European Journal of Pharmacology 765 (2015) 402–405

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European Journal of Pharmacology

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Immunopharmacology and inflammation

Olopatadine hydrochloride suppresses hot flashes induced by topicaltreatment with tacrolimus ointment in rats

Kyosuke Satake a,n, Junichi Ikeda b, Tadafumi Tamura a, Toru Amano a, Katsuya Kobayashi a

a Immunology & Allergy Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japanb Nephrology Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan

a r t i c l e i n f o

Article history:Received 13 March 2015Received in revised form2 September 2015Accepted 3 September 2015Available online 8 September 2015

Keywords:Tacrolimus ointmentOlopatadineAtopic dermatitisHot flashesVascular permeabilityNGF

Chemical compounds studied in this article:Olopatadine hydrochloride (PubChem CID:5282402)Cetirizine hydrochloride (PubChem CID:55182)Tacrolimus hydrate (PubChem CID: 445643)

x.doi.org/10.1016/j.ejphar.2015.09.00899/& 2015 Elsevier B.V. All rights reserved.

esponding author. Fax: þ81 55 986 7430.ail address: kyosuke.satake@kyowa-kirin.co.jp

a b s t r a c t

Tacrolimus ointment is prescribed for patients with atopic dermatitis, although it is known to causetransient burning sensations and hot flashes in the applied skin. The aim of this study was to evaluate theeffects of olopatadine hydrochloride (olopatadine), an antiallergic agent with a histamine H1 receptor(H1R) antagonistic activity, on the incidence of hot flashes induced by topical treatment with tacrolimusointment in rats. Consequently, the skin temperature was increased by the topical application of ta-crolimus ointment in rats, and the rise in skin temperature was inhibited by pretreatment with olopa-tadine in a dose-dependent manner. Inhibitory effect of olopatadine on tacrolimus-induced skin tem-perature elevation was significantly more potent than that of cetirizine hydrochloride, other antiallergicagent with H1R antagonistic activity, at doses in which both agents exhibit comparable H1R antagonisticactivity in rats. These results suggest that H1R antagonistic activity-independent mechanism contributeto the inhibitory effect of olopatadine on tacrolimus-induced skin temperature elevation. Olopatadinealso significantly inhibited increases in vascular permeability and nerve growth factor production in theskin induced by topical tacrolimus treatment. Thus, the onset of hot flashes in rats is quantitativelydetermined by measuring the skin temperature and olopatadine attenuates hot flashes induced by to-pical tacrolimus ointment in rats, suggesting that the combination application with olopatadine andtacrolimus ointment is useful for improving medication adherence with tacrolimus ointment treatmentin patients with atopic dermatitis.

& 2015 Elsevier B.V. All rights reserved.

1. Introduction

Atopic dermatitis is a chronic relapsing eczematous skin dis-ease characterized by pruritus and inflammation and accompaniedby signs of cutaneous physiological dysfunction (dry, barrier-dis-rupted skin). Tacrolimus ointment is non-steroid topical calci-neurin inhibitor and is known to inhibit activated T cell-dependentproinflammatory cytokine production (Williams, 2002). Therefore,tacrolimus ointment is frequently employed when the effects ofexisting treatments, such as topical corticosteroids are insufficient,or the physician hesitates to administer these drugs due to theirtopical side-effects (Saeki et al., 2009). However, tacrolimus oftencauses symptoms of stimulation, such as a transient burningsensation and hot flashes in the applied regions (Reitamo et al.,2000), which is the most common reason for premature studydiscontinuation (Reitamo et al., 2007). Therefore, achieving pal-liation of these symptoms is important for preventing the need to

(K. Satake).

withdraw therapy with tacrolimus ointment.Olopatadine hydrochloride (olopatadine, Kyowa Hakko Kirin

Co., Ltd., Japan) is an antiallergic agent with a histamine H1 re-ceptor (H1R) antagonistic action that is applied to treat the signsand symptoms of allergic rhinitis, chronic urticaria and eczemadermatitis (Ohmori et al., 2002). In the present study, we in-vestigated the effects of olopatadine treatment on the incidence ofhot flashes induced by the topical application of tacrolimus oint-ment. First, we demonstrated that the elevation of temperatureinduced by the administration of topical tacrolimus ointment tothe rat abdominal skin can be measured using a thermometer.Next, olopatadine was administered orally to the rats, and theeffects on the temperature changes induced by tacrolimus oint-ment were examined. In general, flare and heat sensations are theresult of an increased blood flow induced by vasodilation, whichsubsequently enhances vascular permeability. It has been reportedthat nerve growth factor (NGF) stimulates plasma extravasation inthe rat skin (Otten et al., 1984). Therefore, we investigated theeffects of olopatadine on the enhanced vascular permeability andNGF production induced by topical treatment with tacrolimusointment.

Fig. 1. Temperature changes in the abdominal skin induced by tacrolimus ointmentin rat. Tacrolimus ointment (0.03% and 0.1%) was applied to the abdominal rat skinone (�0.5 h), two (�0.5, �24 h) or three (�0.5, �24, �48 h) consecutive times.The temperature changes in the abdominal skin were measured before and 0.5 hafter the last treatment with tacrolimus ointment. Each column represents themeanþS.E.M. of six to 18 rats. #Po0.05, ##Po0.01, ###Po0.001 compared to thewhite petrolatum group according to Dunnett test.

K. Satake et al. / European Journal of Pharmacology 765 (2015) 402–405 403

2. Materials and methods

2.1. Experimental animals

Male 6-week-old Hairless Wister Yagi (HWY) rats were pur-chased from Japan SLC (Shizuoka, Japan). The rats were kept in aspecific pathogen-free animal facility with a maintained tem-perature of 19–25 °C, humidity of 30–70% and 12-h day/night cycleand given access to food and water ad libitum. The experimentswere conducted in accordance with the Guiding Principles for theCare and Use of Laboratory Animals, and the experimental pro-tocol used in this study was approved by the Committee for Ani-mal Experiments at Kyowa Hakko Kirin Co., Ltd. (Shizuoka, Japan).

2.2. Materials

Olopatadine was synthesized at Yokkaichi Plant, Kyowa YukaCo., Ltd. (Mie, Japan). Cetirizine hydrochloride (cetirizine) waspurchased from Sigma-Aldrich. Olopatadine and cetirizine weredissolved in distilled water. Tacrolimus ointment (0.03% and 0.1%,Protopics) was purchased from Astellas Pharmaceuticals (Tokyo,Japan).

2.3. Drug treatment

Olopatadine at doses of 0.01 and 0.1 mg/kg or cetirizine at adose of 10 mg/kg was administered orally to the rats once daily forfour consecutive days. Thirty min after the final administration ofolopatadine or cetirizine, tacrolimus ointment (200 mg/body) wasapplied to the tape-stripped abdominal skin of rats.

2.4. Measurement of skin temperature

The skin temperature of the rats was measured using a Ther-moTracer (TH5100, NEC avio) before and 30 min after the appli-cation of tacrolimus ointment. The temperature change was de-fined as the difference between the skin temperature before andafter tacrolimus application.

2.5. Measurement of vascular permeability in the skin

One hour after the topical application of tacrolimus ointment tothe abdominal skin, the anesthetized rats were intravenously in-jected with 1.0 w/v% Evans blue solution at a dose of 25 mg/kg.Thirty min later, the animals were killed, and the tacrolimus-treated skin was removed. Evans blue dye that had leaked into thetissue was extracted with formamide at 45 °C, and its absorbanceat 625 nm was determined with a spectrophotometer (THERMO-maxTM; Molecular Devices, USA). The data were analyzed as perthe protocol of each organization.

2.6. Measurement of NGF in the skin

One hour after the topical application of tacrolimus ointment,the tacrolimus-treated skin was collected. The skin samples wereminced and homogenized in phosphate-buffered saline containinga protease inhibitor (CompleteTM; Roche Diagnostics) with aPolytron tissue homogenizer. The precipitate was removed viacentrifugation, and the supernatant was collected. The NGF (Pro-mega) and protein (Thermo Scientific) concentrations were ana-lyzed with an assay kit according to the manufacturer's protocol.

2.7. Statistical analysis

The data are presented as the meanþS.E.M. Student's t-test orthe Aspin–Welch test following the F test were used for the

analysis of differences between two groups. Multiple comparisonsamong treatment groups were made using a one-way analysis ofvariance followed by Tukey test, Dunnett test or Williams test, orthe Kruskal–Wallis followed by Steel–Dwass test. In all tests, a Pvalue of o0.05 was considered to be statistically significant.

3. Results

3.1. Temperature changes in the skin induced by tacrolimus ointment

First, we investigated whether hot flashes are induced by to-pical treatment with tacrolimus ointment in rats. In order to mi-mic the skin condition of patients with atopic dermatitis, the skinbarrier function of the abdominal skin in the rats was disrupted bytape stripping prior to the application of tacrolimus ointment. Theincidence of hot flashes in the rats was evaluated by measuring thetemperature at sites of treatment with tacrolimus ointment. Asshown in Fig. 1, the skin temperature was significantly increasedby a single application of tacrolimus ointment (0.03% and 0.1%),compared with single treatment with white petrolatum. No skintemperature changes were observed after the third treatment withtacrolimus ointment, indicating that the elevation of the skintemperature induced by tacrolimus treatment was transient.

3.2. Effects of olopatadine and cetirizine on the temperature changesin the skin induced by tacrolimus ointment

In order to investigate whether olopatadine has the potential toinhibit the elevation of skin temperature induced by topicaltreatment with tacrolimus ointment in rats, olopatadine was ad-ministered orally once daily for four consecutive days prior to ta-crolimus ointment application. As shown in Fig. 2A, althougholopatadine had no significant effect on skin temperature in thenormal rats, the skin temperature elevation induced by topicaltreatment with tacrolimus ointment was significantly suppressedin the olopatadine-treated rats compared with that observed inthe olopatadine-untreated rats. Olopatadine at doses of 0.01 and0.1 mg/kg, which inhibited histamine-induced plasma leakage inrats by 63.4% and over 75% respectively (Tamura, 2011), sup-pressed the increase in the skin temperature induced by topicaltacrolimus ointment dose dependently (Fig. 2B). Cetirizine at10 mg/kg, which inhibited histamine-induced plasma leakage in

Fig. 2. Effects of olopatadine and cetirizine on the temperature changes in theabdominal skin induced by tacrolimus ointment. (A) Olopatadine 0.1 mg/kg,(B) olopatadine 0.01 mg/kg or 0.1 mg/kg and (C) olopatadine 0.1 mg/kg or cetirizine10 mg/kg were administered orally to rats for four consecutive days. Tacrolimusointment was applied to the abdominal skin 0.5 h after the final olopatadine orcetirizine treatment. The temperature changes in the abdominal skin were mea-sured before and 0.5 h after treatment with tacrolimus ointment. Olo: olopatadine.Each column represents the meanþS.E.M. of six rats. ##Po0.01, ###Po0.001compared to the white petrolatum-treated group according to Tukey test.***Po0.001 compared to the tacrolimus ointment-treated group according to Tukeytest (A). ##Po0.01 compared to the white petrolatum-treated group according toStudent's t-test. ***Po0.001 compared to the tacrolimus ointment-treated groupaccording to Williams test (B). *Po0.05 compared to the cetirizine-treated groupaccording to Student’s t-test (C).

Fig. 3. Effects of olopatadine on the tacrolimus ointment-induced increase invascular permeability in the abdominal rat skin. Olopatadine (0.1 mg/kg) was ad-ministered orally to rats for four consecutive times. Tacrolimus ointment 0.1% wasapplied 0.5 h after the final olopatadine treatment. One hour after treatment withtacrolimus ointment, the anesthetized rats were intravenously injected with Evansblue dye. Thirty min after injection, the tacrolimus-applied skin was removed, andthe Evans blue dye was extracted and quantified photometrically. Each column andvertical bar represents the meanþS.E.M. of six rats. ##Po0.01 compared to thewhite petrolatum-treated group according to Student's t-test. *Po0.05 comparedto the tacrolimus ointment-treated group according to Aspin–Welch test.

K. Satake et al. / European Journal of Pharmacology 765 (2015) 402–405404

rats by over 75% (data not shown), also suppressed tacrolimus-induced skin temperature elevation by 55%. Inhibitory effect ofolopatadine at 0.1 mg/kg on tacrolimus-induced skin temperatureelevation was significantly higher than that of cetirizine at 10 mg/kg (Fig. 2C).

3.3. Effects of olopatadine on the increase in vascular permeability inthe skin induced by topical tacrolimus ointment

We next examined whether tacrolimus ointment induces vas-cular permeability at sites of application. Topical treatment withtacrolimus ointment induced an increase in the amount of Evansblue dye extravasation in the treated rat skin (Fig. 3), whereastreatment with olopatadine prior to topical tacrolimus ointmentsignificantly suppressed vascular permeability (Fig. 3).

3.4. Effects of olopatadine on the production of NGF in the skintreated with topical tacrolimus ointment

Finally, we evaluated the production of NGF in the skin treatedwith tacrolimus ointment. As shown in Fig. 4, the levels of NGF inthe skin were significantly increased by topical tacrolimus treat-ment, compared with that observed after topical white petrolatumtreatment. Moreover, olopatadine significantly suppressed the in-crease in the NGF production in the skin induced by treatmentwith 0.03% tacrolimus ointment (Fig. 4).

4. Discussion

The most common adverse effects associated with the appli-cation of tacrolimus ointment in patients with atopic dermatitisare hot flashes and a burning sensation at the site of application(Saeki et al., 2009). The aim of this study was to evaluate the ef-fects of olopatadine on the incidence of hot flashes induced by

Fig. 4. Effects of olopatadine on the production of NGF in the abdominal skintreated with tacrolimus ointment. Olopatadine (0.1 mg/kg) was administered orallyto rats for four consecutive days. Tacrolimus ointment was applied 0.5 h after thefinal olopatadine administration. The abdominal skin was isolated 1 h after treat-ment with tacrolimus ointment, and the NGF levels in the homogenized skin tissuewere measured using ELISA. Olo: olopatadine. Each column represents themeanþS.E.M. of six rats. #Po0.05 compared to the white petrolatum-treatedgroup according to the Steel–Dwass test. *Po0.05 compared to the 0.03% tacroli-mus ointment-treated group according to the Steel–Dwass test.

K. Satake et al. / European Journal of Pharmacology 765 (2015) 402–405 405

topical treatment with tacrolimus ointment in rat abdominal skin.However, it has not been previously reported whether hot flashesare induced by tacrolimus ointment in rats. In the current study,we first demonstrated that the skin temperature is significantlyelevated following the topical application of tacrolimus ointmentin rats. Consistent with the findings of a previous report showingthat a burning sensation occurs during the first weeks of treat-ment with tacrolimus ointment and subsequently decreases overtime in patients with atopic dermatitis (Remitz et al., 2007), ele-vation of the skin temperature in rats was not observed after therepeated application of tacrolimus ointment. These results suggestthat tacrolimus induces hot flashes in rats resembling those that inhumans and that tacrolimus-induced hot flashes can be evaluatedin rats by measuring the skin temperature.

The present study clearly demonstrated that olopatadinetreatment suppresses the increase in skin temperature induced bytacrolimus ointment in rats. The other antiallergic agent cetirizinealso suppressed tacrolimus-induced skin temperature elevation ata dose which exhibits significant H1R antagonistic activity in rats.Although olopatadine at 0.1 mg/kg and cetirizine at 10 mg/kg ex-hibited comparable inhibitory effects on histamine-induced plas-ma leakage in rats, inhibitory effect of olopatadine at 0.1 mg/kg ontacrolimus-induced skin temperature elevation was significantlymore potent than that of cetirizine at 10 mg/kg (Fig. 2C). Theseresults suggest that olopatadine attenuate tacrolimus-induced hotflushes in rats through both H1R antagonistic activity-dependentand H1R antagonistic activity-independent mechanisms.

In general, heat sensation is caused by an increased blood flowdue to vasodilation. Vasodilation results in the opening of junc-tions between vascular endothelial cells, followed by the inductionof vascular permeability. In the current study, tacrolimus ointmentinduced not only elevation of the skin temperature, but also vas-cular permeability, in the skin of the rats. In addition, we showedthat the production of NGF in the skin was induced by topicaltacrolimus ointment. NGF has been reported to enhance vasodi-lation and vascular permeability (Bennett et al., 1998; Otten et al.,1984), suggesting that NGF is partially involved in the increase invascular permeability and skin temperature caused by tacrolimustreatment. As shown in Fig. 4, olopatadine significantly suppressedNGF production induced by topical tacrolimus ointment in thisstudy. It has been reported that olopatadine, but not cetirizine,inhibited the increase in NGF induced by toluene-2,4-diisocyanate

in the nasal lavage fluid of rats (Tamura and Komai, 2008). Ac-cordingly, olopatadine may suppress the hot flashes and vascularpermeability induced by tacrolimus as a result of its inhibitoryeffects on the production of NGF in the skin.

The topical application of tacrolimus is followed by the initialrelease of substance P (SP) and calcitonin gene-related peptides(CGRP) from primary afferent nerve fibers in murine skin duringthe early inflammatory response (Ständer et al., 2007). The releaseof neuropeptides and their subsequent binding to mast cells leadsto mast cell degranulation, as regularly observed in tacrolimus-treated mice. Although the precise mechanisms underlying theburning sensations induced by tacrolimus are unclear, mediatorsderived from mast cells, such as histamine and tryptase, may in-duce pruritus and burning by binding to their corresponding re-ceptors on sensory nerve fibers (Ständer et al., 2007). CGRP is avery potent vasodilator and its production is induced by NGF(Supowit et al., 2001). In addition to the inhibitory effect on NGFproduction, olopatadine suppresses SP release and mast cell de-granulation (Tamura et al., 2005). Further studies are needed toclarify the mechanisms of inhibitory effects of olopatadine on theskin temperature elevation induced by tacrolimus.

In conclusion, in the present study, olopatadine attenuated thehot flashes induced by tacrolimus ointment through the me-chanism that is different from H1R antagonistic activity such asthe inhibition of NGF production, suggesting that combinationtreatment with olopatadine and tacrolimus ointment is useful forimproving medication adherence with tacrolimus ointment inpatients with atopic dermatitis.

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