Characterization of the chloroquine-induced mouse model of pruritus using an automated behavioural system.

Pruritus is a major symptom of several dermatological diseases but has limited therapeutic options available. Animal models replicating the pathophysiology of pruritus are needed to support the development of new drugs. Induction of pruritus by chloroquine (CQ) in mice is widely used, although, as with similar models, it has low throughput and does not distinguish between antipruritic effects and confounding factors such as sedation. To overcome these issues, we incorporated into the model an automated system that measures both scratching and locomotor behaviour simultaneously. We combined this system with the determination of CQ levels in different tissues to understand the impact of the route of CQ administration on the pruritogenic response. We concluded that whereas oral CQ does not induce pruritus due to insufficient skin levels, the bell-shaped curve of pruritus observed following subcutaneous administration is due to toxicity at high doses. We validated the model with several drugs currently used in humans: nalfurafine, aprepitant, cyproheptadine and amitriptyline. By comparing the effects of the drugs on both scratching and locomotor activity, we concluded that nalfurafine and aprepitant can exhibit efficacy at doses devoid of central effects, whereas central effects drove the efficacy of the other two drugs. This was further confirmed using non-brain-penetrant drugs. Moreover, as anticipated, anti-inflammatory drugs showed no efficacy. In conclusion, the use of an automated integrated behavioural assessment in CQ-induced pruritus makes the assay suitable for screening purposes and allows for a correct interpretation of the antipruritic effect of the compounds evaluated.

Selective Sphingosine 1-Phosphate Receptor 1 Agonist Is Protective Against Ischemia/Reperfusion in Mice.

BACKGROUND AND PURPOSE: Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke. METHODS: Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion. RESULTS: In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups. CONCLUSIONS: The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.

Discovery of a novel class of zwitterionic, potent, selective and orally active S1P1 direct agonists.

Amido-1,3,4-thiadiazoles have been identified as a novel structural class of potent and selective sphingosine-1-phosphate receptor subtype 1 agonists. Starting from a micromolar HTS hit with the help of an in-house homology model, robust structural-activity relationships were developed to yield compounds with good selectivity and excellent in vivo efficacy in rat models.

Biaryl analogues of teriflunomide as potent DHODH inhibitors.

The structure-activity relationships of a novel series of biaryl dihydroorotate dehydrogenase (DHODH) inhibitors related to teriflunomide are disclosed. These biaryl derivatives were the result of structure-based design and proved to be potent DHODH inhibitors which in addition showed good antiproliferative activities on peripheral blood mononuclear cells and good efficacies in vivo in the rat adjuvant-induced-arthritis model.

ß2-adrenoreceptors control human skin microvascular reactivity.

Topical α1- and α2-adrenoreceptor (ADRA1 and 2) agonists are effective in alleviating permanent vasodilation and facial erythema associated with rosacea by inducing skin vasoconstriction. Although ß-adrenoreceptor (ADRB) antagonists are used off-label for rosacea, pharmacological and pharmacodynamic data pertaining to these receptors in skin micro-vessels are lacking. Objectives: To analyse the expression of different adrenergic receptors and their contribution to vasoreactivity in skin micro-vessels. Small arteries (500-800 µm) and arterioles (<200 µm) were studied in human foreskin tissue. Specifically, ADR-A1, -A2, -B1 and -B2 expression was assayed by immunofluorescence, polymerase chain reaction (PCR), and western blotting. Small skin artery reactivity was evaluated using ex vivo myography (500-800 µm) or a visible microscope perfusion system with precision-cut skin slices (<200 µm). ADRB2 was the most highly expressed receptor in small skin arteries and arterioles, followed by ADRA2. ADRA2 activation via brimonidine-induced vasoconstriction was greater in skin arterioles than in small skin arteries, and more potent than that with norepinephrine (NE). The use of prazosin (ADRA1 inhibitor) partially attenuated brimonidine-induced vasoconstriction, indicating some activation of ADRA1 by brimonidine, at least at 10-µM concentrations. Small skin arteries and arterioles, pre-treated with prazosin and stimulated with NE, exhibited ADRB2-mediated vasodilation, which was inhibited by the beta blockers, propranolol or timolol. This study shows that ADRB2 is predominantly expressed in small skin arteries and arterioles, and that ADRBs plays a functional role in vasodilation. The data presented here indicate that ADRBs can be a therapeutic target for the treatment of rosacea.

Rac mediates TNF-induced cytokine production via modulation of NF-kappaB.

TNF is a key factor in a variety of inflammatory diseases. Here we report that TNF induced pro-inflammatory cytokine synthesis of IL-6 and IL-8 is mediated by the Rho GTPase Rac. TNF induces p42/p44, p54 and p38 MAPK kinase; these kinases have been implicated in control of cytokine synthesis. However, over-expression of a dominant negative form of Rac strongly inhibited TNF-induced p42/44 MAPK kinase activation, but had little effect upon JNK and no effect upon p38 MAPK activity. Another key signalling pathway controlling cytokine expression is NF-kappaB. When analyzing TNF-induced NF-kappaB activity via luciferase-reporter assays or via EMSA, we were able to show that the dominant negative version of Rac could completely abrogate TNF-induced NF-kappaB activity. In addition, we also observed that inhibition of the ERK pathway led to a reduction in TNF-induced NF-kappaB transcriptional activity; this was accompanied by an ablation of TNF-induced p65 phosphorylation at serine 276. This would suggest that TNF-induced activation of Rac, lies upstream of NF-kappaB activation, and that the inhibition of this pathway results in inhibition of cytokine production.

Prediction of the skin permeability of topical drugs using in silico and in vitro models.

The main challenge of topically applied drugs is to overcome the skin barrier to reach the site of action at the concentration needed for efficacy. In the research of new topical drugs, design of molecules with optimized properties for skin penetration is a key factor and assays for its characterization are needed. A group of 20 representative topical molecules of clinical use were studied in two in silico models (Potts & Guy and Barratt), and an in vitro assay with artificial membrane (Skin-PAMPA). A subset of 9 drugs were also evaluated in the Franz cells assay, formulated in a solvent and in a marketed formulation. Each assay allowed us to grade compounds according to their permeability value. Globally good alignments were found for the studied compounds when comparing models, although discrepancies for some compounds such as tazarotene, tacrolimus, ketoconazole and metronidazole were observed. Overall, the studied in silico and the in vitro models are useful tools to support selection and characterization of research compounds in terms of skin permeability.

Therapeutic Effect of a Novel Phosphatidylinositol-3-Kinase δ Inhibitor in Experimental Epidermolysis Bullosa Acquisita.

Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal-epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases.

Discovery and characterization of 4'-(2-furyl)-N-pyridin-3-yl-4,5'-bipyrimidin-2'-amine (LAS38096), a potent, selective, and efficacious A2B adenosine receptor antagonist.

A novel series of N-heteroaryl 4'-(2-furyl)-4,5'-bipyrimidin-2'-amines has been identified as potent and selective A(2B) adenosine receptor antagonists. In particular, compound 5 showed high affinity for the A(2B) receptor (Ki = 17 nM), good selectivity (IC(50): A(1) > 1000 nM, A(2A) > 2500 nM, A3 > 1000 nM), displayed a favorable pharmacokinetic profile in preclinical species, and showed efficacy in functional in vitro models.

Chemokine receptors: attractive targets for drug discovery.

Studies of two antibodies, efalizumab and natalizumab, have recently demonstrated that the blockade of leukocyte migration is of therapeutic benefit for the treatment of diseases such as psoriasis and multiple sclerosis. The role of chemokines in the control of cell traffic led to their receptors being considered one of the most promising family of targets aimed at disrupting cell recruitment in chronic inflammatory processes. Choosing the appropriate chemokine receptor for each disease was not easy, and the interpretation of target validation studies proved to be extremely difficult. Despite an intense effort in the search for chemokine receptor antagonists in the last decade, no compounds in advanced clinical trials exist as such. The inherent complexity of the family, the differences between the chemokine system in mice and men, and the species selectivity of small-molecule compounds could account for this fact. Pharmaceutical companies still believe in chemokine receptors as therapeutic targets, as demonstrated by the number of compounds reported to be in development. In the next years, the developmental progression of these compounds will reveal which target within the chemokine family is of real therapeutic value.

Epigenetic regulation of IL-12-dependent T cell proliferation.

It is well established that the cytokine IL-12 and the transcription factor STAT4, an essential part of the IL-12 signaling pathway, are critical components of the Th1 differentiation process in T cells. In response to pathogenic stimuli, this process causes T cells to proliferate rapidly and secrete high amounts of the cytokine IFN-γ, leading to the Th1 proinflammatory phenotype. However, there are still unknown components of this differentiation pathway. We here demonstrated that the expression of the histone methyltransferase Mll1 is driven by IL-12 signaling through STAT4 in humans and mice and is critical for the proper differentiation of a naïve T cell to a Th1 cell. Once MLL1 is up-regulated by IL-12, it regulates the proliferation of Th1 cells. As evidence of this, we show that Th1 cells from Mll1(+/-) mice are unable to proliferate rapidly in a Th1 environment in vitro and in vivo. Additionally, upon restimulation with cognate antigen Mll1(+/-), T cells do not convert to a Th1 phenotype, as characterized by IFN-γ output. Furthermore, we observed a reduction in IFN-γ production and proliferation in human peripheral blood stimulated with tetanus toxoid by use of a specific inhibitor of the MLL1/menin complex. Together, our results demonstrate that the MLL1 gene plays a previously unrecognized but essential role in Th1 cell biology and furthermore, describes a novel pathway through which Mll1 expression is regulated.

Drug discovery and chemokine receptor antagonists: eppur si muove!

The blockade of leukocyte migration has been demonstrated to be a valid option for the treatment of several autoimmune diseases. Chemokines play an active role in regulating cell infiltration into inflammatory sites and disrupting chemokine-receptor interactions has emerged as an alternative therapeutic approach. Pharmaceutical companies have developed an intense activity in the drug discovery of chemokine receptor antagonists in the last 10 years. Potent and selective compounds have been obtained and some of them are currently being evaluated in the clinic. The success of these trials will demonstrate whether the blockade of a single receptor is of therapeutic benefit. Alternative approaches, such as pan-receptor antagonists or inhibitors of the signalling pathways evoked by chemokines, are also being explored. In the meantime, new relationships between chemokines and receptors will be revealed, increasing our knowledge of such a fascinating field.

Chemokines in autoimmunity: from pathology to therapeutics.

Leukocyte recruitment, accumulation, and activation have been both a unifying and enigmatic feature of a variety of autoimmune pathologies. While these processes were not well-known for decades, recent scientific discoveries have underscored the importance of specific chemokines in the evolution of autoimmunity. This has been supported by in vivo data from clinical studies and animal model experiments. Although chemokines are an attractive target for drug development, there are hurdles that need to be cleared. Nonetheless, the quest to understand chemokine biology and develop effective therapies continues to capture the imagination of scientists in academia and pharma alike.

Synthesis and biological evaluation of 2-phenylpyran-4-ones: a new class of orally active cyclooxygenase-2 inhibitors.

A series of 2-phenylpyran-4-ones were prepared and evaluated for their ability to inhibit cyclooxygenase-2 (COX-2). Extensive structure-activity relationship work was carried out within this series, and a number of potent and selective COX-2 inhibitors were identified. Compounds having a p-methylsulfone group at the 2-phenyl ring showed the best COX-2 inhibitory activity. The introduction of a substituted phenoxy ring at position 3 enhanced both the in vitro and in vivo activity within the series. A selected group of 3-phenoxypyran-4-ones exhibited excellent activity in an experimental model of pyresis. The in vivo antiinflammatory activity of these compounds was confirmed with the evaluation of their antiarthritic and analgesic effectiveness. Moreover, their pharmacokinetic profile in rats is compatible with a once a day administration by oral route in humans. Within this novel series, compounds 21, 31, 34, and 35 have been selected for further preclinical and clinical evaluation.

Inhibition of delta-like ligand 4 decreases Th1/Th17 response in a mouse model of multiple sclerosis.

Notch is a family of receptors involved in the differentiation of several tissues, including the central nervous system and the immune system. One of the Notch ligands, delta-like 4 (Dll4), has been implicated in the differentiation of Th1 cells and the development of Th17 responses, which are involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Our results show that a single administration of an anti-Dll4 antibody is not enough to avoid the development of EAE or to ameliorate the already established clinical signs, despite the treatment reduces the proliferative T cell responses and decreases Th1/Th17 immune responses.

Differential pharmacological behaviour of p38 inhibitors in regulating the LPS-induced TNF-α production in human and rat whole blood in vitro.

p38 inhibitors are potent TNF-α suppressors in LPS-stimulated human whole blood and promote efficacy in the rat adjuvant arthritis model. However, the anti-TNF-α activity of p38 inhibitors in rat whole blood has not been explored, preventing the establishment of a potential relation between in vitro and in vivo activity data in the same species. We have pharmacologically characterized a rat whole blood assay based on LPS stimulation. While p38 inhibitors showed good activity in the human assay, they failed to inhibit TNF-α in the rat whole blood assay. At high LPS concentration some compounds even potentiated TNF-α production in the rat assay, which could be reverted in the presence of the ERK pathway inhibitor U0126. Our results suggest that p38 contributes directly to TNF-α production in human whole blood while playing a negative regulatory role in rat blood which can be overridden by p38 inhibition in the presence of high stimulus concentration.

The sphingosine-1-phosphate receptor-1 antagonist, W146, causes early and short-lasting peripheral blood lymphopenia in mice.

Agonists of the sphingosine-1-phosphate (S1P) receptors, like fingolimod (FTY720), are a novel class of immunomodulators. Administration of these compounds prevents the egress of lymphocytes from primary and secondary lymphoid organs causing peripheral blood lymphopenia. Although it is well established that lymphopenia is mediated by S1P receptor type 1 (S1P1), the exact mechanism is still controversial. The most favored hypothesis states that S1P1 agonists cause internalization and loss of the cell surface receptor on lymphocytes, preventing them to respond to S1P. Hence, S1P1 agonists would behave in vivo as functional antagonists of the receptor. For this hypothesis to be valid, a true S1P1 antagonist should also induce lymphopenia. However, it has been reported that S1P1 antagonists fail to show this effect, arguing against the concept. Our study demonstrates that a S1P1 antagonist, W146, induces a significant but transient blood lymphopenia in mice and a parallel increase in CD4+ and CD8+ lymphocytes in lymph nodes. Treatment with W146 also causes the accumulation of mature T cells in the medulla of the thymus and moreover, it induces lung edema. We show that both the S1P1 antagonist and a S1P1 agonist cause lymphopenia in vivo in spite of their different effects on receptor expression in vitro. Although the antagonist purely blocks the receptor and the agonist causes its disappearance from the cell surface, the response to the endogenous ligand is prevented in both cases. Our results support the hypothesis that lymphopenia evoked by S1P1 agonists is due to functional antagonism of S1P1 in lymphocytes.

Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist.

The structure-activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels.

Understanding autoimmune disease: new targets for drug discovery.

A more complete understanding of the mechanisms that drive autoimmune diseases has begun to be translated into therapeutic options with significant clinical consequences. A clear example of this is the introduction of biological therapies, which have provided new therapeutic avenues, as well as validated the mediators (TNFalpha, IL-6), mechanisms (T cell costimulation, leukocyte migration), and cellular players (T and B lymphocytes) of the disease process itself. New discoveries into the role of Th17 and regulatory T cells and the epigenetic regulation of cytokine expression may offer novel intervention strategies to satisfy the unmet medical needs that still exist in these diseases.

Design, synthesis, and structure-activity relationships of aminopyridine N-oxides, a novel scaffold for the potent and selective inhibition of p38 mitogen activated protein kinase.

A novel series of aminopyridine N-oxides were designed, synthesized, and tested for their ability to inhibit p38alpha MAP kinase. Some of these compounds showed a significant reduction in the LPS-induced TNFalpha production in human whole blood. Structure-activity relationship studies revealed that N-oxide oxygen was essential for activity and was probably a determinant factor for a marked selectivity against other related kinases. Compound 45 was identified as a potent and selective p38alpha inhibitor with an appropriate balance between potency and pharmacokinetics. In vivo efficacy of 45 was demonstrated in reducing TNFalpha levels in an acute murine model of inflammation (ED(50) = 1 mg/kg in LPS-induced TNFalpha production when dosed orally 1.5 h prior to LPS administration). The oral efficacy of 45 was further demonstrated in a chronic model of adjuvant arthritis in rats with established disease when administered orally (ED(50) = 4.5 mg/kg).