Therapeutic potential of a phospholipase D1 inhibitory peptide fused with a cell-penetrating peptide as a novel anti-asthmatic drug in a Der f 2-induced airway inflammation model

Asthma is a chronic lung disease that causes airflow obstruction due to airway inflammation. However, its therapeutics remain inadequate. We previously reported that phospholipase D1 (PLD1) is a key enzyme involved in the production of pro-inflammatory cytokines in airway inflammation induced by the house dust mite allergen Dermatophagoides farinae 2 (Der f 2). We also revealed that PLD1 is specifically inactivated by AP180 (assembly protein, 180 kDa) and identified the PLD1-specific binding motif (TVTSP) of AP180. Therefore, the aims of this study were to develop a novel anti-asthmatic agent that could suppress airway inflammation by inhibiting PLD1 and examine its acute and chronic toxicity. We designed TAT-TVTSP, a PLD1-inhibitory peptide fused with a cell-penetrating peptide (CPP) delivery system. TAT-TVTSP was efficiently delivered to bronchial epithelial cells and significantly reduced Der f 2-induced PLD activation and Interleukin 13 (IL-13) production. Intranasally administered TAT-TVTSP was also efficiently transferred to airway tissues and ameliorated airway inflammation in a Der f 2-induced allergic asthma mouse model. Moreover, we investigated the safety of TAT-TVTSP as a therapeutic agent through single- and repeated-dose toxicity studies in a mouse model. Taken together, these results indicated that a PLD1-inhibitory peptide fused with a cell-penetrating peptide may be useful for treating allergic inflammatory asthma induced by house dust mites (HDMs). A drug that targets a key enzyme involved in airway tissue inflammation shows promise in the treatment of allergic asthma. The enzyme phospholipase D1 (PLD1) triggers airway inflammation in allergic asthma brought on by house dust mites. Joong-Soo Han at Hanyang University in Seoul, Eung-Gook Kim at Chungbuk National University, Cheongju, South Korea, and co-workers have developed a treatment aimed at suppressing PLD1 and trialed the drug on mouse models of dust-mite allergy. The team designed a carrier system capable of accurately delivering a PLD1-inhibitory peptide to airway tissues and cells. They found that airway inflammation was significantly reduced in the treated mice. The drug appeared to be relatively safe when used in repeated doses, although further investigations are needed to verify this. The team hope their treatment will improve therapies for allergic asthma.


Introduction
Allergic asthma is a heterogeneous inflammatory lung disease that affects~334 million people worldwide 1,2 . The disease is caused by individual genetic factors and is aggravated by environmental insults such as viral infection, irritants, and allergens in dust, animal fur, and pollen 1 . In most cases, patients with asthma are prescribed inhaled corticosteroids with or without longlasting ß agonists, theophyllines, or leukotriene-receptor antagonists, according to their symptoms 3 . However, these anti-asthmatic therapies are limited to symptomatic treatment and may incur severe side effects. Furthermore, despite receiving multiple therapies,~40% of patients with asthma remain symptomatic, and up to 5% have difficultto-control asthma 3,4 . In this context, many researchers have focused on improving disease control by studying methods to reduce the administration of corticosteroids to patients with severe allergic asthma. Nevertheless, the number of patients with asthma has not fallen much.
House dust mites (HDMs) are the most prevalent triggers of asthma, and~85% of asthmatics are allergic to HDMs 5 . In particular, Dermatophagoides pteronyssinus (Der p) and D. farinae (Der f) stimulate the production of Th2 pro-inflammatory cytokines, including Interleukin 4 (IL-4) and Interleukin 13 (IL-13) 5,6 . We have reported that phospholipase D1 (PLD1) is a key enzyme in the production of IL-13 in response to Der f 2 and have suggested that it is a potential therapeutic target in allergic disorders 7,8 . PLD1 is a membrane-associated enzyme that catalyzes the hydrolysis of phosphatidylcholine to yield the lipid second messenger phosphatidic acid and free polar head group choline 9 . However, targeting of PLD1 can incur systemic adverse effects because it is a ubiquitous enzyme involved in membrane trafficking 10 , cytoskeletal rearrangement 11 , differentiation 12,13 , survival 14,15 , and autophagy 16 . Although small-molecule PLD1 inhibitors showed significant therapeutic effects in cancer 17,18 , autoimmunity 19 , and kidney disease 20 , their use is limited because of side effects, selectivity, and toxicity. We previously reported that PLD1 is specifically inactivated by binding to AP180 (assembly protein, 180 kDa), an important factor in clathrin-mediated endocytosis of synaptic vesicles 21,22 . Moreover, we identified the PLD1specific binding motif of AP180 and demonstrated that the pentapeptide (TVTSP) in this motif was effective as an anticancer adjuvant 23 . Thereafter, based on these reports, we registered two domestic patents 24,25 . However, it is not known whether this peptide has any therapeutic potency in allergic diseases, including asthma.
Efficient delivery of therapeutic biomolecules to target tissues is a challenging problem because they poorly penetrate the cellular membrane. Cell-penetrating peptides (CPPs), small cationic peptides derived from proteins that can cross phospholipid bilayers, offer one possible solution 26 . CPPs, as a helpful tool for the intracellular delivery of various molecules, such as peptides, proteins, siRNAs, and therapeutic agents, have been studied in various fields 27 . In particular, TAT (trans-activator of transcription) is internalized via clathrin-mediated endocytosis and micropinocytosis and is assumed to be biologically inert 28 . It has been used in preclinical models to study pain 29 , ischemia 30 , cancer 31 , and inflammation 32 , and there have been over 20 phase I and phase II clinical trials involving more than 2000 patients treated with CPPfused therapeutics 33 . These results imply that CPPs can be used as an efficient drug delivery system.
Here, we aimed to develop a novel agent that suppressed airway inflammation by targeting PLD1 and to assess its in vivo toxicity. We designed TAT-TVTSP, a PLD1-specific inhibitory peptide fused with TAT, as the delivery vehicle. TAT-TVTSP effectively penetrated bronchial epithelial cells and airway tissues and significantly reduced Der f 2-induced PLD activation and airway inflammation in vitro as well as in vivo. Furthermore, TAT-TVTSP was found to be relatively safe in single-and repeated-dose toxicity studies of a mouse model. These findings indicate that therapy using a PLD1inhibitory peptide fused with a CPP delivery system can be an efficient and promising strategy to prevent allergic asthma induced by HDMs.

Materials
The house dust mite allergen Der f 2 was provided by the Arthropods of Medical Importance Resource Bank, Yonsei University, College of Medicine (Seoul, Korea), as described previously 8 . Peptides were synthesized by Cos-moGenetech (Seoul, Korea) and were validated by HPLC and mass spectrometry (data not shown). PLD inhibitors (FIPI and VU0155069) were supplied by Tocris (Oxford, UK).

Determination of PLD activity
PLD activity was determined in [ 3 H]-palmitic acidlabeled cells by measuring the amount of [ 3 H]-phosphatidylbutanol (PBt) produced by PLD-catalyzed transphosphatidylation in the presence of 1-butanol, as described previously 34 . BEAS-2B cells were labeled with 2 µCi/ml [ 3 H]-palmitic acid (DuPont-New England Nuclear, Boston, MA, USA) in serum-free medium for 18 h. They were then pretreated with 0.3% (v/v) 1-butanol for 15 min before stimulation with Der f 2. Cells were pre-incubated with TAT, TAT-TVTSP, FIPI, or VU0155069 for 2 h after labeling with [ 3 H]-palmitic acid. After treatment with Der f 2 for 30 min, the cells were washed with ice-cold PBS and suspended in ice-cold methanol. Lipids were extracted, and PBt was separated by thin layer chromatography using an ethylacetate/isooctane/acetic acid/water (26:4:6:20, v/v) solvent system. Authentic PBt bands (R f = 0.76) were identified with 0.002% (w/v) primulin dissolved in 80% (v/v) acetone, scraped, and counted in a liquid scintillation counter (PerkinElmer, Norwalk, CT, USA).

Real-time PCR and reverse transcription (RT)-PCR
Total cellular RNA was isolated using RNAiso Plus (Takara Bio, Ohtsu, Japan). Next, 2 µg of isolated total RNA was mixed with the Reverse Transcription Master premix kit (Elpis Bio, Daejeon, Korea), and the mixture was incubated for 60 min at 37°C. The transcribed products were mixed with each primer set and GoTaq DNA polymerase (Promega, Madison, WI, USA) and were amplified in a PCR machine (Takara Bio). The primer sequences were as follows: IL-13 sense (5′-GAGTGTG TTTGTCACCGTTG-3′) and antisense (5′-TACTCGTT GGTCGAGAGCTG-3′); GAPDH sense (5′-CATGAGAA GTATGACAACAGCCT-3′) and antisense (5′-AGTCC TTCCACGATACCAAAGT-3′). The PCR conditions for IL-13 and GAPDH were as follows: denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 40 s (35 cycles and 26 cycles). PCR products were analyzed on a 1.5% agarose gel. For real-time PCR, an aliquot of 1 µl of cDNA was amplified in duplicate in a final volume of 20 µl using the SensiFAST™ SYBR® NO-ROX Kit (Bioline, London, UK). The thermocycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 56°C for 1 min. The primer sequences for real-time PCR were the same as those for RT-PCR.

ELISA
Cell supernatants were collected after Der f 2 treatment, and the concentrations of IL-13 were measured using a commercial ELISA kit (Bender Medsystems, Vienna, Austria) following the manufacturer's instructions. The concentrations of samples were determined from a standard curve ranging from 3.13 to 200 pg/ml. For mouse experiments, lung tissues were homogenized in 20 ml/g tissue protein extraction reagent (ThermoFisher, Rockford, IL, USA) using a tissue homogenizer (Biospec Products, Bartlesville, OK, USA). Homogenates were incubated at 4°C for 30 min and centrifuged at 1000×g for 10 min. The supernatants were collected and passed through a 0.45-micron filter (Gelman Sciences, Ann Arbor, MI, USA) for the measurement of cytokine levels.

Measurement of delivery efficiency
Cells were plated onto coverslips in 24-well plates, and TAT-TVTSP-FAM (Supplementary Figure 2) was added for the indicated times. After removal of the medium, the cells were fixed with 4% PFA (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. The medium was replaced by 0.1% BSA in PBS, and the cells were mounted on slides with Vectashield (DAKO, Glostrup, Denmark). FAM signals were photographed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). The tissue permeability of TAT-TVTSP was also examined in a mouse model. Five-week-old female mice habituated for 1 week were intranasally administered TAT-TVTSP-FAM or saline. The tracheas and bronchioles were excised 2 h later and were immediately frozen in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and sectioned at a thickness of 10 µm. The sections were fixed with 4% PFA for 15 min, washed with 0.1% BSA in PBS, and mounted with Vectashield. Intracellular fluorescence signals were observed using a fluorescence microscope.

Animals
Female 6-week-old BALB/c mice (Orient, Gyeonggi-do, Korea) were used to generate the mouse model of allergic airway inflammation. For the in vivo toxicity studies, male and female 5-week-old ICR mice (Koathech, Gyeonggi-do, Korea) were used. They were maintained under specific pathogen-free conditions, fulfilling the standards of facilities approved by the American Association for the Accreditation of Laboratory Animal Care. All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines.

Mouse model of allergic airway inflammation
The protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Hanyang Laboratory Animal Research Center (2015-0197A). The mice were assigned to the saline, Der f 2, Der f 2 + TAT or Der f 2 + TAT-TVTSP groups. As shown in Fig. 2a, TAT or TAT-TVTSP was administered intranasally 2 h prior to the intranasal injection of Der f 2 (1.2 mg/kg), and this was repeated 7 times over 2 weeks.

Histology
Lung tissues were fixed in 10% formalin, embedded in paraffin, and sectioned. Sections were counterstained with H&E or PAS for histological examination using standard techniques. After staining, the slides were rinsed in distilled water, mounted with Vectashield, and photographed using an optical microscope (Olympus, Tokyo, Japan).

AHR measurement
Airway hyperresponsiveness (AHR) was measured using a whole-body plethysmographic chamber (OCP-3000; Allmedicus, Gyeonggi-do, Korea). The mice were exposed to the indicated concentrations of methacholine (Sigma-Aldrich) using an ultrasonic nebulizer (Omron Healthcare, Kyoto, Japan). After placing the mouse in the chamber, airway responsiveness was measured by evaluating enhanced pause (Penh). Penh, as an index of airway hyperresponsiveness, was calculated as previously described 35 .

BALF assay
Bronchoalveolar lavage fluid (BALF) was obtained as previously described 36 . Lungs were lavaged with 1 ml of Hank's balanced salt solution via a tracheostomy tube. The total cell numbers were counted using a hemocytometer (Sigma-Aldrich). BALF was then centrifuged at 1500×g for 3 min at 4°C, and smears of BAL cells were prepared by cytocentrifugation (Cytospin3; Thermo, Billerica, MA, USA) at 1000 rpm for 3 min. BAL cells were stained using a Hemacolor Staining Kit (Merck, Darmstadt, Germany), counted and classified as neutrophils, eosinophils, lymphocytes, or macrophages.

Single-dose toxicity study
Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Hanyang Laboratory Animal Research Center (2016-0038A). Forty animals aged 5 weeks were randomly assigned to four different dose groups (Supplementary Table 1), and each concentration of TAT-TVTSP was injected intranasally. Changes in the clinical signs, body weight, and blood analysis indices were evaluated. All the animals were euthanized on day 14, except for four mice that had to be sacrificed earlier. Mortality was recorded, and tissues from The results are expressed as the means ± SD of three independent experiments. All differences were assessed by Student's t-test the heart, kidney, liver, stomach, spleen, lung, intestine, and brain were collected for immediate necropsy. After fixation, organs sampled for histology were stained with H&E according to the standard procedures. In addition, blood samples were freshly drawn from each heart and were analyzed at the Korea Conformity Laboratories (Incheon, Korea). The following standard hematology markers were analyzed: red blood cell count, hemoglobin, hematocrit, mean corpuscle volume, mean corpuscle hemoglobin concentration, platelet count, and white blood cell count. Additionally, blood samples were centrifuged twice at 3000 rpm for 10 min to separate sera. Liver function was evaluated from the serum levels of total bilirubin (T-BIL) and alkaline phosphatase (ALP). Albumin (ALB) was assayed as a measure of tissue damage or inflammation. Gamma glutamyl transferase (GGT) and total protein (TP) were also quantified. These parameters were determined using a Biochemical Autoanalyzer (Type 7170; Hitachi, Japan).

Repeated-dose toxicity study
Protocols were reviewed and approved by the Institution Animal Care and Use Committee of the Hanyang Laboratory Animal Research Center (2016-0038A). Forty animals (20 males and 20 females) were randomly allocated to four different dose groups (Supplementary Table 1). TAT-TVTSP (or saline) was administered intranasally every day for 4 weeks. Other procedures were similar to those in the "Single-dose toxicity study" section.

Statistics
Statistical analysis for a complex sampling design was performed using SPSS software, version 18.0 (SPSS Inc., Chicago, IL, USA). The data are expressed as the means ± SD of at least three independent experiments; P < 0.05 was considered statistically significant. Comparisons between groups were made using unpaired Student's t-tests, and multi-group comparisons of means were carried out by one-way repeated-measures analysis of variance (ANOVA).

TAT-TVTSP is efficiently delivered into BEAS-2B cells and reduces Der f 2-induced PLD activation and IL-13 production
We investigated whether a peptide in the PLD1-specific binding motif of AP180 was applicable as an inhibitor of airway inflammation induced by the house dust mite allergen Der f 2. As shown in Fig. 1a, we designed a PLD1inhibitory peptide (TVTSP) coupled with TAT as a delivery vehicle. To verify whether this peptide (TAT-TVTSP) regulated PLD activity, human bronchial epithelial cells (BEAS-2B) were exposed to Der f 2 (20 µg/ml) for 30 min after pretreatment with TAT-TVTSP (10 or 20 µM) or TAT (10 µM) for 2 h. Der f 2-induced PLD activation was repressed by TAT-TVTSP in a dosedependent manner (Fig. 1b). Moreover, as shown in Supplementary Figure 1, TAT-TVTSP efficiently decreased Der f 2-induced PLD activation compared with excess treatment (50 µM) or Lipofectamine-mediated transfection of TVTSP, indicating that TAT powerfully acted as a transcellular delivery cargo. To further examine the efficiency of delivery of TAT-TVTSP, we labeled its Cterminus with the fluorescent dye FAM (Supplementary Figure 2) and measured the intracellular fluorescence over a time course. Green fluorescence was detected in almost all cells 1 h after exposure to TAT-TVTSP-FAM, and the signals were strongest after 2 h (Fig. 1c). Next, we found that Der f 2-induced IL-13 mRNA expression was significantly reduced by TAT-TVTSP (Fig. 1d, e), and the same was true for IL-13 production itself (Fig. 1f). Moreover, exposure to TAT-TVTSP had no effect on cell viability (Fig. 1g). Interestingly, the inhibitory effect of TAT-TVTSP on IL-13 expression was similar to that of other PLD inhibitors, FIPI and VU0155069 (Supplementary Figure 3). In summary, TAT-TVTSP was efficiently delivered into bronchial epithelial cells and reduced Der f 2-induced PLD activation and IL-13 production without any cytotoxicity.

Intranasally administered TAT-TVTSP is efficiently transferred into airway tissues and blocks airway inflammation in a mouse model of Der f 2-induced acute allergic asthma
To confirm whether TAT-TVTSP was also effective in vivo, we made a HDM-induced airway inflammation mouse model of acute allergic asthma 8 . Female 6-weekold mice were intranasally injected with TAT or TAT-TVTSP (10 mg/kg) for 2 h prior to the administration of Der f 2 (1.2 mg/kg), and this was repeated 7 times over 2 weeks (Fig. 2a). We first assessed the delivery efficiency of TAT-TVTSP to target tissues by analyzing in vivo FAM fluorescence 2 h after intranasal injection of TAT-TVTSP-FAM. Considerable accumulation of green fluorescence was observed in both bronchioles and tracheas (Fig. 2b). Next, we investigated whether TAT-TVTSP affected airway inflammation in vivo. Lung histology was examined to evaluate the extent of inflammation, epithelial hyperplasia, and mucus secretion by goblet cells in airway tissues. As shown in Fig. 2c, after the injection of TAT (or saline), the intranasal administration of Der f 2 stimulated the infiltration of peribronchial inflammatory cells, resulting in thickening of the airway tissues as well as increased secretion of mucus around the bronchial airways. Remarkably, these effects were prevented by intranasal challenge with TAT-TVTSP. Moreover, compared with pretreatment with TAT (or saline), TAT-TVTSP almost completely blocked Der f 2-induced IL-13 production (Fig. 2d). We also evaluated airway hyperresponsiveness (AHR) by analyzing lung resistance to methacholine (Fig. 2e). Intranasal administration of TAT-TVTSP substantially attenuated Der f 2-developed AHR to nebulized methacholine, implying that it blocked allergic inflammation and AHR. The number of immune cells in BALFs was also significantly decreased by TAT-TVTSP (Fig. 2f). Taken together, we conclude that targeting of PLD1 with TAT-TVTSP has powerful antiinflammatory effects on the Der f 2-induced allergic airway inflammation model and may be an effective therapeutic for allergic diseases, including asthma.

Single-dose toxicity study
We next investigated whether TAT-TVTSP is clinically applicable as a novel anti-asthmatic agent. In the doseprobing pilot study for the toxicity test, 3 mice in the 250 mg/kg group, 5 in the 500 mg/kg group, and 7 in the 1000 mg/kg group died, indicating that the median lethal dose (LD 50 ) of TAT-TVTSP was 453.8 mg/kg, when calculated by the Behrens-Kärber method (Supplementary Figure 4 and Fig. 3a). In addition, 6 mice (3 male and 3 female animals) were challenged with TAT (1000 mg/kg). Two of these (1 male and 1 female) died on day 0. At full necropsy, we found that they had suffocated to death, indicating that intranasal administration caused serious stress. However, the 4 remaining mice showed no any abnormal behavior, and all survived to day 14, implying that TAT is not harmful per se (data not shown).
Based on this pilot study, a single-dose toxicity study was conducted (Fig. 3b). At 14 days after exposure, there was less body-weight gain in the TAT-TVTSP-treated groups than in the control group (Supplementary  Table 2). In the low-dose groups, 2 of the 5 female mice had several symptoms such as loss of appetite, no stool, crouching and lacrimation, while the 5 males displayed no abnormal behavior, and all survived. Of two females in this group, one showed symptoms, including alopecia, crouching, edema, and gasping behavior on day 4 and was dead by day 6. Another mouse also had the same symptoms on days 8-11 and died on day 12. In the mediumdose groups, none of the animals had abnormal behavior, and all survived to day 14. In the high-dose groups, 5 (2 male and 3 female) of the 10 mice showed appetite loss and gasping on day 1. Two of them (1 male and 1 female) had serious symptoms, including lacrimation, edema, and gasping; the female died on day 5, and the male died on day 13 (Supplementary Table 3). Next, blood samples obtained from each mouse were analyzed in detail for serum chemistry and hematology parameters. As shown in Table 1, the serum levels of measured factors were within normal ranges and showed no significant difference. Moreover, the hematological parameters did not change significantly, although the platelet (PLT) levels appeared to rise slightly in female mice, and white blood cell (WBC) and eosinophil (EOS) levels tended to fall with increasing dose (Table 2). Finally, histopathological analysis was conducted by microscopic observation of each tissue in detail. As shown in Fig. 3c, the lungs, as well as other observed organs, did not undergo any change in appearance and micro-morphology in any of the TAT-TVTSP groups.

Repeated-dose toxicity study
Based on the single-dose toxicology study and examination of in vivo airway inflammation, we investigated the toxicity of repeated intranasal administration of TAT-TVTSP (Fig. 4a). All the TAT-TVTSP-treated female groups showed a tendency to gain a little less weight than the control group, whereas all the male groups gained weight similar to the saline group (Supplementary Table 4). None of the animals showed any clinical manifestations, and all survived to day 28 (Supplementary  Table 5). Moreover, the weights of the liver, lung, spleen,  GGT (IU/l) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Data are expressed as means ± SD (n = 5 per each group). Differences were assessed by one-way ANOVA ALB albumin, ALP alanine aminotransferase, GGT gamma (γ)-glutamyl transferase, TP total protein, T-BIL total bilirubin kidney, stomach, and heart were not affected in any of the TAT-TVTSP-treated groups ( Fig. 4b and Supplementary  Table 6). However, the brain weight in the high-dose group was significantly reduced compared with that in the control group (Fig. 4b). Intranasal injection is frequently used for the delivery of drugs into brain tissues, and PLD1 was previously reported as an important mediator of neurogenesis in brain tissues such as the hippocampus and cortex 12,37 . Therefore, it might be anticipated that repeated intranasal injection of TAT-TVTSP would have some effect on brain development by suppressing PLD1. Finally, histological analysis was conducted by microscopic observation of each tissue, and no organs were affected in the TAT-TVTSP-treated groups (Fig. 4c).

Discussion
Asthma is a chronic lung disease characterized by airway inflammation, airflow obstruction, and airway hyperresponsiveness 1 . Allergic diseases, including asthma, are generally caused by individual genetic factors and are aggravated by environmental insults 1 . House dust mites are the major inhalant allergens of chronic allergic diseases, including childhood asthma 5,38 . HDMs induce airway inflammation by stimulating the production of Th2 pro-inflammatory cytokines 6,39 . We previously demonstrated that PLD1 is activated in T lymphocytes of Dermatophagoides farinae-specific allergic patients 40 . Furthermore, we showed that Der f 2 stimulated IL-13 production by activating PLD1 in human bronchial epithelial cells 7,8 , indicating that targeting PLD1 might PLD1 is an important enzyme responsible for the production of the lipid second messenger phosphatidic acid by catalyzing the hydrolysis of phospholipids 9 . Primary alcohols (for example, n-butanol) act as competitive nucleophiles to water, resulting in a transphosphatidylation to produce phosphatidylalcohols (for example, phosphatidylbutanol), instead of phosphatidic acid 18 . Based on this principle, n-butanol has been used as a PLD inhibitor in various studies 41 , but it has several problems, such as indirect targeting, intracellular complementary systems, low efficiency, and side effects. Consequently, new specific PLD inhibitors need to be developed 18 . Several small-molecule PLD inhibitors have been shown to be effective in various diseases, but they also have limitations in terms of selectivity, side effects, and toxicity. In these circumstances, we recently developed a small peptide consisting of five amino acids that binds to and inactivates PLD1 23 . However, the peptide may be quickly degraded by intracellular functions, including peptidase effects or autophagy 42 , and the delivery into cells or tissues can be limited by the lowpenetrating efficiency in crossing the cellular membrane.
These issues evoked the necessity for the appropriate topical targeting of PLD1 and application of efficient drug delivery systems. Therefore, we considered the utilization of a CPP as a delivery cargo and constructed TAT-TVTSP, a PLD1-specific inhibitory peptide fused with TAT. As expected, TAT-TVTSP was efficiently transferred into airway tissues and strongly suppressed Der f 2induced airway inflammation indices, indicating that it has powerful possibilities as an anti-asthma agent.
Although TAT was first regarded as a biologically inert vehicle, recent studies have reported that it can affect various cellular processes 43 . In HeLa cells, a TAT-derived peptide inhibited tumor necrosis factor-mediated signal transduction 44 , and a microarray study revealed altered gene expression patterns after the application of HIV-1 TAT 45 . Moreover, in human lung epithelial cells, TAT modulated inflammatory responses by inhibiting the production of multiple cytokines and induced apoptosis 46 . However, despite these reports, TAT appeared to only act as a delivery vehicle in the present study. It was not cytotoxic, and the previously reported Der f 2 effects, such as PLD activation and IL-13 upregulation in vitro, as well as bronchial inflammation stimulation in vivo, were faithfully reproduced (Figs. 1 and 2).
Toxicological analysis of a pro-drug is critical in evaluating its clinical potential. The general toxicity of drugs needs to be appraised through single-and repeated-dose The organs were weighed, and the values were expressed as the organ weight/body weight and presented as the means ± SD of 10 mice per group. Differences were assessed by one-way ANOVA. *P < 0.05 compared with the brain weight of the untreated control. c Fixed organ samples were dehydrated, paraffinized, and embedded. Sections of 4 µm were stained with H&E and observed under a microscope. Scale bar, 100 µm experiments because toxicants may induce acute and chronic effects depending on the dose and duration of drug exposure. Therefore, we investigated the safety of TAT-TVTSP as a therapeutic agent through single-and repeated-dose toxicity test in a mouse model. The median lethal dose (LD 50 ) of TAT-TVTSP was calculated to bẽ 450 mg/kg. In the single-dose toxicology study, TAT-TVTSP induced acute toxicity, including the loss of body weight and several clinical signs (crouching and lacrimation). Nevertheless, we observed no statistically significant effects on hematology, serum biochemistry, and histopathology. In the repeated-dose toxicology study, TAT-TVTSP also did not display strong toxicity. However, the brain weight was significantly reduced in the high-dose group, suggesting that repeated intranasal injection of TAT-TVTSP might cause the suppression of brain functions via unexpected targeting. Further studies of the pharmacokinetics of TAT-TVTSP are required to address its unexpected brain targeting. Nonetheless, we may conclude that TAT-TVTSP as a novel and effective PLD1 inhibitor suppresses HDM-induced airway inflammation in vitro as well as in vivo. Furthermore, topical application of TAT-TVTSP as a novel inhaled antiasthmatic drug is expected to reduce the adverse effects that can be caused by the systemic inhibition of PLD.