Histamine H4 receptor antagonism reduces hapten-induced scratching behaviour but not inflammation

Authors


Wolfgang Bäumer, Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany, Tel.: ++49 (0) 511 9538732, Fax: ++49 (0) 511 9538581, e-mail: wolfgang.baeumer@tiho-hannover.de

Abstract

Abstract:  Effects of the histamine H4 receptor antagonist JNJ 7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) were tested in two models of allergic contact dermatitis. Dermatitis was induced by 2,4-dinitrochlorobenzene and toluene-2,4-diisocyanate, which differ in their Th1–Th2 profile in that way that 2,4-dinitrochlorobenzene is a classical contact allergen with a pronounced Th1-mediated inflammation, while the respiratory chemical allergen toluene-2,4-diisocyanate induces a Th2-dominated inflammation. JNJ 7777120 (15 mg/kg) administered 2 h and 30 min before and 1 h after challenge did not reduce the hapten-induced ear swelling determined 24 h after challenge. This was confirmed by histological evaluation of the ear skin. A repeated administration of the haptens to the rostral part of the back of sensitized animals resulted in a frequent scratching behaviour. An administration of JNJ 7777120 (15 mg/kg) 30 min before challenge reduced this hapten-induced scratching significantly. The H1 receptor antagonist cetirizine also reduced the scratching bouts in sensitized mice. A combination of H1 and H4 receptor antagonists resulted in the strongest inhibition of scratching behaviour associated with allergic dermatitis. These results indicate that H4 receptor antagonism fails to reduce the allergic inflammatory response but strongly inhibits allergen-induced itch. Thus, a combination of H4 and H1 receptor antagonism might be a new strategy to treat pruritus related to allergic diseases like atopic dermatitis.

Introduction

Allergic skin diseases like atopic dermatitis or allergic contact dermatitis are characterized by local inflammation and pruritus. Although histamine is one of the major mediators in allergic diseases the classical H1 antihistamines show insufficient efficacy in treatment of pruritus and allergic inflammatory processes (1). The histamine H2 receptor seems not to be involved in pruritus as H2 receptor agonists do not induce scratching and cimetidine does not reduce histamine-induced scratching (2). The role of the H3 receptor in pruritus is still debated in literature. Intra-dermal injection of the H3/H4 receptor antagonist thioperamide induced scratching behaviour in mice (3). On the other hand, systemically administered thioperamide dose-dependently reduced histamine- or clobenpropit-induced scratching behaviour in mice, which is in agreement to our own observations. In 2000, a new histamine receptor subtype (H4) has been cloned. This H4 receptor is expressed on several haematopoietic cells and it has been shown to be important in mast cell, eosinophil, monocyte, dendritic cells (DC) and T-cell function, as well as in allergic inflammation in vivo (4–7). Thus, an incomplete anti-inflammatory action by H1-antihistamines in allergic diseases like atopic dermatitis might be interpreted as an action of histamine via the H4 receptor. Interestingly, there are studies indicating a central role for the H4 receptor in pruritus, as the H4 receptor agonist clobenpropit induced scratching in mice (2). Furthermore, H4 receptor antagonists are superior to traditional antihistamines in the attenuation of experimental pruritus, supporting this point of view (8). This study was performed to test the H4 receptor antagonist JNJ 7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) in models of allergic contact dermatitis which differ in their Th1–Th2 response and which were established to compare the anti-inflammatory actions of calcineurin inhibitors, glucocorticoids and phosphodiesterase 4 inhibitors (9–11). Apart from ear swelling as an endpoint of inflammation, effects of antihistamines on allergen-induced pruritus were also tested.

Material and methods

Reagents

Toluene-2,4-diisocyanate (TDI), 2,4-dinitrochlorobenzene (DNCB), thioperamide and diphenhydramine were purchased from Sigma (Deisenhofen, Germany). Clobenpropit was obtained from Calbiochem (La Jolla, CA, USA). Cetirizine drop solution (10 mg/ml) was obtained from Aliud pharma (Laichingen, Germany) and diluted with phosphate buffered saline to the end concentration of 15 mg/kg body weight. Ranitidine solution was purchased from Ratiopharm (Ulm, Germany). JNJ 7777120 was synthesized by K. Sander and H. Stark from the Johann Wolfgang Goethe University in Frankfurt/Main as described previously (12). JNJ 7777120 was solved in dimethyl sulphoxide (2.5 mg in 50 μl) and diluted with phosphate buffered saline to the appropriate end concentrations. Vehicle-treated mice received phosphate buffered saline + 2.5% dimethyl sulphoxide (corresponding to the highest concentration used for JNJ 7777120).

Mice

Female BALB/c and NMRI mice were obtained from Charles River (Sulzfeld, Germany) at the age of 8 weeks (20 g body weight). All animals were healthy and were housed in groups of six mice per cage at 22°C with a 12-h light/dark cycle. Water and a standard diet (Altromin, Lage/Lippe, Germany) were available ad libitum. The animal experiment has been approved by the LAVES, Oldenburg, Germany (AZ. G33-42502-06/1126).

Sensitization procedure

NMRI mice (Charles River) were sensitized to toluene-2,4-diisocyanate (TDI) and 2,4-dinitrochlorobenzene (DNCB) as described previously (10) with slight modifications. In brief, the abdominal skin of the mice was shaved and depilated, then the abdominal skin was stripped with adhesive tapes. For active sensitization, 100 μl of 5% TDI in acetone were administered to the stripped epidermis on the first day, followed by 50 μl of 5% TDI on three consecutive days.

The allergic reaction was boosted 21 days later by administration of 20 μl of 0.5% TDI in acetone on the left ears to examine the sensitization status. Before as well as 24 h after challenge, the ear thickness was measured with a cutimeter (model 7309; Mitutoyo, Neuss, Germany). The mice were equally distributed to the treatment groups (n = 6) according to their swelling intensity, so that each group contained animals which had responded to varying extents. They had to rest until the ear thickness had reached almost a normal level after 7 days. To exclude residues of the allergen on the ears, the untreated right ears were used for the main experiment.

The sensitization procedure for DNCB was as follows: 100 μl 0.5% DNCB (acetone/dimethyl sulphoxide, 9:1) was administered to the stripped epidermis on two consecutive days. The animals were treated with 50 μl Freund′s complete adjuvant i.d. to induce a Th1 response. On day 8, the reaction was challenged by administration of 20 μl of 0.5% DNCB on the ears.

Mice received JNJ 7777120 or other histamine antagonists 2 h, 30 min before and 1 h after challenge.

Systemic treatment during sensitization

One group was treated with JNJ 7777120 (15 mg/kg) i.p 1 h before, 1 and 3 h after sensitization with DNCB. The dose was selected from former studies, where JNJ 7777120 was effective in models of peritonitis and experimental pruritus (8,13). A sensitized second group (n = 6) was treated with vehicle at the same times. Ear thickness was determined 24 h after challenge (day 8) and the mice were killed for histological evaluation of ear inflammation.

Systemic treatment during challenge

One group was treated with JNJ 7777120 (15 mg/kg) i.p 2 h, 30 min before and 1 h after challenge with TDI and DNCB, respectively. A sensitized second group (n = 6) was treated with vehicle at the same times. Ear thickness was determined 24 h after challenge and the mice were killed for histological evaluation of ear inflammation. As a positive control, a group of mice (n = 6) was treated with dexamethasone (5 mg/kg i.p.) 2 h, 30 min before and 1 h after challenge with TDI and DNCB, respectively.

To test, whether a H4 receptor agonist increases the allergic inflammatory response, one group of mice received 15 mg/kg clobenpropit (H4 receptor agonist/H3 receptor antagonist) 30 min before TDI challenge and one group received 15 mg/kg clobenpropit 30 min before and 1 h after DNCB challenge. In an additional experiment, clobenpropit was administered topically (500 μm/ear solved in 20 μl acetone/dimethyl sulphoxide, 9:1) 1 h before, 1 and 3 h after TDI challenge. Vehicle treated mice received 20 μl acetone/dimethyl sulphoxide (9:1) at the indicated times.

For blocking all four known histamine receptors, mice received diphenhydramine (H1R antagonist, 30 mg/kg), ranitidine (H2 receptor antagonist, 30 mg/kg) and thioperamide (H3/H4 receptor antagonist, 15 mg/kg) 30 min before as well as 1 h after DNCB challenge. The indicated doses were taken from Satoh et al. (14) for diphenhydramine and Bell et al. (2) for thioperamide. For ranitidine, the dose was selected according to results obtained by Griswold et al. (15).

Histolological examination

One part of the ear tissue collected 24 h after the challenge was fixed in 4% formaldehyde (Fluka, Deisenhofen, Germany) and embedded in paraffin blocks for histological section and stained with haematoxylin–eosin with respect to oedema formation and granulocyte accumulation. The analysis was performed semiquantitatively (‘−’, no inflammatory cell influx/oedema to ‘+++’, high inflammatory cell influx/oedema). These parameters were measured in 10 high-powered fields at 20 times magnification by a blinded examination.

Hapten-induced scratching

Female NMRI or BALB/c mice were sensitized on the abdominal skin with TDI or DNCB as described above. NMRI mice were sensitized to DNCB. NMRI mice were taken as it was demonstrated in previous experiments, that these mice are more susceptible to H4 receptor-mediated scratching (16). On the other hand, several studies concerning scratching behaviour are performed with BALB/c mice (2,17). Thus, to compare results obtained in this experimental setting with other studies, BALB/c mice were sensitized to TDI. The mice rested for 3 weeks followed by a repeated sensitization with 20 μl TDI (1%) or DNCB (0.5%) onto the rostral part of the mouse back twice a week until the mice showed a stable itch reaction (after the fourth boost reaction). This protocol was adapted from that of Inagaki et al. (17). The mice received the histamine antagonists 30 min before topical administration of the indicated haptens. Scratching bouts were recorded for 20 min after administration of the hapten in analogy to ref. (2). Vehicle-treated and hapten-challenged mice showed about 30–70 scratching bouts within 20 min observation period. To compare different treatments and results obtained with NMRI mice with those of BALB/c mice, the counts observed in vehicle groups were set as 100%.

Statistics

Figures are presented as mean (± SEM). Statistically significant differences between the vehicle and histamine antagonist treatment were assessed by a one-way ANOVA followed by a post hoc test (Dunnett′s test). A comparison of only two groups was done by means of a t-test. A P-value of less than 0.05 was regarded as significant. For the statistical analysis, the program Graph Pad prism version 4.03 (GraphPad software, Inc., San Diego, CA, USA), was used.

Results

JNJ 7777120 failed to inhibit the inflammatory response, when administered in the challenge or during sensitization phase

Vehicle-treated NMRI mice had a mean ear swelling of 110 μm (± 23) 24 h after DNCB challenge. Mice treated three times with JNJ 7777120 (15 mg/kg) directly before and after challenge had no diminished ear swelling (110 ± 29 μm). Dexamethasone (5 mg/kg) significantly reduced the DNCB-induced ear swelling.

TDI-sensitized, vehicle-treated mice showed a mean swelling of 68 μm (± 15). Again, no diminished response was observed in JNJ 7777120 treated animals (76 ± 25 μm) in the challenge reaction, whereas dexamethasone strongly reduced the TDI-induced inflammatory reaction (Fig. 1). These results are confirmed by a histological evaluation of oedema formation and inflammatory cell influx (Table 1).

Figure 1.

 Ear swelling in NMRI mice 24 h after challenge with DNCB (grey bars) and TDI (cross hatched bars). The H4 receptor antagonist JNJ 7777120 (JNJ, 15 mg/kg i.p.) did not reduce the allergen-induced ear swelling, whereas systemically administered dexamethasone (5 mg/kg i.p.) reduced the inflammatory response provoked by both haptens. The H4 receptor agonist clobenpropit (Cloben, 15 mg/kg i.p.) did not enhance the allergic inflammatory reaction, when administered directly before and after challenge (dark grey bars and grey hatched bars, respectively), mean ± SEM for six mice, *P ≤ 0.05, **P ≤ 0.01.

Table 1.   Histological evaluation of inflammatory cell influx and oedema formation (mean values) 24 h after toluene-2,4-diisocyanate (TDI) and 2,4-dinitrochlorobenzene (DNCB) challenge, respectively
 TDI + vehicleTDI + JNJ 7777120DNCB + vehicleDNCB + JNJ 7777120DNCB + H1 – H4 antagonists
  1. NMRI mice were treated with 15 mg/kg JNJ 7777120 directly before and after challenge as indicated in Material and methods. For H1 to H4 antagonism, diphenhydramine (30 mg/kg), ranitidine (30 mg/kg) and thioperamide (15 mg/kg) were administered directly before and after challenge. Ears of six mice per group have been evaluated semiquantitatively (‘−’, no inflammatory cell influx/oedema to ‘+++’, high inflammatory cell influx/oedema).

Cell influx++++/+++++++++/+++
Oedema++/+++++++++/+++++

For blocking all four known histamine receptors mice received diphenhydramine (H1 receptor antagonist), ranitidine (H2 receptor antagonist) and thioperamide (H3/H4 receptor antagonist). However, this total blockade of histamine receptors did also not reduce the inflammatory reaction significantly (vehicle + DNCB: 103 ± 21 μm vs antihistamines + DNCB: 88 ± 20 μm).

JNJ 7777120 also failed to reduce an inflammatory response, when administered in the sensitization phase. Mice receiving JNJ 7777120 1 h before, 1 and 3 h after sensitization, had a comparable inflammatory response when the allergic reaction is challenged 8 days later (vehicle + DNCB: 91 ± 24 μm vs JNJ 7777120 +  DNCB: 125 ± 36 μm).

The H4 receptor agonist clobenpropit did not enhance the allergic response

The H4 receptor agonist/H3 receptor antagonist clobenpropit was administered directly before challenge with TDI. This administration did not enhance the allergic inflammatory response. In the DNCB model, clobenpropit was then administered additionally 1 h after challenge, but again, no change in inflammatory response was observed (Fig. 1). Topically applied clobenpropit (500 μm per ear) administered 1 h before, 1 and 3 h after TDI challenge also had no impact on ear swelling measured 24 h after TDI challenge (vehicle + TDI: 90 ± 18 μm, clobenpropit + TDI: 87 ± 15 μm).

JNJ 7777120 dose-dependently inhibited allergen-induced scratching in sensitized mice

JNJ 7777120 dose-dependently reduced the DNCB- and TDI-induced scratching bouts. The reaction is more pronounced in NMRI mice, sensitized to DNCB than in BALB/c mice sensitized to TDI (Fig. 2). The ED50 value for JNJ 7777120 calculated for inhibition of TDI-induced scratching in BALB/c mice was 5.4 mg/kg whereas the ED50 for the DNCB-induced scratching in NMRI mice was 1.2 mg/kg. The histamine antagonists tested for effects on allergic inflammatory response were also studied in allergen-induced pruritus. The H1 receptor antagonist diphenhydramine also inhibited the scratching bouts, whereas the H2 receptor antagonist ranitidine failed to inhibit allergen-induced scratching behaviour in both sensitization procedures. The H3/H4 receptor antagonist thioperamide reduced the allergen-induced scratching in BALB/c mice to a similar extend as that of the H4 receptor antagonist JNJ 7777120 (Fig. 3). As diphenhydramine is a well-known sedative, which might influence scratching behaviour, a newer antihistamine with virtually no sedative component, cetirizine was also tested. Cetirizine inhibited the allergen-induced scratching in DNCB (NMRI mice, not shown) and TDI (Fig. 4, BALB/c mice) sensitized animals. A combination of cetirizine and JNJ 7777120 resulted in the most pronounced inhibition of scratching (Fig. 4). The combination is significantly more effective compared with cetirizine (15 mg/kg) alone (P = 0.016) and also more effective compared with JNJ 7777120 (15 mg/kg) alone (P = 0.053).

Figure 2.

 Data illustrating effects of the H4 receptor antagonist JNJ 7777120 on TDI- (A, BALB/c mice) and DNCB- (B, NMRI mice) induced scratching during the 20 min posthapten administration. JNJ 7777120 (i.p.) dose-dependently reduced the allergic itch response. The inhibitory action was more pronounced in the DNCB model performed with NMRI mice. Mean ± SEM for 6–7 mice each group, *< 0.05 compared with vehicle-treated animals.

Figure 3.

 The histamine antagonists tested for their possible anti-inflammatory action were tested separately in allergen-induced scratching provoked by TDI (scratching bouts were counted 20 min posthapten administration). The H1 receptor antagonist diphenhydramine (Diphen; 15 mg/kg i.p.) inhibited the scratching bouts significantly, whereas the H2 receptor antagonist ranitidine (Ranit; 30 mg/kg i.p.) failed to inhibit allergen-induced scratching behaviour. The H3/H4 receptor antagonist thioperamide (Thiop, 15 mg/kg i.p.) reduced the allergen-induced scratching significantly, mean ± SEM for six BALB/c mice, *< 0.05 compared with vehicle-treated animals.

Figure 4.

 The H1 receptor antagonist cetirizine (Cetir, 5–15 mg/kg i.p.) dose-dependently reduced allergen-induced scratching provoked by TDI (scratching bouts were counted 20 min posthapten administration). Cetirizine (15 mg/kg i.p.) has a similar impact on scratching as JNJ 7777120 (15 mg/kg i.p.). A combination of cetirizine and JNJ 7777120 resulted in the most pronounced reduction. Mean ± SEM for six BALB/c mice, *P < 0.05 compared with vehicle-treated animals.

Discussion

Histamine is a well-known and prominent mediator in allergic diseases like asthma and atopic dermatitis. However, the therapeutic value of classical antihistamines in the control of pruritus, seems to reside principally in their sedative properties (1). The lack of efficacy of H1 (and H2) antihistamines for the treatment of atopic skin lesions might partly be because of the lack of blocking of the H4 receptor mediated histamine actions. H1 antihistamines (e.g. diphenhydramine, cetirizine, loratidine) do not bind to the H4 receptor (18,19). In this study, it is demonstrated that the H4 receptor plays a pivotal role in scratching behaviour associated with allergic dermatitis. However, the inflammatory response seems not to be modified via H4 receptor antagonism, neither in the more Th1-pronounced allergic inflammation (DNCB), nor in the more Th2-dominated allergic inflammatory response provoked by the respiratory chemical allergen TDI (10). This finding is supported by the fact, that the H4 receptor agonist (and H3 receptor antagonist) clobenpropit does not influence the TDI-induced allergic response. The pruritic response to the haptens DNCB as well as TDI was dramatically attenuated by treatment with JNJ 7777120, a selective H4 receptor antagonist. As there exist histamine specific C-fibres, it is postulated that histamine plays a central role in human pruritus (20). Intra-dermal injection of histamine as well as of the H4 receptor agonist clobenpropit induces scratching behaviour in BALB/c (2) and NMRI (16) mice, indicating a central role for histamine in pruritus also for mice. These findings are in line with a study of Dunford et al. (8). They could demonstrate that JNJ 7777120 attenuated H4 receptor-mediated pruritus and also of pruritus provoked in an IgE-dependent mouse model of passive cutaneous anaphylaxis. At least for the TDI model used in this study, a participation of TDI-specific IgE was demonstrated (21). The study of Ingaki et al. (17) shows that a repeated sensitization with DNFB, which is closely related to the DNCB used in this study, results of in enhanced serum IgE levels. Thus, in mice sensitized over a long term with TDI and DNCB, a part of the itch reaction is probably provoked by IgE-mediated mast cell degranulation (thus, the Th1–Th2-polarization valid for the sensitization procedure to evaluate the impact on inflammatory reaction (Fig. 1) must be qualified for the scratching experiments). A direct excitatory effect of histamine via H1, H2, H3, and also H4 receptor has been demonstrated for human enteric neurons. The authors conclude, that mast cells, which are located in proximity to nerves might be the source for histamine (22). This is supported by the fact, that frequency and severity of abdominal pain correlates with the proximity of activated mast cells to nerves (22). A similar role for the mast cell-sensory nerve end interface can be postulated for the skin. However, to clearly point out the role of mast cell-derived histamine, hapten-induced scratching experiments have to be performed in mast cell-deficient mice.

The cytokine mRNA expression in lesional skin of mice long-term sensitized with 2,4-dinitrofluorobenzene demonstrates a participation of IFNγ, IL-4 and IL-5 (17). Own cytokine and chemokine determinations revealed a variety of enhanced cytokines and chemokines involved in TDI-induced allergic inflammation (enhanced concentrations of IL1-β, IL-4, IL-5, IL-6, GMCSF, KC and MIP-1 (23)). In both models (TDI and DNCB) of allergic dermatitis the T-cell attracting chemokines RANTES and TARC are also enhanced (10). However, the precise direct or indirect role of cytokines and chemokines in pruritus is still unclear, although for some cytokines like IL-2 and IL-31 a direct role for pruritus in atopic dermatitis is postulated (24).

Most interestingly, pruritus induced by compound 48/80, a substance which can induce scratching behaviour in mice independent of mast cell mediators (25) was also attenuated by JNJ 7777120 indicating that H4 receptor antagonism may modulate conduction of other pruritic signals. Furthermore, it can be excluded that the antiprurinergic potential of JNJ 7777120 is because of any sedative properties (8). There are many hints that JNJ 7777120 acts on the peripheral sensory nerves as mast cells are not necessary for H4 receptor-mediated scratching (8). However, a parallel expression and activity of the H4 receptor in the central nervous system and an influence by JNJ 7777120 cannot totally be ruled out as this H4 receptor antagonist is described to cross the blood brain barrier (8), but H4 receptor density has been described to be low. There is an obvious difference concerning efficacy of JNJ 7777120 in attenuating the allergen-induced itch response between the two models used in this study (Fig. 2). This might be due to the different haptens used or due to strain-specific differences concerning H4 receptor-mediated pruritus. NMRI mice are much more susceptible to H4 receptor-mediated scratching by clobenpropit than BALB/c mice (16). Thus, further experiments were conducted (TDI-induced scratching in NMRI mice and DNCB-induced scratching in BALB/c mice), which revealed that again the DNCB-induced scratching was attenuated by lower doses of JNJ 7777120 (data not shown), indicating that the hapten and not the strain is the relevant factor. Nevertheless, the higher susceptibility of NMRI mice to H4 receptor-mediated scratching (16) was the reason, why all experiments concerning effects of JNJ 7777120 on allergic inflammatory reaction were performed in NMRI outbred mice with generally similar responses to allergens/haptens compared with BALB/c mice (26). However, the inflammatory reaction was not enhanced by the H4 receptor agonist clobenpropit, nor reduced by the highly selective H4 receptor JNJ 7777120, although a s.c. administered dose of 10 mg/kg JNJ 7777120 showed anti-inflammatory efficacy in a mouse zymosan-induced peritonitis model, which is described to be mast cell dependent (13). In a rat model of carrageenan paw oedema, JNJ 7777120 attenuated the early inflammatory reaction but failed to influence the late responses (27). JNJ 7777120 displayed also some anti-inflammatory effects in a rat colitis model induced by trinitrobenzene sulphonic acid. However, very high concentrations (100 mg/kg) of JNJ 7777120 were used in this study (28). The half-life of JNJ 7777120 determined in mice after oral administration was 1 h (13). It was thus decided to administer JNJ 7777120 several times around challenge and sensitization, respectively. However, even a repeated administration of a dose, which had strong impact on allergen-induced scratching behaviour, did not reduce the allergic inflammatory response in either of the two selected models. This is confirmed by experiments using clobenpropit. The H4 receptor agonist did not enhance the inflammatory response irrespective of administration (systemically or topically). In addition, simultaneous blockade of all four known histamine receptor subtypes did also not affect the inflammatory response (Table 1). These findings are in line with results obtained with histidine decarboxylase knockout mice (29). These mice lacking histamine, showed a reduced scratching behaviour caused by contact dermatitis. However, the histological examination revealed that both histidine decarboxylase knockout mice and wild type mice displayed the similar extent of inflammatory cell infiltration (29).

In conclusion, this study supports recent findings, that the histamine H4 receptor is involved in allergen/histamine associated pruritus (2,8). However, anti-inflammatory properties of JNJ 7777120 were not seen in TDI- or DNCB-induced allergic contact dermatitis, two models in which calcineurin inhibitors, phosphodiesterase 4 inhibitors and glucocorticoids displayed strong anti-inflammatory effects (9–11,30).

Acknowledgements

This study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG BA2071/2-1). S. Wendorff was supported by a grant of the H. Wilhelm Schaumann-Stiftung. Wolfgang Bäumer is appointed as an endowed professor in ‘Veterinary Dermatopharmacology’ granted by Bayer HealthCare AG.

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