Thyrotropin-Releasing Hormone Analog Taltirelin Inhibits Acute and Chronic Itch in Mice

Abstract

Itch is an unpleasant sensation that induces a desire to scratch. Chronic itch is accompanied by inflammatory skin diseases and causes repetitive scratching leading to tissue damage. The thyrotropin-releasing hormone analog taltirelin exerts analgesic effects on acute and chronic pain by activating the descending inhibitory systems. However, little is known regarding its effects on acute and chronic itch. In this study, we examined the effects of taltirelin on acute and chronic itch. Subcutaneous injection of chloroquine increased the number of scratching bouts in 30 min, whereas intraperitoneal injection of taltirelin reduced these episodes in a dose-dependent manner. In addition, chronic itch induced by diphenylcyclopropenone treatment was alleviated by taltirelin injection. These findings indicate that taltirelin alleviates acute and chronic itch in mice, and can be a potent therapeutic antipruritic drug.

INTRODUCTION

Itch is an unpleasant sensation that induces a desire to scratch. Long-lasting itch, known as chronic itch, leads to repeated scratching, which disrupts the skin barrier and induces inflammation.¹⁾ Chronic itch is associated with emotional disorders such as depression and reduces the QOL of patients.²⁾

Thyrotropin-releasing hormone (TRH) is released from the hypothalamus and induces the secretion of thyroid-stimulating hormone from the anterior pituitary gland, thereby contributing to metabolic regulation.³⁾ In addition, TRH can directly regulate the activity of neurons expressing TRH receptors in various brain regions, resulting in changes in animal behavior. TRH and its analog taltirelin, marketed in Japan as a drug, act on neurons in the locus coeruleus and increase their activity.⁴⁾ In addition, activation of locus coeruleus neurons by taltirelin suppresses acute and chronic pain via α2-adrenergic receptors in the spinal cord.^5,6) However, it is unknown whether TRH and taltirelin alleviate acute and chronic itch.

In this study, we examined the effects of taltirelin on acute and chronic itch by conducting behavioral experiments in mice. We adopted the chloroquine (CQ)-induced acute itch model, which shows transient scratching behavior after CQ injection, and the diphenylcyclopropenone (DCP)-induced chronic itch model, which shows persistent scratching behavior. We demonstrated that CQ-induced scratching behavior was suppressed by taltirelin in a dose-dependent manner. Moreover, chronic itch induced by DCP treatment was suppressed by taltirelin treatment. These results indicate that taltirelin is a potent inhibitor of acute and chronic itch in mice.

MATERIALS AND METHODS

This study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals prescribed by the Japanese Physiological Society. All the animal experiments were approved by the Kitasato University Institutional Animal Care and Use Committee (Approval Number: EiKen 20-11-3).

Animals

Adult male C57BL/6 mice older than 8 weeks were purchased from Japan SLC. All the mice were housed in cages with ad libitum access to food and water. The room was maintained at about 25 °C on a 12-h light/12-h dark cycle.

Mouse Model of Acute Itch

One day before developing an animal model of acute itch, the cheek of each mouse was shaved with electric clippers (Natsume Seisakusho Co., Ltd., Tokyo, Japan). Next, 30 µL CQ (Sigma-Aldrich, St. Louis, MO, U.S.A.) was subcutaneously injected into the cheek to create an acute itch model. Immediately after CQ injection, the mouse behavior was recorded using a web camera (C920n; Logicool, Tokyo, Japan) for 30 min. Saline was injected into the cheek of mice in a control group. Taltirelin (Tocris, Minneapolis, MN, U.S.A.) or saline was injected intraperitoneally 30 min before CQ injection. Scratching bouts were defined as paw lifting and scratching behavior and were manually counted offline.

Mouse Model of Chronic Itch

One day before treatment with DCP (Sigma-Aldrich), the hair on the neck of each mouse was shaved with electric clippers (Natsume Seisakusho). Fifty microliters of DCP (1%) dissolved in acetone was applied to the back of the neck. Seven days after the first application of DCP, the same amount of DCP (1%) was applied to the same region. One and two days after 2nd application of DCP, the mice were placed in a small plastic cage and their behavior was recorded using a web camera (C920n; Logicool, Tokyo, Japan) for 1 h. Saline and taltirelin were intraperitoneally applied 30 min before starting behavioral recording. Scratching bouts were manually analyzed offline.

Open Field Test

The open field tests were performed in an open field apparatus (40 × 40 × 40 cm). Each mouse was placed in the center of the open field apparatus, and the behavior was recorded using a web camera (C920n: Logicool) for 10 min. Locomotion activities were analyzed using video tracking software (ANY-Maze, Stoelting Co., Wood Dale, IL, U.S.A.).

Statistical Analysis

Data are presented as the mean ± standard error of the mean (S.E.M.). Statistical comparisons were performed using Origin PRO 8.6 J (OriginLab, Northampton, MA, U.S.A.). Data on scratching bouts induced by CQ injection were analyzed using one-way ANOVA followed by Bonferroni’s test. Data from scratching bouts induced by DCP treatment were analyzed using a paired or unpaired Student’s t-test. Differences were considered statistically significant at p < 0.05.

RESULTS AND DISCUSSION

First, we examined the effect of taltirelin or saline on acute itch induced by subcutaneous injection of CQ into the cheeks of mice. The mice barely scratched before CQ injection and after saline injection into the mouse’s cheek (saline + saline, 11.0 ± 3.93 bouts/30 min, n = 6) (Figs. 1A, B). After CQ administration, the number of scratching bouts increased, and the effect persisted for 30 min (Fig. 1A). However, 1.0 mg/kg taltirelin reduced the number of CQ-induced scratching bouts compared to saline-injected mice (Fig. 1A). Next, we examined the dose-dependency of the antipruritic effect of taltirelin. At 0.1 mg/kg, taltirelin did not change CQ-induced scratching bouts compared to saline-injected mice (CQ + taltirelin 0.1 mg/kg, 94.7 ± 9.23 bouts/30 min, n = 7; CQ + saline, 98.0 ± 13.6 bouts/30 min n = 9, p > 0.05), while high concentrations of taltirelin (1.0 and 3.0 mg/kg) significantly reduced the number of scratching bouts (CQ + taltirelin 1.0 mg/kg, 54.4 ± 11.6 bouts/30 min, n = 14, p < 0.05; CQ + taltirelin 3.0 mg/kg, 5.38 ± 2.08 bouts/30 min, n = 8, p < 0.05) (Fig. 1B), suggesting that taltirelin reduced the number of scratching bouts induced by CQ in a dose-dependent manner. These data suggest that taltirelin prevents CQ-induced acute itch.

Fig. 1. Taltirelin Alleviated CQ-Induced Acute Itch in a Dose-Dependent Manner

(A) Time course of the mean number of scratching bouts after saline or CQ injection into the cheek. Tal, taltirelin. (B) Effects of intraperitoneal injection (i.p.) of drugs on the number of scratching bouts in 30 min (Saline + Saline (i.p.), n = 6; CQ + Saline (i.p.), n = 9; CQ + taltirelin 0.1 mg/kg (i.p.), n = 7; CQ + taltirelin 1.0 mg/kg (i.p.), n = 14; CQ + taltirelin 3.0 mg/kg (i.p.), n = 8). Data represent the mean ± S.E.M. * p < 0.05 vs. saline. Data were analyzed by one-way ANOVA with Bonferroni post hoc test.

Since it has been reported that taltirelin can alter locomotion activities of mice,⁷⁾ it is possible that this effect may affect pruritic behaviors. Thus, we performed open field tests to examine the effect of 1 mg/kg taltirelin on locomotion activities. The locomotion activity of mice with saline was consistent with that with taltirelin (1 mg/kg) (saline, 36.9 ± 3.30 m, n = 5, taltirelin, 33.9 ± 1.53 m, n = 5, p > 0.05). Thus, this result suggests that taltirelin (1 mg/kg) does not affect locomotion activities measured by open field tests.

To confirm whether taltirelin alleviates chronic itch, we examined its effect on chronic itch induced by DCP treatment. DCP was applied to the back of the neck of mice on days 0 and 7 (Fig. 2A). Since the effect of DCP treatment may be different for each mouse, at first, we confirmed whether DCP treatment caused chronic itch in each mouse in the experiment (Pre) on day 8, and then we examined the effect of drugs on the scratching behavior in the experiment (Post) on day 9. DCP treatment induced robust spontaneous scratching behavior on day 8 and saline injection did not change the number of scratching bouts induced by the DCP treatment (Pre, 176.4 ± 26.9 bouts/60 min, n = 4; Post, 208.1 ± 43.0 bouts/60 min, n = 8, p > 0.05) (Fig. 2B). However, intraperitoneal injection of 1.0 mg/kg taltirelin significantly reduced the number of scratching bouts induced on day 9 after the 1st DCP treatment (Post) compared with that before taltirelin injection (Pre) (Pre, 176.6 ± 26.8, n = 8, Post, 66 ± 12.2 bouts/60 min, n = 8, p < 0.05) (Fig. 2C). Moreover, relative scratching bouts were decreased by taltirelin treatment compared with saline treatment (DCP + saline, 1.55 ± 0.44, n = 8, DCP + taltirelin 0.41 ± 0.07, n = 8, p < 0.05) (Fig. 2D). These results suggest that taltirelin alleviates chronic itch induced by DCP treatment.

Fig. 2. Taltirelin Alleviated DCP-Induced Chronic Itch

(A) Diagram of experiments. Exp, behavior experiment. (B) Number of scratching bouts before (Pre) and after (Post) saline injection (n = 8). (C) Number of scratching bouts before (Pre) and after (Post) 1.0 mg/kg taltirelin injection (n = 8). * p < 0.05. Paired Student’s t-test. (D) Relative scratching bouts calculated from data in (B) and (C). Data represent the mean ± S.E.M. * p < 0.05. Unpaired Student’s t-test.

Itch-related behavior is regulated by the noradrenergic descending inhibitory system in the spinal cord. Depletion of noradrenergic neurons reduces the content of noradrenaline in the spinal cord, which in turn enhances itch-related behavior.⁸⁾ In addition, intrathecal injection of α1- and α2-adrenergic receptor agonists reduces itch-related responses, respectively.⁸⁾ Furthermore, direct activation of locus coeruleus noradrenergic neurons inhibits CQ-induced itch-related behavior by activation of the inhibitory neurons of the spinal cord via α1-adrenergic receptors.^9,10) Thus, both α1- and α2-adrenergic receptors in the spinal cord are involved in the regulation of itch behavior by the noradrenergic descending inhibitory system. In the present study, we demonstrated that acute itch induced by CQ injection was inhibited by taltirelin in a dose-dependent manner. This effect may be a consequence of the activation of the descending inhibitory pathway. Taltirelin directly excites noradrenergic neurons in the locus coeruleus⁴⁾ and increases the release of noradrenaline in the spinal cord, resulting in alleviating acute pain via α2-adrenergic receptors.⁵⁾ Thus, the antipruritic effect of taltirelin we observed in the present study may be induced by activation of the noradrenergic descending inhibitory system in the spinal cord. Further research is needed to clarify the mechanisms underlying the antipruritic action of taltirelin in CQ-induced acute itch.

Chronic itch is induced by chronic inflammatory skin diseases such as dry skin and contact dermatitis.¹¹⁾ DCP, which causes persistent itch, is used as a model of contact dermatitis.¹²⁾ Repeated scratching of the itchy area exacerbates inflammation, and as the inflammation worsens, astrocytes are activated in the spinal cord.¹²⁾ These activated astrocytes release the inflammatory factor lipocalin-2 and increase neuronal activity in the spinal cord, leading to the induction of chronic itch.^13,14) The descending inhibitory system inhibits neuronal activity and alleviates chronic itch. Activation of noradrenergic neurons in the locus coeruleus using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) suppresses DCP-induced chronic itch.⁹⁾ Herein, we revealed that taltirelin suppresses chronic itch induced by DCP application. Taltirelin suppresses chronic inflammatory pain by activating the descending inhibitory pathway, including noradrenergic and serotonergic neurons.⁶⁾ Therefore, the antipruritic effect of taltirelin on DCP-induced chronic itch may be mediated by the activation of noradrenergic and serotonergic neurons in the brainstem, which in turn inhibits neuronal activity in the spinal dorsal horn and alleviates the condition.

In the present study, we observed the scratching behavior induced by DCP treatment to the back of the nape. Scratching behavior basically indicates itch in animal experiments. However, the behavior can be induced by injection of capsaicin, an algogen, into the back of the neck.¹⁵⁾ Since the pruritic stimulation and the nociceptive stimulation to the back of the neck can induce scratching behavior, the scratching behavior induced by DCP treatment to the back of the neck may be evoked by itch sensation and pain sensation. Therefore, further study is needed to clarify the precise effect of taltirelin on chronic itch using different models of chronic itch.

In summary, the present study revealed that the TRH analog, taltirelin, exerts an antipruritic effect on acute and chronic itch. Taltirelin is more stable than TRH and its effect lasts longer.⁵⁾ Therefore, these results suggest that taltirelin may be a good antipruritic drug candidate for chronic itch.

Acknowledgments

The study was supported by Kitasato University.

Conflict of Interest

The authors declare no conflict of interest.

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