398.1.S1).
Dermaprazole Temporally Upregulates the Expression of HO1 in Dermatitis Model
As expected, treatment of mice with low- or high-dose dermaprazole in a prophylactic or therapeutic course significantly upregulated the gene expression of HO1 at the disease peak (i.e., day 16). This was mirrored by relatively low expression of the major pro-oxidant enzymes NOX2 and NOX4 (Supplementary Fig. S12; https://dx.doi.org/10.1667/RR15398.1.S1). However, this upregulation of antioxidant defense mechanism was temporal and subsided after the animals recovered from the effects of radiation (i.e., day 30). Intriguingly however, the animals that still manifested radiation dermatitis by day 30 (e.g., steroid group) had elevated levels of HO1, a cytoprotective molecule known to be induced by cellular stress (25), at this time point (Supplementary Fig. S12: https://dx.doi.org/10.1667/RR15398.1.S1). Consistent with the phenotype of increased cellular stress are the very high levels of NOX2 and NOX4 in the steroid-treated group by day 30 (Supplementary Fig. S12). Collectively, these data sets suggest that the animals in the steroid group were still coping with the radiation-induced oxidative burden in part through heightened antioxidant defense mechanism.
DISCUSSION
Dermaprazole Mitigates Ionizing Radiation-Induced Dermatitis
Radiation dermatitis is a dose-limiting normal tissue toxicity to the skin that occurs in a large proportion of cancer patients who receive radiation therapy. Radiation dermatitis manifests as an inflammatory reaction at the site of irradiation and may include redness, desquamation, loss of hair and necrotic changes. In moderate-to-severe cases, the inflammatory skin reaction may progress to fibrosis that permanently scars the irradiated tissue. This may result in significant compromise in the quality of life of the affected patients and may force the discontinuation of radiation therapy and threaten relapse of the underlying cancer (26).
Despite intensifying research efforts, to date, there is no effective therapy for radiation-induced dermatitis. As described above, several agents have been tested for their efficacy in treating or reversing radiation dermatitis. Nonetheless, there still lacks an effective drug to mitigate this commonly occurring complication of cancer therapy. In light of the central role of oxidative stress and inflammation in radiation-induced normal tissue toxicities, there are ongoing efforts to test and develop antioxidants and anti-inflammatory molecules to protect normal tissues from radiation toxicities including dermatitis. In this regard, several candidate drugs have been evaluated for their therapeutic potential in ameliorating radiation dermatitis. Included among these candidates that share common mechanisms of action to that of Dermaprazole are pharmacological activators of Nrf2 (21, 27) and HO1 inducers (22, 28). The attenuation of radiation-induced oxidative stress, inflammation and overall dermatitis score by these agents suggest that targeting of the Nrf2/HO1 pathway is an attractive therapeutic strategy in the search for agents that protect normal tissue from the effects of radiation. Accordingly, the emerging role of PPIs as polypharmacological molecules with simultaneous modulation of oxidative stress, inflammation and fibrosis makes this class of drug an ideal candidate to be repurposed for cancer therapy-induced complications including dermatitis, mucositis, pneumonitis, proctitis and esophagitis. In the current study, we reformulated esomeprazole into a topical product that is able to penetrate into the dermis and protect the skin from harmful effects of ionizing radiation, including ulceration, necrosis, inflammation and fibrosis (Figs. 5 and 6B; Supplementary Figs. S5–S11; https://dx.doi.org/10.1667/RR15398.1.S1), resulting in nearly complete closure of the wounds in most of the dermaprazole-treated animals (Fig. 5).
Dermaprazole Retains the Biological Activity of Esomeprazole
The formulation of esomeprazole powder into a Lipoderm®-based cream produced a topical product, dermaprazole, which retained the biological activity of esomeprazole. Esomeprazole, the S-enantiomer of omeprazole, is a PPI widely used for the treatment of gastroesophageal reflux. Emerging studies indicate that omeprazole, esomeprazole and other PPIs possess biological activities that extend beyond suppression of gastric acidity. Some of the recently reported effects of PPI include regulation of oxidative stress, inflammation and fibrosis. The antioxidant effect of PPIs is due to direct scavenging of ROS and restoration of depleted endogenous antioxidants (29). The anti-inflammatory effect of PPIs is in part due to downregulation of classic pro-inflammatory molecules and impaired migration of neutrophils (30). More recently, we have shown the antifibrotic effect of PPIs to be due to upregulation of HO1, downregulation of extracellular matrix components and direct inhibition of fibroblast proliferation (18). Intriguingly, all of these extra-gastric effects of PPIs could not be reproduced with other antacids such as the histamine H2-receptor antagonists (18, 31). Accordingly, the effect of PPIs on extra-gastric targets is likely due to the presence of benzimidazole moiety in their structure. Benzimidazoles are considered as privileged scaffolds and form the basis for approximately 25% of the 100 top-selling drugs (32). In the current study, dermaprazole maintained the antioxidant, anti-inflammatory and antifibrotic effects that are also possessed by its unformulated analog, esomeprazole. In addition, dermaprazole was well tolerated with no adverse effects on body weight or the weight of the heart, lungs, kidneys and liver (Supplementary Fig. S13; https://dx.doi.org/10.1667/RR15398.1.S1).
Dermaprazole Downregulates DDAH-iNOS Pathway in Dermal Tissue In Vitro and In Vivo
Recently published studies indicate that DDAH, an enzyme expressed by every nucleated mammalian cell, supports pro-inflammatory and profibrotic activities. For example, Pullamsetti et al. (33) used a mouse model that genetically overexpresses human DDAH and showed that exposure of these transgenic mice to the chemotherapeutic drug bleomycin exaggerates the fibrotic response, including greater accumulation of collagen, while DDAH inhibition with a small molecule suppresses fibrotic changes. Similarly, other studies have noted a pathobio-logical role of the NOS pathway associated with increased proliferation of fibroblasts and augmented fibrotic tissue remodeling (34). Our recently published data demonstrate that PPIs modulate both DDAH and iNOS (18), and systemic administration of esomeprazole in a mouse model of bleomycin-induced lung injury suppresses lung inflammation and fibrosis (18). Similarly, our current data show that the expression of both DDAH and iNOS are upregulated by ionizing radiation and dermaprazole significantly downregulates their expression in the dermal tissue (Supplementary Figs. S9 and S11; https://dx.doi.org/10.1667/RR15398.1.S1). This effect is reflected by decreased levels of circulating NO and tissue nitrotyrosine in the dermaprazole treated groups (Supplementary Fig. S8; https://dx.doi.org/10.1667/RR15398.1.S1). Previously, we have reported that esomeprazole regulates circulating NO levels in a mouse model of lung injury (35).
Esomeprazole and other Proton Pump Inhibitors Chemosensitize Tumor Cells
The potential future use of dermaprazole in cancer patients affected with dermatitis raises the question of whether concomitant use of PPIs impair the anti-tumor effect of chemoradiation therapy in part by protecting the tumor from the anticancer treatment. Part of this concern may be addressed from existing in vitro and in vivo data available in the literature. For example, Luciani et al. (36) assessed the sensitivity of 28 chemotherapy-resistant human cancer cell lines upon pretreatment with the PPIs omeprazole and esomeprazole. They found that pretreatment of cancer cells with the PPIs resulted in order-of-magnitude reduction in the half maximal inhibitory concentration (IC50) values for the chemotherapeutic agents cisplatin, vinblastine and 5-fluorouracil compared to no PPI pretreatment control.
In conclusion, dermaprazole, the topically formulated PPI esomeprazole, is able to macroscopically and microscopically reduce inflammation and scarring induced by ionizing radiation in a preclinical model of radiation-induced dermatitis. The anti-inflammatory and antifibrotic effects of dermaprazole appear to be due to the early induction of endogenous antioxidant defense mechanisms and downregulation of pro-inflammatory and profibrotic mechanisms. Some of the dermaprazole-regulated key molecular pathways include HO1-Nrf2, DDAH-iNOS and collagen. Early induction of the HO1 and Nrf2 molecules by dermaprazole is expected to prepare the skin tissue to handle the early surge in oxidative stress perpetrated by ionizing radiation. Subsequently, downstream targets of ROS, superoxide and hydroxyl radicals, including pro-inflammatory and profibrotic cytokines, are favorably regulated. The remarkable efficacy of dermaprazole on dermal tissue inflammation and fibrosis, as well as the antitumor activity of PPIs (37), may well be good foundations for the dual use of dermaprazole to reduce cancer therapy-induced inflammation while chemosensitizing the underlying tumor. Meanwhile, we are investigating to extend the potential clinical utility of dermaprazole in other skin conditions characterized by inflammatory and/or fibrotic phases. In particular, the profound effect of dermaprazole on classic pro-inflammatory molecules such as TNFa, innate immune signaling such as TLR4, and components of extracellular matrix (ECM) such as collagen and fibronectin (Supplementary Figs. S9–11; https://dx.doi.org/10.1667/RR15398.1.S1) invites categorical evaluation of the efficacy of the formulation beyond radiation-induced ailments into diseases of the connective tissue.
Supplementary Material
Table S1. Measurement of esomeprazole concentration from dermaprazole cream by LC-MS.
Table S2. Experimental design of ionizing radiation-induced dermatitis in a 30-day mouse model.
Fig. S1. Representative LC-MS chromatogram of dermaprazole.
Fig. S1. Representative LC-MS chromatogram of dermaprazole.
Fig. S2. Atomic Force Microscopy (AFM) scan showing the topography of dermaprazole cream in comparison to cream only control.
Fig. S3. Skin permeation/retention of dermaprazole ex vivo using Franz diffusion cell technique.
Fig. S4. Western blot analysis of Kelch-like ECH-associated protein 1 (Keap1) in homogenates derived from irradiated EpidermFT tissue.
Fig. S5. Topical application of dermaprazole improves dermatitis scoring in a mouse model of fractionated radiation-induced dermatitis. Animals were irradiated 2 × 15 Gy on days 0 and 7, and treated with dermaprazole in a prophylactic or therapeutic course. Vehicle (base) cream and steroid (1% hydrocortisone) treatment were included as controls. The degree of dermatitis was scored by a blinded dermatologist using CTCAE criteria: 0 = normal skin appearance; 1 = mild erythema; 2 = moderate-to-severe erythema; 3 = desquamation of 25–50% of irradiated area; 4 = desquamation of >50% irradiated area; and 5 = frank ulcer.
Fig. S6. Dermaprazole reduces histopathological changes in the dermal tissue of radiation-induced dermatitis model. H&E stained skin tissues were evaluated by a board-certified dermatopathologist who was unaware of the treatment groups. Topical dermaprazole significantly reduced inflammation, epidermal thickening and parakeratosis at day 16 (panel A), as well as inflammation, epidermal thickening, ulcer, necrosis and parakeratosis by day 30 (panel B) of the study.
Fig. S7. Immunohistochemical staining of irradiated skin tissue for CD11b and F4/80 showing that topical application of dermaprazole inhibits pro-inflammatory cells.
Fig. S8. Panel A: Immunohistochemical staining of irradiated skin tissue for 3-nitrotyrosine showing that topical application of dermaprazole targets the iNOS pathway to inhibit accumulation of nitrotyrosine in irradiated skin tissue. Panel B: Relative changes in circulating levels of nitric oxide in serum samples obtained at baseline and after irradiation. Relative changes from baseline in total nitrite levels are shown for comparison.
Fig. S9. Dermaprazole regulates radiation-induced changes in the expression of pro-inflammatory molecules in the dermal tissue of radiation dermatitis model. Quantitative RT-PCR data are shown for the gene expression profile of TNFα, IL1β, IL6, iNOS, NFκB, TLR4, VCAM1 and ICAM1 is shown. Data are expressed as the mean ± SEM from duplicate experiments.
Fig. S10. Dermaprazole reduces collagen thickening/ fibrosis in a mouse model of radiation-induced dermatitis. Masson's trichrome-stained skin tissues were evaluated by a board-certified dermatopathologist who was unaware of the treatment groups. Topical dermaprazole significantly reduced dermal fibrosis compared to the vehicle or steroid (1% hydrocortisone) group at day 30 (P < 0.05).
Fig. S11. Dermaprazole regulates radiation-induced changes in the expression of profibrotic molecules in the dermal tissue of a radiation dermatitis model. Quantitative RT-PCR data are shown for the gene expression profile of TGFβ, collagen 1, collagen 3, collagen 5, fibronectin and DDAH. Data are expressed as the mean ± SEM from duplicate experiments.
Fig. S12. Dermaprazole temporally regulates radiation-induced changes in the expression of oxidative stress-related genes in the dermal tissue of radiation dermatitis model. Quantitative RT-PCR data are shown for the gene expression profile of HO1, NADPH oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4) at the anticipated disease peak time (day 16) and at the study completion (day 30). Data are expressed as the mean ± SEM from duplicate experiments.
Fig. S13. Mouse model of radiation-induced dermatitis. Panel A: Measurement of total body weight over time. Panel B: Organ weights for the heart, lungs, liver and kidneys normalized to the respective body weights at the time of necropsy. Lung and kidney weights represent combined total weight for the left and right tissues. Treatment with dermaprazole in a prophylactic or therapeutic course did not have adverse effects on the body or organ weights. Data are expressed as the mean ± SEM.
ACKNOWLEDGMENTS
We acknowledge the NMR and Drug Metabolism Core Facility at Baylor College of Medicine, which is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01 HD087157-01A1) and Cancer Prevention and Research Institute of Texas (RP160805) to Dr. Martin M. Matzuk. This study was supported in part by the National Institutes of Health (NIH grant no. P30 CA125123, which supports the Dan L. Duncan Comprehensive Cancer Center at Baylor College of Medicine). JMN acknowledges financial support from the NIDCR F31 NRSA (grant no. F31DE026682) of the NIH. AGS acknowledges support from the FDA (no. 1R01FD005109-01A1) and the Veterans Affairs Administration (no. I01 BX004183-01A1). YTG is supported by grants from the NHLBI (grant nos. K01HL118683; R01HL137703), American Heart Association (grant no. 17GRNT33460159) and by intramural funding from Baylor College of Medicine (project ID 2690000104). This content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors. YTG is an inventor on patents, owned by Stanford University and Baylor College of Medicine, that protect the use of agents, including proton pump inhibitors (PPIs), for therapeutic use of new indications. MSL and MDB are inventors on the patent owned by Baylor College of Medicine.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1. Measurement of esomeprazole concentration from dermaprazole cream by LC-MS.
Table S2. Experimental design of ionizing radiation-induced dermatitis in a 30-day mouse model.
Fig. S1. Representative LC-MS chromatogram of dermaprazole.
Fig. S1. Representative LC-MS chromatogram of dermaprazole.
Fig. S2. Atomic Force Microscopy (AFM) scan showing the topography of dermaprazole cream in comparison to cream only control.
Fig. S3. Skin permeation/retention of dermaprazole ex vivo using Franz diffusion cell technique.
Fig. S4. Western blot analysis of Kelch-like ECH-associated protein 1 (Keap1) in homogenates derived from irradiated EpidermFT tissue.
Fig. S5. Topical application of dermaprazole improves dermatitis scoring in a mouse model of fractionated radiation-induced dermatitis. Animals were irradiated 2 × 15 Gy on days 0 and 7, and treated with dermaprazole in a prophylactic or therapeutic course. Vehicle (base) cream and steroid (1% hydrocortisone) treatment were included as controls. The degree of dermatitis was scored by a blinded dermatologist using CTCAE criteria: 0 = normal skin appearance; 1 = mild erythema; 2 = moderate-to-severe erythema; 3 = desquamation of 25–50% of irradiated area; 4 = desquamation of >50% irradiated area; and 5 = frank ulcer.
Fig. S6. Dermaprazole reduces histopathological changes in the dermal tissue of radiation-induced dermatitis model. H&E stained skin tissues were evaluated by a board-certified dermatopathologist who was unaware of the treatment groups. Topical dermaprazole significantly reduced inflammation, epidermal thickening and parakeratosis at day 16 (panel A), as well as inflammation, epidermal thickening, ulcer, necrosis and parakeratosis by day 30 (panel B) of the study.
Fig. S7. Immunohistochemical staining of irradiated skin tissue for CD11b and F4/80 showing that topical application of dermaprazole inhibits pro-inflammatory cells.
Fig. S8. Panel A: Immunohistochemical staining of irradiated skin tissue for 3-nitrotyrosine showing that topical application of dermaprazole targets the iNOS pathway to inhibit accumulation of nitrotyrosine in irradiated skin tissue. Panel B: Relative changes in circulating levels of nitric oxide in serum samples obtained at baseline and after irradiation. Relative changes from baseline in total nitrite levels are shown for comparison.
Fig. S9. Dermaprazole regulates radiation-induced changes in the expression of pro-inflammatory molecules in the dermal tissue of radiation dermatitis model. Quantitative RT-PCR data are shown for the gene expression profile of TNFα, IL1β, IL6, iNOS, NFκB, TLR4, VCAM1 and ICAM1 is shown. Data are expressed as the mean ± SEM from duplicate experiments.
Fig. S10. Dermaprazole reduces collagen thickening/ fibrosis in a mouse model of radiation-induced dermatitis. Masson's trichrome-stained skin tissues were evaluated by a board-certified dermatopathologist who was unaware of the treatment groups. Topical dermaprazole significantly reduced dermal fibrosis compared to the vehicle or steroid (1% hydrocortisone) group at day 30 (P < 0.05).
Fig. S11. Dermaprazole regulates radiation-induced changes in the expression of profibrotic molecules in the dermal tissue of a radiation dermatitis model. Quantitative RT-PCR data are shown for the gene expression profile of TGFβ, collagen 1, collagen 3, collagen 5, fibronectin and DDAH. Data are expressed as the mean ± SEM from duplicate experiments.
Fig. S12. Dermaprazole temporally regulates radiation-induced changes in the expression of oxidative stress-related genes in the dermal tissue of radiation dermatitis model. Quantitative RT-PCR data are shown for the gene expression profile of HO1, NADPH oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4) at the anticipated disease peak time (day 16) and at the study completion (day 30). Data are expressed as the mean ± SEM from duplicate experiments.
Fig. S13. Mouse model of radiation-induced dermatitis. Panel A: Measurement of total body weight over time. Panel B: Organ weights for the heart, lungs, liver and kidneys normalized to the respective body weights at the time of necropsy. Lung and kidney weights represent combined total weight for the left and right tissues. Treatment with dermaprazole in a prophylactic or therapeutic course did not have adverse effects on the body or organ weights. Data are expressed as the mean ± SEM.