
Crotamiton (
Itch is a sensation felt on the skin that causes a desire to scratch. Previous studies have revealed that the sensation is transmitted via sensory peripheral neurons by the activation of certain molecular receptors and/or ion channels (Kittaka and Tominaga, 2017). One of the most popular pruritogen (itch-inducing agent) is histamine (Ohsawa and Hirasawa, 2014). It is an endogenous pruritogen that is mostly released from mast cells (Cho
Moreover, chloroquine (an anti-malarial agent) causes a severe itching that is not related to histamine receptor activation (Olatunde and Obih, 1981). The molecular entities for chloroquine-induced itching have been identified to be Mas-related G-protein-coupled receptor A3 (MRGPRA3) and transient receptor potential A1 (TRPA1) ion channel (Wilson
Recently, Kittaka
All reagents including crotamiton, histamine, chloroquine, capsaicin, allyl isothiocyanate (AITC), and permethrin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Crotamiton, capsaicin, and AITC were dissolved in ethanol, histamine and chloroquine were dissolved in water, and permethrin was dissolved in dimethyl sulfoxide.
Lumbar and thoracic DRG neurons were isolated from 10-week-old mice and cultured as described previously (Malin
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, St. Louis, MO, USA), supplemented with 10% FBS and 100 U/mL penicillin–streptomycin solution (Hyclone), at 37°C in a humidified atmosphere of 5% CO2-95% air. The cells were sub-cultured every 3 or 4 days with fresh DMEM medium for maintenance. Further, the cells were transfected with selected genes using a FuGENE® HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Each gene was transfected to give a final amount of 1 μg and all experiments were performed one day after transfection. The genes used in this study—mouse H1R (NM_001252643), mouse TRPV1 (NM_001001445), mouse MRGPRA3 (NM_153067), and mouse TRPA1 (NM_177781)—were cloned from mouse dorsal root ganglia and matched 100% for sequences with those in the NCBI GenBank database. All the genes were subcloned into pcDNA3.1 (Invitrogen).
Intracellular Ca2+ was measured using an inverted microscope (Nikon, Eclipse Ti-U, Kanagawa, Japan) imaging technique (“calcium imaging”), following a previously described method (Pradhananga and Shim, 2015). Briefly, HEK293T cells or primary culture of DRG neurons were plated on poly-lysine-coated 8-well chambers (Lab-Tek™), and the incubated cells were transfected with H1R and/or TRPV1 cDNA. One day after transfection, the culture media were replaced with normal bath solution [140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 5.5 mM HEPES (pH 7.4)]. For intracellular calcium detection, the cultures were incubated with the calcium-specific fluorescent dye Fluo-3 AM (5 μM, Invitrogen) for 1 h in the presence of 0.1% Pluronic F-127 (Invitrogen) at 37°C. Following incubation, the media were washed out, and histamine or chloroquine were applied to induce calcium influx. The calcium-specific fluorescence was observed at 488/515 nm (excitation/emission wavelengths). Microscopic images were captured using Nikon NIS Elements software (Nikon) at 1.5 s intervals. Changes in intracellular calcium levels were expressed as F/F0 ratio, where F0 is the initial fluorescence intensity. The ratio was calculated using the ImageJ program (Schindelin
All animal experimental procedures were performed in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee at Lee Gil Ya Cancer and Diabetes Institute (LCDI), Gachon University, Korea (Approved animal protocol number: LCDI-2014-0068). Male ICR mice (aged 10–11 weeks) were purchased from Orient Laboratory Animals (Seoul, Korea). The animals were allowed free access to food and water under a 12:12 h light:dark cycle. The scratching behavior test was performed as previously described (Jang
All data are presented as mean ± SEM. For comparison between two groups, the unpaired student’s
We first investigated whether crotamiton has the potential to inhibit signals induced by histamine. HEK293T cells that transiently express both H1R and TRPV1 (H1R/TRPV1), were used to develop an experimental model for calcium imaging, because histamine-induced itching is mostly triggered by calcium influx after coordinated activation of H1R and TRPV1 (Shim
As shown in Fig. 1A and 1B, intracellular calcium levels were significantly increased upon histamine (10 µM) stimulation in H1R/TRPV1 cells (“Con”, 874 cells), suggesting that the experimental model was successfully developed. However, upon pretreatment with crotamiton (1 mM) for 5 min, a negligible increase in intracellular calcium levels (“+crotamiton”, 838 cells, Fig. 1A-1C) was observed after subsequent histamine treatment, suggesting that crotamiton inhibits histamine-induced calcium influx in H1R/TRPV1 cells. Moreover, the inhibitory effect of crotamiton was concentration-dependent (Fig. 1D, IC50=101.2 µM), indicating a receptor-mediated inhibitory effect, probably via the H1R/TRPV1 pathway. To further investigate the effect of crotamiton, cells expressing only TRPV1 were used. As shown in Fig. 1E, pretreatment with crotamiton (1 mM) barely decreased the calcium influx induced by capsaicin (a TRPV1 agonist, 1 µM, 543 cells) compared to control (704 cells). Overall, it was found that crotamiton inhibits histamine-induced calcium influx via the H1R/TRPV1 pathway; however, it does not directly involve TRPV1.
To further verify whether crotamiton also inhibits chloroquine-induced itching, a similar experimental approach was followed using HEK293T cells transiently expressing both MRGPRA3 and TRPA1 (MRGPRA3/TRPA1).
The calcium influx induced by chloroquine (1 mM) significantly reduced upon pretreatment with crotamiton (1 mM) for 5 min (“Con”: 1065 cells
It was further investigated whether all the effects of crotamiton were due to non-specific reasons, such as cytotoxicity. The MTT assay revealed that incubation of HEK293T cells with crotamiton (1 mM) for 30 min did not cause any significant changes in cell viability (Control: 103.5 ± 2.334%
Scabies is accompanied by severe itching. Therefore, it is plausible that most anti-scabies agents also have similar anti-pruritic effects. For this purpose, the effects of permethrin, an anti-scabies agent with a different molecular structure, were compared with those of crotamiton by calcium imaging. However, pretreatment with permethrin (1 mM) did not exert any inhibitory effect on the calcium influx induced by either histamine- (103.2 ± 3.947%, 325 cells) or chloroquine (125.9 ± 7.323%, 193 cells), as shown in Fig. 3B. However, pretreatment with crotamiton (1 mM) significantly inhibited both histamine- (12.43 ± 4.026%, 441 cells) and chloroquine-induced calcium influx (12.89 ± 2.374%, 189 cells). Thus, this result strongly indicated that the inhibitory effects of crotamiton on calcium influx induced by histamine or chloroquine are intrinsic and specific because permethrin failed to inhibit histamine- and chloroquine-induced calcium influx.
To further verify the inhibitory effect of crotamiton on histamine- and chloroquine-induced responses in sensory neurons, primary cultures of mouse DRG neurons were used, and calcium influx was measured by using calcium imaging techniques. It was found that crotamiton (1 mM) significantly reduced the number of histamine (100 µM)-responsive DRG neurons (Fig. 4A). However, the calcium influx induced by capsaicin (1 µM) was not altered by crotamiton pretreatment (Fig. 4B), which is in agreement with the findings shown in Fig. 1.
Similar results were obtained in chloroquine (1 mM)-stimulated DRG neurons. Crotamiton (1 mM) significantly reduced the chloroquine-induced calcium influx in DRG neurons and the number of chloroquine-responsive neurons (Fig. 4C). However, the calcium influx induced by AITC (100 µM) was not affected by crotamiton pretreatment (Fig. 4D), consistently with the findings shown in Fig. 2.
Therefore, these data strongly suggest that crotamiton inhibits histamine- and chloroquine-induced calcium influx in sensory neurons.
Finally, the
Scabies is a contagious skin infection caused by the scabies mite, and its hallmark clinical symptom is a severe itching. Although crotamiton has long been used for the treatment of scabies, other drugs such as permethrin and ivermectin are preferred (Larson, 2008; Goldust
It had been vaguely predicted that crotamiton might inhibit histamine-induced itching by inhibiting H1R (Sekine
A recent study showed that crotamiton also inhibits the activation of TRPV4 by a specific TRPV4 agonist, GSK1016790A (Kittaka
Researchers are beginning to embrace the concept of G-protein-coupled receptor (GPCR)–TRP axis, which explains the common molecular mechanisms underlying transmission of various sensory signals, including itching (Veldhuis
Our results showed that crotamiton does not directly inhibit TRP ion channels. Specifically, it was found that it does not significantly inhibit capsaicin-induced TRPV1 activation (Fig.1D). Likewise, crotamiton failed to inhibit chloroquine-induced TRPA1 activation (Fig. 2D). Kittaka
Although TRPV1 and TRPA1 contribute to itch transmission in sensory neurons, the major function of these ion channels is sensing nociceptive stimuli, such as pain and temperature (Vay
Overall, it was found that crotamiton, an anti-scabies agent, has novel inhibitory effects on both histamine- and chloroquine-dependent itch pathways in sensory neurons. In addition, it suppressed scratching behaviors induced by both histamine and chloroquine in mice. Therefore, the present study suggests that crotamiton can be used as a novel anti-pruritic agent in addition to being a scabicidal agent.
This work was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1F1A1060704).
The authors declare that they have no conflicts of interest.
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