Neuromedin B Induces Acute Itch in Mice via the Activation of Peripheral Sensory Neurons.

Neuromedin B is expressed in nociceptive and itch-sensitive dorsal root ganglia neurons, but its peripheral pruritogenic potential is not well described. The potential of neuromedin B as a pruritogen and pro-inflammatory peptide in the skin was tested in vivo in an acute model in mice and monkeys as well as an allergic dermatitis model in mice. To identify the underlying mechanisms in vitro real time PCR analysis for neuromedin B and its receptor expression in murine mast cells and dorsal root ganglia as well as functional calcium imaging in the ganglia was applied. Neuromedin B induces itch when injected intradermally, and the peripheral signal is likely transmitted through the activation of dorsal root ganglia. Thus, neuromedin B could be an interesting new therapeutic target for peripheral processing of itch at the level of sensory neurons.

Diphenhydramine pharmacokinetics after oral and intravenous administration of diphenhydramine and oral administration of dimenhydrinate to healthy dogs, and pharmacodynamic effect on histamine-induced wheal formation: a pilot study.

BACKGROUND: Histamine type-1 (H1) receptor antagonists such as diphenhydramine are frequently used for treatment of pruritus in dogs, yet therapeutic efficacy for allergic disorders is reported to be highly variable. Dimenhydrinate is a salt of diphenhydramine and 8-chlorotheophylline, and has been reported to produce superior oral absorption of diphenhydramine. HYPOTHESIS/OBJECTIVE: To determine the pharmacokinetic and pharmacodynamic properties of diphenhydramine in dogs after intravenous (1 mg/kg) and oral (5 mg/kg) administration, and when given orally as dimenhydrinate at a dose of 10 mg/kg (≈5 mg/kg diphenhydramine). ANIMALS: Each drug was administered to six healthy, fasted mixed-breed dogs in a research facility, using a cross-over design. METHODS AND MATERIALS: Blood samples were collected for pharmacokinetic analysis of diphenhydramine and chlorotheophylline at defined intervals. Pharmacodynamic response was measured by histamine-mediated cutaneous wheal formation. RESULTS: There was great variability in the data and one dog was an extreme outlier. The mean systemic availabilities of diphenhydramine were 7.8% and 22.0% after oral administration of diphenhydramine and dimenhydrinate, respectively, whereas the mean maximum concentrations were 36 (± 20) and 124 (± 46) ng/mL. The terminal elimination half-lives of diphenhydramine and dimenhydrinate were 5.0 (± 7.1) and 11.6 (± 17.7) h, respectively. Plasma diphenhydramine concentrations did not correlate with the percentage reduction in histamine-induced wheal formation. Theophylline reached plasma concentrations considered to be therapeutic for dogs. CONCLUSION: Oral absorption of diphenhydramine was approximately three times greater with a longer half-life when it was administered as the combination product dimenhydrinate.

To scratch an itch: Establishing a mouse model to determine active brain areas involved in acute histaminergic itch.

BACKGROUND: Strategies to efficiently control itch require a deep understanding of the underlying mechanisms. Several areas in the brain involved in itch and scratching responses have been postulated, but the central mechanisms that drive pruritic responses are still unknown. Histamine is recognized as a major mediator of itch in humans, and has been the most frequently used stimulus as an experimental pruritogen for brain imaging of itch. OBJECTIVE: Histaminergic itch via histamine and the selective histamine H4 receptor (H4R) agonist, ST-1006, recruit brain nuclei through c-fos activation and activate specific areas in the brain. METHODS: An acute itch model was established in c-fos-EGFP transgenic mice using ST-1006 and histamine. Coronal brain sections were stained for c-fos immunoreactivity and the forebrain was mapped for density of c-fos + nuclei. RESULTS: Histamine and ST-1006 significantly increased scratching response in c-fos-EGFP mice compared to vehicle controls. Mapping c-fos immunostained brain sections revealed neuronal activity in the cortex, striatum, hypothalamus, thalamus, amygdala, and the midbrain. CONCLUSIONS: Histaminergic itch and selective H4R activation significantly increased the density of c-fos + nuclei in the medial habenula (MHb). Thus, the MHb may be a new target to investigate and subsequently develop novel mechanism-based strategies to treat itch and possibly provide a locus for pharmacological control of pruritus.

Comparison of topical tofacitinib and 0.1% hypochlorous acid in a murine atopic dermatitis model.

BACKGROUND: Topical administration of PR022, 0.05% hypochlorous acid (HOCl) in gel has been demonstrated to be beneficial in a chronic murine atopic dermatitis model. In a follow up study we tested a higher concentration (0.1%) of PR022 HOCl gel in comparison to the Janus kinase inhibitor tofacitinib, both of which are currently in clinical phase studies for treatment of human atopic dermatitis. METHODS: The effect of topically administered HOCl (0.1%) in gel was compared to a topical formulation of tofacitinib (0.5%) in a therapeutic setting on atopic dermatitis-like lesions in NC/Nga mice as well as itch behaviour. NC/Nga mice were sensitized with house dust mite allergen. After reaching visible lesions, mice were treated either topically with HOCl or tofacitinib or gel vehicle for 17 days. After termination of the study, dorsal root ganglia were isolated for ex vivo stimulation and skin samples were taken for cytokine determination in inflamed skin. RESULTS: When administered onto lesional skin of NC/Nga mice, both HOCl and tofacitinib reduced lesions and scratching behaviour. The reduced inflammatory response by HOCl and tofacitinib treatment was demonstrated by diminished inflammatory cytokines in affected skin tissue from NC/Nga mice. Dorsal root ganglia neurons re-stimulated with a range of mediators of itch showed a reduced response compared to the vehicle control mice, when isolated from tofacitinib or HOCl treated mice. CONCLUSIONS: These data indicate a similar beneficial potential of topical high dose PR022 HOCl (0.1%) in gel and tofacitinib, in a translational murine model of atopic dermatitis.

Impact of synthetic canine cerumen on in vitro penetration of auricular skin of dogs by florfenicol, terbinafine, and betamethasone acetate.

OBJECTIVE To determine the pharmacokinetics of florfenicol, terbinafine, and betamethasone acetate after topical application to canine auricular skin and the influence of synthetic canine cerumen on pharmacokinetics. SAMPLE Auricular skin from 6 euthanized shelter dogs (3 females and 3 neutered males with no visible signs of otitis externa). PROCEDURES Skin adjacent to the external opening of the ear canal was collected and prepared for use in a 2-compartment flow-through diffusion cell system to evaluate penetration of an otic gel containing florfenicol, terbinafine, and betamethasone acetate over a 24-hour period. Radiolabeled 14C-terbinafine hydrochloride and 3H-betamethasone acetate were added to the gel to determine dermal penetration and distribution. Florfenicol absorption was determined by use of high-performance liquid chromatography-UV detection. Additionally, the effect of synthetic canine cerumen on the pharmacokinetics of all compounds was evaluated. RESULTS During the 24-hour experiment, mean ± SD percentage absorption without the presence of synthetic canine cerumen was 0.28 ± 0.09% for 3H-betamethasone acetate, 0.06 ± 0.06% for florfenicol, and 0.06 ± 0.02% for 14C-terbinafine hydrochloride. Absorption profiles revealed no impact of synthetic canine cerumen on skin absorption for all 3 active compounds in the gel or on skin distribution of 3H-betamethasone acetate and 14C-terbinafine hydrochloride. CONCLUSIONS AND CLINICAL RELEVANCE 3H-betamethasone acetate, 14C-terbinafine hydrochloride, and florfenicol were all absorbed in vitro through healthy auricular skin specimens within the first 24 hours after topical application. Synthetic canine cerumen had no impact on dermal absorption in vitro, but it may serve as a temporary reservoir that prolongs the release of topical drugs.

Distinct neuronal populations in the basolateral and central amygdala are activated with acute pain, conditioned fear, and fear-conditioned analgesia.

Fear-conditioned analgesia (FCA) is modulated by brain areas involved in the descending inhibitory pain pathway such as the basolateral (BLA) and central amygdala (CEA). The BLA contains Ca2+/calmodulin-dependent protein kinase II (CaMKII) and parvalbumin (PV) neurons. CEA neurons are primarily inhibitory (GABAergic) that comprise enkephalin (ENK) interneurons and corticotropin-releasing factor (CRF) - neurons that project to the periaqueductal grey. The purpose of our experiment was to determine the pattern of activation of CaMKII/PV and ENK/CRF neurons following the expression of acute pain, conditioned fear, and FCA. A significant reduction was observed in nociceptive behaviors in mice re-exposed to a contextually-aversive environment. Using NeuN and cFos as markers for activated neurons, CaMKII, PV, ENK, or CRF were used to identify neuronal subtypes. We find that mice expressing conditioned fear displayed an increase in c-Fos/CaMKII co-localization in the lateral amygdala and BLA compared to controls. Additionally a significant increase in cFos/CRF co-localization was observed in mice expressing FCA. These results show that amygdala processing of conditioned contextual aversive, nociceptive, and FCA behaviors involve different neuronal phenotypes and neural circuits between, within, and from various amygdala nuclei. This information will be important in developing novel therapies for treating pain and emotive disorders in humans.

Allergic inflammation is augmented via histamine H4 receptor activation: The role of natural killer cells in vitro and in vivo.

BACKGROUND: Natural Killer cells (NK cells) are identified as pivotal mediators in allergic skin diseases and accumulate in lesions of atopic dermatitis (AD) patients. Histamine levels are increased in these lesions and histamine is involved in chemotaxis in dendritic cells and NK cells. OBJECTIVE: The aim of this study was to determine if the histamine H4 receptor (H4R) mediates NK cell chemotaxis and whether it influences interplay between NK cells and dendritic cells during the early phase of allergic inflammation. METHODS: Chemotactic function of the H4R as well as the influence of the H4R on the cytokine profile of an NK cell-dendritic cell co-culture was studied in vitro. The effect of H4R activation on NK cell migration, NK cell-dendritic cell interaction and cytokine levels in the skin was further characterized in the murine TDI model of allergic dermatitis. Additionally, the impact of the H4R on dermal NK cells was determined in the ovalbumin (OVA)- induced allergic dermatitis model, comparing wild type and H4R knockout mice. RESULTS: The selective H4R agonist ST-1006 induced NK cell chemotaxis in vitro, which was inhibited with the H4R antagonist JNJ7777120. In vivo, mice treated with TDI plus ST-1006 topically onto the ear, showed significantly enhanced ear swelling and an increased number of NK cells compared to just allergen challenged ears. CCL17 levels in the ear were also significantly increased 8h after allergen challenge. Histology revealed that the main source for increased CCL17 were dendritic cells. These effects could be blocked using the H4R antagonist JNJ7777120. In the chronic model of allergic dermatitis, OVA induced NK cell migration into lesional skin sites. The number of NK cells was lower in OVA-sensitized H4R knockout mice compared to wild type mice. CONCLUSIONS: These results identify the H4R as a new target controlling NK cell migration and NK cell-dendritic cell interaction in the skin during early allergic inflammation. These results further suggest that blocking the H4R in the skin might be beneficial in diseases like AD.

Topically Administered Janus-Kinase Inhibitors Tofacitinib and Oclacitinib Display Impressive Antipruritic and Anti-Inflammatory Responses in a Model of Allergic Dermatitis.

The prevalence of allergic skin disorders has increased rapidly, and development of therapeutic agents to alleviate the symptoms are still needed. In this study, we orally or topically administered the Janus kinase (JAK) inhibitors, tofacitinib and oclacitinib, in a mouse model of dermatitis, and compared the efficacy to reduce the itch and inflammatory response. In vitro effects of JAK inhibitors on bone marrow-derived dendritic cells (BMDCs) were analyzed. For the allergic dermatitis model, female BALB/c mice were sensitized and challenged with toluene-2,4-diisocyanate (TDI). Each JAK inhibitor was orally or topically applied 30 minutes before and 4 hours after TDI challenge. After scratching bouts and ear thickness were measured, cytokines were determined in challenged skin and the cells of the draining lymph node were analyzed by means of flow cytometry. In vitro, both JAK inhibitors significantly inhibited cytokine production, migration, and maturation of BMDCs. Mice treated orally with JAK inhibitors showed a significant decrease in scratching behavior; however, ear thickness was not significantly reduced. In contrast, both scratching behavior and ear thickness in the topical treatment group were significantly reduced compared with the vehicle treatment group. However, cytokine production was differentially regulated by the JAK inhibitors, with some cytokines being significantly decreased and some being significantly increased. In conclusion, oral treatment with JAK inhibitors reduced itch behavior dramatically but had only little effect on the inflammatory response, whereas topical treatment improved both itch and inflammatory response. Although the JAK-inhibitory profile differs between both JAK inhibitors in vitro as well as in vivo, the effects have been comparable.

Effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine primary hepatocytes.

OBJECTIVE: To investigate the cytotoxic effects of azathioprine, 6-mercaptopurine, and 6-thioguanine on canine hepatocytes. SAMPLE: Commercially available cryopreserved canine primary hepatocytes. PROCEDURES: The study consisted of 2 trials. In trial 1, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 6 concentrations (0.468, 0.937, 1.875, 3.750, 7.500, or 15.000 µmol/L) for 24, 48, or 72 hours. At each time, cell viability and lactate dehydrogenase (LDH) activity were determined for each thiopurine-concentration combination, and alanine aminotransferase (ALT) activity was determined for cells incubated with each thiopurine at a concentration of 15 µmol/L. In trial 2, hepatocytes were incubated with azathioprine, 6-mercaptopurine, or 6-thioguanine at 1 of 3 concentrations (18.75, 37.50, or 75.00 µmol/L) for 24 hours, after which the free glutathione concentration was determined for each thiopurine-concentration combination and compared with that for hepatocytes incubated without a thiopurine (control). RESULTS: Incubation of hepatocytes with each of the 3 thiopurines adversely affected cell viability in a time- and concentration-dependent manner; however, this decrease in cell viability was not accompanied by a concurrent increase in LDH or ALT activity. Likewise, free glutathione concentration for hepatocytes incubated for 24 hours with supratherapeutic thiopurine concentrations (> 18.75 µmol/L) did not differ significantly from that of control cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that thiopurines adversely affected the viability of canine hepatocytes in a time- and concentration-dependent manner but had a nonsignificant effect on the LDH and ALT activities and free glutathione depletion of those hepatocytes.

Histamine induces proliferation in keratinocytes from patients with atopic dermatitis through the histamine 4 receptor.

BACKGROUND: Epidermal hyperproliferation resulting in acanthosis is an important clinical observation in patients with atopic dermatitis, and its underlying mechanisms are not completely understood. OBJECTIVE: Because increased levels of histamine are present in lesional skin, we investigated the effect of histamine, especially with regard to histamine 4 receptor (H4R) activation, on the proliferation of human and murine keratinocytes. METHODS: The expression of H4R on human and murine keratinocytes was detected by using real-time PCR. Keratinocyte proliferation was evaluated by using different in vitro cell proliferation assays, scratch assays, and measurement of the epidermal thickness of murine skin. RESULTS: We detected H4R mRNA on foreskin keratinocytes and on outer root sheath keratinocytes; H4R mRNA was more abundant in keratinocytes from patients with atopic dermatitis compared with those from nonatopic donors. Stimulation of foreskin keratinocytes, atopic dermatitis outer root sheath keratinocytes, and H4R-transfected HaCaT cells with histamine and H4R agonist resulted in an increase in proliferation, which was blocked with the H4R-specific antagonist JNJ7777120. Abdominal epidermis of H4R-deficient mice was significantly thinner, and the in vitro proliferation of keratinocytes derived from H4R-deficient mice was lower compared with that seen in control mice. Interestingly, we only detected H4R expression on murine keratinocytes after stimulation with LPS and peptidoglycan. CONCLUSION: H4R is highly expressed on keratinocytes from patients with atopic dermatitis, and its stimulation induces keratinocyte proliferation. This might represent a mechanism that contributes to the epidermal hyperplasia observed in patients with atopic dermatitis.