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We investigated in vivo efficacies of the newly synthesized VLA-4 antagonist Compound A {trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid} on Ascaris antigen–induced airway inflammation and hyperresponsiveness in a murine asthmatic model. Oral administration of Compound A significantly inhibited eosinophil infiltration into BALF and airway hyperresponsiveness 48 h after the antigen challenge. Histologic analysis of the lung sections confirmed the BALF result and revealed suppression of edema and mucus hyperplasia at 8 and 48 h after the challenge, respectively. These findings clearly show that orally active Compound A has therapeutic potential for treatment of asthma. Keywords:: VLA-4, eosinophil, airway hyperresponsiveness

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.

For the purpose of obtaining orally potent VLA-4 inhibitors, we have carried out structural modification of the (N'-phenylureido)phenyl group in compound 1, where the group was found to be attributed to poor pharmacokinetic profile in our previous research. Through modification, we have identified several compounds with both potent in vitro activity and improved oral exposure. In particular, compound 7e with 7-fluoro-2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazolyl group as a novel replacement of the (N'-phenylureido)phenyl group significantly inhibited eosinophil infiltration into bronchoalveolar lavage fluid at 15mg/kg in an Ascaris-antigen-induced murine bronchial inflammatory model, and its efficacy was comparable to that of the anti-mouse α(4) antibody (R1-2).

A series of benzoic acid derivatives was synthesized as VLA-4 antagonists. Introduction of chlorine or bromine into the 3-position on the central benzene of the diphenylurea portion as in lead compound 2 led to improvement in the pharmacokinetic properties. In particular, 12l demonstrated an acceptable plasma clearance and bioavailability in mice and rats as well as dogs (mice, CL = 18.5 ml/min/kg, F = 28 % ; rats, CL = 5.2 ml/min/kg, F = 36 % ; dogs, CL = 3.6 ml/min/kg, F = 55 % ). Additionally, 12l exhibited potent activity with an IC50 value of 0.51 nM and efficacy by oral administration at a dosage of 10 mg/kg in a rat pleurisy model.

A novel series of benzoic acid derivatives as VLA-4 antagonists were synthesized. Optimization, focusing on activity and lipophilicity needed for cell permeability, resulted in the identification of 15b and 15e with good activity (IC50 = 1.6 nM each) and moderate lipophilicity (Log D = 2.0, 1.8). Furthermore, 15e demonstrated efficacy in murine asthma model by an oral dose of 30 mg/kg.

Class IA and IB phosphoinositide 3-kinases (PI3Ks) have been shown to regulate mast cell functions such as proliferation, development, survival and degranulation, but the functional redundancy between these two PI3K signaling pathways in mast cells remains unclear. Here, we have generated mice deficient in both class IA regulatory subunit p85α and class IB catalytic subunit p110γ, and show that p85α(-/-)p110γ(-/-) mice exhibit a more severe defect in mast cell development than single-knockout mice. In addition, the in vivo passive cutaneous anaphylaxis reaction of p85α(-/-)p110γ(-/-) mice was nearly completely abrogated, whereas single-knockout mice exhibit just marginal reduction. Pharmacological inactivation of Akt in wild-type bone marrow-derived mast cells (BMMCs) led to partial reduction of degranulation, while over-expression of a constitutively active Akt partially restored the impaired degranulation in p85α(-/-)p110γ(-/-) BMMCs. We also found that the extracellular signal-regulated kinase (ERK) signaling pathway was activated in a PI3K-dependent manner upon FcεRI stimulation and that simultaneous inhibition of Akt and ERK resulted in nearly complete blockade of FcεRI-induced degranulation. Our data provide evidence that Akt and ERK pathways play redundant roles in FcεRI-induced degranulation.

We have focused on optimization of the inadequate pharmacokinetic profile of trans-4-substituted cyclohexanecarboxylic acid 5, which is commonly observed in many small molecule very late antigen-4 (VLA-4) antagonists. We modified the lipophilic moiety in 5 and found that reducing the polar surface area of this moiety results in improvement of the PK profile. Consequently, our efforts have led to the discovery of trans-4-[1-[[2,5-dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid (14e) with potent activity (IC(50) = 5.4 nM) and significantly improved bioavailability in rats, dogs, and monkeys (100%, 91%, 68%), which demonstrated excellent oral efficacy in murine and guinea pig models of asthma. Based on its overall profile, compound 14e was progressed into clinical trails. In a single ascending-dose phase I clinical study, compound 14e exhibited favorable oral exposure as expected and had no serious adverse events.

Optimization of benzoic acid derivatives by introducing substituents into the diphenyl urea moiety led to the identification of compound 20l as a potent VLA-4 antagonist. Compound 20l inhibited eosinophil infiltration into bronchial alveolar lavage fluid in a murine asthma model by oral dosing and its efficacy was comparable to anti-mouse alpha4 antibody (R1-2). Furthermore, this compound significantly blocked bronchial hyper-responsiveness in the model.

Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection when immature T cells are strongly activated through interaction with self peptide-MHC complexes. The expression of Nur77 in thymocytes and T cell lines leads to apoptosis in a manner dependent on its transcriptional activity. It is well established that Nur77 function is negatively regulated by post-translational modification. Here we demonstrate that the MAPK-induced phosphorylation of Nur77 during T cell activation plays a critical role in the induction of apoptosis. Upon T cell receptor (TCR) stimulation, ERK5 (also known as big MAP kinase 1, BMK1), a member of the MAPK family, phosphorylates Nur77, leading to its transcriptional activation. In contrast, the activation of the ERK2 signaling pathway failed to activate Nur77 although ERK2 is also able to phosphorylate Nur77. Furthermore, the blockade of ERK5 signaling pathway suppressed TCR-induced cell death. These results indicate that ERK5 regulates Nur77 function through its phosphorylation.

An investigation into the structure–activity relationship of a lead compound, prolyl-5-aminopentanoic acid 4 , led to the identification of a novel series of 4-piperidinylacetic acid, 1-piperazinylacetic acid, and 4-aminobenzoic acid derivatives as potent VLA-4 antagonists with low nanomolar IC 50 values. A representative compound morpholinyl-4-piperidinylacetic acid derivative ( 13d : IC 50 = 4.4 nM) showed efficacy in the Ascaris -antigen sensitized murine airway inflammation model by oral administration.