Methods for Preclinical Assessment of Antipruritic Agents and Itch

Vol. 38, No. 5 Biol. Pharm. Bull. 38, 635–644 (2015) 635 Review

Methods for Preclinical Assessment of Antipruritic Agents and Itch Mechanisms Independent of Mast-Cell Histamine Yasushi Kuraishi Laboratory of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama; Toyama 930–0194, Japan. Received January 26, 2015

Itch is a sensation that provokes a desire to scratch. Mast-cell histamine was thought to be a key itch mediator. However, histamine and mast-cell degranulation were reported not to elicit scratching in animals. It was difficult to investigate the pathophysiology of itching and to evaluate the antipruritic efficacy of chemical agents in the early 1990 s. We showed that hind-paw scratching and biting were elicited by stimulation with pruritogenic agents in mice. Those results demonstrated for the first time that cutaneous itching could be evaluated behaviorally in animals. We established various animal models of pathological itch of the skin (dry skin, mosquito allergy, surfactant-induced pruritus, and herpes zoster) and mucus membranes (pollen allergy). Mast-cell histamine did not play a key role in itching in any animal model examined except for the

pollen allergy model. Histamine is not an exclusive itch mediator of mast cells; tryptase and leukotriene B4 released from mast cells also act as itch mediators. Epidermal keratinocytes release several itch mediators,

such as leukotriene B4, sphingosylphosphorylcholine, thromboxane A2, nociceptin, nitric oxide, and histamine, which may play important roles in pathological itching. Appropriate animal models of pathological itching are needed for pharmacological evaluation of the antipruritic efﬁcacy of chemical agents. Key words pruritus; animal model; scratching; biting; keratinocyte; itch mediator

1. INTRODUCTION itch-related responses in mice and animal models of pathological itch. This review describes our studies on the development The literature on the peripheral and neuronal mechanisms of methods for the preclinical assessment of antipruritic agents of itching, while still limited in scope, is increasing each and the mechanisms of pruritus, independent of mast-cell his- year. Animal studies in the past decade have revealed new tamine. endogenous itch mediators and shed light on the underlying mechanisms of pathological itching.1–3) Two decades ago, 2. ITCH-RELATED RESPONSES IN ANIMALS Greaves wrote, “Itching remains an orphan symptom, frustrat- ing to patients and their physicians and sadly neglected by 2.1. Scratching Behaviors As mentioned previously, it neurophysiologist and pharmacologists.” 4) At that time, the was difficult to evaluate the efficacy of antipruritic agents in methodology for the study of itching in animals was lacking; animal experiments in the early 1990 s. Since itch induces a therefore, there were no animal models of physiological and desire to scratch in humans, observing scratching behaviors is pathological itch. Mast-cell histamine is a classical endo- central to the study of itch in animals. Since compound 48/80 genous itch mediator, and itch induces a desire to scratch in was shown to produce itching via mast-cell degranulation in humans.5) Cutaneous stimulation was shown to elicit hind-paw humans,14) we first examined its effects. An injection of com- scratching (scratching reflex) in spinal dogs as early as the pound 48/80 into the rostral backs of mice elicited hind-paw 1900 s.6) However, in 1965, studies showed that intradermal scratching.15) Substance P was also shown to cause itching injections of histamine and the mast-cell degranulator com- via mast-cell degranulation in humans,16) and we found that pound 48/80 did not elicit scratching in dogs, although they this peptide could also elicit hind-paw scratching in mice.15) induced erythema and wheal.7) An intradermal injection of Interestingly, histamine did not cause hind-paw scratching in histamine was also reported not to elicit scratching in mice in mice,15) suggesting that mast-cell histamine is not the final 1974.8) To compound the issue further, a 1987 study claimed common itch mediator. As mentioned previously, “scratching” that the “scratching” behavior observed in arthritic rats was behavior was claimed to be a sign of chronic pain.9) There- due to underlying chronic pain.9) Electrophysiological studies fore, we examined whether algesiogenic agents would elicit showed that cutaneous stimulation with cowhage spicules, scratching in mice and found that injections of the algesio- which elicit itching in humans,5) activated C-polymodal noci- genic agents capsaicin and formaldehyde into the rostral back ceptors and myelinated mechanoreceptors in cats.10,11) Hista- did not elicit hind-paw scratching.15) We were thus able to mine was shown to activate most of the C-polymodal nocicep- demonstrate for the first time that cutaneous itching could be tors responsive to bradykinin (an inflammatory mediator that evaluated behaviorally in animals. Since then, the literature on causes pain in humans)12) in a skin-nerve preparation from animal studies of the peripheral and neuronal mechanisms of rats.13) Thus, it was difficult to investigate the pathophysiology itch has been gradually increasing (Fig. 1), in most of which of itching and to evaluate the antipruritic efficacy of chemi- hind-paw scratching was used as an index of itching. cal compounds in the early 1990 s. Therefore, we investigated Since histamine is a classical itch mediator and H1 hista-

Fig. 2. Mouse Strain Differences in Scratching Responses to Intrader- mal Injections of Histamine and Serotonin * p<0.05 vs. saline; n=8 in each group.

2.2. Scratching Behaviors and Opioid Receptors Itch evokes scratching in animals as well as in humans, but scratching is not a behavioral response exclusive to pruritogenic stimulation. For example, humans and some monkeys Fig. 1. Number of Original Articles on Animal Itch Studies scratch their heads with hands as an emotional response, and Papers in clinical veterinary medicine that describe itching as a specific symp- rodents scratch their flanks with their hind paws for groom- tom are included. Itch-related articles published by other groups from 1965 to 1995: ing. Therefore, the behavioral distinction between itch-related the failure of histamine and compound 48/80 to elicit scratching in dogs (1965)7); 8) scratching and non-itch-related scratching is not easy, and we the failure of histamine to elicit scratching in mice (1974) ; scratching induced by intracerebroventricular and intrathecal injections of bombesin in rats (1983); re- needed to develop new means of verifying when scratching is sponses of primary afferents to cowhage stimulation in cats (1987)10,11); scratching an itch response. We directed our attention to the effects of induced by injections of opioids into the medullary dorsal horn in monkeys (1992, 1993); and scratching induced by injection of morphine into the medullary dorsal opioid antagonists on itching in humans. Opioid antagonists horn in rats (1995). were reported to inhibit experimentally induced itching in healthy individuals18) and pruritus and scratching in patients mine receptor antagonists are generally used to treat pruritus, with dermatoses and systemic disorders.19–21) In contrast, epi- results from itch experiments in which histamine-insensitive dural and intrathecal opioids were known to induce pruritus mice are used may not be applicable to humans. In our first at analgesic doses.22) We found that opioid antagonists sup- itch experiment, histamine did not elicit scratching in ddY pressed hind-paw scratching induced by pruritogens17,23) and mice15); it also did not elicit scratching in Sprague-Dawley rats associated with allergy24) and chronic dermatitis25) in mice. To (unpublished observation). Therefore, we searched for a strain date, many studies have shown that opioid antagonists inhibit of mice that would show scratch responses to histamine injec- hind-paw scratching in mouse models of pathological itch.26) tion. Serotonin, on the other hand, elicited hind-paw scratch- Thus, opioid antagonists may be useful to verify that scratching in ddY mice17) and therefore was used as the positive ing is an itch-related response. control. An intradermal injection of serotonin (100 nmol/site) The opioid antagonist naloxone and the µ-opioid receptor induced hind-paw scratching in all strains of mice tested. In antagonist naltrexone inhibit hind-paw scratching induced by contrast, histamine (100 nmol/site) induced hind-paw scratch- intradermal injections of pruritogens in healthy mice.17,23,27) ing only in the ICR strain of mice, and other mice tested were In healthy humans, naloxone increases itch thresholds to his- insensitive to histamine (Fig. 2). Therefore, we generally used tamine.18) In contrast, µ-opioid receptor agonists elicit facial ICR mice in our animal studies of itch. scratching following intracisternal injection and body scratch-

Dr. Yasushi Kuraishi was born in Fukuoka, Japan, in 1948. He received a Ph.D. from Kyoto Uni- versity in 1979 under the supervision of Professor Hiroshi Takagi. After completion of the doctoral program in pharmacology, Kyoto University Graduate School of Pharmaceutical Sciences, he joined the Faculty of Pharmaceutical Sciences, Kyoto University, as an Assistant Professor in 1977 and became an Associate Professor in 1987. He moved to the Research Institute for Wakan-yaku, Toyama Medical and Pharmaceutical University, as a Full Professor in 1992, and to the Faculty of Pharma- ceutical Sciences in 1996. Toyama Medical and Pharmaceutical University was reorganized as the University of Toyama in 2005, when he was appointed an Executive Vice President. After the comple- Yasushi Kuraishi tion of his term, he was appointed as a Full Professor at the University of Toyama Graduate School of Medicine and Pharmaceutical Sciences in 2009 and retired in 2014. He received the Pharmaceuti- cal Society of Japan Award for Young Scientists (1990) and the Pharmaceutical Society of Japan Award for Divisional Scientific Contributions (2014). His major research field is pharmacology with particular interest in pain and itch. Vol. 38, No. 5 (2015) Biol. Pharm. Bull. 637 ing following intrathecal injection, suggesting that both spinal scratch with their hind paws is an itch-associated behavioral cord and brain are sites of the pruritic side effect of opioid response. In murine models of pathological pruritus, caudally analgesics.28–30) However, opioid antagonists may act on the directed biting was shown to be inhibited by opioid antago- lower brainstem rather than on the spinal dorsal horn and the nists,31,45) supporting the idea that caudally directed biting is periphery to inhibit itch signaling.31,32) These findings, taken an itch-associated behavior. together, suggest that µ-opioid receptors are involved in the Using biting behavior as an index of itch response, we transmission or positive modulation of itch signals in the brain found that the descending noradrenergic system regulates itch under normal conditions. transmission in the spinal cord mediated through the α1 and 46,47) Opioid peptides have been suggested to be involved in the α2-adrenoceptors. The α2-adrenoceptor agonist clonidine pruritus of cholestasis. Opioid antagonists reduce scratch- and the serotonin-noradrenaline reuptake inhibitor milnacip- ing activity and pruritus perception in patients with pruritus ran inhibited pruritogen-induced biting through their action on of cholestasis.20,33) However, opioid antagonists cause opioid the spinal cord.48,49) Under normal conditions, the descending withdrawal-like side effects in these patients.20) µ-Opioid serotonergic system does not seem to exert tonic inhibition of receptor binding is decreased in several brain regions in rats itch transmission.47) that have undergone bile duct resection.34) Total opioid activ- 2.4. Pruriceptive Activity of Primary Sensory Nerves ity and methionine-enkephalin, an opioid peptide, increase in Observation of the activity of sensory nerves may provide plasma from bile duct-resected rats.35) In addition, a micro- important information on how pruritus is induced, and injection of plasma from patients with pruritus of cholestasis whether antipruritic agents exert their effects peripherally or into the medullary dorsal horn induces facial scratching in centrally. We compared the responses of cutaneous nerves healthy monkeys, but plasma from nonpruritic cholestatic in ICR and ddY mice to intradermal injections of histamine patients does not.36) Thus, the idea that an increase in opioid and serotonin. The activity of cutaneous nerves was as low peptidergic activity in the brain causes cholestatic pruritus is as 0.1 impulse/s without stimulation. In ICR mice, both seductive and plausible. However, the release of total opioid histamine and serotonin markedly increased the activity of peptide from brain regions such as the striatum does not dif- primary afferents, the time-courses of which were similar to fer between bile duct-resected and sham-operated rats.37) On those of scratching behaviors.50) In ddY mice, histamine did the other hand, the expression of preproenkephalin mRNA not increase the activity of primary afferents or hind-paw has been shown to increase in the liver of bile duct-resected scratching, although serotonin increased both.50) Those find- rats.38) It was proposed that peripheral neuroinflammation is a ings suggested that histamine does not elicit scratching in ddY cause of cholestatic pruritus.39) Thus, there has been no report mice because of the lack of functional expression of histamine on pathological pruritus that is attributed to the increased ac- receptors in the cutaneous nerves. tivity of the opioid peptidergic systems in the central nervous NC mice develop severe dermatitis when they are housed system. for long periods under pathogen-uncontrolled conditions.25,51) 2.3. Biting Behaviors Similar to pain information, The frequencies of spontaneous scratching of the rostral part itch information from the periphery may be modulated in of the body and biting of the caudal part of the body were the dorsal horn, and an understanding of the mechanisms of found to be positively correlated with the spontaneous ac- modulation is crucial for the development of centrally acting tivities of the cutaneous nerves innervating the corresponding antipruritic agents. When pruritogenic stimuli are applied to cutaneous areas.31) This suggests that spontaneous scratching the rostral back, sensory information enters the dorsal horn and biting are primarily due to pruriceptive inputs. Systemic at the higher thoracic and lower cervical levels. It is difficult administration of naltrexone inhibits spontaneous scratching to administer agents locally to these regions in freely moving and biting behaviors without affecting the increased activity rodents. In contrast, intrathecal administration to the lumbar of the cutaneous nerves, suggesting that the μ-opioid receptor dorsal horn through a lumbar puncture is feasible in freely antagonist inhibits itching by acting on the central nervous moving rodents. The lumbar dorsal horn receives sensory in- system.31) On the other hand, a single topical application of formation from the hindlimbs and caudal portions of the body. E6005, a phosphodiesterase 4 inhibitor, decreases both spon- In some studies, licking and biting behaviors were interpreted taneous scratching and the activity of the cutaneous nerves, as a single category of (nociceptive) response.40–42) In addi- suggesting that E6005 inhibits itching by acting primarily in tion, abdomen scratching and caudally directed biting/licking the periphery.52,53) behaviors induced by the intrathecal injection of substance P In ICR mice, acute cutaneous allergy increased hind-paw were reported to be inhibited by the systemic administration scratching and cutaneous nerve activity, the time courses of 54) of morphine, and these behaviors were suggested to be noci- which were similar. Terfenadine, a non-sedative H1 hista- ceptive responses.43) Therefore, the behavioral distinction of mine receptor antagonist,55) inhibited neither the increased itch responses from pain responses was indispensable to the scratching nor nerve activity.54) In contrast, azelastine, a 55) investigation of the modulation mechanisms of itch signaling less-sedative H1 histamine receptor antagonist, suppressed in the spinal dorsal horn. We injected serotonin, a pruritogen, both increased scratching and nerve activity.54) The inhibitory into the hind paws of mice and recorded biting and licking effects of azelastine were shown to be due, at least in part, to behaviors separately. Although serotonin injection induced its direct inhibitory action on the primary sensory neurons.54) biting and licking of the injection site, only the biting was Observation of sensory nerve activity may therefore provide suppressed by opioid and serotonin antagonists.44) In contrast, important information on whether antipruritic agents exert an injection of formaldehyde solution elicited marked licking, their effects peripherally or centrally. but not biting, behaviors.44) On the basis of those results, we 2.5. Pruriceptive Activity of Dorsal Horn Neurons hypothesized that biting of the regions that the rodents cannot Spinothalamic tract neurons that responded to cutaneous 638 Biol. Pharm. Bull. Vol. 38, No. 5 (2015) stimulation with histamine and cowhage were identified in 3. PRURITUS INDEPENDENT OF MAST-CELL HIS- the superficial dorsal horn of cats and primates.56,57) We iden- TAMINE tified dorsal horn neurons that were mechanosensitive and responded to acute cutaneous allergy in ICR mice.58) Acute 3.1. Substance P Substance P is one of the most potent allergy-responsive neurons were found in the superficial layers pruritogenic endogenous peptides.14,66) It releases histamine but not in the deep layers of the dorsal horn and constituted from cutaneous mast cells,67) which is due to a direct activa- only about 10% of mechanosensitive neurons, half of which tion of G proteins rather than a receptor-mediated process.68) responded to noxious heat stimulation.58) The pruriceptive Itching induced by intradermal substance P was shown to responses of superficial dorsal horn neurons were shown to be be inhibited by local pretreatment with compound 48/80 and suppressed by cutaneous noxious stimuli including scratching systemic pretreatment with H1 histamine receptor antago- in primates and mice.59,60) nists in humans.14,16) Taken together, these findings suggest The main pruritogenic component of cowhage is mucunain, an important role for mast-cell histamine in substance P- which acts on proteinase-activated receptors (PAR) 2 and induced itching. However, paradoxically, we also observed 4.61) In humans, cowhage and histamine activate distinctly that intradermal substance P elicited hind-paw scratching in different C-fibers,62) and cowhage-induced itching may be histamine-insensitive ddY mice.15) These discrepancies let us signaled by capsaicin-sensitive and histamine-insensitive C- to investigate the mechanisms of substance P-induced itch in fibers.63) In the primate dorsal horn, 25% of the spinothalamic histamine-responsive ICR mice. Although an intradermal in- tract neurons tested responded to histamine, 15% responded jection of substance P released histamine from the skin (Fig. to cowhage, and none responded to both.57) Consistent with 3, unpublished observation), hind-paw scratching induced those findings, we observed that cutaneous stimulation with by substance P was not inhibited by chlorpheniramine, an histamine or a PAR2 agonist (and induction of acute cutane- H1 histamine receptor antagonist, at a dose that suppressed ous allergy) induced Fos expression in different regions in the histamine-induced scratching69) (Fig. 3). Local pretreatment superficial dorsal horn in ICR mice; Fos-positive cells were with compound 48/80 markedly inhibited substance P-induced mainly distributed in the inner aspect of lamina II after hista- scratching, but this inhibition was observed in mast cell- mine stimulation and lamina I and the outer aspect of lamina deficient mice and substance P elicited hind-paw scratching II after stimulation with the PAR2 agonist or induction of in these mice as well.23) These findings suggested that the acute cutaneous allergy.64) However, in the superficial dorsal inhibition of itching by compound 48/80 pretreatment does horn of ICR mice, most of the histamine-responsive neurons not simply indicate the involvement of mast cells. Substance responded to PAR2 and noxious stimulation, and most PAR2- P-induced scratching in ICR mice was inhibited by NK1 23) responsive neurons responded to histamine and noxious stimu- tachykinin receptor antagonists. In contrast, NK1 agonists, 65) lation. It is difficult to differentiate electrophysiologically but not NK2 and NK3 agonists, elicited hind-paw scratching between the dorsal horn neurons responsible for histamine- in these mice.23) Therefore, our findings suggest that mast-cell independent itching and the dorsal horn neurons responsible histamine is not a primary factor in substance P-induced itch- for histamine-mediated itching in mice. ing in mice.

Fig. 3. Substance P Elicits Hind-Paw Scratching by Mechanisms Independent of Mast Cells and Histamine in Mice A) Intradermal injections of histamine and substance P elicited hind-paw scratching in ICR mice. n=8 in each group. B) Intradermal injection of substance P releases histamine in the skin. Histamine release was measured by dermal microdialysis in ICR mice. n=5. C) Chlorpheniramine inhibited scratching induced by histamine, but not by substance P, in ICR mice. n=8 in each group. D) Substance P elicited hind-paw scratching in mast cell-deﬁcient (WBB6F1 W/W v) mice as well as control (WBB6F1 +/+) mice. n=8 in each group. Values are mean and standard error of the means. Graphs were drawn from the data in refs. 23 and 61 and additional data. Vol. 38, No. 5 (2015) Biol. Pharm. Bull. 639

Substance P-induced scratching allowed the preclinical as- rolidone carboxylate, and urea) in addition to lipid components sessment of compounds for antihistamine-resistant pruritus from the stratum corneum,83) gradually increased spontaneous for the first time. Toray Industries, Inc. (Kamakura, Japan) hind-paw scratching.82) Skin dryness was observed from the was developing the κ-opioid receptor agonist TRK-820 (later day after the start of the treatment, and an increase in spon- named nalfurafine) as an analgesic at that time, but our re- taneous scratching became obvious after 3 d.82) An increase in search on itch changed the developmental target of TRK-820 spontaneous scratching was not obvious in a high-humidity from pain to antihistamine-resistant pruritus. TRK-820 was environment (unpublished observation), a feature similar to shown to inhibit scratching induced by intradermal substance seasonal xerosis in winter. The numbers of total mast cells P and intracisternal morphine in mice.70,71) It was also shown and degranulated mast cells did not increase in this animal to be effective against severe pruritus in hemodialysis pa- model of pruritic dry skin and an increase in spontaneous tients.72) Nalfurafine has been approved in Japan since 2009 scratching was also observed in mast cell-deficient mice.82) for the treatment of uremic pruritus in hemodialysis patients. The daily removal of lipid and aqueous components was also 3.2. Mast-Cell Itch Mediators In addition to histamine, shown to increase spontaneous hind-paw scratching in rats.84) many proteinases (endopeptidases) have long been known to 4.2. Mosquito Allergy Although the bites of many in- elicit itching when administered to the human skin.73) It was sects generally elicit pain as well as itch, the bite of female shown in 1955 that an intraepidermal injection of the protein- mosquitoes mainly causes itching in humans.85,86) Mosquito ase papain elicited itching without causing wheal-and-flare re- bites induce both immediate and delayed cutaneous reac- actions, although these reactions were induced after intrader- tions.86) When bitten by a mosquito for the first time in their mal injection.73) In the early 1970 s, it was reported that itching life, humans develop a delayed cutaneous reaction but no im- induced by trypsin and chymase was mediated by histamine mediate reaction, including itching.85) However, repeated mos- released from mast cells, while papain- and kallikrein-induced quito bites within a short period of time change the delayed itching was not.74,75) The involvement of proteinases in pruri- cutaneous reactions to immediate reactions.85) ICR mice bitten tus went unnoticed for many years thereafter. In the 1990 s, by female mosquitoes (Aedes albopictus) for the first time molecular cloning identified four PAR family members, in their life did not develop a hind-paw scratching response PAR1–4; PAR1, PAR3, and PAR4 are thrombin receptors, within a 1-h period, but repetition (twice a week) was found to while PAR2 is activated by mast-cell tryptase.76) We wondered increase hind-paw scratching of the affected skin gradually.24) whether tryptase played an active role in mast cell-mediated Repeated pretreatment with cetirizine, a nonsedative H1 itching. Intradermal injections of tryptase, but not chymase, histamine receptor antagonist that inhibits the chemotaxis of elicited scratching in ICR mice, and the action of tryptase T cells and monocytes,87) was reported to inhibit immedi- was inhibited by the serine protease inhibitor nafamostat, ate itching following mosquito bites in humans.86) Although anti-PAR2 antibody, and the PAR2 antagonist FSLLRY.77) plasma extravasation was minute following mosquito bites in Scratching induced by intradermal compound 48/80 was also naïve ICR mice, it was markedly increased in sensitized mice inhibited by nafamostat, anti-PAR2 antibody, and FSLLRY, and suppressed by pretreatment with terfenadine.24) Hind- and coadministration of nafamostat and terfenadine exerted paw scratching following mosquito bites was not inhibited an additive inhibitory effect on compound 48/80-induced by terfenadine at a dose that suppressed plasma extravasation scratching.77) Those results suggested that tryptase participates and histamine-induced scratching.24) As mentioned above, in mast cell-mediated itching in cooperation with histamine. azelastine inhibited mosquito bite-induced scratching, which PAR2 activation by mast-cell tryptase may play an important may be due in part to its direct inhibitory action on primary role in scratching in NC mice with chronic allergic dermati- sensory neurons.54) An extract from the salivary glands of tis.78,79) female mosquitoes also increased hind-paw scratching as the intradermal injection was repeated. This was not affected by 4. ANIMAL MODELS OF PATHOLOGICAL ITCH a deficiency in mast cells.24) Mosquito allergy scratching may involve lipoxin A4 (a 5-lipoxygenase metabolite) and the serine Animal models of pathological itch are necessary for anti- protease granzyme A, both of which are T-cell mediators.88–90) pruritic drug development and elucidation of the pathophysi- After repeated mosquito bites, the serum concentration of ological mechanisms of itching in pruritic diseases. Therefore, total immunoglobulin G1 (IgG1) markedly increased, and that we sought to develop mouse models of pathological itch. of total IgE also tended to increase.24) The high-affinity IgG 4.1. Dry Skin Itching is a common symptom in various receptor FcγRI was found to be present in small-sized dorsal dermatoses characterized by dry skin, such as senile xerosis, root ganglion neurons.91) Challenge with antigen activated seasonal xerosis, and atopic dermatitis. Dry skin is also a dorsal root ganglion neurons that were previously exposed to common cutaneous manifestation in pruritic internal diseases, antigen-specific IgG.91) Therefore, direct activation of primary such as chronic renal failure and chronic cholestasis. However, afferents with mosquito allergen may also play a role in mos- it was unclear whether dry skin is an underlying cause of itch- quito allergy itching. ing in patients with these conditions.80,81) Therefore, we tried 4.3. Pollen Allergy Although mosquito bite allergy in- to establish an animal model of pruritic dry skin. Disruption duced immediate itch responses, type I allergy and mast cells of the cutaneous barrier function by the removal of lipid com- may not play important roles. Pollinosis including allergic ponents in the stratum corneum or by the mechanical removal conjunctivitis is a typical type I allergy, and its symptoms are of the stratum corneum caused skin dryness, but daily repeti- due to the release of histamine and other active substances tion of these treatments did not increase hind-paw scratching by mast cells.92) To confirm the roles of mast cells and hista- in ICR mice.82) In contrast, a daily regimen consisting of the mine in pollinosis-associated pruritus, we sought to develop a removal of aqueous components (including amino acids, pyr- mouse model of pruritic pollen allergy. One study showed that 640 Biol. Pharm. Bull. Vol. 38, No. 5 (2015) applying a solution of ovalbumin onto the ocular surface elic- application increased the histamine content of the epidermis, ited hind-paw scratching in sensitized BALB/c mice, whereas but not that of the dermis, in normal and mast cell-deficient application of a ragweed pollen suspension did not.93) We mice.99) The increase in epidermal histamine was at least found that a subconjunctival injection of an extract of ragweed partly due to increased processing of the 74-kDa precursor pollen elicited hind-paw scratching in ICR mice that had been L-histidine decarboxylase to the 53-kDa active form in epider- immunized with ragweed pollen in combination with an alu- mal keratinocytes.99,100) minum hydroxide adjuvant, but not in mice immunized with Sodium dodecyl sulfate (SDS) is a neutral anionic surfac- ragweed pollen alone.94) In contrast to mosquito bite allergy, tant, and a single topical application of SDS did not induce de- pollen allergy-induced scratching was almost completely abol- layed scratching.101) However, daily topical application of 10% ished by mast-cell deficiency.94) Terfenadine showed a statisti- SDS gradually increased hind-paw scratching and skin surface cally significant but partial inhibition of scratching at a dose pH (from 5.0 to 6.0).101) Daily topical application of 10% N- that almost completely suppressed plasma extravasation.94) lauroylsarcosine sodium salt did not increase scratching and 101) Leukotriene B4 has been shown to be produced in RBL-2H3 skin surface pH. Chronic scratching induced by SDS was cells (a cell line with mucosal mast-cell characteristics) fol- also markedly inhibited by terfenadine but not affected by lowing immunoglobulin E receptor activation95) and to be mast-cell deficiency.101) The gene expression and posttransla- increased in the tears of patients with giant papillary conjunc- tion processing of L-histidine decarboxylase and histamine 96) tivitis. We found that leukotriene B4 was also involved in content in the epidermis were increased after daily treatment pollen allergy-induced scratching in mice.94) with SDS.101) 4.4. Cleanser Eczema The use of toiletries and cos- 4.5. Herpes Zoster Pain and itch are two frequent metics can cause adverse effects such as skin irritation and complaints associated with herpes zoster.102) We established itching.97) Many cleansers contain surfactants, which irritate a mouse model of herpes zoster pain; transdermal inocula- the skin and change cutaneous functions. These effects of sur- tion of human herpesvirus 1 to the hind paw causes herpes factants are thought to be due to their interaction with stratum zoster-like lesions in the inoculated dermatome and mechani- corneum proteins and lipids, and the negative charge on their cal allodynia.103,104) We observed that herpetic mice bit and hydrophilic head plays an important role in the interaction.98) licked the lesional skin, raising the possibility that they felt To elucidate the mechanisms of surfactant-associated itching, spontaneous itch and pain. Therefore, to observe scratching we attempted to develop an animal model of topical surfac- as an itch-related behavior, we inoculated the herpesvirus on tant-induced pruritus. Single topical application of the surfac- the midflank, a region that mice can scratch. A rash erupted tants 10% sodium laurate and 10% N-lauroylsarcosine sodium on day 4 after inoculation and extended within the dermatome salt, pH 10.1 and 7.7, respectively, to the shaved skin of ICR on days 5 to 6. After onset on day 4, scratching and licking mice immediately increased hind-paw scratching, which grad- of the lesional skin peaked on days 5 and 6, respectively.105) ually decreased within 1.5 h after application. Sodium laurate Spinal neurons expressing BB2 bombesin receptors (receptors increased scratching again 2 to 3 h after application.99) Two- for gastrin-releasing peptide) were shown to play a key role in 106) hours topical application of 10% sodium laurate significantly itch signaling. Ablation of BB2 receptor-expressing spinal increased the pH of the skin surface from 5.1 to 6.0, but the neurons decreased the scratching but not the licking.105) Oxi- pH nearly normal (pH 5.0–5.2) 2 h after the application of 10% dative stress in the affected skin and TRPA1 channels may be N-lauroylsarcosine sodium salt.99) Delayed scratching after involved in herpes-associated itch and pain.107) sodium laurate application was markedly inhibited by terfenadine but not affected by mast-cell deficiency.99) Sodium laurate

Fig. 4. Epidermal Keratinocytes Release Several Itch Mediators, Many of Which Act in an Autocrine or Paracrine Manner to Enhance Itching

5-LOX, 5-lipoxygenase; BLT1-R, BLT1 leukotriene B4 receptor; GC, guanylyl cyclase; LTB4, leukotriene B4; NO, nitric oxide; NOS1/2, nitric oxide synthase 1 or 2; ORL1-R; ORL1 nociceptin receptor; TP-R, TP prostanoid receptor; TXA2, thromboxane A2. Vol. 38, No. 5 (2015) Biol. Pharm. Bull. 641

5. ROLES OF EPIDERMAL KERATINOCYTES IN in mice because of their low histamine sensitivity. However, PRURITUS animal experiments on itch have revealed many itch mediators, the roles of which vary depending on the etiology of As mentioned above, substance P-induced scratching was pruritus. Therefore, appropriate animal models of pathological not primarily mediated by mast cells and their mediator his- itch are needed for the pharmacological evaluation of the anti- tamine and was inhibited by the NK1 tachykinin receptor pruritic efficacy of chemical agents. antagonist in mice.23) Therefore, we further investigated the mechanisms underlying substance P-induced scratching. It is Acknowledgments I would like to express sincere grati- possible that primary afferents are a site of action of intrader- tude to all the collaborators contributing to our research de- mal substance P because low levels of NK1 receptor mRNA scribed here, many of whom are cited in this review. I also are expressed in the dorsal root ganglion.108) However, since express cordial thanks to all the members of our research we found that leukotriene B4 had potent pruritogenic activ- group. This work was supported financially by Grants-in-Aid ity after intradermal injection in mice,109) we examined the for Scientific Research from the Japan Society for the Promo- involvement of leukotriene B4 in substance P-induced scratch- tion of Science. ing. Although substance P increased the levels of leukotriene

B4 and prostaglandin E2 via the activation of NK1 receptors Conflict of Interest The author declares no conflict of in the skin, the former, but not the latter, was found to be interest. involved in substance P-induced scratching.110) Blockade of leukotriene B4 may contribute to the antipruritc effects of REFERENCES some H1 histamine receptor antagonists such as azelastine, emedastine, and bepostatine.69,111,112) Epidermal keratinocytes 1) Ikoma A. Updated neurophysiology of itch. Biol. Pharm. Bull., 36, may play an important role in the production of pruritogenic 1235–1240 (2013). 110) 2) Kuraishi Y. Potential new therapeutic targets for pathological pruri- leukotriene B4 (Fig. 4). Under normal conditions, mRNA encoding the BLT1, but not BLT2, leukotriene B receptor was tus. Biol. Pharm. Bull., 36, 1228–1234 (2013). 4 3) Tominaga M, Takamori K. An update on peripheral mechanisms found to be expressed in the dorsal root ganglion and skin.113) 113) and treatments of itch. Biol. Pharm. Bull., 36, 1241–1247 (2013). Leukotriene B4 acted on dorsal root ganglion neurons, and 4) Greaves MW. Itching—research has barely scratched the surface. N. leukotriene B4 produced by epidermal keratinocytes may be a Engl. J. Med., 326, 1016–1017 (1992). final itch mediator. Leukotriene B4 is involved in the scratch- 5) Shelley WB, Arthur RP. The neurohistology and neurophysiology of inducing actions of other keratinocyte-producing substances the itch sensation in man. AMA Arch. Derm., 76, 296–323 (1957). such as sphingosylphosphorylcholine and nociceptin27,114) (Fig. 6) Sherrington CS, Laslett EE. Observations on some spinal reflexes

4). Leukotriene B4 was also shown to be involved in patholog- and the interconnection of spinal segments. J. Physiol., 29, 58–96 ical scratching in mouse models of chronic allergic dermatitis (1903). and dermatophyte-associated itching.115,116) 7) Schwartzman RM. The reaction of canine skin to histamine and An intradermal injection of U-46619, a stable analogue 48/80. J. Invest. Dermatol., 44, 39–42 (1965). 8) Varadi DP. Pruritus induced by crude bile and purified bile acids. of thromboxane A2, elicited scratching mediated by TP 117) Experimental production of pruritus in human skin. Arch. Derma- prostanoid receptors in mice. Thromboxane synthase was tol., 109, 678–681 (1974). expressed in keratinocytes, and the TP receptor was mainly 9) De Castro-Costa M, Gybels J, Kupers R, Van Hees J. Scratching expressed in nerve fibers in the skin and epidermal keratino- behaviour in arthritic rats: a sign of chronic pain or itch? Pain, 29, cytes, suggesting that thromboxane A2 synthesized by kerati- 123–131 (1987). nocytes acts on primary afferents to induce itching and acts 10) Tuckett RP, Wei JY. Response to an itch-producing substance in on keratinocytes themselves to enhance itching117) (Fig. 4). cat. I. Cutaneous receptor populations with myelinated axons. Brain Substance P acted on keratinocytes to produce nitric oxide Res., 413, 87–94 (1987). 11) Tuckett RP, Wei JY. Response to an itch-producing substance in cat. via the activation of NK1 receptors and nitric oxide synthase 1, which enhanced substance P-induced scratching118,119) (Fig. II. Cutaneous receptor populations with unmyelinated axons. Brain 4). Nitric oxide synthase 2 was induced in the epidermal ke- Res., 413, 95–103 (1987). 12) Mitchell JC, Krell R. A study of the cutaneous effects of bradyki- ratinocytes of mice with pruritic dry skin, and nitric oxide 120) nin. J. Invest. Dermatol., 43, 177–180 (1964). was involved in spontaneous scratching. Collectively, those 13) Lang E, Novak A, Reeh PW, Handwerker HO. Chemosensitivity of results suggested that epidermal keratinocytes release several fine afferents from rat skin in vitro. J. Neurophysiol., 63, 887–901 itch mediators, many of which act in an autocrine or paracrine (1990). manner to enhance itching. Considering that the cutaneous 14) Fjellner B, Hägermark Ö. Studies on pruritogenic and histamine- receptor level for itching is located very superficially5) and releasing effects of some putative peptide neurotransmitters. Acta removal of the epidermis and the subepidermal nerve network Derm. Venereol., 61, 245–250 (1981). abolishes itching,121) itch mediators of epidermal keratinocytes 15) Kuraishi Y, Nagasawa T, Hayashi K, Satoh M. Scratching behavior may play important roles in pathological itch. induced by pruritogenic but not algesiogenic agents in mice. Eur. J. Pharmacol., 275, 229–233 (1995). 16) Hägermark Ö, Hökfelt T, Pernow B. Flare and itch induced by sub- 6. CONCLUSION stance P in human skin. J. Invest. Dermatol., 71, 233–235 (1978). 17) Yamaguchi T, Nagasawa T, Satoh M, Kuraishi Y. Itch-associated Itch can be evaluated behaviorally in animals; hind-paw response induced by intradermal serotonin through 5-HT2 receptors scratching and biting of the rostral and caudal parts of the in mice. Neurosci. Res., 35, 77–83 (1999). body, respectively, can be used as an index of itching. The 18) Bernstein JE, Swift RM, Soltani K, Lorincz AL. Antipruritic effect role of histamine in pathological itch may be underestimated 642 Biol. Pharm. Bull. Vol. 38, No. 5 (2015)

of an opiate antagonist, naloxone hydrochloride. J. Invest. Derma- study by the use of in vivo microdialysis. Life Sci., 61, 1169–1175 tol., 78, 82–83 (1982). (1997). 19) Monroe EW. Efﬁcacy and safety of nalmefene in patients with se- 38) Bergasa NV, Sabol SL, Young WS III, Kleiner DE, Jones EA. Cho- vere pruritus caused by chronic urticaria and atopic dermatitis. J. lestasis is associated with preproenkephalin mRNA expression in Am. Acad. Dermatol., 21, 135–136 (1989). the adult rat liver. Am. J. Physiol., 268, G346–G354 (1995). 20) Bergasa NV, Alling DW, Talbot TL, Swain MG, Yurdaydin C, 39) Belghiti M, Estevez-Herrera J, Gimenez-Garzo C, Gonzalez-Usano Turner ML, Walker EC, Jones EA. Effects of naloxone infusions in A, Montoliu C, Ferrer-Montiel A, Felipo V, Planells-Cases R. Po- patients with the pruritus of cholestasis. A double-blind, random- tentiation of the transient receptor potential vanilloid 1 channel ized, controlled trial. Ann. Intern. Med., 123, 161–167 (1995). contributes to pruritogenesis in a rat model of liver disease. J. Biol. 21) Peer G, Kivity S, Agami O, Fireman E, Silverberg D, Blum M, Chem., 288, 9675–9685 (2013). Iaina A. Randomised crossover trial of naltrexone in uraemic pruri- 40) Dubuisson D, Dennis SG. The formalin test: a quantitative study tus. Lancet, 348, 1552–1554 (1996). of the analgesic effects of morphine, meperidine, and brain stem 22) Ballantyne JC, Loach AB, Carr DB. Itching after epidural and spi- stimulation in rats and cats. Pain, 4 (Suppl. C), 161–174 (1977). nal opiates. Pain, 33, 149–160 (1988). 41) Sakurada T, Kuwahara H, Sakurada S, Kisara K, Ohba M, Muneka- 23) Andoh T, Nagasawa T, Satoh M, Kuraishi Y. Substance P induction ta E. Behavioural assessment as substance P antagonists in mice.

of itch-associated response mediated by cutaneous NK1 tachykinin Neuropeptides, 9, 197–206 (1987).

receptors in mice. J. Pharmacol. Exp. Ther., 286, 1140–1145 (1998). 42) Abbott FV, Hong Y, Blier P. Activation of 5-HT2A receptors potenti- 24) Ohtsuka E, Kawai S, Ichikawa T, Nojima H, Kitagawa K, Shirai ates pain produced by inﬂammatiory mediators. Neuropharmacol- Y, Kamimura K, Kuraishi Y. Roles of mast cells and histamine in ogy, 35, 99–110 (1996). mosquito bite-induced allergic itch-associated responses in mice. 43) Hylden JL, Wilcox GL. Intrathecal substance P elicits a caudally- Jpn. J. Pharmacol., 86, 97–105 (2001). directed biting and scratching behavior in mice. Brain Res., 217, 25) Yamaguchi T, Maekawa T, Nishikawa Y, Nojima H, Kaneko M, 212–215 (1981). Kawakita T, Miyamoto T, Kuraishi Y. Characterization of itch-asso- 44) Hagiwara K, Nojima H, Kuraishi Y. Serotonin-induced biting of ciated responses of NC mice with mite-induced chronic dermatitis. the hind paw is itch-related response in mice. Pain Res., 14, 53–59 J. Dermatol. Sci., 25, 20–28 (2001). (1999). 26) Kuraishi Y. Animal models of pathological itch. Pain Models: 45) Akiyama T, Carstens MI, Carstens E. Spontaneous itch in the ab- Translational Relevance and Application. (Handwerker HO, Arebdt- sence of hyperalgesia in a mouse hindpaw dry skin model. Neuro- Nielsen L eds.) IASP Press, Washington DC, pp. 391–402 (2013). sci. Lett., 484, 62–65 (2010).

27) Andoh T, Saito A, Kuraishi Y. Leukotriene B4 mediates sphingo- 46) Gotoh Y, Andoh T, Kuraishi Y. Noradrenergic regulation of itch sylphosphorylcholine-induced itch-associated responses in mouse transmission in the spinal cord mediated by α-adrenoceptors. Neu- skin. J. Invest. Dermatol., 129, 2854–2860 (2009). ropharmacology, 61, 825–831 (2011). 28) Tohda C, Yamaguchi T, Kuraishi Y. Intracisternal injection of opi- 47) Gotoh Y, Omori Y, Andoh T, Kuraishi Y. Tonic inhibition of aller- oids induces itch-associated response through μ-opioid receptors in gic itch signaling by the descending noradrenergic system in mice. mice. Jpn. J. Pharmacol., 74, 77–82 (1997). J. Pharmacol. Sci., 115, 417–420 (2011). 29) Kuraishi Y, Yamaguchi T, Miyamoto T. Itch-scratch responses in- 48) Gotoh Y, Andoh T, Kuraishi Y. Clonidine inhibits itch-related re-

duced by opioids through central mu-opioid receptors in mice. J. sponse through stimulation of α2-adrenoceptors in the spinal cord in Biomed. Sci., 7, 248–252 (2000). mice. Eur. J. Pharmacol., 650, 215–219 (2011). 30) Andoh T, Yageta Y, Konno M, Yamaguchi-Miyamoto T, Takahata 49) Andoh T, Gotoh Y, Kuraishi Y. Milnacipran inhibits itch-related H, Nojima H, Nemoto H, Kuraishi Y. Evidence for separate involve- responses in mice through the enhancement of noradrenergic trans- ment of different μ-opioid receptor subtypes in itch and analgesia mission in the spinal cord. J. Pharmacol. Sci., 123, 199–202 (2013). induced by supraspinal action of opioids. J. Pharmacol. Sci., 106, 50) Maekawa T, Nojima H, Kuraishi Y. Itch-associated responses of 667–670 (2008). afferent nerve innervating the murine skin: different effects of his- 31) Maekawa T, Yamaguchi-Miyamoto T, Nojima H, Kuraishi Y. tamine and serotonin in ICR and ddY mice. Jpn. J. Pharmacol., 84, Effects of naltrexone on spontaneous itch-associated responses in 462–466 (2000). NC mice with chronic dermatitis. Jpn. J. Pharmacol., 90, 193–196 51) Matsuda H, Watanabe N, Geba GP, Sperl J, Tsudzuki M, Hiroi J, (2002). Matsumoto M, Ushio H, Saito S, Askenase PW, Ra C. Development 32) Kuraishi Y, Yageta Y, Konno M, Andoh T, Yamaguchi-Miyamoto T, of atopic dermatitis-like skin lesion with IgE hyperproduction in Nojima H. Intracisternal, but not intrathecal, injection of naloxone NC/Nga mice. Int. Immunol., 9, 461–466 (1997). inhibits cutaneous itch-related response in mice. Biol. Pharm. Bull., 52) Andoh T, Yoshida T, Kuraishi Y. Topical E6005, a novel phospho- 31, 2143–2145 (2008). diesterase 4 inhibitor, attenuates spontaneous itch-related responses 33) Bergasa NV, Alling DW, Talbot TL, Wells MC, Jones EA. Oral in mice with chronic atopy-like dermatitis. Exp. Dermatol., 23, nalmefene therapy reduces scratching activity due to the pruritus of 359–361 (2014). cholestasis: a controlled study. J. Am. Acad. Dermatol., 41, 431–434 53) Andoh T, Kuraishi Y. Antipruritic mechanisms of topical E6005, a (1999). phosphodiesterase 4 inhibitor: Inhibition of responses to proteinase- 34) Bergasa NV, Rothman RB, Vergalla J, Xu H, Swain MG, Jones EA. activated receptor 2 stimulation mediated by increase in intracel- Central mu-opioid receptors are down-regulated in a rat model of lular cyclic AMP. J. Dermatol. Sci., 76, 206–213 (2014). cholestasis. J. Hepatol., 15, 220–224 (1992). 54) Ohtsuka E, Kawai S, Nojima H, Andoh T, Kamimura K, Kuraishi 35) Swain MG, Rothman RB, Xu H, Vergalla J, Bergasa NV, Jones EA. Y. Inhibitory effect of azelastine on allergic itch-associated re- Endogenous opioids accumulate in plasma in a rat model of acute sponse in mice sensitized with mosquito salivary glands extract. J. cholestasis. Gastroenterology, 103, 630–635 (1992). Pharmacol. Sci., 91, 263–266 (2003). 36) Bergasa NV, Thomas DA, Vergalla J, Turner ML, Jones EA. Plasma 55) Yanai K, Tashiro M. The physiological and pathophysiological roles from patients with the pruritus of cholestasis induces opioid recep- of neuronal histamine: an insight from human positron emission tor-mediated scratching in monkeys. Life Sci., 53, 1253–1257 (1993). tomography studies. Pharmacol. Ther., 113, 1–15 (2007). 37) Bergasa NV, Mohajer B, Maisonneuve IM, Ho A, Maidment NT, 56) Andrew D, Craig AD. Spinothalamic lamina I neurons selectively Olive F, Gunduz M, Kreek MJ. Basal total opioid peptide release sensitive to histamine: a central neural pathway for itch. Nat. Neu- in the striatum of rats with cholestasis from bile duct resection: a rosci., 4, 72–77 (2001). Vol. 38, No. 5 (2015) Biol. Pharm. Bull. 643

57) Davidson S, Zhang X, Yoon CH, Khasabov SG, Simone DA, tors: theme and variations. Oncogene, 20, 1570–1581 (2001). Giesler GJ Jr. The itch-producing agents histamine and cowhage ac- 77) Ui H, Andoh T, Lee JB, Nojima H, Kuraishi Y. Potent pruritogenic tivate separate populations of primate spinothalamic tract neurons. action of tryptase mediated by PAR-2 receptor and its involvement J. Neurosci., 27, 10007–10014 (2007). in anti-pruritic effect of nafamostat mesilate in mice. Eur. J. Phar- 58) Omori Y, Andoh T, Shirakawa H, Ishida H, Hachiga T, Kuraishi Y. macol., 530, 172–178 (2006). Itch-related responses of dorsal horn neurons to cutaneous allergic 78) Tsujii K, Andoh T, Lee JB, Kuraishi Y. Activation of proteinase- stimulation in mice. Neuroreport, 20, 478–481 (2009). activated receptors induces itch-associated response through hista- 59) Davidson S, Zhang X, Khasabov SG, Simone DA, Giesler GJ Jr. mine-dependent and -independent pathways in mice. J. Pharmacol. Relief of itch by scratching: state-dependent inhibition of primate Sci., 108, 385–388 (2008). spinothalamic tract neurons. Nat. Neurosci., 12, 544–546 (2009). 79) Tsujii K, Andoh T, Ui H, Lee JB, Kuraishi Y. Involvement of trypt- 60) Akiyama T, Carstens MI, Carstens E. Transmitters and pathways ase and proteinase-activated receptor-2 in spontaneous itch-associ- mediating inhibition of spinal itch-signaling neurons by scratching ated response in mice with atopy-like dermatitis. J. Pharmacol. Sci., and other counterstimuli. PLoS ONE, 6, e22665 (2011). 109, 388–395 (2009). 61) Reddy VB, Iuga AO, Shimada SG, LaMotte RH, Lerner EA. Cow- 80) Morton CA, Lafferty M, Hau C, Henderson I, Jones M, Lowe JG. hage-evoked itch is mediated by a novel cysteine protease: a ligand Pruritus and skin hydration during dialysis. Nephrol. Dial. Trans- of protease-activated receptors. J. Neurosci., 28, 4331–4335 (2008). plant., 11, 2031–2036 (1996). 62) Namer B, Carr R, Johanek LM, Schmelz M, Handwerker HO, 81) Kato A, Hamada M, Maruyama T, Maruyama Y, Hishida A. Pru- Ringkamp M. Separate peripheral pathways for pruritus in man. J. ritus and hydration state of stratum corneum in hemodialysis pa- Neurophysiol., 100, 2062–2069 (2008). tients. Am. J. Nephrol., 20, 437–442 (2000). 63) Johanek LM, Meyer RA, Hartke T, Hobelmann JG, Maine DN, 82) Miyamoto T, Nojima H, Shinkado T, Nakahashi T, Kuraishi Y. Itch- LaMotte RH, Ringkamp M. Psychophysical and physiological evi- associated response induced by experimental dry skin in mice. Jpn. dence for parallel afferent pathways mediating the sensation of itch. J. Pharmacol., 88, 285–292 (2002). J. Neurosci., 27, 7490–7497 (2007). 83) Jokura Y, Ishikawa S, Tokuda H, Imokawa G. Molecular analysis 64) Nakano T, Andoh T, Lee JB, Kuraishi Y. Different dorsal horn neu- of elastic properties of the stratum corneum by solid-state 13C- rons responding to histamine and allergic itch stimuli. Neuroreport, nuclear magnetic resonance spectroscopy. J. Invest. Dermatol., 104, 19, 723–726 (2008). 806–812 (1995). 65) Akiyama T, Carstens MI, Carstens E. Excitation of mouse superfi- 84) Nojima H, Carstens MI, Carstens E. c-fos expression in superficial cial dorsal horn neurons by histamine and/or PAR-2 agonist: poten- dorsal horn of cervical spinal cord associated with spontaneous tial role in itch. J. Neurophysiol., 102, 2176–2183 (2009). scratching in rats with dry skin. Neurosci. Lett., 347, 62–64 (2003). 66) Thomsen JS, Sonne M, Benfeldt E, Jensen SB, Serup J, Menne T. 85) Mellanby K. Man’s reaction to mosquito bites. Nature, 158, 554– Experimental itch in sodium lauryl sulphate-inflamed and normal 554 (1946). skin in humans: a randomized, double-blind, placebo-controlled 86) Reunala T, Lappalainen P, Brummer-Korvenkontio H, Coulie P, study of histamine and other inducers of itch. Br. J. Dermatol., 146, Palosuo T. Cutaneous reactivity to mosquito bites: effect of cetiri- 792–800 (2002). zine and development of anti-mosquito antibodies. Clin. Exp. Al- 67) Lowman MA, Rees PH, Benyon RC, Church MK. Human mast lergy, 21, 617–622 (1991). cell heterogeneity: histamine release from mast cells dispersed 87) Jinquan T, Reimert CM, Deleuran B, Zachariae C, Simonsen C, from skin, lung, adenoids, tonsils, and colon in response to IgE- Thestrup-Pedersen K. Cetirizine inhibits the in vitro and ex vivo dependent and nonimmunologic stimuli. J. Allergy Clin. Immunol., chemotactic response of T lymphocytes and monocytes. J. Allergy 81, 590–597 (1988). Clin. Immunol., 95, 979–986 (1995). 68) Mousli M, Bronner C, Landry Y, Bockaert J, Rouot B. Direct 88) Kuraishi Y, Ohtsuka E, Nakano T, Kawai S, Andoh T, Nojima H, activation of GTP-binding regulatory proteins (G-proteins) by sub- Kamimura K. Possible involvement of 5-lipoxygenase metabolite in stance P and compound 48/80. FEBS Lett., 259, 260–262 (1990). itch-associated response of mosquito allergy in mice. J. Pharmacol. 69) Andoh T, Kuraishi Y. Inhibitory effects of azelastine on substance Sci., 105, 41–47 (2007). P-induced itch-associated response in mice. Eur. J. Pharmacol., 89) Nakano T, Andoh T, Kamimura K, Kuraishi Y. Involvement of

436, 235–239 (2002). lipoxin A4 in itch-associated response of mosquito allergy in mice. 70) Togashi Y, Umeuchi H, Okano K, Ando N, Yoshizawa Y, Honda T, Acta Derm. Venereol., 87 (Suppl.), 473P (2007). Kawamura K, Endoh T, Utsumi J, Kamei J, Tanaka T, Nagase H. 90) Andoh T, Akiyama T, Enokida A, Kuraishi Y. Granzyme A and Antipruritic activity of the κ-opioid receptor agonist, TRK-820. Eur. proteinase-activated receptor 2 are involved in the induction of J. Pharmacol., 435, 259–264 (2002). itch-associated responses to mosquito allergy in mice. Acta Derm. 71) Umeuchi H, Togashi Y, Honda T, Nakao K, Okano K, Tanaka T, Venereol., 91 (Suppl.), 638P (2011). Nagase H. Involvement of central µ-opioid system in the scratch- 91) Andoh T, Kuraishi Y. Direct action of immunoglobulin G on pri- ing behavior in mice, and the suppression of it by the activation of mary sensory neurons through Fc gamma receptor I. FASEB J., 18, κ-opioid system. Eur. J. Pharmacol., 477, 29–35 (2003). 182–184 (2004). 72) Kumagai H, Ebata T, Takamori K, Muramatsu T, Nakamoto H, Su- 92) Okubo K, Kurono Y, Fujieda S, Ogino S, Uchio E, Odajima H, zuki H. Effect of a novel kappa-receptor agonist, nalfuraﬁne hydro- Takenaka H, Baba K; Japanese Society of Allergology. Japanese chloride, on severe itch in 337 haemodialysis patients: a Phase III, guideline for allergic rhinitis. Allergol. Int., 60, 171–189 (2011). randomized, double-blind, placebo-controlled study. Nephrol. Dial. 93) Shii D, Oda T, Shinomiya K, Katsuta O, Nakamura M. Cyclospo- Transplant., 25, 1251–1257 (2010). rine A eye drops inhibit the early-phase reaction in a type-I al- 73) Arthur RP, Shelley WB. The role of proteolytic enzymes in the pro- lergic conjunctivitis model in mice. J. Ocul. Pharmacol. Ther., 25, duction of pruritus in man. J. Invest. Dermatol., 25, 341–346 (1955). 321–328 (2009). 74) Hägermark Ö, Rajka G, Bergqvist U. Experimental itch in human 94) Andoh T, Sakai K, Urashima M, Kitazawa K, Honma A, Kuraishi

skin elicited by rat mast cell chymase. Acta Derm. Venereol., 52, Y. Involvement of leukotriene B4 in itching in a mouse model of 125–128 (1972). ocular allergy. Exp. Eye Res., 98, 97–103 (2012). 75) Hägermark Ö. Studies on experimental itch induced by kallikrein 95) Yamashita M, Kushihara M, Hirasawa N, Takasaki W, Takahagi H, and bradykinin. Acta Derm. Venereol., 54, 397–400 (1974). Takayanagi M, Ohuchi K. Inhibition by troglitazone of the antigen- 76) O’Brien PJ, Molino M, Kahn M, Brass LF. Protease activated recep- induced production of leukotrienes in immunoglobulin E-sensitized 644 Biol. Pharm. Bull. Vol. 38, No. 5 (2015)

RBL-2H3 cells. Br. J. Pharmacol., 129, 367–373 (2000). 109) Andoh T, Kuraishi Y. Intradermal leukotriene B4, but not prosta-

96) Akman A, Irkec M, Orhan M, Erdener U. Effect of lodoxamide on glandin E2, induces itch-associated responses in mice. Eur. J. Phar- tear leukotriene levels in giant papillary conjunctivitis associated macol., 353, 93–96 (1998). with ocular prosthesis. Ocul. Immunol. Inﬂamm., 6, 179–184 (1998). 110) Andoh T, Katsube N, Maruyama M, Kuraishi Y. Involvement of

97) de Groot AC, Nater JP, van der Lender R, Rijcken B. Adverse leukotriene B4 in substance P-induced itch-associated response in effects of cosmetics and toiletries: a retrospective study in the gen- mice. J. Invest. Dermatol., 117, 1621–1626 (2001).

eral population. Int. J. Cosmet. Sci., 9, 255–259 (1987). 111) Andoh T, Kuraishi Y. Involvement of blockade of leukotriene B4 98) Ananthapadmanabhan KP, Moore DJ, Subramanyan K, Misra M, action in anti-pruritic effects of emedastine in mice. Eur. J. Phar- Meyer F. Cleansing without compromise: the impact of cleansers macol., 406, 149–152 (2000). on the skin barrier and the technology of mild cleansing. Dermatol. 112) Andoh T, Kuraishi Y. Suppression by bepotastine besilate of sub- Ther., 17 (Suppl. 1), 16–25 (2004). stance P-induced itch-associated responses through the inhibition of

99) Inami Y, Andoh T, Sasaki A, Kuraishi Y. Topical surfactant- the leukotriene B4 action in mice. Eur. J. Pharmacol., 547, 59–64 induced pruritus: involvement of histamine released from epidermal (2006).

keratinocytes. J. Pharmacol. Exp. Ther., 344, 459–466 (2013). 113) Andoh T, Kuraishi Y. Expression of BLT1 leukotriene B4 receptor 100) Inami Y, Andoh T, Kuraishi Y. Prevention of topical surfactant- on the dorsal root ganglion neurons in mice. Brain Res. Mol. Brain induced itch-related responses by chlorogenic acid through the Res., 137, 263–266 (2005). inhibition of increased histamine production in the epidermis. J. 114) Andoh T, Yageta Y, Takeshima H, Kuraishi Y. Intradermal noci-

Pharmacol. Sci., 121, 242–245 (2013). ceptin elicits itch-associated responses through leukotriene B4 in 101) Inami Y, Sasaki A, Andoh T, Kuraishi Y. Surfactant-induced chron- mice. J. Invest. Dermatol., 123, 196–201 (2004). ic pruritus: Role of L-histidine decarboxylase expression and hista- 115) Andoh T, Haza S, Saito A, Kuraishi Y. Involvement of leukotriene

mine production in epidermis. Acta Derm. Venereol., 94, 645–650 B4 in spontaneous itch-related behaviour in NC mice with atopic (2014). dermatitis-like skin lesions. Exp. Dermatol., 20, 894–898 (2011).

102) Bhala BB, Ramamoorthy C, Bowsher D, Yelnoorker KN. Shingles 116) Andoh T, Takayama Y, Kuraishi Y. Involvement of leukotriene B4 and postherpetic neuralgia. Clin. J. Pain, 4, 169–174 (1988). in dermatophyte-related itch in mice. Pharmacol. Rep., 66, 699–703 103) Takasaki I, Andoh T, Shiraki K, Kuraishi Y. Allodynia and hyper- (2014). algesia induced by herpes simplex virus type-1 infection in mice. 117) Andoh T, Nishikawa Y, Yamaguchi-Miyamoto T, Nojima H, Na-

Pain, 86, 95–101 (2000). rumiya S, Kuraishi Y. Thromboxane A2 induces itch-associated 104) Sasaki A, Serizawa K, Andoh T, Shiraki K, Takahata H, Kuraishi responses through TP receptors in the skin in mice. J. Invest. Der- Y. Pharmacological differences between static and dynamic al- matol., 127, 2042–2047 (2007). lodynia in mice with herpetic or postherpetic pain. J. Pharmacol. 118) Andoh T, Kuraishi Y. Nitric oxide enhances substance P-induced Sci., 108, 266–273 (2008). itch-associated responses in mice. Br. J. Pharmacol., 138, 202–208 105) Sun YG, Zhao ZQ, Meng XL, Yin J, Liu XY, Chen ZF. Cellular (2003). basis of itch sensation. Science, 325, 1531–1534 (2009). 119) Andoh T, Al-Akeel A, Tsujii K, Nojima H, Kuraishi Y. Repeated

106) Sasaki A, Adhikari S, Andoh T, Kuraishi Y. BB2 bombesin treatment with the traditional medicine Unsei-in inhibits substance receptor-expressing spinal neurons transmit herpes-associated itch P-induced itch-associated responses through down-regulation of the

by BB2 receptor-independent signaling. Neuroreport, 24, 652–656 expression of nitric oxide synthase 1 in mice. J. Pharmacol. Sci., (2013). 94, 207–210 (2004). 107) Adhikari S, Sasaki A, Kuraishi Y. Involvement of oxidative stress 120) Shinkado T, Miyamoto T, Nojima H, Fukuda M, Kuraishi Y. In- in herpes-associated itch and pain in mice. Acta Derm. Venereol., volvement of nitric oxide in itch-associated responses induced by 91 (Suppl.), 616P (2013). cutaneous barrier disruption in mice. Society for Neuroscience,

108) Andoh T, Nagasawa T, Kuraishi Y. Expression of tachykinin NK1 Neuroscience Meeting Planner, Online, Program No. 719.1 (2001). receptor mRNA in dorsal root ganglia of the mouse. Brain Res. 121) Keele CA, Armstrong D. Substances producing pain and itch. Wil- Mol. Brain Res., 35, 329–332 (1996). liams & Wilkins, Baltimore, MD (1964).