Identification of Novel Bisamide-Decorated Benzotriazole Derivatives as Anti-Phytopathogenic Virus Agents: Bioactivity Evaluation and Computational Simulation.

As a notorious phytopathogenic virus, the tobacco mosaic virus (TMV) severely reduced the quality of crops worldwide and caused critical constraints on agricultural production. The development of novel virucides is a persuasive strategy to address this predicament. Herein, a series of novel bisamide-decorated benzotriazole derivatives were elaborately prepared and screened. Biological tests implied that the optimized compound 7d possessed the most brilliant antiviral inactive profile (EC50 = 157.6 µg/mL) and apparently surpassed that of commercial ribavirin (EC50 = 442.1 µg/mL) 2.8-fold. The preliminary antiviral mechanism was elaborately investigated via transmission electron microscopy, microscale thermophoresis (MST) determination, RT-qPCR, and Western blot analysis. The results showed that compound 7d blocked the assembly of TMV by binding with coat protein (Kd = 0.7 µM) and suppressed TMV coat protein gene expression and biosynthesis process. Computational simulations indicated that 7d displayed strong H-bonds and pi interactions with TMV coat protein, affording a lower binding energy (ΔGbind = -17.8 kcal/mol) compared with Ribavirin (ΔGbind = -10.7 kcal/mol). Overall, current results present a valuable perception of bisamide decorated benzotriazole derivatives with appreciably virustatic competence and should be profoundly developed as virucidal candidates in agrochemical.

Identification of natural Rutaecarpine as a potent tobacco mosaic virus (TMV) helicase candidate for managing intractable plant viral diseases.

BACKGROUND: Naturally occurring alkaloids are particularly suitable for use as pesticide precursors and further modifications due to their cost-effectiveness, unique mechanism of action, tolerable degradation, and environmental friendliness. The famous tobacco mosaic virus (TMV) is a persistent plant pathogenic virus that can parasitize many plants and severely reduce crop production. To treat TMV disease, TMV helicase acts as a crucial target by hydrolyzing adenosine triphosphate (ATP) to provide energy for double-stranded RNA unwinding. RESULTS: To seek novel framework alkaloid leads targeting TMV helicase, this work successfully established an efficient screening platform for TMV helicase inhibitors based on natural alkaloids. In vivo activity screening, enzyme activity detection, and binding assays showed that Rutaecarpine from Evodia rutaecarpa (Juss.) Benth exhibited excellent TMV helicase inhibitory properties [dissociation constant (Kd ) = 1.1 µm, half maximal inhibitory concentration (IC50 ) = 227.24 µm] and excellent anti-TMV ability. Molecular docking and dynamic simulations depicted that Rutaecarpine could stably bind in active pockets of helicase with low binding energy (ΔGbind = -17.8 kcal/mol) driven by hydrogen bonding and hydrophobic interactions. CONCLUSION: Given Rutaecarpine's laudable bioactivity and structural modifiability, it can serve as a privileged building block for further pesticide discovery.

Novel spiro[chromanone-2,4'-piperidine]-4-one derivatives as potential inhibitors of fatty acid synthesis in pathogens: Design, synthesis, and biological evaluation.

Bacterial survival depends on membrane lipid homeostasis that enables to regulate lipid composition to adapt and optimize their growth in diverse environments. Therefore, the development of inhibitors that interfere with the bacterial fatty acid synthesis process is considered to be a promising tactic. In this study, 58 novel spirochromanone derivatives were prepared and their structure-activity relationship (SAR) was investigated. The bioassay results showed that all most of the compounds showed excellent biological activities, exampled by compounds B14, C1, B15, and B13, which had outstanding inhibitory activities toward various pathogenic bacteria with EC50 values of 0.78 µg/mL ∼3.48 µg/mL. Preliminary antibacterial behavior was studied by a series of biochemical assays including, but not limited to, fluorescence imaging patterns, GC-MS analysis, TEM images, and fluorescence titration experiments. Notably, compound B14 decreased the lipid content of the cell membrane, and increased cell membrane permeability, thereby destroying the integrity of the bacterial cell membrane. Further qRT-PCR results indicated that compound B14 interfered with the mRNA expression levels of fatty acid synthesis process-related genes including ACC, ACP, and Fab family genes. Herein, we highlight the promising bactericidal skeleton based on the spiro[chromanone-2,4'-piperidine]-4-one as a potential inhibitor of fatty acid synthesis.

[Research progress in medicines on protease-activated receptor 2].

Protease-activated receptor 2(PAR2) is a member of protease-activated receptors(PARs). PAR2 distributed in tissues and cells(such as skin, airway epithelial cell, pancreas, etc.) has a broad biological effects, and is involved in pathogenesis of many diseases, such as mechanical pain, asthma, pain of pancreatic cancer, inflammation, pruritus, etc. Intervention of PAR2 will help us to identify the role of PAR2 in the mechanisms of diseases and in the development of new drugs. This article concentrates on the research progress of agonist, antagonist, and pepducin on PAR2.

Automated On-Line Microdialysis Sampling Coupled with HPLC for Synchronous Determination of Puerarin in Subcutaneous Tissue and Plasma Following Topical Administration.

BACKGROUND: Studies on transdermal administration have shown that puerarin can permeate rat skin rapidly with long-term drug delivery, but there are no reports demonstrating whether topical use of puerarin can provide a steady plasma concentration to produce therapeutic effects. The aim of the study is to evaluate the percutaneous penetration and plasma concentration of puerarin after transdermal administration in experimental rats. METHODS: The skin and plasma concentration of puerarin was quantified by microdialysis, and the recovery was determined by retrodialysis. Puerarin microdialysate concentrations were measured by on-line high-performance liquid chromatography (HPLC). Puerarin release from gels was determined by analysis of the amount of remaining drug after dermal application to hairless skin. RESULTS: The average recoveries of puerarin in the skin and plasma over an 8-hour period were 31.49% and 15.5%. Puerarin was rapidly absorbed with transdermal administration, with the C(max) values of 30.64 µg/mL and 3.53 µg/mL, the AUC0 t-values of 11.60 and 1.48 µg/mL per minute, for skin and plasma, respectively. CONCLUSIONS: The results indicate that the automated on-line microdialysis technique can be used to detect the skin and plasma pharmacokinetics of puerarin and that the use of skin gel can provide an effective means of puerarin administration.

Curcumin inhibits proliferation of interleukin-22-treated HaCaT cells.

The interleukin (IL)-23/IL-17A/IL-22 cytokine axis plays a critical role in the pathogenesis of psoriasis, wherein IL-22 effects on epidermal alternations by inhibiting differentiation and inducing keratinocyte proliferation. In this study, we investigated effects of curcumin on IL-22-induced proliferation in a human keratinocyte cell line (HaCaT) in vitro experiment. The HaCaT cells growth was measured by a Cell Counting Kit-8. The cyclin D1 and cyclin E was detected by real-time RT-PCR and weatern blot. The STAT3 and phosphorylation of STAT3 was tested by weatern blot also. Our results show that curcumin exhibited a significant anti-proliferation effect on HaCaT cells, even in the presence of IL-22. Since STAT3 is crucial for IL-22 signal transduction, we examined the level of phosphorylation of STAT3 in all of the experimental groups. As expected, curcumin inhibited IL-22 induced phosphorylation of STAT3; furthermore, curcumin down regulated cyclin D1 and cyclin E. We can reach a conclusion that curcumin can suppress the proliferation of keratinocytes even with IL-22 treatment. Therefore, we have confidence in future curcumin research about psoriasis treatment.

Tryptase and protease-activated receptor-2 stimulate scratching behavior in a murine model of ovalbumin-induced atopic-like dermatitis.

The aim of the current study was to investigate the involvement of tryptase and protease-activated receptor-2 (PAR2) in the pathogenesis of itch using a recently developed murine model of atopic dermatitis (AD) elicited by epicutaneous sensitization with ovalbumin (OVA). We also examined whether tacrolimus exerts an antipruritic effect. Epicutaneous sensitization of BALB/c mice with OVA led to a significant increase in the number of scratches. Notably, PAR2 mRNA and protein levels as well as cutaneous levels of tryptase were significantly enhanced in epicutaneously sensitized mice. Pretreatment with the protease inhibitor, leupeptin, PAR2 antibody, and tacrolimus significantly reduced the number of degranulated mast cells and tryptase content, and consequently alleviated scratching behavior. Cetirizine (10mg/kg) exerted a significant inhibitory effect on the scratching behavior of mice, but did not affect the number of degranulated mast cells and induction of tryptase. Our results collectively suggest that tryptase and PAR2 are involved in OVA allergy-induced scratching behavior.

[Mechanism of psoriasis generation in animal models].

Psoriasis is a chronic inflammatory disease related to genome-wide and surroundings, it is important to develop a suitable animal model to research psoriasis pathogenesis and evolve pharmacotherapeutics. With the development of transgenetic technology in the past few years, psoriasis virulence gene animal model become a hotspot. Research of animal model of human psoriasis genes is reviewed in the paper.

[The preparation of paeonol transdermal delivery systems based on the microemulsion-based gels and its pharmacokinetics characters].

Investigation of the pharmacokinetics of paeonol microemulsion, microemulsion-based gels and marketed paeonol ointments by the skin-blood synchronous microdialysis coupled with LC/MS is reported in this study. The microdialysis systems were established by linear probes and concentric circles probes. In vivo recovery of paeonol in skin is (69.7 +/- 4.8) % and in blood is (51.6 +/- 7.2)%. The paeonol microemulsion, microemulsion-based gels and marketed paeonol ointments were administered to rats. PBS (pH 7.4) served as perfused solution. The perfusion rate was 5 microL x mL(-1) and the microdialysis samples were collected every 20 min intervals. The paeonol concentration in perfused solution was determined by LC/MS. The results showed that paeonol microemulsion and microemulsion-based gels significantly raised the drug concentrations in skin more than that of paeonol ointments. The paeonol microemulsion-based gels has similar bioavailability as the paeonol ointments in blood, but its blood drug concentrations were steadier. The paeonol microemulsion-based gels may be developed into a new preparation for dermis eczema. The skin-blood synchronous microdialysis technique proved to be a new method for the pharmacokinetics study of transdermal delivery systems.

A modified LC-MS/MS method for determination of tetramethylpyrazine in microdialysis samples and calibration of home-made linear probes.

Tetramethylpyrazine (TMP) is one of the most important active ingredients of a Chinese herb Ligusticum wallichii Franchat, which is widely used for the treatment of cardiovascular diseases. Several factors may affect TMP exposure after topical administration, resulting in large variability and demanding further elucidation of drug distribution. This paper describes a new efficient reliable LC-MS/MS assay for the determination of TMP in dermal microdialysate, where TMP was separated on an Agilent C(18) column (3.5 µm, 100 mm × 2.1 mm i.d.) using a mixture of methanol, water and acetic acid (50:50:0.6, v/v/v) at a flow-rate of 0.3 mL/min. The retention time was 1.89 min for TMP and 1.17 min for the internal standard (caffeine). Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for TMP was not altered through the implant lifetime. The validation and sample analysis results showed that the method is precise, accurate and well suited to support dermal microdialysis experiments.

Transient receptor potential vanilloid-1 participates in the inhibitory effect of ginsenoside Rg1 on capsaicin-induced interleukin-8 and prostaglandin E2 production in HaCaT cells.

OBJECTIVES: Ginsenoside Rg1 (GRg1), one of the major active constituents of Panax notoginseng, has shown anti-inflammatory and antinocioceptic activity, but its role in keratinocytes needs further study. We have examined the inhibitory effect of GRg1 on transient receptor potential vanilloid-1 (TRPV1) activation in keratinocyte HaCaT cells and explored its involved mechanism. METHODS: HEK 293T cells over-expressing exogenous TRPV1 were constructed and named HEK 293T-TRPV1 cells. The effects of GRg1 on production of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2) ), calcium influx, the expression of cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) transcriptional activity in HEK 293T-TRPV1 and HaCaT cells were examined by ELISA, Fluo 3-AM fluorescence probe, Western blot and Dual-Luciferase Reporter Assay, respectively. KEY FINDINGS: The results showed that GRg1 blocked intracellular calcium by both capsaicin and proton activation in a TRPV1-dependent manner. Furthermore, GRg1 inhibited the expression of COX-2 and NF-κB transcriptional activity induced by capsaicin in keratinocytes. The inhibitory effect of GRg1 was similar to capsazepine, an antagonist of TRPV1. More importantly, GRg1 dose-dependently inhibited capsaicin-induced PGE(2) and IL-8 secretion in HaCaT cells and HEK 293T-TRPV1 cells. CONCLUSIONS: These data showed that GRg1 could inhibit TRPV1 mediated responses in HaCaT cells, indicating that GRg1 acted as a TRPV1 antagonist.

Acid inhibition effect of ilaprazole on Helicobacter pylori-negative healthy volunteers: an open randomized cross-over study.

OBJECTIVE: The aim of this study was to evaluate the effects and safety of different doses of ilaprazole on healthy volunteers without a Helicobacter pylori infection. METHODS: A total of 12 healthy Chinese volunteers were enrolled and divided into four groups randomly, with a 5-day treatment of oral ilaprazole 5mg, 10mg and 20mg or omeprazole 20mg, respectively. After an interval of a 14-day washout phase, each was switched to another dose group and eventually completed all four regimens. The percentage time of intragastric pH>4 was the major index. The polymorphisms of the metabolic enzyme CYP2C19 in these volunteers were also detected. RESULTS: The percentage time of intragastric pH>4 in the ilaprazole 5, 10 and 20mg groups were 80.4%, 88.1% and 91.0%, respectively, during the first 24h, compared to that of the 20mg omeprazole group (76.6%, P>0.05). Ilaprazole 20mg provided a significant higher mean 24-h pH than that of the same dose of omeprazole both on Day 1 (7.78 vs 6.67, P<0.01) and Day 5 (7.95 vs 7.44, P<0.05). No CYP2C19-dependent difference or obvious adverse effect were found in any ilaprazole groups. CONCLUSION: Low dose ilaprazole offers a gastric acid inhibition comparable to a routine dose of omeprazole, and further investigations in patients with acid-associated diseases are needed.

Pharmacokinetics of aconitine in rat skin after oral and transdermal gel administrations.

The purpose of this study was to evaluate percutaneous penetration and arrhythmogenic effects of aconitine after transdermal administration, compared with the oral route. Skin penetration of aconitine was tested by a microdialysis technique in rats and in vivo recovery was determined by retrodialysis. After oral and transdermal administration of aconitine, dialysate was sampled at 20 min intervals until the end of the experiment for the determination of concentration of aconitine in skin. Blood samples were collected and analyzed using a validated HPLC-MS/MS method. In addition, we concurrently recorded the electrocardiogram (ECG). The in vivo recovery of aconitine in the skin was calculated to be 39.59%. The C(max) values for aconitine absorbed into the skin after oral and transdermal administration were 1.51 ± 0.53 and 2723.8 ± 848.8 ng/mL, respectively, and within the plasma, 215.86 ± 79.29 and 20.92 ± 3.15 ng/mL. The C(max) value for the plasma concentration of aconitine after oral administration was approximately 10 times higher than with the transdermal route. For oral administration, the ECG revealed various types of arrhythmias at a period of T(max) , which is normal in transdermal gel administration. These results indicate that transdermal aconitine gel is a safe formulation that can deliver the drug in sufficient amounts and safe concentrations to produce therapeutic action in rats.

[Pharmacokinetics of scopolamine hydrobromide oral disintegrative microencapsule tablets in Beagle dogs determined with LC-MS/MS].

The study aims to elucidate the characteristics of pharmacokinetics of scopolamine hydrobromide oral disintegrative microencapsule tablets in healthy Beagle dogs. Chromatographic separation was performed on a C18 column (100 mm x 3.0 mm, 3.5 microm) with methanol - 2 mmol x L(-1) ammonium formate (25 : 75) as the mobile phase. A trip-quadrupole tandem mass spectrum with the electrospray ionization (ESI) source was applied and positive ion multiple reaction monitoring mode was operated. Six Beagle dogs were randomly devided into two groups. They received oral single dose of scopolamine hydrobromide oral disintegrative microencapsule tablets 0.6 mg (test tablet) or scopolamine hydrobromide normal tablets (reference tablet). Plasma samples were collected at designed time. Plasma concentration of scopolamine hydrobromide was determined by LC-MS/MS and pharmacokinetic parameters were calculated. The pharmacokinetic parameters of test tablet vs reference tablet were as follows: C(max): (8.16 +/- 0.67) ng x mL(-1) vs (3.54 +/- 0.64) ng x mL(-1); t1/2: (2.83 +/- 0.45) h vs (3.85 +/- 0.82) h; t(max): (1.25 +/- 0.27) h vs (0.42 +/- 0.09) h; AUC(0-12h): (25.06 +/- 3.75) h x ng x mL(-1) vs (9.59 +/- 1.02) h x ng x mL(-1); AUC(0-infinity): (26.30 +/- 3.92) h x ng x mL(-1) vs (10.80 +/- 1.45) h x ng x mL(-1); MRT(0-12h): (3.38 +/- 0.34) h vs (3.86 +/- 0.26) h; MRT(0-infinity): (3.98 +/- 0.63) h vs (5.37 +/- 1.00) h. The absorption rate and AUC of test tablet is different from that of reference tablet. The bioavailability of test tablet is better than those of reference tablet.

A novel spray-dried nanoparticles-in-microparticles system for formulating scopolamine hydrobromide into orally disintegrating tablets.

Scopolamine hydrobromide (SH)-loaded microparticles were prepared from a colloidal fluid containing ionotropic-gelated chitosan nanoparticles using a spray-drying method. The spray-dried microparticles were then formulated into orally disintegrating tablets (ODTs) using a wet granulation tablet formation process. A drug entrapment efficiency of about 90% (w/w) and loading capacity of 20% (w/w) were achieved for the microparticles, which ranged from 2 µm to 8 µm in diameter. Results of disintegration tests showed that the formulated ODTs could be completely dissolved within 45 seconds. Drug dissolution profiles suggested that SH is released more slowly from tablets made using the microencapsulation process compared with tablets containing SH that is free or in the form of nanoparticles. The time it took for 90% of the drug to be released increased significantly from 3 minutes for conventional ODTs to 90 minutes for ODTs with crosslinked microparticles. Compared with ODTs made with noncrosslinked microparticles, it was thus possible to achieve an even lower drug release rate using tablets with appropriate chitosan crosslinking. Results obtained indicate that the development of new ODTs designed with crosslinked microparticles might be a rational way to overcome the unwanted taste of conventional ODTs and the side effects related to SH's intrinsic characteristics.

[Preparation of scopolamine hydrobromide nanoparticles-in-microsphere system].

This study is to prepare scopolamine hydrobromide nanoparticles-in-microsphere system (SH-NiMS) and evaluate its drug release characteristics in vitro. SH nanoparticles were prepared by ionic crosslinking method with tripolyphosphate (TPP) as crosslinker and chitosan as carrier. Orthogonal design was used to optimize the formulation of SH nanoparticles, which took the property of encapsulation efficiency and drug loading as evaluation parameters. With HPMC as carrier, adjusted the parameters of spray drying technique and sprayed the SH nanoparticles in microspheres encaposulated by HPMC was formed and which is called nanoparticles-in-microsphere system (NiMS). SH-NiMS appearances were observed by SEM, structure was obsearved by FT-IR and the release characteristics in vitro were evaluated. The optimized formulation of SH nanoparticles was TPP/CS 1:3 (w/w), HPMC 0.3%, SH 0.2%. The solution peristaltic speed of the spray drying technique was adjusted to 15%, and the temperature of inlet was 110 degrees C. The encapsulation product yeild, drug loading and particle sizes of SH-NiMS were 94.2%, 20.4%, and 1256.5 nm, respectively. The appearances and the structure of SH-NiMS were good. The preparation method of SH-NiMS is stable and reliable to use, which provide a new way to develop new dosage form.

Anchoring of ulex europaeus agglutinin to chitosan nanoparticles-in-microparticles and their in vitro binding activity to bovine submaxillary gland mucin.

Focused on the natural biodegradable material of chitosan (CS), this investigation concerned its spray-dried nanoparticles-in-microparticles (NiMPs) modified with ulex europaeus agglutinin (UEA). Chitosan nanoparticles were obtained by ionotropic gelation process with pentasodium tripolyphosphate as gelatinizer. Then UEA lectin was bound onto the CS nanoparticles activated by glutaraldehyde. The conjugated spherical UEA-CS-NiMPs, prepared by spray drying method, exhibited 12-85% coupling efficiency of UEA depending upon the amount of activator glutaraldehyde. And the UEA-grafted particles showed additional higher binding tendency with bovine submaxillary gland mucin as compared to the plain chitosan microparticles. Furthermore, the activity and intrinsic fucose-specificity of UEA were still maintained after the covalent modification. It is thus evident that the UEA anchored CS-NiMPs might be used as a potential drug delivery system targeted to the specific regions of gastrointestinal tract.

Mannose 6-phosphate-modified bovine serum albumin nanoparticles for controlled and targeted delivery of sodium ferulate for treatment of hepatic fibrosis.

OBJECTIVES: The aim was to prepare neoglycoprotein-based nanoparticles for targeted drug delivery to hepatic stellate cells, and to evaluate their characteristics in vitro and in vivo. METHODS: The neoglycoprotein of bovine serum albumin modified with mannose 6-phosphate was synthesised from mannose, and used as wall material to nanoencapsulate the model natural antifibrotic substance sodium ferulate using a desolvation method. The morphology, drug loading capacity, release in vitro and biodistribution in vivo of the nanoparticles were studied. Selectivity of the nanoparticles for hepatic stellate cells was evaluated by immunohistochemical analysis of fibrotic rat liver sections. KEY FINDINGS: The spherical nanoparticles were negatively charged with zeta potential ranging from -2.73 to -35.85 mV, and sizes between 100 and 200 nm with a narrow size distribution. Drug entrapment efficiency of about 90% (w/w) and loading capacity of 20% (w/w) could be achieved. in vitro, the nanoparticles showed an initial rapid continuous release followed by a slower sustained release. After intravenous injection into mice, the nanoparticles showed a slower elimination rate and a much higher drug concentration in liver compared with the sodium ferrate solution, and less distribution to the kidneys and other tissues. Immunohistochemistry indicated that the neoglycoprotein-based nanoparticles were taken up specifically by hepatic stellate cells. CONCLUSIONS: The nanoparticles may be an efficient drug carrier targeting hepatic stellate cells.

Induction of leukotriene B(4) and prostaglandin E(2) release from keratinocytes by protease-activated receptor-2-activating peptide in ICR mice.

Protease-activated receptor-2 (PAR2) has been shown to play a key role in the pathophysiology of itch. However, the precise mechanism of PAR2-mediated itch remains largely unknown. In the present study, we investigated the effects of several agents on the scratching behavior induced by PAR2-activating peptide (SLIGRL-NH2). Pretreatment of experimental animals with tacrolimus or the 5-lipoxygenase inhibitor zileuton significantly reduced SLIGRL-NH2-induced scratching behavior, whereas histamine H(1) receptor antagonist cetirizine or the cyclooxygenase inhibitor indomethacin had little effect. Furthermore, intradermal injection of SLIGRL-NH2 increased cutaneous levels of LTB(4) and PGE(2). In vitro, SLIGRL-NH2 treatment enhanced LTB(4) and PGE(2) release from primary keratinocytes in a concentration-dependent manner. Preincubation of keratinocytes with zileuton resulted in a significant decrease of LTB(4) release and treatment of indomethacin led to a significant decrease of PGE(2) in response to SLIGRL-NH2 stimulation. In addition, SLIGRL-NH2-induced secretion of LTB(4) and PGE(2) was significantly inhibited by tacrolimus, whereas cetirizine had no effect. These results indicate that SLIGRL-NH2 stimulates LTB(4) and PGE(2) release from mouse keratinocytes and that enhancement of LTB(4) and PGE(2) secretion contributes to SLIGRL-NH2-induced scratching behavior in ICR mice.

Participation of proteinase-activated receptor-2 in passive cutaneous anaphylaxis-induced scratching behavior and the inhibitory effect of tacrolimus.

Proteinase-activated receptor-2 (PAR2) may be an important regulator of skin mast cell function during cutaneous inflammation and hypersensitivity. However, little is known of the role of PAR2 in allergic pruritus, because mast cells, which are thought to be responsible for this symptom, can release a number of different pruritogens. In the present study, we investigated the effects of several agents on passive cutaneous anaphylaxis-induced scratching behavior in ICR mice. As a result, cetirizine and ketanserin produced dose-dependent inhibition of scratching behavior induced by passive cutaneous anaphylaxis. Combined cetirizine with ketanserin exhibited significant inhibitory effects for the number of passive cutaneous anaphylaxis-induced scratching behavior. Pretreatment of the experimental animals with PAR2-neutralizing antibody and protease inhibitor leupeptin significantly inhibited passive cutaneous anaphylaxis-induced scratching behavior. Furthermore, we found that topical application of tacrolimus significantly reduced the number of scratching behavior induced by passive cutaneous anaphylaxis in a dose-dependent manner. Combined cetirizine with tacrolimus also exhibited significant inhibitory effects for the number of passive cutaneous anaphylaxis-induced scratching behavior. Tacrolimus in doses of 3% and 10% significantly inhibited tryptase-induced scratching behavior. These results suggest that PAR2 may be involved in passive cutaneous anaphylaxis-induced scratching behavior and tacrolimus produces an anti-allergic pruritus effect in ICR mice.