Several dermatoses, including psoriasis, atopic dermatitis, and rosacea, alter the expression of the innate immune effector human cathelicidin antimicrobial peptide (CAMP). To elucidate the roles of aberrant CAMP in dermatoses, we performed cDNA array analysis in CAMP-stimulated human epidermal keratinocytes, the primary cells responding to innate immune stimuli and a major source of CAMP LL37 in skin. Among LL37-inducible genes, IL-1 cluster genes, particularly IL36G, are of interest because we observed coordinate increases in CAMP and IL-36γ in the lesional skin of psoriasis, whereas virtually no CAMP or IL-36γ was observed in nonlesional skin and normal skin. The production and release of IL-36γ were up to 20–30 ng/ml in differentiated keratinocytes cultured in high-calcium media. G-protein inhibitor pertussis toxin and p38 inhibitor suppressed IL-36γ induction by LL37. As an alarmin, LL37 induces chemokines, including CXCL1, CXCL8/IL8, CXCL10/IP-10, and CCL20/MIP3a, and IL-36 (10–100 ng/ml) augments the production of these chemokines by LL37. Pretreatment with small interfering RNA against IL36γ and IL-36R IL36R/IL1RL2 and IL1RAP suppressed LL37-dependent IL8, CXCL1, CXCL10/IP10, and CCL20 production in keratinocytes, suggesting that the alarmin function of LL37 was partially dependent on IL-36γ and its receptors. Counting on CAMP induction in innate stimuli, such as in infection and wounding, IL-36γ induction by cathelicidin would explain the mechanism of initiation of skin inflammation and occasional exacerbations of psoriasis and skin diseases by general infection.

Mycosis fungoides (MF) starts as an indolent disease, progresses from a patch stage to confluent plaques and ultimately develops skin tumors. Tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment in MF. The purpose of this study was to elucidate the involvement of TAMs in the lesional skin of different stages of MF. First, we immunohistologically examined the percentage of CD163+ macrophages and CD206+ cells, as well as the levels of periostin and IL-4 in cancer stroma. The percentage of CD206+ cells increased in parallel with tumor progression, while there was no significant difference in the percentage of CD163+ cells. Periostin was prominent in the stromal area at the patch and plaque stages but decreased at the tumor stage. In contrast, IL-4 was prominently stained at both plaque and tumor stages. To further elucidate the molecular mechanisms of the effects of these stromal factors on TAMs, we examined their effects on mRNA expression in monocyte-derived macrophages in vitro. Based on microarray analysis and gene ontology, we examined a series of chemokines and MMPs whose expression was strongly connected with periostin stimulation. The DNA microarray results were verified in M2 macrophages using real-time PCR. We further examined the mRNA expression of these chemokines and MMPs in the presence of periostin and IL-4 to simulate the advanced stages of MF and validated their protein expression by ELISA. Our present report suggests possible roles of periostin on TAMs in establishing the tumor microenvironment in MF.

A novel class of potent, selective, and orally active non-peptide bradykinin (BK) B2 receptor antagonists were designed and synthesized starting from 8-benzyloxyimidazo[1,2-a]pyridine derivative 2. The unique screening lead (2) was discovered by a two-step intentional random screening process, involving recognition of the relationship between BK and angiotensin II (Ang II) and the common structural features. Systematic chemical modification of 2 elucidated the structural requirements essential for B2 binding affinity leading to the identification of 8-[[3-(N-acylglycyl-N-methylamino)-2,6-dichlorobenzyl]oxy]-3-halo- 2- methylimidazo[1,2-a]pyridine skeleton as the basic framework of this new series of B2 antagonists. A molecular modeling study suggested the key role of the N-methylanilide moiety at the 3-position of the 2,6-dichlorobenzene ring to allow these compounds to adopt the characteristic active conformation. The representative lead compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors, with nanomolar IC50S and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at an oral dose of 1 mg/kg. Pharmacokinetic studies of compounds 47c and 50b in rats highlighted their excellent oral bioavailabilities, indicating that they represent the first orally active non-peptide B2 antagonists reported to date.

An orally active, nonpeptide bradykinin (BK) B2 receptor antagonist, FR173657 (E)‐3‐(6‐acetamido‐3‐pyridyl)‐N‐[N‐[2‐4‐dichloro‐3‐[(2‐methyl‐8‐quinolinyl) oxymethyl]phenyl]‐N‐methylaminocarbonyl‐methyl]acrylamide) has been identified. This compound displaced [3H]‐BK binding to B2 receptors present in guinea‐pig ileum membranes with an IC50 of 5.6 × 10−10 M and in rat uterus with an IC50 of 1.5 × 10−9 M. It did not inhibit different specific radio‐ligand binding to other receptor sites. In human lung fibroblast IMR‐90 cells, FR173657 displaced [3H]‐BK binding to B2 receptors with an IC50 of 2.9 × 10−9 M and a Ki of 3.6 × 10−10 M, but did not reduce [3H]‐des‐Arg10‐kallidin binding to B1, receptors. In guinea‐pig isolated preparations, FR173657 antagonized BK‐induced contractions with an IC50 of 7.9 × 10−9 M, but did not antagonize acetylcholine or histamine‐induced contractions even at a concentration of 10−6 M. FR173657 caused parallel rightward shifts of the concentration‐response curves to BK at concentrations of 10−9 M and 3.2 × 10−9 M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration‐response curve at a concentration of 10−8 M. Analysis of the data yield a pA2 of 9.2 ± 0.2 (n = 5) and a slope of 1.5 ± 0.2 (n = 5). In vivo, the oral administration of FR173657 inhibited BK‐induced bronchoconstriction dose‐dependently in guinea‐pigs with an ED50 of 0.075 mg kg−1, but did not inhibit histamine‐induced bronchoconstriction even at 1 mg kg−1. FR173657 also inhibited carrageenin‐induced paw oedema with an ED50 of 6.8 mg kg−1 2 h after the carrageenin injection in rats. These results show that FR173657 is a potent, selective, and orally active bradykinin B2 receptor antagonist.

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We describe the receptor binding and antagonistic properties of two novel nonpeptide antagonists, FR167344 (3-bromo-8-[2,6-dichloro-3-[N-[(E)-4-(N,N-dimethylcarbamoyl)cinnamido acetyl]-N-methylamino]benzyloxy]-2-methylimidazo[1,2-a]pyridine hydrochloride) and FR173657 (8-[3-[N-[(E)-3-(6-acetamidopyridin-3-yl)acryloylglycyl]-N-m ethylamino]-2,6-dichlorobenzyloxy]-2-methylquinoline), for the human bradykinin receptor subtypes (B1 and B2). In competitive experiments using membranes prepared from Chinese hamster ovary cells expressing the bradykinin receptor subtypes, FR167344 and FR173657 showed a high affinity binding to the B2 receptor with IC50 values of 65 and 8.9 nM, respectively, and no binding affinity for the B1 receptor. FR167344 and FR173657 inhibited the B2 receptor-mediated phosphatidylinositol (PI) hydrolysis and produced a concentration-dependent rightward shift in the dose-response curve to bradykinin. This shift was accompanied by a progressive reduction of maximal response. Estimated pA2 values for the antagonism of bradykinin-induced PI hydrolysis by FR167344 and FR173657 were 8.0 and 9.0, respectively. FR167344 and FR173657 showed no stimulatory effects on PI hydrolysis. Therefore, FR167344 and FR173657 are potent, highly selective, and insurmountable antagonists for the human bradykinin B2 receptor.

In the course of our studies on non-peptide bradykinin (BK) B(2) receptor ligands, it was suggested that the 4-substituent of the quinoline ring may play a critical role in determining binding affinities for human and guinea pig B(2) receptors, as well as agonist/antagonist properties. We carried out an extensive investigation to elucidate the structure-activity relationships (SAR) for this key pharmacophore. Introduction of lower alkoxy groups to the 4-position of the quinoline ring of 3 led to the identification of 4-ethoxy derivative 22b as a unique partial agonist. This compound significantly stimulated inositol phosphates (IPs) formation in Chinese hamster ovary cells expressing the cloned human B(2) receptor at concentrations greater than 10 nM and displayed one-tenth of the intrinsic activity of BK. The agonist activity of 22b was selective for the B(2) receptor and was inhibited by selective peptide and non-peptide B(2) antagonists. On the other hand, 22b strongly suppressed BK-induced IPs formation through the cloned human B(2) receptor. Further studies on the key pharmacophore led to identification of a 2-picolyloxy moiety as a powerful agonist switch, leading to the discovery of a potent and efficacious non-peptide B(2) agonist, 19a. Successive optimization of the acyl side chain afforded 38, which exhibited full agonist activity on stimulation of IPs formation. Furthermore, this strategy could be applied successfully to the benzimidazole series. The representative 1-(2-picolyl)benzimidazole derivative 47c increased PGE(2) production at a 1 microM concentration to the same level as the maximum effect of BK. Thus, we have established the medicinal chemistry modifications required to convert our highly potent non-peptide B(2) antagonists to agonists with potent efficacy.

Recently we reported on overcoming the species difference of our first orally active non-peptide bradykinin (BK) B2 receptor antagonists, incorporating an 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton, leading to identification of the first clinical candidate 4a (FR167344). With this potent new lead compound in hand, we then investigated further refinement of the basic framework by replacement of the imidazo[1,2-a]pyridine moiety and discovered several bioisosteric heterocycles. Extensive optimization of these new heteroaromatic derivatives revealed the detailed structure-activity relationships (SAR) around the imidazo[1, 2-a]pyridine ring and the 2,6-dichlorobenzyl moiety, leading to the discovery of our second clinical candidate 87b (FR173657) which inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50's of 1.4 and 0.46 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.075 mg/kg by oral administration. Further modifications of the terminal substituents on the pyridine moiety led to a novel pharmacophore and resulted in the identification of 99 (FR184280), whose IC50 value for human B2 receptors (0.51 nM) was comparable to that of the second-generation peptide B2 antagonist Icatibant.