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We examined transcript expression and post-transcriptional regulation of human ADAM33, a recently identified asthma gene. A detailed messenger RNA (mRNA) expression profile was obtained using Northern, reverse transcription polymerase chain reaction, and in situ hybridization analyses. ADAM33 mRNA was expressed significantly in smooth muscle-containing organs, minimally in immune organs and hematopoietic cells, and highly in repairing duodenal granulation tissue. Expression was seen in asthmatic subepithelial fibroblasts and smooth muscle but not in respiratory epithelium. In all tissues, transcripts of approximately 5 kb predominated over those of approximately 3.5 kb by 2- to 5-fold. The effect of the 3' untranslated region (UTR) on ADAM33 protein expression and maturation was examined. The presence of the 3'UTR in untagged full-length constructs promoted prodomain removal, detected as mature approximately 100 kD protein by ADAM33-reactive antibodies; in its absence, maturation was 2- to 3-fold less in HEK293 cells. His-tagged and untagged constructs lacking the 3'UTR demonstrated that lack of maturation was not a result of tag-mediated effects. Minimal maturation of ADAM33 occurred in primary lung and MRC5 fibroblasts following adenoviral-mediated expression of ADAM33 lacking the 3'UTR. In contrast, prodomain removal was observed with plasmids and adenovirus encoding only the pro- and catalytic domains. Thus, the 3'UTR of ADAM33 and domains downstream of the catalytic domain regulate potential ADAM33 activity. Mechanisms of regulation of ADAM33, distinct from closely related ADAMs, thus include mRNA localization and processing and protein maturation.

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AimsTwo clinical studies were conducted to determine possible drug−drug interactions between apremilast and a strong CYP3A4 inhibitor, ketoconazole, or a potent CYP3A4 inducer, rifampicin. The main objectives of these two studies were to evaluate the impact of multiple doses of ketoconazole on the pharmacokinetics of apremilast and its metabolites, and the effect of multiple oral doses of rifampicin on the pharmacokinetics of apremilast.MethodsThese single centre, open label, sequential treatment studies in healthy subjects included two treatment periods for ketoconazole and three treatment periods for rifampicin. Apremilast was administered as a 20 mg (ketoconazole study) or 30 mg (rifampicin study) single oral dose.ResultsKetoconazole increases overall exposure (AUC(0,∞)) of apremilast by ≈36% (2827 vs. 2072 ng ml−1 h, 90% CI = 126.2, 147.5) and peak exposure (Cmax) by 5% (247 vs. 236 ng ml−1). Multiple doses of rifampicin increase apremilast clearance ≈3.6-fold and decrease apremilast mean AUC(0,∞) by ≈72% (3120 vs. 869 ng ml−1 h, 90% CI = 25.7, 30.4) and Cmax (from 290 vs. 166 ng ml−1) relative to that of apremilast given alone. A 30 min intravenous infusion of rifampicin 600 mg had negligible effects on the overall exposure (AUC(0,∞)) of apremilast (2980 vs. 3120 ng ml−1 h, 90% CI = 88.0, 104.1).ConclusionKetoconazole slightly decreased apremilast clearance, resulting in a small increase in AUC which is probably not meaningful clinically. However, the effect of CYP3A4 induction by rifampicin on apremilast clearance is much more pronounced than that of CYP3A4 inhibition by ketoconazole. Strong CYP3A4 inducers may result in a loss of efficacy of apremilast because of decreased drug exposure.

1. Pig nasal mucosal strips were incubated with alpha-adrenoceptor antagonists followed by alpha2-adrenoceptor agonist concentration-response curves. 2. Contractions elicited by the alpha2-adrenoceptor agonists BHT-920 (pD2 = 6.16 +/- 0.07), UK 14,304 (pD2 = 6.89 +/- 0.13) and PGE-6201204 (pD2 = 7.12 +/- 0.21) were blocked by the alpha2-adrenoceptor antagonist yohimbine (0.1 microm). In contrast, the alpha1-adrenoceptor antagonist prazosin (0.03 microm) had no effect on the BHT-920-, UK 14,304- and PGE-6201204-induced contractions, but blocked the contractile response to the alpha(1)-adrenoceptor agonist phenylephrine (pD2 = 5.38 +/- 0.04) and the mixed alpha1- and alpha2-adrenoceptor agonist oxymetazoline (pD(2) = 6.30 +/- 0.22). 3. The alpha2-adrenoceptor antagonist yohimbine (0.01-0.1 microm, pA2 = 8.04), alpha2B/C-adrenoceptor antagonist ARC 239 (10 microm, pK(b) = 6.33 +/- 0.21), alpha2A/C-adrenoceptor antagonist WB 4101 (0.3 microm, pK(b) = 8.01 +/- 0.24), alpha2A-adrenoceptor antagonists BRL44408 (0.1 microm, pK(b) = 6.82 +/- 0.34) and RX 821002 (0.1 microm, pKb = 8.31 +/- 0.35), alpha2C-adrenoceptor antagonists spiroxatrine (1 microm, pKb = 7.32 +/- 0.32), rauwolscine (0.1 microm, pKb = 8.16 +/- 0.14) and HV 723 (0.3 microm, pKb = 7.68 +/- 0.14) inhibited BHT-920-induced contractions in pig nasal mucosa. 4. The present antagonist potencies showed correlations with binding affinity estimates (pKi) obtained for these antagonists at the human recombinant alpha2A- and alpha2C-adrenoceptors (r = 0.78 and 0.83, respectively) and with binding affinity estimates (pKd) obtained in pig native alpha2A- and alpha2C-monoreceptor assays (r = 0.85 and 0.78, respectively). No correlation was observed for the alpha2B-subtype. 5. In conclusion, contractile responses to phenylephrine, BHT-920, UK 14,304, PGE-6201204 and oxymetazoline indicate that alpha1- and alpha2-adrenoceptors are present and mediate vasoconstriction in pig nasal mucosa. Furthermore, correlation analysis comparing antagonist potency in pig nasal mucosa with affinities for human recombinant alpha2-adrenoceptors and native pig alpha2-adrenoceptors suggest that alpha2A- and alpha2C-adrenoceptor subtypes constrict pig nasal mucosa vasculature.

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We define the pharmacological and pharmacokinetic profiles of a novel ␣ 2C -adrenoceptor agonist, compound A [N- [3,4-dihydro-4-(1H-imidazol-4-ylmethyl -ethyl-NЈ-methylurea]. This compound has high affinity (K i ) for the human ␣ 2C -adrenoceptor (K i ϭ 12 nM), and 190-to 260-fold selectivity over the ␣ 2A -and ␣ 2B -adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC 50 ϭ 166 nM) and efficacy (E max ϭ 64%) responses at the ␣ 2C -adrenoceptor, much lower potency and efficacy at the ␣ 2A -adrenoceptor (EC 50 ϭ 1525 nM; E max ϭ 8%) and ␣ 2B -adrenoceptor (EC 50 ϭ 5814 nM; E max ϭ 21%) subtypes, and low or no affinity and functional activity at the ␣ 1A -, ␣ 1B -, and ␣ 1D -adrenoceptor subtypes. In the human saphenous vein postjunctional ␣ 2C -adrenoceptor bioassay, compound A functions as a potent agonist (pD 2 ϭ 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC 50 ϭ 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/ kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating ␣ 2C -adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.