Pharmacological characterization of the new histamine H₄ receptor agonist VUF 8430

Abstract

Background and purpose:

We compare the pharmacological profiles of a new histamine H₄ receptor agonist 2-(2-guanidinoethyl)isothiourea (VUF 8430) with that of a previously described H₄ receptor agonist, 4-methylhistamine.

Experimental approach:

Radioligand binding and functional assays were performed using histamine H₄ receptors expressed in mammalian cell lines. Compounds were also evaluated ex vivo in monocyte-derived dendritic cells endogenously expressing H₄ receptors and in vivo in anaesthetized rats for gastric acid secretion activity.

Key results:

Both VUF 8430 and 4-methylhistamine were full agonists at human H₄ receptors with lower affinity at rat and mouse H₄ receptors. Both compounds induced chemotaxis of monocyte-derived dendritic cells. VUF 8430 also showed reasonable affinity and was a full agonist at the H₃ receptor. Agmatine is a metabolite of arginine, structurally related to VUF 8430, and was a H₄ receptor agonist with micromolar affinity. At histamine H₃ receptors, agmatine was a full agonist, whereas 4-methylhistamine was an agonist only at high concentrations. Both VUF 8430 and agmatine were inactive at H₁ and H₂ receptors, whereas 4-methylhistamine is as active as histamine at H₂ receptors. In vivo, VUF 8430 only caused a weak secretion of gastric acid mediated by H₂ receptors, whereas 4-methylhistamine, dimaprit, histamine and amthamine, at equimolar doses, induced 2.5- to 6-fold higher output than VUF 8430.

Conclusions and implications:

Our results suggest complementary use of 4-methylhistamine and VUF 8430 as H₄ receptor agonists. Along with H₄ receptor antagonists, both agonists can serve as useful pharmacological tools in studies of histamine H₄ receptors.

Introduction

Histamine is a chemical mediator that controls many physiological functions through the interaction with four histamine receptor subtypes, which are all members of the large multigene family of G-protein coupled receptors (Parsons and Ganellin, 2006). The histamine H₁ and H₂ receptors represent very successful therapeutic targets (Parsons and Ganellin, 2006; receptor nomenclature follows Alexander et al., 2008), while the H₃ receptor and H₄ receptor have emerged as potential targets for future treatment of central nervous system (CNS) disorders (Leurs et al., 2005) and inflammatory diseases (de Esch et al., 2005; Lim et al., 2006b) respectively. The potential therapeutic use is strongly related to the relative selective tissue distribution of both the H₃ receptor and H₄ receptor. Whereas the H₃ receptor is mainly present in the nervous system, the H₄ receptor is primarily localized on haematopoietic cells (Oda et al., 2000; Liu et al., 2001a; Morse et al., 2001; Zhu et al., 2001). The H₄ receptor has been demonstrated to be involved in the chemotaxis of mast cells, eosinophils and monocyte-derived dendritic cells (MoDCs) (Hofstra et al., 2003; Ling et al., 2004; Gutzmer et al., 2005), to control mediator production, such as interleukin (IL)-16 release by human CD8⁺ T cells (Gantner et al., 2002), leukotriene B₄ production by mast cells (Takeshita et al., 2003; Thurmond et al., 2004), and suppression of IL-12p70 production by MoDCs (Gutzmer et al., 2005). Various studies suggest that the H₄ receptor is a potential new drug target for inflammatory diseases, including chronic allergy, asthma, atopic dermatitis and inflammatory bowel diseases (Thurmond et al., 2004; Dunford et al., 2006; 2007). Moreover, the H₄ receptor has been detected in primary synovial culture obtained from rheumatoid arthritis patients and colorectal cancer tissues, suggesting a possible role for H₄ receptors in these diseases as well (Cianchi et al., 2005; Ohki et al., 2007).

Despite a growing body of evidence, validation of the histamine H₄ receptor as a drug target is mandatory. For this purpose, selective and potent agonists and antagonists for this receptor are needed. The H₃ receptor and H₄ receptor proteins are each other's closest relatives and show a relatively high level of homology (Hough, 2001), especially within the seven transmembrane domains, which are thought to bind small molecule agonists and antagonists. As might be expected on the basis of such a high homology, the H₄ receptor binds many imidazole-based H₃ receptor ligands with high affinity (Lim et al., 2005; Gbahou et al., 2006). The previously presumed H₃ receptor-selective, inverse agonist, thioperamide (Arrang et al., 1987) binds to the related H₄ receptor with equal affinity and is therefore now classified as a non-selective H₃/H₄ receptor inverse agonist (Lim et al., 2005). Some pharmaceutical companies have meanwhile started to focus on the discovery of selective non-imidazole H₄ receptor antagonists. This involvement has resulted in a number of patent applications for potent H₄ receptor antagonists (Lim et al., 2006b). Currently, JNJ 7777120 (Jablonowski et al., 2003) and the related VUF 6002 (Terzioglu et al., 2004; Venable et al., 2005) should be considered as prototypic non-imidazole H₄ receptor antagonists.

Considering their high value in research, we have focused on the discovery of selective non-imidazole H₄ receptor agonists, as well. Previously, we have described the histamine analogue, 4-methylhistamine (Durant et al., 1975) as a potent H₄ receptor agonist (Lim et al., 2005). Moreover, we recently reported on the synthesis and initial structure–activity relationships (SAR) of the non-imidazole H₄ receptor agonist 2-(2-guanidinoethyl)isothiourea (VUF 8430) (Lim et al., 2006a). In the present paper, we compare the potential of the new H₄ receptor agonist VUF 8430 as a pharmacological tool, with the standard H₄ receptor agonist 4-methylhistamine and other histamine receptor ligands. Our results clearly indicate the usefulness of VUF 8430 as a complementary pharmacological tool in dissecting the functions of the histamine H₄ receptor.

Methods

Cell culture and transfection

SK-N-MC cell lines, which stably express either the human H₃ or H₄ receptors as well as a cAMP responsive element (CRE)-driven β-galactosidase reporter gene (Lovenberg et al., 1999; Liu et al., 2001a), were cultured in Eagle's minimal essential medium (EMEM) supplemented with 5% fetal calf serum, 0.1 mg·mL⁻¹ streptomycin, 100 µ·mL⁻¹ penicillin and 600 µg·mL⁻¹ G418 at 37°C in 5% CO₂ and 95% humidity. COS-7 and HEK 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% and 10%, respectively, fetal calf serum, 0.1 mg·mL⁻¹ streptomycin, and 100 µ·mL⁻¹ penicillin. Approximately 10⁶ COS-7 cells were seeded in a 10 cm dish, 1 day prior to transfection. Plasmid DNA was mixed in 0.9% NaCl solution, wherafter 25 kDa polyethyleneimine (PEI) solution (1 mg·mL⁻¹, pH 7.0) was added to obtain a 2:1 mass ratio PEI : DNA. The mixture was incubated for 10 min, and it was then added to the COS-7 cell monolayer.

Radioligand binding assays

Cell homogenates of SK-N-MC cells expressing human H₃ receptors were incubated for 40 min at 25°C with approximately 1 nmol·L⁻¹[³H]N^α-methylhistamine in 25 mmol·L⁻¹ potassium phosphate buffer and 140 mmol·L⁻¹ NaCl (pH 7.4 at room temperature), whereas cell homogenates of SK-N-MC expressing human H₄ receptors were incubated 1 h at room temperature with 10 nmol·L⁻¹[³H]histamine in 50 mmol·L⁻¹ Tris-HCl (pH 7.4 at 37°C), with or without competing ligands. Bound radioligand was collected on 0.3% PEI-pretreated 96-well GF/C filters, which were washed three times with 3 mL of ice-cold washing buffer (4°C) containing 25 mmol·L⁻¹ Tris-HCl and 140 mmol·L⁻¹ NaCl (pH 7.4 at 4°C) for the H₃ receptor and 50 mmol·L⁻¹ Tris-HCl (pH 7.4 at 4°C) for the H₄ receptor). The binding analysis for mouse and rat H₄ receptors were performed as described above for human H₄ receptors. The binding analysis of [³H]mepyramine and [¹²⁵I]iodoaminopotentidine binding to human H₁ receptors and human H₂ receptors, respectively, was performed according to Bakker et al. (2004). The binding data were analysed with Prism 4.0 (Graphpad Software, Inc.) and data are presented as mean ± SEM.

Reporter gene assay

A CRE-β-galactosidase reporter gene assay was employed to determine the activity of the tested ligands at either the human H₃ or H₄ receptor. Approximately 4 × 10⁶ cells per 96-well plate of SK-N-MC cells were exposed for 6 h to the tested compounds in serum-free EMEM medium containing 1 µmol·L⁻¹ forskolin. Thereafter, the medium was discarded, the cells were lysed in 100 µL assay buffer [100 mmol·L⁻¹ sodium phosphate buffer at pH 8.0, 4 mmol·L⁻¹ 2-nitrophenol-β-D-pyranoside (ONPG), 0.5% Triton X-100, 2 mmol·L⁻¹ MgSO₄, 0.1 mmol·L⁻¹ MnCl₂, 40 mmol·L⁻¹β-mercaptoethanol], incubated overnight at room temperature, and the β-galactosidase activity was determined at 420 nm with a PowerwaveX340 plate reader (Bio-Tek Instruments, Inc., USA). To measure activity of the compounds at H₂ receptors, approximately 4 × 10⁶ resuspended HEK 293T cells were transiently cotransfected with a mixture containing 2.5 µg CRE-β-galactosidase reporter gene and 2.5 µg cDNA of the human H₂ receptor, and 35 µL of 1 mg·mL⁻¹ 25 kDa linear PEI, and transferred into a 96-well plate (4 × 10⁴ cells per well). After incubation of 24 h, the cells were exposed with the tested ligands for 6 h. The β-galactosidase activity was determined as described above. For the H₁ receptor, HEK 293T cells were contransfected with NFAT-luciferase reporter gene and cDNA of the human H₁ receptor, with a method as described above, and incubated 24 h before the cells were exposed with the tested compounds for 6 h. Subsequently, the luciferase was determined as described previously (Bakker et al., 2004). The intrinsic activity of agonists was determined relative to the activity of histamine.

Generation of monocyte-derived dendritic cells

Peripheral blood mononuclear cells (PBMC) were separated from heparinized buffy coats by density gradient centrifugation on Lymphoprep (Fresenius Kabi Norge AS, Norway). Adherent cells were obtained by plastic adherence: 1 × 10⁸ PBMC were plated in 80 cm² culture flasks (Nuclon™Δ, Nunc GmbH & Co AG, Wiesbaden, Germany) in Iscove medium supplemented with 5% v/v AB serum, 1% w/v non-essential salts, 2 mmol·L⁻¹ L-glutamine, 100 µ·mL⁻¹ penicillin, 100 mg·mL⁻¹ streptomycin and 0.5% w/v gentamycin (all from Biochrom AG, Berlin, Germany) for 1 h (37°C, 5% CO₂, humidified atmosphere). The non-adherent cells were removed by vigorous washing with phosphate buffered saline and visual inspection. The adherent cells (enriched monocytes, purity at least 85%) were further cultured in RPMI 1640 medium supplemented with 5% v/v FCS, 12 mmol·L⁻¹ HEPES, 2 mmol·L⁻¹ L-glutamine, 100 U·mL⁻¹ penicillin, 100 mg·mL⁻¹ streptomycin, interleukin-4 (10 ng·mL⁻¹, R&D Systems, Wiesbaden, Germany) granulocyte-monocyte-colony stimulating factor (50 ng·mL⁻¹, Berlex Pharmaceutical Company, Montville, USA). Half of the medium was replaced by fresh medium on days 3 and 5 of culture. Non-adherent cells were harvested at day 7 of culture and considered as MoDC as described previously (Gutzmer et al., 2005).

Chemotaxis assay

Chemotaxis of MoDC was measured over polycarbonate membranes with 5 µm pore diameter (Corning Inc., Costar, NY, USA). CCL2 (positive control, 0.8 nmol·L⁻¹), histamine, 4-methylhistamine and VUF 8430 (at 10 µmol·L⁻¹) were used as a chemoattractant in the lower chamber. The upper chamber with the membrane was filled with 250 µL medium containing 10⁶ MoDCs. Chemotaxis was allowed for 1.5 h and the number of transmigrated cells was counted after staining with Trypan blue in a Burker-Türk counting chamber.

Gastric acid secretion in vivo

All animal procedures were in accordance with international guidelines governing animal experimentation, which was approved by the Ethics Committee of the University of Parma. Experiments were carried out with adult male Wistar rats (7–9 weeks, 200–250 g), purchased from Harlan-Italy (MI) and housed at constant temperature (20°C) and humidity (50–55%), with alternating 12-h light and dark cycles, and fed with standard laboratory chow and tap water. Gastric secretion in anaesthetized rats was measured by the stomach-lumen perfusion technique of Bertaccini et al. (1968) with minor modifications. The rats were used after 18-h fasting with free access to water. After anaesthesia with ethyl-urethane (1.25 g·kg⁻¹ intraperitoneally), the stomach was perfused (60 mL·h⁻¹) through an oesophageal cannula with warm saline (NaCl 154 mmol·L⁻¹, 37°C, pH 5.5) at a rate of 1 mL·min⁻¹, using a peristaltic pump. The perfusion fluid was collected in 10-min periods via a duodenal cannula, and titrated to pH 7 with 10 mmol·L⁻¹ NaOH, using an automatic titration system (Radiometer, Copenhagen). In separate experiments, the histamine receptor ligands, amthamine (0.1–1000 µmol·kg⁻¹), histamine (0.01–1000 µmol·kg⁻¹), 4-methylhistamine (3–300 µmol·kg⁻¹), dimaprit (3–300 µmol·kg⁻¹) or VUF 8430 (30–300 µmol·kg⁻¹) were injected intravenously (i.v.) to investigate the effects on basal acid secretion. The H₂ receptor inverse agonist ranitidine (3 mg·kg⁻¹ or 8.6 µmol·kg⁻¹ i.v.) or the H₄ receptor antagonist JNJ 7777120 (10 mg·kg⁻¹ or 36 µmol·kg⁻¹ i.v.) was given 30 min before secretagogues. Acid responses to secretory compounds were calculated for each rat by subtracting basal acid output (average of two collection periods before the stimulant injection) from the maximal acid response (average of two collection periods) and expressed as ΔµEq HCl·kg⁻¹·min⁻¹. Amthamine, dimaprit, histamine, 4-methylhistamine, ranitidine, ethyl-urethane and VUF 8430 were dissolved in saline. DMSO 100% was used as the vehicle to dissolve JNJ 7777120. Each agent was prepared immediately before use and administered in a volume of 0.1 mL per 100 g body weight. Control animals received the vehicle in place of the active agent.

Data analysis

Results are expressed as the means ± SEM of 5–8 rats per group. Comparisons between two groups were made by using the Student's t-test for unpaired data. A value of P < 0.05 was considered statistically significant. The software package Prism GraphPad 4.0 (GraphPad Software Inc., San Diego, CA) was used to process data.

Materials

Amthamine dihydrobromide, dimaprit dihydrobromide, JNJ 7777120, 4-methylhistamine dihydrochloride, thioperamide fumarate and VUF 8430 [S-(2-guanidylethyl)-isothiourea dihydrobromide] were synthesized at the Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, while [¹²⁵I]iodoaminopotentidine was labelled at the Department of Nuclear Medicine and PET Research, Vrije Universiteit Medical Centre, Amsterdam. Forskolin, histamine dihydrochloride, mepyramine (pyrilamine maleate), N^α-methylhistamine dihydrochloride, Pertussis toxin, 750 kDa PEI, ranitidine hydrochloride, agmatine sulfate, putrescine dihydrochloride, spermine, spermidine and Trypan blue were purchased from Sigma RBI (USA). ONPG and G418 were purchased from Duchefa (The Netherlands), and [³H]N^α-methylhistamine (85 Ci·mmol⁻¹), [³H]histamine (12.4 Ci·mmol⁻¹), [³H]mepyramine (23 Ci·mmol⁻¹) were from Perkin-Elmer Life Science, Inc. (USA), and 25 kDa PEI (for transfection) was from Polyscience, Inc. (Germany). Gifts of SK-N-MC cell lines expressing human H₃ receptors, human H₄ receptors, mouse H₄ receptors, rat H₃ receptors and rat H₄ receptors from Dr Lovenberg are greatly acknowledged (Liu et al., 2001a,b).

Results

Binding of VUF 8430 and related compounds to human and rodent H₄ receptors

In radioligand binding studies using [³H]histamine and SK-N-MC cells stably expressing the human H₄ receptor (Liu et al., 2001a), the newly discovered VUF 8430 (Lim et al., 2006a) was almost as potent as histamine and the known histamine H₄ receptor agonist, 4-methylhistamine (Lim et al., 2005) in binding to the H₄ receptor (Figure 1A, Table 1). Moreover, the structurally related H₂ receptor agonist dimaprit was approximately 100-fold less effective than VUF 8430 in displacing [³H]histamine binding to the human H₄ receptor (Figure 1A, Table 1).

Figure 1.

Binding of a variety of amines to the human H₄ receptor. (A) Histamine, dimaprit, VUF 8430 and agmatine, but not arginine, and (B) the polyamines putrescine, cadaverine, spermidine and spermine dose-dependently displaced binding of [³H]histamine to the human H₄ receptor stably expressed in SK-N-MC cells.

Table 1.

Affinity (pK_i) of H₄ receptor ligands for different species variants of H₄ receptor stably expressed in SK-N-MC cells

Ligand	Structure	Human	Mouse	Rat
Histamine		7.9 ± 0.1	7.4 ± 0.1	7.3 ± 0.1
4-methyl-histamine		7.6 ± 0.1	7.2 ± 0.1	6.7 ± 0.1
Dimaprit		6.8 ± 0.1	6.2 ± 0.1	6.0 ± 0.1
VUF 8430		7.5 ± 0.1	7.0 ± 0.1	6.9 ± 0.1
Agmatine		5.6 ± 0.1	5.1 ± 0.1	5.1 ± 0.2
L-arginine		<3	n.d.	n.d.
Putrescine		4.9 ± 0.1	n.d.	n.d.
Cadaverine		4.7 ± 0.1	n.d.	n.d.
Spermidine	NH2-(CH2)4-NH-(CH2)3-NH2	3.7 ± 0.1	n.d.	n.d.
Spermine	NH2-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2	4.3 ± 0.1	n.d.	n.d.

Data shown are mean ± SEM of at least three independent experiments.

n.d., not determined.

The endogenous amine, agmatine, has a clear structural resemblance to VUF 8430 (Table 1) and is formed by decarboxylation of L-arginine by arginine decarboxylase (ADC) and hydrolyzed by agmatinase to putrescine. Agmatine binds to several receptors in the brain and has been proposed as a novel neurotransmitter (Li et al., 1994; Reis and Regunathan, 2000). In our experiments, agmatine showed a micromolar affinity for the human H₄ receptor (Figure 1A, Table 1). As can be seen in Figure 1A, its amino acid precursor, arginine, was totally inactive at the H₄ receptor, whereas its metabolite, putrescine, still showed moderate affinity (Figure 1B, Table 1). Following this observation, we tested a variety of other polyamines (Figure 1B) and found that the putrescine homologue cadaverine, spermine and spermidine all showedmoderate to weak affinities for the human H₄ receptor (Figure 1B, Table 1).

Agonist activities of VUF 8430 and agmatine at human histamine receptors

We subsequently evaluated the functional activities of histamine, VUF 8430, 4-methylhistamine and agmatine at the four human histamine receptor subtypes. As reported previously (Lim et al., 2005; 2006a), both histamine (α = 1 by definition), 4-methylhistamine (α = 1) and VUF 8430 (α = 1) act as full agonists at the human H₄ receptor as measured by the G_i-protein mediated inhibition of the forskolin-induced (1 µmol·L⁻¹) CRE-mediated transcription of β-galactosidase in SK-N-MC cells, stably expressing the human H₄ receptor (B_max = 1.8 pmol·mg⁻¹ protein) and a CRE-β-galactosidase reporter gene (Figure 2A). In this assay, the endogenous agonist histamine was slightly more potent (pEC₅₀ = 7.7 ± 0.1, n = 16) compared with VUF 8430 (pEC₅₀ = 7.3 ± 0.1, n = 6) or 4-methylhistamine (pEC₅₀ = 7.4 ± 0.1, n = 5). Agmatine, under these experimental conditions, was a partial agonist (α = 0.65 ± 0.05, n = 4) with a pEC₅₀ value of 5.4 ± 0.1 (n = 4). At the related human H₃ receptor (Figure 2B), both histamine (α = 1 by definition) and VUF 8430 (α = 1) were full agonists, as measured by the inhibition of the 1 µmol·L⁻¹ forskolin-induced CRE-mediated transcription of β-galactosidase in SK-N-MC cells stably expressing the human H₃ receptor (B_max = 475 fmol·mg⁻¹ protein) and a CRE-β-galactosidase reporter gene. Agmatine also acts as a full agonist (α = 1) at the H₃ receptor (Figure 2B) with a pEC₅₀ value of 6.1 ± 0.1 (n = 3); 4-methylhistamine was clearly less effective at the human H₃ receptor (Figure 2B) and was a partial agonist (α = 0.38 ± 0.12, n = 3) with a pEC₅₀ value of 6.1 ± 0.1 (n = 3).

Figure 2.

Functional activity of histamine, 4-methylhistamine (4-MeHA), VUF 8430 and agmatine at the human H₄ receptor (A), human H₃ receptor (B) expressed in SK-N-MC cells and human H₁ receptor (C) and human H₂ receptor (D) expressed in HEK 293T cells. The NFAT-luciferase reporter gene assay was used to measure H₁ receptor activity, while the CRE-β-galactosidase was employed to measure activation of cAMP production by H₂ receptor or inhibition of forskolin-induced cAMP generation by the H₃ and H₄ receptors.

VUF 8430 did not interact with the histamine H₁ and H₂ receptor subtypes. Histamine displayed a pEC₅₀ value of 7.0 ± 0.1 (n = 4) at human H₁ receptors, measured using HEK 293T cells transiently expressing the human H₁ receptor and a NFAT-luciferase reporter gene (Figure 2C). In the same experimental set-up, 4-methylhistamine, VUF 8430 and agmatine showed no agonist activity at the H₁ receptor at concentrations up to 100 µmol·L⁻¹ (Figure 2C). At human H₂ receptors, histamine displayed a pEC₅₀ of 7.2 ± 0.1 (α = 1, n = 4), measured using COS-7 cells transiently expressing the human H₂ receptor and a CRE-β-galactosidase reporter gene. In this assay, 4-methylhistamine (α = 1) exhibited a pEC₅₀ of 6.8 ± 0.1 (n = 4), whereas VUF 8430 and agmatine only weakly activated the human H₂ receptor at 100 µmol·L⁻¹ (Figure 2D).

Affinity of H₄ receptor agonists at rodent histamine receptors

Previously, significant species differences were reported for the binding of histamine to rodent H₄ receptors (Liu et al., 2001b). We therefore tested VUF 8430 and agmatine for their affinity at mouse and rat H₄ receptors (Figure 3A,B, Table 1). As previously reported (Liu et al., 2001b), histamine showed an three- to fivefold lower affinity for the mouse and rat H₄ receptor compared with the human H₄ receptor, measured by displacement of [³H]histamine binding to homogenates of SK-N-MC cells stably expressing the various H₄ receptor orthologs (Figure 3A, Table 1). Similarly, both VUF 8430 and agmatine showed a somewhat lower affinity at the rodent receptor (Figure 3B, Table 1). At both the mouse (Figure 3C) and rat H₄ receptor (Figure 3D), VUF 8430 was as effective as histamine in inhibiting the 1 µmol·L⁻¹ forskolin-induced CRE activation, displaying pEC₅₀ values of 6.5 ± 0.1 (α = 1, n = 3) and 6.4 ± 0.1 (α = 1, n = 4) respectively. Furthermore, agmatine equipotently and partially activates the mouse and rat H₄ receptors with a pEC50 value of 4.6 ± 0.1.

Figure 3.

Interaction of VUF 8430 with the H₄ receptor species variants. Histamine (A) and VUF 8430 (B) inhibits binding of [³H]histamine to human, mouse and rat H₄ receptors stably expressed in SK-N-MC cells. VUF 8430 inhibited forskolin-induced cAMP responsive element (CRE) through mouse H₄ receptor (C) and rat H₄ receptor (D), to the same extent as histamine.

To understand the action of the various H₄ receptor agonists in in vivo rodent models we have also tested histamine, VUF 8430 and 4-methylhistamine at the four different rat histamine receptors (Table 2). As expected, both 4-methylhistamine and VUF 8430 do not show significant affinity (pK_i < 4) for the rat H₁ receptor and each display a similar affinity for the rat H₂ receptor compared with histamine (Table 2). As observed for the human H₃ receptor, VUF 8430 binds with higher affinity to rat H₃ receptors, compared with 4-methylhistamine, whereas both H₄ receptor agonists show a similar affinity for rat H₄ receptors (Table 2). In the same set of experiments, we also tested the H₄ receptor antagonists thioperamide, JNJ 7777120 (Jablonowski et al., 2003) and VUF 6002 (Terzioglu et al., 2004; Venable et al., 2005) at the four rat histamine receptors. As observed previously for the human histamine receptors (Lim et al., 2005), thioperamide was slightly more active at the rat H₃ receptor (pK_i = 7.8) compared with the rat H₄ receptor (pK_i = 7.2), whereas JNJ 7777120 and VUF 6002 showed a good selectivity (>100 fold) for the rat H₄ receptor (Table 2).

Table 2.

Affinity (pK_i) of H₄ receptor ligands at rat histamine receptors

Ligand	pKi at rat histamine receptor
	H1	H2	H3	H4
Histamine	4.5 ± 0.2	4.3 ± 0.1	8.1 ± 0.1	7.4 ± 0.1
4-methylhistamine	<4	4.1 ± 0.1	5.2 ± 0.1	6.8 ± 0.1
Dimaprit	4.4 ± 0.2	4.5 ± 0.3	6.3 ± 0.1	6.1 ± 0.1
VUF 8430	<4	3.7 ± 0.1	6.5 ± 0.1	6.9 ± 0.1
Thioperamide	4.1 ± 0.1	3.7 ± 0.1	7.8 ± 0.1	7.2 ± 0.1
JNJ 7777120	5.1 ± 0.1	4.9 ± 0.1	5.3 ± 0.1	7.8 ± 0.1
VUF 6002	4.7 ± 0.1	4.5 ± 0.1	5.3 ± 0.1	6.8 ± 0.1

Data shown are mean ± SEM of at least three independent experiments.

In vivo evaluation of VUF 8430 as a pharmacological tool for studies of H₄ receptors

In view of the difference in agonist activity at H₂ receptors, of 4-methylhistamine and VUF 8430 (Figure 2D) we compared VUF 8430 and 4-methylhistamine for their potential to stimulate H₂ receptor function in vivo. We therefore measured gastric acid secretion in unconscious rats after application of histamine, the H₂ receptor agonists dimaprit and amthamine, VUF 8430, or 4-methylhistamine. Both histamine and amthamine are potent inducers in vivo of gastric acid secretion in the rat (Figure 4A). Moreover, dimaprit and 4-methylhistamine also induce gastric acid secretion, although higher doses were needed, whereas VUF 8430 only marginally induced gastric acid secretion at the highest tested doses (Figure 4A). These data are in good accordance with our in vitro findings in transfected cells (Figure 2D). Gastric acid secretion induced by VUF 8430 (30 mg·kg⁻¹ or 50 µmole·kg⁻¹, i.v.) and 4-methylhistamine (10 mg·kg⁻¹ or 93 µmole·kg⁻¹, i.v.) was suppressed by administration of the selective H₂ receptor antagonist ranitidine (3 mg·kg⁻¹, i.v.) (Figure 4B). The H₄ receptor antagonist JNJ 7777120 was not able to reduce 4-methylhistamine or VUF 8430-induced gastric acid secretion (Figure 4B).

Figure 4.

Analysis of gastric acid secretion in anaesthetized rats. The histamine H₂ receptor agonists amthamine and 4-methylhistamine (4-MeHA) induced acid secretion, but VUF 8430 only showed minimal effects at high doses (A). The effects of 4-methylhistamine (30 mg·kg⁻¹ or 93 µmole·kg⁻¹ i.v.) (B) or VUF 8430 (30 mg·kg⁻¹ or 50 µmole·kg⁻¹ i.v.) (C) were inhibited by ranitidine, but no significant inhibition by JNJ 7777120 (D and E) was observed. Values represent the mean ± SEM of responses in 6–8 animals for each experimental group. Comparisons between two groups were made by using the Student's t-test for unpaired data. **P < 0.01 versus 4-methylhistamine (B) or VUF 8430 (C).

Ex vivo evaluation of VUF 8430

We also evaluated the H₄ receptor-mediated effects of VUF 8430 in an ex vivo assay of migration of human MoDCs. The H₄ receptor has been shown to be expressed on the MoDCs and plays a role in histamine-induced chemotaxis (Gutzmer et al., 2005). Histamine-induced migration of MoDCs as efficaciously as the chemokine, CCL2 and this effect was also mimicked by H₄ receptor agonists 4-methylhistamine and VUF 8430 (Figure 5).

Figure 5.

Chemotaxis of human monocyte-derived dendritic cells (MoDCs). MoDCs were loaded in upper wells of migration chambers and allowed to migrate towards chemotactic agents in the lower well through polycarbonate membrane with 5-µm pore diameter. The positive control (0.8 nmol·L⁻¹ CCL2) and the tested compounds [histamine, 4-methylhistamine (4-MeHA), and VUF 8430, all at 10 µmol·L⁻¹] were diluted in cell culture medium as described in Methods.

Discussion and conclusions

In this study, we evaluated VUF 8430 as a pharmacological tool for the study of histamine H₄ receptors, using in vitro, ex vivo and in vivo models. VUF 8430 was originally synthesized in a receptor programme looking for close analogues of the H₂ receptor agonist dimaprit (Durant et al., 1977). However, with the discovery of new histamine receptor subtypes (H₃ and H₄ receptors), we have recently tested a large number of histamine receptor ligands for their activity at the human H₄ receptor (Lim et al., 2005). This detailed evaluation resulted in the discovery of the moderately potent H₂ receptor agonist, 4-methylhistamine, as a potent H₄ receptor agonist (Lim et al., 2005). Moreover, we also observed that dimaprit showed reasonable H₄ receptor agonist activity (Lim et al., 2005) and subsequently described a new efficient synthetic pathway and an initial SAR of the related VUF 8430 (Lim et al., 2006a). This dimaprit derivative shows high affinity at the human H₄ receptor and acts as a full agonist (Lim et al., 2006a; this study). As shown in this study, both 4-methylhistamine and VUF 8430 also act as H₄ receptor agonists at endogenously expressed H₄ receptors in MoDCs. Expression of H₄ receptors was detected in MoDCs at both the mRNA and protein level and these receptors were involved in the activation of the transcription factor AP-1, the inhibition of IL-12p70 secretion and chemotaxis (Gutzmer et al., 2005). We found that both VUF 8430 and 4-methylhistamine induced chemotaxis of MoDCs as effectively as histamine or the chemokine CCL2.

The discovery of VUF 8430 as an H₄ receptor agonist led us to investigate whether agmatine is an H₄ receptor ligand as well. Agmatine is a metabolite of L-arginine, occurs naturally in the body and has been considered to act as an important chemical messenger at other sites such as imidazoline receptors and α₂-adrenoceptors (Li et al., 1994; Reis and Regunathan, 2000; Raasch et al., 2001). In our study, we showed that replacement of the isothiourea group of VUF 8430 with an amine function, as in agmatine, resulted in a sharp drop in binding affinity for H₄ receptors. Nevertheless, in contrast to its precursor, L-arginine, agmatine binds to the H₄ receptor with a K_i value of 2 µmol·L⁻¹. In the reporter gene assay, agmatine showed partial agonist activity at the human H₄ receptor and full agonism at human H₃ receptors. Micromolar concentrations of agmatine are considered to have physiological importance (Lortie et al., 1996; Raasch et al., 2001) and are found in the brain, kidney and other tissues (Li et al., 1994; Lortie et al., 1996; 2004). Hence, agmatine may be one of the endogenous ligands for the H₃ and H₄ receptor. These data also show the importance of the isothiourea group of VUF 8430 in the binding to H₄ receptors. Previously, we reported that the isothiourea group most likely forms hydrogen bonds with residues E5.46 (1.82 Å) and S6.52 (2.21 Å) in the receptor protein (Jongejan et al., 2008). The resulting H-bonding network is identical to the pattern observed for the imidazole ring of histamine, and an essential structural feature for the binding of histamine to the H₄ receptor (Lim et al., 2005).

Besides agmatine, we also observed that other polyamines bind to H₄ receptors; putrescine, cadaverine, spermidine and spermine all showed affinity for H₄ receptors. Cadaverine is commonly found in microorganisms, while putrescine, spermidine and spermine are metabolites of arginine endogenously found in the human body. Like agmatine, concentrations of the polyamines are tightly controlled and they increase under certain conditions, such as liver regeneration, sepsis, brain ischaemia and acute excitotoxic brain damage (Anehus et al., 1986; Gilad et al., 1996; Noguchi et al., 1996; Vivo et al., 2002). The relevance of this action of polyamines on H₄ receptor remains to be elucidated.

A detailed in vitro comparison of the actions of VUF 8430, agmatine and the earlier identified H₄ receptor agonist 4-methylhistamine, indicate that all three compounds, like histamine (Liu et al., 2001b), show a somewhat reduced affinity at the rat and mouse H₄ receptors compared with the human H₄ receptor. In radioligand binding and functional studies, VUF 8430 also shows reasonable affinity and full agonist efficacy at the related H₃ receptors. Similarly, agmatine acts as an efficacious agonist at the H₃ receptor, whereas 4-methylhistamine only shows some agonistic action at H₃ receptors, at high concentrations. Both VUF 8430 and agmatine are very weakly active at the H₂ receptor and inactive at the H₁ receptor. In contrast, despite the relatively low affinity of 4-methylhistamine at the H₂ receptor, 4-methylhistamine is as active as histamine at this receptor. Interestingly, the differential effects of 4-methylhistamine and VUF 8430 at the H₂ receptor were also observed in vivo. In anaesthetized rats, we observed that 4-methylhistamine was effective in stimulating gastric acid secretion in vivo, thus confirming a considerable activity at the H₂ receptor (Durant et al., 1975). Compared with 4-methylhistamine, VUF 8430 is considerably less potent and less efficacious in triggering gastric acid secretion in the rat. Only at very high dosages, VUF 8430 stimulated gastric acid secretion and then only to a very small extent. The stimulatory effect of both H₄ receptor agonists was effectively blocked by the H₂ receptor inverse agonist ranitidine, but not by the H₄ receptor antagonist JNJ 7777120. Based on these data, we conclude that 4-methylhistamine in vivo shows considerable H₂ receptor agonistic activities at dosages >3 mg·kg⁻¹ i.v. For VUF 8430 only at considerable higher dosages (30 mg·kg⁻¹ i.v.) some minor H₂ receptor-mediated effects were observed in vivo.

In conclusion, our search for new ligands for the histamine H₄ receptor has resulted in the discovery of the potent H₄ receptor agonist VUF 8430, which shows a pharmacological profile clearly different from that of the other high affinity H₄ receptor agonist, 4-methylhistamine. Whereas 4-methylhistamine still showed considerable H₂ receptor agonistic activity (in vitro and in vivo), but was devoid of H₁ receptor activity and only weakly activated the H₃ receptor, VUF 8430 on the other hand showed a limited selectivity towards the H₃ receptor, but was very selective with respect to the H₁ or H₂ receptors. Consequently, both high affinity H₄ receptor agonists are complementary and together with the available selective H₄ receptor antagonists can serve as pharmacological tools in future studies to validate the H₄ receptor as a new drug target. Furthermore, identification of VUF 8430 also led to the discovery of agmatine as potential endogenous H₄ receptor agonist. The physiological relevance of agmatine and the polyamines as histamine H₄ receptor ligands remains to be elucidated.

Glossary

Abbreviations:

CRE

cAMP responsive element

JNJ 7777120

1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine

MoDC

monocyte-derived dendritic cell

VUF 8430

2-(2-guanidinoethyl)isothiourea

Conflict of interest

None.